Lipid Metabolism and Health - EBOOKOID PDF

Lipid
Metabolism
and
Health
Lipid
Metabolism
and
Health
Edited by
Robert J. Moffatt
Bryant Stamford
Boca Raton London New York
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Taylor & Francis Group, the academic division of T&F Informa plc.
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Lipid metabolism and health / [edited by] Robert J. Moffatt and Bryant Stamford.
p. cm.
Includes bibliographical references and index.
ISBN 0-8493-2680-X (alk. paper)
1. Lipids--Metabolism. 2. Health. I. Moffatt, Robert J. II. Stamford, Bryant A.
QP751.L5475 2005
612.3’97--dc22 2005053181
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Contributors
Sofiya Alhassan, Ph.D. Stanford University School of Medicine, Stanford,

California
Theodore J. Angelopoulos, Ph.D., M.P.H. Exercise Physiology Laboratory,

University of Central Florida, Orlando, Florida
Vic Ben-Ezra, Ph.D. D e p a r t m e n t o f K i n e s i o l o g y, Te x a s Wo m e n ’ s

University, Denton, Texas
Robert Carter III, Ph.D. Laboratory of Adaptation Physiology, Thermal

and Mountain Medicine, United States Army Research Institute of
Environmental Medicine, Natick, Massachusetts
Sarah Chelland, M.S. Department of Nutrition, Food and Exercise

Sciences, Florida State University, Tallahassee, Florida
Yumei Coa, B.S. Department of Nutritional Sciences, Pennsylvania State

University, University Park, Pennsylvania
Stephen F. Crouse, Ph.D. Texas A&M University, College Station, Texas
Paul G. Davis, Ph.D. Department of Exercise and Sport Science, University

of North Carolina at Greensboro, Greensboro, North Carolina
Jacqueline L. Dupont, Ph.D., Hazel K. Stiebeling Professor, Department

of Nutrition, Food and Exercise Sciences, Florida State University,
Tallahassee, Florida
J. Larry Durstine, Ph.D. Department of Exercise Science, University of

South Carolina, Columbia, South Carolina
Sarah Gebaur, B.S. Department of Nutritional Sciences, Pennsylvania

State University, University Park, Pennsylvania
Peter W. Grandjean, Ph.D. Department of Health & Human Performance,

Auburn University, Auburn, Alabama
Amy E. Griel, M.Ed. Department of Nutritional Sciences, Pennsylvania

Kirsten F. Hilpert, B.S. Department of Nutritional Sciences, Pennsylvania
Harlan P. Jones, Ph.D. Laboratory of Psychoneuro-Immunology, Department

of Psychiatry and Behavioral Sciences, Emory University School of Medicine,
Atlanta, Georgia
William B. Kannel, M.D., M.P.H., F.A.C.C. Boston University School of

Medicine/Framingham Heart Study, Framingham, Massachusetts
Penny M. Kris-Etherton, Ph.D., R.D. Department of Nutritional Sciences,

Pennsylvania State University, University Park, Pennsylvania
Michael R. Kushnick, Ph.D. School of Recreation and Sport Sciences, Ohio

University, Athens, Ohio
Tom LaFontaine, Ph.D. PREVENT Consulting Services LLC, Columbia,

Missouri
Robert J. Moffatt, Ph.D., MPH, Georgia A. Stamford Professor of Exercise

Physiology, Department of Nutrition, Food and Exercise Sciences, Florida
State University, Tallahassee, Florida
Sachin M. Navare, M.D. Division of Cardiology in the Henry Low Heart

Center, University of Connecticut, School of Medicine, Hartford,
Connecticut
Lynn B. Panton, Ph.D. Department of Food, Nutrition and Exercise

Sciences, Florida State University, Tallahassee, Florida
Tricia Psota, B.S. Department of Nutritional Sciences, Pennsylvania State

University, University Park, Pennsylvania
Jeffrey L. Roitman, Ed.D. Research Medical Center, Kansas City, Missouri
Bryant A. Stamford, Ph.D. Professor and Chair, Department of Exercise

Science, Hanover College, Hanover, Indiana
Andrea C. Summer, B.S. Department of Exercise Science, University of

South Carolina, Columbia, South Carolina
Paul D. Thompson, M.D. Division of Cardiology in the Henry Low Heart

Center, University of Connecticut, School of Medicine, Hartford,
Connecticut
Jason D. Wagganer, M.S. Department of Exercise and Sport Science,
University of North Carolina at Greensboro, Greensboro, North Carolina
Table of Contents
1 Lipids and Health: Past, Present, and Future.............................. 1

Bryant A. Stamford and Robert J. Moffatt
2 Cardiovascular Risk Assessment ................................................ 13

William B. Kannel
3 Basic Lipidology ........................................................................... 31

Jacqueline L. Dupont
4 Lipid and Lipoprotein Metabolism ............................................ 47

Paul G. Davis and Jason D. Wagganer
5 The Vascular Biology of Atherosclerosis ................................... 61

Robert Carter III and Harlan P. Jones
6 Exercise Training and Endothelial Function in Patients

at Risk for and with Documented Coronary
Artery Disease ............................................................................... 85
Tom LaFontaine and Jeffrey L. Roitmann
7 Essential Laboratory Methods for Blood Lipid and

Lipoprotein Analysis .................................................................. 117
Peter W. Grandjean and Sofiya Alhassan
8 Metabolic Syndrome................................................................... 147

Vic Ben-Ezra
9 Obesity, Lipoproteins, and Exercise ......................................... 173

Theodore J. Angelopoulos
10 Pharmacological Treatments of Lipid Abnormalities............. 183

Sachin M. Navare and Paul D. Thompson
11 New Insights on the Role of Lipids and Lipoproteins
in Cardiovascular Disease: The Modulating Effects
of Nutrition ................................................................................ 211
Kirsten F. Hilpert, Amy E. Griel, Tricia Psota, Sarah Gebauer,
Yumei Coa, and Penny M. Kris-Etherton
12 Physical Activity, Exercise, Blood Lipids, and

Lipoproteins................................................................................. 265
J. Larry Durstine and Andrea C. Summer
13 Acute Changes in Lipids and Lipoprotein-Lipids

Induced by Exercise.................................................................... 283
Stephen F. Crouse
14 Smoking, Heart Disease, and Lipoprotein Metabolism......... 299

Robert J. Moffatt, Sara Chelland, and Bryant A. Stamford
15 Lipid and Lipoprotein Concentrations in Americans:

Ethnicity and Age ....................................................................... 315
Michael R. Kushnick and Lynn B. Panton
Index ..................................................................................................... 349

1
Lipids and Health: Past, Present, and Future
Bryant A. Stamford and Robert J. Moffatt
CONTENTS
Introduction .............................................................................................................1
The Cholesterol Risk Factor ..................................................................................3
Lipoproteins.............................................................................................................4
Atherosclerosis ........................................................................................................6
Past, Present, and Future.......................................................................................7
Lipids and Health...................................................................................................8
References ................................................................................................. 9
Introduction
The German philosopher, Arthur Schopenhauer (1788–1860), once said that
when new ideas are first introduced they are likely to be dismissed out of
hand, then ridiculed, and finally, accepted as self evident. This natural pro-
gression is particularly applicable to the scrutinizing mind of the scientist
who must dismiss new ideas as unacceptable, thus ensuring that acceptabil-
ity will occur only when ample empirical evidence is provided. The accep-
tance of serum cholesterol as causally related to coronary artery disease
(CAD) has traversed just such a gauntlet, and is now accepted as self-evident.
Moreover, vigorous research efforts have revealed a relationship and inter-
actions that are substantially more complex than imagined when this concept
was introduced more than a half century ago.
In the 1930s, medical researchers were aware that extraordinarily high
levels of serum cholesterol were associated with pathology. Xanthomatosis
was known to be related to symptoms of heart disease (angina pectoris) and
likely was a contributor to myocardial infarction.1 Such cases were rare,
however, and a relationship between cholesterol and CAD in those with
1
2 Lipid Metabolism and Health
high, but lesser, levels of cholesterol was dismissed. This, even though it was
known that cholesterol was found in atherosclerotic lesions in persons not
suffering from xanthomatosis. The cholesterol in lesions was considered to
be incidental, as the lesions were, it was assumed, caused by degenerative
alteration of the arterial wall.
At the time, the normal range for cholesterol was determined in the similar
manner employed to judge other blood-borne components. The mean of the
general population was assessed, standard deviations were calculated, and
the normal range extended from minus two standard deviations of the mean,
to plus two. This meant that only those individuals with blood cholesterol
levels beyond two standard deviations above the mean were diagnosed as
hypercholesterolemic.
The normal range extended to 300 mg/dl (7.76 mmol/L), and, thus, only
approximately 2.5% of the population would be viewed as having a danger-
ously high level of cholesterol (greater than 300 mg/dl). And, given the
tendency of practitioners to allow older patients a greater margin of error,
it was not uncommon to extend the normal range by 10% in those at or
above retirement age. Thus, persons 65 and older could be told that their
cholesterol test results were “normal,” notwithstanding an incredibly high
level, reaching 330 mg/dl!
This interpretation logically excused serum cholesterol as a causal factor
in CAD, because while only a tiny fraction of patients were labeled as
hypercholesterolemic, legions were dying of CAD. Moreover, factors includ-
ing cigarette smoking, high blood pressure, and diabetes had been identified
and indicted as causal, pushing cholesterol further into the background. The
well-respected 1948 text, Quantitative Clinical Chemistry, by Peters and
VanSlyke2 stated the case unequivocally: “There is no satisfactory evidence
that the incidence of atherosclerosis bears any relationship to the concentra-
tion of cholesterol in the blood.”
Once a seed is planted and takes root, it is difficult to stamp it out com-
pletely, even though the strength of evidence to the contrary is formidable.
Indeed, today, although the role of cholesterol in the progression of CAD is
taken for granted, not long ago offspring of the original seed continued to
flourish, and many still questioned the extent of impact of cholesterol on
atherosclerosis. And among those who accepted the basic premise, it was
not clear that reducing cholesterol reduced mortality from CAD; and if it
did, how much reduction was required? In addition, if there were a bona
fide relationship, was the use of powerful medications warranted, or did
available medications impose risks that were greater than those imposed by
the high level of cholesterol itself?
Lipids and Health: Past, Present, and Future 3
The Cholesterol Risk Factor

The Framingham Heart Study was the key to elevating serum cholesterol to
the status of CAD risk factor.3–5 Thousands of men and women were studied
prospectively and it was determined that, indeed, a relationship exists between
cholesterol and CAD. As the concentration of blood cholesterol increases, the
risk of CAD increases as well, the risk relationship defined as a continuous
and curvilinear function (of the concentration of blood cholesterol). Many
additional large-scale studies solidified the Framingham findings.
Despite the volume of data supporting blood cholesterol as problematic,
skeptics required data supporting positive outcomes arising from interven-
tion. Specifically, if cholesterol is a risk factor for CAD, reducing the concen-
tration of cholesterol in the blood should reduce risk. The first step was
determining safe and effective ways to reduce cholesterol. Dietary and drug
intervention was studied and found to be effective.6–9 Results from the Cor-
onary Primary Prevention Trial10 demonstrated that a drop in blood choles-
terol of 9% reduced CAD risk by 19%. Results of this study combined with
several others gave rise to national guidelines that replaced use of the “nor-
mal range” approach.
Impetus for change can be credited largely to efforts of the National Cho-
lesterol Education Program (NCEP), launched by the National Heart, Lung
and Blood Institute (NHLB) of the National Institutes of Health (NIH) in
1985. New guidelines set stricter goals as blood cholesterol levels below 200
mg/dl were deemed “healthy” and desirable, while those exceeding 240
mg/dl were viewed as clinically significant. Awareness of the risks associ-
ated with hypercholesterolemia increased greatly thanks to efforts of the
NCEP and by 1995, 70–80 million more Americans sought to have their blood
cholesterol concentrations determined, a 40% increase in ten years.
The above guidelines have been in place for nearly two decades. Many
experts argue that such guidelines, while an improvement on the “normal
range” approach, are far too liberal (given that the average total cholesterol
level in the U.S. is 205 mg/dl). Moreover, such guidelines are viewed as
deficient in many ways, particularly when considering the interplay between
cholesterol and other risk factors. In addition, ample evidence has accumu-
lated in recent years attesting to the clinical efficacy of so-called “statin”
drugs to dramatically overhaul the cholesterol profile and, in turn, reduce
the incidence of heart attacks and CAD deaths.
It would appear that continual redefining of guidelines would be the order
of the day as new evidence accumulates. However, it must be taken into
consideration that the creation, establishment, and acceptance of new guide-
lines add up to a ponderous and painstaking process. And when new guide-
lines are introduced, confusion often reigns because, in effect, at least for a
while, two (or more) sets of guidelines are operating. The older guidelines
continue to be followed faithfully by many practitioners on the front lines,
while news of updated guidelines is disseminated directly to the public

through various media outlets. Confronting such ominous circumstances
ensures that the approach to new guidelines is calculated and cautious in
the extreme.
This is a universal dilemma and is not peculiar to cholesterol. Serum
triglycerides have traversed similar terrain. Traditionally, serum triglycerides
have been viewed as lacking clinical significance until reaching 275–300 mg/
dl, and even then there was some question as to the importance of such
elevated levels. This is akin to the previous acceptance of the “normal range”
criterion for cholesterol values. While it was known that triglycerides are
adversely impacted by increased body fatness and uncontrolled diabetes,
the facts that the role of triglycerides in CAD is controversial, and triglycer-
ides are not recognized as an independent CAD risk factor, have inspired
continued tolerance of such high levels.
Most recently, however, as metabolic syndrome has attracted increased
attention, and owing to exploration of definitive diagnostic strategies, serum
triglycerides seem to have elevated in status, leading to a tightening of
guidelines. Diagnostic criteria for metabolic syndrome have been set forth
that include a cluster of five characteristics. One of these is serum triglycer-
ides (fasting) in excess of 150 mg/dl.
Regarding serum cholesterol and triglycerides as important health threats,
each has gone through a stage of benign neglect in which very high levels
were considered “normal.” And now, each has captured the spotlight with
emphasis focused on reducing levels to a fraction of what was previously
deemed acceptable.
Efforts have been ongoing to further improve upon cholesterol guidelines
for clinicians. New cholesterol guidelines have been proposed that address
the need for placing blood cholesterol levels within the context of a global
heart disease risk profile.11 A risk score is computed referencing the proba-
bility of a heart attack within 10 years. The new guidelines recommend
recurring assessment at five-year intervals beginning in young adulthood.
Efforts in this direction not only broaden the scope of factors considered,
they also have enhanced the sophistication of risk analysis by requiring a
lipoprotein profile. Attention also has been focused on the interaction of
serum triglycerides and lipoproteins.
Lipoproteins
Cholesterol is insoluble and, therefore, transportation of cholesterol in the
blood is challenging. Over the years, considerable research has been con-
ducted on cholesterol transport.12–14 It was found that cholesterol is trans-
ported in combination with other substances as lipoproteins, with a
hydrophobic lipid core, and a surrounding layer of apolipoproteins and
phospholipids. The apolipoproteins were found to vary in size and density

(labeled as high, low, and very low), and this, in turn, was found to be
significant in determining the metabolic fate of the complex.15–17
The relative proportion of alpha (high-density HDL) to beta (low-density
LDL) lipoproteins was found to be critical to the cholesterol/CAD relationship.
The fraction of blood cholesterol transported as LDL contributes to atheroscle-
rosis, whereas HDL is inversely related to risk. Vigorous research efforts have
uncovered several more classes and subclasses of lipoproteins.18 For utilitarian
reasons in the clinical setting, the ratio of total cholesterol to HDL typically is
employed, because direct assessment of LDL is difficult, and there exists a
high correlation between LDL and total cholesterol.
But still, many questions remained unanswered as numerous exceptions
to the rule surfaced. Intermediate-density lipoproteins (IDL) have been
found to increase CAD risk, especially when IDL is the major lipoprotein.19
Very-low-density lipoproteins (VLDL) also may be influential. However, the
role of VLDL may be important because of the inverse relationship with
HDL, and may reflect metabolic disorders (insulin resistance and diabetes,
for example), rather than a direct impact.19
Despite progress, many inconsistencies associated with the prediction of
CAD risk based upon serum cholesterol levels and the blood lipid profile
remained. For example, if all risk factors are equal (or reasonably so), why is
it that individuals with similar levels of LDL can have substantially different
levels of risk for CAD? This would seem to be inconsistent with the notion
that LDL entrance into the interior arterial wall (the endothelium) is gradient
driven. The more LDL that is present the more interaction there will be
between LDL and the arterial wall, resulting in greater LDL penetration of the
endothelium, greater oxidation of LDL, and thus greater atherogenesis.
Unexplored until recently is the size and density of lipoprotein particles,
and such explorations offer revealing insights.20,21 At any given level of serum
LDL, the size and density of LDL particles may be the determining factor
that promotes CAD risk, because small, dense particles may enter the arterial
wall more readily than larger, “fluffy” particles.22,23 Those with small LDL
particles have a substantially larger number of LDL particles and, thus,
despite equal levels of serum LDL, gross differences in the particle size and
density would appear to preserve the gradient driven aspects of atherogen-
esis. Unfortunately, when assessing LDL with a conventional blood lipid
profile approach, the size and density of LDL particles escapes detection.
HDL is responsible for reverse cholesterol transport — the removal of
cholesterol from developing lesions, which would reduce CAD risk.24
Enzymes carried by HDL may also play a protective role, acting to retard
oxidation of LDL (discussed below).25 This would suggest that a high level
of HDL is always helpful, and that a low level of HDL is always destructive.
This is not the case, however, as inconsistencies have again been observed.
Particle sizing may be relevant to HDL as well as LDL and may help to
explain some of these inconsistencies.22,23 Larger HDL particles may be more
effective in reverse cholesterol transport, and may interfere with interaction
between LDL and the endothelium. Smaller HDL particles may be ineffective
in this regard. Moreover, small HDL particles may actually contribute to
atherosclerosis. Thus, a patient with a preponderance of larger HDL particles
may be at lower risk than another patient with fewer large HDL particles,
even though conventional blood lipid assessment reveals that the two are
equal on the HDL scale.
Particle sizing may also have relevance with regard to VLDL.22 Larger
VLDL particles may increase CAD risk, because when insulin resistance is
present, excess carbohydrate increases production of triglyceride. This
results in VLDL that are loaded with triglyceride, which can lead to metab-
olism of large VLDL particles into small LDL and small HDL which, in turn,
can promote atherosclerosis.
Atherosclerosis
The “injury” hypothesis of atherosclerosis was proposed by Ross in 1970.26
The driving event in the process was thought to be damage to the endothe-
lium, progressing to denuding of the delicate endothelial lining, and even-
tually progressing to the status of fibrous plaques. Major emphasis of the
injury hypothesis was placed on smooth muscle proliferation.
Attention was focused on fatty streaks and the foam cells loaded with lipids.
Because of the emphasis on smooth muscle proliferation, it was assumed that
fatty streaks, the earliest of lesions, were associated with foam cells that were
derived from smooth muscle cells exclusively. Later, it was determined that
while some foam cells originate from smooth muscle, most arise from mono-
cytes in the bloodstream. This finding challenged the injury hypothesis,
because monocytes can penetrate an intact and functioning endothelium
where they take up residence as macrophages and attract cholesterol.
Subsequent research efforts by Ross and Glomser26 and others postulated
the utility of both hypotheses — the endothelial injury, and monocyte (lipid
infiltration) hypotheses in the progression of atherosclerosis.27,28 Cholesterol
may enter an uninjured endothelium that is fully functioning, and this could
lead to the accumulation of foam cells. Damage to the endothelium may
result, owing to secretion of local factors (such as cytokines and growth
factor), and to an inflammatory response. This, in turn, would promote
fibrous plaque development.
Progress in defining the steps of atherosclerosis was stymied, however,
when it was discovered that isolation and incubation of monocytes in a
medium loaded with cholesterol did not cause the monocytes to soak up
cholesterol, and thus produce foam cells.29 The same finding occurred with
smooth muscle cells. This led to research that revealed the need for alteration
of cholesterol prior to being taken up and accumulating. The cholesterol
must experience oxidative damage.30 In turn, animal research has indicated
that antioxidants can retard progression of lesions substantially.31 Research

efforts into the impact of antioxidants (specifically vitamin E) in humans is
ongoing, with mixed results.32
Integrity of the endothelium is a hot topic currently. Improved endothelial
function has many advantages in that platelets and inflammatory cells are
less likely to adhere, and the natural balance between locally derived vasodi-
lating and vasoconstricting substances is preserved. Nitric oxide (NO) is a
natural vasodilator, and it has been reported that in the presence of endo-
thelial dysfunction, there is a paradoxical vasoconstriction response to
vasodilator substances. This may be an important factor in initiating athero-
sclerosis.33
With all of the complexities associated with initiation and progression of
atherosclerosis, it is clear that several factors conspire, conflict, and contrib-
ute. At first glance, it might appear that as the research movement in this
area advances, the role of blood lipids has been demeaned. The role of a
dysfunctional endothelium, the impact of NO, and the intricacies of the
inflammatory response, have seized the focus. However, blood lipids retain
their position in the spotlight as several studies have reported improved
endothelial function when blood lipids are reduced.34–36 And a profound and
acute improvement in endothelial function was observed following LDL
apheresis.37
Past, Present, and Future

Historically, in Japan decades ago, dietary fat intake was low, serum choles-
terol levels were low (160 mg/dl),and the incidence of CAD was low.38 This,
despite a high incidence of hypertension and the immense popularity of
cigarette smoking. Is it possible that a very low cholesterol level precluded
atherosclerosis and development of CAD, even in the face of other significant
CAD risk factors? Is there a protective threshold for cholesterol, and LDL in
particular? Or, are other factors operating that have yet to be uncovered and
elucidated.
Much still needs to be determined in the realm of lipoproteins and their
role in promoting atherosclerosis, such as the role of lipoprotein (a), and
specifics surrounding the increased risk associated with high serum triglyc-
eride levels and low HDL. Further examination is needed of the notion that
at any given level of serum LDL, the size and density of LDL particles may
be the determining factor that promotes CAD risk. Particle sizing may be
relevant to HDL as well as LDL, and may help to explain some of the current
inconsistencies. A better understanding of homocysteine, HS-Crp, as a
marker of the inflammatory response, the nature of receptor activity, the
significance of nitric oxide, and elucidation of the roles of cytokines and
growth factors, may lead to revision of current hypotheses and creation of

new clinical strategies.
Lipids and Health

The purpose of this volume is to provide an overview and historical per-
spective of the evolution of serum lipids and lipoproteins from a mere
curiosity, to acceptance as an established and major CAD risk factor, and,
ultimately, to formulation of present clinical guidelines. Speculation regard-
ing future developments and the further potential evolution of guidelines
will be discussed.
Considerable attention has been focused on the fundamentals, such as
basic lipidology. Lipids are the structural components of all living cells, and
they play a number of critical roles. Lipid/lipoprotein metabolism is dis-
cussed with regard to the regulation, absorption, synthesis and excretion of
cholesterol. The biology of atherosclerosis emphasizes arterial adaptations
and the inflammatory response, as well as the impact of atherosclerosis on
cerebral vascular and peripheral artery disease. A chapter on endothelial
function as impacted by nitric oxide and exercise is included.
Clinical methodologies for measuring lipoproteins are a critical consider-
ation given the many challenges associated with accurate determination of
the number and size of circulating LDL (and HDL) particles and the CAD
risk they confer. A critique of commonly employed assessment techniques
and the implications of their potential inaccuracies is discussed. Clinical
strategies, with emphasis on pharmacological treatments, are discussed with
regard to managing unhealthy lipid levels.
Lipids and lipoproteins can be impacted by a number of factors, including
obesity, diabetes and metabolic syndrome, diet/nutrition, exercise (acute and
chronic effects), cigarette smoking and environmental tobacco smoke, alco-
hol consumption, heredity, age, gender, and race. These factors are discussed
in detail.
In summary, the relationship between lipids and CAD risk is well estab-
lished. The complexities associated with this relationship are continually
being revealed and addressed, which has, among other things, instigated a
shift toward more aggressive clinical management of unhealthy lipid levels.
This is a highly positive step, and represents the first prong of a comprehen-
sive approach. The second prong entails primary preventive intervention
strategies that include emphasis on improving a variety of lifestyle factors
(weight management, healthy dietary practices, daily exercise, etc.). Progress
in these areas is greatly needed and is critical to reducing the incidence of
the number one cause of death in the industrialized world today.
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108–119.
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2
Cardiovascular Risk Assessment
William B. Kannel
CONTENTS
Introduction ...........................................................................................................13
Incidence of Atherosclerotic Cardiovascular Disease.....................................14
Current Status of Risk Factors — Hypertension .............................................15
Dyslipidemia..........................................................................................................19
Diabetes and the Metabolic Syndrome .............................................................21
Obesity....................................................................................................................22
Indicators of Pre-Symptomatic Arterial Ischemia ...........................................23
Indicators Suggesting Unstable Lesions ...........................................................23
Novel Risk Factors................................................................................................23
Multivariable Risk Assessment ..........................................................................24
Preventive Implications .......................................................................................25
References ............................................................................................... 27
Introduction
Five decades of epidemiologic research provides health workers with valu-
able insights into the factors predisposing to atherosclerotic cardiovascular
disease (CVD), stimulating world-wide interest in preventive cardiology.
This provoked public heath initiatives against smoking in the 1960s, hyper-
tension in the 1970s and dyslipidemia in the 1980s.1 It also stimulated clinical
trials to demonstrate the efficacy of risk factor modification. The epidemio-
logic population approach provided an undistorted appraisal of the way
CVD evolves in the population indicating that coronary heart disease (CHD)
is extremely common, afflicting one in five persons before age 60 years.2,3 It
was demonstrated to be a highly lethal disease with unanticipated sudden
death as a prominent feature of the mortality and a substantial fraction of
13
myocardial infarctions that are silent or unrecognized.2,4 Because of this

clinical presentation, a more vigorous preventive approach is now advocated
using multivariable risk assessment and trial data demonstrating the benefits
of risk factor correction. Epidemiological research indicates that atheroscle-
rotic vascular disease is multifactorial, giving rise to the risk factor concept.
Certain living habits promote atherogenic traits in genetically susceptible
persons that, after prolonged exposure, produce a compromised arterial
circulation leading to clinical cardiovascular events. Atherosclerotic cardio-
vascular disease is now regarded as a multifactorial process involving a
variety of factors each of which is best considered as an ingredient of a
cardiovascular risk profile.
Incidence of Atherosclerotic Cardiovascular Disease

Data from the Framingham Study encompassing 44 years of surveillance of
the original cohort and 20 years of the offspring indicate the hazard of this
leading cause of death. Coronary disease is the most common manifestation,
equaling in incidence all the others combined for persons aged 35–64 years.
It is also dominant above age 65 (Table 2.1). Women lag behind men in
incidence of CVD with a diminishing gap in incidence with advancing age.
Because of the half-century duration of follow-up of the Framingham cohort
it was possible to determine the actual lifetime risk of developing a coronary
event. This indicates that a 40-year-old man has a 48.6% lifetime risk and
for women the risk is 31.7%, which is three times the risk of breast cancer
(Table 2.2). The lifetime risk diminishes the longer one survives without
acquiring it, but even at age 70 the risk is 34.9% for men and 24.2% for
women. Because the short-term mortality after onset of an initial myocardial
infarction is so high (25% 1-year mortality for men and 38% for women),
TABLE 2.1
Incidence of Atherosclerotic Cardiovascular Events
in the Framingham Study: 44-Year Follow-up of
Original Cohort and 20-Year Follow-up of Offspring
Average Annual Incidence per 1000
Age 35–64 Years Age 65–94 Years
Men Women Men Women
CVD (all types) 17 9 44 30
Coronary disease 12 5 27 16
Stroke 2 2 13 11
Heart failure 2 1 12 9
Peripheral artery 3 2 8 5
Average annual incidence rates are age adjusted.
Cardiovascular Risk Assessment 15
TABLE 2.2
Lifetime Risk of Initial Coronary Events:
Framingham Study Participants
Lifetime Risk (95% Confidence Intervals)
Age (Years) Men Women
40 48.6% (45.8–51.3) 31.7% (29.2–34.2)
50 46.9% (44.0–49.8) 31.1% (28.6–33.7)
60 42.7% (39.5–45.8) 29.0% (26.3–31.6)
70 34.9% (31.2–38.7) 24.2% (21.4–27.0)
Source: From Lloyd-Jones DM, et al. Lancet 1999;
34:381–385. With permission.
this disease must be diagnosed on its way to clinical expression and its
predisposing risk factors corrected. One in six coronary events presents with
sudden death as the first, last and only symptom.
Current Status of Risk Factors — Hypertension

Hypertension (> 140/90 mmHg) afflicts one in four Americans. About 4%
of persons less than 30 years of age have the condition and it increases in
prevalence to 71% beyond age 80 years.5 Estimates of the prevalence of
isolated systolic hypertension that rises with age, vary because of age, eth-
nicity, the definition used and whether clinical or population data are sur-
veyed. Data from the SHEP trial, which used > 160/90 mmHg to define the
condition, estimates the prevalence at 8% for age 60–69 years, 11% at 70–79
years and 22% at 80 years and over.6 About 65% of hypertension in the elderly
is of the isolated systolic variety, the prevalence in women exceeding that in
men beyond age 55 years.7
Framingham Study estimates of the rate of progression to hypertension
from non-hypertensive blood pressures indicate that over a 4-year period
about 50% of the elderly with high–normal blood pressure can be expected
to progress to hypertension (> 140/90 mmHg) a rate five times that of
persons with optimal (120/80 mmHg) blood pressure.8 The average blood
pressure of the Framingham cohort declined progressively over five decades
so that elevated blood pressure is currently only one-third as prevalent as
formerly.9 However, if treatment-normalized pressures are included hyper-
tension prevalence appears to have increased. This is likely a result of earlier
detection and institution of therapy at lower blood pressures. No decline in
blood pressure over time was observed in participants not receiving treat-
ment. Half a century of follow-up of Framingham Study participants indi-
cates that the lifetime probability of receiving antihypertensive medication
is 60% for men and 57% for women.10
TABLE 2.3
Percent of Hypertensive Persons Developing Overt
CVD Prior to Indications of Target Organ
Involvement: 20-year Follow-up to Framingham
Study
Age (Years)
35–44 45–54 55–64 65–74 All Ages
Men 75% 58% 48% 33% 50%
Women 33% 56% 38% 33% 39%
Target organ involvement: proteinuria, ECG abnormality,
cardiomegaly.
The primary variety of hypertension was formerly believed to be benign

and the rise in blood pressure with age essential in order to perfuse vital
organs. Initiation of treatment was often delayed until there was evidence
of target organ involvement. Framingham Study data indicated that this
practice was imprudent because 40–50% of hypertensive persons developed
overt CVD prior to evidence of proteinuria, cardiomegaly or ECG abnormal-
ities (Table 2.3). Its cardiovascular consequences were believed to derive
chiefly from the diastolic pressure and the isolated systolic hypertension of
the elderly was regarded as an innocuous accompaniment of arterial stiff-
ening. Treatment of this entity was considered fruitless, intolerable and
dangerous. Blood pressures assigned as normal for the elderly (100 plus age
mmHg) were substantially higher than for the middle-aged. Women were
believed to tolerate hypertension better than men. It was believed that there
were age-related critical thresholds for blood pressure regarding hyperten-
sive cardiovascular hazards. Isolated systolic hypertension was considered
an innocuous accompaniment of advanced age.11,12
Based on epidemiological data, the current concept of an acceptable blood
pressure is now based on what is optimal for avoiding hypertension-related
CVD rather than on what is usual. Epidemiological data from the Framing-
ham Study and elsewhere clearly indicate that at all ages and in both sexes,
CVD risk increases incrementally with the systolic blood pressure and at any
given blood pressure the hazard is greater in the elderly (Table 2.4). Similar
graded relationships of blood pressure to CHD and all-cause mortality have
been reported in several other cohorts.13–15 There is no threshold for blood
pressure risk as claimed by some, and in the Framingham cohort, 45% of
the CVD events in men occurred at a systolic blood pressure < 140 mmHg,
the value recently claimed by some to be the threshold of risk.17 Large data-
sets are needed to precisely estimate CVD incidence trends at low blood
pressures. Both the MRFIT data on > 350,000 male screenees followed for
CVD mortality, and the Prospective Studies Collaboration involving almost
1 million participants and 56,000 vascular deaths, found no indication of a
threshold of risk down to 115/75 mmHg.16,18 Persons aged 40–69 years had
a doubling of stroke or CHD mortality with every 20 mmHg increment
TABLE 2.4
Average Annual Cardiovascular Disease Incidence by Systolic Blood
Pressure: 30-Year Follow-up to Framingham Study
Systolic Blood Pressure Men (Years Old) Women (Years Old)
(mmHg) 45–54 55–64 65–74 45–54 55–64 65–74
74–119 8 16 16 3 6 12
120–139 11 18 23 5 9 17
140–159 19 31 37 9 16 22
160–179 29 43 52 9 24 20
180–300 35 62 78 16 36 45
All systolic blood pressure CVD incidence trends statistically significant.
Source: From Cupples LA, D’Agostino RB. In: Kannel WB, Wolf PA, Eds. The
Framingham Study: an Epidemiological Investigation of Cardiovascular Disease. Wash-
ington DC: NHLBI National Printing Office; 1971; section 34.
increase of systolic (or 10 mmHg diastolic) throughout the entire range of

blood pressure. Recent examination of the relation of non-hypertensive blood
pressure to the rate of development of CVD in the Framingham Study found
a significant graded influence of blood pressure from optimal (< 120/80
mmHg) to normal (120–129/80–84 mmHg) to high–normal (130–139/85–89
mmHg) among untreated men and women.19 Compared with optimal,
high–normal blood pressure conferred a 1.6- to 2.5-fold age- and risk factor-
adjusted risk of a CVD event.
The tenaciously held belief that the adverse consequences of hypertension
derive chiefly from the diastolic blood pressure component has long been
convincingly refuted by prospective epidemiological data demonstrating
that the impact of systolic pressure is greater than the diastolic compo-
nent.12,16,20 Examination of the increment in CVD risk per standard deviation
increment in systolic vs. diastolic blood pressure, to take into account the
different range of values for each, at all ages in both sexes in the Framingham
Study indicates a consistently greater impact for the systolic blood pressure
(Table 2.5).
Although risk ratios are no larger than for other cardiovascular events, the
most common hazard for hypertensive patients of all ages is coronary dis-
ease, equaling in incidence all the other hypertensive atherosclerotic conse-
quences combined (Table 2.6). Although the CVD incidence rates appear to
be higher for most adverse outcomes in men, the risk ratios comparing those
with and without hypertension in each sex are no higher in men than women
(Table 2.6). Hypertension predisposes to all clinical manifestations of CHD
including myocardial infarction, angina pectoris and sudden death, impos-
ing a 2- to 3-fold increased risk.
Because of progressive arterial stiffening with advancing age, isolated sys-
tolic hypertension comprises about two thirds of the hypertension of the
elderly. Its chief determinants are a high–normal systolic blood pressure in
middle-age and prior diastolic blood pressure elevation that disappears as the
arteries lose compliance with advancing age.11 Isolated systolic hypertension
TABLE 2.5
Risk of Cardiovascular Disease by Systolic vs.
Diastolic Blood Pressure: Framingham Study
38-Year Follow-up
Risk Factor-Adjusted Increment
per Standard Deviation Increase
Systolic Diastolic
Age (Years) Men Women Men Women
35–64 40% 38% 37% 29%
65–94 41% 25% 25% 15%
Covariates: cholesterol, glucose, cigarettes, ECG-left
ventricular hypertrophy.
All differences significant at P < 0.001.
Source: From Kannel WB. Hypertension. In: Aronow
WS, et al., Eds. Vascular Disease in the Elderly. Futura;
1997. With permission.
TABLE 2.6
Risk of Atherosclerotic Cardiovascular Events in Hypertensive Persons: 36-Year
Follow-up. Framingham Study
Age 35–64 Years Age 65–94 Years
Biennial Ratea Risk Ratioa Biennial Ratea Risk Ratioa
CVD Events Men Women Men Women Men Women Men Women
CHD 45 21 2.0* 2.2* 72 44 1.6* 1.9*
Stroke 12 6 3.8* 2.6* 36 38 1.9* 2.3*
PAD 10 7 2.0* 3.7* 17 10 1.6* 2.0*
H.F. 14 6 4.0* 3.0* 33 24 1.9* 1.9*
*P < 0.0001. Biennial rates per 1000.
CHD, coronary heart disease; PAD, peripheral artery disease; H.F., heart failure.
a Age-adjusted.
Source: Kannel WB. Drugs Aging 2003; 20(4):277–286. With permission.
is associated with excess development of CHD, stroke, heart failure, and

peripheral artery disease, increasing cardiovascular events and mortality rates
2- to 3-fold.11 The disproportionate rise in systolic blood pressure that results
in isolated systolic hypertension produces a widening of the pulse pressure.
This increase in pulse pressure is definitely not an innocuous accompaniment
of advancing age as previously believed. Risk of cardiovascular events
increases progressively by 20–23% per 10 mmHg increment in pulse pressure
in men and 11–21% in women21 (Table 2.7).
TABLE 2.7
Risk of Cardiovascular Events by Pulse Pressure (Age-
Adjusted Rate per 1000): 30-Year Follow-up to Framingham
Study
Pulse Pressure (mmHg)
2–39 40–49 50–59 60–69 70–182
Men 35–64 years 9 13 16 22 33
Men 65–94 years 4 16 32 39 58
Women 35–64 years 4 6 7 10 16
Women 65–94 years 17 19 22 25 32
Increment per 10 mmHg: Men 35–64 years 20%; Men 65–94 years
23%; Women 35–64 years 21%; Women 65–94 years 11%.
Source: From Kannel WB. Am J Cardiol 2000; 85:251–255. With per-
mission.
Dyslipidemia
Dyslipidemia is a fundamental aspect of accelerated atherogenesis.22 Epide-
miological, clinical, angiographic and postmortem investigations establish a
causal relationship between dyslipidemia and vascular atherosclerosis and
demonstrate that treating it reduces its occurrence.23 Despite dietary advice,
and the variety of lipid-modifying medications available, its prevalence in
the general population remains unacceptably high. Each of the blood lipids
influence the risk of CHD in a continuous graded fashion even within the
range considered normal. Data from the MRFIT involving 356,000 screenees
showed a continuous graded relationship of serum total cholesterol to cor-
onary mortality.24 The average lipid values at which CHD occurs in Framing-
ham Study middle-aged men is only 227 mg/dl for total cholesterol, 43 mg/
dl for high-density lipoprotein cholesterol (HDL-C), 151 mg/dl for low-
density lipoprotein cholesterol (LDL-C) and 5.6 for the total/HDL cholesterol
ratio.25 The average lipid values at which CHD occurs are higher in women,
decrease with age, and have been declining over the past five decades.
Epidemiological population-based data and clinical trials suggest that each
1% increase in total cholesterol throughout its range confers a 2% increment
in CHD and comparable reduction in LDL-C yields the same reduction in
initial and recurrent CHD events.26 The most efficient lipid profile for esti-
mating CHD potential is the total/HDL cholesterol ratio that affords a prac-
tical reflection of the net effect of the cholesterol entering the arterial intima
in the LDL and being removed in the HDL (Table 2.8). This ratio determines
the CHD risk whether the total cholesterol is above or below 240 mg/dl.
Optimal treatment of dyslipidemia should improve this ratio to a goal of
3.5 that corresponds to half the high average CHD risk. A high triglyceride
TABLE 2.8
Efficiency of Specified Blood Lipids in
Predicting Coronary Disease in
Framingham Study Subjects Aged 50–80
Years
Age-Adjusted Q5/Q1
Risk Ratio
Men Women
Total cholesterol 1.9 2.5
LDL cholesterol 1.9 2.5
HDL cholesterol 0.4 0.5
Total/HDL cholesterol 2.5 3.1
LDL/HDL cholesterol 2.5 2.6
Q, quintiles of the distribution of the lipids.
Source: From Kannel WB, Wilson PWF. Am Heart
J 1992; 124:768–774. With permission.
(> 150 mg/dl) in association with a reduced HDL-C signifies presence of

insulin resistance and more atherogenic small-dense LDL.
The dyslipidemic CHD risk imposed is strongly influenced by the burden
of associated risk factors.27 Measurement of other risk factors such as blood
pressure blood glucose, and weight is important because these cluster with
dyslipidemia about 80% of the time and profoundly influence the risk
imposed by dyslipidemia (Table 2.9). Dyslipidemia also tends to cluster with
thrombogenic risk factors such as PAI-1, and when associated with inflam-
matory markers such as CRP, is especially dangerous.28,29 The lack of a clear
demarcation of high-risk coronary candidates based solely on lipid values
indicates the need to evaluate dyslipidemia in the context of multivariable
risk assessment. Global risk assessment is recommended by the ATP III
guidelines for evaluation of dyslipidemic risk and treatment.30 Multivariable
CHD risk assessment using Framingham Study multivariable risk formula-
tions is recommended to estimate the 10-year probability of CHD events for
TABLE 2.9
20-Year Risk of Coronary Disease Associated with Lipid Abnormalities by
Extent of Risk Factor Clustering in Framingham Study Offspring: Age-
Adjusted Incidence per 1000
High Cholesterol High Triglyceride Low HDL-C
No. of Risk Factors Men Women Men Women Men Women
None 103 110 88 37 172 21
One 232 141 190 52 228 74
≥ Two 313 177 309 186 306 179
Associated risk factors: other lipids, elevated blood pressure, body mass index,
glucose.
dyslipidemic persons depending on the burden of other risk factors. Dys-

lipidemic persons at high global risk (> 20% 10 year risk) are assigned more
stringent goals for LDL cholesterol.30
Diabetes and the Metabolic Syndrome

Atherosclerotic cardiovascular disease is a major hazard of Type 2 diabetes.
Its prevalence in the general population has been rising.31 Persons with the
metabolic syndrome, a condition characterized by disturbed glucose and
insulin metabolism, visceral adiposity, dyslipidemia and hypertension,
appear to have a prediabetic state.32
Diagnostic criteria for this metabolic syndrome were promulgated by the
3rd Adult Treatment Panel,33 requiring three or more of the aforementioned
variables as specified in Table 2.10. Recent estimates indicate that the con-
dition is highly prevalent in the U.S. population, affecting 24% of adults.34
About 50% of diabetic men and 62% of women in the Framingham Study
had the metabolic syndrome whereas among persons with the metabolic
syndrome, only 15% of men and 17% of women had overt diabetes. The
hazard for CVD in persons with the metabolic syndrome is estimated to be
substantial: 3-fold increased risk of CHD and stroke, and 5-fold increased
risk of CVD mortality.35
TABLE 2.10
NCEP ATP III Criteria for Designation
of the Metabolic Syndrome
Risk Factor Defining Level
3 or More of the Following:
Abdominal obesity Waist circumference
Men > 40 inches (102 cm)
Women > 35 inches (88 cm)
Triglycerides 150 mg/dl
HDL-cholesterol
Men < 40 mg/dl
Women < 50 mg/dl
Blood pressure ≥ 130/85 mmHg
Fasting glucose ≥ 110 mg/dl
Source: From NCEP ATP III. Circulation 2002;
106:3134–3421. With permission.
Obesity
Overweight (body mass index [BMI] 25–29) and obesity (BMI > 29) are now
epidemic, posing a major threat to the public health. Adiposity is the most
prevalent metabolic disorder in the United States. At any given time 40% of
women and 25% of men are attempting to lose weight.36 The high average
weight of Americans carries a substantial health penalty of hypertension,
dyslipidemia, diabetes, cardiovascular disease, gallstone disease, and pros-
tate and colon cancer. Following considerable prior skepticism about obesity
it is no longer regarded as an innocent accompaniment of CVD risk factors.
After longer periods of observation, consideration of patterns of adiposity
and the clear and consistent demonstration that changes in weight are mir-
rored by changes in multiple atherogenic risk factors,37 the true role of obesity
as a cardiovascular hazard has become accepted. The burden of cardiovas-
cular risk factors is substantially greater in the obese than in lean persons
and the greater the adiposity the higher the blood pressure, level of dyslip-
idemia, blood glucose insulin resistance and left ventricular hypertrophy.
The average number of risk factors that cluster with any particular risk factor
increases, the greater the associated BMI (Table 2.11). Weight gain leading
to abdominal obesity promotes many of the atherogenic traits that in aggre-
gate have been characterized as the metabolic syndrome. The hazard of obesity
varies widely depending on the accompanying burden of CVD risk factors.
Weight loss improves insulin sensitivity and reduces the amount of athero-
genic risk factor clustering. If optimal weight is defined as that weight which
optimizes the cardiovascular risk profile it would correspond to a BMI of
22.6 for men and 21.1 for women.38 Estimates from the Framingham Study
indicate that if everyone could be maintained at optimal weight there would
be 25% less CHD and 35% fewer strokes and heart failure.39 Unfortunately,
sustained weight reductions in the obese have been difficult to achieve.
TABLE 2.11
Risk Factor Clustering with Elevated Cholesterol According to BMI:
Framingham Offspring Cohort Subjects Aged 30–70 years
Men Women
BMI Av. No. of Risk Factors BMI Av. No. of Risk Factors
<23.6 1.5 <20.6 1.7
23.7–25.4 1.7 20.6–22.2 1.6
25.5–27.1 2.0 22.3–23.8 1.9
27.2–29.3 2.1 23.9–26.5 1.9
>29.3 2.5 >26.5 2.6
BMI, body mass index. Elevated cholesterol: men 232 mg/dl; women 224
mg/dl (upper quintile values).
Indicators of Pre-Symptomatic Arterial Ischemia

There are a number of indicators of arterial ischemia that greatly affect the
risk of established risk factors. A compromised coronary arterial circulation
may be manifested prior to symptoms by ECG abnormalities such as non-
specific repolarization abnormalities, left ventricular hypertrophy, blocked
intraventricular conduction and myocardial infarction. Myocardial infarc-
tions in hypertensive persons are surprisingly often silent or unrecognized.
In the Framingham Study, 49% of myocardial infarctions are unrecognized.40
In order not to overlook these myocardial infarctions, that are hazardous
despite lack of symptoms, biennial ECG examinations should be done.
Carotid and femoral vascular bruits usually signify diffuse atherosclerosis
and are associated with not only stroke and intermittent claudication, but
CHD as well.41 Framingham Study data indicated that it is imprudent to
await indications of target organ involvement because 40–50% of hyperten-
sive persons developed overt CVD prior to evidence of proteinuria, cardi-
omegaly or ECG abnormalities.
Indicators Suggesting Unstable Lesions

Elevated fibrinogen and leukocyte count within the purported “normal” range
and level of C-reactive protein (CRP) tend to coexist and predict atherosclerotic
CVD events. They appear to reflect the presence of unstable lesions that are
undergoing inflammatory lipid infiltration and fissuring portending throm-
botic arterial occlusion.42–44 Levels of these risk factors are likely to be elevated
in persons who smoke, are hypertensive, diabetic or dyslipidemic.
Novel Risk Factors

It has been claimed that only half the coronary disease incidence is explained
by the standard major risk factors, but a recent report based on 120,000
patients enrolled in clinical CHD trials indicated that at least one major risk
factor is present in 85% of men and 81% of women.45 Another report derived
from 400,000 persons enrolled in three cohort studies showed that among
those suffering fatal CHD events, 87–100% had exposure to at least one major
risk factor.46 Nevertheless, epidemiological research continues to find and
evaluate additional risk factors that contribute to the occurrence of CVD,
and warrant further evaluation.
Subgroups of HDL and LDL are shown to be associated with CHD but
the utility of these refinements over the standard lipoprotein determinations
is not established.47,48 Similarly, lipoprotein (a) is associated with CHD and
stroke in some, but not all studies.49 Elevated triglyceride is consistently
associated with increased CHD risk, but its predictive power is often lost or
attenuated when HDL cholesterol and triglycerides or diabetes are taken
into account. However, a recent meta-analysis of 17 studies strongly suggests
an independent incremental triglyceride CHD risk, particularly when the
LDL or total/HDL cholesterol ratio is high.50
C-reactive protein is a confirmed risk factor for CHD along with other
circulating markers of inflammation.51 A recent meta-analysis of CRP inves-
tigations indicates that the independent relative risk of an elevated CRP is
less than suggested in earlier reports. It appears that the predictive value of
CRP adds relatively little to multivariable risk estimation based on the stan-
dard risk factors.52
Homocysteine, an amino acid regulated by vitamins B-12 and folate, found
in higher concentration in 29% of the elderly Framingham Study participants
and 5% of subjects in the Physicians’ Health Study has been found to be asso-
ciated with increased risk of cardiovascular disease.53,54 However, no clinical
trials have thus far tested whether reducing homocysteine by vitamin supple-
mentation decreases risk of developing atherosclerotic cardiovascular disease.
Multivariable Risk Assessment

Epidemiological investigation has long contended that atherosclerotic CVD
is of multifactorial etiology. There are faulty lifestyles that promote athero-
genic traits in susceptible persons, indicators of unstable lesions, and signs
of a compromised arterial circulation that strongly indicate impending clin-
ical events. The cardiovascular risk factors seldom occur in isolation of each
other because they are metabolically linked, tending to cluster, and the extent
of this clustering profoundly influences the CVD hazard of any particular
risk factor. Weight gain leading to visceral adiposity promotes most of the
components of the cluster of risk factors characterized as the insulin-resistant
metabolic syndrome. The hazard of obesity varies widely depending on the
burden of atherogenic risk factors that accompany it. Multivariable analysis
of the influence of established and potential risk factors is undertaken to
explore clues to the pathogenesis of CVD, and for estimation of the inde-
pendent effect of risk factors and the global risk of candidates for CVD
events. The set of risk factors employed for the former is constrained by the
hypothesis to be tested, and for the latter by the availability of reliable non-
invasive tests for the risk factors, cost, and whether the risk factors used can
be safely modified with the expectation of benefit.
Major established risk factors have been synthesized into composite scor-
ing algorithms based on Framingham Study data for the cardiovascular
disease outcomes of coronary disease, stroke, peripheral artery disease and
heart failure.55–58 The risk factors chosen had to be independent contributors
to risks that are not highly intercorrelated, and obtainable by ordinary office
procedures and readily available reliable laboratory tests.
When confronted with a patient with any particular CVD risk factor it is
essential to test for the others that are likely to coexist with it. Such coexist-
ence can be expected 80% of the time. Now that guidelines for dyslipidemia,
hypertension and diabetes recommend treating modest elevations of risk
factors, candidates for treatment are best targeted by global risk assessment
to reduce the number needed to treat to prevent one event.
Preventive Implications
Awaiting overt signs and symptoms of cardiovascular disease before initi-
ating treatment of hypertension, dyslipidemia or glucose intolerance is no
longer justified. The occurrence of symptoms is more properly regarded as
a medical failure than the first indication for therapy. As stated by Chobanian,
“Intensified efforts to alter modifiable cardiovascular risk factors such as
blood pressure, lipoprotein levels, smoking, blood glucose levels, weight and
physical activity levels must become a national priority.”59
Overwhelming evidence indicates a continuous incremental influence of
systolic blood pressure on CVD mortality at all ages in both men and women.
Optimal blood pressure for avoiding CVD is less than 140/90 mmHg with
no discernible critical blood pressure that delineates normal from abnormal.
For cost-effective treatment of blood pressure in the high–normal prehyper-
tensive range multivariable risk assessment is required and the goal of ther-
apy should be to improve global CVD risk.60 Because the hazard of any
degree of blood pressure elevation is increased when there is concomitant
dyslipidemia, diabetes, proteinuria, or the metabolic syndrome, more rigor-
ous blood pressure control is recommended. In this circumstance and when
there is already existing overt renal disease, coronary disease or peripheral
artery disease, the importance of what appear to be trivial increases in blood
pressure, even within the high–normal rage, should not be underestimated.
The effort needed to lower the blood pressure to the goals recommended for
avoiding CVD is worthwhile.
Notwithstanding the abundant evidence of the benefits of treatment of
systolic hypertension, this is the type of hypertension least likely to be
treated, and when treated is seldom treated to the recommended goal.61,62
Despite the variety of antihypertensive agents for controlling elevated blood
pressure by different mechanisms, many patients are failing to reach the JNC
7 recommended goals of therapy.63 This is due to failure to achieve goals for
TABLE 2.12
Control of Systolic and Diastolic Blood Pressure in
Framingham Study Participants 1990–1995
Control of All Hypertensives On Treatment
Systolic (<140 mmHg) 33% 49%
Diastolic (<90 mmHg) 83% 90%
Both (<140/90 mmHg) 30% 48%
(n = 1995) (n = 1189)
Source: Lloyd-Jones DM, Evans JC, Larson MG, et al. Differen-
tial control of systolic and diastolic blood pressure: factors as-
sociated with lack of blood pressure control in the community.
Hypertension 2000; 36:594–599.
systolic blood pressure. The Framingham Study reports that, whereas 90%
of hypertensive persons on treatment have their diastolic blood pressure
reduced to below 90 mmHg, only 48% have their systolic pressure controlled
to below 140 mmHg (Table 2.12). This failure applies to all subgroups of
hypertensive persons including African Americans, the elderly, and patients
with diabetes. Failure to control systolic pressure adequately in the elderly
and diabetics is particularly unfortunate because the benefits are greatest in
these patients.
Poor control of blood pressure is attributable to the need for long-term
adherence to treatment of an asymptomatic condition, adverse symptomatic
side effects when there were none to begin with, and the high cost of medi-
cations. About half of patients prescribed blood pressure medications stop
taking them by the end of the first year.64 Physicians’ knowledge and attitudes
concerning vigorous control of blood pressure also appear to be problematic.
Lipid correction has been consistently shown by numerous trials to bestow
benefit for atherosclerotic CVD. Lipid-modifying agents, particularly the
statins, reduce coronary and stroke events, modify endothelial dysfunction,
decrease platelet aggregation, stabilize plaques and promote coronary
vasodilation.22,23 The benefit of correcting dyslipidemia for decreasing the
hazard of coronary morbidity and mortality has been demonstrated with
and without established coronary disease, and whether lipids are distinctly
abnormal or only average. Recently reported results of the Heart Protection
Study indicate that high-risk patients, including those with LDL-cholesterol
values under 100 mg/dl, benefit from aggressive lipid-modifying therapy.65
The VA-HIT trial showed that even a modest fibrate-induced increase in
HDL cholesterol results in a reduction in CHD events.66
As clinicians cope with the task of implementing preventive measures for
avoiding or delaying development of atherosclerotic CVD by modifying
correctable risk factors, they must remember that the goal is to target high-
risk persons and reduce their global (multivariable) risk as well as the par-
ticular risk factor under scrutiny. This requires getting familiar with multi-
variable risk factor algorithms to assess risk before, during and after
treatment.
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3
Basic Lipidology
Jacqueline L. Dupont
CONTENTS
Introduction ...........................................................................................................31
Lipid Classes..........................................................................................................32
Acylglycerides ..............................................................................................32
Fatty acids .....................................................................................................32
Phospholipids and Sphingolipids.............................................................34
Sterols and Steroids .....................................................................................36
Lipid Digestion......................................................................................................37
Regulation of Cholesterol Synthesis ..................................................................38
Essential Fatty Acids and Eicosanoids..............................................................41
Lipid Peroxidation ................................................................................................43
References ............................................................................................... 45
Introduction
Lipids are structural components of all living cells. They are integral to
membranes, providing a water barrier that gives form to cellular compo-
nents. Major classes and functions of lipids are: (a) acylglycerides, energy
source and storage, (b) phospholipids, metabolically active cellular lipids,
(c) fatty acids, essential metabolites and precursors of autocoids, and
(d) sterols and their metabolites including hormones and bile acids. The
metabolism of lipids includes ingestion of lipids from foods, digestion and
transport, and functions of cholesterol and essential fatty acids. Lipid trans-
port is not included in this chapter as it is considered extensively elsewhere.
31
Lipid Classes
Acylglycerides
The most abundant form of lipids in plants and animals is triacylglycerides
made up of fatty acids esterified to glycerol (Figure 3.1). Di- and monoacyl-
glycerides exist in small quantities and are important in metabolic transfor-
mations of the glycerides. The fatty acids may be attached to any of the three
glycerol carbons and their positions are designated by the stereospecific
numbering of the glycerol molecule. In the esterified form the molecules
have no polar constituents and are thus quite hydrophobic. The character-
istics of the fatty acyl chains determine the nature of the acylglycerides.
Fatty acids
Fatty acids are hydrocarbon chains of from 2 to 20 and more carbons with
a carboxyl at one end. The nomenclature has evolved over time from known
chemistry and from sources from which they are isolated (Table 3.1). Those
of two or three carbons are volatile, 4–6 carbons are called short chain, 8–12
carbons are medium chain and 14–18 carbons are long chain. A group of
20 carbon and longer chain fatty acids is important in metabolism and is
referred to as a very long chain or by their individual names. The short-
chain fatty acids are water soluble, and as the chains lengthen, the water
solubility declines. The melting point is the opposite, with melting points
rising as chain length increases. Fatty acids may be saturated, i.e., having all
carbons in single linkage to other carbons or hydrogens, or monounsat-
urated, or polyunsaturated (Figure 3.2). The natural double bonds formed
FIGURE 3.1
Space-filling and conventional models of triacylglycerols: (A) space filling; (B) conformational;
(C) stereospecific numbering (sn) of glycerides. If the R3 substituent is PO4, the compound is
phosphatidic acid.
Basic Lipidology 33
TABLE 3.1
Fatty Acids Important in Nutrition
Melting
Symbola Systematic Nameb Common Name Point (oC) Sources
Saturated Fatty Acids (SFA)
2:0 n-Ethanoic Acetic 16.7 Many plants

3:0 n-Propanoic Propanoic −22.0 Rumen
4:0 n-Butanoic Butyric −7.9 Rumen and milk fat
6:0 n-Hexanoic Caproic −8.0 Milk fat
8:0 n-Octanoic Caprylic 12.7 Milk fat, coconut
10:0 n-Decanoic Capric 29.6 Milk fat, coconut
12:0 n-Dodecanoic Lauric 42.2 Coconut, palm kernel
14:0 n-Tetradecanoic Myristic 52.1 Milk fat, coconut
16:0 n-Hexadecanoic Palmitic 60.7 Most common SFA in
plants and animals
18:0 n-Octadecanoic Stearic 69.6 Animal fat, cocoa butter
20:0 n-Eicosanoic Arachidic 75.4 Widespread minor
22:0 n-Docosanoic Behenic 80.0 Minor in seeds
24:0 n-Tetracosanoic Lignoceric 84.2 Minor in seeds
Monounsaturated (Monoenoic) Fatty Acids
10:1 n-1 cis-9-Decanoic Caproleic Milk fat

12:1 n-3 cis-9-Dodecanoic Lauroleic Milk fat
14:1 n-5 cis-9-Tetradecanoic Myristoleic Milk fat
16:1 n-7t trans-Hexadecanoic Palmitelaidic HVOc
16:1 n-7 cis-9-Hexadecanoic Palmitoleic 1 Most fats and oils
18:1 n-9 cis-9-Octadecanoic Oleic 16 Most fats and oils
18:1 n-9t trans-9-Octadecanoic Elaidic 44 Ruminant fat, HVO
18:1 n-7t trans-11-Octadecanoic trans Vaccenic 44 Ruminant fat
20:1 n-11 cis-9-Eicosanoic Gadoleic Fish oils
20:1 n-9 cis-11-Eicosanoic Gondoic 24 Rapeseed, fish oils
22:1 n-9 cis-13-Docosanoic Erucic 24 Rapeseed, mustard oil
Polyunsaturated (Polyenoic) Fatty Acids
Dienoic
18:2 n-9 cis,cis-6,9- −11 Minor in animals
Octadecadienoic
18:2 n-6 cis,cis-9,12- Linoleic −5 Most plant oils
Octadecadienoic
Trienoic
18:3 n-6 All-cis-6,9,12- γ-Linolenic Evening primrose,
Octadecatrienoic borage oils
18:3 n-3 All-cis-9,12,15- α-Linolenic −11 Soybean and Canola oils
Octadecatrienoic
20:3 n-6 All-cis-8,11,14- Dihomo-
Eicosatrienoic gammalinolenic
(continued)
TABLE 3.1 (CONTINUED)

Fatty Acids Important in Nutrition
Melting
Symbola Systematic Nameb Common Name Point (oC) Sources
Tetra-, Penta-, Hexanoic

20:4 n-6 All-cis-8,11,14- Arachidonic −49.5 Meat
Eicosatetranoic
20:5 n-3 All-cis-5,8,11,14,17- EPA, Timnodonic Fish oils
Eicosapentanoic
22:4 n-6 All-cis-7,10,13,16- Adrenic Brain
Docosatetranoic
22:5 n-6 All-cis-7,10,13,16,19- DPA, Brain
Docosapentanoic Clupanodonic
22:6 n-3 All-cis-4,7,10,13,16,19- DHA Fish
Docosahexanoic
a Number of carbons: number of double bonds, location of first double bond from the
methyl carbon; t = trans.
b Geometric isomer-∆ positions of double bonds.
c Hydrogenated vegetable oil.
by removal of hydrogens are primarily in the cis configuration and in methyl

interrupted positions (three carbons apart) rather than in conjugated posi-
tions. Enzymes exist in mammalian systems to insert double bonds between
the C-9 position and the carboxyl carbon. Plant enzymes synthesize 18 car-
bon fatty acids with double bonds at the n-3 (methyl carbon minus 3) or ω-
3 (omega carbon minus 3) and the n-6 positions. Because those fatty acids
are required in metabolism, they are essential components of human diets.
Some trans fatty acids are naturally occurring but most in the human diet
are formed by chemical hydrogenation of vegetable oils. The cis or trans
configuration (Figure 3.3) is very important in metabolism, conveying major
aspects of physical conformation and therefore reactivity to the molecule
(Figure 3.2). The melting points of cis unsaturated fatty acids are lower than
saturated or trans unsaturated fatty acids (Table 3.1).
Phospholipids and Sphingolipids

The glycerol molecule is the backbone of the major group of phospholipids
or glycerophosphatides (Figure 3.4). Phosphatidic acid is the building block
to which fatty acids are esterified at the C1 and C2 positions, with the C1
position having a saturated and the C2 position an unsaturated fatty acid.
Individual phospholipids have different patterns of fatty acids and are further
characterized by their derivatives with the compounds choline, ethanolamine,
serine, inositol and others. Phosphatidylcholine is better known by its com-
mon name of lecithin. The presence of polar constituents makes phospholip-
ids both neutral and polar and they are therefore amphipathic, reacting at
hydrophobic and hydrophilic interfaces.
Basic Lipidology 35
FIGURE 3.2
Space-filling and conventional models of fatty acids: (A) stearic acid (18:0), space-filling; (B)
stearic acid, conformational; (C) elaidic acid (18:1n-9t) trans, conformational; (D) α-linolenic
acid, all-cis, conformational.
FIGURE 3.3
Hydrogenation of cis and trans double bonds.
FIGURE 3.4
Structure of common phospholipids.
Another group of phosphorus-containing lipids is formed from the

18-carbon amino alcohol sphingosine (Figure 3.5). Linked to a long-chain
fatty acid, it forms ceramide and with the inclusion of sugar molecules it
forms cerebrosides and gangliosides. Some of the glycosphingolipids have
sialic (N-acetyl neurominic) acid linked to one or more of the sugar residues
of a ceramide oligosaccharide. These compounds are functional in mem-
branes, particularly in nervous tissue.
Sterols and Steroids

Steroids are hydrocarbons with a rigid ring structure and, having few sites
with reactive groups, are quite hydrophobic (Figure 3.6). Plant sterols are
called phytosterols and the major animal sterol is cholesterol. The structure
FIGURE 3.5
General structure of sphingolipids.
Basic Lipidology 37
PSYCHOSINE
SPHINGOSINE
CH3(CH2)12 CEREBROSIDE
C=C
CERAMIDE
H CH–CH–CH2–O–Sugar–Sugar–Sugar NEUTRAL
OH NH CERAMIDE
C=O
(CH2)13
CH3
FIGURE 3.6
Space-filling and conventional models of cholesterol. (A) conventional; (B) space-filling.
accounts for its dielectric characteristic and its functions in nerve tissues and
membranes. Steryl esters do not cross membranes, whereas free cholesterol
crosses by passive diffusion. Cholesterol is converted to bile salts by hydrox-
ylation and conjugation. It is the precursor to steroid-based hormone
systems, sex hormones and adrenocorticoid hormones. As 7-dehydrocholes-
terol, cholesterol is the precursor to vitamin D, the only biological conversion
of cholesterol that breaks the ring structure. Because it cannot be degraded,
cholesterol must be excreted as free cholesterol or bile acids.
Lipid Digestion
The average American diet contains about 150 g of fat daily, mainly as
triacylglycerols with cholesterol making up 300–600 mg. Digestive enzymes,
being proteins, are water soluble and the dietary fat is hydrophobic. The
beginning of digestion is the secretion of lingual lipase, which acts on mainly
short-chain acylglycerols in the mouth and stomach. Partial emulsification
occurs by muscular action and the presence of phospholipids, polysacchar-
ides and peptides. The major digestive activity occurs in the duodenum and
the ilium. There the presence of bile acids and pancreatic phospholipids
effects further emulsification and formation of micelles (Figure 3.7). Bile salts
adhere to the surface of lipid droplets and prevent access by lipase (Figure
3.8). Lipolysis occurs because procolipase is secreted with lipase and is
activated to colipase by pancreatic trypsin; the colipase complexes with
lipase and also binds to the lipid droplets, permitting lipase to come in
FIGURE 3.7
Orientation of fatty acids in micellar configuration.
contact with the triacylglycerols. Hydrolysis of fatty acids occurs, yielding

free fatty acids and sn2-monoaclyglycerols. Bile micelles are formed when
bile acids and phospholipids reach a critical micellar concentration. Water-
soluble micellar aggregates engulf free fatty acids and monoacylglycerols
and the micelles diffuse through the unstirred water layer. Passage through
the unstirred water layer is the rate-limiting step in lipid absorption. The
free fatty acids and monoacylglycerols diffuse freely through the mucosal
membrane and are re-esterified into triacylglycerols that do not passively
diffuse in the reverse direction.
Phospholipids are hydrolyzed by pancreatic phospholipase A2, cleaving
the fatty acid at the sn2 position. The resulting lysophospholipid, along with
the free fatty acids, diffuse into the mucosal cell where the remaining fatty
acid is cleaved from the lysophospholipid. Inside the mucosal cell the phos-
pholipid is reformed by acylation. Cholesterol from the diet is hydrolyzed
and free cholesterol in micelles from biliary secretion is subjected to the same
micellar transport through the unstirred water layer and into mucosal cells
as fatty acids. Free cholesterol is re-esterified and the cholesteryl ester does
not diffuse in the reverse direction.
Regulation of Cholesterol Synthesis

The metabolism of cholesterol contains a system of controls that are, of
course, maintained by genetic coding. The metabolic systems are described
Basic Lipidology 39
FIGURE 3.8
Processes of digestion and absorption of dietary fat. Hydrolyzed fatty acids and monoacyl-
glycerides are made water soluble by incorporation into micelles and thereby cross the unstirred
water layer, then diffuse into mucosal cells where they are re-esterified.
here and much research is underway to determine the genetic regulatory

elements. Cholesterol synthesis occurs in the liver, although all mammalian
cells examined have the capacity to synthesize cholesterol and extrahepatic
tissues account for most synthesis in humans. The necessary precursor is
acetyl coenzyme A (CoA). It is generated within the mitochondria and con-
verted into citrate, which diffuses into the cytosol and is hydrolyzed by
citrate lyase to yield acetyl-CoA and acetoacetate (Figure 3.9). Three mole-
cules of acetyl CoA are converted to β-hydroxy-β-methyl glutaryl (HMG)
CoA. The action of HMG CoA reductase produces mevalonic acid, and that
enzymic step is the major site of regulation of cholesterol synthesis. Statin
drugs are inhibitors of HMG CoA reductase. Mevalonate is phosphorylated
FIGURE 3.9
Synthesis of cholesterol. Three molecules of acetyl CoA form HMG CoA which is reduced to
mevalonate by an irreversible step; three mevalonate molecules condense to form farnesyl
pyrophosphate, and two of those condense to form squalene. Squalene is cyclized to form
lanosterol. Many reactions remove three methyl groups and transfer the double bond from the
8–9 to the 5–6 position.
and three molecules are combined to produce farnesyl pyrophosphate and

two molecules of it unite to produce the 30-carbon squalene. Squalene is
oxidized and cyclized to form the steroid ring lanosterol in a reaction that
is not reversible in mammalian systems. Loss of three methyl groups com-
pletes the formation of cholesterol, a total process involving more than
20 steps.
The other significant step in the regulation of cholesterol synthesis is the
formation of bile acids (Figure 3.10). The irreversible synthetic enzyme of
bile salts is 7α-hydroxylase. Bile acids are secreted via the gallbladder and
Basic Lipidology 41
FIGURE 3.10
Structural formulas of cholesterol, cholanoic acids, and two commonly occurring primary bile
acids.
bile duct into the intestine and a large portion is reabsorbed. This enterohe-
patic circulation is a factor in regulation of cholesterol synthesis by control-
ling the demand for bile acid formation for digestion. Cholesterol itself is
also reabsorbed and the system of synthesis and absorption constitutes a
continuous self-regulating cycle of cholesterol metabolism. Factors that
impinge on one factor have cascading effects on the complete cycle to main-
tain a constant whole body cholesterol balance.
Essential Fatty Acids and Eicosanoids

Linoleic and α-linolenic (18:2 n-6 and 18:3 n-3) are called essential fatty acids.
They cannot be synthesized by mammalian organisms because of the absence
of enzymes that can introduce double bonds beyond the ∆9 (9th carbon from
FIGURE 3.11
Families of fatty acids formed from C18 precursors by desaturation (D) and elongation (E). The
n-9 pathway is exhibited only in mammals deficient in dietary n-6 fatty acids.
the carboxyl) position. The n-minus 3 or 6 nomenclature is used instead of

delta because it makes reference to the fatty acid families clearer (Figure
3.11). Linoleate is essential in skin integrity and other membrane functions.
The elongation and desaturation of 18:2 n-6 to the 20 carbon fatty acid
arachidonic acid (20:4 n-6) is the precursor for the family of autocoids known
as eicosanoids (Figure 3.12). Oxygenation of the arachidonate by cyclooxy-
genase results in cyclization to form prostaglandins and thromboxanes.
Action of lipoxygenase in linear reactions produces leukotrienes. These com-
pounds are formed in extremely small amounts and have potent metabolic
functions.
They participate in physiological reactions regulating blood pressure,
diuresis, blood platelet aggregation, the immune system, gastric secretions,
reproduction, smooth muscle contractions and others. Excess production is
associated with adverse reactions such as inflammation. Their study is a
major activity in lipid metabolism research.
Basic Lipidology 43
FIGURE 3.12
Eicosanoid synthesis from arachidonate (C20:4n-6). HETE (hydroxyeicosatetranoic acid), PETE
(peroxyeicosatetranoic acid), PG (prostaglandin), GSH (glutathione), and MDA (malondialde-
hyde).
Lipid Peroxidation
The presence of methyl-interrupted double bonds creates a methyl group
in the polyunsaturated fatty acid chain that is vulnerable to oxidation.
Removal of a hydrogen results in formation of a free radical that is very
reactive and starts a chain reaction that propagates in a cascade (Figure
FIGURE 3.13
Peroxidation of polyunsaturated fatty acids. Autoxidation results when a hydrogen atom is
removed by an oxidizing agent from a methylene group between two double bonds leaving a
resonating free radical that can be reduced by an antioxidant such as tocopherol. The free radical
can propagate additional free radicals or be oxidized by molecular oxygen to a peroxide that
can degrade to many smaller compounds or polymerize.
3.13 and Figure 3.14). This process is called peroxidation and it is con-
trolled in vivo by antioxidants. The products of the oxidation can react
with proteins and DNA, causing deleterious changes. Oxidation is obvi-
ously necessary for such reactions as formation of eicosanoids, but if there
is not sufficient antioxidant capacity at the cellular level metabolic damage
is done. Tocopherols are major cellular antioxidants. and many newly
described plant components are being investigated as contributors to
control of peroxidation.
Basic Lipidology 45
FIGURE 3.14
Peroxidation of polyunsaturated fatty acids.
References
1. Dupont, J. Lipids, in Present Knowledge in Nutrition, Brown, M.L., Ed. Interna-
tional Life Sciences, Washington, DC, 1990, chap.7.
2. Dupont, J. Saturated and hydrogenated fats in food in relation to health, J. Am.
Coll. Nutr., 10:577–592, 1991.
3. Jones, P.J.H. and Papamandjaris, A. Lipids: Cellular metabolism, in Present
Knowledge in Nutrition, Bowman, B.A. and Russell, R.M., Eds. International Life
Sciences, Washington, DC, 2001, chap. 10.
4. Lichtenstein, A.H. and Jones, P.J.H. Lipids: Absorption and transport, in Present
Knowledge in Nutrition, Bowman, B.A. and Russell, R.M., Eds. International Life
Sciences, Washington, DC, 2001, chap. 9.
5. Gropper, S.S., Smith, J.L. and Groff, J.L. Advanced Nutrition and Human Metab-
olism, 4th ed. Thomson Wadsworth, Belmont, CA, 2005, chap. 6.
6. Gurr, M.I., Harwood, J.L. and Frayn, K.N. Lipid Biochemistry, An Introduction,
5th ed. Blackwell Science, Iowa State Press, Ames, IA, 2002.
4
Lipid and Lipoprotein Metabolism
Paul G. Davis and Jason D. Wagganer
CONTENTS
Introduction ...........................................................................................................47
Lipoprotein Classification....................................................................................50
Lipid Transport .....................................................................................................51
Lipid Transport: Exogenous Pathway......................................................51
Lipid Transport: Endogenous Pathway ...................................................53
Reverse Cholesterol Transport............................................................................54
Formation of HDL .......................................................................................54
Reverse Cholesterol Transport: Direct Pathway.....................................56
Reverse Cholesterol Transport: Indirect Pathway..................................57
Other Anti-Atherogenic Roles of HDL ....................................................58
References ............................................................................................... 58
Introduction
Due to their hydrophobic nature (insoluble in water), cholesterol and tri-
glyceride (or triacylglycerol), the major lipids in the blood, must be trans-
ported within lipoproteins. Lipoproteins constitute a packaging of
electrically neutral lipid (triglyceride and esterified cholesterol) within a
monolayer shell consisting of mostly phospholipids and also of proteins
(apolipoproteins) and a small amount of unesterified or “free” cholesterol.
The components of this outer shell are amphipathic (charged on one end
and neutral on the other). With their neutral ends facing the hydrophobic
core and the charged ends facing outward, the outer shell components make
the lipoprotein particles water soluble, allowing them to be transported
through the circulatory system.
47
Lipoproteins are classified by density (high-density, low-density, etc.). The

density is dictated by both the lipid content (i.e., the amount of cholesterol
and triglyceride) and the ratio of the amounts of lipid and protein in the
lipoprotein. For example, high-density lipoprotein (HDL) contains approxi-
mately equal amounts of lipid and protein, while lipoproteins of lower
density contain much larger amounts of lipid than protein, making them
larger and more buoyant. Lipoproteins are most commonly classified based
on their density gradients following ultracentrifugation. Characteristics of
the lipoproteins and their major subfractions isolated through this method
are listed in Table 4.1. Other common methods of lipoprotein classification
include (1) non-denaturing polyacrylamide gradient gel electrophoresis, in which
lipoproteins of different sizes migrate different distances across the gel;
(2) agarose gel electrophoresis, in which lipoproteins migrate different distances
based upon their different surface charges; and (3) nuclear magnetic resonance
(NMR) spectroscopy, in which “signals” emitted from the terminal methyl
groups of the lipids within the lipoproteins are read and lipoproteins are
classified based upon this lipid content.
Although lipoproteins are generally classified by density, it is primarily
the apolipoprotein content of a lipoprotein that determines both its function,
as well as types and quantities of its lipid content. In addition to serving as
important structural components of the different lipoproteins, different apo-
lipoproteins also serve as ligands for specific receptors and as cofactors for
specific enzymes. Conversely, change in the lipid content (and size) of a
given lipoprotein particle can alter the tertiary structure of an apolipoprotein,
resulting in either an increased or decreased affinity for a particular receptor
or enzyme. Therefore, both the lipid and the apolipoprotein contents are key
factors in determining a lipoprotein’s “behavior.” The major plasma apolipo-
proteins are described in Table 4.2 and will be discussed below. All, with the
exception of the B class, are “exchangeable,” meaning that they may leave
one lipoprotein particle and be incorporated into particle of another.1
The remainder of this chapter begins with a brief description of the com-
position of the different lipoproteins and will then discuss the coordinated
role of these lipoproteins in transport of lipid to tissues throughout the body
(including atherosclerotic plaque) and the process of reverse cholesterol trans-
port, in which lipid is harvested from atheroma and catabolized. This chapter
discusses atherosclerosis only to the extent that lipid transport is involved.
More detailed mechanisms of atherosclerosis, as well as the effects of various
interventions on lipoprotein metabolism, are detailed in other chapters of
this text.
TABLE 4.1
Characteristics of Major Lipoproteins
Lipoprotein Density Electrophoretic Diameter Approximate NMR % Composition
Class (g/ml) Mobility (nm) Classification TG CE FC PL apo
Chylomicron <0.94 Origin 75–1200 – 86 3 2 7 2
VLDL 0.94–1.006 Pre-beta 30–80 V6-V1 55 12 7 18 8
IDL 1.006–1.019 Slow pre-beta 25–35 IDL 23 29 9 19 19
LDL 1.019–1.063 Beta 18–25 L3-L1 6 42 8 22 22
HDL2 1.063–1.125 Alpha 9–12 H5-H3 5 17 5 33 40
HDL3 1.125–1.21 Alpha 5–9 H2-H1 3 13 4 35 55
VLDL, very low-density lipoprotein; IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein; HDL, high-
Lipid and Lipoprotein Metabolism
density lipoprotein; TG, triglyceride; CE, cholesteryl ester; FC, free cholesterol; PL, phospholipids; apo, apolipoprotein.
Source: Adapted from Burnett, J.R., Barrett, P.H.R., Crit. Rev. Clin. Lab. Sci., 39, 89, 2002. With permission.
49
TABLE 4.2
Major Apolipoproteins
Major Sites Associated
Apolipoprotein of Synthesis Lipoproteins Major Functions
A-I Liver, intestine Chylomicron, Accepts cholesterol from peripheral
HDL cells through ABCA1; cofactor for
LCAT; facilitates lipid uptake
through SR-BI
A-II Liver HDL Facilitates lipid uptake through SR-
BI; displaces apo A-I from HDL
B-48 Intestine Chylomicron Structural component
B-100 Liver VLDL, IDL, Facilitates lipid uptake through LDL
LDL receptor
C-I Liver, lung, Chylomicron, Inhibits HL activity; activates LPL
skin, testes, VLDL, HDL activity; inhibits apo E-mediated
spleen lipid uptake by LDL receptor and
LRP
C-II Liver, intestine Chylomicron, Cofactor for LPL
VLDL, HDL
C-III Liver, intestine Chylomicron, Inhibits LPL and HL activity; may
VLDL, HDL stimulate CETP activity
E Liver, brain, Chylomicron, Facilitates lipid uptake through LDL
skin, testes, VLDL, HDL receptor and LRP
spleen
(a) Liver Lp(a) Most likely inhibits fibrinolysis
through competing with
plasminogen for binding with fibrin
HDL, high-density lipoprotein; VLDL, very low-density lipoprotein; IDL, intermediate-den-
sity lipoprotein; LDL, low-density lipoprotein; ABCA1, adenosine triphosphate-binding cas-
sette-A1; LCAT, lecithin:cholesteryl acyltransferase; SR-BI, scavenger receptor BI; HL, hepatic
lipase; LPL, lipoprotein lipase; LRP, LDL receptor-related protein; CETP, cholesteryl ester
transfer protein.
Lipoprotein Classification
The major lipoproteins are chylomicrons, very low-density lipoprotein
(VLDL), intermediate-density lipoprotein (IDL), low-density lipoprotein
(LDL), and high-density lipoprotein (HDL) (Table 4.1). In short, chylomi-
crons and VLDL are the major carriers of triglyceride and are synthesized
and released by the intestine (chylomicrons) and liver (VLDL). Chylomicrons
and VLDL contain approximately 85% and 55% triglyceride, respectively,
with smaller amounts of cholesterol, protein, and phospholipids. As
explained below, the primary apolipoproteins are apolipoprotein (apo) B-48
for chylomicrons and apo B-100 for VLDL. Apo C-II and apo C-III also have
important regulatory roles. NMR spectroscopy divides VLDL into six sub-
classes (V1–V6), with V6 being the largest and most triglyceride-laden. With
Lipid and Lipoprotein Metabolism 51
the exception of those with exceptionally high triglyceride concentrations,

chylomicrons are present in only trace amounts in fasted plasma or serum
samples.
After a brief transition through IDL, which has a relatively small concen-
tration in plasma/serum, VLDL may be converted to LDL. LDL is the most
cholesterol-rich of the lipoproteins, containing approximately 50% choles-
terol and 4% triglyceride, although these proportions vary with different-
sized LDL particles. All VLDL, IDL, and LDL particles, regardless of size,
contain exactly one apo B-100. Apo E also resides on both VLDL, IDL, and
LDL, as well as HDL. NMR spectroscopy divides LDL into three subclasses
(L1–L3), with L1 being the smallest, densest, and containing the most tri-
glyceride and least esterified cholesterol. LDL subfractions (as many as six)
can also be derived by polyacrylamide gradient gel electrophoresis.
HDL contains the largest amount of protein (~50%) and smallest amount
of lipid of all the lipoproteins. The vast majority of lipid contained in HDL
is cholesterol ester. Ultracentrifugation preparations typically divide HDL
into two main subfractions, HDL2 and HDL3, with HDL3 being the more
dense of the two (a less dense “HDL1” has been described, but is not com-
monly found in discernable amounts). All HDL particles contain at least one
apo A-I, and most HDL3 particles also contain apo A-II. It is important to
note that, since lipoprotein fractions and subfractions are classified by den-
sity, they exist on a continuum, meaning that a certain amount of heteroge-
neity exists (e.g., apolipoprotein content) within each classification. NMR
spectroscopy divides HDL into five subclasses (H1–H5), with H1 and H2
corresponding roughly to HDL3 and H3–H5 approximating the HDL2 sub-
fraction.2
Lipid Transport
Lipid transport to tissues via the lipoproteins is summarized in Figure 4.1.
Separate pathways exist for the transport of exogenous (dietary) and endog-
enous (hepatic) lipids. The two pathways are similar, in that large lipid-laden
lipoproteins secreted from the intestine or liver are “trimmed” into smaller
lipoproteins or remnants, which provide lipid to the liver or other tissues
through receptor-mediated mechanisms.
Lipid Transport: Exogenous Pathway

In the exogenous pathway, apo B-48 is synthesized by intestinal cells and,
incorporated with mostly triglyceride, enters the lymphatic system and even-
tually the circulatory system in the form of chylomicrons. Although they
play a more minor role in the structure and metabolism of chylomicrons,
Small
B-100 VLDL E
Intestine
LPL
Peripheral Tissue
Liver FFA
B-100 C
IDL
LDL-R
HL
E
LPL
C E
FFA
FFA
B-100 LDL C
LRP Chylomicron
Chylomicron
E
Remnant
LPL A
B-48 E B-48
Apo A & C
to HDL
FIGURE 4.1
Lipid transport. Dietary lipid is secreted from the small intestine in chylomicrons. The triglyc-
erides of chylomicrons are hydrolyzed by lipoprotein lipase (LPL). Free fatty acids (FFA) are
then taken up by numerous body tissues while chylomicron remnants are catabolized primarily
by the liver through both the low-density lipoprotein (LDL) receptor (LDL-R) and the LDL
receptor-related protein (LRP) through recognition of apolipoprotein (apo) E. De novo synthe-
sized lipid is released from the liver primarily through very low-density lipoprotein (VLDL).
LPL hydrolyzes the triglyceride of VLDL, reducing the lipoprotein to an intermediate-density
lipoprotein (IDL), which is subsequently hydrolyzed by LPL and hepatic lipase (HL) into LDL.
Hepatic catabolism of LDL occurs mainly through apo B-100 recognition by the LDL receptor.
apo A-I and A-IV are also included in these lipoproteins when released from
the intestine, while apo C-I, C-II, C-III, and E are incorporated into the
lipoprotein within the circulation as a result of transfer from HDL.3 The
incorporation of apo C-II into the chylomicron particle is essential for the
catabolism of triglyceride, as this apolipoprotein is a cofactor for the enzyme
lipoprotein lipase (LPL). LPL, which is attached to the luminal surface of
capillary endothelial cells via heparin sulfate-proteoglycans, hydrolyzes the
fatty acids of triglyceride at the first and third positions, allowing them to
be taken up by adjacent tissue (usually muscle or adipose tissue) or to be
bound and transported within circulation to other tissues, including the liver,
by albumin.4 Hepatic lipase (HL; also termed hepatic triglyceride lipase) also
performs this function, although its activity does not depend upon an apo-
lipoprotein co-activator, as does LPL’s.5 Left over are “remnants” consisting
of cholesterol, phospholipids, apolipoproteins, and much less triglyceride.
Most of the apo A and a portion of the apo C are transferred to HDL and
the remaining remnants are available for catabolism by the liver. This can
occur through an LDL receptor or through an LDL receptor-related protein

(LRP), both of which endocytose the remnants into lysosomes, where hydrol-
ysis of the remaining lipoprotein components takes place.3 Apo E serves as
the ligand for both of these receptors. Three major alleles for apo E exist,
with ε3 being the most common. Protein expressed by the ε2 allele does not
bind the LDL receptor and results in increased plasma VLDL-cholesterol
concentration. Conversely, ε4 produces a protein with abnormally strong
binding to the LDL receptor. The prolonged binding inhibits other apo
E-containing from binding the receptor, resulting in increased LDL-choles-
terol concentration.6 In addition, apo C-I inhibits apo E-mediated lipid
uptake by both the LDL receptor and LRP.7 Apo B-48 is not recognized by
the hepatic receptors. However, an apo B-48-specific receptor has been found
to be expressed by macrophages, indicating a possible mechanism for hyper-
triglyceridemia-induced atherosclerosis.8 Nearly all chylomicrons are absent
from circulation within 12 h following a fatty meal.9
Lipid Transport: Endogenous Pathway

Transport of cholesterol and triglyceride synthesized by the liver (endog-
enous pathway) takes place through release of VLDL, which contain apo
B-100 (a “longer” version of apo B-48) and apo C-I, C-II, C-III, and E. As
with chylomicrons, apo C-II serves as a cofactor for LPL, which hydrolyzes
much of the triglyceride within VLDL. This hydrolysis results in a less
buoyant IDL particle. Further hydrolysis by both LPL and hepatic lipase (HL)
results in the loss of most of the triglyceride, as well as apo E, leaving an
even less buoyant LDL particle, which contains esterified cholesterol as its
major lipid component and apo B-100 as its primary apolipoprotein. Apo
B-100 is recognized by the LDL receptor on the liver and other tissues, which
internalizes the lipoprotein, making the cholesterol available for cell mem-
brane structure and steroid hormone synthesis. Regarding both chylomicron
and VLDL metabolism, just as apo C-II is a cofactor for LPL, apo C-III inhibits
LPL and HL activity.7 Therefore, the ratio of apo C-II to apo C-III is important
in regulating plasma concentrations of triglyceride, as well as VLDL and
LDL.
When an insufficient number of LDL receptors are synthesized or the
receptors do not possess proper affinity for apo B (genetic abnormalities
resulting in familial hypercholesterolemia), or when dietary fat intake is high
(which causes down-regulation of LDL receptor synthesis), plasma choles-
terol concentration is abnormally high. This excess cholesterol contained in
apo B-containing lipoproteins, particularly LDL, can be internalized in mac-
rophages and foam cells in the vascular intima through scavenger receptors
(CD36, SR-A), which do not require specific apolipoprotein ligands. These
scavenger receptors have a much higher affinity for LDL in the oxidized
form. Oxidized LDL also contributes to vascular inflammation and inhibits
nitric oxide, a potent vasodilator.10
In addition to the above, lipoprotein (a) [Lp(a)] transports cholesterol, is

often present in atherosclerotic plaque, and may be particularly atherogenic.
Lp(a) is a variant of LDL and is characterized by the covalent attachment of
the glycoprotein apo(a) to apo B-100. Due to one or more proposed mecha-
nisms, apo(a) is most likely responsible for Lp(a)’s atherogenic effect. Apo(a)
consists of repeated coils of protein called “kringles” with amino acid
sequencing very similar to that of plasminogen. Because of this homology,
Lp(a) may inhibit fibrinolysis through interference at the binding site of
fibrin.11 Second, Lp(a) may accelerate wound healing by transporting lipid
to the vascular intima to combine with extracellular matrix components.12
As such, Lp(a) may act as a “repairer” of endothelial injury. Third, the LDL
receptor does not appear to play a major role in clearance of Lp(a) from
plasma.13 This may result in the cholesterol of Lp(a) being more available
for uptake by scavenger receptors. In addition, the LDL component of Lp(a)
is the smaller, denser phenotype associated with higher cardiovascular dis-
ease risk (see Reverse Cholesterol Transport section). Finally, apo(a) may con-
tribute to atherogenesis by increasing monocyte chemotactic activity in the
vasculature,14 possibly through up-regulation of intracellular adhesion mol-
ecule-1 (ICAM-1).15 Lp(a)’s relationship with cardiovascular disease varies
across races. For example, blacks commonly have higher plasma Lp(a) con-
centrations than whites, yet Lp(a) concentrations are not typically related to
cardiovascular disease risk in blacks.16 The number of kringle repeats present
in Lp(a) is highly variable and may explain differences in the relation of
Lp(a) to cardiovascular disease among races.17
Reverse Cholesterol Transport

While excess cholesterol carried by LDL and VLDL is associated with
atherosclerosis due to its uptake by scavenger receptors in vascular lesions,
HDL may protect against atherosclerosis through a process termed reverse
cholesterol transport, whereby HDL harvests cholesterol from arterial plaque
(and other body tissues, as well) and transports it to the liver where it may
be catabolized and secreted as bile. As simple as this concept appears,
successful reverse cholesterol transport relies upon the availability and
activity of a collection of apolipoproteins, enzymes, transfer proteins, and
receptors. The process of reverse cholesterol transport is summarized in
Figure 4.2.
Formation of HDL
The formation of HDL is dependent upon release of apo A-I from the liver
and intestines and upon formation of remnants containing apo A-I following
Peripheral
Tissue or Liver
Macrophage:
FC / PL SR-BI
TG-rich
lipoproteins
ABCA1
TG
Pre-fl 1 HDL Pre-fl 2 HDL CE
HDL2
HDL3 CETP
LCAT
TG CE CE TG
LCAT
FIGURE 4.2
Reverse cholesterol transport. Lipid-poor pre-beta1 high-density lipoprotein (pre-β1 HDL), com-
posed primarily of apolipoprotein (apo) A-I, sequesters free cholesterol (FC) and phospholipid
(PL) from peripheral tissues and arterial macrophages via the adenosine triphosphate-binding
cassette-A1 (ABCA1) transporter. Incorporation of the FC and PL allows HDL to assume a more
discoidal shape (pre-β2 HDL). HDL associated apo A-I and apo C-I permit lecithin:cholesteryl
ester transferase (LCAT) and facilitate esterification and internalization of cholesterol, allowing
HDL to become more spherical (HDL3). Further esterification of cholesterol by LCAT transforms
HDL3 into the less dense HDL2. Cholesteryl ester transfer protein (CETP) facilitates transfer of
CE from HDL to triglyceride (TG)-rich lipoproteins in exchange for TG. Adding and removing
CE to/from HDL, LCAT and CETP, respectively, cause HDL particles to be in constant flux
between HDL2 and HDL3. Hepatic catabolism of HDL occurs mainly through apo A-I and apo
A-II recognition by the scavenger receptor B-I (SR-BI).
chylomicron, VLDL, and HDL catabolism. While most analytical techniques

quantify HDL primarily in its spherical forms (e.g., HDL2, HDL3), formation
of HDL begins with an immature, non-spherical particle that migrates to
form a small pre-beta band via agarose gel electrophoresis (as opposed to
the alpha band formed by the larger HDL species). The pre-beta1 HDL particle,
which is often formed in lymph and delivered to circulation, is a loosely
formed apo A-I bound to small amounts of phospholipid and cholesterol.
The apo A-I may be newly synthesized or may be derived from previous
catabolism of chylomicrons, VLDL, or HDL. The lipid-poor pre-beta1 HDL
crosses the vascular endothelium and enters both interstitial spaces and the
vascular intima where it can interact with various peripheral cells and arte-
rial plaque components, respectively. With the help of the adenosine triphos-
phate-binding cassette-A1 (ABCA1) transporter present in various cells,
including arterial macrophages, pre-beta1 HDL then begins to accumulate
phospholipid and cholesterol from these cells. Through mechanisms not
fully understood, ABCA1 transports phospholipid and cholesterol from
intercellular depots to extracellular apolipoproteins, allowing phospholipid
and cholesterol to become incorporated into the HDL particle. The major
HDL apolipoproteins (A-I, A-II, A-IV, C, and E), all of which contain amphi-
pathic alpha helices, can participate in cholesterol and phospholipid efflux
through ABCA1.18 However, apo A-I of the immature HDL particle, which
can freely traverse the endothelium, is the major acceptor of cholesterol and
phospholipid from peripheral tissues and arterial macrophages. Tangier Dis-
ease patients, who do not synthesize ABCA1, have abnormally low plasma
concentrations of HDL-cholesterol and are at high risk for cardiovascular
disease. ABCA1’s role in cellular cholesterol removal has been reviewed in
detail by Oram.19
As the pre-beta1 HDL incorporates more cholesterol and phospholipid, it
transforms into pre-beta2 HDL, which has a more discoidal shape. In pre-beta2
HDL, apo A-I takes on a more “formal” shape and, along with the phospho-
lipids, “wraps around” the outer edge of the lipoprotein while the cholesterol
is incorporated into the inner “core.” This alteration of apo A-I’s tertiary
structure allows it to become a stronger cofactor for the enzyme lecithin:cho-
lesterol acyltransferase (LCAT). Apo C-I, to a lesser extent, also stimulates
LCAT activity.7 LCAT facilitates transfer of a fatty acid from a phospholipid
to free cholesterol, resulting in esterified cholesterol (cholesteryl ester). Since
cholesteryl ester is particularly hydrophobic, the LCAT reaction results in
further “packaging” of cholesterol within HDL and the previously discoidal
pre-beta2 HDL particle takes on a more spherical shape. This HDL3 particle
(along with HDL2) is one of the two main HDL species typically analyzed
in human plasma.
The end result of reverse cholesterol transport is the delivery of cholesteryl
ester and other lipoprotein components to the liver. As described below, this
is accomplished both through direct and indirect pathways.
Reverse Cholesterol Transport: Direct Pathway

In the direct pathway, LCAT continues to facilitate loading of cholesteryl ester
into the HDL3 particle, converting it to the less dense HDL2. Phospholipase
transfer protein (PLTP) also participates in conversion of HDL3 to HDL2 by
displacing apo A-I and phospholipid from HDL3 to form pre-beta particles.
Although PLTP’s role in HDL3 to HDL2 conversion has not been fully elu-
cidated, loss of some of the apo A-I may result in “unstable” particles. It has
been hypothesized that these unstable particles may combine to form the
less dense HDL2.20 Alternatively, these unstable particles may combine with
pre-beta apo A-II-containing particles.21
HDL delivers cholesterol into cells mainly through the scavenger receptor
class B type I (SR-BI). SR-BI participates in selective uptake of cholesterol,
meaning that it binds HDL and takes in the cholesterol without degrading
the lipoprotein.22 This receptor can bind most of the various lipoproteins,
but has particularly high affinity for both apo A-I and A-II, although con-
flicting reports exist as to which of these two apolipoproteins has the highest
binding affinity and which facilitates the most cholesterol uptake.23,24 Since
SR-BI operates on a concentration gradient, the larger, less dense HDL par-
ticles are able to deliver more cholesterol to the cells. In addition to hepatic
cells, SR-BI is also expressed in steroid-producing cells and in sub-endothe-
lial macrophages. Since SR-BI can facilitate cholesteryl ester transport both
into and out of cells, depending upon the concentration gradient, cholesterol
efflux out of macrophages and foam cells may be an additional mechanism
that SR-BI can contribute to reverse cholesterol transport and protect against
atherosclerosis.25
Reverse Cholesterol Transport: Indirect Pathway

The indirect pathway of reverse cholesterol transport involves the transfer
of cholesteryl ester to the apo B-containing lipoproteins, which then deliver
the cholesterol to the liver as described in the previous section. Cholesteryl
ester transfer protein (CETP), which has a particularly high affinity for HDL,
transfers cholesteryl ester from one lipoprotein to another in exchange for
triglyceride. Since the lipids are transported by CETP on a concentration
gradient, the most common action of this protein is to transfer cholesteryl
ester from HDL to VLDL and LDL in exchange for triglyceride. By exchang-
ing cholesteryl ester for triglyceride, a given HDL particle will become more
dense. A portion of the triglyceride added to HDL through CETP is hydro-
lyzed by hepatic lipase. Through the opposite actions of LCAT and CETP
(i.e., adding and removing cholesteryl ester to or from HDL), it is possible
for HDL to shift back and forth between density ranges (e.g., from HDL3 to
HDL2 and vice versa).
Barter et al. have reviewed the metabolism of CETP and its potential roles
in atherosclerosis in detail.26 Briefly, in plasma with normal lipid concentra-
tions, much of the cholesteryl ester from HDL is transferred to LDL. How-
ever, in hypertriglyceridemic conditions, which result in greater VLDL
concentrations, more lipid exchange takes place between HDL and VLDL,
leaving smaller HDL particles which do not transport as much cholesterol
to the liver for catabolism.27 In addition, more exchange takes place between
VLDL and LDL (cholesteryl ester to VLDL; triglyceride to LDL), leaving less
cholesterol to be taken up through apo B-100 recognition.
The above scenario results in a smaller, denser LDL particle that may be
more atherogenic. A plasma profile consisting of more small dense LDL
(pattern B) than large buoyant LDL (pattern A) is a byproduct of the athero-
genic combination of high triglyceride and low HDL-cholesterol concentra-
tions. In fact, although persons having a pattern B profile are more prone to
cardiovascular disease, this relationship has been shown to disappear when
controlling statistically for triglyceride concentration.28 However, other lon-
gitudinal research has shown the pattern B profile to be independently
related to cardiovascular disease incidence.29 Potential mechanisms by which
small dense LDL may contribute to cardiovascular disease include
(1) increased ability to cross the vascular endothelium and enter the intima,
(2) increased ability to be oxidized, and (3) lower affinity for the LDL receptor
due to altered conformation of apo B-100.30
Other Anti-Atherogenic Roles of HDL

Although reverse cholesterol transport is HDL’s most recognized and pos-
sibly most dominant role, this lipoprotein likely participates in the preven-
tion of atherosclerosis through other mechanisms as well. Protective
mechanisms of HDL independent of reverse cholesterol transport have been
reviewed by Barter et al.31 HDL-associated paraoxonase (a detoxifying
enzyme) can inhibit oxidation of LDL32 and various cell membranes, includ-
ing those of erythrocytes.33 HDL can also inhibit expression of vascular and
intercellular adhesion molecules for monocytes.34 In addition, HDL increases
prostacyclin synthesis35,36 and apo A-I stabilizes its existence in plasma.37,38
Prostacyclin may protect against CAD through vasodilation, inhibition of
platelet aggregation, and inhibition of endothelin-1 synthesis.39 (Endothelin-
1 is a potent vasoconstrictor and smooth muscle mitogen.) Furthermore, in
patients with early development of atherosclerosis, Zeiher et al. demon-
strated less acetylcholine-induced vasoconstriction in patients with the high-
est HDL-cholesterol concentrations.40
In conclusion, lipoproteins represent a complicated, multi dimensional
pathway of lipid transport to and away from various body tissues. The
metabolism of lipoproteins and their effect in contributing to or protecting
against atherosclerosis are highly dependent upon genetic and behavioral
factors that alter the availability and activity of lipids, various apolipopro-
teins, enzymes, and transfer proteins.
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16. Howard, B.V., Le, N.A., Belcher, J.D., et al., Concentrations of Lp(a) in black
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33. Ferretti, G., Bacchetti, T., Busni, D., et al., Protective effect of paraoxanase
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34. Cockerill, G.W., Rye, K.A., Gamble, J.R., et al., High density lipoproteins inhibit
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36. Tamagaki, T., Sawada, S., Imamura, H., et al., Effects of high-density lipopro-
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37. Aoyama, T., Yui, Y., Morishita, H., Kawai, C., Prostacyclin I2 half-life regulated
by high density lipoprotein is decreased in acute myocardial infarction and
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38. Yui, Y., Aoyama, T., Morishita, M., et al., Serum prostacyclin stabilizing factor
is identical to apolipoprotein A-I (apo A-I): a novel function of apo A-I, J. Clin.
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39. Prins, B.A., Hu, R.-M., Nazario, B., et al., Prostaglandin E2 and prostacyclin
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40. Zeiher, A.M., Schächinger, V., Hohnloser, S.H., et al., Coronary atherosclerotic
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89, 2525, 1994.
5
The Vascular Biology of Atherosclerosis
Robert Carter III and Harlan P. Jones
CONTENTS
Introduction ...........................................................................................................61
Anatomical Structure of the Normal Human Artery .....................................63
Endothelial Dysfunction ......................................................................................64
A Tale of Two Hypotheses: Lipids vs. Endothelium ......................................66
Chronic Endothelial Injury Hypothesis ...................................................66
Lipid Hypothesis .........................................................................................67
Stages of Atherosclerosis .....................................................................................68
Initiation of LDL-Mediated Atherogenesis (Lipid Accumulation)......68
LDL Oxidative Modification and Fatty Streak Formation ...................68
Foam Cell Formation (Intracellular Lipid Accumulation by
Macrophages)...................................................................................72
Immigration of Smooth Muscle Cells ......................................................73
Immune Responsiveness during Atherosclerotic Development..........74
Plaque Formation ........................................................................................75
Summary ................................................................................................................77
Acknowledgments ................................................................................................77
References ............................................................................................... 77
Introduction
Cardiovascular disease is the leading cause of mortality in the United States,
Europe, the vast majority of Asia, and is likely to be the greatest threat to
overall health worldwide.1,2 As a major cause of cardiovascular disease, the
development of atherosclerosis starts early in childhood.3 Despite this fact,
most individuals are asymptomatic until many decades later. Autopsy stud-
ies of coronary arteries from healthy, young American soldiers killed during
61
Progressive Narrowing of Artery During Atherosclerosis

FIGURE 5.1
The progression of atherosclerosis. As the atheroma matures the lumen diameter is reduced
which leads to decreased blood flow, thrombosis complications, and unstable plaques. The
clinical presentations may be peripheral artery disease, cerebrovascular disease, or ischemic
heart disease.
the Korean conflict revealed surprisingly advanced atherosclerotic lesions.4

Intimal lesions were discovered in more than 50% of the right coronary
arteries of the youngest group (15–19 years of age). More recently, fatty
streaks, an early marker of atherosclerosis, have been found in the intima of
infants.5 More advanced atherosclerotic lesions are first identified in the
intima of three primary target vessels: the carotid and coronary arteries and
the aorta.6,7 Figure 5.1 illustrates the progressive narrowing of the artery
during atherosclerosis. Although there is significant disparity in the evolu-
tion of lesion formation, ischemic coronary disease, stroke, peripheral artery
disease, and transient ischemic attacks are among the clinical presentations
of matured lesions and ruptured plaques.8, 9
Emerging epidemiologic studies1,10 have shown that elevated low-density
lipoprotein (LDL), male gender, increased homocysteine, and ethnicity are
among the many risk factors and markers involved in the pathogenesis of
atherosclerosis (Table 5.1). In a recent study of 557 first-generation immi-
grants, it was concluded that acculturation into western societies may also
be an independent risk factor for coronary artery disease and atherosclerotic
lesion development.11 Nevertheless, among the consequences of accultura-
tion are stress, dietary patterns, and physical inactivity which also have been
identified as major risk factors for atherosclerosis and cardiovascular disease.
This chapter reviews the recent literature regarding the biology of athero-
sclerosis and considers in detail: (1) anatomical structure of the normal and
diseased artery, (2) chronic endothelial injury and lipid hypotheses, and
(3) the events that contribute to formation of the atherosclerotic lesion. The
authors hope that this chapter will serve as a basic tutorial for the under-
standing of the biology of atherosclerosis, and provide an appreciation for
the complexity of this disease by introducing new and exciting research
contributions to this area.
The Vascular Biology of Atherosclerosis 63
TABLE 5.1
Risk Factors for Atherosclerotic
Lesion Formation
Physical inactivity
Smoking
Infectious agents
Family history
Elevated LDL and VLDL
Low levels of HDL
Elevated lipoprotein (a)
Hypertension
Diabetes mellitus
Male gender
Homocysteine
Ethnicity
Obesity
Age
LDL, low-density lipoprotein; VLDL,
very low-density lipoprotein; HDL,
high-density lipoprotein.
Anatomical Structure of the Normal Human Artery

The structure of the normal artery consists of three layers: the intima, the
media, and the adventitia (Figure 5.2). The intima, the innermost layer, is
composed of an endothelial monolayer lying on the basement membrane
with elastic fibers comprised of type IV collagen, laminin, and heparin sulfate
proteoglycans.12 This layer also contains smooth muscle cells (SMCs) embed-
ded in sulfated polysaccharide, hyaluronic acid intimal thickenings.13
The endothelium of a normal, healthy artery functions as a non-thrombo-
genic surface and serves as a selectively permeable barrier, which regulates
the transport of solutes across the arterial wall. Importantly, the vascular
endothelium is also essential in the regulation of vascular tone, coagulation,
and inflammatory responses.14–16 Changes in shear stress and blood flow lead
to phosphorylation of endothelial nitric oxide synthase (eNOS), which gen-
erates nitric oxide (NO), which then produces vasodilation.17 The intima is
separated from the media by an internal elastic lamina comprised primarily
of the protein polymer elastin.12
The tunica media, the middle layer, is primarily comprised of SMCs sur-
rounded by its own basement membrane. The media’s basement membrane
is anchored within an interstitial matrix composed of type I collagen,
fibronectin, dermatan, and chondroitin sulfate proteoglycans.12,18 This inter-
stitial matrix is intertwined with perforated sheets of elastic fibers.
FIGURE 5.2
Anatomical structure of the normal artery. This illustration displays the three distinct layers of
the vessel wall: intima, media, and adventitia as well as the endothelium and the external and
internal elastic lamina.
The adventitia attaching the vessel to the surrounding tissue is made up

of capillaries, fibroblasts, fat cells, proteoglycans, connective tissue, and elas-
tic and collagen bundles. The adventitia is separated from the tunica media
by the external elastic lamina.12 The connective tissue in the adventitia is
very compressed where it borders the tunica media, but it changes to loose
connective tissue near the periphery of the vessel.19
Endothelial Dysfunction
In humans, the normal endothelium has many unique anti-atherosclerotic
properties, including vasoregulation of conductive and resistance vessels,
monocyte disadhesion, and vessel growth.14,20 The pathophysiological con-
sequences of disruption of these factors serve as hallmarks of endothelial
dysfunction. Endothelial dysfunction as a result of injury leads to compen-
satory responses that modify the normal physiological characteristics of the
endothelium and become the foundation for the disease process.13
Endothelial dysfunction is characterized as a systemic, reversible disorder

and is associated with an impairment in endothelium-dependent vasodila-
tion and recruitment of inflammatory cells to the vessel wall.14,21 Potential
causes of endothelial dysfunction include hypercholesterolemia, diabetes,22
smoking, 23 hypertension, 24 and infectious microorganisms 25 such as
Chlamydia pneumoniae,26 cytomegaloviral infection, Helicobacter pylori infec-
tion, and herpes virus infection,27 many of which are associated with a
reduction in availability of vasodilators such as NO, decreased flow-induced
vasodilation, and increased endothelium-derived contracting factors.23 Lipid
and cell permeability, lipoprotein oxidation, inflammation, platelet activa-
tion, and thrombus formation are all promoted by endothelial dysfunc-
tion. 2 8 , 2 9 The paradigm of endothelial dysfunction propagates a
proatherogenic milieu that favors atheroma formation.30
Ludmer and colleagues, using a selective agonist acetylcholine test, pro-
vided the first evidence in humans of impaired endothelium-dependent
vasodilation in the presence of atherosclerosis,31 which is now attributed to
a reduced bioavailability of NO.15,32,33 In large arteries of humans,23 rabbits,34
pigs,35 and monkeys,36 reduced endothelium-dependent vasodilation due to
atherosclerosis and hypercholesterolemia has been reported. However, the
sensitivity of injured endothelial cells is not homogeneous for all vasoactive
agonists.37 For example, the responsiveness of the endothelial cells to acetyl-
choline, substance P, serotonin, and alpha-adrenergic agonists is severely
decreased, while the responsiveness to bradykinin and adenosine diphos-
phate is only mildly attenuated. In contrast, endothelium-independent
vasodilation to nitro-containing vasodilators is not altered.37
Endothelium dysfunction is also involved in the activation of endothelial-
leukocyte adhesion molecules.38,39 Specifically, P-selectin, E-selectin, intracel-
lular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule
(VCAM-1) are adhesion molecules known to be involved with the recruit-
ment of leukocytes.40 VCAM-1 plays a role in the binding of both monocytes
and leukocytes to endothelial cells. In lesion-prone areas (e.g., endothelial
cells exposed to long duration, high shear stress), VCAM-1 is up-regulated
and occurs in response to inflammatory cytokines.38 Increased expression of
ICAM-1 on endothelial cells has been detected in both lesion-prone areas as
well as on endothelial cells exposed to normal shear stress.41 In humans,
E-selectin is only upregulated on injured endothelial cells and is important
in the regulation of adhesive interactions between certain blood cells and
the endothelium,40,42 whereas P-selectin is involved in adhesion of certain
leukocytes and platelets to the endothelium.43–45 The importance of P-selectin
during atherosclerosis has also been demonstrated in animal models.46 For
example, P-selectin is expressed on endothelial cells overlying active athero-
sclerotic plaques, and inactive atherosclerotic plaques lacking in P-selectin
expression.43 Furthermore, animals lacking P-selectin have a decreased ten-
dency to form atherosclerotic plaques.40
Several potential mechanisms by which statin therapy, angiotensin recep-
tor blockers, and aspirin might improve endothelial dysfunction have been
suggested, including up-regulation of nitric oxide production, reduction of

oxidative stress, and increased adhesion molecule expression.12,33,47 More
recently, the finding that the insulin-sensitizing thiazolidinediones (TZDs),
peroxisome proliferator-activated receptor-gamma (transcription factor)
agonists have antiproliferative and anti-inflammatory effects has led to the
investigation of their possible role in the treatment of endothelial dysfunction
and atherosclerotic lesion formation.12,48
A Tale of Two Hypotheses: Lipids vs. Endothelium

The chronic endothelial injury and the lipid hypotheses are the two main
proliferative mechanisms postulated to explain the underlying pathogenesis
of atherosclerosis. These two hypotheses are not mutually exclusive and are
closely linked by the culmination of molecular and cellular events. The roles
of cell types of the vessel wall in healthy and diseased (atherosclerosis) states
are summarized in Table 5.2. Although others49–53 have postulated alternative
hypotheses about the development of atherosclerosis, the chronic endothe-
lium injury hypothesis is the one most widely accepted.
Chronic Endothelial Injury Hypothesis

Based on pathophysiological evidence in animals and humans, Ross and
Glomset introduced the endothelial injury hypothesis of atherosclerosis,
which initially postulated that endothelial cell uncovering was the initial
step in the development of atherosclerosis.54 However, endothelial dysfunc-
tion is presently considered to be the precursor that initiates the atheroscle-
rotic process and is associated with increased lipoprotein accumulation at
TABLE 5.2
Role of Cell Type of the Vessel Wall in Healthy and Diseased
(Atherosclerosis) States
Cellular Components Healthy Diseased
Endothelial cell NO production Loss of NO production
Vasoreactivity Paradoxical vasoconstriction
Anti-adhesive Leukocyte adhesion
T-cell Inflammatory signals Macrophage stimulation
Cytokine production
Macrophage Lipid uptake Cytokine release
MMP production
Smooth muscle cell Structural Intimal migration
Vasoreactivity Proliferation
NO, nitric oxide; MMP, matrix metalloproteinases.
the site of injury.13,29 The response to the chronic endothelial injury hypoth-
esis or “response to injury hypothesis” of atherosclerosis states that the
protective, inflammatory response followed by the formation of fibroprolif-
erative response begins as a protective mechanism that with time and con-
tinuing insult may become excessive.55,56 Due to release of chemoattractants
and growth regulatory molecules by the altered endothelium,57 leukocytes,58
monocytes, and T lymphocytes59 attach to the endothelial cell surface. The
leukocytes migrate to the subendothelial space, between the tiny junctions
of the endothelial cells, and aggregate within the intima.56 The presence of
elevated levels of oxidized low-density lipoproteins (oxLDL) is the basis of
conversion of monocytes to macrophages, and is a fundamental factor
responsible for injury to the vascular wall.60 Through scavenger cell recep-
tors, macrophages accumulate modified lipid particles and become foam
cells. As the process persists, foam cell and lymphocyte accumulation forms
the basis for the fatty streak.61,62 It is believed that fatty streaks frequently
form at sites with significant intimal smooth muscle accumulation.63 More
advanced lesions develop as a result of continued cell migration and
proliferation56 which eventually turn into a fibrous plaque.64 This hypothesis
is based on the notion that repeated insult to the endothelium leads to
dysfunction, which is followed by a cascade of pathophysiological conse-
quences.
Lipid Hypothesis
In 1913, Nikolai N. Anitschkow demonstrated that cholesterol feeding of
rabbits could induce vascular lesions consistent with the characteristics of
human atherosclerotic lesions.65,66 Unknowingly, his research and others
established the principles of what is now commonly referred to as the “lipid
hypothesis.” Through decades of research and much controversy, the “lipid
hypothesis” is still believed to be one of the prominent mechanisms contrib-
uting to atherosclerosis.66 Based upon its principles, many discoveries have
been made in understanding the pathogenesis of atherosclerosis and the
fight against cardiovascular disease.
Although elevated LDL cholesterol is associated with increased risk for
cardiovascular disease and the pathogenesis of atherosclerosis, LDL has an
essential biological role to transport cholesterol to peripheral tissues.67 Serum
cholesterol is transported by lipoprotein particles that perform important
tasks of carrying both dietary and endogenously produced lipids.68 While
the transport of endogenous lipids is mediated by LDL, very low-density
lipoproteins (VLDL), and high-density lipoprotein (HDL), the dietary lipids
are carried primarily by chylomicrons. For the most part, LDL particles
transport the vast majority of serum cholesterol.
The lipid hypothesis postulates that an elevation in LDL levels results in
penetration of LDL into the arterial wall, leading to lipid accumulation in
SMCs and in macrophages (foam cells).69 LDL also augments smooth muscle
cell hyperplasia and migration into the subintimal and intimal region in
response to growth factors. LDL is modified or oxidized in this environment
and is rendered more atherogenic. Small dense LDL cholesterol particles are
also more susceptible to modification and oxidation. The modified or oxi-
dized LDL is chemotactic to monocytes, promoting their migration into the
intima, their early appearance in the fatty streak,28 and their transformation
and retention in the subintimal compartment as macrophages. Scavenger
receptors on the surface of macrophages facilitate the entry of oxidized LDL
into these cells, transferring them into lipid-laden macrophages and foam
cells. As cell migration and proliferation continues, advanced lesions are
formed which leads to plaque formation.
Stages of Atherosclerosis
Initiation of LDL-Mediated Atherogenesis (Lipid Accumulation)
As postulated by the “lipid hypothesis,” atherosclerotic lesion development
begins with the accumulation of LDL cholesterol levels within the circula-
tion. The studies of Brown and colleagues70 elucidated that the molecular
mechanisms controlling LDL-cholesterol uptake were instrumental in this
determination. Under pathologic conditions where LDL levels are elevated,
lipid accumulation is noticeable along the lining of the arterial wall termed
the tunica lamina (Figure 5.3). The aggregates of lipid particles form intimate
associations with epithelia moieties such as proteoglycans and become
embedded in the tunica lamina structure (Figure 5.4). In defense, the arterial
epithelium fortifies itself with self-protective structural and biochemical
mechanisms that maintain a homeostatic environment in the presence of
lipid accumulation. The expression of molecules such as heparin sulfate
constituents, which provide arterial integrity and blood fluidity and the
expression of many antithrombin molecules,70 are instrumental in protection
against atherogenesis.70 However, under hypercholesterolemic conditions,
the protective integrity of the epithelium falls prey to initiation of lesion
development.
LDL Oxidative Modification and Fatty Streak Formation

Atherosclerotic lesions present initially in the form of fatty streaks forming
along the endothelium of arteries (Figure 5.4). The major contributing event
believed to be responsible in fatty streak development is oxidative modifi-
cations of the lipid and apolipoprotein B (apo B) components of LDL.71
The precise molecular mechanisms responsible for LDL oxidation are
largely unknown. Studies have identified several plausible mechanisms sup-
portive of LDL modification. The enzymatic activity of nitric oxide synthase,
FIGURE 5.3
Initiation of LDL-mediated atherogenesis (lipid accumulation). Atherosclerotic lesion develop-
ment begins with the accumulation of LDL. Lipid accumulation is noticeable along the lining
of the arterial wall.
15-lipoxengenase activity,72 as well as nitric oxide production by epithelial

cells and macrophages73 have been shown to be capable of LDL modification.
Recent findings supporting their proatherogenic role have been documented
using gene knockout models.74–76 Despite formidable evidence that LDL
oxidation confers lesion formation, data regarding antioxidant therapy to
date have not shown promise.77 In broad terms, atherosclerosis can be char-
acterized as a chronic inflammatory disease. As such, cellular responses such
as cellular adhesion and recruitment during lesion development are central
components as in other chronic inflammatory diseases.
The recruitment of monocytes occurs at the sites of lipid accumulation and
function in uptake of various lipids and apolipoprotein components pro-
duced from oxidative stress and other biochemical breakdown products of
LDL (Figure 5.4). Such recruitment is known to be regulated by chemotactic
factors10 as well as being attracted by oxidative-LDL species.10 Chemokines
are small proteins subdivided into three major groups based upon the struc-
tural positions of the first two cysteines at the amino terminus of the mole-
cule.78–80 Chemokines stimulate the migration and activation of cells,
especially phagocytic cells and lymphocytes. Most notable is the release of
macrophage chemotactic protein 1 (MCP-1) found to be produced locally by
endothelial cells71 and the coordinate expression of chemokine receptor 2
(CCR2), the receptor for MCP-1 by monocytes. In fact, it has been shown
FIGURE 5.4
LDL oxidative modification and monocyte recruitment (fatty streak formation). The initial sign
of atherogenic development is the formation of the fatty streak, underlying the endothelium of
large arteries. The primary cellular events contributing to the fatty streak formation are the
recruitment of monocytes which are converted to macrophages, which uptake LDL. Recruitment
of monocytes to lesion probe areas is regulated by adhesion molecules that are expressed on
the endothelium cell surface.
that hypercholesterolemia patients exhibit increased MCP-1 production.81

Furthermore, disruption of MCP-1 and its receptor CCR2 genes was shown
to reduce the development of atherosclerosis in mice.81 Other chemokines
such as interleukin-8 (IL-8), RANTES, and IP-10 have also been implicated
in monocyte recruitment.10 Current research in this area offers the potential
for therapeutic use in deterring atherogenic processes by impairing leuko-
cyte trafficking.
It has been speculated that macrophage-mediated uptake of modified LDL
species may be an initial attempt to dampen the inflammatory environment
produced by oxidative LDL species.10 Ultimately, however, the response and
uptake of oxidized LDL species leads to progressive inflammation and ath-
erosclerotic lesions. The uptake of LDL occurs mainly via macrophage LDL
receptors or by scavenger receptor-mediated uptake.10 The mode of LDL
uptake is determined by the nature of LDL modification. Studies show that
while native LDL is normally endocytosed via specific LDL receptors, highly
modified LDL, such as certain apolipoproteins, are not recognizable by the
LDL receptors and are relegated to uptake by scavenger receptors. The latter
is most associated with macrophage foam cell formation, a topic to be
FIGURE 5.5
Foam cell formation (intracellular lipid accumulation) by macrophages. A hallmark of early
atherosclerotic lesion development is conversion of the macrophage to foam cells that contain
amounts of oxLDL, which is mediated primarily by scavenger receptors.
discussed in a subsequent section of this chapter. As a result of macrophage

recruitment and uptake of LDL constituents, fatty streaks form and become
what is the initial site of atherosclerotic lesions (Figure 5.5).
Another mechanism responsible for the initiation of atherosclerotic lesions
is the increase in adhesion molecules present on endothelial cells. Under
normal circumstances, the arterial endothelium is highly resistant toward
cellular adhesion. However, studies have shown that hypercholesterolemia
induces leukocyte adherence to the endothelium allowing diapedesis
between the endothelial cell and entry into the lamina.10 Several adhesion
molecules have been implicated to significantly foster translocation of leu-
kocytes across the endothelium. Vascular cell adhesion molecule-1 (VCAM-
1), a member of the immunoglobulin superfamily, is expressed by endothelial
cells and regulates the adherence of monocytes and T cells. VCAM-1 has
been found to interact with very late antigen-4 (VLA-4) and influence mono-
cyte adherence during the initial stages of atheroma formation.82 Selectins P
and E have also been implicated in monocyte adhesiveness to the endothe-
lium. Quantitative decreases in atherosclerosis were shown in apo E mice
lacking their respective genes.83
Foam Cell Formation (Intracellular Lipid Accumulation by Macrophages)

As mentioned in a previous section, macrophages play an important role in
LDL metabolism by uptake of native LDL cholesterol and modified species
of LDL via two major receptor mechanisms, LDL-specific receptor and scav-
enger receptor endocytosis, respectively. As the accumulation and modifica-
tion of LDL ensues, macrophages within the subendothelium begin to
incorporate large amounts of oxidized LDL species via scavenger receptor
uptake, resulting in a phenotype given the term “foam cell” (Figure 5.5). The
most notable scavenger receptors identified to date that have been demon-
strated to have a significant impact on atherosclerotic development are the
scavenger receptor A (SR-A) and the receptors of the cluster differentiation
36 surface molecules (CD36) receptors.84 In particular, it was shown that in
apo E-deficient murine models deficient in SR-A or CD36, gene receptor
expression resulted in a significant reduction in lesion formation.84,85
As determined by the studies of Brown and colleagues, homeostatic con-
trol of cholesterol uptake is under strict mediation through LDL-specific
receptor feedback mechanisms regulated by the SREBP transcription factors
required for LDL receptor expression.70 In the presence of elevated mem-
brane-bound cholesterol, inactivation of SREBP occurs, inhibiting LDL recep-
tor expression. In contrast, however, uptake of oxidative LDL species via
scavenger receptors, SR-A or CD36 or by macrophage-mediated phagocyto-
sis is not under such regulatory control. Instead, prevention of cholesterol
intracellular overload is dependent on mechanisms of active efflux out of
the cell. The vast majority of oxidized LDL entering macrophages via the
scavenger receptors consists of free cholesterol or esterified cholesterol. There
are several fates of native cholesterol metabolism, which include Acyl CoA
esterification and the storage of lipid droplets containing cholesterol esters
that characterize the phenotype of foam cells. Excretion of excess cholesterol
by foam cells is believed to occur through processes that transform choles-
terol into a more soluble form through enzymatic modifications.
A major pathway of cholesterol efflux is called the “reverse cholesterol
transport” pathway that involves HDL as an acceptor molecule. The HDL-
reverse cholesterol transport mechanism received much attention when
studies found an inverse relationship between risk for atherosclerosis and
HDL content.86 A genetic basis for HDL-mediated cholesterol transport is
shown in patients afflicted with Tangier disease, which is characterized by
extremely low levels of HDL and accumulation of cholesterol within mac-
rophages. Mutations in ABCA1, which encodes a member of the ATP binding
cassette family of HDL transporters, were found to cause the genetic defect.
Although the precise mechanism that is disrupted by this aberration is
unclear, studies suggest that mutation in ABC A1 alters cholesterol transport
to the HDL acceptor molecules.87 Under normal conditions, HDL-cholesterol
is esterified via lecithin-cholesterol acyltransferase (LCAT) or is directly
transported to the liver via SR-B1 binding. Thus, it is clear that macrophages
play a paramount role in cholesterol maintenance within its surrounding
environment, but more important is its ability to control the fate of internal-
ized cholesterol for self-preservation.
Immigration of Smooth Muscle Cells

A hallmark of advanced lesion development is the immigration of smooth
muscles cells from the arterial wall into the subepithelial space (Figure 5.6).
The factors that lead to the mobilization of smooth muscle cells are not well
understood, but it is believed to be due to preexisting stimuli. For example,
macrophages have been shown to secrete the chemokine platelet-derived
growth factor (PDGF), which is a chemoattractant for smooth muscle.88 In
fact, studies have demonstrated PDGF expression to be elevated in individ-
uals with atherosclerosis.89 Smooth muscle cells found within the atheroscle-
rotic region were found to have distinct characteristics from normal smooth
muscle cells. These cells exhibit characteristics of clonal expansion. Studies
have demonstrated that the slow but steady proliferation can be attributed
FIGURE 5.6
Immigration of smooth muscle cells and immune responsiveness during atherosclerotic devel-
opment. A hallmark of advanced lesion development is the immigration of smooth muscle cells
from the arterial wall into the subepithelial space, which may also contribute to foam cell
development. As with many chronic inflammatory diseases, immune surveillance will ultimate-
ly make a significant contribution to the progression and disease outcome. Circulating leuko-
cytes and lymphocytes of mainly T-cells respond to the site of injury.
to a single cell.90 Smooth muscle cells in developing atheroma also are capa-
ble of taking up modified lipoproteins.89 Not only does their proliferative
capacity augment atheroma development, but apoptotic cell death of smooth
muscle cells participates in lesion progression. Apoptosis of smooth muscle
cells is believed to be associated with the presence of inflammatory cytokines
at the lesion site.91 Thus, smooth muscle cell immigration plays a significant
role in progression of atheromas (Figure 5.6). Current research is aimed at
developing molecular strategies targeting both proliferative and apoptotic
pathways.
Immune Responsiveness during Atherosclerotic Development

With the exception of macrophage activation, lymphocyte activation does
not appear to have a major impact on the initial stage of atherosclerotic
formation. Studies using RAG-1 recombinase-deficient mice illustrated that
the lack of functional B and T lymphocytes had no bearing on atherosclerotic
development in the presence of elevated cholesterol.92 However, as with
many chronic inflammatory diseases, immune surveillance will ultimately
make a significant contribution to the progression and outcome of disease.
Circulating leukocytes and lymphocytes of mainly T lymphocytes respond
to endothelial injury. At such stages of lesion development, a multitude of
secreted and cell-associated mediators are accessible to lymphocyte recog-
nition. For example, endothelial cell-associated adhesion molecules such as
VCAM-193 can also increase the avidity for monocytes to enter lesion sites.
Also, as previously mentioned, chemokines produced by activated macro-
phages can attract T-cells to the lesion site. As T-cells begin to accumulate
in the surrounding lesion, they become activated and can modulate athero-
sclerotic development through the release of cytokines (Figure 5.7). Through
the release of cytokines, T-cells can elicit both pro-atherogenic and anti-
atherogenic responses. Such dichotomy is due to the presence of T subpop-
ulations capable of secreting distinct cytokines that display opposing func-
tionality. These populations of T-cells are commonly referred to as T-helper
cells, subdivided into Th1 and Th2 subpopulations.94 Th1 cells mainly secrete
IL-2, interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Th1-associated
cytokines mediate pro-inflammatory responses and delayed hypersensitivity
responses. On the other hand, the Th2 subpopulation preferentially secretes
IL-4, IL-5, IL-6, IL-10 and IL-13.94 Th2 cells function in anti-inflammatory
responses and immune tolerance.
Studies that examined the role of Th1 versus Th2 cytokine responses in
the progression of atherosclerosis have shown that T-helper cell cytokine
mediation is not as clearly defined along the two divergent functions
between Th1 and Th2. In fact, IFN-γ has been shown to suppress scavenger
receptor expression and proliferation of smooth muscle cells, suggesting an
anti-atherogenic potential.10 On the other hand IFN-γ is capable of activating
macrophages. In studies utilizing apo E-deficient mice that lacked a
FIGURE 5.7
Plaque formation. Plaques develop from initial fatty streaks that progress into advanced lesions
comprised of inflammatory cells, smooth muscle cells, extracellular lipids, and fibrous tissues.
Their continued accumulation proliferation and activation within the lesion leads to plaque
expansion. Consistent with the earlier events of atherosclerotic lesion development, plaque
formation involves the participation of cytokines, chemokines, hydrolytic enzymes, and growth
factors in this process. During the advanced stages of plaque formation, lipid moieties, leuko-
cytes and necrotic materials are walled off by a fibrous cap. An accumulation of these cellular
constituents and fibrotic tissues leads to further expansion and can lead to ischemic heart disease
or stroke, which is due mainly to plaque rupture and thrombosis.
functional IFN-γ receptor, atherosclerosis was decreased as compared to nor-

mal mice.95 The role of Th2 cytokine mediation is also very complex. While
IL-4 cytokine production by Th2 cells acts antagonistically toward IFN-γ
production, IL-4 has been shown to induce LDL oxidation through induction
of 15-LO enzymatic activation.10 IL-10 production by Th2 cells seems to be
the most consistent in opposing pro-atherogenic processes such as macro-
phage deactivation96,97 and plaque stability. Thus, the implication of T-cells’
activation offers a complex environment in determination of their specific
roles in atherosclerotic development and progression.
Plaque Formation
Plaques develop from initial fatty streaks that progress into advanced lesions
comprised of inflammatory cells, extracellular lipid, and fibrous tissues (Fig-
ure 5.7). Their continued accumulation proliferation and activation within
the lesion leads to plaque expansion. Consistent with the earlier events of
atherosclerotic lesion development, plaque formation involves the partici-
pation of cytokines, chemokines, hydrolytic enzymes, and growth factors in
this process.98 During the advanced stages of plaque formation, lipid moi-
eties, leukocytes and necrotic materials are walled off by a fibrous cap. An
accumulation of these cellular constituents and fibrotic tissues leads to fur-
ther expansion. At a particular threshold, the compensatory dilation of the
artery is overcome by the intrusion of the lesion into the lumen resulting in
eventual alterations in blood flow and plaque rupture.
While the initial events of atherogenesis involve mainly the disruption of
the endothelia and leukocyte accumulation, the formation of the more
advanced plaque includes smooth muscle cells (Figure 5.7). As mentioned
previously, smooth muscle cells migrate via chemotactic regulation into the
arterial intimal lesion site and become active participants in atheroma devel-
opment. The smooth muscle cells involved in atheroma exhibit an altered
phenotype in comparison to normal arterial tunica media smooth muscle
cells. These smooth muscle cells proliferate at a higher rate within athero-
sclerotic plaques versus normal intimal regions of the aorta.99 Further justi-
fication for the importance of smooth muscle cell proliferation demonstrated
that clonal expansion of smooth muscle cells was likely and is the basis for
lesion progression.90 It is still unclear, however, what initiates medial smooth
muscle proliferation versus normal smooth muscle cells. It is believed that
growth factors in conjunction with additional stimuli promote the prolifer-
ative response by smooth muscle cells at the lesion site. For example, vascular
smooth muscles cells (VSMCs) in the presence of serum show minimal
mitogenic capacity.100 Other studies substantiate this finding.100,101 One pos-
sibility for the lack of mitogenicity could be due to the presence of suppres-
sive factors. Based upon the evidence of this study it has been postulated
that basement membrane constituents such as heparin can suppress smooth
cell proliferation.102,103 Thyberg, Hedin and colleagues also showed that the
basement membrane component, laminin, inhibits while the interstitial
matrix component, fibronectin, promotes phenotypic modulation of smooth
muscle cells.88,104 In contrast, the metalloproteinases that are induced by
inflammatory cytokines105,106 were found to induce smooth cell prolifera-
tion.107 In addition to smooth muscle proliferation, the apoptosis of smooth
muscle cells participates in advanced lesion development. Cell death may
be the result of cytokine regulation present within the lesion site.108 Also,
interaction with fas-expressing T-cells can lead to cell death.109 Therefore,
understanding the regulation of smooth muscle expansion and depletion
with regard to progression of plaque formation will likely have a great
impact on the innovation of new therapies to combat atherosclerosis.
A large proportion of the developing atheroma includes connective tissue
consisting of extracellular matrix macromolecules. Among the matrix pro-
teins, the class collagens and proteoglycans are commonly associated with
plaque development. Matrix proteins are produced by vascular smooth mus-
cle cells and can accumulate within the developing plaque upon stimulation
by transforming growth factor-β and platelet-derived growth factor.110

Matrix molecules have an important regulatory function. For example,
fibronectin and heparan sulfate are found to inhibit cell cycle and cell–matrix
interactions and influence chemokine expression by macrophages.111–114
Matrix accumulation within the intima is under control of matrix metallo-
proteinases (MMPs).112 MMPs act in degradation of matrix molecules and
therefore control lesion accumulation. Matrix molecules also contribute to
the outward growth of the lumina. Thus, the extracellular matrix is a key
component in plaque development.
Summary
Cardiovascular disease is the leading cause of mortality in the United States,
Europe, and a vast majority of Asia and is likely to be the greatest threat to
overall health worldwide. This chapter emphasizes the biological process of
atherosclerosis and what is known about the cells and molecules that are
associated with the evolution of this multifaceted disease. While evidence
suggests that elevated lipids and endothelial dysfunction both play an
important role in atherogenesis, more research is needed to determine the
molecular and cellular interactions of these factors in promoting the patho-
genesis of atherosclerosis. Whereas atherosclerosis has long been an area of
significant biomedical, clinical, and epidemiological research emphasis, there
is considerable evidence that the quantitative determinants of disease vul-
nerability must be identified.
Acknowledgments
Special thanks to Scott B. Robinson of scienceinflash.com for graphic illus-
trations. The authors express their deep appreciation to Drs. Samuel N.
Cheuvront and Sangeeta Kaushik for reviewing the manuscript.
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6
Exercise Training and Endothelial Function in
Patients at Risk for and with Documented
Coronary Artery Disease
Tom LaFontaine and Jeffrey L. Roitmann
CONTENTS
Introduction ...........................................................................................................85
Chronic Physical Activity, Exercise Training, and Reduced
Morbidity and Mortality ............................................................................87
Chronic Physical Activity: Enhanced Endothelial Function/Reversal
of Endothelial Dysfunction ........................................................................88
Overview of Animal Studies .....................................................................88
Exercise Training and Endothelial Function in Humans ...............................91
Correction of Endothelial Dysfunction in Youth.............................................97
Mechanisms of Improved Endothelial Function Following
Exercise Training..........................................................................................98
Case Study ...........................................................................................................102
Summary ..............................................................................................................104
References ............................................................................................. 106
Introduction
Numerous interventions, aside from exercise, have been shown to attenuate
endothelial dysfunction in humans (see Table 6.1).1 Several studies demon-
strate improved endothelial function following lipid altering therapy.2–6
A recent study demonstrated improved endothelial function in elderly Type
2 diabetes mellitus patients with just 3 days of cerivastatin therapy.6 Other
studies have shown improved endothelial function following LDL pheresis
and antioxidant therapy with Vitamin C.7,8 The data on Vitamin E is
85
TABLE 6.1
Important Vasoactive, Inflammatory,
and Thromboactive Molecules
Produced by the Intact Endothelium
Nitric oxide (NO)
Angiotensin II
Bradykinin
Endothelins
Prostacyclin
Anti-inflammatories
Monocyte chemotactic factor-1 (MCP-1)
Adhesion molecules (VCAM-1, selectins)
Interleukins 1, 6, & 18
Tumor necrosis factor
Tissue plasminogen activator
Plasminogen activator inhibitor-1 (PAI-1)
Prostaglandins
Von Willebrand’s factor
Endothelial hyperpolarizing factor
equivocal although a recent report found that 2 weeks of Vitamin E therapy

decreased P-selectin in dyslipidemic patients suggesting an improved envi-
ronment favoring vasodilation.9 However, recent studies have shown no
benefit of antioxidant therapy on long-term morbidity and mortality among
patients with or at risk for cardiovascular disease (CVD).10 Several reports
have shown that interventions, including ACE inhibitors, HMG-CoA reduc-
tase inhibitors, folic acid supplementation in hyperhomocysteinemic
patients, grape juice, tea, dark chocolate, walnuts, L-arginine in high doses,
iron chelation, tetrahydrobiopterin, and recently ACEII Receptor 1 blockers
attenuate coronary endothelial dysfunction in patients with coronary artery
disease (CAD).2,11–16
Exercise training, particularly in combination with comprehensive lifestyle
changes, has been shown to reduce the risk for the development and pro-
gression of atherosclerosis and related manifestations, including sudden
death and recurrent revascularizations.17–22 Numerous studies have reported
that physically active or fit persons have a significantly lower risk for car-
diovascular morbidity and mortality even in the presence of major and
traditional CAD risk factors.23,24 However, the mechanism for these observed
benefits is incompletely understood. Recent evidence has demonstrated that
exercise training with or without diet and weight loss improves endothelial
function among populations of children and adolescents with CAD risk
factors and patients with long-standing diseases such as hypertension, dys-
lipidemia, diabetes, CAD, and congestive heart failure. This chapter dis-
cusses the particular role of exercise training in preserving normal
endothelial function and in improving or reversing endothelial dysfunction
in patients with or at risk for cardiovascular disease.
Exercise Training and Endothelial Function 87
Chronic Physical Activity, Exercise Training, and Reduced

Morbidity and Mortality
Wannamethee et al., in a prospective, 5-year cohort study, reported approxi-
mately a 50% reduction in CVD mortality and morbidity in patients with
documented CAD who became or remained active compared with those who
remained sedentary.25 A recent 1-year follow-up investigation of post-CAD
patients in cardiovascular rehabilitation (CVR) by Steffen-Batey et al. reported
similar results.26 The results of the ETICA study showed after 33 months of
follow-up that 11.9% of patients who exercise trained (26% increase in VO2
peak) after PTCA and/or stent implantation had a recurrent event compared
with 32.2% among patients who did not exercise.22
Several meta-analyses have confirmed a 20–25% reduction in CVD mor-
tality following participation in CVR.27,28 Similar findings were reported in
a recent meta-analysis that included data from trials for emergency throm-
bolysis and rescue percutaneous coronary interventions (PCI).29
In a single-center randomized trial in Finland, Hammalainen et al., after
a 10-year follow-up of CVR following myocardial infarction, reported a 37%
reduction in sudden death among patients participating in a multifactorial
intervention program.30 At 15 years, 16.5% of patients who were initially
randomized to CVR experienced sudden death compared with 28.9%
(p < 0.006) of patients randomized initially to the control group.31 Coronary
mortality was 47.9% and 58.5% (p < 0.04) in the CVR and control groups,
respectively. Recently, Witt et al., in a study of community CVR following
myocardial infarction, observed a 72% reduction in risk of mortality among
participants versus non-participants in CVR.32 Thus, it is clear that exercise
training and comprehensive CVR in CAD patients improves mortality and,
perhaps, morbidity. However, the mechanisms for these observed benefits
have not been completely elucidated.
Numerous studies have shown that regular exercise training in patients
at risk for or with CAD improves lipids, diabetes, hypertension, obesity, and
thrombogenic risk factors.33–35 However, the benefit of exercise on mortality
and morbidity in CAD patients is independent of, and sometimes dispro-
portionate to the effect on risk factors.23,24 Exercise training improves myo-
cardial perfusion, but has limited effect on the size and extent of
atherosclerotic lesions.17,21 Recent studies suggest that a mechanism by which
exercise training in CAD patients reduces progression of atherosclerosis and
risk for recurrent events is improvement in vascular tone and endothelial
function.36–45
Chronic Physical Activity: Enhanced Endothelial Function/

Reversal of Endothelial Dysfunction
Overview of Animal Studies
The animal research provides substantial support for the beneficial influence
of regular physical activity on endothelial function, particularly in the pres-
ence of established risk factors, endothelial dysfunction, and atherosclerosis.
Numerous studies have shown that systematic exercise training can improve
endothelium dependent vasodilation (EDD) in several animal species.46–48
This section will provide a review of several pertinent investigations. For
excellent and more comprehensive summaries of the basic animal research
relating to exercise and its effect on endothelial and smooth muscle cellular
function, the reader is referred to three excellent reviews.46–48
Muller and colleagues examined EDD in isolated arteries from the hearts
of pigs and reported that arterioles and slightly larger resistance arteries
from trained pigs showed significantly better EDD than the same arteries
from untrained pigs.49 These authors further demonstrated that this benefi-
cial response was lost when endothelial nitric oxide synthase (eNOS) was
blocked. Laughlin et al. and Woodman et al. also reported that exercise
training increased mRNA for the eNOS gene in coronary arterioles and eNOS
content in coronary arterioles and small resistance arteries.50,51
One hypothesis of the mechanism of how regular exercise improves EDD
is through the effects of increased blood flow and shear stress acting on
endothelial cells.52 Results of studies in rats and dogs with an arterial-venous
fistula that produced increased blood flow and shear stress demonstrated
increased eNOS expression and improved endothelial function.53,54 Wood-
man et al. reported that e-NOS expression is increased in isolated coronary
arteries of pigs exposed to increased intraluminal flow for 4 h.55 Other studies
have shown that hindlimb unweighting of the rat soleus muscle for 2 weeks,
a procedure that would reduce intraluminal blood flow and shear stress,
resulted in decreased EDD in the feed arteries and 1A arterioles, but no
change in 2A arterioles.56–58
Laughlin and colleagues addressed the non-uniformity across vascular
beds in EDD and eNOS protein in response to exercise training.47,59 In general
it appeared that EDD was improved more in smaller coronary resistance
than in larger conduit arteries, although eNOS protein content per gram of
total protein was greater in the large coronary arteries.
Wang et al. reported that EDD was enhanced in large conduit coronary
arteries but not in coronary resistance arteries of dogs.60 In contrast, Laughlin
and colleagues have reported increased EDD in coronary resistance arteries,
but not in conduit arteries following longer-term training.47 Laughlin et al.
recently reported that short-term training (1 h 3.5 mph × 7 days) resulted in
improved EDD in the conduit arteries of pigs, but not arterioles and
that these short-term effects were not associated with changes in eNOS or
superoxide dismutase (SOD-1).61 These contrasting results suggested that

the early adaptive response in the arterial tree may differ depending on the
length of the exercise training period. Thus, EDD of conduit arteries appears
to be enhanced early (7–10 days) in the adaptive process, but returns towards
the pre-training state after several weeks of training. The responses in resis-
tance or smaller order arterioles may involve a longer-term adaptation pro-
cess. There also is the possibility of species differences. Perhaps with
sustained training, EDD in conduit arteries normalizes as structural adap-
tations such as increased vessel diameter become manifest.
Laughlin and colleagues have reported improved EDD following exercise
training in hypercholesterolemic pigs and in pigs subjected to progressive
coronary artery occlusion and atherosclerosis induced by a high fat diet.47,62–65
Henderson et al. hypothesized, for example, that exercise training would
improve endothelial function of coronary arterioles of pigs in the early stages
of coronary artery atherosclerosis induced by a high-fat, high-cholesterol
diet.66 Yucatan miniature swine were randomized to a normal fat diet (8%
fat calories) or a high-fat diet (46% fat calories). Both groups were then
subdivided into sedentary or exercise-trained groups. Responses to EDD
induced by bradykinin, ADP, and increased blood flow were similar among
groups after 20 weeks. However, EDD in response to aggregating platelets
in the presence of indomethacin and ketanserin was attenuated in the high-
fat sedentary animals whereas this attenuated response was prevented or
reversed in the high-fat exercise trained animals. The mechanism of the
observed improved EDD with exercise training may be related to increased
eNOS expression and NO bioavailability, attenuation of a bradykinin
induced release of an indomethacin-sensitive prostanoid constrictor, and/or
decreased release of a vasoconstrictor substance produced by the action of
the cyclooxygenase enzyme.47
Davis et al. demonstrated that 3 weeks of exercise training in c-SRC defi-
cient mice resulted in no effect on either eNOS gene expression or the
expression of SOD-1.67 This finding further supported NO as the mediator
of enhanced EDD. Graham and Rush recently reported that gp91phox-
dependent oxidative stress and reduced antioxidant capacity contributed to
impaired EDD in spontaneously hypertensive rats.68 In contrast, exercise
training reduced gp91phox-dependent oxidative stress, enhanced endothe-
lial eNOS-derived NO and contributed to restored EDD. Yamashita et al.
reported that exercise provided cardio-protection via activation of manga-
nese SOD that decreases oxidative damage in endothelial cells.69
Several additional studies have shown that eNOS inhibition and decreased
NO bioavailability promotes atherosclerosis.70–75 Thus, one mechanism for
how exercise training may prevent the development of atherosclerosis and/
or retard its progression and prevent atherosclerotic events is through
increasing NO bioavailability. Neibauer et al. investigated the effect of exer-
cise in hypercholesterolemic, Apo-E deficient mice.76 The mice were assigned
to four groups: sedentary, exercise, sedentary plus eNOS inhibition, and
exercise plus eNOS inhibition. The mice were trained on treadmills, 6 days
per week, 2 h per day. Arteries of the sedentary, eNOS inhibited mice man-
ifested a threefold greater atherosclerotic lesion formation compared with
the exercise trained eNOS inhibited mice. In fact, there was no lesion forma-
tion in the exercise trained eNOS-inhibited animals, suggesting that regular
exercise training completely counteracted the atherogenic effect of decreased
NO bioavailability.
Fogarty et al. hypothesized that exercise training improves endothelial
dysfunction by enhancing NO-mediated vasodilator responses to vascular
endothelial growth factor (VEGF-165) in arteries exposed to chronic coronary
occlusion.77 Using female Yucatan miniswine, these investigators induced
chronic proximal left circumflex occlusion with the ameroid occluder tech-
nique. Eight weeks post-surgery, the animals were randomized to either
14 weeks of inactivity or 5 days/week of exercise training on a treadmill. In
non-occluded arteries, exercise training had no effect. However, exercise
training markedly enhanced VEGF-165-induced vasodilation of collateral-
dependent left circumflex arterioles. Furthermore, VEGF-165-induced
vasodilation of the occluded left circumflex of exercise-trained swine
exceeded that of non-occluded exercise-trained or sedentary left anterior
descending artery (LAD) arterioles. Enhanced vasodilation of exercise-
trained left circumflex arterioles was abolished by inhibition of eNOS. Com-
bined inhibition of eNOS and cyclo-oxygenase decreased VEGF-165 induced
vasodilation of all vessels. These results further support the conclusion that
exercise-induced improvement in EDD is mediated via increased NO.
Laughlin et al. recently reported that sprint training (six bouts of treadmill
running, 2.5 min at 60 m/min with 4.5 min rest periods, 5 days/week)
improved ACH-induced vasodilation in rat white gastrocnemius second-
order arterioles, but not in 2A or 3A arterioles in red muscle fibers.78 This
study suggested that training-specific effects may occur predominantly in
white anaerobic versus red aerobic muscle fibers.
Several additional studies supported the role of exercise training in
improving EDD via increasing eNOS and NO bioavailability. Tanabe et al.
reported that exercise training of aging rats attenuated the aging-related
decrease in eNOS and NO production and release in the aorta.79 Regarding
gender differences, Laughlin et al. found that baseline eNOS expression is
greater in female compared with male pigs but that the increase in eNOS
expression following exercise training is similar among genders.80
Recently, Suvorava et al. investigated the effects of physical inactivity on
endothelial dysfunction in young healthy male mice.81 The mice were ran-
domized to either continue living in large groups of five in large cages where
they were running, climbing, fighting, etc. or to live alone in small cages
where they were primarily resting (sedentary). Aerobic capacity of skeletal
muscle decreased significantly in the inactive mice (p < 0.05) as did EDD
(p < 0.001) and eNOS protein expression (p < 0.01). These alterations in
aerobic capacity, EDD, and vascular eNOS expression were completely
reversible when the mice living alone underwent regular exercise training.
This study nicely demonstrated in mice the dramatic negative effect of
physical inactivity on vascular function and muscle aerobic capacity that is

completely reversible by a short period of moderate exercise training.
In an interesting study, Hayward et al. investigated the effects of exercise
training (30 min/day, for 8 weeks at a moderate to high intensity) in rats on
endothelial function following exposure to the chemotherapeutic agent
5-fluorouracil (5-FU).82 It had been demonstrated previously that 5-FU
induces endothelial-independent vasoconstriction of vascular smooth mus-
cle.83 The results showed that exercise training enhanced EDD after 5-FU-
induced vasoconstriction due, at least in part, to an increase in aortic eNOS
protein content and activity.
Recently, an association between atherosclerosis, endothelial dysfunction,
and low levels of bone marrow-derived endothelial progenitor cells (EPCs)
has been reported.84 Progenitor cells are primitive bone marrow cells that
have the capacity to proliferate, migrate, and differentiate into various
mature cell types including endothelial cells. EPCs may play a critical role
in endothelial cell maintenance and repair and have been implicated in
promoting both re-endothelialization and neovascularization.85 Thus, EPCs
may prevent endothelial dysfunction by contributing to the repair of dam-
aged endothelial cells, maintaining vascular homeostasis, and promoting
angiogenesis. One study of mice randomized to exercise training on running
wheels or no running demonstrated that exercise training increases the pro-
duction and circulating number of EPCs, inhibits neointimal formation after
vascular injury, promotes angiogenesis, and decreases apoptosis of endothe-
lial cells.86 In addition, these authors also demonstrated increased circulating
EPCs and reduced EPC apoptosis in the blood of exercise-trained humans
with CAD.
Numerous additional studies in several species have demonstrated
improved coronary and peripheral artery and arteriole EDD, increased
expression of eNOS, increased NO bioavailability, and reduced oxidative
stress following exercise training.46–48,87,88 In conclusion, the animal evidence
provides overwhelming support for the beneficial effects of exercise training
on EDD in most vascular beds.
Exercise Training and Endothelial Function in Humans

Haskell et al. compared coronary vascular reactivity in ultra-distance runners
and sedentary gender and age-matched men and women using quantitative
angiography.89 There was no significant difference among groups in basal
diameter of epicardial coronary arteries. However, during angiography,
when intravenous nitroglycerin, a stimulator of EDD was injected, the cor-
onary arteries of the ultra-distance runners demonstrated a 200% greater
increase in vasodilation than in the coronary arteries of sedentary men and
women. This was one of the first studies to suggest improved endothelial
function in highly physically active persons compared with sedentary peers.

Kingwell et al. and others have reported improved EDD in healthy physically
active subjects compared with sedentary young and middle-aged men and
women and following aerobic exercise training in these same groups 90–93
Cross-sectional studies show that age-related increases in arterial stiffness
are attenuated in endurance-trained adults.40,41 Tanaka et al. demonstrated
that exercise training (walking/jogging, 40–45 min/session, 4–6 days/week
at 70–75% of maximum heart rate) significantly improved arterial compli-
ance.40 These effects were independent of changes in body mass, adiposity,
blood pressure, or peak oxygen uptake. In a second study, Tanaka and
colleagues demonstrated that regular aerobic exercise for 3 months (prima-
rily walking) restored age-related reductions in central arterial compliance
in previously sedentary but healthy middle-aged and older men (18–77 years
of age).41 Clarkson et al. reported that a 10-week program of anaerobic and
aerobic exercise in young military recruits resulted in improved EDD of the
brachial artery as measured by flow mediated dilation (FMD) but there were
no changes in endothelial-independent dilation (EID) assessed by nitroglyc-
erine infusion.91 DeSouza et al. found no age-related decline in EDD in
response to acetylcholine in endurance-trained men.42 In a sub-study of
13 middle-aged sedentary men who trained for 12 weeks, FMD increased
30% (p < 0.01) and was found to be similar to levels found in young adults
and middle-aged and older endurance-trained men.
In another cross-sectional study, Taddei et al. reported that endurance
training can prevent the age-associated endothelial dysfunction through the
restoration of NO availability consequent to the prevention of oxidative
stress.43 These reports provided provocative evidence that endurance exer-
cise training may prevent or attenuate the age-related decline in endothe-
lium-dependent vasodilation and restore levels in previously sedentary
middle-aged and older men.
Maiorana et al., in a randomized crossover study of combined aerobic and
resistance training in healthy middle-aged men, found that exercise training
did not affect either EDD or EID.92 This is one of the few published studies
that demonstrated no beneficial effect of exercise training on endothelial
function and, according to Maiorana et al., raises the possibility that aug-
mentation of endothelial function may occur more readily in patients with
endothelial dysfunction.46 In contrast, these same authors reported improved
EDD and EID in CHF patients and EDD in Type 2 diabetes mellitus patients
in response to combined aerobic and resistance training.39,94
Exercise training has been shown to improve insulin sensitivity and gly-
cemic control in patients with diabetes mellitus and blood pressure in
patients with Stages 1 and 2 hypertension.34,35 Patients with diabetes, abnor-
mal lipids, or hypertension or who are overweight, with or without central
obesity, who are physically active and/or physically fit, have lower rates of
CVD and all-cause mortality.95,96 However, the mechanisms facilitating these
benefits of exercise are incompletely understood. Recently, several groups
have reported improved EDD following exercise training in patients with
diabetes, hypertension, and documented coronary and/or peripheral ath-

erosclerosis.
Higashi et al. studied the effects of exercise training on forearm blood flow
in 17 patients with Stage 1 hypertension.38 After 12 weeks, FMD during
reactive hyperemia increased significantly (p < 0.05) in the exercise training
group compared with the control group. There also was an increase in
acetylcholine-stimulated NO release. This study also demonstrated
improved EDD following exercise training in Stage 1 hypertensive patients
that was mediated through increased endothelial release of NO. A study of
combined aerobic and resistance exercise for 8 weeks in patients with Type
2 diabetes mellitus demonstrated enhanced EDD in conduit and resistance
vessels.39
Guan-Da investigated the effects of aerobic exercise training (subjects pro-
gressed to 40–45 min of walking, 4–6 days per week at 70–75% of heart rate
maximum) on EDD in 30 sedentary Chinese men with impaired fasting
glucose (IFG) (mean age = 63 years).97 After 6 months of training, EDD was
significantly improved. Fuchsjager-Mayerl et al. reported similar findings in
persons with Type 1 diabetes mellitus.98 These authors exercise trained
26 persons with Type 1 diabetes mellitus with no evidence of overt angiop-
athy for 4 months. Peak oxygen uptake increased 27% and FMD was signif-
icantly enhanced. After 8 months of detraining, the vascular benefits were
completely reversed. Lavrencic et al. also reported that physical training
improves FMD in patients with the metabolic syndrome.99
Studies have shown that persons with high normal or Stage 1 hypertension
who exhibit an exaggerated blood pressure response to aerobic exercise are
at a significantly increased risk for worsening blood pressure.100,101 Recently,
Stewart et al. investigated the relationship between an exaggerated blood
pressure response to exercise and EDD as assessed by FMD among 38 men
and 44 women, 55–75 years of age with high normal blood pressure (pre-
hypertension) or mild Stage 1 hypertension.102 Overall, FMD accounted for
11% and 10%, respectively, of the variance in maximal SBP and maximal
pulse pressure. The authors concluded that the results suggest that endothe-
lial dysfunction may be a mechanism contributing to an exaggerated exercise
blood pressure response and may be a link between exercise hypertension
and worsening resting hypertension. Chang et al. reported similar findings
in a study comparing 25 men and women with an exaggerated blood pres-
sure response (defined as ≥ 210 mmHg in men and ≥ 190 mmHg in women)
to 25 men and women with a normal blood pressure response during
dynamic treadmill exercise testing.103
A recent study by Hambrecht et al. demonstrated improved vasodilatory
capacity in the coronary arteries of patients with documented CAD and
endothelial dysfunction.44 Patients were randomized to an exercise training
group and to a control group. Exercise training consisted of 4 weeks of
6 times/day, supervised 10-min exercise sessions on bicycle ergometers at
80% of peak heart rate. Paradoxical vasoconstriction of coronary arteries in
response to acetylcholine infusion was reduced by 54% in the exercise group
compared with the control group. Exercise training also resulted in signifi-
cantly improved coronary blood-flow reserve (p < 0.01) and EDD (p < 0.01)
compared with the control group. This study was the first to demonstrate
improved endothelial function following aerobic exercise training in the
coronary arteries of patients with CAD and documented endothelial dys-
function without evidence of CHF.
Linke et al. recently investigated the systemic effects of lower body exercise
training on radial artery endothelial function.45 Twenty-two male patients
with CHF (left ventricular ejection fractions = 24 ± 2%) were randomized to
either exercise training or an inactive control group. After 4 weeks, exercise
trained patients demonstrated a significant increase in the baseline corrected
internal diameter of the radial artery in response to acetylcholine infusion.
In the training group, increases in agonist-mediated FMD correlated with
changes in functional work capacity.
Hambrecht et al. also investigated the effects of 6 months of training on
endothelial function in congestive heart failure (CHF) patients.104 After training,
EDD increased significantly (p < 0.05) in response to acetylcholine compared
with the control group. Increased peak oxygen uptake was correlated with
increased EDD. Hambrecht et al. reported similar findings in another study of
CHF patients.37 In this study, administration of L-arginine concurrent with exer-
cise training further improved EDD. This suggested that an increase in NO
bioavailability via supplementation with L-arginine, the substrate for NO for-
mation, complemented the shear stress-induced increase in eNOS activity. Sev-
eral other investigators have reported similar findings in CHF patients.94,105,106
An ongoing debate in CVR centers around the role of home-based com-
pared with hospital or clinic-based programming. Gielen et al. recently inves-
tigated the effects of home-based versus hospital-based exercise training on
coronary vasomotion.107 Nineteen patients with CAD and documented cor-
onary endothelial dysfunction were randomized to an in-hospital exercise
training program (n = 10) for 4 weeks (60 min per day in 10–15-min sessions
of bicycle ergometry) or a control group (n = 9). After 4 weeks, all training
patients were enrolled in a 5-month home-based exercise program (20 min
per day of bicycle ergometry plus one 60-min group session per week).
Coronary artery endothelial function was assessed by ACH infusion and
quantitative angiography at 4 weeks and 6 months. Endothelial dysfunction
was significantly improved in the exercise training compared with the con-
trol group at 4 weeks and at 6 months. However, endothelial function was
worse at 6 months compared with 4 weeks in the exercise-trained group.
ACH-induced increases in coronary blood flow were also improved signif-
icantly at 4 weeks and 6 months but it was attenuated at the 6-month
evaluation. Although this study did not directly compare a home-based
(non-randomized and the home-based program involved a 1-hour session
each week at the hospital) versus a hospital-based program, it suggested
that there is a relation between daily training duration (60 min daily in the
hospital-based versus 20 min daily in the home-based program) and
improved coronary vasomotion.
An unresolved issue involves the systemic versus local nature of the effects
of exercise training on endothelial function. Walsh et al. randomized ten
persons with CAD in a crossover design to 8 weeks of combined lower body
aerobic and resistance training.108 FMD in response to reactive hyperemia
was assessed before and after training. Baseline function in the exercise-
trained group was compared with a convenience sample of ten controls
without documented cardiovascular disease. Both EDD and EID of the bra-
chial artery were impaired in untrained persons with CAD. Training signif-
icantly improved FMD, but not endothelial-independent vasodilation (EID).
The effect of exercise training on EDD appeared to be generalized or systemic
rather than limited to the exercise trained vascular beds.
In contrast, in patients with CHF, Kobayashi et al. recently reported that
exercise training appeared to improve EDD only in the trained extremities.109
These investigators randomized 28 persons with CHF to either a control
group or an aerobic exercise training group that underwent 3 months of
cycle ergometry. Walking performance, as assessed by the 6-min walk test,
increased significantly only in the exercise group. FMD of the posterior tibial
artery improved but there were no changes in the brachial artery in the
exercise-trained group. Neither brachial nor posterior tibial artery EDD
improved in the control group. Thus, exercise training appeared to correct
endothelial dysfunction predominantly via a local effect in the trained limbs.
Gokce et al. reported similar findings in a study of the effects of aerobic
exercise on EDD in the peripheral circulation in 58 patients with CAD (mean
age = 59 years).110 FMD in response to reactive hyperemia and nitroglycerine-
mediated vasodilation was assessed before and after 10 weeks of standard
CVR (mostly lower limb exercise, 30 min, moderate intensity, 3 days/week)
in 40 patients and 18 matched patients who remained sedentary. Exercise
training resulted in a 29% increase in maximal METs and improved EDD in
the leg, but not the arm vasculature. No changes occurred in nitroglycerine-
mediated EID.
Edwards et al. recently reported the results of exercise training in a stan-
dard (upper and lower body aerobic exercise) CVR program on endothelial
function in persons with documented CAD.111 After 12 weeks, FMD of the
brachial artery improved by 11.1%. Exercise training increased plasma nitrite
and nitrate levels and SOD-1 activity and decreased oxidative stress. Belar-
dinelli, in an excellent review of the role of vasomotor reactivity and the
effects of exercise training on FMD in CVR patients, concluded that shear
stress produced by pulsatile blood flow during exercise may be the most
important factor inducing e-NOS activation, increased NO synthesis, and
improved endothelial function.112
The benefits of exercise training on EDD are well documented. However,
little is known about the optimal exercise intensity, frequency, duration,
mode, volume, and other factors involved in an effective exercise prescrip-
tion. Goto et al. recently investigated the effects of exercise intensity on EDD
in 26 healthy, young men.113 Subjects were randomized to 12 weeks of bicycle
ergometry training, 30 min, 5–7 days/week at 25%, 50%, or 75% of maximal
oxygen uptake. Forearm blood flow response to ACH (EDD) and isosorbide
dinitrate (EID) was assessed before and after training. Only the group that
exercised at 50% of maximal oxygen uptake demonstrated improved EDD.
No group showed improved EID. The administration of L-NMMA (an inhib-
itor of eNOS) abolished the enhanced EDD in the 50% group. High intensity
(75% of maximal oxygen uptake) appeared to increase, while moderate inten-
sity (50% of maximal oxygen uptake) appeared to decrease oxidative stress.
Mild exercise (25% of maximal oxygen uptake) did not alter any parameters.
The finding that training at 75% of maximal oxygen uptake appeared to
increase oxidative stress and did not improve endothelial function is pro-
vocative. Thus, intense exercise may impair EDD by increasing oxidative
stress. However, Goto et al. were unable to demonstrate a relationship
between EDD and oxidative stress in their healthy young subjects. Matsu-
moto et al. previously reported that NO increases with increasing intensity
of exercise perhaps in response to the increased oxidative stress resulting in
maintained endothelial function.114 Although the Goto et al. study is provoc-
ative, further work with larger samples and in patients with risk factors and
CAD is needed before reasonable conclusions may be drawn regarding the
mode, frequency, intensity, duration, and volume of exercise necessary to
enhance endothelial function.
Bergholm et al. reported that 3 months of high-intensity running (80% of
maximal oxygen uptake, 1 h/session, 4 days/week) decreased circulating
antioxidant levels resulting in impaired EDD, but not EID.115 These authors
postulated that high intensity training-induced decreases in circulating anti-
oxidants may adversely affect EDD. It is also possible that these effects are
transient, and that long-term (6–12 months or longer) exercise training may
lead to increased antioxidative capacity and changes in vascular structure
and function that facilitate improved blood flow to working muscles (i.e.,
increased vessel size and enhanced capillarization).
Previous studies investigated the effects of exercise training on endothelial
function in stable CAD or CHF patients and in patients with CAD risk
factors. Hosakawa et al. performed a non-randomized study of the effect of
regular aerobic exercise training on endothelial function in 41 post-MI
patients who also had undergone percutaneous coronary angioplasty
(PTCA).116 The investigators examined a non-infarct related coronary artery
via ACH infusion at baseline and 6 months post-MI. Endothelial function
was significantly improved in the regular exercise group (n = 24) compared
with the non-exerciser group (n = 17). Regular exercise was the only signif-
icant predictor of improvement in endothelial function. Recently, Vona et al.
examined the effects of 3 months of moderate aerobic training (40 min of
cycle ergometry, 3 days/week, at 75% of peak exercise heart rate or ~ 60%
of maximal oxygen uptake) in 54 patients with documented CAD and a
recent MI.117 Compliance to the training program was 88%. FMD of the
brachial artery was significantly impaired at 3 weeks post MI. After exclud-
ing patients on statins or ACE inhibitors that have documented beneficial
effects on endothelial function, patients with classic risk factors known to
impair endothelial function, and patients with left ventricular dysfunction,

only 54 of 968 patients (5.6%) were randomized to the exercise or the control
group. This study therefore allowed for an independent evaluation of the
effects of exercise training on endothelial dysfunction in patients with a
recent MI. The results showed that FMD significantly improved in the
trained compared with the control group. In addition, FMD was correlated
with increased maximal oxygen uptake, as has been demonstrated in other
studies.93,104,118
Although this review has focused on NO and enhanced endothelial
vasodilatory capacity, exercise may also improve EDD via mechanisms
related to reduced vasoconstrictive factors. Endothelin-1 (ET-1) and angio-
tensin II are the primary endothelial-derived substances that induce vaso-
constriction.18,119 Therefore, lower levels of ET-1 are associated with less
vasoconstrictive tone and a greater capacity for unopposed vasodilation.
Maeda and colleagues have reported significantly higher ET-1 concentration
in young athletic humans after 30 min of cycle ergometry above (130%) and
below (90%) the ventilatory threshold and that aerobic exercise training
results in decreased ET-1 concentration in healthy, young adults.119,120 In a
recent study of healthy older women (61–69 years of age), 3 months of aerobic
exercise training using bicycle ergometers at 80% of ventilatory threshold
for 30 min, 5 days/week significantly decreased plasma ET-1.121 This reduc-
tion in ET-1 may have beneficial effects on EDD and may contribute to the
prevention of hypertension and the development and progression of athero-
sclerosis. Callaerta-Vegh et al. reported no change in ET-1 in CHF patients
after myocardial infarction following 8 weeks of exercise training.122 In addi-
tion, nitrate elimination which mirrors NO production was decreased over
2 months following myocardial infarction. However, exercise training
reversed this trend suggesting increased NO bioavailability. Maeda and
colleagues interestingly have reported an increase in ET-1 production in
inactive muscles of humans during exercise and also a reduction in the
magnitude of the decrease in blood flow to the splanchnic bed after ET-1
blockade in rats.123,124 It is beyond the scope of this chapter to thoroughly
discuss the effect of exercise training on ET-1. For more information we
recommend reviewing the articles by Maeda et al.119,120
Correction of Endothelial Dysfunction in Youth

Atherosclerosis often begins in childhood and endothelial dysfunction
appears to be a necessary stage in the transition from normal vascular struc-
ture and function to plaque formation and obstructive atherosclerotic dis-
ease. Consistent with the recognition of the role of endothelial dysfunction
in the early onset of atherosclerotic vascular disease, several studies have
shown that overweight and obese children and adolescents demonstrate
impaired endothelial function as assessed by FMD.125,126 Three recent studies

demonstrate that exercise training improves endothelial dysfunction in over-
weight and obese children and adolescents.127–130
Watts et al. studied the influence of aerobic exercise on endothelial function
in 14 obese children (8.9 ± 0.4 years of age).127 All subjects initially demon-
strated impaired FMD. After 8 weeks of exercise training, FMD significantly
improved in the obese children compared with a matched non-exercise
trained control group. In another study of 19 obese adolescents, mean age
of 14.3 years, with impaired endothelial function, Watts et al. reported that
8 weeks of circuit training (combined aerobic and resistance training) nor-
malized FMD.128 Finally, Woo et al. reported that diet and exercise training
in 82 9–12-year-old obese children with impaired endothelial function
resulted in improved FMD at 6 weeks and a reduction in carotid intima-
media thickness at 1 year.129,130
Mechanisms of Improved Endothelial Function Following

Exercise Training
Although the literature clearly supports a direct effect of exercise training in
both animals and humans on improving endothelial function, particularly
in the presence of impaired EDD, the mechanisms of this benefit are incom-
pletely understood. Numerous investigators have examined the role of sev-
eral observed vascular adaptations to exercise training that may explain the
exercise training-induced improvements in EDD.
Exercise training has been repeatedly shown to improve myocardial perfu-
sion in CAD and CHF patients and to reduce clinical events.17–22,26–33 Hambrecht
suggests that four mechanisms are important mediators of the observed reduc-
tion in morbidity and mortality with exercise training and CVR: improved
endothelial function, reduced progression of coronary lesions, reduced throm-
bogenic risk, and improved coronary collateral circulation.131
Regression of coronary atherosclerotic lesions has been reported following
high-volume exercise training, particularly when combined with low-fat
diet, lipid-lowering medications, and other lifestyle changes such as tobacco
cessation.17–19,21 However, the magnitude of the change in coronary stenoses
is small (~ 41–37% after 1 year, for example in the Lifestyle Heart Trial).17 It
is unlikely that this degree of change can explain the observed improvement
in myocardial perfusion and reduction in clinical events following sustained
exercise training.
Studies have shown that coronary diameter increases with exercise train-
ing but again this cannot adequately explain the improved myocardial per-
fusion following exercise training.89 Additionally, human studies do not
document any significant increases in coronary collateral circulation in
response to exercise training.132
The vascular endothelium releases tissue-type plasminogen activator (t-

PA) that is critical for effective endogenous fibrinolysis. A recent study of 62
men, aged 22–35 or 50–75 years, who were either sedentary or endurance
exercise trained, found that net t-PA release was significantly blunted in the
older men.133 At the highest dose of bradykinin, the increase in t-PA release
was 35% less in the older, sedentary men compared with the younger sed-
entary men. In contrast, the endurance trained older men did not demon-
strate an age-related decline in net t-PA release and activity. In a subgroup
of sedentary older men who completed 3 months of endurance exercise
training, the capacity of the endothelium to release t-PA increased 55% over
baseline and was comparable with that observed in younger sedentary men.
This study suggests that other aspects of endothelial function such as anti-
thrombotic functions can also be enhanced with regular endurance exercise
and may be partially responsible for the reduction in cardiovascular mortal-
ity and morbidity observed in physically active and fit individuals.
Thus, improvement in endothelial function favoring vasodilation, fibri-
nolysis, less oxidative stress, and reduced arterial inflammation may explain
observed improvements in myocardial perfusion and reduced clinical events
following a regular aerobic exercise program. It also is possible that the shear
stress associated with increased blood flow during exercise results in vascu-
lar remodeling leading to an increased heart and skeletal muscle capillarity
(angiogenesis) and enlargement of conduit vessels (arteriogenesis).134,135
However, the following discussion will focus on factors which result in
improved endothelial function, particularly EDD as a mechanism for reduc-
tion in risk of clinical cardiovascular disease and events.
Cell culture studies and animal experiments suggest that shear stress
increases endothelial L-arginine uptake and enhances eNOS activity and
expression. These changes result in increased NO bioavailability, as well as
increased activity of SOD-1 thus preventing premature NO breakdown.136,137
Maiorana et al. and Green et al., in excellent review articles cited earlier,
concluded that the animal literature suggests that initially (1–4 weeks) exer-
cise training enhances eNOS expression and NO bioavailability in order to
buffer the increased shear stress associated with exercise hyperemia.46,48 With
prolonged training of greater than 4 weeks, structural adaptations become
manifest resulting in increased lumen diameter, thus normalizing shear
stress. Endothelial-derived NO then returns towards baseline. In support of
this hypothesis, Oltman et al. reported that after 16 weeks of training (pigs)
that vasodilator response to adenosine in large epicardial arteries remains
augmented even after the endothelium was removed.138
Fukai et al. and Gielen both conclude that exercise training may improve
EDD and myocardial perfusion by increasing eNOS and SOD-1 expression
and NO bioavailability.139,140 Gielen speculated that these changes occur rap-
idly after initiating an exercise program and that, if prospective studies
confirm that endothelial function is an independent prognostic marker of
future CVD events, exercise training would be a key intervention to treat
symptomatic atherosclerosis and an important preventive strategy with both
short- and long-term prognostic benefits.141 In a recent review on the effect

of exercise training on endothelial function in cardiovascular diseases in
humans, Walther et al. concluded that exercise training through repetitive
increases in laminar shear stress leads to an increase of NO bioavailability
via increased NO production and a reduction in NO inactivation through
reduced reactive oxygen species.142
Kemi et al. recently examined the effect of 13 weeks of high intensity
training followed by 4 weeks of detraining on cardiomyocyte contractile
activity and endothelial function in adult rats.143 Multiple regression analysis
revealed that cell length, relaxation, and calcium decay were the main
explanatory variables related to increased maximal oxygen uptake (r2 = 0.87,
p < 0.02). With detraining, maximal oxygen uptake decreased 50% at 2 weeks
and stabilized at 5% above baseline at 4 weeks. Cardiomyocyte size regressed
in parallel with the increase in maximal oxygen uptake and remained 9%
greater than control at 4 weeks. Cardiomyocyte shortening, calcium transit
time course, cardiomyocyte contraction/relaxation, and EDD regressed com-
pletely within 2–4 weeks of detraining. Multiple regression analysis identi-
fied cardiomyocyte length and vasorelaxation as the main determinants of
regression of maximal oxygen uptake during detraining (r2 = 0.76, p < 0.02).
EDD and enhanced vascular sensitivity to NO returned to baseline within
2 weeks of detraining. Although the time course of the onset of enhanced
EDD could not be measured in this study, the authors speculate that
improvements in endothelial function occur rapidly with training in parallel
to the return to baseline following detraining.
There is little work in humans elucidating the time course of the improve-
ment of EDD. A recent, non-randomized small study suggested that exercise
acutely improves FMD.144 In part one of this study, brachial artery FMD was
assessed during exercise to exhaustion on five consecutive days. FMD
improved daily, was significantly improved by day 3 (p = 0.012), reached
maximal improvement on day 6 and returned to baseline by day 9. In the
second part of this study, 17 subjects (38 ± 2 years of age) trained for 4 weeks,
30 min/session, 3 days/week at 70% of maximal heart rate, on bicycle
ergometers. FMD improved significantly (p = 0.028) post-training, but
returned to baseline following 2 weeks of detraining. The results suggested
that the beneficial effects of aerobic exercise training on endothelial function
are rapid but short-lived. This study, however, was non-randomized,
involved a small number of male subjects, and was short-term, thus great
care must be used in interpreting these data. Longer (6–12 months), random-
ized, and well-controlled studies with larger samples in clinical populations
are required before conclusions can be drawn regarding the frequency, dura-
tion, and intensity of exercise necessary to induce and sustain improvements
in endothelial function.
Hambrecht et al. investigated the molecular mechanisms mediating the
observed exercise training-induced improvements in EDD.145 Using left
internal mammary artery (LIMA) tissue harvested during coronary artery
bypass surgery of 17 exercise-trained (ET) and 18 control (C) patients, eNOS
expression and content of phospho eNOS–Ser1177, AKT, and phospho-AKT

were determined. EDD improved 56% in response to ACH in the ET group
after 4 weeks. There were no changes in the control patients. The improve-
ment in ACH-induced vasodilation of the LIMA was closely related to a
shear stress-induced AKT-dependent phosphorylation of eNOS on Ser1177.
Recently, Erbs et al. examined the effects of polymorphisms in the promoter
(T-786c) and exon 7 (G894T) of the eNOS gene on endothelial function and,
more specifically, on the endothelial response to physical training in patients
with CAD.146 Both of these polymorphisms have been shown to be associated
with reduced vascular NO production and/or proteolytic cleavage of eNOS.
The results of this study suggested that patients with either the promoter or
exon 7 polymorphism of the eNOS gene demonstrate attenuated EDD, but
only the promoter polymorphism attenuated the training-induced improve-
ment in EDD.
The anti-inflammatory effects of exercise training represent another pos-
sible mechanism that may partially explain how regular exercise training
improves endothelial dysfunction. Hingorani et al. reported that systemic
inflammation promotes endothelial dysfunction and others have reported
that hsCRP, a marker of systemic arterial inflammation, directly contributes
to endothelial dysfunction.147–149 Several recent studies have shown that aer-
obic exercise training exerts anti-inflammatory effects in older healthy adults
and in patients with the metabolic syndrome, risk factors for CVD, and
CHF.150–154
As indicated earlier in this chapter, a recent area of interest has been the
discovery of endothelial progenitor cells (EPCs). Progenitor cells are primi-
tive bone marrow cells that have the capacity to proliferate, migrate, and
differentiate into various mature cells.84–86 For example, EPCs have the ability
to mature into cells that line the lumen of blood vessels or the endothelium.
In adult humans, EPCs primarily appear to contribute to rapid re-endothe-
lialization at sites of endothelial cell damage to prevent migration of leuko-
cytes and other cells into the intima, and de novo formation of blood vessels
and promotion of angiogenesis.84–86
Studies demonstrate an inverse correlation between CAD risk factors and
the number and migratory activity of EPCs.155,156 Smoking seems to be the
major independent predictor of reduced EPC levels while impairment of
EPC migration was most influenced by hypertension. The level and migra-
tory activity of EPCs may serve as surrogate biological markers for vascular
function and cumulative CVD risk.84 Rauscher and colleagues also demon-
strated that chronic treatment with bone marrow-derived progenitor cells
from young mice without atherosclerosis prevented atherosclerosis progres-
sion in older mice despite persistent high fat diet-induced hypercholester-
olemia.157 Treatment with cells from older mice with atherosclerosis was
much less effective. These authors concluded that a progressive decline in
progenitor cells may contribute to the development of atherosclerosis. These
findings suggest that interventions to increase EPCs may prove beneficial in
preventing the development and progression of atherosclerosis.
Recently, acute exercise has been shown to increase circulating levels of

EPCs in humans.158,159 Rehman et al. investigated whether a single episode
of exercise acutely increases the number of EPCs and cultured/circulating
angiogenic cells (CACs).158 Twenty-two middle-aged (mean age = 54 years)
men and women without known CAD underwent exhaustive dynamic exer-
cise on a bicycle ergometer. The results indicate that circulating EPCs increase
fourfold in peripheral blood and circulating CACs increase 2.5-fold. Adams
et al. studied whether a maximal stress test in patients with known CAD (n
= 28) and in healthy subjects (n = 11) acutely increases the number of circu-
lating EPCs.159 Sixteen of the CAD patients had exercise-induced ischemia.
Circulating EPCs were monitored for 144 h post-exercise. EPCs increased
only in the ischemic patients. This was in contrast to the study by Rehman
et al., which showed an increase in EPCs in patients without evidence of
exercise-induced ischemia. However, these studies suggested, given the abil-
ity of EPCs to promote angiogenesis and vascular repair that exercise-
induced increases in EPCs may serve as a physiological repair or compen-
sation mechanism that may partially explain the preventive effects of exercise
on atherosclerosis development and progression.
Case Study
A 50-year-old man presented to the Emergency Room (ER) with a recent
30–60 min history of substernal chest pain radiating to the left shoulder and
scapula precipitated by exertion. He rated the pain 3–4 on a 0–10 scale. He
has had several of these episodes in the past 3–4 months that usually resolved
after a few minutes of rest. However, this episode was persistent. He denied
nausea, dyspnea, or diaphoresis. He had become progressively more anxious
about these episodes, which together with the persistence of this episode
brought him to the ER. A resting ECG showed mild ST elevation with T-wave
inversion in anterior leads. Troponin and CKMB band were mildly elevated,
suggesting a possible evolving myocardial infarction. He reported a history
of untreated dyslipidemia, pre-hypertensive blood pressure of 134/88
mmHg, and lack of regular physical exercise.
A lipid profile at the time of the presentation revealed a total cholesterol
of 221 mg/dl, triglycerides of 211 mg/dl, high-density lipoprotein of 39 mg/
dl, and low-density lipoprotein of 140 mg/dl. Homocysteine (8 mg/dl) and
lipoprotein (a) [Lp(a)] (15 mg/dl) were within normal limits. Highly sensi-
tive C-reactive protein (hsCRP) was slightly elevated at 3.4 mg/L. Body mass
index was 28.2 with a waist circumference of 40.25 inches. His father had
died of a myocardial infarction at age 58 years and his mother, who is alive,
had a transient ischemic attack (TIA) at age 72 years. His only sibling, a
48-year-old brother, has a history of dyslipidemia, hypertension, and is over-
weight and physically inactive.
A cardiac catheterization was done within 90 min of the onset of chest

pain that revealed an 85% proximal left anterior descending stenosis with
irregularities distal to the lesion, and two areas of 20–30% narrowing in the
right and left circumflex coronary arteries. Left ventricular ejection fraction
was 58%.
A percutaneous coronary angioplasty with implantation of a sirolimus
eluting stent was performed across the LAD lesion. The patient tolerated
this procedure without complications and was discharged after 48 h of
observation. Prior to discharge, a sestamibi exercise test was administered
to 85% of maximal predicted heart rate. Results showed a 55% left ventricular
ejection fraction and mild reversible perfusion abnormalities in the anterior
and inferior walls. Medically, the patient was placed on Atenolol, 25 mg BID,
Ramipril, 10 mg QD, 81 mg ASA QD, and Zocor, 20 mg QD. He was advised
to take a daily multivitamin with 400 µg of folic acid and no iron, 200 IU
Vitamin E, 250 mg Vitamin C, and 100 µg selenium. He was referred to
Outpatient Cardiovascular Rehabilitation (CVR) to start within 1 week of
discharge.
On entry into the CVR program, he underwent cardiopulmonary exercise
testing and a prescription was prepared based on the heart rate at the ven-
tilatory threshold. He was advised to consume an anti-atherogenic diet con-
sisting of 20–25% fat, < 7% saturated fat, < 150 mg/day cholesterol, 25–40 g
fiber/day, and < 2000 mg/day sodium. He also was advised to consume
more coldwater fish and foods rich in monounsaturated and omega-3 fatty
acids such as olives, olive oil, canola oil, flaxseed, avocados, walnuts,
almonds, peanuts, peanut butter and other nut butters.
During the first exercise session, he experienced mild substernal chest pain
rated 1 on a 0–4 pain scale, which was similar to his historical pain. The
patient was hemodynamically stable and the pain resolved with rest. He
was referred to Cardiology and a repeat sestamibi exercise test was admin-
istered which was similar to the post-discharge test. The patient was referred
back to CVR and advised to exercise within tolerance for chest pain (not >
1–1.5 on a 0–4 scale). Support for reentering CVR in the presence of mild
but stable ischemia comes from a recent report by Hambrecht et al. that
showed, in stable patients with documented CAD, 1-year outcomes superior
in patients randomized to CVR compared with those under PTCA with stent
implantation.20 He was advised to continue to aggressively manage risk
factors for atherosclerotic progression.
This case illustrates a relatively common scenario in CVR programs. One
explanation for the persistent, though less severe symptoms in light of an
apparent successful revascularization procedure, is endothelial dysfunction.
The patient has several risk factors for endothelial dysfunction including
dyslipidemia, upper normal blood pressure, slightly high hsCRP level, pos-
itive family history, high-fat diet, overweight, known CAD, and sedentary
lifestyle. As discussed previously, studies in patients with single-vessel,
occlusive disease demonstrate paradoxical coronary vasoconstriction in
response to intracoronary injection of vasodilators such as acetylcholine in

epicardial arteries with “visually” normal lumens.160
Further support for the role of endothelial dysfunction in recurrent
ischemia following a successful percutaneous intervention (PCI) comes from
a report by Caramori et al.161 It is well documented that catheter-based
coronary interventions are associated with extensive arterial injury resulting
in endothelial dysfunction. Caramori et al. performed intracoronary acetyl-
choline infusion in 39 CAD patients treated with a PCI for a LAD stenosis
6 months earlier. Twelve had received a PTCA with stent implantation, 15
had PTCA only, and 12 had directional coronary atherectomy. The results
showed that although all patients demonstrated some degree of endothelial
dysfunction, the LAD constricted more in response to acetylcholine in the
stented patients, suggesting that stenting may be associated with more resid-
ual endothelial impairment. Monnink et al. also recently demonstrated that
exercise-induced ischemia after a successful PCI was due to endothelial
dysfunction distal to the interventional site.162
Summary
The growing knowledge that the luminal diameter of coronary epicardial
and resistance vessels and major peripheral arteries is highly dynamic in
response to flow-mediated (shear stress) and agonist-mediated (nitric oxide
and endothelin-1) factors has greatly advanced the understanding of athero-
sclerosis. Ludmer et al. first observed a paradoxical vasoconstriction of ath-
erosclerotic segments of coronary arteries in response to the infusion of
acetylcholine.160 This paradoxical vasoconstriction was observed in angio-
graphically “normal” arteries. It is also known that a large portion of the
control of luminal diameter resides in the endothelium, the single cell lining
of the vascular system.
In addition, it has been observed that persons with major coronary risk
factors often demonstrate endothelial dysfunction even before anatomical
atherosclerotic lesions are observed.163–169 Thus, it appears that endothelial
dysfunction is a key pathological feature in the early stages of atherosclerosis.
Additionally, endothelial dysfunction also plays a significant role in acute
coronary syndrome (ACS), by the relative inability of the vascular surface
to inhibit platelet aggregation and the increased endothelial permeability to
influx of cellular material leading to both ACS and/or progression of ath-
erosclerotic lesions. This dysfunction may also contribute to plaque rupture
as the initiating event in ACS. Recent studies demonstrate that treatment
and management of major risk factors improves endothelial function. This
has been observed for dyslipidemia, weight loss, hypertension, enhanced
management of diabetes mellitus, and smoking cessation. Statins, ACE inhib-
itors, purple grape juice, folic acid, L-arginine, dark chocolate, and other
TABLE 6.2
Risk Factors Documented to Be Associated with Endothelial
Dysfunction
Presence of oxidized LDLs Post-menopausal state
Presence of small, dense LDLs Insulin resistance/diabetes mellitus
Hypertension Impaired fasting glucose
Type 1 and 2 diabetes mellitus Acute postprandial hypertriglyceridemia
Hyperhomocysteinemia Active and passive smoking
Elevated lipoprotein (a) Psychosocial stress
Low HDLs Aging
Overweight and obesity Physical Inactivity
Impaired glucose tolerance Inflammation (hsCRP)
Lipoprotein remnant particles Metabolic syndrome
TABLE 6.3
Interventions Demonstrated to Improve Endothelial Dysfunction
LDL lowering by statins Purple grape juice
LDL lowering by low-fat diet Iron chelation
ACE/ACE II receptor inhibitors Black and green tea
L-Arginine High monounsaturated diet
Moderate alcohol intake Smoking cessation
Premenopausal status Anti-oxidant therapy
Exercise training Weight loss and loss of central body fat
Increasing HDL Dark chocolate
LDL lowering with pheresis Thermal therapy
substances have been shown to correct or improve impaired endothelial

function (Tables 6.2 and 6.3).
Exercise training has been shown to improve endothelial function in
patients with congestive heart failure, hypertension, diabetes mellitus, and
CAD. Hambrecht et al. were the first to demonstrate that just 4 weeks of
daily moderate intensity endurance exercise training in patients with CAD
attenuates paradoxical coronary vasoconstriction in response to acetylcho-
line.44 Linke et al. demonstrated that the enhanced endothelial function fol-
lowing exercise training in CHF patients is systemic in nature, although other
studies failed to substantiate this finding.45,108–111 These data provide compel-
ling support for an important mechanism by which regular exercise may
improve endothelial function, enhance coronary blood flow in patients with
or at risk for CAD, and reduce the risk for progression of atherosclerosis and
recurrent events.
Hambrecht et al. recently reported that regular physical activity improves
endothelial function in persons with CAD by increasing the phosphorylation
of nitric oxide synthase.145 Erbs et al. have shown that the improvement in
EDD following 4 weeks of exercise training in stable CAD patients is atten-
uated in the presence of the promoter polymorphism of the eNOS gene, but
not in patients with the wild-type or the exon 7 (G894T) polymorphisms.146
In addition, it has been shown that EPCs, which can differentiate into endo-
thelial cells and may also be involved in vascular repair and angiogenesis,
increase with acute exercise in humans and with short-term exercise training
in animals.84–86, 155–159
It has been consistently demonstrated that both acute exercise and exercise
training enhance endothelial function in animals and humans. Exercise
clearly improves endothelial function in arteries that are dysfunctional sec-
ondary to the presence of risk factors or atherosclerotic vascular disease or
other mechanisms (e.g., aging). Endothelial function (EDD and FMD) is
enhanced in fit and/or physically active compared with unfit and/or phys-
ically inactive populations, and exercise may potentiate the effects of other
agents that enhance endothelial function.
At this time the appropriate intensity, duration, and frequency of exercise
to optimize enhancement of endothelial function are unknown. Goto et al.
suggested that moderate-intensity exercise improves EDD whereas higher-
intensity exercise may impair EDD, but more work is necessary to determine
the generalizability of this finding.113 More studies with larger sample sizes
including women, middle-aged and older adults and persons with CAD and
CVD are necessary before definitive recommendations can be made. The few
studies that have been completed suggest that a common prescription of
30–45 min of moderate intensity (50–75% of maximal oxygen uptake reserve
or heart rate reserve), 4–5 days per week is effective. This exercise should
be coupled with other daily activity, because the benefits of exercise on the
endothelium may be acute and related to recent exercise.
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endothelial progenitor cells and monocyte/macrophage-derived angiogenic
cells, J Am Coll Cardiol, 43, 2314, 2004.
159. Adams V, Lenk K, Linke A, et al. Increase of circulating endothelial progenitor
cells in patients with coronary artery disease after exercise-induced ischemia,
Arterioscler Thromb Vasc Biol, 24, 684, 2004.
160. Ludmer PL, Selwyn AP, Shook TL, et al. Paradoxical vasoconstriction induced
by acetylcholine in atherosclerotic coronary arteries, N Engl J Med, 315, 1046,
1986.
161. Caramori PR, Lima VC, Seidelin PH, et al. Long-term endothelial dysfunction
after coronary artery stenting, J Am Coll Cardiol, 34, 1675, 1999.
162. Monnink SH. Exercise-induced ischemia after successful PTCA is related to
distal endothelial dysfunction, J Inves Med, 51, 221, 2003.
163. Tuzcu EM, Kapadia SR, Tutar E, et al. High prevalence of coronary atheroscle-
rosis in asymptomatic teenagers and adults: Evidence from intravascular ul-
trasound, Circulation, 103, 2705, 2001.
164. Quinones MJ, Hernandez-Pampaloni M, Schelbert H, et al. Coronary vasomo-
tor abnormalities in insulin-resistant individuals, Ann Intern Med, 140, 700,
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165. Reddy KG, Nair RN, Sheehan HM, et al. Evidence that selective endothelial
dysfunction may occur in the absence of angiographic or ultrasound athero-
sclerosis in patients with risk factors for atherosclerosis, J Am Coll Cardiol, 23,
833, 1994.
166. McGill HC, McMahan CA, Zieske AW, et al. Associations of coronary heart
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7
Essential Laboratory Methods for Blood Lipid
and Lipoprotein Analysis
Peter W. Grandjean and Sofiya Alhassan
CONTENTS
Introduction ......................................................................................................... 118
Plasma and Serum Preparation........................................................................ 119
Quantifying Blood Lipid Concentrations .......................................................120
Materials......................................................................................................121
General Procedures ...................................................................................121
Quantifying Apolipoprotein Concentrations .................................................123
Materials......................................................................................................123
General Procedures ...................................................................................123
Methods for Isolating Lipoproteins .................................................................123
Sequential Ultracentrifugation ................................................................124
Materials .........................................................................................124
General Procedures .......................................................................124
Calculating the Plasma Density..................................................125
Calculating the NaBr Needed to Adjust Plasma Density ......126
Lipoprotein Precipitation..........................................................................126
Materials .........................................................................................127
General Procedures ......................................................................127
Solutions .........................................................................................127
Lipoprotein Separation .............................................................................128
Gel Electrophoresis....................................................................................128
Materials .........................................................................................129
Solutions .........................................................................................129
Pre-Electrophoresis Procedures ...............................................................130
Lipoprotein Isolation.................................................................................131
Lipoprotein Analysis .................................................................................132
Analyses of Intravascular Enzyme and Transfer Protein Activities ..........133
Lipoprotein Lipase and Hepatic Lipase Activities...............................133
117
Materials .........................................................................................134
Solutions .........................................................................................135
Preparation .....................................................................................136
Preliminary Procedures................................................................136
Final Preparations .........................................................................137
Assay Procedures ..........................................................................137
Calculations....................................................................................138
Example Calculation.....................................................................138
Estimating the Rate of Cholesterol Ester Exchange.............................139
Materials .........................................................................................139
Preliminary Procedures (Determining Assay Linearity) ........140
Developing the Standard Curve (Determining Neutral
Lipid from Fluorescence Intensity)..............................140
Assay Procedure............................................................................141
Calculations....................................................................................141
Summary ..............................................................................................................142
References ............................................................................................. 142
Introduction
Contemporary blood lipid and lipoprotein analytical techniques were born
largely from early observations that these blood constituents were strongly
related to the development of cardiovascular diseases and the need to stan-
dardize analytical methods so that population standards could be estab-
lished.1,2 The methodologies described in this chapter are employed in
clinical settings or may be found cited in the scientific literature describing
different facets of blood lipid transport in humans.
Obviously, the full array of analytical methods available to lipid research-
ers and complete descriptions of each analytical method are well beyond the
scope of this chapter. We will outline the essential and most common meth-
ods for determining blood lipid and apolipoprotein concentrations, lipopro-
tein size and densities, and activities of intravascular enzymes and transfer
proteins in human blood. We will explain the underlying principles, identify
the necessary instrumentation and supplies, and describe a general approach
for each technique. Thus, the purpose of this chapter is to provide informa-
tion that is essential for establishing these methods in your laboratory. Read-
ers are encouraged to access additional resources for more specific
information on these and other analytical techniques.1,3,4
Essential Laboratory Methods for Blood Lipid and Lipoprotein Analysis 119
Plasma and Serum Preparation

Daily variation in blood cholesterol concentrations can range from 5% to
10% and may be attributed to such factors as dietary intake, alcohol con-
sumption, menstrual cycle fluctuations, recent physical exertion, hydration
status, illness and acute inflammation.5–7 Even greater variation (5–25%) is
observed for triglyceride concentrations.6 Therefore, these factors should be
taken into consideration when interpreting blood lipid data and, certainly,
every effort should be made to control or account for these factors if serial
samples are being obtained from an individual for comparison or monitoring
purposes. To reduce measurement variation due to environmental influ-
ences, blood samples for routine blood lipid analyses should be obtained
after an 8- to 12-hour fast. The fasting blood samples should also be obtained
at the same time of day in order to encourage consistent conditions under
which serial blood samples are obtained.
Most assay procedures for lipid and apolipoprotein concentrations recom-
mend the use of serum, although plasma may be used in some assays with
similar results.8–11 Likewise, plasma or serum may be used when isolating
lipoproteins for particle size and density determination.3 When quantifying
the activities of intravascular lipases, the methods employed for obtaining
the blood samples dictate that plasma samples be utilized.12,13 In all instances,
it is recommended that the collection tubes be chilled either in a refrigerator
at 4°C or on ice for several minutes prior to blood collection. Serum from
fasting blood samples can be obtained from collection tubes containing no
additive. Immediately after collection, the blood samples should once again
be stored on ice and allowed to clot prior to centrifugation. If plasma samples
are desired, the use of Na+–EDTA collection tubes are generally recom-
mended because heparin additive in collection tubes will interfere with
lipoprotein isolation techniques such as gel electrophoresis.14,15 The collection
tubes are then chilled immediately after blood sampling and prior to under-
going centrifugation. The use of Na+–heparin collection tubes, however, is
recommended when the purpose of the blood sampling is to determine lipase
activities.13
The collection tubes are centrifuged at 1500–3000 × g for 15–30 min at 4°C
and serum or plasma is obtained by pipette. Serum samples obtained for
measuring lipid and apolipoprotein concentrations can be aliquoted and
frozen prior to analyses. Serum that will be used for high-density lipoprotein
(HDL) separation by precipitation can be stored at 4°C for up to 72 h after
blood sampling and should not be frozen until after completing the separa-
tion procedures. Likewise, plasma obtained for lipoprotein isolation should
not be frozen.16
Blood lipid transport is a dynamic process, involving lipid and apoprotein
exchanges between lipoprotein fractions as well as lipoprotein, apoprotein
and lipid modifications. These exchanges and modifications continue in the
blood after collection. Therefore, in addition to maintaining chilled collection

tubes throughout the blood sampling process, preservatives may be intro-
duced to the serum or plasma prior to storage.17 Protease activity can be
inhibited by the addition of phenylmethylsulfonylfluoride (PMSF) at a final
sample concentration of 0.015%. Sodium azide (NaN3) may be added to the
plasma or serum at a final concentration of 0.04% to inhibit bacterial decom-
position. Ethylenediaminetetraacetic acid (EDTA) can be added to the sam-
ples at a final concentration of 0.4% to prevent oxidation and bacterial
phospholipase activity.3,17
Under all circumstances it is important to establish a plan for blood col-
lection, processing and storage so that samples are handled consistently.
Sample analyses should be carried out as soon after collection as possible.
However, in longitudinal research, multiple serum or plasma samples are
collected from an individual over a period of time. In these research settings,
a common practice is to collect all blood samples from an individual prior
to lipid and lipoprotein analyses. As the samples are collected and processed,
they are stored frozen at –20 to –70°C and all samples from an individual
are assayed in a single run in order to avoid inter-assay variation.
Quantifying Blood Lipid Concentrations

Enzymatic procedures are the most commonly employed method for esti-
mating cholesterol and triacylglycerol concentrations in plasma or serum.
The enzymatic reagents can be prepared by the researcher as previously
described;8,10 however, these reagents are produced commercially, are fairly
inexpensive and, once reconstituted, have a shelf life of up 30 days.
The cholesterol reagent contains three enzymes, and the dye necessary for
the indirect quantification of cholesterol concentration. These enzymes are:
cholesterol esterase, cholesterol oxidase, and peroxidase. The enzymatic reactions
that take place in the serum–reagent mixture generally occur as follows:
(1) cholesterol esters are hydrolyzed by cholesterol esterase to free cholesterol
and fatty acids; (2) free cholesterol is oxidized by cholesterol oxidase to cholest-
4-en-3-one and hydrogen peroxide; (3) a quinoneimine dye is produced
when hydrogen peroxide oxidizes p-hydroxybenzenesulfonate and 4-ami-
noantipyrine in the presence of peroxidase. The dye has a maximum absor-
bance in the visible light spectrum at approximately 500 nm. Cholesterol is
indirectly quantified by reading the sample absorbance using a spectropho-
tometer. The intensity of the color produced by these reactions is propor-
tional to the total cholesterol concentration.
The contemporary enzymatic method for estimating serum triacylglycerol
concentration actually employs a series of reactions designed to quantify the
glycerol concentration in the serum.10 Thus, triacylglycerol concentrations
are determined indirectly using a reagent containing four enzymes. The
enzymes in the reagent are: lipoprotein lipase, glycerol kinase, glycerol phosphate
oxidase, and peroxidase. The enzymatic reactions occur as follows: (1) triglycer-
ides are hydrolyzed by lipoprotein lipase into fatty acids and glycerol;
(2) through the action of glycerol kinase, glycerol is phosphorylated to glyc-
erol-1-phosphate and adenosine-5-diphosphate (ADP); (3) glycerol phosphate
oxidase oxidizes G-1-P to dihydroxyacetone phosphate and hydrogen perox-
ide and; (4) a quinoneimine dye is formed when the hydrogen peroxide
reacts with 4-amino-antipyrine and 5-dichloro-2-hydroxybenzene sulfonate.
The dye has an optimal absorbance in the visible light spectrum at 540 nm.
As with cholesterol, triacylglycerol is indirectly quantified by reading the
sample absorbance since the intensity of the color produced by these reac-
tions is proportional to the total triacylglycerol concentration.
As with cholesterol and triacylglycerol concentrations, there are several
methods that may be employed for determining plasma free fatty acids.3 The
enzymatic approach for determining free fatty acid concentration is generally
recommended due to the reduced cost — in terms of time and necessary
equipment — and comparable precision and accuracy versus gas chroma-
tography.18 The reagents for determining free fatty acid concentrations are
available commercially, and the approach is similar to that described for
cholesterol and triglyceride.19
Materials
Constant temperature incubator, spectrophotometer, pipettes with vol-
umes from 10 to 100 µl and from 100 to 1000 µl, stopwatch or timing mech-
anism, matched cuvettes suitable for wavelength analysis in the visible light
spectrum, lipid reagent, calibrator standard serum with known concentra-
tion values, control and test sera are needed.
General Procedures
It is important to collect blood samples according to the manufacturer’s
recommendations in order to avoid contaminants and substances that may
interfere with the enzymatic reagents. The procedures described in the pack-
age inserts should be followed so as to minimize technician error and sample
variability. Reagents should be prepared and stored according to manufac-
turer instructions in order to optimize the performance and shelf life. Assay
reliability should be quantified by including control serum at several inter-
vals within a single assay run.
Prior to each assay, the reagent is removed from refrigerated storage and
allowed to warm to room temperature. Matched cuvettes are labeled in
duplicate and a temperature-controlled incubator is set at 30 or 37°C. The
reagent is then introduced into each cuvette. Next, distilled water, control
serum or test serum is introduced into appropriately labeled cuvettes (usu-
ally in a 1:100 ratio with the reagent), gently mixed by inversion and placed
in the incubator at regularly timed intervals (30-s to 1-min intervals are often
used). The cuvettes remain in the incubator for the specified time (e.g., 10
min at 37°C or 15 min at 30°C) and are removed from the incubator and
read in the spectrophotometer in keeping with the timed intervals.
The cuvettes containing reagent and distilled water are used as “blanks”
and read first in order to “zero” the absorbance reading in the spectropho-
tometer. Duplicate cuvettes containing control serum are placed at the begin-
ning, middle and end of each assay run. For quality control measures, the
duplicate control and test sample absorbances should be within 0.01 of each
other. Duplicate samples that meet this criterion are averaged and the lipid
concentration is determined against calibrator standard values.
The lipid concentrations may be determined from a single calibrator stan-
dard or a regression equation developed from several levels of calibrator
standards. If a regression equation is developed, the range of calibrator
standard values should encompass all test serum values. If a single calibrator
standard is used, the lipid concentration may be determined as follows:
 Test Sample absorbance 

 Calibrator Standard ab ∗
bsorbance 
Known Lipid Concentration Value of the Calibrator Standard
Reference ranges for assessing lipid concentrations, comparing lipid concen-

trations to population-based standards and determining level of cardiovas-
cular disease risk have undergone considerable revision in recent years.20,21
Table 7.1 provides a summary of the most recent classification for blood
lipids as established by the report from the National Cholesterol Education
Program Adult Treatment Panel III.21
TABLE 7.1
National Cholesterol Education Program: Adult Treatment Panel Recommendations
for Blood Lipid Levels
Total Cholesterol LDL-C TG HDL-C
Optimal < 100 Lowa < 40
Desirable < 200 Near optimal 100–129 Normal < 150
Borderline high 200–239 Borderline high 130–159 Borderline high 150–199
High ≥ 240 High 160–189 High 200–499 High 60
Very high ≥ 190 Very high ≥ 500
LDL-C, low-density lipoprotein cholesterol; TG, triglyceride; HDL-C, high-density lipoprotein
cholesterol. All lipid concentrations are given in mg/dl.
a HDL-C less than 40 mg/dl is considered low for men. HDL-C of 50 mg/dl is considered
low for women.
Source: National Cholesterol Education Program. J.A.M.A., 285, 2486, 2001. Visit http://
www.nhlbi.nih.gov/guidelines/cholesterol for the most recent report from the National Cho-
lesterol Education Program’s Adult Treatment Panel III.
Quantifying Apolipoprotein Concentrations

Apolipoprotein concentrations are commonly determined using various
immunoassay techniques. Radioimmunoassay, radial immunodiffusion,
enzyme-linked immunosorbent assay, fluorescence immunoassay, nephelo-
metric immunoassay, electroimmunoassay, and immunoturbidimetric assays
have each been employed with a different degrees of sensitivity and speci-
ficity for apolipoproteins A-I, A-II, B, C-III, E and lipoprotein (a).22–26 Of these
methods, immunoturbidimetric assays are probably the most widely used
procedures for quantifying apolipoproteins due to the technical simplicity,
robust results for serum and plasma specimens, and high degree of reliabil-
ity.27 Population-based reference values remain dependent on the method
for quantifying apolipoproteins.28
Materials
Auto-analyzer, commercially available reagents, apolipoprotein calibrators,
control plasma or serum, test plasma or serum samples are needed.
General Procedures
Test plasma or serum is introduced into the reagent containing anti-human
apolipoprotein antibodies. The antibodies bind the apolipoproteins in the
sample causing an insoluble aggregate. The degree of turbidity that results
in the sample is proportional to the specific apolipoprotein concentration
and may be determined spectrophotometrically at ~ 700 nm.26,27
Methods for Isolating Lipoproteins

A number of analytical techniques may be used for isolating lipoproteins.
The most common approaches for isolating lipoprotein classes include var-
ious ultracentrifugation techniques, precipitation and gel electrophoresis.3,4
Recent advances in lipoprotein separation by nuclear magnetic resonance
(NMR) spectroscopy provide more comprehensive lipoprotein and lipid
information than can be obtained from any of the methods described here,
and show tremendous promise for clinical and research purposes.29,30
Although the methods for NMR spectroscopy have been described, the
means of completing such analysis are beyond the scope and resources for
most laboratories. Currently, lipoprotein and lipid profiles can be obtained
by NMR through commercial contract (www.liposcience.com).
Sequential Ultracentrifugation
Sequential ultracentrifugation, first described by Havel et al.31 in the 1950s,
is a principal method for characterizing lipoproteins. Lipoproteins can be
separated by ultracentrifugation because these macromolecules have lower
hydrated densities than other plasma proteins and because of differences in
the relative percentages of lipids and proteins among the lipoprotein
classes.16 Lipoprotein isolation by ultracentrifugation is a relatively slow and
labor-intensive process. Moreover, it is recognized that apolipoproteins
redistribute among lipoproteins and lipid peroxidation may occur during
these procedures.16 However, the major lipoprotein fractions as we describe
them today — very low-density, intermediate-density, low-density and high-
density lipoprotein classes — were first described by ultracentrifugation
separation techniques. Today, a variety of ultracentrifugation procedures
exist, some of which reduce the time and effort required by earlier meth-
ods.16,32
Materials
Ultracentrifuge and appropriate rotor(s), weight scale sensitive to 0.0000 g,
balance scales for determining plasma densities, wax paper or disposable
weighing trays (for weighing solutes), glass containers for mixing and stor-
ing the prepared solutions, capped ultracentrifuge tubes, tube racks (capable
of holding sample preparation tubes), pin light or small flashlight, syringes
or capillary pipette for removing top lipoprotein fractions, NaN3 and PMSF
solutions, NaBr are needed.
General Procedures
Plasma samples are obtained and the total volume and density of the samples
are determined. Additives, as described previously, are introduced to pre-
serve the plasma sample. Next, a volume of sodium bromide (NaBr) is added
to the plasma sample in order to adjust the sample to the appropriate density
(g/ml) and the sample is mixed thoroughly (see Table 7.2). After eliminating
trapped air in the plasma while it is under a light vacuum, aliquot the
adjusted plasma into ultracentrifuge tubes and tightly cap each tube. The
tubes undergo ultracentrifugation at constant temperature and speed for
18–24 h.3 The top plasma fraction is then removed by capillary pipette. This
process is repeated in order to isolate each lipoprotein class from the lowest
to the highest densities. A thorough description of the equipment, materials
and procedures required for sequential ultracentrifugation is provided by
Schumaker and Puppione.16 The general steps and calculations for determin-
ing the density of plasma and the NaBr needed to adjust plasma density is
explained below.
TABLE 7.2
Characteristics of Major Lipoprotein Classes in Human Plasma
Density Lipoprotein Concentration Range
Lipoprotein Electrophoretic Range Size (mg/dl)
Class Class (g/ml) (nm) Males Females
Chylomicron Origin < 0.940 80–500 12 ± 13 2 ± 3
VLDL Pre-β 0.940–1.006 30–80 129 ± 122 59 ± 63
IDL Pre-β & β 1.006–1.019 25–30 40 ± 23 24 ± 14
LDL β 1.019–1.063 16–25 399 ± 81 365 ± 56
HDL α 1.063–1.210 9–13 300 ± 83 457 ± 115
VLDL, very low-density lipoprotein; IDL, intermediate-density lipoprotein; LDL,
low-density lipoprotein; HDL, high-density lipoprotein.
Source: Adapted from Schumaker, V.N., Puppione, D.L., in Methods in Enzymology
Vol. 128, Albers, J., Segrest, J., Eds., Academic Press, Philadelphia, 1986, p. 155.
Calculating the Plasma Density

First, determine the weight of a volume of water (pipette 600–700 µl of water
into a capillary tube and cap the tube. Place the capillary tube on the ultra-
sensitive scale and tare the weight so that the display reads 0.0000 g. Next,
withdraw 100 µl of water and return the capillary tube to the scale. Allow
the display to stabilize, and record the weight. Tare the scales again so that
the display reads 0.0000 g. Repeat the weight measurements at least five
times, making sure there is no more than 0.05% error in the weights. Use
the average of the five measurements as the weight of water.
Second, determine the weight of an equal volume of plasma. (Repeat the
same procedure outlined above.) The density of the plasma sample may now
be calculated as follows:
w pl ∗ d H 2 O
d pl =
w H2 O
where dpl = density of the plasma (g/ml); dH2O = density of water (specific
for temperature); wpl = determined weight of the plasma (g); wH2O = deter-
mined weight of water (g). For example: If the water temperature is 24°C,
the water density is 0.997327 g/ml.
If the average weight of water was determined to be 0.10027 g and the
average weight of plasma was determined to be 0.10413 g, then:
0.10413 g ∗ 0.997327 g/ ml
d pl = = 1.0357 g/ ml
0.10027 g
Calculating the NaBr Needed to Adjust Plasma Density

First, calculate the volume of NaBr needed to adjust the plasma to the desired
density as follows:
d desired− d pl
V NaBr = V pl ×
d NaBr − d desired
where: VNaBr = volume of NaBr; V pl = volume of plasma sample; ddesired

= desired density of the plasma; dNaBr = density of solid sodium bromide
= 3.21 g/ml.
For example, if the volume of our plasma sample is 90 ml and the current
plasma density is 1.0357 g/ml (from above), the volume of NaBr required
to achieve a desired density of 1.063 g/ml is calculated as:
1.063 − 1.0357
V NaBr = 90 ml ×
3.21 − 1.063
= 90 ml × 0.012715
= 1.1444
After determining the volume of NaBr, we must calculate the weight of

NaBr needed to adjust the plasma sample to the desired density as follows:
w NaBr = V NaBr × mol . wt . NaBr
= 1.444 × 3.21 g/ ml
= 3.67335 g
Lipoprotein Precipitation
Precipitation methods for separating plasma lipoproteins were first intro-
duced in the 1970s33 and have since been refined so that lipoprotein fractions
can be isolated in just a few hours of obtaining blood samples.34 Precipitation
methods for isolating high-density lipoproteins have shown good correlation
with the more time-consuming and labor-intensive benchmark technique,
preparative ultracentrifugation.34,35 In addition, these methods are compati-
ble with the enzymatic procedures for cholesterol determination.3 As such,
precipitation methods are commonly employed in both clinical and research
laboratories for quantifying high-density lipoproteins and subfractions of
this lipoprotein class.
Although a number of precipitating agents for apolipoprotein B (apo B)-
containing lipoproteins exist, there are two commonly used precipitation
procedures for obtaining high-density lipoproteins: the use of heparin/man-

ganese or magnesium/phosphotungstic acid solutions.36 Of note, the hep-
arin/manganese-chloride (MnCl 2 ) method is recommended by the
International Federation of Clinical Chemistry and was the methodology
used in the Lipid Research Clinic Prevalence Study.1
Both methods work by precipitating apo B-containing lipoproteins from the
whole plasma or serum. The precipitate forms a pellet in the bottom of the
reaction tube after addition of these solutions and subsequent centrifugation.
High-density lipoproteins remain in the supernatant or soluble fraction for
lipoprotein or cholesterol quantification. If the heparin/MnCl2 solution is used,
a second precipitation step involving dextran/sulfate may be employed to
precipitate the larger high-density lipoproteins (HDL2 fraction). The dextran/
sulfate precipitates the larger high-density lipoproteins by polyanion precip-
itation, leaving HDL3 lipoproteins in the soluble fraction.34
The high-density lipoprotein cholesterol values obtained from the precip-
itation procedures may be influenced by the blood samples (pH, the final
concentration of EDTA in collection tubes), characteristics of the lipoproteins
(protein–lipid ratios), and the presence of heparin and MnCl2 in the super-
natant.36 HDL-cholesterol concentrations are systematically about 10% lower
when precipitation is carried out using the MgCl2/phosphotungstic acid
solution as compared with heparin and MnCl2.36 Nonetheless, cholesterol
concentrations obtained by precipitation are in close agreement with those
obtained by preparative ultracentrifugation.34,35 The double-precipitation
procedures, using heparin/MnCl2 and dextran sulfate, are outlined below.
These procedures have been described previously by Gidez et al.34 and
Warnick and Albers,37 and the outside influences on the methodological
results have been discussed in detail.36
Materials
Refrigerated centrifuge, pipettes with volumes from 10 to 100 µl and from
100 to 1000 µl, weight scale sensitive to 0.0000 g, wax paper or disposable
weighing trays (for weighing solutes), magnetic stir plate and stir bars, glass
containers for mixing and for storing the prepared solutions, ice trays for
sample tubes, polypropylene sample tubes (12 × 75 mm), distilled water,
heparin (from porcine intestinal mucosa — 170 USP units/mg), MnCl2 ⋅
4H2O, dextran sulfate (Mr 15,000) are needed.
General Procedures
Solutions
A. Heparin–MnCl2 Solution: Add 20.0 g MnCl2 ⋅ 4H2O to distilled water

and dissolve completely using a magnetic plate and stir bar. Dilute
the solution to 100 ml with water. Next, dissolve 81.6 mg of heparin
in 5.0 ml of 1.01 M MnCl2.
B. Dextran/Sulfate Reagent: Prepare 500 ml of 0.15 M NaCl solution

by adding 4.383 g NaCl to distilled water and diluting to 500 ml.
Next, dissolve 0.143 g (143 mg) of dextran sulfate in 10 ml of 0.15
M NaCl solution.
Lipoprotein Separation
Plasma or serum samples are obtained as described previously. If plasma
samples are obtained from EDTA collection tubes, make sure that the tubes
were filled to capacity during collection so that the final concentration of
EDTA is proper and consistent throughout all samples.36 If plasma or serum
samples appear turbid, as occurs with elevated triglyceride concentrations,
the triglyceride-rich lipoproteins must be removed by ultracentrifugation at
d 1.006 g/ml before undergoing precipitation. Otherwise, incomplete pre-
cipitation will result and HDL-cholesterol values may be greatly overesti-
mated.36
Place all samples and solutions on ice and maintain them on ice during
the separation procedure. Introduce a volume of plasma or serum into the
appropriately labeled reaction tubes (usually 3.0 ml). Next, add one-tenth
volume (0.3 ml or 300 µl) of the heparin-MnCl2 solution. Mix thoroughly
and let the tubes stand at room temperature for 20 min. Centrifuge the tubes
at 1500 × g in a clinical centrifuge for 1 h at 4°C.
After centrifugation, withdraw the supernatant immediately. The super-
natant will be used to determine total HDL-cholesterol and, if so desired, to
precipitate HDL2. Therefore, dispense the supernatant into a tube marked
for HDL-cholesterol analysis. (Use 2.0 ml of the supernatant for HDL2 pre-
cipitation and retain the remainder of the supernatant for the subsequent
HDL-cholesterol determination.)
The HDL2 subfraction can be precipitated by adding one volume (usually
2.0 ml) of the supernatant obtained above to an appropriately marked reac-
tion tube. Add one-tenth volume (usually 0.2 ml or 200 µl) of the dextran
sulfate solution. Mix thoroughly and allow the tubes to stand at room tem-
perature for 20 min. Next, centrifuge as before for 30 min at 4°C (or you may
centrifuge in a refrigerated super-speed centrifuge at 10,000 rpm at 4°C for
10 min). Remove the supernatant immediately after centrifugation and trans-
fer into a storage tube marked for HDL3-cholesterol analysis.
If the cholesterol analysis is to be performed within a few days, samples
can be refrigerated at 4°C. If the analysis is to be performed within a few
months, samples should be frozen at –20 to –70°C. Cholesterol concentrations
may be determined using the enzymatic procedures described previously.
Gel Electrophoresis
Differences in size and electrical charge among lipoprotein classes and sub-
classes allow them to be separated by electrophoresis.14,15,38 Separation of
lipoproteins by polyacrylamide gradient gel electrophoresis (PAGE) yields dis-

tinct regions in which very low-density, low-density, and high-density lipo-
proteins and their subclasses can be resolved. In the past, different
polyacrylamide gradient gels were used for low-density lipoprotein subclasses
and high-density lipoproteins. However, the introduction of composite gels
allows electrophoretic separation of low- and high-density lipoprotein sub-
classes in the same gel.39 PAGE has also shown great utility in identifying
different apolipoproteins from delipidated lipoprotein samples.3 A thorough
discussion of the methodology and common variations in electrophoresis pro-
cedures for separating lipoprotein classes have been published elsewhere.14
Materials
Fume hood for mixing solutions, cold room or refrigerator (capable of tem-
peratures < 10°C), gel chamber and constant voltage power source, pipettes
(0.5–10 µl, 10–100 µl, 100–1000 µl), gel staining trays, digital camera, flatbed
scanner and gel-scanning software or densitometer, microcentrifuge tubes,
polyacrylamide gradient gels (these may be prepared or purchased commer-
cially), Sudan black B stain (lipid stain), colloidal Coomassie blue (protein
stain), bromophenol blue (0.2% w/v bromophenol blue, 40% w/v sucrose)
tracking dye, high molecular weight (HMW) standard and latex micro-
spheres of known diameter (25–30 nm) are needed.
General Procedures
Serum or plasma samples are applied to a polyacrylamide gradient gel and
electrophoresed at a constant voltage. Lipoproteins in the sample migrate
through the gel according to electrical charge until reaching gel pores that
are smaller than the lipoprotein particle diameter. After electrophoresis, the
gel is stained with a lipid- or protein-specific stain. The stained lipoprotein
bands that result can be quantified by densitometry. When lipid stain is used,
the integrated optical density obtained for each lipoprotein band is directly
related to the cholesterol ester and triglyceride contained in the lipopro-
teins.14 The procedures described below are for commercially available com-
posite gels. These procedures may vary, depending on the gels,
electrophoresis equipment and laboratory preference; however, the general
electrophoretic approach to identifying specific lipoprotein classes and sub-
classes remain similar.
Solutions
A. TBE Running Buffer: Add 250 ml of stock Tris Borate EDTA (TBE)
solution to 2.25 L of deionized water and mix thoroughly. The stock
TBE solution (90 mM Tris-Base, 80 mM boric acid, 2.5 mM
Na2–EDTA; pH 8.3) is prepared by adding 109 g Tris, 49.5 g boric
acid, 9.3 g Na2EDTA into 1 L deionized water and stirring to mix.
Adjust the pH of the solution to 8.35 with HCl or K2CO3. The stock
TBE solution can be stored indefinitely at room temperature. The
TBE running buffer should be stored at 40°C.
B. 50% Ethylene Glycol Monoethyl Ether (Cellosolve) Solution: Mix
Cellosolve with deionized water in a 1:1 ratio (500 ml of Cellosolve
and 500 ml of deionized water) and invert to mix. This stock solution
will be used to prepare the Sudan black B post-stain and will serve
as the de-staining solution.
C. Sudan Black B Post-Stain: First, combine 8.0 g zinc acetate with
400 ml of deionized water and heat to ~ 100°C for 30 min with slow
stirring until the zinc acetate is dissolved. Next, add 200 ml of Cel-
losolve and maintain the preparation temperature for an additional
30 min. Add another 200 ml of Cellosolve while continuing to stir
the solution at 100°C. Slowly add 5.0 g of Sudan black B when the
total volume is reduced to ~ 700 ml and continue to stir and maintain
temperature until the final volume reaches ~ 450 ml. Filter the solu-
tion while it is hot using medium pore quantitative grade filter
paper. Repeat the filtering process after the solution has cooled to
room temperature. Maintain this as the Sudan black B stock solution.
Prepare the final post-stain solution by diluting 20 ml of the Sudan
black B stock solution with 80 ml of 50% Cellosolve solution. Mix
by gentle inversion and store at room temperature.
D. Coomassie Blue Post-Stain & De-Stain: Add 40 ml methanol and
10 ml glacial acetic acid to 50 ml deionized water and mix by slow
stirring. Next, add 7.5 mg Coomassie Blue and stir to dissolve. The
de-stain solution is prepared by adding 100 ml methanol and 15 ml
glacial acetic acid to 85 ml of deionized water and mixing by slow
stirring.
E. Fixing Solution (10% trichloroacetic acid): Add 10 g trichloroacetic
acid to 100 ml deionized water and mix by stirring until dissolved.
Pre-Electrophoresis Procedures
Prepare the electrophoresis chamber in the cold room or refrigerator and
add chilled TBE running buffer to the lower reservoir. Position the gels
appropriately, fill the top reservoir and clear the top of the gels of trapped
air bubbles with the TBE running buffer. The gels are then pre-electrophore-
sed for a period (10–60 min) at 100–120 V.
The test and control samples are prepared by mixing 15 µl of bromophenol
blue tracking dye and 10 µl of sample. Plasma or serum samples may be
used for PAGE. (Avoid heparinized plasma, as the heparin activates lipo-
protein lipase and modifies the lipid content in the lipoprotein fractions.
Thus, heparinized plasma interferes with lipoprotein migration and
will result in streaks and non-specific bands forming in the sample lanes.)
Reconstitute the HMW standard with 100 µl of TBE running buffer and then
combine 60 µl of bromophenol blue tracking dye with 40 µl of reconstituted
HMW standard. Prepare the latex beads by adding 10 µl of the latex beads
to 90 µl of bromophenol blue tracking dye.
Lipoprotein Isolation
Again, clear any trapped air bubbles from the top of the gels with the TBE
running buffer and then carefully pipette the plasma samples into designated
gel lanes. Prepare a separate gel for the HMW standards and latex beads.
Introduce only the HMW standard to each lane at this time. Electrophorese
the gels progressively (15 V for 15 min, 70 V for 20 min) and then achieve
a constant voltage for ~ 3000 volt-hours (e.g., 120 V for 24 h). Introduce 10 µl
of latex bead mixture to the HMW standard lanes after approximately 3 h
of electrophoresis. This insures that the HMW proteins do not combine with
the beads prior to migrating down the gel lanes.
Once electrophoresis is completed, carefully remove the gels and place
them in a tray containing enough deionized water to cover the gel and
provide occasional gentle shaking for 1 h. Decant the water and add 50 ml
of 10% trichloroacetic acid. Again, provide occasional gentle shaking for 1 h
and then decant and rinse the gel with deionized water. Next, place the gel
into 50% Cellosolve solution for 1 h with gentle shaking. Discard the solution,
introduce fresh Cellosolve solution and repeat shaking for an additional
hour. Remove the gel and place it in a new tray with enough Sudan black
B post-stain to completely immerse the gel. Stain the gel overnight (~ 18 h).
The gel with the HMW standards undergoes the same procedures, except
that this gel is placed in a tray containing the Coomassie blue stain and
stained for 1.5–2 h.
After staining, decant the post-stain and rinse the gel several times with
deionized water. Next, add the Cellosolve solution and de-stain for 1 h with
gentle shaking. Decant and add new Cellosolve solution and repeat the de-
staining each hour for an additional 3 h. De-staining takes approximately
4 h in total; however, if bands are not distinguishable from the gel back-
ground or the gel background is not clear after 3–4 changes of de-staining
solution, then the gel may continue to undergo de-staining overnight. The
gel with the HMW standards is placed immediately in the de-stain solution
(prepared as described for solutions in the section Quantifying Apolipoprotein
Concentrations).
After de-staining, rinse all gels several times with deionized water until
the acid smell from the de-staining solution is no longer evident on the gels.
Place the gels in a new tray with TBE running buffer and provide gentle
shaking until the gels regain their original shape. The gels may be stored in
TBE running buffer indefinitely in sealed trays (see Figures 7.1 and 7.2).
VLDL
LDL
HDL
albumin
FIGURE 7.1
Whole plasma was introduced into pre-cast gradient gels (2–27% polyacrylamide) and under-
went electrophoresis at constant voltage. Gels were stained with Sudan black B, photographed
and scanned for further analysis.
HMW Standa rd
Thyroglobulin (17.0 nm)
Ferri tin (12.2 nm)
Catalase (10.4 nm)
LDH (8.1 nm)
Al bum in (7.1 nm)
FIGURE 7.2
A HMW standard was introduced to multiple lanes of a single pre-cast gradient gel (2–27%
polyacrylamide) and underwent electrophoresis at constant voltage. Gels were stained with
Coomassie blue, photographed and scanned for further analysis.
Lipoprotein Analysis
Quantitation of gel bands may be carried out with a densitometer or photo
system equipped with a background light source. If the latter technique is
employed, digital photos are either scanned electronically or by flatbed
80
70
60
Electrophoretic Mobility
[relative to albumin(Rf)]
50
40 LDH CATALASE FERRITIN THY ROGLOBULIN
30
20
HDL Particle Diameter = 35.6267 – (0.36729 x Rf%)
10
5 7 9 11 13 15 17
Hydrated Diameter (nm)
FIGURE 7.3
Relationship between hydrated diameter (nm) and electrophoretic mobility relative to albumin
(Rf) was determined using an HMW standard containing protein with known hydrated diam-
eters (bovine serum albumin 7.1 nm, lactate dehydrogenase 8.1 nm, catalase 10.4 nm, ferritin
12.2 nm, and thyroglobulin 17.0 nm) (#17-0445-01, Amersham Pharmacia Biotech).
scanner and imported into gel-scanning software. The resulting scans are
typically analyzed by determining the area under the curve or the peaks for
each of the bands. Particle size determination is carried out by plotting the
relative migration of the HMW standards and latex beads against the known
hydrated diameters for each of the proteins in the standard (see Figure 7.3).
Since albumin in the plasma or serum will have the smallest particle diam-
eter, the migration of albumin is typically designated as 100% migration and
all lipoproteins will migrate as a percentage of the albumin. The resulting
regression equation is then used to estimate lipoprotein particle sizes from
the known relative migration for each of the lipoprotein band peaks
(see Figure 7.4). Additional information for quantifying lipoprotein bands is
available.40
Analyses of Intravascular Enzyme and Transfer Protein

Activities
Lipoprotein Lipase and Hepatic Lipase Activities
Both hepatic lipase and endothelial-bound lipase hydrolyze triglyceride from
triglyceride-rich lipoproteins. The hydrolysis of triglyceride is important
physiologically for the uptake and subsequent metabolism of free fatty acids.
The remnant material generated from this hydrolysis is integral for the
intravascular formation of HDL precursors and the transformation of HDL3
to HDL2.41,42 Post-heparin plasma contains endothelial-bound lipase derived
250
LDL 1
VLDL
LDL 2 albumin
Intensity (pixels)
150 HDL 1 HDL 2
Background Threshold
50
0 7 31 37 74 82 100
Electrophoretic Mobility [expressed relative to albumin (Rf )]
FIGURE 7.4
Representative analysis of a single lane in a composite gel (2–27% polyacrylamide). Lipoprotein
particle diameter peaks were quantified on the basis of their electrophoretic mobility (Rf) relative
to albumin. HDL particle profile scores were calculated using the Rf and the intensity (as a
multiple of the threshold intensity [represented by the trace]) of each HDL peak.
from adipose tissue, muscle tissue, the liver (hepatic triglyceride lipase), and
phospholipid lipase.43
The plasma protein concentrations of lipoprotein lipase are determined
using immunoassays that are similar in approach to those described previ-
ously for apolipoproteins.27,44 Plasma lipase activities are generally quanti-
fied by incorporating a radioactive or fluorescence label in a triglyceride
substrate and measuring the label remaining after exposure to the lipase in
a test sample.43,45–49 Today, versions of the fluorescence techniques for quan-
tifying lipase protein concentrations and lipase activities may be carried out
using commercially available kits. Here we outline procedures for estimating
plasma lipase activity using radio-labeled substrate and post-heparin plasma
samples.43,49
Materials
Weight scale sensitive to 0.0000 g, magnetic stir plate and magnetic stir bars,
pH meter, sonicator, cell disruptor, water bath with metabolic shaker, refrig-
erated super-speed centrifuge, scintillation counter, fume hood (for storage
and equipped with nitrogen gas and multiple sample lines), adjustable
pipettes (10–100 µl, 100–1000 µl, and 1–5 ml), 2 ml cryovials with rubber
o-ring seal (sample and label storage), 15 ml polypropylene reaction tubes,
bullet tubes with snap cap, tube racks (capable of holding 15 ml conical
reaction tubes), 20 ml scintillation vials with foil-lined caps, scintillation
cocktail, absorbent paper, radioactive detergent, various sizes of cylindrical
beakers from 25 ml to 2 L capacities, stoppered glass containers (250 ml to

1 L in size), Tris(hydroxymethyl aminomethane), NaCl, heparin (grade 1A
from porcine intestinal mucosa), K2CO3 (potassium carbonate, anhydrous
ACS reagent), H3BO3 (boric acid), HCl (hydrochloric acid) and KOH (potas-
sium hydroxide) for pH adjustment of solutions, MeOH (methanol, research
grade), CHCl3 (chloroform, research grade), pH calibrators (pH 7.0 and pH
10.0), C7H14 (heptane, research grade), Triton X-100, triolein, phosphatidyl-
choline (L-α lecithin from egg yolk), FA-free albumin, [3H]triolein (glycerol
tri[9,10(n)-3H]oleate in toluene solution) (1 ml = 5 µCi), toluene, plasma or
serum in at least 1-ml aliquots for the apolipoprotein CII source are needed.
General Procedures
In the presence of 3H-labeled lipid substrate and supplied apolipoprotein
CII (as a cofactor), the rate of hydrolysis of the labeled substrate is measured
in a timed assay at physiologic temperature.43,49 The reaction is immediately
stopped by denaturing the plasma lipase proteins and the labeled free fatty
acids are partitioned from the mono-, di-, and triglycerides.50 The radioac-
tivity of the labeled free fatty acids is then measured in a liquid scintillation
counter. Distinction of total triglyceride lipase activity is determined in low
sodium buffer, while only hepatic triglyceride lipase is active in a high
sodium buffer.43 Thus, the activity of endothelial-bound lipase can be calcu-
lated as the difference between the determined total lipase activity and that
of hepatic triglyceride lipase.
Solutions
A. Buffer 1; used for post-heparin lipase activity (PHLA): Add 2.35 g

Tris, 1.11 g NaCl, and 0.015 g (15 mg) heparin to 75 ml of distilled
water while stirring. Adjust the pH to 8.6 with concentrated HCl
and bring to a final volume of 100 ml (0.194 M Tris–HCl buffer,
pH 8.6, containing 0.19 M NaCl and heparin).
B. Buffer 2; used for hepatic triglyceride lipase activity (HTGLA): Add
2.35 g Tris, 13.5 g NaCl, and 0.015 g (15 mg) heparin to 75 ml of
distilled water while stirring. Adjust the pH to 8.6 with concentrated
HCl and bring to a final volume of 100 ml (0.194 M Tris–HCl buffer,
pH 8.6, containing 2.31 M NaCl and heparin).
C. Buffer 3; used for substrate and diluent preparation: Add 2.35 g Tris
and 0.88 g NaCl to 75 ml of distilled water while stirring. Adjust the
pH to 8.6 with concentrated HCl and bring to a final volume of 100
ml (0.194 M Tris–HCl buffer, pH 8.6, containing 0.15 M NaCl).
D. Solution 4; Triton X-100 for substrate: Add 1 ml of Triton X-100 to 100
ml of distilled water and place in a sonicator until dissolved com-
pletely. Avoid shaking or allowing this solution to foam. [1% (v/v)
aqueous solution of Triton X-100].
E. Solution 5; Lipid solvent: Under a fume hood, combine 282 ml

MeOH (methanol), 250 ml CHCl3 (chloroform) and 200 ml C7H14
(heptane). Mix by gentle swirling.
F. Solution 6; FFA extraction: Add 13.8 g K2CO3 to 1 L of distilled water
while stirring. In a separate container, add 3.1 g H3BO3 to 500 ml of
distilled water while stirring. Next, add 200 ml of the 0.1 M H3BO3
solution to 800 ml of the 0.1 M K2CO3 solution while stirring (0.1 M
K2CO3–H3BO3 buffer, pH 10.5).
All solutions should be stored in glass containers. Solutions 1 and 2 should

be stored at 4°C. Other solutions can be stored at room temperature.
Preparation
Post-heparin blood samples should be drawn into Na+–heparin tubes and
placed on ice, centrifuged cold, and the plasma is either analyzed immedi-
ately or placed at –70°C until analysis.
Obtain approximately 20 ml of plasma from a single donor for the apoli-
poprotein CII source. Allocate the plasma into 1.1-ml aliquots and store at
–70°C.
Aliquot 15 µl of the labeled triolein into cryovials for storage at –20°C
immediately after receiving the 3H-labeled triolein. Only one 15-µl aliquot
will be used for each substrate preparation.
Preliminary Procedures
Heat a water bath to 56°C. A separate water bath with metabolic shaker
should be heated to 37°C. Reconstitute the 15 µl of the labeled triolein with
300 µl of toluene and vortex vigorously for 5 min. The vortexing is repeated
two more times at 30-min intervals.
Remove the test plasma samples from cold storage and place on ice for
slow thawing. Remove one of the plasma samples to be used as the apo CII
source from cold storage and allow it to thaw on ice. Label 15 ml conical
reaction tubes in duplicate for total (PHLA) and hepatic lipase activity
(HTGLA) and the test sample identification. (Four reaction tubes, two for
PHLA and two for HTGLA will be labeled for each of the test samples.)
Label blanks for total (BT) and hepatic activities (BH) in triplicate. Also label
microcentrifuge tubes for each subject and/or time point.
Prepare the plasma sample diluent by adding 5 ml of buffer #3 (0.194 M
Tris–HCl buffer, pH 8.6, containing 0.15 M NaCl) into a 15-ml container.
While stirring, add 0.3 g of fatty acid-free albumin. After the solution is
homogenous, place the diluent on ice.
Aliquot 10 ml of scintillation fluid into 20-ml scintillation vials. Prepare a
scintillation vial for each of the reaction tubes prepared for test samples and
blanks.
The substrate is prepared by separately preparing aqueous and lipid por-

tions. Prepare the aqueous portion as follows: Add 10 ml of buffer #3 (0.194 M
Tris–HCl buffer, pH 8.6, containing 0.15 M NaCl) into a 25-ml glass container.
While stirring, add 0.6 g of fatty acid-free albumin. After solution is homo-
geneous, place on ice. Next, place the plasma apo CII source in the water
bath at 56°C for 10 min. Withdraw 1.0 ml from the aqueous solution and
introduce 1.0 ml of the heat-deactivated plasma into the aqueous solution
while stirring. Replace the aqueous solution on ice.
Prepare the lipid portion of the substrate solution by pipetting 206 µl of
unlabeled triolein, 8 µl of phosphatidylcholine (lecithin) and 125–130 µl of
the 3H-labeled triolein into a 20-ml scintillation vial. Evaporate the lipid
solvents under N2 gas. (The solvents are evaporated when the contents of
the scintillation vial are odorless.) Add 600 µl of the 1% (v/v) solution of
Triton X-100 to the scintillation vial.
The final substrate solution is prepared by adding the aqueous portion to
the lipid portion in the scintillation vial. Cap the solution and vortex vigor-
ously. Emulsify the contents of the substrate solution by placing the probe
from the cell disruptor into the substrate solution for 1 min. (The setting on
the cell disruptor should be set at a moderate energy level.) Remove the
substrate for 30 s and then repeat the emulsification for an additional minute.
Place the substrate emulsion on ice until proceeding with the assay. The
substrate is stable for approximately 6 h after emulsification.
Final Preparations
Pipette 100 µl of the diluent and 50 µl of plasma sample into the appropriately
labeled microcentrifuge tubes. Cap the tubes, vortex and place on ice.
Into the appropriately labeled 15-ml reaction tubes, pipette 80 µl of buffer
#1 (0.194 M Tris–HCl buffer, pH 8.6, containing 0.19 M NaCl and heparin)
into tubes marked for PHLA. Pipette 80 µl of buffer #2 (0.194 M Tris–HCl
buffer, pH 8.6, containing 2.31 M NaCl and heparin) into tubes marked for
HTGLA. Next, add 20 µl of the 3:1 diluted sample from the bullet tubes into
appropriately labeled reaction tubes. Add 20 µl of distilled water to the blank
tubes (BT and BH). Place all tubes on ice. Finally, pipette 20 µl of the substrate
into 20-ml scintillation vials (in triplicate) for determining the total activity
of the 3H-labeled substrate.
Assay Procedures
Turn on the metabolic shaker in the water bath heated to 37°C. Pipette 100
µl of substrate into each reaction tube at 30-s intervals. After introducing the
substrate, gently vortex the tube and place it in the water bath for 45 min.
Following the 45-min incubation, remove the reaction tubes in keeping with
the 30-s intervals. As the reaction tubes are removed, introduce 3.25 ml of
solution # 5 (methanol: chloroform: heptane) immediately followed by 1.05
ml of solution #6 (potassium carbonate: borate). Vortex the tubes and place

them on ice until all reaction tubes have been removed from the water bath.
Centrifuge the reaction tubes at 2500 × g and 4°C for 15 min. A liquid
(alkaline methanol–water): liquid (chloroform–heptane organic phase) parti-
tion should be visible after centrifugation. Withdraw 1 ml of the top phase
(alkaline methanol–water phase) from each sample and introduce into the
appropriately labeled 20-ml scintillation vials. Cap the vials and vortex vigor-
ously. The scintillation vials should be placed in a dark place overnight (6 h).
Count the activity of each sample (cpm) in the scintillation counter for 10 min.
Calculations
A. Average the total counts of the three substrate vials and the blanks
(BT and BH). Do the same for each of the duplicate total lipase and
hepatic lipase sample vials.
B. Adjust the sample cpm by subtracting the background cpm from
each of the sample averages (sample cpm - blank cpm). BT counts
are subtracted from PHLA sample counts and BH counts are sub-
tracted from HTGLA sample counts.
C. Calculate the lipase activity: (Adjusted sample cpm/Substrate cpm) ×
(1/incubation time) × (1/specific activity of the substrate) × volume of
the organic phase × (1/partition coefficient) × (1/volume of the plasma
analyzed), where: (1/incubation time) is = 1/0.75 h; (1/specific activity)
= (3.0817 µmol free fatty acid/substrate cpm × 5); volume of the organic
phase = 2.45 ml; (1/partition coefficient) = 1/0.76 h;(1/plasma volume)
= 1/0.02. Each of the terms above may be combined (3.0817 µmol free
fatty acid × 2.45 ml)/(5 × 0.75 h × 0.76 × 0.02 ml). Thus, the ratio of the
adjusted sample counts to the substrate counts is multiplied by the
constant, 132.4275.
D. Multiply both PHLA and HTGLA results by 3.
E. Subtract HTGLA from PHLA to estimate the endothelial-bound
lipase activity LPLA.
Example Calculation
In this example, substrate average = 50,000 cpm; PHLA sample average =
3000 cpm; HTGLA sample average = 2000 cpm; BT average = 150 cpm; BH
average = 100 cpm.
PHLA = (3000 – 150)/50,000 × 132.4275 = 7.5484

7.5484 × 3 = 22.65 µmol FFA/ml/h
HTGLA = (2000–100)/50,000 × 132.4275 = 5.0322
5.0322 × 3 = 15.10 µmol FFA/ml/h
LPLA = 22.65 – 15.10 = 7.55 µmol FFA/ml/h
Estimating the Rate of Cholesterol Ester Exchange

Cholesterol ester transfer protein (CETP) is one of several lipid transfer
proteins that have been isolated in human plasma.51–53 CETP is of particular
interest to those studying reverse cholesterol transport because it facilitates
the exchange of triglycerides and cholesterol esters between lipoproteins.53,54
Both CETP activity — the CETP-mediated transfer of lipids among lipopro-
teins — and CETP protein concentrations have been measured previously.
CETP concentrations, which correlate well with CETP activity, are commonly
measured by radioimmunoassay and ELISA techniques.55–57 CETP-mediated
transfer of lipids is estimated by quantifying the lipid exchange between
donor (HDL) and acceptor (VLDL and LDL) lipoprotein fractions.55 Previous
methodology for measuring CETP activity required the isolation of donor
and acceptor lipoprotein fractions by sequential ultracentrifugation, radio-
labeled cholesterol to be esterified and incorporated into the donor lipopro-
tein fraction, the determination of an optimal donor:acceptor mass ratio and
an optimal incubation time for the linear rate of cholesterol ester exchange
between donor and acceptor lipoproteins.54,55,58 CETP activity was then mea-
sured as the transfer of 3H-HDL3–cholesterol ester to the d < 1.063 g/ml
lipoproteins [VLDL + LDL (V + LDL)] during incubation at 37°C. The donor
(3H-HDL3) and acceptor lipoproteins (V + LDL) were separated by lipopro-
tein precipitation and the radioactivity in the HDL3-containing supernatant
was determined. A recently developed method for quantifying CETP activity
is available as a commercial kit.59 Lipid transfer between donor and acceptor
is quantified by fluorescence, the donor and acceptor sources are not influ-
enced by variations in endogenous lipoproteins and the specific activity of
the donor is not influenced by HDL concentrations. The procedures,
described below, require less preparatory time and exhibit improved reli-
ability and specificity over previous methodology.
Materials
Fluorescence spectrophotometer, multi-channel pipette (10–100 µl), multi-
channel sample loading tray, 96-well black microplate, 12 × 75 mm culture
tubes, book tape, microcentrifuge tubes, 15 ml reaction tubes, 50 ml sample
tubes, parafilm, CETP Activity Kit (Roar Biomedical Inc., No. RB-CETP), iso-
propanol, Tris-Base (hydroxymethyl aminomethane), Na2EDTA (ethylenedi-
aminetetraacetic acid, disodium salt dihydrate), NaCl, pooled plasma samples
(from at least three different individuals to use as control) are needed.
General Procedures
The optimal incubation time and donor acceptor ratio for the test kit is
determined and a standard curve is developed for calculating CETP activity
from the measured fluorescence. CETP activity is determined by incubating
the test sample with donor and acceptor particles resulting in the transfer of
fluorescent-neutral lipid from donor to acceptor particles. The CETP activity
is then measured as the increase in fluorescence intensity as the fluorescent-

neutral lipid is exchanged from the donor to the acceptor fraction.59
Preliminary Procedures (Determining Assay Linearity)

Each of the three pooled “control” plasma samples are combined with buffer
(included in the CETP Activity Kit) in microcentrifuge tubes by pipetting 90
µl of buffer and 10 µl of plasma, capping the tube and mixing by gentle
vortex.
Assign microplate wells in triplicate for progressively greater volumes of
each diluted plasma sample. For example, assign wells A1 to A3 as “blank
wells” for control plasma 1. Assign wells A5 to A7 for 2 µl, A9 to A11 for 5
µl, B1 to B3 for 8 µl, B5 to B7 for 10 µl of diluted plasma. Assign microplate
wells D and E for control plasma 2 and G and H for control plasma 3 in the
same manner. (Skip a row of wells in the microplate between the assigned
rows for each of the diluted control plasmas.)
Prepare a mixture of the buffer, donor and acceptor by combining 204 µl
of donor, 204 µl of acceptor and 4.692 ml of buffer in a 15 ml reaction tube.
Mix the solution by gentle vortex.
Using a multi-channel pipette, introduce into the appropriately assigned
microplate wells the following contents so that the total assay volume for
each well is 200 µl. Diluted control plasma sample 1:
Microplate Well Buffer Diluted Control Buffer + Donor + Acceptor

Assignment (µl) Plasma (µl) Mixture (µl)
A1 to A3 10 0 190
A5 to A7 8.0 2.0 190
A9 to A11 5.0 5.0 190
B1 to B3 2.0 8.0 190
B5 to B7 0 10 190
Repeat the same procedure for each of the diluted control plasma samples.
After the microplate wells have been prepared, cover the microplate with
book tape and incubate for 1–3 h at 37°C in a water bath. Fluorescence
intensities will be read each hour of the incubation in order to determine
optimal assay linearity for time and concentration. Fluorescence intensity is
read at an excitation of 465 nm and an emission of 535 nm. The book tape
is removed prior to reading the fluorescence and new book tape is used to
cover the plate prior to re-incubation.
Developing the Standard Curve (Determining Neutral Lipid from

Fluorescence Intensity)
Label 12 × 75-mm culture tubes from T0 to T5. Add 1 ml of isopropanol to
each tube. Next, add an additional 1 ml of isopropanol and 5 µl of donor
into the tube marked T5, cover with parafilm and mix by gentle inversion.
Remove 1 ml of solution from T5 and add to the tube marked T4, cover with
parafilm and mix by gentle inversion. Repeat the process by removing solu-
tion from T4 and adding to the tube marked T3, from T3 to T2 and T2 to T1.
Assign microplate wells in triplicate for each of the T tubes (T0 through T5),
skipping a well between each. For example, assign wells A1 to A3 for T0, A5
to A7 for T1, etc. Next, pipette 200 µl of solution from each tube into the
appropriately assigned microplate wells. Cover the microplate with book tape
and place on ice until reading. The book tape is removed prior to reading and
the fluorescence intensity is read at an excitation of 465 nm and an emission
of 535 nm. The book tape is removed prior to reading the fluorescence.
A standard curve is developed by plotting the fluorescence intensity
against the fluorescent donor substrate in ρmoles (which is dispersed pro-
portionally in isopropanol).
Assay Procedure
Add 90 µl buffer and 10 µl test plasma into appropriately labeled microcen-
trifuge tubes, cap the tubes and mix by gentle vortex. Prepare the
buffer–donor–acceptor solution by adding 4 µl of donor, 4 µl of acceptor and
92 µl of buffer for each of the test samples, controls and blanks into a 15-ml
reaction tube. Assign microplate wells in triplicate for each of the test sam-
ples, controls and blanks as before. All test samples from an individual
should be determined on the same microplate due to variations in the fluo-
rescence intensity that may occur between different microplates.
Into each of the appropriately labeled microplate wells, pipette 95 µl of
buffer and 5 µl of buffer (for blanks) or diluted plasma sample (for tests).
Next, introduce 100 µl of the buffer–donor–acceptor solution into each well
for a final well volume of 200 µl. Cover the plate with book tape, incubate
in a water bath at 37°C for 3 h and read the fluorescence intensity as before
after 3 h of incubation.
Calculations
A. Average the test and blank fluorescence intensity units (FIU) from
the triplicate measures of each.
B. Subtract the blank FIU from the test sample FIU.
C. Use the standard curve (described previously) to determine the
remaining neutral lipid in the donor fraction of the test sample.
D. Calculate the CETP activity in ρmol cholesterol ester transferred/µl
plasma/hr as follows:
CETP Activity (µmol/µl/h) =

[(m × (sample FIU – blank FIU)) + b)/0.5 µl plasma/3 h)
where: m = slope; b = intercept; 0.5 µl = diluted sample volume; 3 h =

incubation time.
Summary
It is our hope that the assays outlined in this chapter are helpful for estab-
lishing these methods and procedures in your laboratory. Although these
methods have been employed in our laboratories as described, we recognize
that variations in our procedures may be incorporated in other laboratories
with equal success. We encourage readers to review other resources on these
and other analytical techniques for researching lipid and lipoprotein meta-
bolism.3,4
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8
Metabolic Syndrome
Vic Ben-Ezra
CONTENTS
Introduction .........................................................................................................147
Dyslipidemia: The Good, the Bad, and the Ugly..........................................149
Waist Circumference: Its Association with Elevated Triglycerides,
Hyperapolipoprotein B, Small Dense LDL, and
Hyperinsulinemia ......................................................................................150
LPL Activity: A Key to Lipid Dysregulation and Insulin Resistance? ......152
Leptin, Lipotoxicity, and PPARs.......................................................................156
Adiponectin: A Player in Lipid Dysregulation? ............................................158
Acylation-Stimulating Pathway (ASP): New Player in the Insulin
Resistance–Dyslipidemic Relationship?.................................................159
Summary and Conclusions ...............................................................................162
References ............................................................................................. 163
Introduction
In the year 2000 poor diet and physical inactivity was the second (16.6%)
estimated actual cause of death in the United States totaling 400,000, second
only to tobacco at 435,000 deaths (18.1%).1 This represents an increase of one-
third from the 1990 data where poor diet and inactivity accounted for an
estimated 300,000 deaths, and the largest increase among all actual causes
of death. The authors suggest that the 2000 data may underestimate the
impact of diet and inactivity and therefore overtake deaths related to smok-
ing.1 These alarming figures when coupled with the NHANES III data which
indicate that approximately 24% of the United States population (47 million)
has the metabolic syndrome (MS),2 present extraordinary challenges to the
U.S. health care system now and in the years to come.
147
TABLE 8.1
National Cholesterol Education Program
(NCEP): The Metabolic Syndrome
Risk Factor Defining Level
Waist circumference
Men > 102 cm (approx. 40 inches)
Women > 88 cm (approx. 35 inches)
Triglycerides > 1.7 mmol/L (150 mg/dl)
HDL-C
Men < 1.03 mmol/L (40 mg/dl)
Women < 1.29 mmol/L (50 mg/dl)
Blood pressure > 130/80 mmHg
Fasting glucose > 6.1 mmol/L (110 mg/dl)
The metabolic syndrome is diagnosed when three or
more of these factors are present.
Dyslipidemia, often referred to as the atherogenic lipid phenotype, the

atherogenic lipid profile,3 or atherogenic dyslipidemia, often characterized
by a combination of hypertriglyceridemia, low high-density lipoprotein
(HDL) levels, a preponderance of small dense low-density lipoprotein (LDL)
particles, and hyperapolipoproteinemia B, is closely linked to the MS and
increased risk for coronary heart disease. In fact the relationship between
the MS and cardiovascular disease, predominantly through lipid dysregu-
lation, is remarkable.
NHANES III, conducted by the National Center for Health Statistics, Cen-
ters for Disease Control and Prevention, examined the relationship between
NCEP-defined MS (see Table 8.1), diabetes, and the prevalence of coronary
heart disease. Among participants age 50 years and older, with and without
diabetes, the prevalence of the MS was 43.5%.4 Although diabetes is surely
associated with increased frequency of coronary heart disease (CHD),
NHANES III data indicate that having diabetes and the MS increases the
CHD risk by approximately 2.5-fold over those with diabetes alone (Figure
8.1). It should also be noted that the incidence of MS in those without diabetes
is almost double that of diabetics with MS and the prevalence of the MS was
approximately 60% greater than the prevalence of Type 2 diabetes in the
same population.4 These data can be interpreted to mean that there is more
to the development of CHD than lipid dysregulation associated with the
metabolic consequences of diabetes. Moreover, many point to insulin resis-
tance (IR) as the main factor for abnormal lipidemia (elevated triglycerides,
decreased HDL, increased apo B and small dense LDL), hyperglycemia,
reduced cholesterol absorption, alterations in hepatic and lipoprotein lipase
activity, and decreased plasma levels of adiponectin and increased levels of
acylation-stimulating protein (ASP). The aforementioned either coexist with
or contribute to dyslipidemia or elevated risk for CHD. This review will
focus on those aspects of the MS, mainly IR and increased waist circumfer-
ence (WC), that alter fatty acid mobilization and produce triglyceride
Metabolic Syndrome 149
25%
19.2%
20%
CHD Prevalence
13.9%
15%
8.7%
10%
7.5%
5%
0%
No MS/No DM MS/No DM DM/No MS DM/MS
54.2% 28.7% 2.3% 14.8%
FIGURE 8.1
The relationship between CHD and MS in patients with diabetes mellitis (NHANES III).
accumulation in many non-adipose tissue sites, and how leptin, adiponectin,

and ASP may play a role in this dysregulation.
Dyslipidemia: The Good, the Bad, and the Ugly

The identification of increased risk for cardiovascular disease (CVD) began
with recognizing cholesterol as a primary lipid contributor of disease devel-
opment. Many studies have since expanded the total cholesterol idea into
examining more carefully other lipid factors such as subfractions of both
HDL and LDL, HDL and LDL particle size, triglycerides, and apolipopro-
teins A and B. Examination of these and other factors such as insulin, and
inflammatory markers like C-reactive protein (CRP) or tumor necrosis factor
alpha (TNF-α) (please refer to review articles regarding details of material
not covered in this monograph5,6), came about as a result of continued CVD
development despite the reduction in total cholesterol. In fact, Sniderman et
al.7 point out that most cases of premature vascular disease found in the
Framingham Study8 had total cholesterol (TC) and LDL levels that were not
very different than those individuals who did not develop premature CVD.
Subsequently, both basic and prospective studies have found that CVD risk
is associated with increased small dense LDL, increased triglycerides,
increased apo B or apo B/apo A-1, low HDL cholesterol levels, high total
cholesterol/HDL, but not as clearly or simply high levels of TC or LDL
cholesterol.8–15 In addition, in studies using LDL-lowering treatments with
the outcome measures of coronary events or progression of disease, decreas-

ing small dense LDL showed the most benefit.16–18 Participants recruited for
the AFCAPS/TexCAPS, a primary prevention study, reported at baseline
with average TC and LDL-cholesterol (LDL-C) with some individuals also
having low HDL-cholesterol (HDL-C) levels. Subjects were assigned lova-
statin or a placebo for 1 year with the intent to compare the rate of first acute
major coronary events. Baseline LDL-C, HDL-C and apo B were significant
predictors of coronary events but apo B and the apo B/apo A-1 were the
best on-treatment predictors of coronary events, not LDL-C.19 Roeters van
Lennep et al.20 treated 848 patients with known coronary disease with statins
and successfully reduced total cholesterol by 30%. On-treatment apo B was
predictive of myocardial infarction and all-cause mortality and the apo B/
apo A-1 was the strongest predictor of future cardiovascular events. On-
treatment levels of TC, LDL-C and triglycerides were not associated with
increased risk of cardiovascular events in these men or women with known
coronary disease; however, HDL-C levels were a predictor in women. Please
note that high TC or LDL-C, and or low HDL-C are not being abandoned
as important risk factors to consider when examining risk for CVD. However,
it is important to note that what the literature may be providing is additional
“best practice” predictors that add, and in some cases, supplant the well-
accepted measures as risk factors.
Waist Circumference: Its Association with Elevated

Triglycerides, Hyperapolipoprotein B, Small Dense LDL,
and Hyperinsulinemia
An enlarged waist, typically identified using NCEP ATP III guidelines of >
102 cm for men and > 88 cm for women (see Table 8.2), is linked to developing
the MS and increased risk for coronary heart disease. The general hypothesis
is that central fat accumulation (abdominal adiposity) results in lipid dys-
regulation (hypertriglyceridemia, low HDL, and increased small dense LDL
levels and apo B). The evidence from a variety of population studies provides
the groundwork for using a simple measure, waist circumference (WC), in
conjunction with or in place of body mass index (BMI), to further or better
identify individuals at risk for the MS and lipid dysregulation.21–24 Lemieux
et al.22 suggest that using waist circumference (a correlate of increased insulin
and apo B levels) and triglyceride levels (a correlate of small dense LDL)
may be a more powerful tool in identifying men with an atherogenic meta-
bolic profile. Critical cut-points for WC (≥ 90 cm) and triglycerides (≥ 2.0
mmol/L) were used to predict CAD risk factors (hyperinsulinemia, hyper-
apolipoprotein B, and small dense LDL). They found that using these cut-
points, greater than 80% of men exhibited the atherogenic metabolic triad,
TABLE 8.2
Criteria for Metabolic Syndrome among U.S. Population ≥ 50 Years
No Diabetes Diabetes
No No
Metabolic Metabolic Metabolic Metabolic
Syndrome Syndrome Syndrome Syndrome Total
Percentage of population 54.2 28.7 2.3 14.8
Criterion
% Waist circumference 34.4 82.0 18.5 86.0 55.0
(M > 102 cm; F > 88 cm)
% Triglycerides ≥ 150 mg/dl 18.0 77.8 5.1 72.1 42.8
% HDL cholesterol (M < mg/dl; 16.5 70.7 2.6 69.7 39.5
F < 50 mg/dl)
Blood pressure ≥ 130/85 mmHg 45.3 86.2 43.0 82.7 62.5
(%)
Fasting glucose > 110 (%) 6.2 30.9 83.0 90.2 27.2
Source: From Alexander, C.M. et al., Diabetes, 52, 1210, 2003. With permission.
while only 50% of men with triglyceride levels < 2.0 mmol/L but with high
WC values (≥ 100 cm) had these risk factors. Validation of this model on a
sample of 287 men with and without CAD found that only men with both
elevated waist and triglycerides were at increased risk for CAD compared
with men with low waist and triglycerides. Lemieux et al.22 suggest that
“hypertriglyceridemic waist” be used as a clinical tool to identify men at
risk for CAD. Along this same line Kahn and Valdez23 applied cut-points for
elevated triglycerides (≥ 1.45 mmol/L) and enlarged WC (men ≥ 95 cm;
women ≥ 88 cm) in examining 8730 adults from NHANES III with the intent
to identify a state of lipid over accumulation and its metabolic consequences.
Persons with enlarged waist and elevated triglycerides had higher fasting
insulin, glucose, apo B, and uric acid, lower HDL cholesterol and greater
prevalence of diabetes than people without enlarged waist and elevated
triglycerides.
It should be noted that the NCEP cut-points for WC were determined
based upon their association with BMI. Since BMI provides a more broad
body view where factors like height or distribution of body fat, for example,
may dilute the quality of risk factor associations, it is therefore of particular
interest to discern if WC independent of BMI predicts CVD/disease risk. In
addition, the NCEP WC cut-offs may in some populations or ethnic groups
be too high. Okosun et al.26 found that at the same levels of BMI (overweight:
25–29.9; obese: ≥ 30 kg/m2) black and Hispanic men tended to have lower
WC values than white men, while women across ethnic groups were similar.
Zhu et al.,25 using NHANES III data, demonstrated that WC may be a
better discriminating variable for increased risk of cardiovascular disease
than BMI. However, in this study obesity-associated risk factors (HDL-C,
LDL-C, glucose, blood pressure) were effectively predicted at WC of 96 cm
and 85 cm for white men and women, respectively. The Canadian Heart
Health Surveys data, showing waist circumference cutoffs for Caucasian men
of ≥ 90 cm and women of ≥ 80 cm may be most successful for prediction of

cardiovascular disease risk factors.24
In a series of studies27–29 using NHANES III data, Zhu et al.29 found that
for white men, BMI in combination with WC better estimated the odds of
having CVD risk factors than either measure alone, and that WC alone
determined the likelihood of having CVD risks in white women. Okosun et
al.27 found that WC was strongly associated with metabolic dysregulation
(hypertension, Type 2 diabetes, dyslipidemia, hypertriglyceridemia, or
hyperinsulinemia) in White, Black, and Hispanic Americans independent of
BMI. Lastly, Janssen et al.28 determined that WC explains obesity-related
health risk not BMI. Therefore health risks are similar across categories of
BMI (normal-weight [18.5–24.9], overweight [25–29.9], class I obese
[30–34.9]) if they have similar WC.
LPL Activity: A Key to Lipid Dysregulation and Insulin

Resistance?
Lipoprotein lipase (LPL) has a number of roles in lipid and lipoprotein
metabolism that include hydrolysis of circulating triglycerides, binding to
LDL receptors and inducing receptor-mediated breakdown of very low-
density lipoprotein (VLDL), and it mediates the selective uptake of choles-
terol esters.29 LPL is the major enzyme responsible for hydrolyzing tri-
glyceride from chylomicrons and very low-density lipoprotein proteins and
thereby providing fatty acids for a variety of tissues. Specifically, and most
importantly, LPL can be found in adipose, heart, and skeletal muscle tis-
sue.30,31 For an excellent overview on LPL, see Preiss-Landl et al.32
LPL activity varies among tissues, adipose, heart and skeletal muscle,
relative to feeding and fasting. LPL increases and decreases dramatically
with feeding and fasting respectively, in adipose tissue,33–36 while heart and
skeletal LPL generally respond in the opposite direction. These changes in
LPL activity promote the uptake of free fatty acids (FFAs) and thereby,
storage of triglyceride in adipose tissue during feeding and diminishing
triglyceride uptake by other tissues (e.g., skeletal muscle, pancreas, etc.),
while the reverse condition (fasting) generally promotes sending FFAs/
triglycerides to the heart and skeletal muscle for energy metabolism. Lithell
et al.36 found a 46% increase in adipose LPL and 32% decrease in skeletal
muscle and heart LPL after eating in humans, while others have found
40–80% decreases in adipose, skeletal muscle, and heart tissue LPL after
caloric restriction.37,38
Fat loss or gain has also been associated with changes in LPL. Eckel et al.39
found a 70% decline in fasting skeletal and heart muscle LPL after a 13%
body weight loss in obese women after 3 months. It should be noted that
the obese women, prior to weight loss, had skeletal and heart muscle LPL
that was 13% lower than normal weight controls. The implication from this
study as well as those that have examined intramuscular triglyceride loss
resulting from weight loss,40,41 is that the predisposition for skeletal muscle
to excessively store triglyceride is decreased, and fat storage is diminished.
The effect of obesity/fat weight gain will be discussed later in association
with fatty acid and/or lipid dysregulation.
As previously mentioned, LPL activity rises and falls with diet. As such,
increases in insulin concurrent with plasma glucose elevation appear to up-
regulate skeletal muscle LPL.37 However, if euglycemia is maintained during
a hyperinsulinemic clamp, skeletal muscle LPL tends to decrease in healthy
subjects. Obesity and/or insulin resistance, however, tends to cause skeletal
LPL to increase in response to a euglycemic hyperinsulinemic clamp.39,42 This
rise should further exacerbate the insulin-resistant condition by increasing
skeletal muscle storage of triglyceride, the result of which is linked to IR at
the skeletal muscle. More recently, Goodarzi et al.43 have found direct evi-
dence that LPL is an IR gene using LPL haplotypes and direct quantitative
measures of IR in Mexican-Americans. This data is part of the UCLA/Cedar
Sinai Mexican American Coronary Artery Disease (MACAD) project that
enrolls families with identified CAD. Two generations are enrolled in the
study: (1) parental generation (the one with CAD and the spouse) where
CAD is apparent; and (2) adult offspring and the spouses of those offspring.
There were 74 families totaling 291 subjects that were both genotyped and
administered the euglycemic-hyperinsulinemic clamp. The results suggest a
common LPL haplotype that is protective against IR and a common haplo-
type that predisposes to IR in Mexican Americans. This group also found
these same LPL haplotypes to protect and predispose to clinical CAD.44
The overexpression of muscle LPL in mice results in hyperinsulinemia and
hyperglycemia. In this mouse model a mismatch occurs that appears to
accelerate plasma triglyceride deposition in muscle tissue.45 In humans it has
been shown that insulin suppression of skeletal muscle LPL activity is
reduced in obese women or those that have non-insulin-dependent diabetes
mellitus.46 This increased muscle LPL activity results in increased triglyceride
uptake and thereby increases the potential for over-accumulation of intra-
muscular triglyceride. This excess triglyceride accumulation, sometimes
referred to as lipotoxicity, has been closely linked to IR.47,48
Houmard et al.49 reported significant increases (+ 360%) in insulin sensi-
tivity after weight loss in morbidly obese men and women resulting from
gastric bypass surgery. The improved insulin action was concurrent with
significant decreases in intramuscular long-chain fatty acyl-CoAs leading the
authors to conclude that, at least in part, changes in intramuscular tri-
glyceride may be responsible for enhanced insulin action. He et al.50 exam-
ined the effects of weight loss through caloric restriction combined with
increased physical activity on muscle lipid content and droplet size in over-
weight and obese men and women. The intervention resulted in a decrease
of 10% and 17% in weight and fat mass, respectively, along with a 16%
increase in VO2max and 49% increase in insulin sensitivity. The overall lipid
content of the muscle did not change; however, they found that as a result
of the intervention program the lipid within the muscle had dispersed into
smaller and more numerous droplets. The decrease in droplet size was
significantly correlated (r = –0.46) with changes in aerobic fitness. They
concluded that the change in the lipid droplet size, combined with increases
in oxidative enzymes, in part explains the change in insulin sensitivity. These
findings may also lend some insight into the paradox of increased intramus-
cular triglyceride content found in endurance trained individuals who typ-
ically demonstrate excellent insulin sensitivity51 or partly explain why
intramuscular lipid is not reduced in obese subjects after diet and exercise
training.52
Non-esterified fatty acids are believed by many to hold the key to most,
if not all, IR and IR associated dyslipidemia (see Figure 8.2). The link here
involves dysregulated adipocyte lipid metabolism and its effects on skeletal
muscle, the liver, and the pancreatic β cells (see Figure 8.3). Data show that
β-cell dysfunction is associated with elevated plasma fatty acid levels, such
that increased (could lead to hyperinsulinemia) or decreased (possibly indi-
cating a failing β cell) insulin secretion results.53–56 However, if one looks at
first-degree relatives of people with Type 2 diabetes, they typically show a
poorer acute insulin response to a glucose challenge, a reduced insulin medi-
ated glucose uptake, and significantly elevated fasting plasma FFA levels
compared with those without a family history of the disease.57,58 These people
are at greater risk of developing diabetes.57–60 Lowering the plasma FFA
concentration with acipimox (i.e., drug that lowers circulating FFA) in first-
degree relatives improved insulin-mediated glucose uptake as well as their
Fat Cells Liver

FFA CE
TG VLDL CETP HDL

Apo B
IR X VLDL TG
Apo A-1
CE CETP TG CETP?
Kidney
Insulin
SD
LDL
LDL
hepatic lipase
FIGURE 8.2
Mechanisms relating to insulin resistance and dyslipidemia.
Adipose
↑ Lipolysis
Muscle ↑ FFA mobilization

Liver
↑ FFA oxidation Pancreas ↑ FFA oxidation

↑ Triglyceride storage ↑ Triglyceride storage
↑ Triglyceride storage
↑ Gluconeogenesis
↑
Glucose utilization ↑
Insulin secretion
Hyperglycemia
FIGURE 8.3
Increased FFA effects on other tissues.
acute insulin response61 to a euglycemic-hyperinsulinemic clamp. A corre-

lation (r = –0.64, p < 0.006) was found between the fall in plasma FFA and
the increase in acute insulin response. These data provide further insight
into the possible role that lipid dysregulation (lipotoxicity) plays in the
insulin-resistance syndrome (IRS). We will also use the term insulin-resistant
(IR) dyslipidemia to refer to IRS. If one examines Figure 8.3, the fat cell’s
oversecretion of fatty acids or underability to sequester fatty acids (discussed
in the next section) appears to stimulate the liver to increase production of
VLDL, triglycerides and apo B, which can lead to increased LDL, and in
particular small dense LDL, while increasing the degradation of HDL and
apo A-1. This mechanism is supported by data that shows increased CEPT
(Guerin) and hepatic lipase activity in Type 2 diabetics.62 The increased CEPT
could promote the transfer of triglyceride to VLDL and then to LDL or from
HDL directly to small dense LDL63 which when acted upon at the liver by
hepatic lipase produces small dense LDL particles.64–66 In addition HDL, in
particular HDL-2, appears to be more readily converted into HDL-3 in part
by the transfer of triglyceride from VLDL to HDL (CEPT action) and its
subsequent arrival at the liver where hepatic lipase hyrolyzes the triglyceride
producing the more dense HDL (HDL-3).63, 66, 67
Leptin, Lipotoxicity, and PPARs

Leptin, a hormone primarily produced by adipocytes, appears to have sig-
nificant regulatory control over food consumption, energy expenditure, fuel
storage and usage, and has been implicated as the (one) link between obesity
and insulin resistance.68–70 Leptin acts by signaling the hypothalamic leptin
receptors, which monitor adipose tissue mass, and produces signals that
control both energy intake and energy expenditure.68,71 Hypothalamic neu-
rons may then be stimulated which act to stimulate sympathetic alpha-
adrenergic neurons and the release of norepinephrine.72,73 Plasma leptin lev-
els rise with the accumulation of fat mass, and decline in response to body
fat loss.71,74 Obesity, however, appears to produce/result in hyperleptinemia
and leptin resistance as evidenced by the lack of weight loss in obese indi-
viduals after exogenous leptin administration.71,72,75 The mechanisms to
explain this are not fully elucidated but may be related to alterations in the
leptin receptor or the ability of leptin to cross the blood–brain barrier once
hyperleptinemia develops.76–78 Moreover, since insulin stimulates leptin pro-
duction,76–79 hyperleptinemia may be “simply” related to hyperinsulinemia.
This relationship cannot explain hyperleptinemia in the face of failing pan-
creatic β cells, which often occurs in late stages of Type 2 diabetes. However,
since leptin also mediates inhibition of insulin secretion through the β-cell
leptin receptor,81,82 this could lead to a potential role of leptin in the β cells’
inability to produce and secrete insulin in sufficient quantity in Type 2
diabetes (see review by Cederberg and Enderback83).
Lipotoxicity (over-accumulation of tissue triglycerides) is often associated
with hyperleptinemia and insulin resistance, but the association is not clear.
Tissue triglyceride accumulation occurs in muscle, liver, and β cells of the
pancreas, in both animal models of Type 2 diabetes, as well as Type 2 diabetic
humans.47,48,64,83 The net effect of this contributes to muscle and liver insulin
resistance, a fatty liver, and pancreatic cells with impaired insulin secretion.83–85
Many studies have found a significant positive correlation between plasma
leptin and insulin in both obese and non-obese men and women with and
without diabetes.86–88 It should also be noted that humans and animals that
lack adipose tissue (lipoatrophy) are insulin-resistant, a state that is partially
reversed with leptin replacement.90–93 Leptin treatment improves insulin sen-
sitivity,83,84,94 possibly through decreasing the amount of intramuscular tri-
glyceride accumulation. In this regard, leptin has been shown to protect non-
adipose tissue from intracellular triglyceride accumulation by increasing the
oxidation of fatty acids. This appears to be accomplished through the down-
regulation of malonyl-CoA production via suppression (phosphorylation) of
acetyl-CoA carboxylase thereby decreasing lipogenesis (triglyceride produc-
tion) and increasing fatty acid oxidation.70,94 The key signaling mechanism
may be through AMP-kinase (AMPK). Leptin inhibits acetyl CoA carboxy-
lase via activation of AMPK. Minokoshi et al.95 found that leptin increases
AMPK in soleus and red gastrocnemius muscle of mice in two ways: directly
by muscle incubated with leptin and indirectly through stimulation of alpha-
adrenergic sympathetic nerves via leptin stimulation of hypothalamic neu-
rons. It should be noted that increased AMPK activation is also associated
with increased GLUT-4 transporter activation in contracted skeletal muscle
in both non-diabetic and diabetic subjects, thus promoting glucose uptake
and improving insulin sensitivity.96–99
AMPK stimulation in liver, muscle and adipose tissue has also been asso-
ciated with glitazone treatment.100, 101 Decreasing triglycerides in islet cells
via troglitazone administration in Zucker diabetic fatty rats85,102 prevents
lipotoxicity, reduces cell apoptosis, and improves insulin sensitivity. Humans
with Type 2 diabetes103–106 or impaired glucose tolerance107,108 also treated
with troglitazone, or the newer thiazolidinediones pioglitazone and rosiglit-
azone, improve glycemic control and insulin sensitivity. After 12 weeks on
troglitazone therapy 18 non-diabetic obese subjects, half of whom had
impaired glucose tolerance, showed the following improvements: 27%
increase in glucose disposal rates, 128% increase in the insulin-sensitivity
index, 48% decrease in fasting insulin, and a 40% decrease in plasma insulin
response to an oral glucose challenge.108 Others have demonstrated an
improved insulin secretion rate in people with IGT after troglitazone treat-
ment.107 Troglitazone also produced a 50% increase in insulin sensitivity in
first-degree relatives of Type 2 diabetics,109 individuals known to demon-
strate insulin resistance.59–61 Thiazolidinediones, other molecular actions are
thought to work through peroxisome proliferator-activated receptors
(PPARs) which regulate genes involved with adipocyte differentiation and
lipid and glucose metabolism (see reviews by Olefsky,110 Ferre,112 and Glide
and Van Bilsen111). The PPARs, α, β, γ, are members of a family of nuclear
transcription factors that are distributed at different levels of expression in
a variety of tissues. PPARα is mainly found in tissues with high rates of fatty
acid oxidation such as the liver, and skeletal and cardiac muscle. It may be
in part responsible for regulating muscle lipid homeostasis through regula-
tion of genes in human skeletal muscle cells that promote fatty acid catab-
olism such as carnitine palmityltransferase, malonyl-CoA decarboxylase,
and pyruvate dehydrogenase kinase.113 These effects on skeletal muscle
would reduce intramyocellular triglyceride accumulation and result in
improved insulin sensitivity. In fact Ye et al.114 found that activating either
PPARα (with a specific chemical agonist) or (with Pioglitazone) increased
whole-body insulin sensitivity and reduced muscle triglyceride and long-
chain acyl-CoAs in rats fed a high-fat diet. Overall insulin sensitivity was
inversely correlated with muscle long-chain acyl-CoAs (r = 0.74) and with
plasma triglycerides (r = 0.77). Troglitazone115 and rosiglitazone106 have both
been shown to decrease plasma fatty acid levels, and all three thiazolidinedi-
ones increased fatty acid uptake in skeletal muscle cells of Type 2 diabetics.116
PPARγ is found predominantly in adipose tissue and to a minor extent in
skeletal muscle, stimulates fatty acid storage and uptake, and is involved
in reducing leptin and up-regulating adiponectin expression. It is through
activation of PPARγ that the glitazone family, in part, has its effects. For a
pharmacological overview of the cellular and metabolic actions of thiazol-
idinediones please see Owens.117
Adiponectin: A Player in Lipid Dysregulation?

Adiponectin, a protein produced by adipocytes, is reported to be involved
in glucose and fatty acid metabolism via decreased hepatic glucose produc-
tion, and increased fatty acid oxidation in skeletal muscle.118 Thus, adiponec-
tin may be a link in the mechanisms involved with insulin resistance since
it decreases triglyceride concentration in liver and skeletal muscle in obese
mice.83,84,118 Low plasma levels of adiponectin are found in individuals with
insulin resistance, obesity, or coronary heart disease.119,120 In an attempt to
discriminate between obesity and IR in their relationship with adiponectin,
Abbasi et al.121 examined plasma adiponectin levels in obese and non-obese
individuals, with and without insulin resistance. Fasting plasma adiponectin
levels were measured in 60 non-diabetic individuals who were divided into
four groups based upon their BMI (≥ 30 or < 27) and insulin sensitivity
(sensitive or resistant). Insulin-resistant subjects had significantly lower adi-
ponectin levels whether they were obese or non-obese (17.1 µg/ml vs. 16.3
µg/ml, respectively) compared with the insulin sensitive obese or non-obese
(34.3 µg/ml vs. 29.8 µg/ml, respectively) subjects. Furthermore, Weyer et
al.119 found similar results when comparing Caucasians and Pima Indians
over a wide range of BMI, body fat, and insulin sensitivity. Plasma adiponec-
tin concentration was negatively correlated with percent body fat (r = –0.43),
fasting plasma insulin (r = –0.63) and positively correlated with insulin
sensitivity (r = 0.59). Faraj et al.122 found that improved insulin sensitivity
was best predicted (r = 0.70) by the increase in adiponectin in morbidly obese
men and women who were either weight stable or reducing in weight after
gastric bypass surgery. Results of these studies point to a closer association
of adiponectin with insulin sensitivity than with obesity. Moreover, circulat-
ing adiponectin levels were significantly reduced in non-obese, insulin-resis-
tant first-degree relatives of Type 2 diabetics, and negatively correlated with
fasting insulin and positively correlated with insulin sensitivity.123 It should
be noted that the underlying mechanism(s) linking insulin-resistant related
variables and adiponectin are as yet undefined. However, adiponectin stim-
ulates AMPK in skeletal muscle124 and troglitazone treatment in normal,
obese, and Type 2 diabetic subjects increased plasma adiponectin concentra-
tion.125 Both of these actions reduce triglyceride accumulation and may ame-
liorate insulin resistance-related lipid dysregulation. Lastly, 21 days of
pioglitazone treatment resulted in a twofold increase in adiponectin while
markedly decreasing endogenous glucose production via increased hepatic
insulin action in Type 2 diabetics.126 These findings provide links to the
involvement of PPARγ in the insulin resistance syndrome. Insulin-resistant

mouse models including lipoatrophic diabetic and obese and Type 2 diabetic
mice all show a reversal of insulin resistance when adiponectin is intro-
duced.127
Acylation-Stimulating Pathway (ASP): New Player in the

Insulin Resistance–Dyslipidemic Relationship?
We introduced earlier in this review concepts that reveal an important met-
abolic “cross-talk” between adipose tissue, insulin, and the adipose tissue
hormones leptin and adiponectin that may combine, in a redundant systems
fashion, to regulate fuel homeostasis and lipid partitioning. Studies exam-
ining cellular signaling pathways such as the AMPK system and the PPAR
network have revealed a promising mechanistic connection between adipose
tissue hormones and insulin sensitivity. Insulin action is effected by a number
of cytokine hormones secreted by adipose tissue (e.g., leptin, adiponectin;
see above) that are involved in the regulation of energy homeostasis and
lipid partitioning.69,117,120 One such adipocyte secretagogue that has received
increased attention is acylation-stimulating protein (ASP).128 Recent review
articles cover other adipocyte secretagogues (e.g., resistin, interleukin-6,
TNF-α) that are not covered in this monograph.5, 6, 83, 84, 118, 148
ASP, which is also known as C3a-des-Arg, is another lipogenic autocrine
secretion that acts similarly to insulin in function (i.e., lipogenic and inhibits
hormone sensitive lipase), and therefore may provide an additional “piece”
to the energy homeostasis–lipid-partitioning “puzzle” that presents as the
IR-dyslipidemic state. The precursor components required to form ASP are
compliment C3, adipsin, and factor B, all of which are synthesized and
secreted by fully differentiated adipose cells.128
Similar to the function of insulin, ASP is an important biological mediator
between the balance of triglyceride synthesis and degradation (lipolysis).129
Upon binding ASP receptors, a G-coupled signal transduction pathway elic-
its the secondary messenger diacylglycerol (DAG) and the eventual down-
stream translocation of protein kinase C.130 Currently it is believed that ASP
stimulation of TG synthesis is effected through a protein kinase C-dependent
mechanism that directly phosphorylates serine/threonine residues on dia-
cylglycerol acyltransferase (DGAT),131 the rate-limiting enzyme in TG syn-
thesis. In addition, ASP may also influence FFA re-esterification through a
phosphodiesterase 4 (PDE4)-mediated mechanism; however, the preceding
upstream signaling pathway has yet to be determined.132 Insulin, on the
other hand, also effects lipid status in adipocytes primarily by TG synthesis,
FFA re-esterification, and inhibition of lipolysis through stimulation of phos-
phatidylinositol 3-kinase in a phosphodiesterase 3 (PDE3)-dependent man-
ner, degrading cAMP thus inhibiting hormone sensitive lipase.132–134
Furthermore, with the recent discovery of an ASP-specific orphan G protein-

coupled receptor (C5L2) associated with a Gi subunit,135, 136 the processes
involved in ASP signaling are becoming more clear. ASP-responsive recep-
tors are expressed in 3T3-L1 cells, human fibroblasts and human adipose
tissue, and possibly in rat skeletal muscle.137 In addition to ASP’s role in TG
synthesis at the adipocyte (very similar to that of insulin), glucose transport
is enhanced through translocation of glucose transporters (Glut1, Glut3, and
Glut4) to the adipocyte plasma membrane surface as well as in rat skeletal
muscle tissue cultures.137–139 In fact, a recent study using the rat knock-out
model139 suggests that ASP in the normophysiological state has differential
effects on non-esterified fatty acids (NEFA) trapping in adipose and skeletal
muscle. Essentially, in adipose tissue ASP increases LPL activity and the
resultant trapping of NEFA to form TG. ASP functions in an “anti-lipotoxic”
fashion at the skeletal muscle, decreasing LPL activity and thereby support-
ing the hydrolysis and lipolysis (i.e., oxidation) of lipid substrate.137 In addi-
tion, ASP facilitates the “trapping” of the glycerol backbone from the glucose
molecule providing an essential substrate for the esterification of fatty acids
and the storage of energy for future metabolic needs. Interestingly, Weyer et
al.140 did not find an association between insulin action and ASP concentra-
tions in an at-risk population (i.e., Pima Indians) for the development of IR
and dyslipidemia. Further development of the obesity concept and the pos-
sible role that ASP “resistance” plays in fatty acid and/or lipid dysregulation
will be discussed later.
The consequence of a dysregulated insulin–peripheral tissue axis (liver,
skeletal muscle, and pancreatic β cell) leads to the well-defined exacerbation
of lipolysis of adipose tissue TG stores. The storage and release of FFA by
adipocytes is an important mechanism for energy availability. As noted
earlier in this chapter, it is well known that there is differential regulation
of LPL activity in adipose tissue and muscle as observed in vivo in the fasting
and fed state with insulin-mediated LPL activity primarily affecting TG
hydrolysis. Similar to LPL, several lines of evidence suggest that ASP action
is also under nutritional regulation; increasing in adipose tissue and decreas-
ing in muscle during the postprandial rise in insulin, with reciprocal changes
observed during fasting.137,141 However, ASP’s main function is to trap the
fatty acids (i.e., liberated during TG hydrolysis through subsequent insulin-
mediated LPL activity) directly into adipocytes.142 It is important to note here
that TG clearance and fatty acid trapping occur concurrently and result from
different signaling pathways (see above). Therefore, analogous to the insu-
lin–LPL relationship with IR dyslipidemia discussed earlier, ASP has begun
to receive marked attention as a possible metabolic marker of obesity-related
disorders and may have considerable pathophysiological significance in the
metabolic syndrome.
Abnormal function of the ASP pathway leads to a state of ineffective “fatty-
acid trapping” in adipose tissue143 comparable to that seen in insulin resis-
tance (i.e., resulting from chronic elevated FFA concentrations). A number
of disorders such as obesity, diabetes and cardiovascular diseases associated
with dyslipidemia have been linked to abnormal adipose tissue metabolism

and elevated FFA, as well as elevated plasma ASP concentrations.144,145
Cianflone144 has demonstrated that ASP levels in gynoid obese individuals
are double in magnitude compared with those in an age-matched control
group. Also in a recent study by the same group,146 surgically induced weight
loss showed a strong correlation between reduced plasma ASP and the
change in apo B (mg/dl) (r = 0.55, p = 0.009). In the same patient group, an
index of postoperative insulin sensitivity correlated well with changes in
adiponectin concentrations (r = –0.70, p = 0.01). In addition, a dysfunctional
ASP pathway has been demonstrated in hyper-apo B subjects.147 Sniderman
et al.147 showed decreased binding of radiolabeled ASP in the face of elevated
circulating ASP concentrations, suggesting reduced receptor number and
tissue responsiveness. Recently, it has been hypothesized that a defective
ASP pathway in some obese subjects, as indicated by ineffective ASP binding
when exposed to elevated FFA, may be representative of an “ASP resistant”
state.148 A decreased ASP tissue responsiveness may lead to insulin resistance
(i.e., secondary to elevated circulating FFA concentrations) as a result of
inefficient FFA trapping and to the eventual development of the previously
noted constellation of metabolic risk factors that lead to peripheral tissue
lipotoxicity. It is plausible to infer that alterations in plasma ASP in Type 2
diabetics and the resultant elevations in FFA due to ineffective fatty acid
trapping may encourage a hyper-apo B lipid profile. Some discrepancy exists
in the literature regarding circulating plasma ASP levels in hyper-apo B
patients.147,149,150 Kildsgaard’s group149 did not find elevated plasma ASP
concentrations in hyper-apo B patients while Zhang et al.150 found elevated
plasma ASP concentrations in a similar patient group. Both the Sniderman147
and Faraj148 reviews note that the use of the arteriovenous difference tech-
nique at the subcutaneous adipose depot of obese and or hyper-apo B
patients may be a better method to determine the existence of elevated ASP.
These authors report that ASP can only be detected in a capillary bed site-
specific manner. Still others have measured elevated whole blood concen-
trations of ASP in these populations.142,143,146 At this time it is important to
note that the different methodological approaches employed throughout the
literature surely contribute to some difficulties in interpreting the currently
available data.
Interestingly, in Pima Indians (a group at high risk for the development
of Type 2 diabetes), a recent study showed a genetic bivariate linkage with
circulating ASP (↑) levels for BMI (↑) and HDL (↓), each an important risk
factor for the development of the metabolic syndrome.151 It should be noted
that ASP normally enhances glucose uptake; however, paradoxically, in Pima
Indians (again a population at increased risk for IR dyslipidemia) ASP levels
did not correlate well with insulin action as determined by the hyperglyce-
mic-euglycemic glucose clamp.140 Neither radiolabeled ASP on whole plasma
nor subcutaneous capillary arteriovenous difference experiments were
obtained in this study so it cannot be ascertained if indeed this group was
in fact ASP resistant. It is important to note, however, that obese individuals
may simply have elevated ASP concentrations because of enlarged adipo-

cytes (similar to hyperleptinemia), therefore having a greater potential to
trap FFAs, thus increasing TG esterification beyond adipocyte capabilities.148
Consequently, additional support to the heterogeneity of overweight and
obesity may be phenotypically presented as either an “ASP resistant” indi-
vidual with overproduction of ASP or as “ASP deficient” as noted in knock-
out rat models where fasting ASP concentrations are normal yet become
abnormally elevated in the postprandial state with a resultant delay in TG
clearance.152 Interestingly, obese women show a marked increase in post-
prandial fatty acid trapping, potentially contributing to adipocyte enlarge-
ment and quite efficient TG clearance.153 Further studies examining
circulating levels of ASP in populations at increased risk for the development
of IR dyslipidemia could provide a useful insight into the potential role that
ASP may play in the development of lipotoxicity-related comorbidities. The
fatty-acid trapping capacity of individuals with different phenotypic presen-
tations of obesity associated with Type 2 diabetes may be a key determinant
in differentiating those individuals at greater risk for developing a hyper-
apoB lipid profile.147 Moreover, continued pursuit of basic science research
using knock-out rat and mouse models may provide useful insights into
the mechanisms behind ASP’s regulatory contribution to IR dyslipidemia.
Summary and Conclusions

The insulin-resistance syndrome manifests itself by involving many tissues
including the liver, skeletal muscle, and adipose tissue. The derangements
that result affect fuel metabolism and energy homeostasis that produces
increased risk for cardiovascular disease. At the center of the dysregulated
metabolism, of both carbohydrate and lipid metabolism, appears to be the
adipocyte where fatty acids are preferentially and “correctly” stored as tri-
glycerides thereby not allowing triglycerides to overaccumulate in other
tissues. This is intimately tied into the adipocyte’s ability to secrete the
hormones leptin, adiponectin, and ASP (Figure 8.4). These hormones are
responsible for food intake and storage as well as influencing energy expen-
diture through both fat and carbohydrate metabolism. This “adipocentric”
view is widely supported by both human and animal studies that present
central fat deposition, and in most cases, visceral fat, as the root of dysreg-
ulated metabolism that can lead to acquiring the metabolic syndrome. So it
sounds pretty simple: avoidance of the metabolic syndrome/insulin-resis-
tance syndrome may merely, in part, lie with keeping one’s waistline in
check, especially as we age where the tendency is to gain weight and the
incidence of the metabolic syndrome is high. Keeping an active lifestyle
combined with prudent dietary considerations may in many cases avoid the
onset of the metabolic syndrome, and associated lipid dysregulation and
Adipocyte
Leptin Adiponectin ASP

Adipose tissue
Glucose uptake
TG storage
HSL
Central Nervous Muscle Liver
System fat oxidation Hepatic glucose production
Food intake Fatty acid oxidation
TG storage TG storage
Energy expenditure
Insulin sensitivity
Plasma FFA
TG
LDL/sd LDL
Plasma HDL
FIGURE 8.4
Adipocentric regulation of metabolism.
increased risk for cardiovascular disease. Failure to do this puts one’s met-
abolic fate in the hands of pharmaceuticals like thiazolidinediones to correct
metabolic dysregulation, or other medications, such as “statins” or niacin,
which return blood lipids and lipoproteins back to the normal range.
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9
Obesity, Lipoproteins, and Exercise
Theodore J. Angelopoulos
CONTENTS
Introduction .........................................................................................................173
Lipoproteins, Obesity and Visceral Adiposity...............................................174
Lifestyle Modification of Dyslipidemias.........................................................177
Conclusion ...........................................................................................................178
References ............................................................................................. 179
Introduction
Obesity refers to excess body fat and develops in response to a chronic energy
surplus. The number of obese, defined as a body mass index (BMI) greater
than or equal to 30 kg/m2,1 is on the rise. Specifically, the overall prevalence
has risen in the past decade to almost one-third of the U.S. population.2
Obesity is a serious health problem in the United States and is a major risk
factor for a number of diseases. Obesity has been linked to an increase in
the prevalence of chronic diseases such as cardiovascular, diabetes mellitus,
cancer (certain types), arthritis, gallbladder and sleep apnea (Figure 9.1).
A common, but interesting finding is that fat location plays a relevant role
in the increased health risk associated with obesity. Vague was the first to
recognize the increased risk of male (android) fat patterning,3 but many since
have expanded on this finding. Waist circumference has been found to be a
better predictor of cardiovascular disease (CVD) risk than BMI.
Alterations in lipoprotein profiles in obese individuals, such as increased
low-density lipoprotein (LDL), are associated with increased risk for coro-
nary heart diseases. Lipoprotein metabolism plays a central role in the eti-
ology of coronary heart diseases. Blood lipid and lipoprotein levels may be
modified with exercise and/or caloric restriction weight loss.4–6 Some species
173
FIGURE 9.1
Body mass index and risk of diseases.
of lipoproteins unfortunately are under strong genetic influence and are not
altered by these lifestyle factors known to influence other lipoproteins.7 By
understanding the mechanism of exercise in the prevention of cardiovascular
diseases, health professionals can better develop a plan to optimize the
exercise regimen.
Lipoproteins, Obesity and Visceral Adiposity

Obesity increases the risk of developing CVD. At a BMI (in kg/m2) of greater
than 30, there are substantial increases in both men and women in reference
to LDL count and significant decreases in high-density lipoprotein
(HDL),8–10 as compared to a BMI of 25. LDL particle size is a good predictor
for coronary heart disease (CHD) risk. Indeed, Schaefer et al.11 found that
CHD patients have smaller LDL particles than control subjects. Increases
in triglycerol (TG) and remnant-like particles, decreases in HDL cholesterol,
and alterations in LDL particle size are common in obese and diabetic
subjects.12,13
Obesity commonly exists as a state of insulin resistance (IR), and a possible
early stage in the development of Type 2 diabetes (referred to here on out
as simply “diabetes”). The link between the two is so strong that the term
“diabesity” has even been proposed.12 A BMI of over 35 has been shown to
Obesity, Lipoproteins, and Exercise 175
FIGURE 9.2
Lipids and lipoproteins and resting BP in insulin-sensitive and insulin-reistant obese subjects.
increase the risk of diabetes to 93 times that of a BMI of less than 20.13
Highlighting the relationship between obesity and the subsequent develop-
ment of diabetes is a study on a group of morbidly obese individuals who
underwent bariatric surgery. After 2 years only 0.2% of the surgery group
had progressed to diabetes (weight loss of 28 kg) compared with 6% who
did not undergo the surgery.14
Due to this close link it should be obvious that similar population trends
exist for diabetes as were already outlined for obesity. Accordingly, in 1995
there were an estimated 135 million Type 2 diabetics worldwide,15 with 12.1
million estimated to reside in the United States in 2002.16 Nationally this
represents an increase of 9% (1 million from 2000), and 17% from 1997. The
same estimates project the number to rise to 17.4 million by 2020, a rise of
24% in regard to percentage of population. At the national level the combined
direct and indirect costs of diabetes for 2002 were estimated at $132 billion.
Pioneering work by Reaven identified several metabolic abnormalities
(i.e., dyslipidemias) that commonly coexist with insulin resistance and the
increased risk associated with them.17 Despite the problems associated with
using the Homeostasis Model Assessment of IR (HOMA) to evaluate IR in
highly insulin-resistant individuals, a solid relationship still exists between
the level of HOMA and clustering of CVD risk factors.18 However, the rela-
tionship between IR and increased CV risk does not seem to rely solely upon
the presence of obesity. Rather, IR has often been shown to be associated
independently with an increased risk of CHD.19,20 IR has been shown to be
positively associated with plasma viscosity. It is possible that this may
explain the link between the metabolic syndrome and vascular complications
that accompany it.21 Once diabetes develops, hyperglycemia per se is an

obvious candidate for the increased risk associated with diabetes due to
structural changes in lipoproteins,22 and the formation of advanced glycation
end products that stimulate vascular injury.23 What should be made clear is
that hyperglycemia is unlikely to be related to the increased risk associated
with non-diabetic IR states due to the fact that it is typically a late develop-
ment in the etiology of diabetes, as will be explained later. Also downplaying
the role of hyperglycemia is the observation that there is a poor correlation
between the duration of diabetes and macrovascular disease.22
The effects of diabetes on plasma lipid composition, often referred to as
“diabetic dyslipidemia,” are highly related to the clustering of metabolic
abnormalities associated by Reaven. They are suspected to contribute to a
significant amount of the elevated CVD risk associated with IR states. It has
more recently been said that increased levels of small dense LDL particles
is the true hallmark of diabetic dyslipidemia. Due to a decreased ability to
bind receptors, they have been shown to confer an increased atherosclerotic
risk.24,25 Furthermore, the presence of small dense LDL particles is associated
with high triglycerides and low HDL-cholesterol (HDL-C) levels.26 It is prob-
able that these abnormalities contribute to the elevated risk of IR, indepen-
dent of diabetic status, due to the fact that diabetic dyslipidemia does not
immediately correct itself with normalization of glycemia, and are also
observed in a prediabetic state.26 It is possible that the role of such plasma
lipid abnormalities has been overestimated as common risk factors, and
accounts for only 25–30% of the observed increased CVD risk.27 Of particular
importance, however, is the observation that plasma total cholesterol levels
are often within normal range in obese individuals with excess visceral
adiposity.
Alterations in lipoproteins are also associated with aging and gender.
Schaefer and colleagues11 had reported an increase in fasting TG concentra-
tions with age. There is an 80% increase between the age of 20 and approx-
imately 50 years. LDL concentrations increased by 30% in the same age
bracket. Noted reasons on these alterations are delayed chylomicron remnant
clearance in the elderly compared with younger populations. These include
a decline in the fractional catabolic rate of LDL, perhaps caused by a decline
in the number of LDL receptors, an age-associated increase in visceral adi-
posity.28 This also may increase the rate of cholesterol synthesis. A noted
increase of dietary fat intake in older populations and inactivity are also to
be blamed. Very low-density lipoprotein apolipoprotein (apo) B-100 secre-
tion being elevated in the elderly coupled with delayed clearance also
accounts for the increases in TG and LDL.
In the very elderly population, those people aged 80 and above, both TG
and LDL are significantly lower compared with middle-aged individuals,11
most probably due to the reduction in BMI and decreased apo B-100 pro-
duction in these subjects.29,30
Gender differences exist with lipoproteins, and it is now accepted that
women have significantly higher concentrations of HDL cholesterol and apo
A-I than men. The ratio of HDL3/HDL2 is gender dependent.31 The hormones
presumably reflect the gender differences in HDL levels: androgens increase
the activity of the enzyme HL and estrogens cause a decrease.32 Even pre-
menopausal women a have higher apo A-I secretion than men.33 In addition,
girls tend to be more susceptible to LDL decreases from weight loss com-
pared with boys. This was concluded in a study of obese children who lost
weight after intervention of diet and exercise.34
Lifestyle Modification of Dyslipidemias

Regular exercise has been recommended as an important strategy for the
prevention and treatment of obesity. Much information has been accumu-
lated regarding the beneficial effects of exercise. It is generally accepted that
exercise training positively affects many of the components of the plasma
lipoprotein-lipid profile that partly determines the person’s CVD risk.35–45
Intervention trials involving aerobic exercise training showed favorable
decreases in TG38,39,46,47 and LDL48–51 only when combined with weight loss,
even with Type 2 diabetic patients.52 Exercise alone in most cases does not
reduce plasma LDL-C.53 Interestingly, Kokkinos et al.54 observed that LDL-C
and TG levels were positively associated with BMI but inversely correlated
with the distance run per week, the frequency of exercise per week and the
durations of the exercise per session. Durstine et al.51 argued that exercise
training does not always alter total cholesterol and LDL-C unless a reduction
in dietary fat intake is brought about and body weight loss is a part of the
exercise training program, or both. Halle et al.55 concluded that increases in
physical activity in hypocholesterolemic men lowered TG and small LDL
concentrations, while Williams and colleagues56 reported that distance per
week and reduced body fat mass correlated significantly with decreases in
small LDL levels. Woolf-May et al.50 reported a decrease in LDL with short
walking of 5–10 min per bout, but performing more than one bout of walking
per day. Finally, LDL oxidation is important in assessing cardiovascular risk.
Decreased LDL oxidation reduces the risk of arteriosclerosis, and a decreased
LDL was observed following a 10-month exercise program.59
Generally, the beneficial effects of regular exercise on blood lipids are
observed even after training at low training volumes. To observe clinically
significant gains, energy expenditures of 1200 to 2200 kcal/week must be
attained.57,58 As such, there is likely a dose–response relationship between
training volume and blood lipid changes.
Endurance exercise training induces significant reductions in TG. Studies
reporting decreases in TG with exercise often show exercise-induced weight
loss and a reduction in TG that is related to baseline values. The higher the
baseline concentration, the greater the exercise-induced reductions.53,54,60,61
Beneficial changes in TG may also occur independent of changes in body

mass.62
A comprehensive review of the effects of exercise on HDL-C has been
undertaken by Durstine and Haskell.63 The majority of studies report favor-
able increases in HDL,64–69 and a dose–response relationship. Thresholds
established from cross-sectional and longitudinal exercise training studies
indicate that 15–20 miles/week of brisk walking or jogging, which elicit
between 1200 and 2200 kcal of energy expenditure per week, increases
HDL-C by 2–8 mg/dl.51
Exercise training volume (kilocalories expended during the exercise train-
ing program) is important for favorable changes in HDL concentration. It
appears that reduction in body weight and fat mass may also be important
for changes in HDL concentration in overweight individuals. Thompson et
al.53 found no significant changes in HDL concentration in overweight men
after prolonged exercise training without weight loss. The same result was
observed by Nicklas and colleagues.70 It appears that the effectiveness of
endurance training, without concomitant weight loss, is blunted in obese
people. Finally, previous studies have shown that exercise training has little
effect on HDL levels in subjects with initially low HDL cholesterol.71,72
In summary, sufficient exercise appears to induce favorable changes in
lipids and lipoproteins in the obese state. Some of the beneficial effects of
exercise, however, may be blunted in obese people if exercise is not combined
with caloric restriction that induces weight loss. Van Gaal and colleagues73
had shown that even 5–10% weight loss, when combined with exercise,
significantly decreases TG levels and increases LDL particle size. A meta-
analysis published by Dattilo et al.74 showed that for every kilogram
decreased in body weight during a weight loss process, HDL increases by
0.009 mmol/L and plasma total cholesterol, LDL and TG decrease by 0.05,
0.02 and 0.015 mmol/L, respectively.
Conclusion
Obesity is accompanied by changes in lipid–lipoprotein profiles that increase
one’s risk for CVD. Lifestyle modifications have shown some beneficial
changes in lipids and lipoproteins of obese individuals. The impact of life-
style modifications may be optimum when sufficient exercise is combined
with caloric restriction that induces weight loss. The level of individual
response may be influenced by training volume, amount of weight loss, and
genetics.
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10
Pharmacological Treatments of Lipid
Abnormalities
Sachin M. Navare and Paul D. Thompson
CONTENTS
Introduction .........................................................................................................183
Targets for Therapy ............................................................................................184
Goals for Therapy ...............................................................................................185
Therapeutic Lifestyle Changes .........................................................................186
Pharmacotherapy ................................................................................................187
Statins....................................................................................................................189
Bile Acid Binding Resins ...................................................................................194
Nicotinic Acid......................................................................................................197
Fibrates .................................................................................................................199
Cholesterol Absorption Inhibitors ...................................................................200
Other Therapies...................................................................................................202
Selection of Drug ................................................................................................204
Monitoring ...........................................................................................................204
Summary ..............................................................................................................205
References ............................................................................................. 205
Introduction
Cholesterol is an essential component of mammalian cell membranes and
also serves as a source of steroid hormones and bile acids. Although vital
for cellular growth and metabolism, its deposition into the arterial wall
produces atherosclerosis.1 Cholesterol is transported primarily as choles-
teryl ester, which is water insoluble, and therefore must be transported in
the water world of the bloodstream in specialized particles called lipopro-
teins. At least three major classes of lipoproteins are involved in transport
183
of cholesterol. Very low-density lipoprotein (VLDL) is a triglyceride-rich

particle that transports triglycerides (TGs) and cholesterol from the liver.
Low-density lipoprotein (LDL) is a cholesterol-rich particle produced by
the delipidation of VLDL. LDL transports cholesterol to various tissues
including the arterial wall and returns cholesterol to the liver via the LDL
receptor. HDL also has a major role in returning cholesterol to the liver
as part of the general process known as “reverse cholesterol transport.”
There is overwhelming evidence that elevation in LDL-cholesterol
(LDL-C) leads to the development of atherosclerosis. There is also strong
evidence that elevations in TG and subnormal levels of HDL increase
atherosclerotic risk. Atherosclerosis is the single most common cause of
mortality in the United States accounting for almost 1.5 million deaths
each year.2 Nearly half of these deaths result from coronary artery disease
(CAD). Thus treatment of the lipoprotein disorders forms the cornerstone
for CAD risk reduction.
Targets for Therapy

The National Cholesterol Education Program (NCEP) Adult Treatment
Panel (ATP) first published guidelines for the detection, evaluation and
treatment of hyperlipidemia in 1988. The latest reiteration of these guide-
lines (NCEP ATP III), published in 2001, provides a simple, evidence-based,
approach to management of dyslipidemia and forms the basis of the current
review.3
ATP III identifies LDL-C as the major atherogenic lipoprotein. Extensive
evidence obtained from animal, genetic, and epidemiological studies as well
as clinical trials shows a strong direct relationship between levels of LDL-C
and CAD events in populations with and without established CAD.4–7 This
relationship is linear and is observed over a broad range of LDL-C levels.8
Moreover, interventions which reduce LDL-C levels reduce both short- and
long-term CAD morbidity and mortality.9 Recent clinical trials have shown
that a 1% reduction in LDL reduces CAD risk by 2%. Angiographic studies
have also demonstrated favorable effects on coronary lesions with LDL
reduction.10 Thus, ATP III uses LDL-C as the primary target for cholesterol-
lowering therapy.
ATP III also recognizes the risk inherent in elevated VLDL levels.11–13
Consequently, in patients at LDL goal, but whose TGs are > 200 mg/dl, non-
HDL cholesterol, calculated as total cholesterol minus the HDL-C, becomes
a secondary target of therapy.
HDL-C is not a distinct target of therapy in ATP III, although the risk
associated with low HDL levels is recognized. Epidemiological studies have
shown that CAD risk correlates inversely with HDL-C levels.14 A 1%
decrease in HDL-C is associated with a 2–3% increase in risk of CAD while
Pharmacological Treatments of Lipid Abnormalities 185
a high HDL-C is considered a negative risk factor. Low HDL is associated

with other atherogenic risk factors such as glucose intolerance and high
TGs. Also, present strategies to increase HDL-C alter other risk factors,
making it impossible to prove that increasing HDL-C alone reduces CAD
events. Nevertheless, despite the absence of conclusive evidence of its value,
ATP III encourages interventions that raise HDL.
Goals for Therapy

The fundamental principle of the ATP III guidelines is that the intensity of
intervention is directly related to the degree of CAD risk. Therefore, the first
step is to determine the 10-year CAD risk using non-LDL traditional risk
factors (Table 10.1) and risk estimates provided by the Framingham Heart
Study (Figure 10.1).
LDL-C treatment goals are categorized into three levels of risk (Table 10.2).
Patients with the highest risk include those with existing CAD and those
whose risk is equivalent to that of patients with CAD (“CAD risk equiva-
lents”). This latter group includes diabetics, patients with non-CAD athero-
sclerotic disease, and those with a 10-year calculated risk > 20% since this
is the approximate risk of a recurrent myocardial infarction or sudden death
in patients with diagnosed CAD. The LDL-C goal for these patients is < 100
mg/dl, although an update released on July 13, 2004 suggests that a goal <
70 mg/dl is an appropriate therapeutic strategy for these high-risk patients.15
Patients with intermediate risk are those with multiple (≥ 2) risk factors. ATP
III designates a LDL-C goal of < 130 mg/dl for this category. This group is
further categorized into two subgroups, one whose 10-year CAD risk is
10–20% and one whose risk is < 10%. Patients with 0–1 risk factors have the
lowest risk and a LDL-C goal of < 160 mg/dl.
TABLE 10.1
Major CHD Risk Factors
Positive risk factors
1. Cigarette smoking
2. Hypertension (BP ≥ 140/90 mmHg or on antihypertensive medication)
3. Low HDL cholesterol (< 40 mg/dl)
4. Family history of premature CHD (CHD in male first degree relative <
55 years; CHD in female first degree relative < 65 years)
5. Age (men ≥ 45 years; women ≥ 55 years)
Negative risk factors
1. High HDL-C > 60 mg/dl
FIGURE 10.1
Score sheet for 10-year CAD risk for men and women using the Framingham risk scores.
Therapeutic Lifestyle Changes

ATP III offers two major modalities for lowering LDL-C: therapeutic lifestyle
changes (TLC) and drug therapy. TLC offers a variety of non-pharmacologic
approaches to lower elevated cholesterol level. ATP III considers TLC as the
most cost-effective means of reducing the risk of CAD. The major compo-
nents of TLC are:
TABLE 10.2
Classification of CHD Risk and LDL Goals
LDL Level to
LDL-C Non-HDL-C LDL Level to Initiate Drug
Risk Goal Goal Initiate TLC Therapy
Category Criteria (mg/dl) (mg/dl) (mg/dl) (mg/dl)
High 1. Existing CHD < 100 < 130 ≥ 100 > 100
2. CHD equivalents
3. ≥ 2 risk factors
(10-year risk)
Intermediate ≥ 2 risk factors < 130 < 160
10-year risk 10–20% < 130 < 160 ≥ 130 ≥ 130
10-year risk < 10% < 130 < 160 ≥ 130 ≥ 160
Low 0–1 risk factors < 160 < 190 ≥ 160 ≥ 190
1. Reduction in dietary saturated fat intake to < 7% of total calories

and cholesterol intake to < 200 mg/day. This may reduce LDL-C by
6–10%.
2. The use of viscous (formerly called “soluble”) fiber and plant stanol/
sterols. A viscous fiber intake of 5–10 g reduces LDL-C by 5% while
2–3 g of stanols/sterols daily reduce LDL-C by 6–15%.
3. Weight reduction.
4. Increased regular physical activity.
The last two modalities are mainly recommended for treatment of metabolic
syndrome but may reduce LDL-C in some patients.
Pharmacotherapy
The initial step in managing serum lipids and CAD risk is to determine if
the lipid levels are abnormal and what element requires treatment. This will
ultimately determine the treatment approach. Screening can be based on a
non-fasting sample, but if the TGs are elevated, a fasting sample must be
obtained. The severity of lipid abnormalities can be classified as in Table 10.3.
The next step is to exclude secondary causes of hyperlipidemia because
treatment of the secondary cause often cures the lipid abnormality (Table
10.4). Hypothyroidism, diabetes, and the nephrotic syndrome should be
excluded by thyroid-stimulating hormone, glucose, hemoglobin A1c, and
urinary protein measurements, respectively. The patient’s medication list
should also be reviewed since many drugs can produce secondary hyper-
lipidemia.
TABLE 10.3
Classification of Severity of
Lipoprotein Abnormalities
Total Cholesterol (mg/dl)
Desirable < 200

Borderline high 200–239
High ≥ 240
Triglycerides (mg/dl)
Normal < 150

High 200–499
Very high ≥ 500
LDL-C (mg/dl)
Optimal < 100

Above optimal 100–129
High 160–189
Very high ≥ 190
HDL-C (mg/dl)
Low < 40
High > 60
TABLE 10.4
Secondary Causes of
Lipoprotein Abnormalities
1. Diabetes mellitus
2. Hypothyroidism
3. Nephrotic syndrome
4. Drugs
a. Alcohol ingestion
b. HIV-protease inhibitors
c. Beta blockers
d. Thiazide diuretics
e. Cyclosporine
f. Glucocorticoids
g. Oral estrogens
h. Isotretinoin
i. Sertaline hydrochloride
The final step is to identify the goals of therapy and to select an appropriate
pharmacological agent if dietary modification, weight loss, and physical
activity fail to correct the problem. Unfortunately, hygienic interventions fail
TABLE 10.5
Summary of Effects of Various Medications
on Lipoprotein Concentrations
Drug Class LDL HDL TG
Statins ↓↓↓↓ ↑↑ ↓↓↓
Sequestrants ↓↓ to ↓↓↓ ↑ ↑↓
Ezetimibe ↓↓ ↑ ↓
Fibrates ↓ to ↓↓ ↑↑↑ ↓↓↓↓
Nicotinic acid ↓ to ↓↓ ↑↑↑↑ ↓↓↓
in most patients with severe lipid abnormalities and pharmacologic therapy

is required.
Lipoprotein disorders are usually chronic conditions requiring lifelong
therapy. Hence, safety, tolerability and cost effectiveness are as important as
efficacy, in selecting drug therapy. Currently five classes of medications are
available for treating lipid disorders. Each class affects the major lipoproteins
differently (Table 10.5).
The initial drug choice should be tailored to the lipoprotein abnormality.
Statins are the drugs of choice for patients with a predominantly LDL abnor-
mality, whereas either a fibrate or niacin could be initial therapy for a patient
with elevated TGs. These medicines can be used as monotherapy or in
combination. Use of drugs in combination increases the efficacy of the ther-
apy and allows use of lower doses of each medication. This strategy may
reduce the side effects of individual therapy, but in some instances, such as
the combination of statins with the fibric acid derivative, gemfibrozil, com-
bined therapy can increase the risk of side effects.
Statins
Introduction
Since the introduction of lovastatin in 1987, statins have revolutionized the
treatment of lipoprotein disorders. They are the most powerful drugs for
reducing LDL-C and have consistently been shown to reduce the risk of all
atherosclerotic clinical events.16–21
Pharmacology
As of June 2004, six statins are FDA approved. Cerivastatin, a seventh drug
approved in 1997, was voluntarily withdrawn from the market in 2001 by
its manufacturer due to a high rate of fatal rhabdomyolysis. The statins owe
their activity to a moiety, resembling hydroxymethyl-glutaric acid, which
TABLE 10.6
Pharmacology of Statins
Half-Life Bioavailability
Drug Form Solubility (h) (%) Elimination
Atorvastatin Active Lipophilic 13–30 14 98% hepatic
Fluvastatin Active Hydrophilic 0.5–3 24 Hepatic
Lovastatin Prodrug Lipophilic 2–4 30 10% renal; 83% hepatic
Pravastatin Active Hydrophilic 2–3 17 20% renal; 70% hepatic
Rosuvastatin Active Hydrophilic 19 20 10% renal; 90% hepatic
Simvastatin Prodrug Lipophilic 1–3 <5 13% renal; 60% hepatic
may be present in open (acid, active) form or closed (lactone, inactive) form.
Of the six statins, only lovastatin and simvastatin are inactive lactones
(prodrugs), which are converted to hydroxy acids (active forms) in the liver.
Lovastatin and pravastatin are fungal derivatives, simvastatin is semi-syn-
thetic while the remainder are purely synthetic compounds. The predomi-
nant route of elimination is through the bile after hepatic transformation. In
addition, the kidneys also eliminate all statins, although the percentage
varies greatly among the different statins (Table 10.6).
Mechanism of Action
All statins are competitive inhibitors of the enzyme 3-hydroxy-3-methylglu-
taryl CoA (HMG CoA) reductase.22 HMG CoA reductase catalyses the con-
version of HMG to mevalonate, the rate-limiting step in cholesterol synthesis.
This decreases hepatic cholesterol content and produces an up-regulation of
LDL receptors on the hepatic cell surface.23 The LDL receptors facilitate the
uptake of apolipoprotein (apo)-B containing lipoproteins, predominantly
LDL-C, by the liver. However, intermediate-density lipoprotein (IDL) and
VLDL remnants are also removed via the LDL receptor and this may account
for some of the triglyceride-lowering effects of the statins.24,25 Statins also
reduce the hepatic production of VLDL by an effect on apo-B secretion.26
Lipid-Lowering Effects
Effects on LDL
Statins reduce LDL-C by 18–63%. There is considerable variation among the
different statins in their effect on LDL-C (Table 10.7). The reduction in LDL-C
with statins is dose-dependent, but the relationship between dose and degree
of LDL reduction is log-linear. In general, each doubling of the statin dose
decreases LDL-C by an additional 6%.27
TABLE 10.7
Summary of Lipid-Lowering Effects of Statins
Equivalent
Starting Maximum FDA Dose (mg) LDL HDL TG
Dose Approved Dose (LDL ↓ by Reduction Increase Reduction
Drug (mg) (mg) 30–35%) (%) (%) (%)
Atorvastatin 10 80 10 37–57 5–13 17–53
Fluvastatin 20 80 80 18–31 3–11 12–25
Lovastatin 20 80 40 24–40 2–10 6–27
Pravastatin 20 80 40 24–34 2–12 15–24
Rosuvastatin 5–10 40 5 45–63 8–14 10–35
Simvastatin 20 80 20 35–46 8–16 12–34
Effects on HDL
Statin therapy increases HDL-C by 5–10%, with greater increases seen in
patients with low HDL and high TGs. Rosuvastatin increases HDL-C up to
10%, an effect greater than the other statins. In general, HDL-C increases
with higher doses of the statin, but with atorvastatin increasing the dose
lessens the increase in HDL-C.28,29
Effects on TG
All statins lower TG concentrations by 7–30%. The effect of statins on TGs
is related to the baseline TG level. In a pooled analysis involving 2689
subjects, statins did not reduce TGs in patients with TGs < 150 mg/dl, but
in patients with baseline TGs > 250 mg/dl, statins produced significant dose-
dependent TG reductions of 22–45%.30 The magnitude of the TG reduction
in hypertriglyceridemic subjects with different statins is directly proportional
to the statin’s effect on LDL.
Administration
All statins can be given in once-daily dosing. High-dose fluvastatin and
lovastatin are slightly more effective when given in divided doses. Statins
are generally administered at night because of their short half-lives and the
fact that cholesterol synthesis is greater at night. Atorvastatin and rosuvas-
tatin are the exceptions and can be administered at any time of the day, or
even every other day, because of their long half-lives. Food increases the
bioavailability of lovastatin, reduces the absorption of pravastatin and has
no effect on the absorption of other statins. Therefore, lovastatin should be
given with food, pravastatin on an empty stomach, whereas other statins
can be given at any time.
Drug Interactions
Various drugs can reduce statin clearance and produce higher statin blood
levels via effects on the cytochrome P450 (CP450) system or on the newly
discovered statin glucuronidation pathway.31,32 Interaction of the statins with
other drugs, therefore, varies with the metabolic pathways of both the statin
and the concomitant medication. Much has been made about the suscepti-
bility of various statins to drug interaction, but caution should be used with
all statins when combined with agents known to affect the CP450 system.
The CY3A4 isoenzyme of the CP450 system is responsible for metabolism
of atorvastatin, lovastatin and simvastatin, while the CY2C9 isoenzyme is
responsible for metabolism of fluvastatin. Pravastatin is metabolized pre-
dominantly by sulfonation, independent of the CP450 system. Drugs, which
inhibit CY3A4 (such as macrolide antibiotics, azole antifungal agents, anti-
depressants, and protease inhibitors), inhibit glucuronidation (such as gem-
fibrozil) or decrease statin excretion from skeletal muscle (such as cyclospor-
ine) (Table 10.8), increase serum levels of statins and predispose to statin-
induced myopathy.33 Individual statins may have special situations either
increasing their safety or risk. For example in the ALERT trial, which ran-
domized 2102 renal transplant recipients on cyclosporine to either fluvastatin
or placebo, the incidence of serious adverse events, such as ≥ 3-fold increase
in alanine aminotransferase or ≥ 5-fold increase in creatinine kinase concen-
trations and non-fatal rhabdomyolysis, did not differ significantly between
groups.34 In contrast, rosuvastatin maximal concentrations and area under
the curve are increased 11- and 7-fold when this agent is administered with
cyclosporine, suggesting that this agent should be avoided in transplant
patients.35
TABLE 10.8
Risk Factors for Statin-Induced Myopathy
1. Advanced age
2. Hypothyroidism
3. Small body habitus and frailty
4. Multi-system disease (renal insufficiency, DM)
5. Multiple medications
6. Perioperative periods
7. Combination with the following medications
a. Fibrates (especially gemfibrozil)
b. Cyclosporine
c. Azole antifungals (ketoconazole, itraconazole)
d. Macrolide antibiotics (erythromycin, clarithromycin)
e. HIV protease inhibitors
f. Nefazodone
g. Verapamil
h. Amiodarone
i. Grapefruit juice ( >1 quart/day)
j. Alcohol abuse
Side Effects
A variety of adverse effects have been attributed to the statins. Major side
effects include hepatotoxicity and myopathy, which are rare. Common side
effects are myalgia and constipation.
Hepatotoxicity is feared by the public, but is extremely rare, occurring in
< 1% of patients at high doses.36 Asymptomatic elevation of transaminase
levels occurs in 0.5–2.0% of patients, is dose dependent, and generally
reverses with either dose reduction or continued administration.37,38 Progres-
sion to permanent liver damage almost never occurs with statins, but
patients with elevated transaminase levels should be monitored, and the
statin should be discontinued if the levels rise > 3 times the upper limit of
normal.
Statins can produce a variety of muscle complaints.33,39 These have been
variously defined by differing authorities, but include:
1. Myositis and rhabdomyolysis (generally defined as CK > 10 × upper

limits of normal [ULN] plus muscle symptoms)
2. Increased CK < 10 ULN with or without symptoms
3. Myalgia with normal CK values
4. Muscle weakness
5. Muscle cramps
The management of statin myopathy has been discussed in detail else-

where.33 In general, physicians should use the lowest dose of statin necessary
to reach the therapeutic goal and warn patients to discontinue the drug and
seek medical care if they develop important muscle pain, weakness or dark
urine. The incidence of asymptomatic small elevations in CK is not known,
since this information is rarely reported in clinical trials. Different
approaches have been recommended to monitoring muscle enzymes and
symptoms during statin therapy. We suggest that statins be stopped if the
CK level is greater than 10 times the upper limits of normal (ULN). CK levels
should be monitored at least monthly if the CK is 5–10 times ULN and the
patient is asymptomatic. CK elevations < 5 times ULN can be treated with
benign neglect in asymptomatic patients. Myalgias are common with a
reported incidence of 5%. Interestingly, the incidence of myalgia in clinical
trial is similar between placebo and drug therapy groups,38 but most clini-
cians are convinced that these medications can induce myalgia. Reducing
the statin dose may relieve the symptoms, as may switching to another statin.
Rare patients may experience severe myositis characterized by muscle pain,
weakness associated with CK > 10 times ULN. Failure to discontinue statin
therapy in this setting can lead to rhabdomyolysis, renal failure and
even death. Fatal rhabdomyolysis is extremely rare (less than 1 death/mil-
lion prescriptions).39 The incidence rate is similar for all statins except for
cerivastatin (16–80 times higher than other statins).39 Risk factors for statin
myopathy have been presented (Table 10.8).
Clinical Trials
Multiple large trials have proven the efficacy of statins in both the primary
and secondary prevention of CAD.16–20,40 They reduce the risk of myocardial
infarction, cardiac mortality, revascularization procedures and peripheral
vascular disease. They also reduce the risk of stroke and overall mortality
in patients with established CAD. The findings of major statin trials are
summarized in Table 10.9.
Bile Acid-Binding Resins

Introduction
These agents, once a mainstay of lipid-lowering therapy, are predominantly
used as adjuncts to other drugs in patients requiring additional LDL-C
reductions and as single agents in children and patients seeking to avoid
long-term statin use.
Pharmacology and Mechanism of Action

The resins currently available in the United States are cholestyramine, colesti-
pol and colesevelam. They are not absorbed into the systemic circulation.
These agents bind bile acids in the gut thus interrupting their enterohepatic
circulation. Depleting the hepatic bile-acid pool increases hepatic cholesterol
7-alpha-hydroxylase activity, the rate-limiting step in bile synthesis. The
increased diversion of cholesterol to bile acid synthesis depletes hepatic
cholesterol, producing up-regulation of hepatic LDL receptors and increased
clearance of LDL-C from the blood.41 However, HMG CoA reductase and
phosphatidic acid phosphatase activity are also up-regulated, increasing
cholesterol and TG synthesis, respectively, because bile acids suppress activ-
ity of these enzymes.42
Administration
Cholestyramine and colestipol are generally administered as powders mixed
in water or fruit juice. A packet or scoopful contains 4 g of cholestyramine
or 5 g of colestipol.3 The usual dose is 2–6 packets daily. Micronized colestipol
is also available as 1 g tablets. Colesevelam is available as 625 mg tablets
and administered in up to six tablets daily. Approximately 5 g of colestipol
TABLE 10.9
Summary of Clinical Trials of Statins
Duration Baseline Coronary Cardiac Total
Trial Agent Patients (years) Lipids LDL HDL TG Events Revascularization Mortality Mortality Stroke
WOSCOPS Pravastatin 6596 4.9 LDL 192 ↓26% ↑5% ↓12% ↓31% ↓37% ↓28% ↓22% ↓11%
40 mg TG 164 (NS)
HDL 44
AFCAPS/ Lovastatin 6605 5.2 LDL 150 ↓25% ↑6% ↓13% ↓25% ↓33% NS NS NS
TexCAPS 20–40 mg TG 158
HDL 36
HPS Simvastatin 20,536 5 LDL 188 ↓29% ↑3% ↓14% ↓27% ↓24% ↓18% ↓12% ↓25%
40 mg TG 135
HDL 46
4S Simvastatin 4444 5.4 LDL 188 ↓35% ↑8% ↓10% ↓34% ↓37% ↓42% ↓30% ↓27%
10/40 mg TG 135
HDL 46
CARE Pravastatin 4159 5 LDL 139 ↓28% ↑5% ↓14% ↓25% ↓27% ↓24% ↓9% ↓31%
40 mg TG 135
HDL 39
Pharmacological Treatments of Lipid Abnormalities
LIPID Pravastatin 9014 6.1 LDL 150 ↓25% ↑5% ↓11% ↓29% ↓24% ↓24% ↓23% ↓19%
40 mg TG 138
HDL 36
NS, not significant; ↓, reduction; ↑, elevation; LDL, low-density lipoprotein; HDL, high-density lipoprotein; TG, triglycerides.
195
are equivalent to 4 g of cholestyramine in their LDL-lowering effect.27

Colesevelam is 4–6 times more potent as a bile-acid sequestrant than the
other two agents.43 The powdered resins are impalatable for many patients
and their physical bulk makes them inconvenient. Colesevelam is more
easily administered and better tolerated than other sequestrants.
Given alone, resins lower LDL-C 10–24% depending on the dose, and pro-
duce an additive effect when combined with a statin.3,44 The statin inhibition
of HMG CoA increases the efficacy of the resin. In a trial comparing LDL
lowering with atorvastatin and colesevelam, the LDL-C reduction with ator-
vastatin 80 mg was not significantly different from the combination of
colesevelam 3.8 g and atorvastatin 10 mg.45 Resins increase HDL-C approx-
imately 3–5%. Resins often increase TG levels by up-regulating activity of
phosphatidic acid phosphatase and are not indicated in patients with hyper-
triglyceridemia.46
Drug Interactions
Resins non-specifically bind coadministered drugs such as warfarin, digi-
talis, thyroxine, non-steroidal anti-inflammatory agents, oral hypoglycemic
agents, statins and gemfibrozil. These interactions can be avoided by admin-
istering the other drugs either 1 h before or 4 h after the administration of
the resin. Colesevelam apparently does not interfere with the absorption of
these drugs and need not be administered separately.47
Side Effects
Up to 39% of patients experience constipation, bloating, epigastric fullness,
flatulence and nausea, giving these drugs a high rate of discontinuation.48,49
Administration of viscous fiber or prune juice may help avoid constipation.
Colesevelam is less likely to cause GI side effects and is better tolerated.50
Clinical Trials
The Lipid Research Clinics Coronary Primary Prevention Trial (LRC-CPPT)
involved 3806 men with hypercholesterolemia but without known coronary
disease.48,49 Cholestyramine treatment reduced mean LDL-C by 12% com-
pared with placebo and decreased non-fatal myocardial infarction and car-
diac death by 19%, new-onset angina by 20% and new positive exercise tests
by 25%, but did not alter total mortality. In the Familial Atherosclerosis
Treatment Study (FATS) coronary atherosclerosis regression trial, intensive
lipid lowering with colestipol in combination with either lovastatin or niacin

reduced the frequency of progression of coronary lesions, increased the
frequency of regression, and reduced the incidence of cardiovascular events
by 70%.51
Nicotinic Acid
Introduction
Niacin or nicotinic acid is a water-soluble compound that functions as a B-
vitamin at low doses and as a lipid-lowering agent at high doses. A related
compound nicotinamide lacks any lipid-lowering effect and functions only
as a vitamin.3
Pharmacology
Niacin is available in two forms: immediate release or crystalline (nicotinic
acid), and sustained release or extended release (Niaspan) forms. The crys-
talline form has to be administered several times in a day and has a high
incidence of flushing. Sustained release formulations cause less flushing than
immediate release preparations, but have a higher incidence of hepatotox-
icity.52
Mechanism of Action
Niacin has multiple potential actions, but its major effect is to inhibit lipolysis
in peripheral tissues thereby reducing the availability of free fatty acids for
hepatic TG synthesis. Reduced substrate for hepatic triglyceride synthesis
ultimately reduces hepatic secretion of apo-B and VLDL.53
The mechanism by which niacin increases HDL is less clear. It appears to
decrease the hepatic uptake of apo AI, thereby reducing HDL catabolism.
Niacin increases HDL-C 15–40%, an effect greater than any other currently
available lipid-lowering drug.3,52,54 The reduction in TGs is more modest, in
the range of 20–35%, with up to 50% reductions seen in patients with hyper-
triglyceridemia. The effect on LDL-C is variable (5–25% reduction). The effect
on HDL-C and TG concentrations is log-linear, with significant changes seen
at low doses whereas high doses are required to produce clinically important
effects on LDL-C.55
Administration
Niacin produces flushing, which can be reduced by slowly increasing the
dose. Crystalline niacin is usually started at doses of 100 mg four times daily
and increased by 100 or 250 mg per dose weekly to 1 g four times a day.
Long-release preparations are started at 500 mg at bedtime and increased
by not more than 500 mg weekly to 1.5–2.0 g/day. The most important niacin
dose is the bedtime dose since niacin inhibits lipolysis, which is greater
overnight and during other periods of fasting.
Side Effects
The most common side effect is prostaglandin-mediated cutaneous vasodi-
latation causing flushing and pruritus.27 Most patients develop tachyphy-
laxis and tolerance to flushing after prolonged use. Flushing can be reduced
by taking the drug with food, or by using aspirin 325 mg or ibuprofen 200
mg 30 min before or with the dose. Niacin can also worsen glucose tolerance,
activate gout and peptic ulcers, and produce conjunctivitis, nasal stuffiness,
acanthosis nigricans, ichthyosis and rarely, retinal edema.3
A serious side effect is hepatotoxicity, including jaundice and fulminant
hepatitis.52–54 The onset of hepatotoxicity is unpredictable, requiring regular
monitoring of serum transaminase levels at 4–6-month intervals throughout
therapy. Niacin should be discontinued if transaminase levels are persis-
tently elevated. Hepatotoxicity should also be suspected if there is a dramatic
reduction in lipoprotein levels.56 The risk of hepatotoxicity is higher with
higher doses and with the use of sustained release formulations.3
Clinical Trials
The Coronary Drug Project (CDP) was a large secondary prevention trial,
which compared five lipid-lowering drugs including nicotinic acid in 8341
patients with known CAD.57 Treatment with nicotinic acid reduced major
coronary events by 25% and on long-term follow-up (15 years) resulted in
an 11% decrease in total mortality.58 In the Stockholm study, 555 patients
with myocardial infarction were randomized to either placebo or the com-
bination of nicotinic acid and clofibrate. Lipid-lowering therapy lowered
serum cholesterol by 13% and TGs by 19% and reduced CAD mortality by
36% and total mortality by 28%.59
Fibrates
Introduction
Fibric acid derivatives or fibrates are effective for lowering TGs and increas-
ing HDL-C. Three fibrates are currently approved in the United States: clo-
fibrate, gemfibrozil and fenofibrate, although clofibrate is difficult to obtain.
Two more fibrates are available in Europe: bezafibrate and ciprofibrate.
Pharmacology
Absorption of gemfibrozil is approximately 44% higher when given 0.5 h
before meals, whereas fenofibrate absorption is 35% higher when given with
meals.60 Both gemfibrozil and fenofibrate should be used cautiously in
patients with renal insufficiency since they are partially renally cleared.
Mechanism of Action
Fibrates activate the nuclear transcription factor, peroxisome proliferator-
activated receptor (PPAR)-alpha producing61–63:
1. Activation of lipoprotein lipase (LPL) and decreased expression of

the LPL inhibitor, apo C-III. Increased LPL activity helps clear tri-
glyceride-rich particles.
2. Increased oxidation of fatty acids in the liver and muscle, reducing
the synthesis of VLDL-TGs.
3. Increased synthesis of apo A-I and A-II, which increases HDL-C.
Fibrates reduce TGs by 30–35% in normal subjects and up to 55% in severely
hypertriglyceridemic patients, and increase HDL-C by 10–15%. These
changes are greater in patients with very high TGs and low HDL-C.60 The
effects on LDL are variable. Fibrates may decrease LDL-C by 10–20% in
normotriglyceridemic patients, but can increase LDL in hypertriglyceridemic
subjects by facilitating the transformation of VLDL to LDL via increased LPL
activity and VLDL delipidation.64,65
Indications
Fibrates are useful for managing elevated TG levels, for increasing HDL-C
especially in hypertriglyceridemic subjects, and in combination with resins
or ezetimibe for statin intolerant patients with elevated LDL-C levels.
Side Effects
The most common side effects of gemfibrozil are upper gastrointestinal
disturbances such as dyspepsia. Both gemfibrozil and fenofibrate can
increase liver function tests (LFTs), which in our experience seems most
common in patients with steatohepatitis. Treatment should be continued
unless the patient is symptomatic or the LFTs are > 3 times ULN. Fibrates
increase the biliary secretion of cholesterol, increasing the likelihood of gall-
stone disease.66 Fibrates have also been associated with myositis and rhab-
domyolysis in patients receiving concomitant therapy with statins especially
with concomitant azotemia.66 Fenofibrate may be safer than gemfibrozil in
combination with statins because it dose not affect the glucuronidation clear-
ance pathway and is not metabolized by the CYP3A4 system.67,68
Fibrates have been implicated in increased carcinogenesis in animal stud-
ies, and there was increased mortality from cancer in the WHO clofibrate
trial.69 However, other human trials have failed to show increased incidence
of cancer with fibrates.
Clinical Trials
Several primary and secondary prevention trials have examined the role of
fibrates in CAD69–72 (Table 10.10). In the WHO clofibrate trial, clofibrate
reduced coronary events. Total mortality was increased, mainly due to the
diseases of gastrointestinal and biliary tracts, reducing the enthusiasm for
the use of fibrates. However, subsequent primary and secondary prevention
trials with gemfibrozil reduced CAD and did not increase total mortality.
Not all trials, however, have shown a reduction in CAD events. The BIP
secondary prevention trial failed to show any reduction in coronary ischemic
events and revascularization with bezafibrate70 and in the Coronary Drug
Project, clofibrate did not reduce recurrent coronary events.57
Cholesterol Absorption Inhibitors

Introduction
Ezetimibe belongs to a new class of lipid-lowering drugs, which selectively
inhibit intestinal absorption of cholesterol and phytosterols.73
TABLE 10.10
Summary of Clinical Trials of Fibrates
Duration Coronary
Trial Agent Type Patients (years) TC HDL TG Mortality Events MI Stroke
WHO Clofibrate Primary prevention 15,745 5.3 ↓11% NA ↓24% ↑43% ↓20% 25% NA
HHS Gemfibrozil Primary prevention 4081 5 ↓14% ↑8% ↓34% ND ↓34% ↓37% ND
BIP Bezafibrate Secondary prevention 3090 6.2 ↓6.5% ↑18% ↓21% ND ND 13% ND
VA-HIT Gemfibrozil Secondary prevention 2531 5 ↓4% ↑6% ↓31% ND ↓22% ↓23% ↓29%
NA, not available; ND, no difference; ↓, reduction; ↑, elevation; HDL, high-density lipoprotein; TG, triglycerides.
Pharmacological Treatments of Lipid Abnormalities
201
Pharmacology
Ezetimibe’s absorption is not affected by food, it can be given daily or even
every other day because of its half-life of 24 h, and it does not interact with
other medications.60
Mechanism of Action
Ezetimibe interacts with the Nieman Pick Like Protein 1 in the intestine to
inhibit absorption of dietary and biliary cholesterol by up to 54%.74 Reduced
delivery of cholesterol to the liver depletes hepatic stores, up-regulates
hepatic LDL-receptor activity thereby increasing LDL-C clearance.
Ezetimibe alone reduces LDL-C by approximately 18%, produces small (5%)
non-significant reductions in TGs and small (3.5%) but significant increases
in HDL-C.75,76 Statin administration increases intestinal cholesterol absorp-
tion. Ezetimibe reduces intestinal cholesterol absorption, making ezetimibe
very effective in combination with statins. For example, ezetimibe 10 mg
plus atorvastatin 10 mg reduces LDL-C 53%, similar to the 54% reduction
achieved with the 80 mg or maximum dose of atorvastatin.77 Similar data
are available with other statins.78–80
Indications
Ezetimibe is useful, therefore, either as single therapy in patients intolerant
of statins or in combination with other lipid-lowering agents for reducing
TC, LDL-C and apo-B in patients with primary hypercholesterolemia.
Other Therapies
Fish Oils
Oils derived from fatty fish (salmon, mackerel, tuna, sardines and herring)
are rich in such n-3 (omega) fatty acids as eicosapentanoic acid (EPA) and
docosahexanoic acid (DHA), which can be used to reduce TG levels.81 Plant
sources of omega-3 fatty acids, including flaxseed, canola oil, soybean oil
and nuts, are not as effective as fish oils in reducing TGs.82
Mechanism of Action
The mechanism by which omega-3 fatty acid (FA) reduces TGs is not known,
but is thought to involve both decreased production and increased catab-
olism of TG-rich lipoproteins.
High omega-3 FA intakes are required to reduce TG levels. An intake of
3 g per day is associated with a 30% TG reduction and 9 g/day reduces
TGs by as much as 50%.83 Reduction in TG levels may be accompanied
by increases in LDL-C levels. Commercial fish oil capsules are approxi-
mately 30% EPA by weight. Postmyocardial infarction patients treated
with 1 g of EPA daily, which is approximately equal to three fish oil
capsules, experienced a 30% reduction in cardiovascular events and a 45%
reduction in sudden death.84 Fish oils also reduce platelet aggregation and
may reduce blood pressure.81
Side Effects
The doses of fish oil required to reduce TGs can produce nausea, abdom-
inal bloating, flatulence and diarrhea. Patients may note a fishy odor and
an after-taste. Platelet inhibition may lead to nosebleeds and easy
bruiseability.81 Also, since each 1 g capsule contains 9 g of fat, patients
often gain weight.
Indications
Fish oils are used to treat hypertriglyceridemia. They can be added to statins
in patients in whom the addition of fibrates or niacin to a statin is contrain-
dicated or not tolerated. The ATP-III panel recommends consumption of fish
oils as an optional treatment modality due to the lack of strong data sup-
porting its use.
Clinical Trials
A fish diet was superior to high-fiber and low-fat diets for reducing cardio-
vascular events in patients with known CAD in the Diet and Reinfarction
trial.85 In the GISSI prevention study, recent myocardial infarction patients
treated with 1 g of EPA from fish oils daily experienced a significantly lower
incidence of cardiovascular events, due to a 30% reduction in cardiovascular
deaths, including a 45% reduction in sudden deaths.84
Selection of Drug
Statins are usually the drugs of first choice for LDL-C lowering therapy.
They are the most effective LDL-C lowering agents currently available,
easy to administer and safe. The different statins produce different degrees
of LDL-C reduction and are metabolized by different routes. Therefore,
the initial choice of statin depends on the magnitude of LDL-C reduction
required to reach the LDL goal and the presence of associated medical
disorders or concomitant drug therapy, since the later may influence statin
metabolism.
For patients requiring large reductions in LDL-C levels, rosuvastatin or
atorvastatin may be the statins of first choice, whereas in patients requiring
only modest reductions in LDL-C, pravastatin or simvastatin may achieve
the therapeutic goals. Pravastatin and fluvastatin may be the preferred drugs
for patients receiving concomitant therapy with drugs that inhibit the
CYP3A4 enzymes, such as macrolide antibiotics and HIV-protease inhibitors.
Similarly, in post-transplant patients receiving cyclosporine, pravastatin or
fluvastatin may be preferred drugs.
Ezetimibe and bile-acid sequestrants are the next most effective LDL-C
lowering drugs. They can be used as a monotherapy, when only mild reduc-
tions in LDL-C are required, especially in young patients. However, their
major use is in addition to a statin to maximize LDL-C reduction. In general,
doubling the dose of a statin produces only an additional 6% LDL-C reduc-
tion, whereas adding either ezetimibe or a bile-acid sequestrant to a starting
dose of a statin can produce reductions in LDL-C similar to those achieved
by the maximal statin dose. This may be beneficial in patients who are unable
to tolerate higher doses of a statin and to reduce the risk of statin side effects.
Adding ezetimibe or a resin to the maximal dose of a statin produces further
reduction in LDL-C. Ezetimibe is better tolerated by patients than resins.
Nicotinic acid and fibrates are not primary LDL-C reducing agents and
hence are usually not the first line of therapy for LDL elevations. However,
these agents may be used if additional LDL-C lowering is required after
maximal doses of statins and absorption inhibitors or if other agents are not
tolerated. Nicotinic acid and fibrates may be used as initial agents in patients
whose primary lipoprotein abnormality is elevated TGs and/or low HDL.
Monitoring
Baseline liver function tests (LFTs) and measurement of creatine kinase,
fasting blood sugar and thyroid-stimulating hormone levels are recom-
mended before therapy. The maximal response to statins occurs within 3
weeks of drug initiation whereas other medications may require 6–8 weeks.
Follow-up lipid levels should be obtained as soon as any effect will be
detectable to provide the patient with appropriate feedback. LFTs with statin
therapy should be obtained after 12 weeks of treatment since LFTs often
increase transiently in the first 12 weeks of therapy. LFTs can then be mea-
sured annually thereafter during statin therapy. We recommend monitoring
LFTs during niacin therapy every 4 months. Statin-treated patients should
be warned of the risk for myopathy and told to stop the drug and report
promptly for a creatine kinase determination if symptoms appear. Once
treatment goals are achieved, follow-up visits should be scheduled every
6–12 months.
Summary
Most patients with important vascular disease or increased risk of vascular
disease will require lipid-lowering therapy to reduce their risk of recurrent
or primary CAD events. Available lipid-lowering medications are extremely
effective in reducing LDL-C and TGs and in increasing HDL-C. These agents
can be used individually or in combination therapy, and most agents have
been documented to reduce CAD events.
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11
New Insights on the Role of Lipids and
Lipoproteins in Cardiovascular Disease: The
Modulating Effects of Nutrition
Kirsten F. Hilpert, Amy E. Griel, Tricia Psota, Sarah Gebauer,

Yumei Coa, and Penny M. Kris-Etherton
CONTENTS
Introduction .........................................................................................................212
The Effect of Nutrients on Lipids and Lipoproteins ....................................213
Total Fat.......................................................................................................213
Saturated Fatty Acids................................................................................217
Trans Fatty Acids .......................................................................................218
Monounsaturated Fatty Acids .................................................................219
Polyunsaturated Fatty Acids ...................................................................220
n-6 PUFA ........................................................................................220
n-3 PUFA ........................................................................................221
Dietary Cholesterol....................................................................................222
Dietary Fiber...............................................................................................222
Epidemiologic Evidence of Fiber and Heart Disease .............222
The Role of Dietary Fiber in Lipid Management ....................224
Glycemic Index/Glycemic Load .............................................................225
Conclusion ..................................................................................................227
Effects of Dietary Patterns on Lipids and Lipoproteins ..............................228
NCEP Recommendations .........................................................................228
Portfolio Diet ..............................................................................................230
Lifestyle Heart Program ...........................................................................231
Mediterranean Diet ...................................................................................232
Dietary Approaches to Stop Hypertension (DASH) Diet...................233
Effects of Weight Loss Diets on Lipids and Lipoproteins ...........................233
Low-Fat Diets .............................................................................................234
Moderate-Fat Diets with MUFA .............................................................234
High-Protein Diets.....................................................................................235
211
Emerging Lipid and Lipoprotein CVD Risk Factors Affected by Diet .....236
Diet Effects on LDL Particle Size ............................................................236
The Effects of a Changing Macronutrient Profile on LDL
Particle Size ....................................................................................237
Effects of High-Fat, Moderate-Fat Diets....................................237
Effects of High-Protein, Low-Carbohydrate Diets ..................237
Other Dietary Interventions – Type of Fat,
Dietary Fiber, and Multiple Dietary Strategies..........239
HDL Particle Size.......................................................................................240
Postprandial TG .........................................................................................242
Lipoprotein (a) ...........................................................................................244
Science-Based Dietary Guidelines for Health ................................................246
Summary ..............................................................................................................246
References ............................................................................................. 248
Introduction
The first lipoprotein particle identified was high-density lipoprotein (HDL)
that was isolated from horse serum in 1929.1 Since the discovery of plasma
lipoproteins (reviewed in Ref. 2), remarkable progress has been made in
understanding their role in the development and progression of cardiovas-
cular disease (CVD). The progress was catalyzed by the development of a
method for the quantitative measurement of different serum lipoproteins
isolated by ultracentrifugation.3 Historically, a panel of plasma lipid and
lipoprotein abnormalities has been a primary intervention target for reduc-
ing risk of CVD.4 With respect to plasma lipids and lipoproteins, an elevated
total and LDL cholesterol (LDL-C) have a long-standing history of being
the major risk factors for CVD. Low HDL cholesterol (HDL-C) and elevated
triglyceride (TG) levels have been identified more recently as important risk
factors for CVD. In addition, other lipoprotein constituents (e.g., apolipo-
proteins A and B) have been shown to have important biological functions
related to CVD. During the past 10 years, it has become abundantly clear
that each lipoprotein fraction is remarkably heterogeneous with respect to
both composition and biological function.5
Numerous epidemiologic studies have shown beneficial effects of single
nutrients as well as dietary patterns on morbidity and mortality related to
CVD. Over the past several decades, a major scientific effort has been
devoted to identifying the mechanisms by which diet impacts overall CVD
risk via modification of lipids and lipoproteins. Research has demonstrated
that nutrition can modulate both lipoprotein composition and function in
ways that span the spectrum from anti-atherogenic to pro-atherogenic.
Dietary factors that affect lipids and lipoproteins include saturated fatty
acids (SFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty
New Insights on the Role of Lipids and Lipoproteins in Cardiovascular Disease 213
acids (PUFA), trans fatty acids (TFA), dietary cholesterol, dietary sterols and
stanols, and soluble fiber. Weight status including gaining or losing weight
markedly affects plasma lipids and lipoproteins. Modifications of lipids and
lipoproteins by these dietary factors can substantially reduce risk of CVD.
Other dietary and lifestyle interventions, such as dietary patterns that
include fruits, vegetables, whole grains, low-fat dairy products, fish and
nuts, along with physical activity, also can significantly reduce risk of CVD
that can be explained mechanistically, in large part, by changes in lipids and
lipoproteins.
The focus of this chapter is to review the current understanding of the
effects that diet has on lipids and lipoproteins with emphasis on a discussion
of the maximal effects that can be expected given ideal adherence to a dietary
intervention that is low in SFA, TFA, dietary cholesterol and high in plant
sterols/stanols and soluble fiber. In addition, observational evidence of the
association between diet and CVD morbidity and mortality is discussed to
provide a rationale for the numerous clinical studies that have evaluated the
effects of diet on lipids and lipoproteins, important risk factors for CVD.
The Effect of Nutrients on Lipids and Lipoproteins

It is well established that elevated levels of total cholesterol (TC), LDL-C,
and TG increase CVD risk, while a high level of HDL-C reduces CVD risk.
These lipid risk factors are the focal point for cholesterol-lowering treatment
guidelines developed by the third Adult Treatment Panel of the National
Cholesterol Education Program.6 The well-known effects of nutrients on TC,
LDL-C, HDL-C, and TG are reviewed in Table 11.1. The total fat content of
the diet is comprised of a mixture of individual fatty acids. Dietary sources
of fat are described in Table 11.2. Furthermore, different food sources of fat
differ substantially in their fatty acid profiles (Figure 11.1). This section
reviews studies demonstrating the effects of total fat, SFA, trans fatty acids,
MUFA, PUFA, dietary cholesterol, fiber, and the glycemic index/glycemic
load on lipids and lipoproteins.
Total Fat
Numerous studies have been conducted that have compared the plasma
lipid and lipoprotein responses to different blood cholesterol-lowering diets
that vary in the amount of total fat and carbohydrate. Low-fat/high-carbo-
hydrate diets (18–30% of kilocalories [kcal] total fat) have been compared
with higher fat diets (30–40% of kcal) that provide similar amounts of SFA
(4–12% of kcal) and dietary cholesterol (< 100–410 mg/day). Consistently, a
low-fat, high-carbohydrate diet compared with a higher fat diet (both
TABLE 11.1
Expected Lipid Response of Selected Dietary Components
Dietary Expected Lipid Response
Component NHLBI Evidence Statement6 LDL-C TC HDL-C TG
Total fat Unsaturated fat does not raise ↔ ↔ ↑ ↓a
LDL-C when substituted for
CHO; it is not necessary to
restrict total fat intake for
LDL-C reduction, provided
SFA are reduced
Saturated fat There is a dose response ↑↑↑ ↑↑↑ ↑ ↓a
relationship between SFA
and LDL-C; diets high in
SFA raise LDL-C and
reducing dietary SFA lowers
LDL-C
Trans fat Trans fatty acids raise LDL-C ↑↑↑ ↑↑↑ ↔a or ↓b ↔
and intake should be kept as
low as possible
Cholesterol Higher intakes of dietary ↑ ↑ ↑ ↔
cholesterol raise LDL-C
n-6 PUFA Linoleic acid, a PUFA, reduces ↓↓ ↓↓ ↔ or ↓ ↓a
LDL-C levels when
substituted for SFA in the
diet; PUFAs also can cause
small reductions in HDL-C
when compared with
MUFA; controlled clinical
trials indicate that
substitution of PUFA for SFA
reduces risk for CHD
n-3 PUFA The mechanisms whereby ↔ or ↑c ↔ ↑ ↓↓↓
they might reduce coronary
events are unknown and
may be multiple; prospective
and clinical evidence suggest
that higher intakes of n-3
fatty acids reduce risk for
CHD events and mortality
MUFA MUFA lowers LDL-C relative ↔ or ↓d ↔ ↑ ↓a
to SFA; mUFA does not
lower HDL-C nor raise TG;
to lower LDL-C, energy
derived from SFA can be
reduced if weight loss is
desirable or replaced with
either CHO or MUFA when
weight loss is not a goal
(continued)

Expected Lipid Response of Selected Dietary Components
Dietary Expected Lipid Response
Component NHLBI Evidence Statement6 LDL-C TC HDL-C TG
Carbohydrate When CHO is substituted for ↓d ↓d ↔ or ↓ ↔ or ↑
SFA, LDL-C decreases;
however, very high intakes
of CHO (>60% total kcal) can
reduce HDL-C and raise TG;
viscous fiber may attenuate
this response
Viscous fiber Use dietary sources of viscous ↓ ↓ ↑ ↔ or ↓
fiber (5–10 g/day) to reduce
LDL-C
Plant stanols/ Use 2–3 g/day to enhance ↓↓↓ ↓↓ ↔ ↔
sterols LDL-C lowering
Soy protein Soy protein can cause small ↓ ↓ ↔ ↔ or ↓
reductions in LDL-C,
especially when it replaces
animal food products
Weight reduction Weight reduction of even a ↓↓ ↓↓ ↑ ↓↓e
(– 10 lb) few pounds will reduce
LDL-C regardless of the
nutrient composition of the
diet, but weight reduction
achieved through a calorie-
controlled low-SFA and
cholesterol diet will enhance
and sustain LDL-C lowering
↑, increase; ↓, decrease; ↔, no change.
a When substituted for dietary carbohydrate.
b Compared with saturated fatty acids.
c Increases LDL size.
d When substituted for saturated fatty acids.
e TG may rebound after maintenance of weight loss, when consuming a high-carbohydrate,
low-fat, low-fiber diet.
relatively low in SFA and cholesterol) decreases LDL-C levels similarly.7–19

Low-fat diets, however, decrease HDL-C and since HDL-C is proportionately
decreased as LDL-C, the ratio of LDL-C to HDL-C does not change.20 On a
moderate-fat diet, however, when unsaturated fat replaces SFA, LDL-C
decreases proportionately more than HDL-C thereby decreasing the
LDL:HDL-C ratio.21 Low-fat, high-carbohydrate diets increase fasting TGs
versus moderate-fat diets, both of which are low in saturated fat. Viscous
fiber may attenuate the hypertriglyceridemic response to dietary carbohy-
drate.6 Within the range of total fat evaluated in the controlled feeding
studies conducted to date, there is a linear dose-response relationship
between total fat content of the diet and the changes in HDL-C and TG.22
In some individuals, low-fat, high-carbohydrate diets, compared with
higher fat diets, induce atherogenic dyslipidemia,6 which is characterized by
TABLE 11.2
Sources of Fatty Acids in the Diet
Type (Chemical Structurea) Dietary Source
Saturated Fat
Butyric (4:0) Butterfat

Lauric (12:0) Coconut oil, palm kernel oil
Myristic (14:0) Butterfat, coconut oil
Palmitic (16:0) Palm oil, animal fat
Stearic (18:0) Cocoa butter, animal fat
Unsaturated Fat
Monounsaturated
Oleic (18:1) Olive oil, canola oil, peanuts, avocado, sunflower oil
Elaidic or trans fat (18:1) Hydrogenated vegetable oil and ruminant fat
Polyunsaturated
n-6 fatty acids
Linoleic (18:2) Vegetable oils (e.g., soybean, corn, safflower)
Arachidonic (20:4) Lard, meat
n-3 fatty acids
α-Linolenic (18:3) Soybean oil, canola oil, walnuts, flaxseed
EPA (20:5) Fish oils, algae
DHA (22:6) Fish oils, algae
EPA, eicosapentanoic acid; DHA, docosahexanoic acid.
a Carbon chain length:double bond.
Canola Oil
Flaxseed Oil
SFA
Walnut Oil
MUFA
mid-oleic Sunflower Oil

Omega-6
Corn Oil
Omega-3
Olive Oil
Soybean Oil
Peanut Oil
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
Fatty Acid content normalized to 100%

a
Source: USDA Nutrient Database, release 15
FIGURE 11.1
Fatty acid composition of common oils. Source: USDA Nutrient Database, release 15.
small dense LDL particles, high TG and low HDL-C levels. In sedentary,
overweight or obese populations, in particular, low-fat, high-carbohydrate
diets increase the prevalence of this phenotype. This phenotype is associated
with increased risk of coronary heart disease (CHD).23
Saturated Fatty Acids

The Seven Countries Study was a classic epidemiologic study that reported
a strong positive correlation between SFA intake and CHD mortality rates,
as well as a significant association between total SFA intake and TC.24 Sub-
sequent epidemiologic studies also have found correlations with classes of
SFA and TC levels and incidence of CHD.25,26 In a more recent analysis of
the study, strong positive associations were reported between 25-year death
rates from CHD and average intake of the four major saturated fatty acids:
lauric, myristic, palmitic, and stearic acid (r > 0.8).27 Specifically, intakes of
lauric acid (12:0) and myristic acid (14:0) were most strongly associated with
TC (r = 0.84, r = 0.81, respectively).
Clinical trials confirm the associations between SFA and TC observed in
epidemiologic studies. The early studies by Keys et al.28 and Hegsted et al.29
in the 1960s evaluated the effect of individual fatty acids on TC in humans
using regression analysis on data from many clinical studies. Predictive
equations estimate that SFA raises TC compared with carbohydrates and
MUFA (which both have neutral effects), while PUFA lowers TC. Clinical
studies also have demonstrated the LDL-C raising effect of SFA.30,31 For every
1% increase in energy from SFA, LDL-C levels will increase approximately
0.033–0.045 mmol/L.21,32,33 In addition to raising TC and LDL-C, SFA also
has been shown to increase HDL-C levels. It is estimated that for every 1%
increase in SFA, HDL-C will increase by 0.011–0.013 mmol/L.21,32,33
In the Dietary Effects on Lipoproteins and Thrombogenic Activity (DELTA)
Study, an average American diet (AAD) (34% kcal total fat, 15% kcal SFA)
was compared with a Step I diet (28.6% kcal total fat, 9% kcal SFA), and a
low-SFA diet (25.3% kcal total fat, 6.1% kcal SFA).34 TC was reduced 5% and
9% on the Step I and low-SFA diets, respectively, compared with the AAD
(both p < 0.01). LDL-C and HDL-C were reduced similarly by 7% and 11%,
respectively, on both the Step I and low-SFA diets versus the AAD (both p
< 0.01).
In addition to equations that incorporate classes of fatty acids, equations
have been generated to predict how alterations in individual dietary fatty
acids affect TC, LDL-C, and HDL-C. Recent regression analyses have dem-
onstrated that stearic acid (18:0) has no affect on TC, LDL-C, and HDL-C,35
while myristic acid (14:0) is more hypercholesterolemic than lauric acid (12:0)
and palmitic acid (16:0).36 A recent meta-analysis of 60 controlled trials deter-
mined the effects of different SFA relative to carbohydrate (CHO) on the
TC/HDL-C ratio.37 Although lauric acid was found to increase LDL-C
the most, it decreased the ratio of TC/HDL-C due to a greater increase in
HDL-C levels relative to TC. Myristic and palmitic acids had little effect on
the ratio due to similar increases in both TC and HDL-C. Stearic acid reduced
the ratio due to slight increases in HDL-C.
Trans Fatty Acids

Elaidic acid (t-18:1) is the predominant trans fatty acid found in some hydro-
genated fats which are used in commercially prepared baked products, fried
foods, and margarine. In the Seven Countries Study, the average intake of
elaidic acid was positively associated with TC (r = 0.70, p < 0.01) and 25-year
mortality rates from CHD (r = 0.78, p < 0.001).27 This association has been
confirmed by other epidemiologic studies as well.38,39 Using follow-up data
from the Nurses’ Health Study, Hu et al.26 found that compared with equiv-
alent energy from CHO, the relative risk (RR) for a 2% increment in energy
from trans fatty acids was 1.93 (p < 0.001). The RR for trans fatty acids was
higher than that for 5% of energy from SFA, and 5% from total fat (RR = 1.17,
p = 0.10, and RR = 1.02, p = 0.55, respectively). Studies have shown that trans
fatty acids increase CHD risk by various lipid-mediated mechanisms includ-
ing raising LDL-C, lowering HDL-C, and raising TG.40 In addition, LDL
particle size is decreased,41 and lipoprotein (a) is increased42–44 by trans fatty
acids (reviewed in a later section).
A recent clinical trial conducted by Judd et al.45 evaluated the effects of
replacing carbohydrates with trans fatty acids on LDL-C. Subjects were fed
diets providing approximately 15% of energy from protein, 39% from total
fat, and 46% from CHO. TC was increased by 5.8% and LDL-C was increased
by 10% when trans fatty acids replaced 8% of the energy provided by CHO.
When 8% of the energy provided by CHO was replaced with a combination
of 4% trans fatty acids and 4% stearic acid, TC was increased by 5.6% and
LDL-C was increased by 8.7%. In a review of the trans fat studies that have
been conducted, a linear dose-dependent relationship was reported between
trans fatty acid intake and the LDL:HDL ratio from intakes of 0.5–10% of
total calories.46 The magnitude of this effect is greater for trans fatty acids
than for SFA.
Several clinical trials have reported an HDL-C-lowering effect of trans fat
when compared with saturated fat. In a study conducted by Mensink et al.,47
subjects were placed on three diets that were identical in nutrient composi-
tion except that 10% of total calories were either from oleic acid, trans isomers
of oleic acid, or SFA. The mean HDL-C level was the same on the SFA and
oleic acid diets, but was 0.17 mmol/L lower on the trans fatty acid diet (p <
0.0001). Likewise, a high trans fat diet (9.2% kcal trans fat, 12.9% kcal SFA)
produced a greater reduction in HDL-C by 0.36 mmol/L compared with a
high SFA diet (0% kcal trans fat, 22.9% kcal SFA).48 These studies and others49
indicate that trans fatty acids are unfavorable due to their HDL-C-lowering
effect in addition to their LDL-C raising effect.
Lichtenstein et al.50 conducted a clinical trial evaluating the effects of

different types of hydrogenated fats on lipids and lipoproteins. The experi-
mental diets provided 30% energy from total fat and were identical except
for fat source. Two-thirds of the fat was provided by soybean oil (< 0.5 g
trans fat per 100 g of fat), semi-liquid margarine (< 0.5 g per 100 g), soft
margarine (7.4 g per 100 g), shortening (9.9 g 100 g), stick margarine (20.1 g
per 100 g), or butter (1.25 g per 100 g). The soybean-oil diet compared with
the butter diet resulted in reductions of TC, LDL-C, and HDL-C of 10%, 12%,
and 3%, respectively. The semi-liquid margarine diet compared with the
butter diet resulted in reductions of 10%, 11%, and 4%, respectively. The stick
margarine diet resulted in reductions of 3%, 5%, and 6%, respectively.
Although all of the vegetable fat diets resulted in decreases in TC, LDL-C,
and HDL-C compared with the butter diet, stick margarine (containing the
highest amount of trans fatty acid) decreased LDL-C the least and decreased
HDL-C the most compared with the other vegetable fats. This resulted in a
4% increase in the TC:HDL-C ratio, whereas the other vegetable fats slightly
decreased the ratio. The soybean oil and semi-liquid margarine diets, which
contained the lowest amount of trans fat, had the most beneficial effects on
blood lipoproteins. The soybean oil caused the greatest reduction in TC and
LDL-C and the smallest reduction in HDL-C, resulting in the greatest
decrease in the TC:HDL-C ratio of 6%. The semi-liquid margarine diet
resulted in a 5% reduction in the TC:HDL-C ratio. This study demonstrates
that increases in trans fat result in a dose–response increase in LDL-C. The
study also indicates that at levels higher than typically consumed in the diet,
which is approximately 2.6% kcal, trans fatty acids decrease HDL-C.
The effects of trans fatty acids on TC, LDL-C, and HDL-C have been
compared with other fatty acids via the development of blood cholesterol
predictive equations. Results indicate that trans fatty acids increase TC and
LDL-C less than SFA, but lower HDL-C more than SFA.36
Monounsaturated Fatty Acids

Unsaturated fat (both MUFA and PUFA) as well as carbohydrate can be used
to replace saturated and trans fatty acid calories, which are both targets for
reduction in cholesterol-lowering diets. Oleic acid, the primary MUFA in the
diet, has been shown to have a neutral effect on TC. Epidemiologic studies
have found inverse associations between MUFA intake and risk of CHD and
ischemic heart disease (IHD) after adjusting for SFA and dietary choles-
terol.26,51,52 Furthermore, the Seven Countries Study showed that rates of
coronary artery disease (CAD) were low despite moderately high total fat
intakes when SFA was replaced with MUFA.24 Mortality rate from CHD is
lower in Mediterranean populations and they consume a diet that differs in
many ways from a Western diet, including widespread use of olive oil, a
major source of oleic acid, as their principal source of fat.
Grundy and Mattson14,30 have demonstrated that replacing SFA with

MUFA lowers LDL-C levels without lowering HDL-C. Kris-Etherton et al.53
demonstrated that replacing SFA with MUFA (37% kcal total fat, 22% kcal
MUFA, 47% kcal CHO) versus CHO (30% kcal total fat, 15% kcal MUFA and
54% kcal CHO) resulted in comparable decreases in LDL-C (6.3% and 7.0%,
respectively). The blood cholesterol-lowering diet high in CHO and low in
fat decreased HDL-C by 7.7% and increased TG by 6.9%, whereas the diet
high in MUFA only decreased HDL-C by 4.1% and decreased TG by 4.6%.54
Furthermore, a meta-analysis conducted by Garg et al.55 found that diets
high in MUFA vs. high in carbohydrate reduce fasting TG levels by 19%,
decrease VLDL-C by 22%, and moderately increase HDL-C without nega-
tively affecting LDL-C.
Polyunsaturated Fatty Acids

Epidemiologic studies from within-population and cross-population studies
provide mixed results regarding whether PUFA (n-3 and n-6) is inversely
associated with CVD mortality. Many studies have shown an association
between dietary PUFA and reduced CVD mortality after adjusting for SFA;56
however other studies, such as the Seven Countries Study, reported no
significant association between PUFA intake and CVD.24,27
n-6 PUFA
Specific associations with linoleic acid (LA), the predominant n-6 fatty acid,
and coronary disease risk also have been inconsistent. A cross-population
study in healthy men found an inverse association between n-6 levels in
adipose tissue and mortality rate from CAD.57 In contrast, a recent study in
an Israeli population consuming PUFA as 10% of total energy did not find an
association between LA intake and acute myocardial infarction (AMI).58 How-
ever, there was a positive association between arachidonic acid, the long chain
derivative of LA, with AMI (p = 0.004). After multivariate adjustment, how-
ever, there was no indication of an adverse association between LA and AMI.
Some of the earliest clinical trials evaluated the effects of diets high in
PUFA, ranging from 13% to 21% of energy, on TC and CHD events.59–62 Three
of these studies reported a 13–15% decrease in TC, which was accompanied
by a 25–43% decrease in CHD events.59–61 Predictive equations have demon-
strated that a 1% increase in PUFA results in a reduction of TC by 0.024
mmol/L.28,29 The TC-lowering effect is approximately half of the cholesterol-
raising effect of SFA.28,29 More recent predictive equations developed for
individual fatty acids demonstrate that LA is the strongest TC and LDL-C-
lowering fatty acid.
Some studies have shown that LA raises HDL-C when compared with
stearic acid (18:0).53 A study by Mattson and Grundy,30 however, reported
an HDL-C-lowering effect (– 5 ± 1.7 mg/dl, p < 0.02) of PUFA at very high
levels (28% of kcal) in normotriglyceridemic individuals. Other studies have

reported no significant change in HDL-C with a high PUFA intake.63
n-3 PUFA
The cardioprotective effects of marine-derived long-chain n-3 fatty acids,
eicosapentanoic acid (EPA) and docosahexanoic acid (DHA), are well estab-
lished.64 An inverse association between n-3 fatty acids and CAD has been
found in numerous epidemiologic studies. In the Seven Countries Study, a
non-significant negative correlation (r = –0.28) was observed between fish
consumption and CAD mortality despite large differences in fish consump-
tion among the cohorts. An inverse correlation also was found in the 25-year
follow-up of the study with n-3 fatty acid intake and 25-year CAD mortality
rates (r = –0.36).27 In the Zutphen Study,65 an increase in fish consumption
from 0 to 45 g/day was associated with a progressive decrease in the risk
of CAD after 20 years (p < 0.05). Epidemiologic studies also have found an
association between α-linolenic acid (ALA), specifically, and CHD risk. In
the Health Professionals Follow-Up Study, a 1% increase in ALA intake was
associated with a 40% lower risk of CHD.66
In the GISSI Prevention Study,67 the largest prospective clinical trial to test
the efficacy of n-3 fatty acids for secondary prevention of CHD, subjects were
randomized to the EPA + DHA supplement group (850 mg/day of omega-3
fatty acid ethyl esters), with and without 300 mg/day of vitamin E. Individuals
in the supplement group compared with the control group experienced a 15%
reduction in the primary endpoint of death, nonfatal myocardial infarct, and
nonfatal stroke (p < 0.02). In addition, all-cause mortality and sudden death
were reduced by 20% (p = 0.01) and 45% (p < 0.001), respectively, compared
with the control group, with Vitamin E providing no benefit.
The mechanisms proposed by which n-3 fatty acids protect against CHD
include increased stabilization of atherosclerotic plaques, decreased produc-
tion of adhesion molecules, chemoattractants, eicosanoids, cytokines, and
increased endothelial relaxation and vascular compliance.68 In addition, n-3
fatty acids are known to influence the lipid profile. Overall, studies have
observed slight increases in LDL-C (5–10%) and HDL-C (1–3%) and substan-
tial decreases in TG levels (25–20%) with marine-based n-3 fatty acid sup-
plementation (< 7 g n-3 fatty acids/day).64
The primary effect of marine sources of n-3 fatty acids on the lipid profile
is due to their TG-lowering effects. In a review of 44 intervention studies by
Harris,69 supplementation of 0.5–25 g of n-3 fatty acids from fish oils for an
average of 6 weeks elicited a substantial decrease in TG levels (10–20%),
while LDL-C and HDL-C concentrations did not change. In addition, a study
of longer duration (16 weeks) found that a low dose of n-3 PUFA from fish
oil (1 g/day) decreased fasting TG levels by 21%.70 Consumption of 3–4 g/
day of EPA and DHA results in a 25% decrease in TG in normolipemic
(TG less than 2 mmol/L) and 34% decrease in TG in hypertriglyceridemic
patients (TG greater than 2 mmol/L).64 The characteristic TG-lowering effect
appears specific to marine sources of n-3 fatty acids and is generally not
observed with plant sources of n-3 fatty acids. However, a TG-lowering effect
was found at very high levels (38 g) of ALA intake.64
In addition, marine-derived n-3 fatty acids have been shown to increase
HDL-C levels 5–15% in recent supplementation trials.71–75 Likewise, slightly
elevated LDL-C levels have been a consistent finding in the n-3 fatty acid
supplementation trials.76–79 In a review, LDL-C was increased by 4.5% in
normolipemic patients and 10.8% in the hypertriglyceridemic patients con-
suming 3–4 g/day of EPA and DHA.64 Several studies suggest that the
elevation of LDL-C probably relates to an increase in LDL particle size.77,79
Dietary Cholesterol
Some epidemiologic studies have shown positive associations between cho-
lesterol intake and CHD risk, including the Seven Countries Study, the
Honolulu Heart Program, and the Western Electric Study,27,80,81 while others
have not.66,82 Numerous studies have demonstrated that there is a positive
linear relationship between dietary cholesterol intake and both TC and
LDL-C. Based on a meta-analysis of 27 controlled feeding studies, each
increase of 100 mg of dietary cholesterol results in an increase in TC by about
0.5 to 1 mmol/L, 80% of which is due to increases in LDL-C.22 In addition,
dietary cholesterol also has a modest HDL-C-raising effect,83,84 especially in
individuals who are hyper-responders.85,86
Dietary Fiber
Epidemiologic Evidence of Fiber and Heart Disease
Several large epidemiologic studies have reported a strong inverse correla-
tion between dietary fiber (refined or whole grain) and CHD. The Health
Professionals Follow-up Study tracked 43,757 U.S. male health professionals,
aged 40–75 years who were initially free of diagnosed CHD and diabetes,
for 6 years.87 The age-adjusted relative risk for total myocardial infarction
was 0.59 among men with the highest quartile of total dietary fiber intake
(median 28 g/day) compared with men with the lowest quartile (median
12.4 g/day). The relative risk for fatal myocardial infarction in the highest
quartile was 0.45 compared with the lowest quartile of fiber intake. In the
Nurses’ Health Study, Wolk et al.88 reported that an increase of 10 g/day
dietary fiber was associated with a 20% reduction in CHD risk. In the Alpha-
Tocopherol, Beta-Carotene Cancer Prevention Study, Pietinen et al.89 fol-
lowed 21,930 male Finnish smokers for 6 years and reported a significant
reduction in both coronary morbidity and mortality associated with
increased intake of dietary fiber. A recent meta-analysis of ten prospective
cohort studies (91,058 men and 245,186 women) found that each 10 g/day
increment of total dietary fiber was associated with a 14% reduction in risk
of all coronary events and 27% reduction in coronary mortality.90 In all four
of these studies, a stronger association was observed between cereal fiber
and CHD risk than between vegetable or fruit fiber. Using data from the
NHANES I Epidemiologic Follow-up Study (n = 9,776), Bazzano et al.91
found that water-soluble dietary fiber intake reduced CHD events 15% and
CVD events 10% when comparing participants with the highest intake (5.9
g/day) to those with the lowest intake (0.9 g/day). However, not every study
shows this relationship. Liu et al.92 studied 39,876 female health professionals
in the Women’s Health Study over 6 years and found a non-significant
inverse association between dietary fiber and risk for CVD and myocardial
infarction after multiple adjustments for smoking, CVD risk factors, and
dietary variables.
The importance of whole grains as a source of fiber has been demonstrated
in several studies.93,94 A study using the Iowa Women’s Health Study data-
base matched women on total grain fiber intake, but differed in the propor-
tion of fiber consumed from whole vs. refined grain.95 Interestingly, after
adjusting for multiple confounding factors, women who consumed a higher
amount of whole grains (4.7 g whole grain/2000 kcal and 1.9 g refined grain/
2000 kcal) had a 17% lower mortality rate (RR = 0.83, 95% CI, 0.73–0.94) than
women who consumed a greater proportion of refined grains (4.5 g/2000
kcal and 1.3 g whole grain/2000 kcal). Death due to CHD was also signifi-
cantly different between groups after adjustment for age and energy intake;
however, this difference lost significance when multiple confounding factors
(i.e., education, hypertension, diabetes, BMI, etc.) were entered into the
model. Furthermore, a prospective cohort study in 3588 men and women
aged 65 years or older reported that dark breads (wheat, rye, pumpernickel)
were associated with a lower risk of CVD (hazard ratio 0.76, 95% CI,
0.64–0.09) compared with cereal fiber from other sources.96 A recent meta-
analysis of 12 population-based cohort studies found that whole-grain foods
significantly reduced the risk of CHD by approximately 26% after adjustment
for multiple CHD risk factors.97 The inverse association of whole grains was
stronger than for cereal fiber, fruits, or vegetables, suggesting that three
servings of whole grains per day may be important to cardiovascular health.
Overall, epidemiologic studies lend convincing support to the hypothesis
that individuals with a higher intake of dietary fiber, especially from whole
grains,93,94,98–102 have a lower risk of CVD than those who consume a diet
poor in fiber. The new recommendations from the National Academy of
Science for fiber intake are 38 and 25 g/day for young men and women,
respectively, based on an intake of 14 g of fiber per 1000 kcal.22 In addition,
several studies suggest that the cardioprotective benefit of regular whole
grain consumption may be conferred via favorable effects on risk factors
associated with CVD, including hypertension,103–105 Type 2 diabetes,104,106–108
and other metabolic risk factors.106,107 Therefore, consistent with the Dietary
Guidelines 2005 Report, the public should consume three servings of whole
grains per day to decrease risk of chronic disease.
The Role of Dietary Fiber in Lipid Management

Numerous studies have demonstrated that diets rich in soluble fiber are more
effective in lowering blood cholesterol levels than are diets rich in insoluble
fiber.109–115 The key soluble fibers are β-glucan (found in oats, barley, and yeast),
psyllium (found in husks of blonde psyllium seed), and pectin (found in fruit).
Several properties of soluble fiber, including viscosity, bile acid-binding capac-
ity, and potential cholesterol synthesis-inhibiting capacity after fermentation in
the colon,116,117 contribute to its cholesterol-lowering effect.118
A meta-analysis of eight studies reported that 10 g/day of psyllium
reduced TC and LDL-C by 4% and 7%, respectively.119 Another meta-analysis
of 67 controlled dietary studies performed by Brown et al.120 found that for
each gram of soluble fiber from oats, psyllium, pectin, or guar gum, TC
concentrations decreased by 0.037, 0.028, 0.070, and 0.026 mmol/L (1.42, 1.10,
2.69, and 1.13 mg/dl), respectively. LDL-C decreased by 0.032, 0.029, 0.055,
and 0.033 mmol/L (1.23, 1.11, 1.96, and 1.20 mg/dl), respectively, demon-
strating that the cholesterol-lowering effects of these soluble fibers are com-
parable. Furthermore, two servings of oats (2.6 g soluble fiber) has been
shown to elicit a 2–3% cholesterol-lowering effect beyond what is achieved
by a blood cholesterol-lowering diet alone.121 Beneficial effects of fiber intake
also have been observed in healthy populations. In a study of normolipi-
demic and normotensive subjects (n = 53,), increased dietary fiber intake
(30.5 g/day total fiber and 4.11 g/day soluble fiber) over 3 months signifi-
cantly reduced LDL-C by 12.8%, while TG and HDL-C did not change.122
Interest in barley as another source of β-glucan is on the rise.123–126 The
addition of 3 or 6 g/day β-glucan from barley to a Step I diet has been shown
to further lower TC (4% and 9%, respectively) and LDL-C (13.8% and 17.4%,
respectively) concentrations in mildly hypercholesterolemic men and
women.123 Overall, these studies achieve modest reductions in TC of 2–18%.
This is important because a reduction in TC of just 1% could reduce CVD
mortality by 2%.127
In addition to lowering blood cholesterol levels, a high-fiber intake pre-
vents or attenuates the hypertriglyceridemic response to a high-carbohydrate
diet.128 The traditional adoption of a high-carbohydrate, low-fat diet can
produce an unfavorable lipid profile by decreasing HDL-C and increasing
TG.129 The mechanism has not been confirmed with some studies concluding
that the hypertriglyceridemic response is the result of reduced VLDL-TG
clearance,130 while others attribute it to increased VLDL-TG secretion because
of increased hepatic fatty acid availability resulting from increased influx of
fatty acids and decreased hepatic fatty acid oxidation.131,132 Elevated levels
of blood TG are considered an independent risk factor for CHD;133,134 a
1 mmol/L increase in fasting blood TG is associated with a 76% and 31%
increase in CVD risk in women and men, respectively.135 Several studies note
that increasing dietary fiber diminishes the adverse effects of a low-fat, high-
CHO diet on HDL-C and TG concentrations.109,120,123,136
An extensive review of 14 studies by Anderson128 found that high-CHO

(60% kcal CHO), low-fiber (6 g/1000 kcal) diets elicited higher fasting serum
TG levels by a mean of 53% (95% CI, 34–71%), compared with low-CHO
(< 45% kcal CHO), low-fiber diets. The opposite was true for high-CHO,
high-fiber (29 g/1000 kcal) diets, which modestly lowered TG by 10% (95%
CI, –2% to –17%) compared with low-CHO (42% kcal CHO), low-fiber (7.5
g/1000 kcal) diets. Even modest increases in dietary fiber from 10 to 22 g/
1000 calories has been associated with a 10% reduction in fasting serum TG
levels in Type 2 diabetic patients consuming a moderate-CHO diet (55% kcal
CHO).137 Garg et al.138 conducted an innovative study comparing the TG
response of two diets matched for fiber content (25 g/day), but varied in
levels of carbohydrate in hypertriglyceridemic diabetic individuals (n = 8).
The high-CHO diet (60% kcal CHO) resulted in a 27.5% (p < 0.002) increase
in plasma TG compared with the low-CHO diet (35% kcal CHO).
Emerging evidence suggests that increases in blood TG levels may con-
tribute to increased concentrations of small, dense LDL particles, which are
atherogenic.139 A recent study of 36 overweight men aged 50–75 years found
that consumption of two large servings of oats daily (about 14 g/day dietary
fiber) substantially decreased small, dense LDL-C (– 17.3%) and LDL particle
number (– 5.0%) compared with the wheat control (+ 60.4% and + 14.2%,
respectively).140 More importantly, although carbohydrate intake increased
and total and saturated fat intakes decrease, HDL-C and TG levels remained
stable in subjects who consumed the high-fiber oat cereals. Other emerging
data suggest that dietary fiber is inversely associated with C-reactive protein,
a marker of inflammation.141,142
The association between increased dietary fiber consumption and
improved lipid profiles indicates a causal relationship between fiber, blood
lipids, and heart disease. Further research is needed to define the optimal
ratio of fiber to carbohydrate in the diet. However, when instituting a low-
fat, high-carbohydrate diet, care should be taken to simultaneously increase
fiber-dense foods with carbohydrate content.
Glycemic Index/Glycemic Load

The traditional approach of classifying carbohydrates is based on their chem-
ical structure (starch, sugars, fiber). Over the past two decades, an alternative
approach, which characterizes dietary CHO on the basis of their effects on
postprandial glycemia has been intensely debated. The glycemic index (GI),
defined as the area under the 2-h glycemic curve after consumption of a food
containing 50 g CHO, divided by the area under the curve for a standard
food (white bread or glucose) also containing 50 g CHO,143,144 may be superior
to the traditional schema in elucidating the effects of CHO-rich foods on
glucose and lipid metabolism. In theory,145 lowering the postprandial rise in
glucose and insulin improves insulin sensitivity and reduces hepatic TG
synthesis and secretion. This results in reduced fasting TG concentrations

and reciprocal increased HDL-C levels.
Excessive postprandial hyperglycemia has been linked to all-cause and
CVD mortality,146–148 increased carotid intima media thickness,149,150 and
impaired endothelial function.151,152 As a consequence of hyperglycemia,
hyperinsulemia has been implicated in the development of dyslipidemia
(i.e., high TG and low HDL-C).153,154 Therefore, controlling postprandial
hyperglycemia via CHO intake may alleviate dyslipidemia associated with
excessive insulin.
Scores for high-GI, moderate-GI, and low-GI foods are > 70, 56–69, and
< 55, respectively, using glucose as the standard.155 Several large-scale epi-
demiologic studies have demonstrated that the average GI and glycemic
load (the product of the GI of a specific food multiplied by its CHO content)
of the diet are significant independent predictors of risk of Type 2
diabetes156–158 and CVD.157 Furthermore, studies show that total carbohydrate
intake or sugar intake is not associated with increased risk. More importantly,
high-GI diets are associated with lower HDL-C concentrations in both
healthy159–161 and diabetic populations.162 In the studies of healthy popula-
tions, reductions in GI range from 16% to 22% when comparing the highest
and lowest quintiles of intake.159–161 This corresponds with significant
increases in HDL-C of 9–20%. Similar results have been observed in indi-
viduals with diabetes.162 The effect of GI on blood TG was evaluated in the
Nurses’ Health Study.159 When comparing the two extreme quintiles of gly-
cemic load (117 vs. 180), TG levels were 144% higher in women with a BMI
> 25 and 40% higher in women of normal weight (BMI ≤ 25).
Clinical studies generally show that when the amount of carbohydrate is
held constant, foods with a higher GI increase fasting blood TG. In 10 of 11
studies reviewed by Miller,163 a reduction in GI of greater than 12 points
lowered TG levels by an average of 9%. Individual variations in lipid
response to low-GI diets has been observed.164 Thirty subjects were treated
with a high-GI diet (GI = 84) for 1 month, a low-GI diet (GI = 73) for 1 month,
and finally a high-GI diet. Individuals diagnosed with type IIa hypercholes-
terolemia showed little lipid response, whereas subjects with a variety of
types of hypercholesterolemia (IIb and IV), characterized by hypertriglycer-
idemia, experienced significant reductions in TG (about 20%), LDL-C
(7–10%), and TC (7–9%).164 These results need to be confirmed, however,
since the diets also differed in fiber, fat, and energy content. In a partially
controlled feeding study, 15 patients with Type 2 diabetes consumed a low-
GI diet (GI = 60) for 6 weeks and a high-GI diet (GI = 87) for 6 weeks.165 The
study investigators provided the starchy food portion of the diet. The diets
were similar in macronutrient profile (23% kcal fat, 57% kcal CHO). Serum
TG increased almost 10% on the high-GI diet and decreased 15% on the low
GI-diet, however due to the low sample size, this difference was not statis-
tically different. In a metabolic ward study, 12 overweight (30–35 BMI) men
consumed two diets ad libitum for 6 days in a crossover fashion. The diets
represented an American Heart Association (AHA) phase 1 diet (30% kcal
total fat, 10% kcal SFA, and 55% kcal CHO) and a low-fat, low-GI diet (32%
kcal total fat, 14% kcal SFA, and 37% kcal CHO).166 The AHA diet induced
a 28% increase in TG levels and a reciprocal 10% reduction in HDL-C,
resulting in a significant increase in TC:HDL-C. The opposite was true for
the low-GI diet; plasma TG fell 35% and LDL particle size increased by 1.6%.
Although difficult to interpret due to differences in the diets, this study
highlights the rapid effects of these diets on lipid metabolism and suggests
that a low-GI, high-protein diet may be beneficial compared with a tradi-
tional low-fat diet.
Low GI-diets also may protect against HDL-C lowering of traditional high-
CHO diets. Luscombe et al.167 conducted a partially controlled feeding study
in which 21 diabetic individuals consumed three diets: a high-GI diet (GI =
63), a low-GI diet (GI = 43), and a high-MUFA, high-GI diet (GI = 59) in a
crossover design. Over each 4-week period, 45% of energy was provided as
key CHO foods, and subjects were instructed on menus for implementing
the intervention diets. This resulted in similar intakes of energy, fiber, and
macronutrients across treatments. The low-GI diet and high-MUFA diet had
comparable effects on the lipid profile, whereas the low-GI diet elicited
higher HDL-C levels (6%) compared with the high-GI diet. TG levels were
18% lower; however, this was not statistically different. In contrast, individ-
uals with impaired glucose tolerance experienced a significant reduction in
HDL-C and no change in TG levels, when they consumed a low-GI diet for
4 months vs. a high-GI diet.168
Studies have also evaluated the impact of GI in the context of weight loss
diets. A study by Heilbronn et al.169 in 55 overweight men and women found
that both high-GI and low-GI diets reduced TG to a similar extent with
concurrent weight loss (~ 5%). HDL-C did not change in either diet; however,
LDL-C levels were reduced more (8%) in subjects on the low-GI diet. A study
comparable in design also reported similar LDL-C-lowering results with no
changes in TG levels in 20 individuals with Type 2 diabetes.170 Other studies
utilizing low-GI diets as a means to lose weight also report no changes in
TG or HDL-C.171,172
The GI can be easily manipulated by food choice selection and offers
promise for treating dyslipidemia. Incorporating low-GI foods into a low-
fat, high-CHO diet may prevent CHO-induced hypertriglyceridemia. The
GI effects on HDL-C are inconclusive and further research is needed. It must
also be noted that results from many studies demonstrate considerable het-
erogeneity in the lipid response to a low-GI diet.145 Although controversial,
the GI concept has been shown to be useful in the dietary management of
diabetes, hyperglycemia, and hyperlipidemia.173,174
Conclusion
Diet plays an important role in modifying CVD risk, partly due to the effects
of nutrients on lipids and lipoproteins. Saturated fatty acids, trans fatty acids,
dietary cholesterol, and simple carbohydrates adversely affect lipids and

lipoproteins, while unsaturated fatty acids and complex carbohydrates rich
in fiber beneficially affect lipids and lipoproteins. It is important to modify
diet appropriately to modify plasma lipids and lipoproteins in a way that
minimizes coronary disease risk.
Effects of Dietary Patterns on Lipids and Lipoproteins

Several dietary patterns have been studied extensively and found to have
specific effects on lipids and lipoproteins, which are reviewed in Table 11.3.
The traditional dietary plans recommended by the National Cholesterol
Education Program (NCEP) to treat hypercholesterolemia are the Step I and
Step II diets.175 Step I diet guidelines include limiting total and saturated fat
and dietary cholesterol, while the Step II diet recommends further reductions
in saturated fat and dietary cholesterol (Table 11.3). Currently, the NCEP
recommends the Therapeutic Lifestyle Changes (TLC) diet, which incorpo-
rates several dietary manipulations to maximally lower LDL-C.6 Other
dietary patterns for managing lipids and lipoproteins include vegetarian
diets, very low-fat diets, and Mediterranean-style diets. Many clinical studies
have evaluated the effects of these diets on blood lipids and lipoproteins.
Typically, these diets lower TC and LDL-C levels, while the effects on TG
and HDL-C levels vary on the basis of diet composition and lifestyle pro-
gram. The availability of several different dietary patterns for the manage-
ment of dyslipidemia provides many options for patients and clinicians. This
aids in optimizing diet adherence, resulting in maximal lipid lowering in
response to diet.
NCEP Recommendations
Numerous free-living and controlled clinical trials have assessed the effects
of Step I and Step II diets on the lipid profile across populations of varying
ages, including individuals with CAD,176 normal blood lipids,177 hypercho-
lesterolemia,178 and combined hypercholesterolemia and hypertriglyceri-
demia.179 The results of these studies consistently show beneficial effects on
TC and LDL-C levels, while the effects on HDL-C and TG levels are specific
to each intervention. When Yu-Poth et al.180 examined 37 dietary intervention
studies in a meta-analysis assessing NCEP guidelines in free-living subjects,
a Step I diet decreased TC, LDL-C, and TG levels by 10%, 12%, and 8%,
respectively, while a Step II diet resulted in decreases of 13%, 16%, and 8%,
respectively, and a 7% decrease in HDL-C levels. Although Step I and Step
II diets decreased HDL-C levels, the TC:HDL-C ratio also decreased 10%,
which reduces overall CVD risk. When following a Step I or Step II diet, a
TABLE 11.3
Lipid Response of Selected Dietary Patterns
Expected Lipid Response
Diet Description TC LDL-C HDL-C TG
Step I175 Total fat < 30%, SFA 8–10%, ↓ ↓ ↓ ↑, ↓ or ↔
cholesterol < 300 mg/day
Step II175 Total fat < 30%, SFA < 7%, ↓↓ ↓↓ ↓ ↑, ↓ or ↔
cholesterol < 200 mg/day
Therapeutic Lifestyle Total fat 25–35%, SFA < 7%, ↓↓↓ ↓↓↓ ↓ or ↔ ↔
Changes6 cholesterol < 200 mg/day
For further LDL-C lowering:
Add plant stanol/sterols
(2 g/day)
Add viscous fiber (10–25 g/day)
Add moderate physical activity
to expend 200 kcal/day
Portfolio182–184 Low-fat vegetarian diet ↓↓↓↓ ↓↓↓↓ ↔ ↔
containing a dietary portfolio
that included a plant sterol
ester-enriched margarine, oats,
barley, and psyllium, soy milk
and soy meat analogs, fruits
and vegetables, and whole
almonds; eggplant and okra
were also used as additional
sources of viscous fiber
Lifestyle Heart185 Low-fat, plant-based diet that ↓↓ ↓↓ ´ ´
uses fruits, vegetables, whole
grains, beans, and soy products
in their natural forms, moderate
quantities of egg whites and
nonfat dairy or soy products
and only small amounts of
sugar and white flour;
moderate aerobic exercise,
stress management training,
smoking cessation, and group
psychosocial support were also
included
Indo-Mediterranean Rich in whole grains (at least ↓ ↓ ↑ ↓
Diet190 400–500 g of whole grains),
fruits, vegetables, walnuts and
almonds, legumes, rice, maize,
wheat, and 3–4 servings/day of
mustard seed or soybean oil
DASH136 Rich in fruits and vegetables (9 ↓ ↓ ↓ ↔
servings/day) and low-fat
dairy products (2–3 servings/
day), with increased fish, nuts,
legumes and is low in saturated
and total fat and sodium
↑, increase; ↓, decrease; ↔, no change.
1-kg decrease in body weight decreased TG by 0.011 mmol/L (r = 0.35,

p < 0.01) and increased HDL-C by 0.011 mmol/L (r = –0.38, p < 0.02). There-
fore, TG levels decrease180 or remain unchanged177–179 when following a Step
I or Step II diet for weight loss. However, TG levels would be expected to
increase if there is no weight loss.176
The TLC diet incorporates several dietary manipulations such as plant
stanols/sterols and viscous fiber and emphasizes weight loss and physical
activity to maximally lower LDL-C.6 The expected combined effect of the
TLC diet is a 20–30% decrease in LDL-C levels. The approximate contribution
of each lifestyle change to a reduction in LDL-C is: 8–10% by limiting SFA
intake to < 7% of calories, 3–5% by limiting dietary cholesterol to < 200 mg,
up to 5% with the addition of 5–10 g/day of viscous fiber, about 5–8% for
a weight loss of approximately 10 pounds, and 6–15% with the consumption
of 2 g/day plant stanol/sterol esters.6
In hypercholesterolemic individuals, consuming a TLC diet (15% kcal
protein, 58% kcal CHO, and 30% kcal total fat: 7% kcal SFA, 10% kcal MUFA,
and 10% kcal PUFA, and 75 mg cholesterol per 1000 kcal) compared with a
Western diet (15% kcal protein, 47% kcal CHO, 38% kcal fat: 16% kcal SFA,
16% kcal MUFA, and 6% kcal PUFA, and 180 mg cholesterol per 1000 kcal)
significantly lowers TC, LDL-C, and HDL-C levels by 9%, 11%, and 7% (all
ps < 0.001), respectively; however VLDL-C levels remain unchanged and TG
levels increase 7% (p = 0.265). Consistent with the effects on LDL-C and
HDL-C, apolipoprotein (apo) B and apo A-I levels were ~ 6% lower after
consumption of the TLC diet compared with the Western diet (p <0.001).178
The influence of gender on lipid response to the TLC diet (26% kcal total
fat, 4% kcal SFA, and 45 mg cholesterol/1000 kcal) vs. an average American
diet (AAD: 35% kcal total fat, 14% kcal SFA, and 147 mg cholesterol/1000
kcal) was evaluated in moderately hypercholesterolemic adults.181 Following
the TLC diet, TC, LDL-C, and apo B levels were significantly decreased when
compared with an AAD (respectively, men: 19%, 21%, and 18%; women:
12%, 15%, and 9%; p < 0.05 for both groups); however, the reductions in TC
and apo B were greater in men than women (p < 0.05). Fasting TG levels
were not affected by the TLC diet in men, but were increased 14% in women
(p < 0.05). Yet, postprandial TG levels measured after a standard fat load
were greater in men than women (p < 0.05). LDL particle size decreased 11%
in men and 21% in women (p < 0.05). These data indicate that middle-aged
men may have a more favorable lipoprotein response to a low-fat, low-
cholesterol diet than postmenopausal women.181
Portfolio Diet
The Portfolio diet is designed to maximally lower LDL-C levels by employ-
ing a variety of dietary interventions, each of which has hypocholesterolemic
properties. The Portfolio diet is a vegetarian diet rich in soy protein, almonds,
plant sterols and viscous fibers primarily from oats, barley, and psyllium.182
When the effects of the Portfolio diet (22.4% kcal protein: 96.8% as vegetable
protein, 50.6% kcal CHO, 27.0% kcal total fat, 4.3% kcal SFA, 11.8% kcal
MUFA, 9.9% kcal PUFA, 10 mg cholesterol/1000 kcal, and 30.7 g fiber/1000
kcal) were evaluated in hypercholesterolemic adults, TC levels decreased by
22.4% and LDL-C levels by 29.0%, respectively (p < 0.001); while HDL-C and
TG levels decreased non-significantly.182,183 Consistent with these changes,
TC:HDL-C and apo B decreased by 19.8% and 24.2%, respectively.
In a follow-up study183 comparing the Portfolio diet (20.0% kcal protein:
99% as vegetable protein, 56.6% kcal CHO, 23.2% kcal total fat, 4.9% kcal
SFA, 9.5% kcal MUFA, 7.9% kcal PUFA, 48 mg cholesterol/1000 kcal, and
37.2 g fiber/1000 kcal) to a Step II diet (19.6% kcal protein: 30% as vegetable
protein, 58.8% kcal CHO, 21.6% kcal total fat, 4.4% kcal SFA, 8.5% kcal
MUFA, 7.5% kcal PUFA, 34 mg cholesterol/1000 kcal, and 26.6 g fiber/1000
kcal), TC and LDL-C levels decreased by 26.6% and 35.0% (p < 0.001),
respectively, on the Portfolio diet but by only 9.9% and 12.1% (p < 0.001),
respectively, on the Step II diet. Consistent with the change in TC and LDL-C
levels, TC:HDL-C and apo B decreased 20.8% and 26.7%, respectively, fol-
lowing the Portfolio diet compared with a reduction of only 2.6% and 8.1%,
respectively, when following the Step II diet (p < 0.001). In addition, TG
levels decreased 6.3% following consumption of the Portfolio diet but
increased 4.9% when following the Step II diet. Neither diet affected HDL-C
or apo A-I levels.
In another study,184 the magnitude of cholesterol reduction observed fol-
lowing the Portfolio diet was similar to low-dose statin therapy. TC and
LDL-C levels decreased by 21.9% and 28.6%, respectively, (p < 0.001, from
baseline) on the Portfolio diet, by 23.3% and 30.9%, respectively, (p < 0.001,
from baseline) on lovastatin treatment, but by only 6.3% and 8.0%, respec-
tively, (p = 0.002, from baseline) on the vegetable-rich control diet. Likewise,
TC:HDL-C and apo B decreased by 17.1% and 22.8%, respectively, following
consumption of the Portfolio diet and by 21.6% and 26.6% on statin treatment
(p < 0.005, compared with control diet). None of the diets affected HDL-C
levels. These results suggest that when several interventions are employed
concurrently, diet can produce favorable effects on the lipid profile that are
similar in magnitude to statin therapy.
Lifestyle Heart Program

The Lifestyle Heart Trial evaluated patients with previous CHD who were
randomized to either a usual-care group or an intensive lifestyle change
group.185 The intervention consisted of a very-low-fat vegetarian diet high in
complex-carbohydrates (15–20% kcal protein, 70–75% kcal CHO, 10% kcal total
fat [PUFA:SFA > 1] and 5 mg/day cholesterol), moderate aerobic exercise, stress
management training, smoking cessation, and group psychosocial support.
After one year on the program, significant reductions occurred in TC, LDL-C,
and apo B levels by 27.6%, 39.8%, and 23.2%, respectively (all ps < 0.005). HDL-C
levels decreased non-significantly by 9.6%, while TG levels non-significantly

increased by 13.4%. The benefits of reducing these CVD risk factors were seen
after five years when the experimental group experienced significant reductions
in average percent diameter stenosis (–3.1% vs. +11.8%, p = 0.001) and angina
(–91% vs. +186%, p < 0.001) and experienced significantly fewer cardiac events
than the control group (25 vs. 45, p < 0.001).
Mediterranean Diet
The dietary patterns characteristic of the Mediterranean region have been
extensively studied to determine why inhabitants of this area have decreased
rates of coronary disease. Diet composition varies in this region but tends
to emphasize fruits, vegetables, breads, cereals, potatoes, beans, nuts, olive
oil, and seeds. Other common characteristics of Mediterranean-style diets
include dairy products (mainly cheese and yogurt), fish, poultry, and wine
consumed in low to moderate amounts, eggs consumed zero to four times
per week, and minimal consumption of red meat.
The Seven Countries Study stimulated interest in the Mediterranean diet
when it was reported that the 15-year mortality rate from CHD in Southern
Europe was two to three times lower than that in Northern Europe or the
United States; yet the mean serum TC values were similar.186 Other epidemi-
ologic studies, such as the ATTICA Study,187,188 have shown that factors, other
than lipids (i.e., physical activity, education, and markers of inflammation and
coagulation), contribute to reduced mortality from CHD. The majority of study
participants lived in areas surrounding Athens and had elevated TC and
LDL-C levels, while one-fifth of the subjects had low HDL-C levels.
In the Lyon Diet Heart Study,189 a randomized, single-blind secondary pre-
vention trial, subjects consumed a Western diet or an experimental diet rich
in ALA (30.5% kcal total fat, 8.3% kcal SFA, 12.9% kcal 18:1, 3.6% kcal 18:2,
0.81% kcal 18:3, and 217 mg/day cholesterol). This Mediterranean-type diet
was rich in whole grains, root vegetables and green vegetables, fish, fruits at
least once daily, and low in red meat (replaced with poultry). Margarine that
had a fatty acid profile similar to that of olive oil, except that it was higher in
linoleic acid and more so in α-linolenic acid, was supplied by the study to
replace butter and cream. Rapeseed and olive oils were used exclusively for
salads and food preparation. After two years on the experimental diet or
Western diet, TC, LDL-C, HDL-C, TG, apo B, and apo A-I levels were
unchanged. Although both groups had similar lipids, lipoproteins, blood pres-
sure, BMI, and smoking status, subjects consuming the ALA-rich Mediterra-
nean diet had a 50–70% lower risk of recurrent coronary events.
The Indo-Mediterranean Diet Heart Study,190 another secondary preven-
tion trial incorporating a diet rich in ALA, showed beneficial effects on blood
lipids, lipoproteins, as well as recurrent coronary events. In this study, the
experimental group was instructed to consume a diet rich in ALA consisting
of 400–500 g of fruits, vegetables, and nuts, 400–500 g of whole grains, and
three to four servings of mustard seed or soybean oil daily; while the control
group was counseled on the Step I diet. After 2 years, TC, LDL-C, and TG
levels decreased in the experimental group by 12.2%, 17.6%, and 19.6%,
respectively; and by 3.1%, 4.2%, and 5.9%, respectively, in the control group
(p < 0.0001 at 2 years between groups). HDL-C levels increased 2.6% in the
experimental group only (p = 0.0288). Like the Lyon Diet Heart Study, sub-
jects consuming the ALA-rich Mediterranean diet had significantly fewer
total cardiac events than the control group (39 vs. 76, p < 0.001).
While epidemiologic evidence and some clinical trial evidence does not
show a beneficial effect of the Mediterranean diet on lipids and lipoproteins,
there is some clinical evidence that demonstrates a beneficial effect. There-
fore, further studies are needed to determine the underlying mechanism(s)
for the reduction in coronary events in coronary patients consuming a diet
characteristic of the Mediterranean region.
Dietary Approaches to Stop Hypertension (DASH) Diet

In a secondary analysis of the Dietary Approaches to Stop Hypertension
(DASH) trial,136 a trial designed to compare the effects of three dietary pat-
terns on blood pressure, the effect of the DASH diet on blood lipids was
assessed. Subjects consumed a control diet high in saturated fat and low in
dietary fiber (15% kcal protein, 52% kcal CHO, 37% kcal total fat, 13% kcal
SFA, 14% kcal MUFA, 7% kcal PUFA, 188 mg/day cholesterol), a fruit and
vegetable diet with a similar macronutrient profile but high in dietary fiber
(29.9 g/day), or a DASH diet emphasizing fruits, vegetables, and low-fat
dairy products (18% kcal protein, 58% kcal CHO, 27% kcal total fat, 7% kcal
SFA, 10% kcal MUFA, 8% kcal PUFA, 141 mg/day cholesterol, 29.7 g/day
fiber). Following the DASH diet, TC, LDL-C, and HDL-C levels decreased
significantly compared with the control group (7.3%, 9.0%, and 7.5%, respec-
tively, p < 0.0001), while non-significant reductions occurred in the fruit and
vegetable group (1.9%, 1.5%, and 0.4%, respectively). TC:HDL-C and TG
levels were unaffected in subjects on the DASH diet.
Different dietary patterns have different effects on lipids and lipoproteins.
Some diets, like the Lifestyle Heart, also show beneficial effects on CHD
events. There are many strategies for diet modification to reduce risk of heart
disease. The optimal dietary pattern is selected in a way that favorably affects
the lipid and lipoprotein profile, body weight, and long-term adherence.
Effects of Weight Loss Diets on Lipids and Lipoproteins

The weight loss diet debate has been ongoing for many years. For some
individuals low-fat diets achieve the most weight loss; while for others,
moderate-fat or low-carbohydrate diets do. Although initial weight loss

associated with different dietary plans is promising, the ability to maintain
a lower weight over an extended period of time defines successful weight
loss.
Low-Fat Diets
The Pritikin Program, a lifestyle intervention program that includes a high-
complex-carbohydrate, high-fiber (35–40 g/1000 kcal), low-fat (< 10% total
calories) and low-cholesterol (< 25 mg/day) diet and exercise component,
reduced weight, TC, and TG by 5%, 21%, and 50%, respectively.191 However,
this weight reduction diet was associated with a 14.6% decrease in HDL-C
levels. When assessing the effects of a low-fat, reduced-calorie diet on weight
and lipids over 10 weeks,192 mean body weight declined by 0.62 ± 0.47 kg/
week during the first 5 weeks and 0.43 ± 0.43 kg/week during the second
5 weeks. Likewise, TC, LDL-C, and apo B levels decreased by 15%, 23%, and
23%, respectively, during the 10-week low-fat, reduced-calorie diet. How-
ever, TG levels increased 22% and HDL-C levels decreased 18%, causing
TC:HDL-C to increase. Thus, while low-fat diets can achieve desired weight
loss and reduce TC and LDL-C levels, they also may exacerbate the hyper-
triglyceridemic response associated with high-carbohydrate, low-fat diets.129
Therefore, when employing a low-fat diet to elicit weight loss, especially in
individuals with hypertriglyceridemia, it is important to replace simple car-
bohydrates with complex carbohydrates and increase dietary fiber to blunt
the hypertriglyceridemic response associated with high-carbohydrate, low-
fat diets.128
Moderate-Fat Diets with MUFA

Although low-fat diets result in weight loss and lower TC and LDL-C levels,
they also are associated with increases in TG levels, decreases in HDL-C
levels, and adherence problems. Several studies have shown that moderate-
fat diets promote weight loss,193–195 lower TG levels,194,195 maintain HDL-C
levels,195 and improve diet adherence193 and weight maintenance.193,195
When comparing a high-carbohydrate weight loss diet with a moderate-
MUFA fat weight loss diet in a controlled clinical setting, TC levels (~ 7%)
and weight (~ 8%) were significantly reduced following both diets; however,
the only moderate-MUFA fat diet significantly lowered LDL-C (8.2%, p <
0.02) and TG levels (21.9%, p < 0.05).194 An innovative controlled feeding
study by Pelkman et al.195 compared the effects of a low-fat diet (28% kcal
total fat, 7.2% kcal MUFA) vs. a moderate-fat diet (32% kcal total fat, 14.2%
kcal MUFA) during 6 weeks of controlled weight loss followed by 4 weeks
of weight maintenance. At the end of the weight loss period, both diet groups
lost equal amounts of weight (moderate-fat diet group, 7.2 ± 0.29 kg; low-
fat diet group, 6.5 ± 0.34 kg; p > 0.10). In addition, TC, LDL-C, TG, apo A-I,
and apo B levels significantly declined during weight loss in both diet groups
(all ps < 0.05). However, individuals following the low-fat diet experienced
significant reductions in HDL-C (12.1%, p < 0.05), while those on the mod-
erate-fat diet maintained HDL-C levels and had significant reductions in
TC:HDL-C (10.9%, p < 0.05). During the weight maintenance period, TG
rebounded to above-baseline levels, while HDL-C remained lower than base-
line levels in the low-fat diet group only. While these results suggest that a
moderate-fat weight-loss diet decreases CVD risk by favorably affecting the
lipid profile, other studies conclude that low-fat diets rich in fiber are as
effective for weight loss and do not cause unfavorable alterations in plasma
lipids.196 Gerhard et al.196 found that a low-fat ad libitum diet (20% kcal total
fat, 8% kcal MUFA) and a moderate-fat ad libitum diet (40% kcal total fat,
26% kcal MUFA) reduced TC and LDL-C similarly (low-fat diet, 9.6% and
10.2%, respectively, and moderate-fat diet, 10.2% and 7.5%, respectively).
However, only the low-fat diet significantly decreased weight (1.53 kg,
p <0.001).
High-Protein Diets
While several studies show some beneficial effects of energy-restricted, high-
protein diets on total weight loss and the blood lipid profile,197–203 others do
not.201,204 In a recent study, researchers reported that a 6-month very-low-
carbohydrate-diet program, similar to the Atkins diet (carbohydrate intake
< 25 g/day), led to a sustained weight loss (10.3%, p < 0.02) with improve-
ments in the lipid profile (decreased TC by 5%, LDL-C by 7% and TG levels
by 43% and increased HDL by 19%, all ps < 0.02).200 Layman et al.198,199
determined that increasing the proportion of protein to carbohydrate in the
diet has beneficial effects on body composition and blood lipids. Subjects
were randomized to either a carbohydrate (CHO) group (diet with a CHO/
protein ratio of 3.5 [68 g protein/day]) or a protein group (diet with a CHO/
protein ratio of 0.4 [125 g protein/day]). The diets were isoenergetic (1700
kcal/day) and provided similar amounts of fat (~ 50 g/day). Both groups
had significant reductions in TC (16.15 mg/dl for protein group and 20.00
mg/dl for CHO group; p < 0.05), while only the protein group experienced
significant reductions in TG (10 mg/dl; p < 0.05). These findings agree with
other studies of high-protein, weight-loss diets, which have also shown
improvements in LDL-C particle size and in postprandial blood-lipid pro-
file.197
These results are somewhat surprising and need to be confirmed in long-
term studies. More importantly, these markedly improved lipid profiles can
be attributed to the substantial simultaneous weight loss and little is known
what occurs when weight loss plateaus. A few studies have shown that when
subjects enter weight maintenance, lipids rebound and adverse effects on
blood lipids persist with time.195,205 In support of these studies, Anderson et
al.202 performed a computer analysis, based on the composition of the
recommended diets, to predict the effects of staying on these diets to main-

tain weight with an energy intake of 2000 calories per day. The results of the
analysis suggest that increases in fatty acid content, increases in dietary
cholesterol and the reduction in soluble fiber implemented in the Atkins diet
would raise serum cholesterol values 9%, 19%, and 2%, respectively. Since
every 1% increase in serum cholesterol values are estimated to increase the
risk of cardiovascular disease 2–3%,206 long-term use of the Atkins diet might
increase the risk of cardiovascular disease by > 50%.
Early work assessing the effects of a high-protein, low-carbohydrate
weight loss diet on lipids and lipoproteins showed that LDL-C levels
increased from baseline by 23% (p < 0.01), while TG levels decreased by 45%
(p <0.01).203 In a multicenter study, Foster et al.201 found that 12 weeks of
weight loss therapy utilizing the Atkins diet resulted in a greater weight loss
(4%) in 63 non-diabetic subjects when compared with a conventional diet.
At three months, individuals following the Atkins diet experienced signifi-
cant increases in TC (+ 5%) and LDL-C (+ 9%) vs. the group on the conven-
tional diet (– 10% and – 15%, respectively); however, by the end of the study
the levels were similar between groups.
Although research has shown that low-fat diets, moderate-fat diets, and
high-protein diets can promote weight loss due to reduced total calorie
intake, they differentially affect lipid metabolism. Selecting a weight loss diet
depends on an individual’s need to lose weight, motivation, and preference
for a specific diet. The available evidence suggests that there is a range of
fat intake that can be used for successful weight loss diets. However, the
long-term effects of high-protein, low-carbohydrate diets on health and dis-
ease prevention are unknown.
Emerging Lipid and Lipoprotein CVD Risk Factors Affected by

Diet
In addition to the major lipid and lipoprotein CVD risk factors, other emerg-
ing lipid risk factors have been identified. These include LDL particle size,
HDL particle size, postprandial TG, and lipoprotein (a).
Diet Effects on LDL Particle Size

LDL subpopulations have been defined on the basis of a number of charac-
teristics, including particle density, size (particle diameter), charge, and
chemical composition.207 The diameter of the most prominent LDL subclass
has been identified and is referred to as LDL peak particle diameter (PPD),
which generally ranges from 22 to 28 nm. Individuals with a predominance
of larger, more buoyant LDL (LDL-I or II) with a peak diameter >25.5 nm
have been defined as pattern or phenotype A, whereas those with a higher

proportion of smaller, more dense LDL (LDL-III or IV) with a peak diameter
< 25.5 nm are referred to as pattern or phenotype B.23,208,209 The size of LDL
particles confers an independent risk of coronary disease.210,211 Individuals
with predominantly small LDL particles (pattern B) experience greater CHD
risk than those with larger LDL particles (pattern A).212–215 About 30–35% of
adult men and 15–25% of postmenopausal women have LDL pattern B, while
the prevalence is much lower in men less than 20 years old and in premeno-
pausal women (5–10%).23,216 Even though there is a genetic basis for LDL
particle size,217 dietary factors also218, 219 have an impact. A number of studies
have reported changes in LDL particle size in response to different dietary
interventions, including, changes in macronutrient composition, type of
dietary fat, type and amount of dietary fiber, and a combination of multiple
dietary factors that achieve maximal LDL-C reduction (Table 11.4).
The Effects of a Changing Macronutrient Profile on LDL Particle Size

Effects of High-Fat, Moderate-Fat Diets
Cross-sectional population analyses220 have demonstrated an association
between reduced LDL particle size and a low-fat, high-carbohydrate diet.
Several clinical studies have been conducted to evaluate the effects of total
fat and carbohydrate on the change in LDL particle size. Krauss and Dreon,221
using a crossover design, studied 105 normolipidemic men who were
instructed to consume a high-fat diet (46% kcal total fat, 39% kcal CHO,
16.2% kcal protein) and a low-fat diet (24% kcal total fat, 60% kcal CHO,
16.1% kcal protein) for 6 weeks. LDL-C was reduced on the low-fat diet in
subjects with either pattern A or B phenotype; however, individuals with
pattern B exhibited a twofold greater reduction than those with pattern A.
Importantly, on the high-fat diet, 87 subjects showed LDL subclass pattern
A and only 18 subjects had pattern B; however, when subjects switched to
the low-fat diet, 36 subjects (41% of pattern A) converted to pattern B while
all subjects with pattern B (when consuming high-fat diet) retained the
classification. In another study, using a very-low-fat diet (about 10.4% kcal
total fat, 75.7% kcal CHO, 14.5% kcal protein) compared with subject’s habit-
ual diet (about 31.8% kcal total fat, 52.1% kcal CHO, 14.0% kcal protein),
Dreon et al.222 found that 26 subjects remained in phenotype A, whereas
12 subjects changed into the denser phenotype B. Collectively, progressive
reductions in dietary fat and increases in carbohydrate increase the propor-
tion of subjects that convert from phenotype A to phenotype B.
Effects of High-Protein, Low-Carbohydrate Diets

Dumesnil et al.166 evaluated the effects of a moderate carbohydrate restricted
diet on LDL particle size. In this study, 12 subjects were randomly assigned
to a Step I diet (15% kcal protein, 55% kcal CHO and 30% kcal total fat) or
TABLE 11.4
238
Effects of High-Fat, Low-Carbohydrate Diets vs. Low-Fat, High-Carbohydrate Diets on LDL Phenotype and Particle Size
High-Fat, Low-Carbohydrate Diet Low-Fat, High-Carbohydrate Diet % of Subjects Who
Subjects Subjects Switched from phA to
Total fat, (n) LDL Total Fat, (n) LDL phB When on the
SFA, CHO, P Particle Size SFA, CHO, P Particle Size Low-Fat, High-CHO Diet
Study (% energy) PhA PhB (nm) (% energy) PhA PhB (nm) (%, n)
Krauss et al.221 46, 18, 39, 16 87 18 phA: 26.8 24, 6, 59, 16 51 54 phA: 26.5* 41%, 36
phB: 25.3 phB: 25.1
Dreon et al.222 31.8, 10.8, 38 0 26.62 10.4, 2.7, 75.7, 26 12 26.05** 32%, 12
52.1, 14.0 14.5
Sharman et al.197 61, 25, 8, 30 10 2 phB: 26.16 25, 12, 59, 15 7 5 phB: 25.28*** 30%, 3
Sharman et al.249 63, 22.3, 8, 28 11 4 27.0 23, 7.7, 56, 20 6 9 26.4*** 45%, 5
Volek et al.250 60, 20.8, 10, 29 a a 27.6 19, 5.6, 62, 17 a a 27.2 a
SFA, saturated fatty acids; CHO, carbohydrate; P, protein; phA, phenotype A; phB, phenotype B.
Significantly different from high-fat, low-carbohydrate diet: *p < 0.0001, **p < 0.001, ***p ≤ 0.05.
a Data not reported.
Lipid Metabolism and Health
a diet that was moderately restricted in carbohydrate (31% kcal protein, 37%
kcal CHO and 32% kcal total fat). The latter diet significantly increased
(p < 0.05) LDL peak particle diameter.
Three studies197,249,250 have evaluated the effects of diets with a greater
restriction in dietary carbohydrate (8 to 10% kcal CHO) on LDL particle size.
All diets were high in protein (29–30% kcal) rather than fat. These studies
reported a significant increase in peak LDL particle diameter in subjects on
the low-carbohydrate diet. In the Sharman et al., 2004 study,249 there were
increases or no changes in LDL particle size for pattern A subjects, whereas
there were pronounced increases in LDL particle size for pattern B subjects
during the very-low-carbohydrate high-protein diet.
Other Dietary Interventions – Type of Fat, Dietary Fiber, and Multiple

Dietary Strategies
In a cross-section study with 291 men conducted in Sweden, Sjogren et al.223
reported that individual fatty acids (C4:0–C10:0 and C14:0) typically found
in dairy products were associated with fewer small dense LDL particles
when assessed over tertile of LDL particle size. Dreon et al.224 reported a
positive correlation between SFA content (specifically, C14:0 and C16:0) and
LDL peak particle diameter in men following a high-fat, high-SFA diet
(46% and 18% kcal, respectively) versus a low-fat diet (24% and 6% kcal,
respectively); however, there was no association of MUFA or PUFA with
LDL particle size. Kratz et al.225 reported that unsaturated fat reduced LDL
particle size relative to SFA. However, Rivellese et al.78 reported neither
MUFA or SFA affected LDL particle size. Increases in LDL particle size from
baseline have been reported with supplementation with 4 g of highly puri-
fied fish oil226 and with 0.95 g EPA + 0.68 g DHA,227 whereas some studies
reported no effect of n-3 fatty acids on LDL particle size.228,229
With respect to dietary fiber, Davey et al.140 examined the effects of large
servings (14 g/day of dietary fiber) of oat cereal or wheat cereal on LDL
size. Subjects with pattern A phenotype (n = 5) had a significant reduction
in LDL size (from 21.0 ± 0.2 to 20.8 ± 0.2 nm; p = 0.05) in the oat group as
did subjects in the wheat group (n = 8) (from 21.1 ± 0.1 to 20.5 ± 0.3 nm;
p = 0.05). However, when examining subjects with pattern B phenotype
(n = 12), oat cereal produced significant decreases in small LDL-C concen-
trations (from 2.41 ± 0.39 to 1.92 ± 0.38 mmol/L; p = 0.05), while the wheat
cereal produced significant increases in small LDL-C concentrations (from
0.13 ± 0.1 to 1.23 ± 0.5 mmol/L; p = 0.05) and in particle concentration (1385
± 89 to 1746 ± 137 nmol/L; p = 0.01). The effects of soy protein and plant
sterols/stanols on LDL particle size also have been examined. Desroches et
al.230 reported that consumption of soy protein was associated with a larger
LDL particle size compared with animal protein. Unesterified plant sterols
and stanols (both 1.8g/day) did not change LDL peak particle diameter or
the small dense LDL levels.231 Lamarche et al.232 examined the effects of
combining plant sterols (1 g/1000 kcal), soybean proteins (23 g/1000 kcal),
viscous fiber (9 g/1000 kcal) and nuts (almond 15 g/1000 kcal) in a very low
SFA diet on LDL particle size. This dietary pattern reduced serum concen-
tration of all LDL fractions: large LDL-C>26.0 nm (– 0.57 mmol/L, p < 0.0001),
medium LDL-C25.5–26.0 nm (– 0.23 mmol/L, p < 0.0001) and small LDL-C<25.5 nm
(– 0.45, p < 0.01).
HDL Particle Size

Based on the NCEP-ATP III Guidelines,233 HDL-C levels are > 40 mg/dl for
men and > 50 mg/dl for women. The anti-atherogenic property of HDL-C
is due to its role in the reverse transport of cholesterol from arterial wall
cells to the liver and steroidogenic organs.234 While a majority of the HDL
particles contain apolipoprotein A-1, there are distinct differences in the
quantitative and qualitative content of the lipid fraction, apolipoproteins,
enzymes, and lipid transfer proteins associated with different HDL sub-
classes. These HDL subclasses are characterized by the differences in their
shape, density, size, charge and anti-atherogenicity,235 and consequently dif-
fer widely in their functional properties. One of the most common classifi-
cations of HDL-C is based on differential precipitation, which classifies HDL
particles based on their density, with HDL2 representing a less dense particle
and HDL3 a more dense subclass of particles.236 Other classifications, based
on particle size, identify five subpopulations of HDL-C using gradient gel
electrophoresis. These subpopulations include HDL2b, HDL2a, HDL3a, HDL3b,
HDL3c.236
Despite epidemiologic evidence from the Physicians’ Health Study, which
indicated that HDL3 levels were the strongest predictor of a reduction in the
risk of myocardial infarction,237 the less dense larger HDL2 particle is con-
sidered the most cardioprotective subpopulation, due to its effect on both
reverse cholesterol transport and the reduction of serum TG levels.238 In
addition, small HDL particle size has been associated with the metabolic
syndrome, including the high plasma TG levels, low HDL-C concentrations,
visceral adiposity, and hyperinsulinemia.239
A strong body of evidence indicates that variations in total dietary fat intake
affect HDL heterogeneity. In a study evaluating the effects of replacing dietary
fat with carbohydrates, individuals consumed a low-fat diet (24% kcal) and a
high-fat diet (46% kcal) in a randomized 2-period crossover design study.240
Following the consumption of the low-fat diet individuals had significantly
decreased levels of HDL3a, HDL2a, and HDL2b. Levels of HDL2b were reduced
significantly more in those individuals who exhibited LDL subclass pattern A,
compared with those who had pattern B phenotype. The results of this study
therefore indicate that a low-fat diet elicits unfavorable changes in HDL sub-
classes, and that these adverse effects are more prominent in individuals with
LDL subclass pattern A. Whereas these results are representative of drastic
reductions in total fat levels, they are not reflective of smaller changes in total
fat intake typical of average food consumption patterns.
The Dietary Effects on Lipoproteins and Thrombogenic Activity (DELTA)

Study was a multicenter controlled feeding study that evaluated the effects
of a stepwise reduction in total and saturated fat on lipids and lipoproteins
in healthy individuals.34 The three diets used were: (1) average American
diet (AAD; 34.3% kcal total fat, 15.0% kcal SFA, 12.8% kcal MUFA, and 6.5%
kcal PUFA), (2) a Step I diet (28.6% kcal total fat, 9.0% kcal SFA, 12.9% kcal
MUFA, and 6.7% kcal PUFA), and (3) a low-fat diet (25.3% kcal total fat,
6.1% kcal SFA, 12.4% kcal MUFA, and 6.7% kcal PUFA). Following a reduc-
tion in total and saturated fat, HDL-C concentrations were decreased and
TG levels were increased. Given this general effect on plasma lipids, further
analyses were conducted to determine whether the stepwise reduction in
total and saturated fat would influence primarily the larger cardioprotective
HDL subpopulations.241 As a result of the stepwise reduction in total and
saturated fat, there were significant reductions (p < 0.001) in the larger, less
dense HDL subpopulations (HDL2 and HDL2b).241 Although HDL3 concen-
trations followed a similar pattern in subjects on the three test diets, the
reductions in HDL2 (17.2%) were greater than the reductions in HDL3 (5.7%).
In addition, the concentrations of these larger HDL subpopulations were
significantly correlated with serum TG levels during all of the test diets
(r = –0.45; p < 0.0001 for low-fat diet), such that as HDL2 concentrations
decreased TG levels increased. These decreases in large HDL particles were
accompanied by a reduction in LDL-C and apo B concentrations. This led
the investigators to speculate that reductions in these larger HDL subpopu-
lations may not be harmful if they are coupled with reductions in LDL-C.
One of the major strengths of the study conducted by Berglund et al.241 was
the use of two different and independent methods (stepwise precipitation pro-
cedure and lipid staining) to assess HDL subpopulation changes, which yielded
similar results. In addition, because of the large and varied subject population
(men and women, African Americans and non-African Americans, pre- and
post-menopausal women, and younger and older men), findings from this
study are applicable to the general population. Therefore, while the dietary
changes employed in the present study may be prudent for a large segment of
the population, they will primarily affect the most anti-atherogenic HDL sub-
populations. These reductions in large HDL particles may not be as harmful if
they are accompanied by a simultaneous decrease in the atherogenic LDL
subpopulation. While it is important to examine the parallel reductions in LDL
and HDL subpopulations, the increase observed in TG levels with a low-fat
diet approach cannot be ignored.
While research has consistently shown a reduction in fasting TG levels
with marine sources of n-3 fatty acids, their effects on different HDL sub-
populations are inconsistent. In a study conducted by Subbaiah and col-
leagues, 242 14 hypercholesterolemic individuals received a fish oil
supplement containing 7.5 g n-3 fatty acids per day, in addition to a back-
ground Step I diet. Following 30 days of supplementation, plasma TG levels
were significantly decreased by 58% (p < 0.005), when compared with base-
line levels. In addition, there was a 41% increase in HDL2 concentrations
(p < 0.005), a 46% increase in the HDL2/HDL3 ratio (p < 0.001), and a 14%
decrease in the LDL/HDL ratio (p < 0.005). These results indicate that sup-
plementation with marine n-3 fatty acids may have a further impact on the
reduction of CVD risk via an increase in the cardioprotective HDL-C sub-
classes.
An increasingly popular strategy for the treatment of low HDL-C levels
is to increase physical activity. Thomas et al.243 designed a study to examine
the effects of combining exercise with n-3 fatty acid supplementation on
lipoprotein subclasses and associated enzymes. Ten healthy, physically active
men were given a supplementation of 4 g/day of EPA and DHA for 4 weeks.
Following 4 weeks of supplementation, total HDL-C and HDL2 concentra-
tions were significantly higher (p < 0.05) and fasting TG levels were signif-
icantly decreased (26%; p < 0.05). Subjects also completed an acute exercise
session before and after supplementation. As a result of an acute exercise
session, total HDL-C, HDL3-C, and LDL-C levels were increased. The com-
bination treatment of n-3 fatty acid supplementation and exercise resulted
in an additive effect on both HDL3-C levels and LDL-C concentrations. The
results, therefore, indicate that the combined treatment of marine-based n-3
fatty acids and physical activity has beneficial affects on fasting levels of the
cardioprotective HDL2 subpopulation. Further research is needed to deter-
mine the chronic impact of the observed increase in HDL3-C levels and LDL-
C following an acute bout of exercise on cardiovascular disease risk.
The results of the research to date indicate that low-fat diets appear to
adversely affect cardioprotective subclasses of HDL-C, and elicit an increase
in fasting TG levels. In addition, preliminary research suggests that supple-
mentation of marine-based n-3 fatty acids may have a positive impact on
these larger less dense HDL particles. Thus, a moderate-fat diet, rich in n-3
fatty acids is recommended for the treatment of low HDL-C levels. This
dietary strategy also will be effective in reducing levels of elevated fasting
TG levels in the presence of low HDL-C.
Postprandial TG
There is growing evidence that an elevated TG level is an independent risk
factor for CVD. ATP III recommends treating an elevated TG level (≥ 150
mg/dl).233 In addition, the delayed clearance of postprandial lipemia is an
independent risk factor for coronary heart disease.244 Abnormal transport
and metabolism of postprandial TG-rich lipoproteins are linked to athero-
sclerosis in the coronary and carotid arteries.139 At a given amount of dietary
fat, postprandial lipids are cleared more slowly in individuals who have a
higher baseline TG level. A delayed clearance of these atherogenic TG-rich
lipoproteins is thought to create a metabolic milieu that results in the pro-
motion of atherogenesis. Thus, a dietary pattern that reduces fasting and
postprandial plasma TG may decrease the accumulation of atherogenic, TG-
rich lipoproteins resulting in decreased risk for atherosclerosis.
A number of studies have been conducted to assess postprandial TG

clearance with respect to n-3 fatty acids. Most studies have been done with
fish-derived n-3 fatty acids and have shown increased postprandial lipid
clearance,245 driven by the potent hypotriglyceridemic effect of n-3 fatty
acids. A study conducted in physically active males illustrated that the
supplementation of 4.0 g of n-3 fatty acids/day for 5 weeks decreased post-
prandial TG area under the curve (AUC) by 42%.246 When a single bout of
exercise (1-h treadmill run) was completed 12 h prior to the high-fat test
challenge, the reduction in the postprandial TG AUC elicited by n-3 fatty
acid supplementation was significantly enhanced (– 58%), beyond that of
the effects seen with n-3 fatty acid supplementation alone.
A recent study247 compared the effects of α-linolenic acid (ALA) and EPA
+ DHA on several cardiovascular disease risk factors, including postprandial
blood lipid concentrations. The study was a placebo-controlled parallel arm
design involving 150 moderately hypercholesterolemic individuals. Subjects
were randomly assigned to receive one of five interventions: (1) 0.8 g EPA
+ DHA/day, (2) 1.7 g EPA + DHA/day, (3) 4.5 g ALA/day, (4) 9.5 g ALA/
day, and (5) n-6 fatty acid control. A postprandial fat challenge was com-
pleted at baseline and following 6 months of treatment. While the decrease
in fasting TG levels following the supplementation of 1.7 g/day EPA + DHA
(– 7.7%) was significantly different (p < 0.05) from the supplementation of
9.5 g/day ALA (10.9%), there were no treatment differences on the postpran-
dial response to a standard fat load. This lack of effect could possibly be
explained by the restricted range of baseline TG levels for the study subjects
and the fact that comparisons were made to a n-6 fatty acid control, rather
than to a standardized SFA load.
A controlled feeding study was conducted in 26 healthy men to evaluate
the effects of n-3 fatty acids, n-6 fatty acids, and saturated fatty acids on
postprandial TG, hemostatic factors and blood lipids and lipoproteins.248 For
the first 3 weeks all subjects consumed a diet high in SFA (30% kcal total
fat, 16% kcal SFA, 8% kcal MUFA, 4% kcal PUFA). Immediately following
this diet period, subjects were randomized to either a high n-3 diet (approx-
imately 5 g/day EPA and DHA) or a high n-6 diet (approximately 5 g/day
linoleic acid) for 3 weeks in a crossover design, with an 8-week washout
period between diets. Both diets contained 30% kcal total fat, 8% kcal SFA,
14% kcal MUFA, 6% kcal PUFA. At the end of each diet period, subjects
participated in a postprandial fat challenge that reflected the diet period that
they had just completed. Following these challenges, postprandial TG levels
were greatest on the n-6, lowest on the n-3 diet (p < 0.001), and intermediary
on the SFA diet. In addition, fasting TG levels were lowest following the n-
3 diet. This study lends further support to the database that demonstrates a
potent hypotriglyceridemic effect of n-3 fatty acids in both the fasting and
postprandial state.
Very few studies have been conducted to evaluate the effects of different
dietary patterns on postprandial TG response. In a recent study,249 the effects
of a hypoenergetic very-low-carbohydrate (< 10% kcal as carbohydrate) and
a hypoenergetic low-fat diet (< 30% kcal as total fat) on fasting blood lipids
and postprandial lipemia were studied in overweight men. Subjects con-
sumed the two diets in a randomized, crossover design for two consecutive
6-week periods. An oral fat tolerance test (86% kcal total fat, 11% kcal car-
bohydrate, 3% kcal protein) was completed at baseline and at the conclusion
of each of the test diet periods. Postprandial TG levels were significantly
reduced following the consumption of both test diets, compared with base-
line, with the greatest reduction following the very-low-carbohydrate diet
(– 34%). These results indicate that short-term use of a hypocaloric very-low-
carbohydrate diet has beneficial effects on postprandial TG levels. This effect
likely is mediated by the reduction in fasting TG levels and a beneficial effect
on other components of the metabolic syndrome, including small dense LDL-
C particles, markers of insulin resistance and the TG:HDL-C ratio. In several
other studies, similar postprandial TG-lowering was observed in normal
weight, normolipidemic men (– 29%) following a ketogenic diet (61% kcal
total fat, 8% kcal carbohydrate, 30% kcal protein),197 and women (– 31%)
following an isoenergetic very-low-carbohydrate diet (60% kcal total fat, 10%
kcal carbohydrate, 30% kcal protein),250 independent of weight loss.
In summary, supplementation with marine sources of n-3 fatty acids elicits
a beneficial reduction on postprandial TG levels. This effect likely is medi-
ated by the potent hypotriglyceridemic properties of marine n-3 fatty acids
in the fasting state. In addition, the use of a very-low-carbohydrate diet may
be an effective means for eliciting a reduction in postprandial TG levels, and
may have additional beneficial effects on risk factors associated with the
metabolic syndrome. In conclusion, the reduction observed in fasting and
postprandial plasma TG decreases atherogenic, TG-rich lipoproteins, result-
ing in a decreased risk for atherosclerosis. Further research is warranted to
develop dietary patterns that will lead to the optimization of both fasting
and postprandial TG levels.
Lipoprotein (a)
Lipoprotein (a) [Lp(a)] is a LDL-modified particle found almost exclusively
in primates. It is composed of an LDL moiety with the apolipoprotein B-100
attached to an apolipoprotein (a) (apo(a), a plasminogen-like protein) by a
disulfide bridge.251 Elevated Lp(a) has been identified as a powerful risk
factor for the development of CHD.252–254 The concentration of Lp(a) is largely
controlled by the apo(a) gene, with Lp(a) levels being inversely associated
with the size of apo(a).255–257 Population studies have noted that African-
Americans generally have higher levels of Lp(a) than other groups, including
Caucasians and Asians;258–260 however, the reasons for this remain unknown.
Although the level of Lp(a) is primarily genetically determined, several
studies have observed changes in Lp(a) with dietary manipulation. SFA
appear superior to other fatty acids in decreasing Lp(a) concentrations. A
study (n = 58) that investigated the effects of trans fatty acid consumption
in amounts typical of Western diets found that a diet high in SFA (16.2%
kcal) significantly reduced Lp(a) levels by 8–11% compared with diets high
in oleic acid (16.7% kcal), moderate in trans fatty acids (3.8% to kcal), and
high in trans fat (6.6% total calories).261 Likewise, Nestel et al.42 reported that
3 weeks of consumption of trans fatty acids at 7% of calories produced a 19%
reduction in Lp(a) when palmitic acid replaced elaidic acid (18:1 trans). These
findings are consistent with previous reports which have shown that the
saturated fatty acids, lauric, myristic, and palmitic acids, resulted in lower
levels of Lp(a) than oleic acid,262 and that substitution of butter for either
partially hydrogenated safflower or partially hydrogenated fish oil resulted
in lower Lp(a) concentrations.263 The DELTA study (n = 103) observed a
stepwise increase in Lp(a) concentrations (15.5, 17.0, and 18.2 mg/dl, p <
0.01) as dietary SFA was reduced from 15% to 9.0% to 6.1%, respectively.34
As the amount of SFA decreased in the diets, the amount of stearic acid
increased, which has been shown to increase Lp(a).264,265 However, when
compared with a diet rich in stearic acid (9.3% kcal), a diet high in trans fat
(8.7% kcal) increases Lp(a) significantly more (10% vs. 30%, respectively).43
A study in healthy young women (n = 25) examined the effect of a high SFA
diet (38.2% kcal total fat, 22.7% kcal SFA) on Lp(a) in direct comparison with
a low SFA diet (19.7% kcal total fat, 10.5% kcal SFA) with no change in the
PUFA to SFA ratio (0.14 and 0.17, respectively).266 Compared with a diet high
in MUFA and PUFA (38.2% kcal total fat, 2.4% kcal SFA, PUFA:SFA = 1.9)
both the higher SFA diets decreased Lp(a), with the highest level of SFA
reducing it the most (13.3%, p < 0.001). Furthermore, the concentrations of
total and LDL-C were similar between the two higher SFA diets.267 A 3-month
intervention in obese African Americans (n = 105) that included both diet
(total fat kcal 25.3%, SFA kcal 7.1%) and exercise components (45–60 min of
treadmill exercise three times per week) increased Lp(a) levels 35%, while
improving other factors (decreasing total and LDL-C, TG, and BMI, and
increasing HDL-C).268
The use of n-3 PUFA to modify Lp(a) levels has received less attention. In
hypertriglyceridemic patients, fish oil has been shown to reduce Lp(a) con-
centrations.269 The combination of a high dose (12 g/day) of fish oil (8.5 g
of omega-3 fatty acids) and a low-fat (30% kcal total fat) low-calorie diet
resulted in a 14% reduction in plasma Lp(a) levels in one study.270 However,
several studies could find no Lp(a)-lowering effect of n-3 fatty acids.248,271
Dietary soy protein has also been shown to modestly increase Lp(a) com-
pared with casein;272 however, this effect is eliminated after alcohol extraction
of soy protein.273 In addition, diets high in fruits and vegetables do not appear
to attenuate the rise in Lp(a) associated with low-SFA diets.274
New evidence also suggests that Lp(a) is differentially affected by specific
individual dietary fatty acids in the postprandial state.275 After 16 young
normolipidemic men consumed a meal rich in trans fat, Lp(a) levels did not
significantly change over 8 h. However, test meals with stearic acid produced
significantly higher Lp(a) levels at 4 h postprandially, followed by palmitic,
oleic, or linoleic acid. Acute changes in Lp(a) following a fat load have not
been consistently shown, however.276,277
Science-Based Dietary Guidelines for Health

Several agencies and organizations such as the National Academies,22 the
National Institutes of Health/National Heart, Lung and Blood Institute,6 the
U.S. Departments of Agriculture/Health and Human Services,278,279 the
American Heart Association,175,280 and the American Diabetes Association,281
have reviewed the scientific literature and developed recommendations for
the prevention and treatment of coronary disease. Overall, the recommen-
dations made for macronutrients, micronutrients, and other dietary constit-
uents by these groups are comparable (Table 11.5). The recommendations
for dietary fat intake (ranging between 20% and 35% of calories) and protein
intake (between 10% and 35% of calories) are similar among agencies. Like-
wise, total carbohydrate intake comprises the remainder of the macronutri-
ent component of the diet. Specific recommendations highlight decreasing
SFA and trans fat intake and to consume adequate amounts of unsaturated
fatty acids, including linoleic acid and α-linolenic acid. Importantly, all rec-
ommendations advise achieving and maintaining a healthy weight and par-
ticipating in regular physical activity.
Summary
The evolution of our understanding of the role that diet plays in modifying
lipid and lipoprotein risk factors for CVD is impressive. For many years, the
research focus was on studying how diet affected established risk factors. It
is clear that saturated and unsaturated fatty acids, as well as dietary choles-
terol, affect the traditional lipid and lipoprotein CVD risk factors. More recent
research has resulted in a growing recognition that diet affects new lipid and
lipoprotein risk factors for CVD such as LDL and HDL size, lipoprotein (a),
and postprandial lipids and lipoproteins. In addition, contemporary research
has identified some dietary factors and strategies that affect both established
and emerging risk factors. These include trans fatty acids, soluble fiber, n-3
fatty acids, glycemic load, and the total diet approach, which incorporates
nutrient-based and food-based dietary interventions. Interventions that tar-
get multiple dietary strategies are expected to have the greatest impact on
reducing total CVD risk. Because LDL-C remains the dominant lipid risk
factor for CVD interventions, a primary goal is to integrate multiple dietary
strategies that maximally lower LDL-C (Table 11.6).
TABLE 11.5
Current Recommendations and Guidelines for Macronutrientsa
American Diabetes NHLBI USDA Dietary AHA Dietary
NAS DRIs22 Association281 NCEP-ATP III6 Guidelines 2005278 Guidelines175,280
Total fat 20–35% < 30% 25–35% Choose fats wisely for good < 30%
health, 20–35%
SFA Low as possible < 10% < 7% < 10% < 10%
< 7%b < 7%b < 7%b
MUFA Remaining fatty acids to – Up to 20% Remaining fatty acids to Unsaturated for SFA
achieve total fat achieve total fat
PUFA 5–10% ~ 10% Up to 10% 5–10% Unsaturated for SFA
Linoleic acid (n-6 PUFA) 5–10% – – 5–10% –
Linolenic acid 0.6–1.2% 2 to 3 servings of fish/week – 0.6–1.2% 2 servings of fatty
fish/week
1 g/day EPA & DHA for
coronary patients
EPA & DHA (n-3 PUFA) Up to 10% can be consumed
as EPA + DHA
Trans fat Low as possible Minimize intake Keep intake low Low as possible Trans fat + SFA < 10%
Cholesterol (mg/day) Low as possible < 300 < 200 < 300 < 300
< 200b < 200b
Carbohydrate 45–65% 60–70% from carbohydrates 50–60% Choose carbohydrates ≥ 6 servings, include
plus MUFA wisely for good health, 3 servings whole
45–65% grains
Protein 10–35% ≤ 20% 15% 10–35%, 2–3 servings 50–100 g/day
Fiber (g/day) 21–38 – 20–30 ≥ 3 servings/day of whole > 25
grains
NAS DRIs, National Academy of Science Dietary Reference Intakes; NHLBI NCEP-ATP III, National Heart Lung and Blood Institute, National Cholesterol
Education Program-Adult Treatment Panel III; USDA, United States Department of Agriculture; AHA, American Heart Association; SFA, saturated fat; MUFA,
monounsaturated fat; PUFA, polyunsaturated fat; EPA, Eicosapentanoic acid; DHA, Docosahexanoic acid.
a Values are expressed as % of calories unless indicated otherwise.
b For individuals at high risk.

TABLE 11.6
Components of the Optimal Diet for LDL Cholesterol Reduction
Dietary Component Reduction in LDL-C
Reduced saturated and trans fat (< 7% calories) 8–10%
Reduced dietary cholesterol (< 200 mg/day) 3–5%
Viscous fiber (5–10 g/day) 3–5%
Plant sterol/stanol esters (2 g/day) 6–15%
Soy protein (25 g/day) 5%
Weight reduction (– 10 lb) 5–8%
Cumulative Estimate 30–48%
Source: Adapted from: National Cholesterol Education Program. NIH
Publication No. 02-5215, 1993; Jenkins, D.J. et al., Curr Opin Lipidol, 11,
49, 2000.
Based on an extensive database as discussed herein, the “optimal diet”

will provide a cornucopia of foods and nutrients that represent dietary
patterns that target multiple CVD risk factors.282,283 Overall, a diet that is
very low in saturated and trans fatty acids and cholesterol, rich in vitamins
and minerals, controlled in calories, high in dietary fiber, moderate in unsat-
urated fats including n-3 fatty acids, and low in simple carbohydrates will
improve the lipid and lipoprotein profile the most. As our knowledge base
increases, it is likely that we will continue to evolve science-based dietary
recommendations that will target a growing number of risk factors. With
this, implementation of multiple dietary strategies will elicit unprecedented
reductions in CVD risk as the result of targeting a growing number of lipid
and lipoprotein risk factors for CVD. The consequence of this will be to
accelerate the decline of CVD as an important cause of mortality and mor-
bidity in the population.
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12
Physical Activity, Exercise,
Blood Lipids, and Lipoproteins
J. Larry Durstine and Andrea C. Summer
CONTENTS
Introduction .........................................................................................................265
Endurance Exercise Training ............................................................................266
Lipids ..........................................................................................................266
Lipoproteins and Lipids ...........................................................................266
Apolipoproteins .........................................................................................270
Resistance Exercise Training .............................................................................271
Postprandial Lipemia .........................................................................................272
Exercise-Induced Mechanisms for Changes in Lipid and
Lipoprotein Metabolism ...........................................................................272
Physical Activity or Physical Inactivity ..........................................................273
Clinical Application............................................................................................274
Conclusions..........................................................................................................275
References ............................................................................................. 275
Introduction
The impact of regular exercise on plasma lipids and lipoproteins in recent
years has been more clearly defined in regard to the interactions between
lipids, lipoproteins, apolipoproteins (apo), and lipoprotein enzymes; and the
influence of various genetic and environmental factors such as aging, body
fat distribution, dietary composition, and cigarette smoking status.1–4 The
purpose of this review is to summarize present information regarding the
exercise training impact on lipid and lipoprotein concentrations.
265
Endurance Exercise Training

Lipids
Cross-sectional and longitudinal exercise training studies usually report
lower plasma triglyceride concentrations but not always.5,6 Large plasma
triglyceride reductions after exercise training are often reported for previ-
ously inactive individuals with higher baseline concentrations,6,7 whereas
subjects with low initial triglyceride concentrations demonstrate small tri-
glyceride reductions after exercise training8–10 (Figure 12.1). Neither cross-
sectional nor longitudinal exercise training studies support an exercise-
induced change in plasma cholesterol concentration6–10 (Figure 12.2). When
reduced plasma cholesterol levels are reported after exercise training, these
reductions were not related to either the initial cholesterol concentrations or
the exercise training program length.11,12 Rather, a reduction in plasma cho-
lesterol is associated with reductions in body weight, percentage body fat,
and dietary fat1,11–13 (see Table 12.1).
Lipoproteins and Lipids

The effect that a single exercise session has on postprandial lipoproteins and
lipids is greater than and different from that attributable to exercise
training14–16 and dietary energy deficiencies.14,17 Chylomicron and very low-
density lipoproteins (VLDL) are usually lower after exercise training.1,10
Physically Active Individuals

85
Sedentary
80 * Runs
Approximately 20
** miles/week
mg • dL-1
75 Runs
Approximately 50
*** miles/week
70 Runs
Approximately 70
miles/week
65
60
Triglyceride
FIGURE 12.1
As the volume of exercise training is increased, the greater is the reduction in triglyceride
concentration.
Physical Activity, Exercise, Blood Lipids, and Lipoproteins 267

180
Sedentary
160 Runs
Approximately 20
miles/week
140
mg • dL-1
Runs
120 Approximately 50
miles/week
100 Runs
Approximately 70
miles/week
80
60
Cholesterol
FIGURE 12.2
Exercise training has little impact on blood cholesterol. Note that even the group running 70
miles per week did not have altered cholesterol concentrations.
Plasma low-density lipoprotein cholesterol (LDL-C), a powerful coronary

artery disease (CAD) risk predictor, is elevated in individuals with diets high
in fat content, especially saturated fats.14 LDL-C concentrations are not usu-
ally lower after aerobic exercise training,6,7,9,10,12,18 though lower LDL-C levels
are reported.14 LDL particles are also categorized according to size. Each size
having a different CAD risk (e.g., the smaller, denser LDL particle directly
correlates with CAD incidences and may depend on elevated triglyceride
concentrations).19 Exercise training may impact these LDL particles differ-
ently. Williams et al.20,21 found no change in small LDL particles after 1 year
of exercise training in overweight men while hypercholesterolemic men
(cholesterol > 240 mg/dl) have reduced triglyceride and small LDL size
particles with increased physical activity levels.22 Overweight subjects com-
pleting 6 months of jogging (~ 20 miles per week at 65–80% of their cardio-
vascular capacity)23 and after 8 months of regular exercise24 exhibited greater
LDL particle sizes along with lower LDL concentrations. Beard et al.25 dem-
onstrated mean LDL particle size increases with 3 weeks of diet and brisk
walking. Cholesterol concentrations decreased in the more dense LDL sub-
fractions and increased in the less-dense LDL fractions, and the change was
correlated with reductions in triglyceride concentrations. A 3-month athero-
sclerosis treatment program that included regular moderate-intensity exer-
cise did not alter LDL particle size in 25 coronary artery disease patients.26
However, the intervention was shown to increase the antioxidant content
and reduce oxidative susceptibility of LDL particles.
Plasma lipoprotein (a) [Lp(a)], an LDL subfraction containing the apolipo-
protein apo(a)27, is highly homologous with plasminogen. As a result, the
TABLE 12.1
Lipid, Lipoprotein, Lipoprotein Enzymes and Transfer Protein
Changes Associated with Exercise
After Regular Exercise Participation
Lipid/Lipoprotein
Triglyceride Decreases of 4 to 37%

Approximate mean change of 24%
Cholesterol No changea
LDL-C No changea
Lp(a) No change
HDL-C Increases of 4 to 18%
Approximate mean change of 8%
Enzyme
LPL
Activity Increased
Mass
HL
Activity No change or reduced (may be reduced with weight loss)
Mass No information
LCAT
Activity Increased/No change
Mass No information
CETP
Activity No change/Increased
Mass Increased
LPL, lipoprotein lipase; HL, hepatic lipase; LCAT, lecithin:cholesterol acyl-
transferase; CETP, cholesteryl ester transfer protein.
a No change if body weight and diet do not change (see text).
apo(a) portions of Lp(a) compete with plasminogen for binding sites on

fibrin, inhibiting fibrinolysis.27 Moreover, Lp(a) concentrations greater than
30 mg/dl have the same negative CAD effects as LDL-C and inhibit throm-
bolysis.27 Lp(a) is an inherited trait and does not appear to change following
regular physical activity participation.2,27–30
Exercise training longer than 12 weeks is more likely to increase plasma
high-density lipoprotein (HDL-C)1,2,10,31 (Figure 12.3) but not always.32 These
chances are usually present in a dose-dependent manner and are associated
with increased energy expenditure.1,2 Several factors deserve consideration
when evaluating the impact of regular exercise participation and include the
exercise training volume measured by kcal expended during the exercise
training program, body weight and composition changes, dietary changes,
and the length of the exercise intervention program. Exercise-induced
increases in HDL-C range from 4% to 22%, while the absolute HDL-C
increases are more uniform and range from 2 to 8 mg/dl. The initial HDL-
C level and HDL-C change are important considerations with an inverse

** **
60 *
Sedentary
Runs
Approximately 20
* miles/week
40 ** **
mg • dL-1
** ** Runs
Approximately 50
miles/week
20 *
Runs
Approximately 70
miles/week
0
HDL-C HDL2-C HDL3-C
FIGURE 12.3
Exercise training is associated with increased HDL-C, and the increase in HDL-C is associated
with an increase in the HDL2-C.
correlation,33 a greater change with higher initial HDL-C levels34 and no

relationship7 being reported. Significant correlations have been established
between distance run per week, the time spent in exercise training and
HDL-C change.6 Collectively these data provide support for a strong rela-
tionship between increased exercise training volume and HDL-C increases.1,2
Wood et al.9 observed an inverse relationship between body fat change and
HDL-C change, but the addition of distance run per week did not improve
the ability to predict HDL-C change. Maintaining body weight and body
fat during exercise training resulted in HDL-C increases of 8 mg/dl7 and
3 mg/dl,35 whereas weight-loss programs using caloric restriction alone or
caloric restriction with exercise training rendered body weight and percent-
age body fat decreases in both groups while HDL-C increased.36 These find-
ings support the finding that exercise training without altered body weight
and/or composition can increase HDL-C, and this increase is augmented by
body fat loss. This is especially true for men with elevated triglyceride and
abdominal obesity.37 Crouse et al.38 reported elevated HDL-C and HDL2-C
during the 24–48 h time period immediately after a single exercise session,
while the overall effect after the exercise training intervention was an
increased HDL2-C and a decreased HDL3-C. HDL2-C is usually increased
after exercise training,7,10,39 while HDL-3C is reduced.39 The HDL3b particle is
directly related to CAD risk, while HDL2a and HDL2b are associated with
reduced CAD risk. Williams et al.40 observed an increased HDL2b and
decreased HDL3b after a 1-year exercise training-program (see Table 12.1).
Apolipoproteins
Apolipoproteins and exercise training have been reviewed.1,41,42 Essentially
increased apo A-I levels are observed7,10,40 but not always.9,12 Whether apo B
levels decrease12,43 or not,8,9,10,12 apo B changes following exercise training
usually parallel LDL-C changes. Wood et al.9 found no overall change in apo
B after 1 year of exercise training, but did find a significant inverse correlation
between distance run and change in apo B levels. Although no differences
in current and former runners were reported,8 higher apo E concentrations
were observed in young but not older runners.44 Lower apo E levels after
exercise have been found. Tanabe et al.45 reported changes after exercise
training in men, but not women. Taimela et al.46 reported in a cross-sectional
study of 1500 subjects between the ages of 9 and 24 evidence that leisure-
time physical activity has a great effect on lipoprotein profiles of apo E2
individuals, a lesser effect on apo E3 subjects, with no effect on apo E4 subjects.
Data from longitudinal studies, however, may provide more direct evidence
of a possible interaction between apo E polymorphism and exercise training.
Nonetheless, St-Amand et al.47 observed in men a significant interaction
between physical activity, lipids and lipoproteins, and apo E phenotypes.
·
An inverse relationship between maximal oxygen consumption (VO2max)
and triglyceride levels for individuals heterozygous for apo E2 or homozy-
gous for apo E3 was observed but not for apo E4 phenotypes. Plasma LDL-
·
C levels were inversely associated with VO2max only in women homozygous
·
for apo E3, whereas VO2max was positively associated with plasma HDL2-C
only in men and women who were apo E3 homozygotes. Though not statis-
tically significant, post-heparin LPL activity was increased in apo E2 subjects.
Recently, Hagberg et al.48 reported decreased triglyceride concentrations that
were significantly greater in apo E2 and E3 groups while HDL-C increases
were greater in only the apo E2 subjects. Thompson et al.49 examined pro-
spectively the impact of the apo E genotyope on lipoprotein responses to
exercise in healthy normolipidemic subjects following 6 months of exercise
training. The results of this study demonstrated that apo E polymorphisms
affect the lipid response to exercise training. Specifically, reductions in LDL-
C/HDL-C and total cholesterol/HDL-C ratio were greater in apo E3 homozy-
gotes producing significantly greater reductions with exercise training in
common markers of CAD risk. Thus, one of apo E’s major functions might
be to facilitate triglyceride clearance. Apo E2 has a low affinity for the apo
E receptor and in the homozygous form can produce marked hyperlipi-
demia. Hence, this information suggests that exercise training is most effec-
tive at reducing plasma triglyceride while increasing HDL-C and LPL
activity in those persons who have an impaired ability to clear triglycerides
or the subjects having apo E2 phenotype. Thereby, increased HDL-C may be
limited by the presence of the apo E2 gene when subjects are sedentary (see
Table 12.1).
Leon et al.,50 on the other hand, found that Apo E polymorphism was
·
not associated with VO2max levels in the sedentary state nor after exercise
training. Nevertheless, VLDL, cholesterol, and triglyceride levels were sig-

nificantly higher in persons with the apo E2 and apo E4 in white men only.
Resistance Exercise Training

Compared with endurance training literature, considerably less information
exists supporting resistance training as a modifier of plasma lipids and
lipoprotein-lipids.51 Present resistance exercise studies are often contradic-
tory, with some showing positive benefits of resistance exercise on the lipid
profile,52–54 while others find no benefit.55–57 Inter-study variations in meth-
odologies can contribute to the many study outcome differences. Although
it is unlikely that differences between studies can be attributed to any single
reason, several possibilities exist with the leading candidate being variation
in the exercise volume (caloric expenditure) completed during resistance
exercise. It may simply be that the caloric threshold for inducing lipid and
lipoprotein changes is typically not reached with resistance training. Indeed,
a concern with the methodology from many resistance exercise studies is
the lack of proper volume quantification and the failure to include a proper
non-exercise control group. Training studies reviewed here meet the follow-
ing requirements: (1) resistance exercise training groups met at least three
times a week, (2) training routines were composed of exercises designed to
train the major muscle groups of the body, performed in one to three sets,
at least six repetitions per set with 15 to 120 s rest between sets, and (3)
resistance training lasted from 8 weeks to 22 months.
Generally, triglyceride concentrations are not altered by resistance exercise
training53,55,57,58 even when initial triglyceride levels were elevated.56 In con-
trast, decreased triglyceride concentrations after resistance training are
reported in elderly women.54 and after moderate-intensity, high-volume
training.59 Plasma cholesterol,56,60 LDL-C,60 and apo B-100 concentrations61
were usually not altered following resistance training when total body mass,
lean body mass, and percentage body fat were not changed.56,57,60,61 However,
decreased body fat percentage and an increase in lean body mass after
resistance exercise training53 are associated with a decrease in plasma cho-
lesterol and LDL-C concentrations. Both LDL-C and total cholesterol have
been shown to be reduced after circuit resistance training.59 In the most
published studies, HDL-C concentrations are unresponsive to resistance
training,56,60 yet increases have been reported.54 When resistance exercise is
combined with aerobic exercise, results are conflicting; HDL-C was variously
reported to be increased62 or unchanged57 after training. Following a single
resistance exercise session, triglyceride is reduced and HDL-C increased,
although the changes appear to be volume dependent.63
Postprandial Lipemia
Exaggerated postprandial lipemia, a comparatively prolonged elevated
blood triglyceride concentration after a meal, is associated with atheroscle-
rosis. Exercise completed within 24 h before a high-fat meal reduces post-
prandial lipemia.64 This triglyceride reduction is found after jogging,65
exercise cycling,66 and resistance exercise.63,67 Current evidence indicates that
the magnitude of the reduction is primarily related to total energy expended
during the preceding exercise session(s), whether the exercise is performed
all at once or intermittently.64,68–70 Neither exercise intensity nor duration
effect postprandial lipemia independent of total energy expenditure.64 Yet to
be clarified are subject characteristics that may influence the magnitude of
the response, such as training status, gender, obesity, and existing athero-
sclerosis. Furthermore, the mechanism responsible for reduced postprandial
lipemia is not well-defined, but is likely related to increased lipoprotein
lipase (LPL) activity in the hours after an exercise session.65,66
To summarize, reduced postprandial lipemia is found when exercise pre-
cedes a meal by up to 24 h. The magnitude of the effect is related to the
exercise volume performed. A measurable, beneficial effect on circulating
lipids and lipoprotein-lipids may be expected by untrained persons after a
single exercise session where 350 kcal are expended, whereas trained indi-
viduals may require 800 kcal or more to elicit comparable changes. Under
these circumstances, the beneficial changes in lipid and lipoprotein-lipid
concentrations after exercise are similar in magnitude to those reported after
the completion of a longitudinal exercise training program. Since blood lipids
and lipoprotein lipids are associated with coronary artery disease, the
changes induced by a single exercise session can be expected to reduce the
risk of this disease. In order for these beneficial lipid and lipoprotein-lipid
changes to be maintained, exercise must be performed on a regular basis,
optimally every other day.
Exercise-Induced Mechanisms for Changes in Lipid and

Lipoprotein Metabolism
Intravenous heparin injections and muscle and adipose tissue biopsy sam-
ples are used to measure LPL activity. High-intensity exercise sessions and
high energy expenditure that deplete intramuscular triglyceride stores are
needed to increase muscle LPL synthesis and release (Table 12.1).26,71
Increased plasma post-heparin LPL activity (PHLPL) is usually not found
until 4 to 18 h post-exercise,72,73 is reported for endurance athletes,74 and is
increased after exercise training,75,76 though not always.5,7,77,78 Ethnic PHLPL
differences exist with higher PHLPL values in white males, but not in black
males after 20 weeks of endurance training.75 Increases in PHLPL were
recently related to endothelial lipase genotype.79
Seip and Semenkovich71 have reviewed the molecular explanations for the
exercise impact on LPL gene expression. In essence, a transient rise in LPL
messenger RNA (mRNA) is present by the fifth day of exercise, while 5–13
consecutive days of exercise training were needed to increase skeletal muscle
LPL mRNA, LPL mass, and total LPL enzyme activity.76,80 Nevertheless, adi-
pose tissue LPL mRNA, protein mass and enzyme activity remained
unchanged.77,80 Rat white skeletal muscle used during voluntary running had
elevated LPL mass, total LPL activity, and heparin-releasable LPL activity with
no change in either white or red muscle not recruited during voluntary exer-
cise.78,81 Thus, exercised white skeletal muscle intrinsically results in elevated
LPL activity by pretranslational mechanisms, and an increase in LPL expres-
sion during exercise training likely requires local contractile activity.
An inverse association exists between resting HL activity and HDL2-C
while HL is directly related to HDL3-C.82 In general, no changes in resting
HL activity are reported between inactive and active individuals,74 and a
single exercise session results in no significant HL activity changes.73,80,83–86
Low CETP activity may provide an anti-atherogenic effect by slowing
hepatic HDL2 catabolism and decreasing the amount of plasma cholesterol-
rich particles.81,87 Elevated plasma CETP activity is found in physically active
individuals,5,82 while decreased CETP activity following exercise training is
reported.77,85,86 In addition, LCAT activity is increased in physically active
men,82,87 but not post-exercise training.5,20,75
Physical Activity or Physical Inactivity

Up to this point our discussion has centered around the positive impact of
regular exercise participation on lipid and lipoprotein metabolism, which
can result in a reduction for cardiovascular disease risk. On the other hand,
it is also known that physical inactivity and lower cardiovascular fitness are
clearly associated with increased heart disease risk.88 The question that
comes to mind concerns the potential for just everyday physical activity, not
including planned exercise programming, and the likelihood for beneficial
improvement in blood lipid and lipoprotein levels and reduced heart disease
risk. Presently, the existing data are conflicting. In the past some published
reviews have suggested that LDL-C and HDL-C are positively influenced
by physical activity,89,90 whereas others3,4 indicate that physical activity has
little effect on LDL-C and only small positive effects on HDL-C. However,
several studies have reported that moderate-intensity physical activity is
associated with positive blood lipid and lipoprotein changes.91–94 Finally,
multiple studies have reported that the volume of physical activity or
exercise performed, regardless of intensity, is an important predictor of HDL-

C change.2–4,41,95,96 Present information does support some positive modifica-
tion in blood lipids and lipoproteins for people maintaining an active life-
style. To maximize the benefits, a person must lead a very active lifestyle.
The current literature is not clear in this area.
Another way to look at the benefits of physical activity on blood lipids
and lipoproteins is to understand how the body adapts to physical inactivity.
Hamilton et al.97 recently proposed a relatively new concept he has termed
“Inactivity Physiology.” He suggests that some health-related proteins like
lipoprotein-lipase are regulated by different metabolic processes over the
physical activity continuum, and this different regulation process is highly
sensitive to physical inactivity. Essentially, physical inactivity could elicit a
physically inactive pathway that in turn would generate a powerful regula-
tion process that can inhibit lipoprotein-lipase. There is evidence that phys-
ical inactivity does develop a biological inhibitory signal that reduces
lipoprotein-lipase activity, and that this process may be independent of lipo-
protein-lipase messenger RNA.98,99 However, much more work in this area
is needed before conclusive statements can be made.
Clinical Application
Current treatment guidelines for the medical management of plasma lipids
and lipoproteins are provided by the National Cholesterol Education Pro-
gram Adult Treatment Panel III (NCEP).100 Combining these guidelines with
an individual risk assessment estimated from Framingham Study data is one
clinical approach to hyperlipidemia management.101 Pharmacologic therapy
is the primary therapy for reducing lipid and lipoprotein levels since it is
highly effective and generally well tolerated.102 Considered adjunctive to
pharmacologic therapy are secondary therapies such as reduced dietary fat
intake, weight loss, and exercise interventions. These therapies are extremely
important, but are greatly limited by patient compliance. Some patients
achieve these desired lipid levels by these interventions, but in clinical prac-
tice this is unfortunately the exception and not the rule. Daily exercise pro-
grams are recommended for all patients with lipid disorders because exercise
can profoundly decrease plasma triglyceride and improve glucose intoler-
ance that contribute to dyslipidemia. Finally, when incorporating physical
activity or exercise programming as an intervention, it must be borne in
mind that the volume of physical activity or exercise completed is a key in
determining whether a positive impact on blood lipid and lipoprotein levels
will be observed. In order for these intervention programs to contribute to
lipid management and optimize blood lipids and lipoproteins, the volume
of physical activity or exercise training completed must be greater than
1200–1500 kcal of energy expenditure each week. The daily energy expen-
diture should be approximately 200 kcal.
Conclusions
Current information supports the favorable impact of exercise training on
the lipid and lipoprotein profile. Regarding hyperlipidemic disorders, the
primary intervention is pharmacological while diet modification, weight
loss, and exercise, though important, are considered adjunctive therapies.
Because much is known about the exercise training-induced plasma lipid
and lipoprotein modifications as well as the responsible mechanisms for
these changes, we are now better able to develop a comprehensive medical
management plan that optimizes pharmacological and adjunctive therapies.
Present scientific investigations are focusing on the molecular basis for lipid
and lipoprotein change as a result of various interventions (e.g., knowing a
person’s apo E genotype). Information from these studies will provide a
better understanding as to why some individuals respond to exercise, while
others do not. Another research concern is the interactive effects between
regular exercise participation and pharmacological inventions. This knowl-
edge coupled with available lipid intervention information, such as diet
modification and weight loss, can aid in optimizing individual medical
management for special lipid disorders.
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13
Acute Changes in Lipids and Lipoprotein-
Lipids Induced by Exercise
Stephen F. Crouse
CONTENTS
Introduction .........................................................................................................283
Acute HDL-C and HDL-C Subfraction Changes with Exercise .................284
Changes in Blood Triglycerides after Exercise ..............................................285
Post-Exercise Changes in Total Cholesterol and LDL Cholesterol.............290
Lipoprotein (a) after Exercise ...........................................................................291
Apolipoproteins and Exercise-Induced Changes ..........................................291
Mechanisms for Changes in Blood Lipids and Lipoproteins
Following Exercise.....................................................................................292
Other Exercise Considerations for Lipid Benefit...........................................293
Summary ..............................................................................................................294
References ............................................................................................. 295
Introduction
Under normal fed conditions in humans, lipids stored in muscle and circu-
lating in blood are an important source of energy to perform physical exer-
cise. The proportion of energy coming from oxidation of lipids, primarily
fatty acids, during exercise is a function of the work intensity. At relatively
low to moderate intensities (< 65% VO2max), lipid oxidation may supply more
than half of the energy required by exercise, but at higher intensities most
energy is provided through oxidation of carbohydrate.1 Regardless of the
energy source during exercise, lipids are the predominant fuel oxidized
during recovery from exercise. Thus an acute change in blood (and muscle)
lipid concentrations is a natural consequence of the use of this energy source
for the work of physical exercise. This is especially the case when the work
283
demands of exercise can be met predominantly by aerobic means. Further-

more, metabolic, hormonal, and physiologic effects of a single session of
exercise, especially intense and prolonged exercise, may take hours or days
from which to fully recover. For example, muscle insulin sensitivity, glucose
transport, and uptake of blood triglycerides (TG) are increased for at least
several hours after a single session of exercise.2,3 Thus, a rationale for acute
changes in blood lipid concentrations is firmly based on known exercise-
induced changes in fuel metabolism.
These acute blood lipid changes lasting for hours or days after exercise
may be at least partly responsible for the anti-atherogenic lipid profile
characteristic of physically trained persons. This hypothesis was proposed
quite some time ago following observations that blood TG and total cho-
lesterol (TC) concentrations were often lower, while high-density lipopro-
tein cholesterol (HDL-C) concentrations were often higher up to 44 h after
exercise.4–8 Attempts to verify these early findings have produced hundreds
of studies with results that vary widely. A careful review of the related
literature will show that factors that could spuriously affect the post-exer-
cise lipid concentrations were not controlled in a majority of the published
studies. There was wide inter-study variability with respect to exercise
mode, intensity, duration, and volume, all factors that could impact post-
exercise lipid concentrations. Often exercise-induced changes in plasma
volume were not considered when reporting blood lipid concentrations.
The timing of blood collection relative to the exercise session was not
uniformly controlled among existing studies. Not surprisingly for human
studies, inter-study variation exists with respect to such potentially con-
founding variables as subject age, training history, gender, menopausal
status, diet, body weight, body fat, and pre-exercise blood lipid concentra-
tions.9,10 The acute exercise studies selected for review in this chapter
include those in which the methods provide some level of control for
changes in plasma volume induced by exercise (Table 13.1).
Acute HDL-C and HDL-C Subfraction Changes with Exercise

It is well documented from case-control study designs that runners show
higher blood HDL-C concentrations as compared to non-runners.9,11–13 How-
ever, in studies in which untrained subjects were trained under controlled
conditions, only about half show significant improvements in HDL-C con-
centrations.13 Several of the early cross-sectional and training studies did not
control for the timing of the last session of exercise before blood samples
were taken. Thus, it is very possible that many studies actually were meas-
uring acute changes in HDL-C rather than a training effect. As evidence that
this could be the case, several well-controlled studies have shown that HDL-
C concentrations were raised by a single session of aerobic exercise. In the
Acute Changes in Lipids and Lipoprotein-Lipids Induced by Exercise 285
majority of published studies related to acute exercise and lipids, a delayed

increase in blood HDL-C concentration was noted 24–72 h after exercise, and
ranged from 4% to 34% over pre-exercise values.14–21 Even individuals with
high blood cholesterol may benefit from this short-term rise in blood HDL-
C.10,22,23 Some data suggest that a volume threshold exists for the HDL-C
change that depends on the functional capacity of the individual. Research
has shown that in trained individuals up to 1000 kcal of exercise energy
expenditure is necessary to produce significant elevations in blood HDL-C
concentrations, whereas 350–400 kcal energy expenditure is sufficient to elicit
significant increases in less well-trained and sedentary persons.10,15,20,21,24
The subfractions of HDL-C identified by ultracentrifugation, namely the
relatively more buoyant HDL2-C and the denser HDL3-C, may confer pro-
tection from cardiovascular disease. Although some differences exist in the
metabolism of these lipoproteins, both have been shown to be inversely
related to heart disease.25 Studies show that both may be acutely elevated
after exercise.10,14–16,19–23,26 As with HDL-C, training status may influence the
response of the HDL subfractions. However, this issue has been addressed
only rarely, so conclusions are tenuous. In a study to compare the exercise
response in untrained and trained men, Kantor et al.18 reported that HDL2-C
concentration was acutely elevated in trained men following aerobic exer-
cise, while in untrained men HDL3-C was higher. In contrast, Crouse et al.10
found that blood HDL3-C, but not HDL2-C, concentration was higher fol-
lowing a single session of aerobic exercise completed by men with elevated
cholesterol before and after 6 months of aerobic training. In support for an
acute increase in the relatively denser HDL3-C subspecies, HDL density
assayed by ultracentrifugation was reportedly reduced for up to 2 days in
physically active women after aerobic exercise.27 This finding demonstrates
that there may be a shift from the less dense (HDL2) to the more dense (HDL3)
HDL subspecies following aerobic exercise. Although several questions
remain unanswered (e.g., which HDL subspecies is changed with exercise
and what volume of exercise is required to produce a beneficial change), it
is clear from these studies that HDL-C and HDL-C subspecies are responsive
to a single session of exercise, and that the response generally results in a
less atherogenic lipid profile.
Changes in Blood Triglycerides after Exercise

In addition to the beneficial effects of exercise on HDL, it is reported in the
majority of controlled acute exercise studies that TG concentrations fall sig-
nificantly after a single session of aerobic exercise. This beneficial fall in
circulating TG persists for up to 72 h.10,14–16,19–23 The acute effect does not
appear to depend on an individual’s training experience, intensity, or mode
of aerobic exercise. There have been relatively few well-controlled studies
TABLE 13.1
286
Acute Exercise Studies Selected for Review

First
Author, APO APO APO APO
Yearref # Subject Timing TC TG VLDL-C LDL-C HDL-C HDL2-C HDL3-C A-I A-II B E LPLa HTGLa LCATa CETPa CETPm
Bounds, 14, m, 28, pre-exercise 160 113 nr 89 48 13.5 35 nr nr nr nr nr nr nr nr nr

200014 t, 50.2
IPE –5.1* –14.1 nr ns 1.6 ns 0 nr nr nr nr nr nr nr nr nr
+24 h 0.5 –16.4* nr ns 10.5* ns 7.7* nr nr nr nr nr nr nr nr nr
+48 h –3.1 –25.9* nr ns 8.1* ns 2.2 nr nr nr nr nr nr nr nr nr
Crouse, 26, m, 47, pre-exercise 251 163 nr 171 46 10.9 36.4 135 nr 95 nr nr nr nr nr nr
199710 t, 42.9
IPE –3.2* 3.7 nr –0.6 0 –13.8 0.5 –2.2 nr –2.1 nr nr nr nr nr nr
+24 h 0 –9.8* nr 5.3* 6.5* 6.4 5.8* 3.0* nr 4.2* nr nr nr nr nr nr
+48 h 1.6* –6.8* nr 5.9* 4.4* 14.7* 3.6* 0.7 nr 1.1 nr nr nr nr nr nr
Crouse, 39, m, 46, pre-exercise 254 177 nr 173 45 6.2 38.5 141 nr 99 nr nr nr nr nr nr
199522 ut, 31
IPE –4.7* –5.7 nr –4.1* –2.2 ns –2.9 ns nr 0 nr nr nr nr nr nr
+24 h 1.2 –18.6* nr 5.8* 6.7* ns 8.3* ns nr 9.1* nr nr nr nr nr nr
+48 h 4.7* –14.7* nr 8.1* 8.9* ns 7.0* ns nr 9.1* nr nr nr nr nr nr
Davis, 10, m, 28, –24 h 150 85 14 80 57 12 45 111 40 78 nr nr nr nr nr nr
199233 t, 62
pre-exercise ns ns ns ns ns ns ns ns ns ns nr nr nr nr nr nr
IPE ns ns ns ns ns ns ns ns ns ns nr nr nr nr nr nr
+1 h ns ns ns ns ns ns ns ns ns ns nr nr nr nr nr nr
Ferguson, 11, m, 26, –24 h 166 101 20 101 45 14 31 nr nr nr nr 13.1 19.5 nr nr nr
199815 t, 56.2
pre-exercise 169 110 22 105 42 14 28 nr nr nr nr nr nr nr nr nr
IPE –6.5 1 0 –18.1* 21.4* 28.6* 17.9 nr nr nr nr –8.4 –15.4 nr nr nr
+24 h –4.7 –36.4* –36.4* –11.4* 28.6* 35.7* 25.0* nr nr nr nr 48.9* –4.6 nr nr nr
+48 h –3 –20* –22.7* –10.5 23.8* 42.9* 14.3 nr nr nr nr 47.3* –18 nr nr nr
Ferguson, 11, m, 27, –24 h 166 101 20 101 45 14 31 nr nr nr nr nr nr nr nr nr
200324 t, 56.2
pre-exercise 4.37 1.24 0.57 2.72 1.09 0.36 0.72 nr nr nr nr nr nr nr nr nr
During Ex 1 –4.81 –3.23 –8.77 –9.56 9.17 –5.56 18.06 nr nr nr nr nr nr nr nr nr
During Ex 2 –6.41* –5.65 –5.26 –15.44* 9.17 0.00 15.28 nr nr nr nr nr nr nr nr nr
During Ex 3 –5.26 –6.45 –5.26 –9.56 4.59 –13.89 15.28 nr nr nr nr nr nr nr nr nr
During Ex 4 –8.47* –1.61 –5.26 –16.18* 6.42 –5.56 11.11 nr nr nr nr nr nr nr nr nr
During Ex 5 –7.78* –4.03 0 –15.44 6.42 0.00 15.28 nr nr nr nr nr nr nr nr nr
IPE –6.41 0.81 0 –18.38* 21.1* 30.56* 18.06 nr nr nr nr nr nr nr nr nr
Foger, 8, m, 34, –48 h 224 132 nr 130 67 12 55 144 53 77 nr nr nr nr 109 1.23
199416 t, nr
+24 –33* –69* nr –38* –9 8 –13 –24* –25* –31* nr nr nr nr –25 –31*
+48 –1 –18* nr –18* 33* 92* 20* 20* 25* 3 nr nr nr nr 6 –15*
+72 2 –5 nr –12* 34* 150* 11 23* 19* 3 nr nr nr nr 8 –10
+120 4 17 nr –3 15 83* 0 19* 19* 5 nr nr nr nr 10 –5
+192 1 19 nr –5 6 42* –2 10* 19* 8 nr nr nr nr 10 1
Goodyear, 12, f, 25, –24 h 197 49 nr 115 72 nr nr nr nr nr nr nr nr nr nr nr
199017 t, 53
+10 min –5.1 159.2* nr –25.2* 5.6 nr nr nr nr nr nr nr nr nr nr nr
+24 h –13.2* 2.2 nr –31.3* 15.3* nr nr nr nr nr nr nr nr nr nr nr
+72 h –9.1* 14.3 nr –13 –5.6 nr nr nr nr nr nr nr nr nr nr nr
+120 h –14.7* 6.1 nr –18.3 –9.7 nr nr nr nr nr nr nr nr nr nr nr
Grandjean, 25, m, 45, –24h 217 144 nr 147 42 9 34 nr nr nr nr 13.1 21.1 nr 31.4 nr
200023 ut, 33.3
IPE –3.2* –2.8 nr –5.4 2.4 –11 2.9* nr nr nr nr 7.6 –6.6 nr –0.3 nr
+24 h 0.5 –11.1* nr –2.0 9.5* 11 8.8* nr nr nr nr 20.6* –0.5 nr 2.9 nr
+48 h 0 –11.1* nr –2.7 14.3* 11 8.8* nr nr nr nr 12.2 –3.8 nr 4.8 nr
Acute Changes in Lipids and Lipoprotein-Lipids Induced by Exercise
Hughes, 32, f, 22, pre-exercise 167 78 nr 98 54 nr nr nr nr nr nr nr nr nr nr nr

1991(30) ut, 39.4
+10 min 2.7 3.0 nr 2.5 4.3 nr nr nr nr nr nr nr nr nr nr nr
+1 h 0 –9.6 nr 0 2.5 nr nr nr nr nr nr nr nr nr nr nr
+24 h –1.3 –18.6* nr –1.0 1 nr nr nr nr nr nr nr nr nr nr nr
+48 h –2.1 –21.6* nr –1.0 1 nr nr nr nr nr nr nr nr nr nr nr
287
288
Acute Exercise Studies Selected for Review

First
Author, APO APO APO APO
Yearref # Subject Timing TC TG VLDL-C LDL-C HDL-C HDL2-C HDL3-C A-I A-II B E LPLa HTGLa LCATa CETPa CETPm
Imanura, 7, f, 22, –1 h 145.1 61.9 nr 78.4 53.6 37.4 15.1 116.7 25 60.4 nr nr nr nr nr nr
200034 ut, 36.2
IPE –0.14 –4.039 nr –0.128 2.61 1.604 –1.987 3.085 1.6 –0.828 nr nr nr nr nr nr
30 min of +.5 h 0.207 –3.716 nr 2.934 3.358 1.872 5.96 4.456 3.2 2.815 nr nr nr nr nr nr
exercise
at 60%
VO2max
+1 h 1.241 –3.231 nr 0.255 0.56 0 1.325 0.771 0.8 –0.166 nr nr nr nr nr nr
+2 h –0.35 –2.908 nr 0 –0.933 –0.535 1.325 0.771 0.8 –0.497 nr nr nr nr nr nr
+24 h 1.103 22.132 nr –0.51 0.933 –1.872 1.325 3.428 2.4 3.642 nr nr nr nr nr nr
–1 h 148.3 64.4 nr 81.4 53.3 36.5 15.2 116.9 25 62.6 nr nr nr nr nr nr

IPE 0.674 –7.453 nr 0.246 1.126 3.288 –3.947 2.31 –0.8 0.479 nr nr nr nr nr nr
60 min of +.5 h –0.41 –6.677 nr 3.194 0.188 1.918 –0.658 0.855 0.8 0 nr nr nr nr nr nr
exercise
at 60%
VO2max
+1 h –0.14 –2.64 nr 0.246 0.563 0 0.658 –0.684 0 –0.958 nr nr nr nr nr nr
+2 h 1.888 –3.106 nr –0.123 1.501 3.014 1.316 0.684 –0.4 –1.118 nr nr nr nr nr nr
+24 h –0.94 –7.919 nr –0.369 0.563 2.74 5.921 0.086 0.4 –1.438 nr nr nr nr nr nr
Kantor, 11, m, 33, –24 h 197 94 nr 126 51 18 33 nr nr nr nr 8.3 13.6 nr nr nr
198718 t, 48
+10 min –2.0 –14.9 nr –4.0* 3.9* nr nr nr nr nr nr –13.4* –2.2 nr nr nr
+24 h 1.5 –6.4 nr 1.0 7.8* nr nr nr nr nr nr 10.8* 2.9 nr nr nr
+48 h 3.6* 2.1 nr 1.6 9.8* 16.7* 3.0 nr nr nr nr 1.2 5.9 nr nr nr
+72 h 3.1 0.0 nr 1.6 9.8* 22.2* 3.0 nr nr nr nr 8.4 8.8* nr nr nr
10, m, 32, –24 h 195 97 nr 136 39 11 28 nr nr nr nr 8.8 15.4 nr nr nr

ut, 34
+10 min –3.6* 6.2 nr –7.4* 0 nr nr nr nr nr nr –12.5* –6.5 nr nr nr
+24 h 1.0 8.3 nr 2.2 7.7* nr nr nr nr nr nr 10.2* –3.3 nr nr nr
+48 h 5.1* 13.4 nr 2.9 7.7* –1.0 14.3* nr nr nr nr 1.1 –5.8 nr nr nr
+72 h 4.1* 7.2 nr 1.5 7.7* –1.0 14.3* nr nr nr nr 2.3 –5.2 nr nr nr
Sady, 198619 10, m, 35, –24 h 197 80 nr 118 63 26 38 156 32 nr nr 10.4 12.2 nr nr nr
t, nr
+18 h –4 –26* nr –8 10* 19* 4 0 –3 nr nr 46* –4 nr nr nr
Visich, 12, m, 26, –24 h 164 67 nr 113 40 16 23 nr nr nr nr 9.9 12.7 nr nr nr
199620 t, 56.4
IPE –3.8 –14.5* nr –3.1 –2.9 –7.1 0 nr nr nr nr nr nr nr nr nr
+1 h 1.0 –13.2* nr 1.4 1 0 3.3 nr nr nr nr nr nr nr nr nr
+6 h 3.3 nr nr nr 2.9 7.1 3.3 nr nr nr nr 2.0 –9.5 nr nr nr
+24 h 3.3 –13.2* nr 3.8 5.8* 0 11.7* nr nr nr nr 19.2* –14.2* nr nr nr
Yu, 199941 22-m, 6-f, –48 h 165 124 43.1 79 43 nr nr 148 34 73 3.4 nr nr nr nr nr
35, t
+15 min –7.3* –22.58* –51.74* –3.40 30.23* nr nr –3.38 3.53 –12.33* –2.94 nr nr nr nr nr
Subject information is: subject number, gender, male (m) or female (f); age in years; training status: mixed trained (mx), trained (t), untrained (ut); and VO2peak (ml/kg/min).
Baseline lipids and lipoprotein-lipids are given as mg/dl, then percent change from baseline. Baseline LPLa (mmol FFA/ml/h), HTGLa (mmol FFA/ml/h), LCATa (mmol
cholest/L/h), CETPa (%CE transferred/4 h), CETP mass (mg/ml).
In Crouse et al.,10 LDL-C was reported for 50% intensity group, and HDL2-C was reported for 80% intensity group.
* Significantly different from baseline, p < 0.05.
Acute Changes in Lipids and Lipoprotein-Lipids Induced by Exercise
289
published on women. Those that exist suggest some gender differences with
respect to acute TG responsiveness after exercise. In premenopausal women,
it has been reported that exercise of low and high intensity resulted in an
increase in TG concentration immediately following aerobic exercise.17,28 The
reason for this different response in women compared with men is not
known, but it may be related to gender differences in LPL activity (discussion
to follow).29 Others have shown that despite an immediate post-exercise rise
in TG concentrations in women, values may fall below pre-exercise values
by 1 h and up to 48 h after exercise.30 Thus, it can be concluded from the
results of existing studies that blood TG concentrations are more resistant
to change with exercise in women than in men.
As with HDL-C, there are published data to suggest that a threshold of
about 350 kcal of energy must be expended before a significant change in
circulating TG will occur; however, this finding is not universal. In this
regard, when higher thresholds have been reported the subjects were rela-
tively well-trained men.15 It has also been reported that those with the highest
pre-exercise TG values show the greatest post-exercise decrease.18,22,31,32
Post-Exercise Changes in Total Cholesterol and LDL

Cholesterol
The response pattern of blood total cholesterol (TC) concentration following
exercise reportedly varies from an increase to a decrease after a single session
of exercise. Increased blood TC concentrations were found in untrained men
with normal and high blood cholesterol for up to 48 h after a single session
of aerobic exercise.10,18,22 In contrast, post-exercise reductions in TC have been
reported in men and women who were trained and untrained.10,14,18,22,23,28
Reductions that have been reported generally do not persist for long (< 24 h),
and are modest at best, ranging from 3% to 5%. Some evidence suggests that
high-volume exercise (e.g., marathon) may produce TC reductions lasting
up to 120 h post-exercise, especially in trained women.16,17 In addition to
these studies in which blood TC was altered, there are several published
reports in which blood TC concentrations did not change after a single
session of exercise.15,19,20,30,33,34 Thus, the weight of the evidence supports the
conclusion that a single session of exercise has little or no effect on circulating
TC. When effects have been shown to occur at all, they were modest and
short-lived.
Similar to study results with respect to blood TC, the published response
to exercise of blood low-density lipoprotein cholesterol (LDL-C) concentra-
tion has been variable. In women LDL-C concentration was reportedly
reduced 24 h after a single session of high-intensity, high-volume exercise.17,28
Others report no change in this lipoprotein concentration in untrained
women after more moderate amounts of aerobic exercise.30,34 Furthermore,
LDL density, as assessed by ultracentrifugation, was not altered in either

normal- or hypercholesterolemic women by a single session of aerobic exer-
cise.27 In support of these findings, Lamon-Fava et al.35 reported that LDL
particle size did not change in women after completion of a marathon, but
increased in men. Reasons for this apparent gender effect are not known.
Following intense endurance events, LDL-C concentrations in men were
variously reduced from 0% to 38% for up to 72 h.14–16,19,20 In men with high
cholesterol, LDL-C concentration has been shown to increase and decrease
from 5% to 8% after exercise.22,23 Reasons for the conflicting findings are not
presently known, but may be related to study differences in the volume of
exercise performed, or to the training status of the subjects. While rare, some
studies exist which suggest exercise may acutely produce harmful effects on
lipoproteins. After long-duration, high-volume exercise (marathon), circu-
lating LDL has been shown to be more susceptible to oxidation making it
more atherogenic.36
Lipoprotein (a) after Exercise

Lipoprotein (a) [Lp(a)] is a modification of LDL in that the apolipoprotein
B-100 of LDL is linked to a glycoprotein, apoprotein (a). An elevated blood
concentration of Lp(a) is a strong risk factor for cardiovascular disease.37,38
Several studies have been conducted to explore the effect of exercise on this
lipid risk marker, since it is thought that lowering Lp(a) would reduce the
risk of cardiovascular disease. Overall, Lp(a) appears to be relatively resistant
to many interventions, including exercise. Lp(a) was not changed in men
and women immediately after and up to 7 days after a single session of cycle
ergometer or treadmill exercise.39,40 However, Lp(a) was reduced 18% when
measured 15 min after men and women completed a triathlon.41 Thus, the
weight of published evidence, although sparse, suggests that Lp(a) is unre-
sponsive to a single session of exercise under normal circumstances, but may
be reduced by exercise when the volume performed is extremely high.
Apolipoproteins and Exercise-Induced Changes

The majority of the circulating apolipoproteins A-1 and B are found in HDL
and LDL, respectively. Apo A-1 is inversely and apo B directly related to CVD
risk.42 Only a very few acute exercise studies have been published to date in
which apo A-1 and apo B were measured. In men with high blood cholesterol,
apo B concentrations rose an average of 4–9% after a single session of exercise
at a moderate intensity.10,22 In contrast, apo B concentrations were not altered

by strenuous exercise performed by well-trained men.16
Foger et al.16 reported that apo A-I and A-II concentrations were reduced
about 25% immediately after completion of a bicycle marathon. Subsequent
measurements made during 8 days of recovery revealed that blood concen-
trations of these apolipoproteins rose 10–20% above pre-exercise values. In
several other studies, increases in apo A-1 were minimal (e.g., 3% rise) or
nonexistent following more modest amounts of exercise.10,19,22,43 In one of the
few studies published to date in which apo E was measured, there was no
change in the blood concentration of this apolipoprotein after a triathlon.41
Whether or not a single session of exercise affects blood concentrations of
other circulating apolipoproteins is currently not known.
Mechanisms for Changes in Blood Lipids and Lipoproteins

Following Exercise
The intravascular biotransformation and metabolism of circulating lipopro-
teins is regulated by a number of proteins and enzymes. The enzymes and
proteins most frequently studied for their potential regulatory effects in
response to exercise include endothelial-bound lipoprotein lipase (LPL),
hepatic triglyceride lipase (HTGL), cholesteryl ester transfer protein (CETP),
and lecithin:cholesteryl acyltransferase (LCAT).
Intravascular remodeling of several lipoproteins, notably VLDL, LDL, and
HDL, is affected by CETP activity. This protein participates in the exchange
of cholesterol and triglyceride among HDL and triglyceride-rich lipopro-
teins. If affected by the exercise stimulus, it could contribute to the modifi-
cation of blood lipoprotein-lipid concentrations noted to occur after aerobic
exercise. Results of a longitudinal exercise training study suggest that CETP
genotype may contribute to the inter-individual differences in blood HDL
subfraction changes that occur with exercise training. However, few acute
exercise studies have been published in which this protein mass or activity
was measured. In those that have been published results were variable, but
it was generally reported that CETP activity was unchanged after a single
session of exercise.23,26 However, there is also evidence that CETP mass may
be reduced up to 2 days after exercise.16 A reduction in CETP mass could
increase the proportion of the total circulating cholesterol carried in HDL,
thus increasing HDL-C concentration, since a lower CETP mass would likely
result in less cholesterol transferred to other lipoproteins in exchange for TG.
With such a paucity of published research, it cannot be conclusively stated
that there is no acute effect of exercise on CETP, but the current evidence
suggests there is not.
Endothelial-bound LPL is generally accepted to be an important modula-
tor of circulating TG concentration and, through its action on TG, a factor
related to the change in HDL-C after exercise. LPL activity is acutely

increased after a single session of exercise, usually reaching maximal activity
by 24–48 h after exercise.15,19,20,23,44 The peak increase in LPL is related in time
to the maximal decrease in post-exercise TG concentration and the peak
increase in HDL-C. Gender differences in the acute response to exercise of
skeletal muscle and adipose tissue LPL activity have been reported. Three
to four hours after 90 min of aerobic cycling exercise, muscle and adipose
tissue LPL activity was higher in men, but unchanged from rest in women.29
This gender-specific response, if verified by additional research, may provide
a mechanistic explanation for the findings that blood TG concentration is
more resistant to change after exercise in women than in men.
Hepatic triglyceride lipase activity, thought to be another important mod-
ulator of circulating lipoprotein-lipids, is generally found to be reduced or
not changed for up to 72 h after exercise.15,18–20,23 Thompson et al.45 reported
that HDL protein circulatory survival time was increased in endurance
trained men compared with untrained men. A decrease in HTGL activity, if
it occurs, could be at least partly responsible for a prolonged survival time
for HDL in the circulation. Such an increased survival time would, in turn,
result in an increase in circulating HDL concentration.
Several lines of research show the importance of LCAT activity in the
reverse cholesterol transport process. LCAT activity is critical for the uptake
of cholesterol from peripheral tissues by nascent HDL and HDL apolipopro-
teins. Such important action makes LCAT a theoretical target through which
the exercise stimulus may mediate an increase in HDL-C and reverse cho-
lesterol transport. Research efforts to measure an increase in LCAT activity
after a single session of exercise have largely been unsuccessful. No change
in LCAT activity has been found after a single session of exercise performed
by recreational runners and untrained men. However, a large volume of
exercise performed by trained men has been shown to result in increased
LCAT activity.23,46–48 Thus, a conclusion to be drawn from the published
literature is that LCAT is relatively unresponsive to a single session of exer-
cise, unless the exercise is of relatively high volume performed by trained
individuals.
Other Exercise Considerations for Lipid Benefit

Aerobic exercise is generally prescribed along the dimensions of mode, fre-
quency, intensity, duration, and volume (caloric expenditure).49 The literature
supports the conclusion that regular exercise must be performed at a fre-
quency of every other day to maintain the beneficial acute effect. This con-
clusion is founded on the published literature which shows that, even after
exhaustive exercise performed by trained athletes, the exercise benefit lasts
only up to 3 days.16 Further evidence to support this conclusion comes from
studies in which exercise was withheld from endurance-trained athletes.

Hardman et al.50 reported that the ability to clear blood lipids after a fat meal
was reduced, and blood TG, very-low density lipoprotein-cholesterol
(VLDL-C), and the TC to HDL-C ratio were higher 60 h and 6.5 days after
well-trained endurance athletes stopped exercising.
The studies related to exercise intensity provide results that are less con-
clusive. Those that do exist often yield questionable results because experi-
menters did not control the volume of exercise among intensity groups.51–53
A review of these studies will show that often subjects exercised at relatively
higher intensities also had a higher caloric expenditure during exercise than
their lower-intensity counterparts. Since the caloric cost of exercise may affect
the acute lipid response, these studies are not helpful in determining the
independent effect of exercise intensity. With this in mind, there are several
controlled studies published in which it was shown that the intensity at
which exercise was performed did not affect the acute lipid response, as long
as the caloric expenditure was equivalent.22,33
Some evidence exists to show that the pattern and magnitude of acute
lipid changes after a single session of exercise may be altered by training.
Crouse et al.10 reported that an LDL-C increase following aerobic exercise in
men with high cholesterol was no longer evident after 6 months of aerobic
training. Also, training status may affect the lipid response pattern to low-
or high-intensity exercise in normocholesterolemic individuals. In studies in
which well-trained runners served as subjects, high intensity compared with
low-intensity exercise produced relatively greater acute increases in HDL-C
concentrations.51,54 Taken as a whole, the published literature generally sup-
ports the conclusion that any effect of exercise intensity cannot be separated
from the training status of those exercising, and the volume (caloric cost) of
exercise performed.
Summary
Research supports the conclusion that blood lipids, lipoprotein-lipids, and
lipid regulatory enzymes can be altered after a single session of aerobic
exercise. The post-exercise changes are consistent with a reduction in heart
and vascular disease risk. Since the benefit is lost after about 72 h, published
recommendations that exercise should be performed at a frequency of every
other day are justified. The intensity at which the exercise is performed is
less important than the volume. Evidence demonstrates that significant acute
effects occur at an exercise volume (dose) of exercise of at least 350 kcal per
session and up to 1000 kcal per session; lower volumes are sufficient for
moderately trained individuals, but higher volumes may be required for
highly trained athletes. The mechanism through which exercise exerts this
beneficial effect is not completely clear. Presently, the most likely mechanism
involves an increase in LPL activity, an increase that is evident for up to 48

h after a single session of exercise. More research is necessary to understand
the mechanisms responsible for the acute lipid response to exercise. Further-
more, additional studies are needed to define the optimal combination of
intensity, mode, and volume of exercise that will most likely cause a mea-
surable and risk-reducing change in blood lipid variables.
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48. Frey, I., Baumstark, M.W., Berg, A., Keul, J. Influence of acute maximal exercise
on lecithin:cholesterol acyltransferase activity in healthy adults of differing
aerobic performance. Eur. J. Appl. Physiol. 1991, 62, 31–35.
49. American College of Sports Medicine. Guidelines for Exercise Testing and Pre-
scription, 6th ed., Lea & Febiger, Philadelphia, 2000.
50. Hardman, A.E., Lawrence, J.E.M., Herd, S.L. Postprandial lipemia in endur-
ance-trained people during a short interruption to training. J. Appl. Physiol.
1998, 84 (6), 1895–1901.
51. Hicks, A.L., MacDougall, J.D., Muckle, T.J. Acute changes in high-density lipo-
protein cholesterol with exercise of different intensities. J. Appl. Physiol. 1987,
63 (5), 1956–1960.
52. Hughes, R.A., Thorland, W.G., Housh, T.J., Johnson, G.O. The effect of exercise
intensity on serum lipoprotein responses. J. Sports Med. Phys. Fitness 1990, 30
(3), 254–260.
53. Tsetsonis, N.V., Hardman, A.E. The influence of the intensity of treadmill walk-
ing upon changes in lipid and lipoprotein variables in healthy adults. Eur. J.
Physiol. 1995, 70, 329–336.
54. Gordon, P.M., Goss, F.L., Visich, P.S., Warty, V., Denys, B.J., Metz, K.F., Robert-
son, R.J. The acute effects of exercise intensity on HDL-C metabolism. Med. Sci.
Sports Exerc. 1994, 26 (6), 671–677.
14
Smoking, Heart Disease, and
Lipoprotein Metabolism
Robert J. Moffatt, Sarah Chelland, and Bryant A. Stamford
CONTENTS
Introduction .........................................................................................................299
CVD and Cigarette Smoking ............................................................................300
Impact of Smoking on Atherosclerosis ...........................................................302
Cigarette Smoking and Lipid Metabolism .....................................................302
Triglycerides and VLDL ...........................................................................302
LDL ..........................................................................................................304
HDL ..........................................................................................................304
Smoking Cessation .............................................................................................306
Metabolic Changes after Smoking Cessation.................................................307
Nicotine and Carbon Monoxide.......................................................................308
Environmental Tobacco Smoke (ETS) .............................................................308
Conclusions..........................................................................................................309
References ............................................................................................. 310
Introduction
Cardiovascular disease (CVD) is the leading cause of mortality and morbid-
ity in the United States, with 1.2 million new and recurrent cases of coronary
attack per year.1 CVD can be attributed to both modifiable and non-modifi-
able risk factors. The non-modifiable factors are those that cannot be changed
and include genetic predisposition to the disease, age, and gender. Modifi-
able risk factors are ones that can be altered by lifestyle intervention, and
include obesity, hypertension, hypercholesterolemia, diabetes, lack of phys-
ical activity, psychological stress, and cigarette smoking.
299
Since the inception of the National Heart Institute and the American Heart
Association in 1948 there have been several large cohort studies, which have
examined the effects of the modifiable risk factors on CVD. The Framingham,
Oslo, and Bogalusa Heart Studies as well as the Multiple Risk Factor Inter-
vention Trials (MRFIT) were all designed to deeply examine the etiology of
CVD and furthermore how to reduce its prevalence.2–5 The Framingham
Heart Study was among the first to reveal that the incidence of atheroscle-
rosis is proportional to higher plasma cholesterol levels, and in fact, was
among the strongest predictors of mortality.2
The MRFIT studies showed an independent contribution to increased mor-
tality for cholesterol and a substantial escalation in risk when elevated cho-
lesterol was combined with other risk factors.5 These results are common to
other epidemiological studies with diverse populations (Lipid Research Clin-
ics, Oslo and Bogalusa Heart Studies). Eventually, investigations broadened
to include examination of the various classes of lipoproteins and their con-
tribution to CVD.6 It was found that LDL had the strongest correlation to
CVD mortality. There was an association between very low-density lipopro-
tein (VLDL) and CVD mortality, but it was not as strong as the relationship
with low-density lipoprotein (LDL). Kostner et al.6 also reported that high-
density lipoprotein (HDL) demonstrated an inverse relationship with CVD.
This information provided additional evidence and helped to solidify the
connection between HDL and CVD that was originally advanced by the
Framingham, Tromso and Honolulu Heart Studies of the 1970s.7–9
Today it is taken for granted that development of CVD is accelerated by
an unfavorable plasma lipid/lipoprotein profile, and it is known that indi-
viduals with multiple lipoprotein abnormalities have a 10-year CVD risk of
greater than 20%. This means that from the time CVD is established, 20 out
of 100 individuals will experience their first coronary event or recurrent event
within 10 years.10 The current definition of an unfavorable lipid/lipoprotein
profile10 is LDL above 130 mg/dl, triglyceride (TG) above 150 mg/dl and
total cholesterol (TC) above 200 mg/dl. In addition, HDL levels below 40
mg/dl are considered to be detrimental.
CVD and Cigarette Smoking
Historically, the death rate associated with cigarette smoking has increased
steadily as smoking gradually was embraced by all segments of U.S. soci-
ety. Cigarette advertisements were seen as early as 1913 and specifically
targeted women, coaxing them to smoke rather than indulge in satisfying
their sweet tooths.11 From humble beginnings, cigarette advertising flour-
ished and smoking increased in popularity. Tobacco products became more
Smoking, Heart Disease, and Lipoprotein Metabolism 301
“palatable” as the former harsh tobaccos from Turkey were being replaced
with milder brands. World War I helped spike the popularity of cigarettes,
because cigarettes were more accessible and convenient for soldiers than
a pipe, and the government provided daily rations to the soldiers free of
charge.
As cigarette smoking took hold in American culture, there was no hint of
health consequences for decades. The first scientific paper supporting an
increased prevalence of CVD due to smoking was not until 1933 and it
concluded that heavy smoking contributed to the degeneration of the arter-
ies, including the coronary arteries.12 The next major indicator of an associ-
ation between CVD and smoking came from a Boston cardiologist named
Samuel Levine,13 who published a book providing evidence that supported
death from coronary artery disease occurring prematurely in smokers com-
pared to non-smokers. Levine indicated smoking as a major risk factor for
acute myocardial infarctions. More crucial evidence was published in 1958
when Hammond and Horn14 reported that men who smoked 20 cigarettes
per day had twice the death rate due to coronary events than non-smokers.
These authors later found that those men who stopped smoking decreased
the mortality rate during a 12-year follow-up.14
Building upon these earlier studies, subsequent research has focused on
two outcomes of smoking. One, an obvious starting point, is mortality rates.
Data suggest that cigarette smoking is directly responsible for as many as
465,000 deaths per year, with more than 40% attributable to CVD (201,000).11
The second is the negative physiological adaptations associated with smok-
ing, including alteration of the lipid/lipoprotein profile. This chapter pro-
vides a review of the recent literature on cigarette smoking with particular
emphasis on alterations in lipoprotein metabolism. The Lipids Research
Clinic and Framingham studies have shown that there is a dose–response
relationship specifically between the number of cigarettes smoked and the
decline in HDL concentration.15,16 A comprehensive meta-analysis by Craig
et al.17 examined published data from 1966 through 1987. They compared
non-smokers, with light (1–9 cigarettes per day), moderate (10–19 cigarettes
per day) and heavy (20 or more cigarettes per day) smokers. Their results
showed that TC (+ 1.8% L, + 4.3% M, and + 4.5% H), TG (+ 10.7% L, + 11.5%
M, and + 18.0% H), VLDL (+ 7.2% L, + 44.4% M, and 39.0% H), LDL (+ 1.1%
L, + 1.4% M, and 11.0% H) increased and HDL (–4.6% L, –6.3% M, –8.9% H)
decreased in a dose-dependent manner.
A smoking-induced decrease in HDL and/or increase in LDL could alter
the LDL/HDL ratio to favor a more atherogenic profile. Freeman et al.18
found that there was a significant difference in the LDL/HDL ratio between
smokers (2.89 ± 1.18) compared to non-smokers (2.38 ± 0.98). This change
in the LDL/HDL ratio among smokers has been subsequently shown in
several study populations.19–22
Impact of Smoking on Atherosclerosis

The impact of smoking on the risk of major coronary events is linked to the
function of the endothelial wall. The endothelial wall is made up of a single
layer, monolayer, of endothelial cells that regulate blood circulation and
metabolism of the vessel. Ross23,24 suggested that injury to these cells is a
critical step in initiating the atherosclerotic process. There are several factors
that can directly affect the stability of the endothelial cells of the coronary
vasculature.
A proposed mechanism for accelerated plaque and lesion formation due
to endothelial injury is free radicals created as a result of cigarette smoking,
in particular those related to increased lipid peroxidation.25 Lipid peroxida-
tion is responsible for the development of reactive species within the vascu-
lature. High levels of these oxygen species can cause a modification of the
LDL particle that will result in lesion formation. Oxidized LDL will cross
the protective barrier and lodge itself in the endothelial wall, which stimu-
lates a natural immune defense with macrophages, leukocytes and mono-
cytes. This immune defense will then release paracrine factors that attract
platelets to the damaged endothelial site. These platelets aggregate and
create foam cells.
Research has shown that a negative correlation also exists between smok-
ing and clotting time as a result of enhanced platelet aggregation.26,27 Fur-
thermore, a smoker’s ability to produce nitric oxide (NO), a compound
responsible for the smooth muscle dilation in the vasculature, is impaired.
The result is an inability to initiate vasodilation during times of hypoxia,
further complicating the smoker’s physiological adaptive responses.
Mustard and Packham28 indicated that carbon monoxide from cigarette
smoke is among those factors having the greatest impact on endothelial
function. Waters et al.29 concluded that smoking will accelerate coronary
progression and can produce new lesions in the arteries. Another indicator
of coronary health is the size of the intima wall relative to the thickness of
the cholesterol plaque. Studies have supported the finding that cigarette
smoking promotes stenosis and a reduced wall-to-plaque ratio.30–32 Further-
more, cigarette smoking promotes extensive production of fatty streaks.33
Cigarette Smoking and Lipid Metabolism

Triglycerides and VLDL
Chronic cigarette smokers have a perturbed TG metabolism especially at the
site of the adipose tissue. Chajek-Shaul et al.34 compared the lipid content
of the adipocytes in the gluteal region of smokers and non-smokers of the
same body mass index (BMI). Smokers had significantly less lipid per cell
than non-smokers (0.48 ± 0.07 vs. 0.64 ± 0.16 µl/cell). The authors suggested
a difference in TG metabolism between smokers and non-smokers.
A later study by Hellerstein et al.35 examined the relationship between the
thermogenic and atherogenic potential of cigarette smoke. They examined
heavy smokers (> 20 cigarettes/day) held on a strict diet for 2 weeks, one
week exposed to cigarette smoke and 1 week not exposed. Stable isotope
diffusions were used to measure free fatty acid (FFA) flux, glycerol flux and
serum FFA concentrations. As a result of smoking, plasma FFA concentration
was increased by 73% and FFA flux increased by 77%. A concurrent increase
in glycerol was noted and there was a threefold increase in hepatic esterifi-
cation of FFA. These results suggest that cigarette smoke can provide a
metabolic mechanism for atherogenesis, which can contribute to the promo-
tion of VLDL formation and release. As already noted, VLDL was found to
be positively correlated to increased risk of CVD.36
TG metabolism is regulated by the action of lipoprotein lipase (LPL), an
enzyme also affected by smoking. Specifically, this enzyme is responsible for
catalyzing TG hydrolysis and clearing TG from the blood. LPL is stimulated
by insulin at the adipose tissue but suppressed by it at the muscular level.
Research has been fairly consistent and suggests that LPL activity at the
skeletal muscle is reduced in smokers compared to non-smokers.34,37 It is
important to note that adipose LPL activity does not appear to differ between
smokers and non-smokers.38,39
LPL activity at the skeletal muscle provides a greater lipid clearance than
at the adipose tissue, therefore an altered activity at the musculature is of
great importance. A recent study by Freeman et al.37 demonstrated a trend
for lower HDL-C levels in smokers as well as higher TG, LDL, and TC levels.
Furthermore, in an attempt to examine the underlying mechanisms, results
of the lipid transport enzymes showed that there was a significant decrease
(33%) in skeletal muscle LPL activity in smokers (3.89 ± 1.58 µmol FFA/ml
per hour) compared with non-smokers (5.85 ± 2.30 µmol FFA/ml per hour).
The authors concluded that the reduced LPL activity may explain the
impaired TG clearance that is commonly seen in smokers due to a slower
metabolism of the TG-rich chylomicrons and VLDL. This perturbation may
in turn decrease the recognition of surface material by the HDL particle and
further delay cholesterol clearance.
LPL at the skeletal muscle also is greatly affected by insulin, and several
studies have confirmed insulin resistance among smokers.38,40,41 Results from
one study showed an inverse relationship between the amount of insulin
released and the amount of LPL activity, therefore those with the greatest
insulin release showed the greatest fall in LPL activity. Furthermore, Elliason
et al.38 assert that the amount of insulin released is positively correlated to
the amount of nicotine consumed per day.
This can present a complicated metabolic dilemma. Cigarette smoke can
create an environment that causes constant stimulation of the sympathetic
nervous system, release of catecholamines, and thus release of fatty acids.
Ultimately this physiological response should stimulate LPL. However, since

LPL is not stimulated, circulating TG concentration is increased. This per-
turbation can cause increased VLDL formation via both the decrease in LPL
and insulin resistance.
LDL
The formation of excess VLDL is of significant importance because it pro-
vides the precursor for LDL formation, a major risk factor for CVD. LDL is
the least affected lipoprotein by smokers.17 However the problem, not uncov-
ered until recently, is the impact of smoking on LDL size, altering it to become
a smaller denser particle that can easily cross the endothelial barrier, lodging
it into the arterial intima.25
Campos et al.42 found that plasma TG concentrations are related to LDL
particle size and HDL concentrations. Support for this claim comes from
Griffin et al.43 who showed a lower ratio of large-to-small LDL particles in
smokers (LDL-I/III = 0.77) compared with non-smokers (LDL-I/III = 1.89).
It is important to note that this disturbance was not found when corrected
for TG, further suggesting that the altered TG metabolism directly influences
the size and nature of the LDL particle.
A second concern regarding the impact of smoking on LDL is oxidation,
which will facilitate an immune response, after it has been lodged in the
arterial intima. This immune response will attract macrophages and initiate
foam cell formation, the beginnings of atherosclerotic plaque. Smoking pro-
motes this process by the abundant free radicals found in smoke. Most of
these are free radicals that cause lipid peroxidation, subjecting the smokers
to increased oxidative stress, as shown by elevated TBARS concentrations
in smokers compared with non-smokers.44
The prime regulator of LDL oxidizability is the vitamin E/protein ratio of
the particle.45 Studies have revealed a difference in oxidizability associated
with a low vitamin E/protein ratio.45,46 Thus it is important to analyze the
LDL particle contents to fully express the danger of oxidation.
HDL
Cigarette smoking imposes a negative impact on HDL, reducing the concen-
tration substantially.17 This is a highly negative consequence, because HDL
is the major lipoprotein responsible for reverse cholesterol transport, a pro-
cess that removes excess cholesterol from the blood and transports it to the
liver for catabolism.
HDL has two important subfractions. HDL2 and HDL3 have different and
distinct roles in HDL metabolism. HDL2 is the larger particle ranging from
8.8 to 13 nm whereas the HDL3 particles are smaller, ranging from 7.3 to
8.7 nm. Concentration of the HDL3 particle seems to be positively correlated
to CVD — the higher the concentration of HDL3, the greater the atherogenic
effect. The relationship between CVD and HDL2 is negative because HDL2
particles accumulate esterified cholesterol to transport to the liver for deg-
radation. Smoking reduces the HDL2 subfraction18,39,47 while exerting a lim-
ited effect on HDL3.48
HDL works in concert with three key enzymes that enable reverse choles-
terol transport to occur efficiently. Lecithin cholesterol acyl-transferase
(LCAT) esterifies free cholesterol in the presence of apolipoprotein (apo) A-I
and promotes the movement of this esterified cholesterol into the HDL core.
Cholesterol ester transfer protein (CETP) transfers esterified cholesterol from
HDL to lower density particles (chylomicrons, VLDL, intermediate-density
lipoproteins [IDL], LDL). Hepatic lipase (HL) is responsible for regulating
the degradation rate of HDL at the liver.
McCall et al.49 analyzed LCAT and HDL response to cigarette smoke expo-
sure. They studied the effect of cigarette smoke on fasted plasma samples
from nonsmoking volunteers. Within 1 h the smoke-exposed plasma had a
44% reduction in LCAT activity; the longer the plasma was exposed to
cigarette smoke the greater was the decrease in activity, and after 6 h LCAT
activity was only at 22% of the normal control. In addition, HDL apolipo-
proteins were cross-linking rapidly (within 1 h) after exposure to the smoke.
In other words, apo A-I, which is responsible for activating LCAT, was
switching with apolipoprotein A-II (apo A-II), known to deactivate LCAT.
This is problematic, because the activity of LCAT is dependent upon the
activation by apo A-I, which in turn governs cholesterol ester flow into the
HDL core. This process “fattens” the HDL making it the larger HDL2 particle,
which can carry the cholesterol to the liver for excretion and/or catabolism.
The researchers suggested that LCAT is sensitive to cigarette smoke and that
this could be responsible for the changes seen in HDL-C in smokers. Other
studies have shown that apo A-I concentration is 4.2% lower in smokers than
non-smokers.48,50
Studies have shown that smoking can activate HL38,39,51 and, as indicated
above, inhibit the activity of LCAT.37,39,48,49 The evidence on CETP is equiv-
ocal; some studies have found that CETP activity is elevated in smokers,52
while others report decreased18 or unchanged activity.37 These results are
also hard to interpret as the full mechanisms behind CETP activity are not
yet elucidated.
Despite conflicting results, correlation analysis supports a strong negative
correlation between total HDL, and HDL2 and HL, while HDL3 was nega-
tively correlated with LCAT.37 These data suggest that these enzymes are
directly responsible for maintaining the balance of HDL in metabolism and
that minor disruptions in the activity of these enzymes will have larger
consequences.
The challenge at hand, then, is to ascertain what all of these interactions
have in common and how smoking promotes the risk for CVD. What is
known is that the type of HDL particle in circulation will directly influence
CVD risk and that LCAT acts to facilitate the movement of cholesterol esters
into the HDL core. What is more, the reverse process of taking cholesterol
ester away from the HDL in exchange for TG is mediated by CETP. In

addition, HL will act on the HDL2 particles and return HDL3 to circulation.
Smoking reduces HDL2 and LCAT in addition to increasing or not chang-
ing the activity of CETP and HL. This suggests that even with normal CETP
and HL activity, an individual will be at greater risk of CVD due to the
decreased clearance of cholesterol via the HDL2 subfraction. In addition, the
movement of cholesterol esters would also be inhibited because the activity
of LCAT is diminished. This combination of events leads to increased cho-
lesterol in circulation, which can be available as substrate for other less dense
particles (VLDL, LDL).
If CETP and HL were to increase along with a reduction in HDL and LCAT,
metabolic problems would escalate. With more cholesterol esters in circula-
tion and not enough LCAT activity to transport them into the HDL core,
there still would be enough CETP to continuously move the esters into VLDL
and LDL. In addition, with increased HL activity the cardioprotective HDL2
particle is being broken down faster than it can be replaced, leaving an excess
of HDL3-C in circulation. Ultimately, this would result in too little HDL-C
to help protect against atherosclerosis.
Smoking Cessation
The World Health Organization reaffirmed in 2000 that cigarette smoking
was one of the most powerful factors contributing to CVD,53 and thus smok-
ing cessation is strongly recommended. Scientific evidence offers strong
support for smoking cessation. Early studies14,54 reported that those individ-
uals who quit smoking had a substantial decrease in risk for acute myocar-
dial infarction. Since then, many studies have shown that among all
preventative interventions, the decrease in CVD and acute myocardial infarc-
tion incidence is greatest among those who stop smoking.55 It has been
observed that smoking cessation can delay the onset of atherosclerosis by
10 years when compared with individuals who continue to smoke.56
Research has shown is a definite adaptation period after smoking cessation
before risk of CVD is diminished, and this can take from as little as 2 years
to as many as 20 years.57 Regardless of the length of the adaptation period,
the risk for CVD will return to the level of those who have never smoked.58–61
Smoking cessation contributes to several physiologic changes that decrease
the risk for CVD, including normalization of the lipid and lipoprotein profile.
A recent meta-analysis62 suggests that with smoking cessation an individual
can experience an increase in HDC, but other lipids and lipoproteins (TC,
LDL, TG) remain unchanged. Increases in HDL can be seen in as little as 17
days.63 A progressive increase in HDL has been observed with sustained
cessation.64–68 These results have important implications, because an increase
in HDL will alter the HDL/LDL ratio — a powerful predictor of CVD —

even though the TC, LDL, and TG may not change.
Strategies that assist in efforts toward smoking cessation have received
considerable attention. Studies show that administration of a nicotine patch
does not alter lipid or lipoproteins.69–71 One study, however, suggests that
HDL can return to baseline on the patch.69
Moffatt et al. 72 examined subjects who were asked to wear a nicotine patch
for the first 35 days of their cessation program, followed by 42 days without
the patch. As expected HDL, HDL2 and HDL3 were all reduced in smokers
compared with non-smokers prior to the start of the program. These differ-
ences were still present after 35 days of cessation while on the nicotine patch.
Normalization of blood values were observed over the succeeding 42 days
when the patch was removed, suggesting that nicotine prevents normaliza-
tion of HDL and its subfractions. This effect is acute and will only persist as
long as the patch is in place. Notwithstanding the negative impact of nicotine
therapy during cessation, it is clearly a more desirable alternative to smoking,
assuming the therapy is short-lived and that cessation will be permanent.
Metabolic Changes after Smoking Cessation

Cessation of smoking will initially result in a metabolic withdrawal syn-
drome that affects 80% of all smokers.73,74 These symptoms include restless-
ness, irritability, anxiety, and confusion. Furthermore, weight gain, an
increase in waist to hip ratio, and an increase in percent body fat due to
increased caloric intake or decreased resting metabolic rate is seen after
smoking cessation.19,66,67,72,75,76
Weight gain typically exerts a negative impact on lipids and lipoproteins.
However, despite the weight gain associated with smoking cessation, HDL
levels improve, progressing gradually to normal values observed in non-
smokers. This suggests that smoking is a more potent mediator of the lipid
and lipoprotein profile than weight gain.
Stamford et al.67 found that following cessation from smoking, subjects
increased their caloric intake by 227 kilocalories (kcal) per day, which
accounted for 69% of the weight gain following cessation. This finding sug-
gests that a substantial percentage (31%) of the weight gain could not be
explained by increased kcal consumption. Stamford et al.67 observed no
chronic change in resting metabolic rate (RMR). However, numerous studies
have reported acute increases in metabolism caused by smoking alone and
in combination with caffeine, food intake, light physical activity, etc. that
when removed would impact caloric balance.66,77–81
Other factors may contribute to weight gain following cessation. Oeser et
al.76 examined circulating leptin levels and the lipid profile. Leptin is known
to be a powerful regulator of appetite and is thought to be altered with
smoking and/or cessation. Results of their study showed, however, that 7

days of nicotine abstinence produced no differences in fasting leptin levels
or plasma concentrations of glucose, insulin or free fatty acids. A limitation
of this study, however, was that it only observed nicotine abstinence for 7
days.
Exercise following cessation is strongly encouraged. Niaura et al.82 found,
similar to the above studies, that smoking cessation caused an increased total
caloric intake in the non-exercise group. Caloric intake did not increase in
the exercise group. The exercise group also demonstrated more favorable
changes in HDL and the HDL2 subfraction.
Nicotine and Carbon Monoxide

Smoking not only exacerbates the lipid profile, it exerts its effects in several
other areas of the body. Cigarettes accomplish this mainly due to their
nicotine and carbon monoxide components. Nicotine is readily able to cross
the blood–brain barrier and can further bind to various receptors. This bind-
ing will cause the release of acetlycholine, norepinephrine, dopamine, sero-
tonin, and vasopressin, which promote sympathetic stimulation, feelings of
fulfillment, and vasoconstriction of the arteries; in turn this can elevate heart
rate and blood pressure. In addition, nicotine has a half-life of approximately
2 h, suggesting that a chronic heavy smoker (> 19 cigarettes/day) may
experience these elevations during his or her sleep cycle.83
In addition to the nicotine delivered in cigarettes, a smoker also takes in
carbon monoxide. This carbon monoxide directly contributes to coronary
hypoxia. This hypoxia will manifest itself in changes that occur to the
concentration of 2,3-diphosphoglycerate (2,3 DPG), an important modulator
of hemoglobin’s affinity for oxygen. The concentration of 2,3 DPG is found
to be elevated in the blood of smokers, signifying the body’s ability to
attempt to compensate for the hypoxic environment created by the carbon
monoxide.84
Environmental Tobacco Smoke (ETS)

Smoking is not only hazardous to the smoker but also to nonsmokers via
environmental tobacco smoke (ETS). Research has established a link between
ETS exposure and CVD,85–90 citing that those who are chronically exposed
are 1.3 times as likely to develop CVD as compared to those who have never
been exposed.86,91 Studies have also provided evidence for ETS exposure and
alterations in the lipid and lipoprotein profile.92,93 Previous research has
shown that chronic ETS exposure elicits an unfavorable lipid/lipoprotein

profile among various populations including hyperlipidemic children,94 ado-
lescents,95 spouses,87 and coworkers.96
More recent data suggest an acute effect of ETS on lipids and lipoproteins
as well. An early study by Moffatt et al.92 examined three groups of women
who were either non-smokers, smokers (≥ 20 cigarettes/day for the last
5 years) or non-smoking women exposed to ETS for 6 h per day, 4 days per
week for at least 6 months. Compared with nonsmokers, both smokers and
ETS-exposed women showed a significant reduction in HDL, HDL2 and apo-
AI. The levels were not different between smokers and ETS-exposed women.
The authors concluded that individuals who do not smoke, but who are
exposed to ETS, will show similar abnormalities in the lipid/lipoprotein
profile and that this can increase CVD risk.
A recent study by Moffatt et al.93 revealed that only 6 h of ETS exposure
is sufficient to reduce HDL and HDL2 by 18% and 37%, respectively. Fur-
thermore, HDL and HDL2 taken 24 h post-exposure were still significantly
lower, 13% and 28% respectively, compared with baseline. Exposure to cigar
smoke has been shown to exert effects similar to those of cigarettes.97 Spe-
cifically, those individuals exposed to cigar smoke, including the cigar
smoker who often reports not to inhaling, for 90 min showed significant
decreases in HDL and HDL2. In addition, the TC/HDL ratio was elevated
at 12 h post-cigar ETS exposure in non-smokers. Together these data suggest
that all types of ETS exposure will have negative impacts on the lipid and
lipoprotein profile.
Conclusions
Smoking is a major risk factor for CVD, due in part to an adverse effect on
the lipid and lipoprotein profile. Similarly, ETS also exerts many of the
negative influences of smoking on innocent bystanders. Smoking decreases
HDL and HDL2, while increasing levels of TC, TG and LDL. Smoking may
negatively alter LDL particle size and negatively impact critical enzymes,
including LCAT, CETP, and HL. Thus, reducing the number of cigarettes
consumed per day is a positive step when complete cessation from smoking,
clearly the preferred outcome, is not attained. With cessation, HDL begins
to normalize quickly, with significant results in as little as 17 days. As ces-
sation continues, HDL increases further.
A negative outcome is associated with smoking cessation. Although
weight gain typically exerts a negative impact on lipids and lipoproteins, an
increase in HDL is observed with smoking cessation in the face of weight
gain, thus offering further support for cessation. Nicotine replacement ther-
apy is helpful to patients attempting to break the addictive stranglehold of
smoking. However, use of the nicotine patch or gum may interfere with the
normalization of HDL. Even so, the ultimate benefits of smoking cessation

on health and reduced risk of CVD would outweigh this potential outcome.
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15
Lipid and Lipoprotein Concentrations in
Americans: Ethnicity and Age
Michael R. Kushnick and Lynn B. Panton
CONTENTS
Introduction .........................................................................................................316
An Overview of Ethnicity .................................................................................316
Asian Americans........................................................................................317
American Indians ......................................................................................318
Hispanics.....................................................................................................318
Black Americans.........................................................................................319
Summary of Ethnicity and Lipid and Lipoproteins in
Adult Americans ...........................................................................320
Lipid and Lipoproteins in Americans through the Life Span ....................321
Developmental Stages...............................................................................322
Early Childhood.........................................................................................322
Total Cholesterol and Low-Density Lipoprotein
Cholesterol .......................................................................322
Triglycerides and High-Density Lipoprotein Cholesterol ......323
Gender and Ethnic Differences ...................................................323
Mid- to Late Adolescence.........................................................................324
Cholesterol .......................................................................324
Gender and Ethnic Differences ...................................................326
Middle Age .................................................................................................326
Cholesterol .......................................................................327
Gender Differences........................................................................ 329
Advanced Age............................................................................................331
Cholesterol .......................................................................331
315

Gender Differences........................................................................ 332
Summary of Lipid and Lipoproteins through the Life Span in
Adult Americans ...........................................................................332
Conclusions..........................................................................................................332
References ............................................................................................. 334
Introduction
The relationship between plasma lipoproteins, their constituents, and the
risk of coronary heart disease (CHD) in adults is well established.1 Choles-
terol plays a central role in the atherosclerotic process and its association
with CHD is reported to be continuous and graded.2 Across ethnicity, increas-
ing cholesterol levels also are linearly related to CHD.3 Furthermore, there
is little dispute that lipid and lipoprotein profiles change over the lifespan,
and there is a clear and positive relationship between blood lipid and lipo-
protein concentrations during childhood and later in life.4
The purpose of this review is to provide an update on the variations in
lipid and lipoprotein concentrations and constituents in human plasma in
major American ethnic groups of the United States (U.S.) during adulthood.
In addition, this review will examine what is currently known about the
changes in lipids and lipoproteins through the lifespan. Those requiring a
comprehensive review of lipid and lipoprotein metabolism can be found in
chapter 4 and elsewhere (5–18).
An Overview of Ethnicity
There is remarkable variation in inter- and intra-individual lipid and lipo-
protein concentrations that no doubt is influenced by a combination of
genetic and environmental factors.19,20 This section will summarize the avail-
able information on differences in lipid and lipoprotein concentrations of
adults of major ethnic American subpopulations and, where evidenced in
the literature, discuss potential reasons for these differences. For the purpose
of this investigation the following ethnic divisions will be made: Asians
(Chinese Americans, Filipino Americans, Japanese Americans, Asian Indian
Americans), American Indians, Hispanic (Mexican Americans), non-His-
panic, African Americans (blacks/Black Americans), and non-Hispanic Cau-
casians (whites/White Americans). These divisions are made with the intent
of marking nationality and regional affiliation.
Lipid and Lipoprotein Concentrations in Americans: Ethnicity and Age 317
Asian Americans
Asians are one of the fastest-growing groups of immigrants in the U.S.
Approximately 11.9 million or 4.2% of the U.S. population indicate “Asian”
origin; defined to include any persons having origins in the Far East, South-
east Asia, or Indian subcontinent. The largest of these groups in the U.S. are
Chinese Americans (2.7 million) followed closely by Filipino Americans
(2.4 million) and then Asian Indian Americans (1.9 million). These combined
groups account for nearly 60% of the total U.S. population reporting Asian
ethnicity.21 However, while there is a great deal of research on lipid and
lipoprotein concentrations in Asian Indian Americans and Japanese Ameri-
cans (1.1 million; ranked 6th in population of Asian Americans), far less
information on other Asian American subgroups is available in the current
literature.21
Chinese and Filipino Americans have been shown to have similar levels
of total cholesterol (TC).22 Choi et al. compared samples of a large group of
older Chinese Americans against an all-white sample from the Framingham
Cohort Study and found that the mean values of TC, low-density lipoprotein
(LDL), LDL cholesterol (LDL-C) and apolipoprotein (apo) B were lower than
whites; however, they further indicated that high-density lipoprotein (HDL),
HDL cholesterol (HDL-C) and apo A-I were also lower than those of their
white counterparts.19,23 Others have suggested that lipoprotein (a) [Lp(a)]
levels of Chinese American women are similar to those of white women (17.5
± 20 and 23.7 ± 29 mg/dl, respectively).24
Japanese Americans have lipoprotein profiles that are characterized by
greater HDL-C than whites.25 Interestingly, Japanese American men appear
to exhibit a greater frequency of the LIPC promoter T allele polymorphism
than other U.S. ethnic groups, which is associated with lower hepatic lipase
(HL) activity.25,26 This polymorphism may help to explain the greater levels
of HDL-C exhibited by this group, since HL activity has been demonstrated
to be inversely related to HDL-C.27 An older report on Japanese American
men’s blood lipid and lipoprotein profiles comes from the Honolulu Heart
Program (HHP) which began in 1965 and reported on CHD risk and risk
factors of 8006 Japanese American men residing in Hawaii over more than
30 years. The report from the HHP found concentrations of TC and LDL-C
were similar to a cohort of age-matched white men from the Framingham
Study.28 However, in contrast to more recent reports, HDL-C concentration
was also similar between the Japanese American men and the white men.28
Asian Indian American men have higher TC and LDL-C, as well as lower
HDL-C than white men.29,30 However, these differences were not reported in
a group of well-educated male physicians of Asian-Indian descent.31 Asian
Indian American women may have greater HDL-C than white women,29,32
but this too is not always reported.30
In addition, Asian Indian American women may have greater Lp(a) than
white women who are not using oral contraceptives,32,33 and both Asian
Indian American men and women appear to have a greater relative
distribution of small, dense LDL particles as compared to white men and

women.31,34,35 However, it has been reported that the abundance of small,
dense LDL may be explained by a greater concentration of triglycerides
(TG)29,30,33,35,36 and a greater visceral accumulation of adipose at similar body
mass indices (BMI) in the Asian Indian Americans compared to whites.36
American Indians
American Indians and Alaskan Natives were reported under one heading
in the 2000 census. Approximately 4.1 million (1.5%) of adults in the U.S.
population reported American Indian or Alaskan Native as their ethnicity.37
Early reports of American Indians suggested that this group had lower CHD
mortality rates than the general population.38 However, these rates appear
to be increasing to levels greater than the average of the general U.S. popu-
lation.39 These results are likely related to findings of the Strong Heart Study
(SHS), an investigation of health in 13 American Indian communities of three
geographically diverse regions of the U.S. A major finding of the SHS was
that the prevalence of diabetes in adult American Indians was > 70% in
Arizona, and > 40% in Oklahoma and North and South Dakota.39,40
American Indians have lower TC and LDL-C than the white adult U.S.
average.39,41–46 An analysis by Robbins et al.46 suggested that the mean LDL-C
concentration of American Indians with diabetes was similar to that of non-
diabetics of this population. Comparisons to the NHANES III data44,45 and
other data47,48 suggest that HDL-C may be lower in American Indians with
or without diabetes as compared to whites. Furthermore, American Indians
have 3–1.5 times lower concentrations of Lp(a) than those of whites (6.1–6.4
mg/dl),49 and 10–5 times lower concentrations than those of blacks (21.5–24
mg/dl).50 In fact, only a small percentage of American Indians from the SHS
(1.73%, 7.37% and 4.34% in Arizona, Oklahoma, and the Dakotas) had Lp(a)
concentrations > 30 mg/dl (the clinical reference considered to be elevated
and at risk for greater CHD events in the current literature).50 Unlike white
and black ethnic groups, however, Lp(a) may be lower in American Indians
with diabetes than those without diabetes.49,51
Another feature of American Indians’ lipoprotein profile is a preponder-
ance of small, dense LDL particles as compared to whites.52 Interestingly,
greater fasting insulin levels, obesity and elevated TG concentrations have
also been reported in American Indians.39,46,52,53 These factors are suggestive
of the increase in insulin resistance and its association to chronic changes in
LDL distribution in this group of Americans.52,54
Hispanics
The 2002 Current Population Survey (CPS) suggests that 37.4 million His-
panic individuals reside in the U.S., representing greater than 13% of the
population.55 Approximately 67% of these Hispanics (or Latinos) are of
Mexican origin (Mexican American). Hispanics in the U.S. have been

reported to have similar or lower rates of CHD56 as compared to whites.
However, more recent reports from the San Antonio Heart Study have not
supported these figures.57,58 The primary difference has been termed the
“healthy migrant effect” by Wei et al.58 to suggest that U.S.-born Mexican
Americans have a greater BMI, a greater prevalence of diabetes and a higher
overall mortality rate than whites or Mexican-born individuals.59,60
Mexican Americans tend to exhibit greater obesity and concomitant ele-
vations in TG and reduced HDL-C,61–67 and these findings are reinforced by
similarly lower levels of apo A-I.68–70 Results from NHANES III suggest that
Mexican American men have greater age-adjusted TC concentrations than
white men.71 Among women, however, the Mexican Americans have lower
TC than white women.71 Others have reported similar levels of TC and
LDL-C,64,65,70,72,73 as well as Lp(a) when compared with white men and
women.70,72,74
Mexican Americans, without diabetes, also have been reported to have
lower levels of LDL-C than whites.56 However, Mexican Americans have a
greater prevalence of small, dense LDL particles.65,75,76 While the small LDL
size in Mexican Americans is significantly related to greater adiposity,67,76 it
has also been demonstrated that Mexican Americans may have greater activ-
ity of cholesteryl ester transfer protein (CETP), potentially helping to pro-
mote these greater levels of small, dense LDL particles.67
Black Americans
Black Americans represent approximately 36.4 million or 13% of the U.S.
population.37 A good deal of literature has been compiled, and our knowl-
edge of the lipid and lipoprotein profile of blacks is well documented. These
profiles would suggest lower CHD risk than whites. However, this is not
the case, as blacks are reported to have the highest overall CHD mortality
rate of any group in the U.S. indicative of other factors besides lipids and
lipoproteins increasing their relative risk.77,78
Black Americans have lower levels of TC and TG and higher levels of
HDL-C than whites as well as a lower prevalence of hypercholester-
olemia.44,79–83 The concentrations of apo B and apo A-I are also consistent
with these findings (lower in the former; greater in the latter).68,81
Another consistent finding in blacks is greater concentrations of Lp(a) than
whites.50,70,74,84 Howard et al. reported on 4125 participants of the Coronary
Artery Risk Development in Young Adults (CARDIA) study (> 1/2 black
men and women).50 This investigation suggested that approximately 80% of
the blacks had levels of Lp(a) that were higher than the median value for
the whites. The median values of Lp(a) in black men and women were 21.5
and 23.9 mg/dl, respectively, compared to 6.1 and 6.4 mg/dl in the white
men and women, respectively.50
Blacks are also reported to have greater overall adiposity,85–87 but lower
accumulation of visceral adiposity than whites.81,88 However, lipid and lipo-
protein differences are still reported to exist between blacks and whites after
adjusting for adiposity indicating a true biologic difference.89 Interestingly,
excess visceral adiposity is often related to reductions in HDL-C through
alterations in HL and lipoprotein lipase (LPL) activity.85 In fact, this may
explain why blacks are often observed to have greater LPL activity, lower
HL activity,81,90,91 and greater HDL2-C concentrations than whites.80,81,91,92
More recently, investigations have determined that blacks may have a
greater mean LDL particle size,65,83 and a smaller portion of small, dense LDL
particles than whites.83 However, we were recently unable to confirm this in
a group of sedentary black and white men. This result may be due to a smaller
number and younger group of subjects, the lack of a statistical difference in
TG concentrations between our white and black groups, and/or greater CETP
activity in the black men, despite greater HDL-C concentrations.93
Summary of Ethnicity and Lipid and Lipoproteins in Adult Americans

Table 15.1 provides an overview of the available data on differences in
plasma lipid and lipoprotein concentrations of the major American ethnic
groups as compared to White Americans.94 Coronary heart disease is the
TABLE 15.1
Overview of the Differences in Lipid and Lipoprotein Concentrations of American
Ethnic Groups Compared to White Americans
Lp LDL
Ethnic Group TC LDL-C TG HDL-C (a) size Other
Asian Americans
Chinese Americans ↓ ↓ – ↓ ↔ – ↓apoB
Filipino Americans ↓ – – – – – –
Japanese Americans ↔ ↔ – ↔↑ – – ↓HLa
Asian Indian Americans ↑↔A ↑↔A ↑ ↓↔A /↑↔B ↑ ↓ –
American Indians ↓ ↓ ↓ ↓ ↓ –
Mexican Americans ↑↔A/↓↔B ↓ ↑ ↓ ↔ ↓ ↓apoA-I;
↑CETPa
Black Americans ↓ ↓ ↓ ↑ ↑ ↑↔ ↑LPLa;
↑HDL2-C;
↓Hla
↑, higher than White Americans; ↓, lower than White Americans; ↔, equal to White Amer-
icans; A in men; B in women; LPLa, lipoprotein lipase activity; HLa, hepatic lipase activity;
CETPa, cholesteryl ester transfer protein activity; TC, total cholesterol; LDL-C, low-density
lipoprotein cholesterol; TG, triglycerides; HDL-C, high-density lipoprotein cholesterol; Lp(a),
lipoprotein (a); LDL size, low-density lipoprotein particle size; apo, apolipoprotein.
Source: Adapted from Watson, K.E., Curr. Cardiol. Rep., 5, 483, 2003. With permission.
leading cause of death among all ethnic groups in the U.S.95 The relationship
between plasma lipoproteins, their constituents, and the risk of CHD in
adults has been established.1 In fact, across ethnicity, increasing concentra-
tions of TC are linearly related to the risk of CHD.3 The current literature
provides only a brief synopsis of lipid and lipoprotein concentrations in
ethnic groups of the U.S. and the current review provides an overview and
reference when considering ethnicity. However, the current literature is lim-
ited in that there are only a few studies that include ethnic populations of
the U.S., and in most cases, they fail to take into consideration important
factors such as social and environmental issues, and age and gender differ-
ences that may further impact lipid and lipoprotein levels. Additionally, the
mechanisms that drive biological differences among ethnic groups are
largely unknown.
Lipid and Lipoproteins in Americans through the Life Span

Plasma lipid and lipoprotein profiles change over the life span, and these
changes begin early in development of the growing fetus and continue into
older age. In fact, it has been demonstrated that lipid and lipoprotein mea-
surements obtained in childhood and adolescence (ages 8–18 years) are
highly predictive of adult levels of TC and LDL-C.4 Lauer et al. reported that
of the children whose TC ranked in the 90th percentile on at least one
measurement between ages 8–18 years, 43% were found to have greater than
the 90th percentile of TC at ages 20–30 years.96 This predictive nature is
important, in the fact that the atherosclerotic process has been demonstrated
to begin early in life97,98 and understandably, it has also been suggested that
lipid and lipoprotein levels at early ages are indicators of long-term risk of
CHD and mortality of CHD.97,99
The prevalence of CHD is positively associated with the level of plasma
cholesterol in both sexes over a wide range of ages.1,4,97,99 While lipid and
lipoprotein concentrations are dynamic throughout the life span, it is typical
to observe the greatest concentrations of LDL-C and TC later in adulthood.
A number of potential explanations have been proposed for the age-associ-
ated changes in lipoprotein concentrations, including dietary patterns,100–104
total and abdominal obesity,103,105–109 aerobic fitness,103,109,110 number and func-
tion of the hepatic LDL receptors,111,112 and altered insulin sensitivity.109 How-
ever, none of these suggested mechanisms that are driving the age-related
changes in lipoprotein levels are well studied or understood.
The next section of the chapter will focus on reviewing the changes in lipid
and lipoprotein concentrations associated with each unique stage of life.
Differences in prospective versus cross-sectional results, the influence of
gender and, where available, the affiliations with various ethnic groups
(prior to adulthood) will be indicated.
Developmental Stages
During gestation, intrauterine changes in TC, LDL-C and HDL-C have been
noted and concentrations are typically greater than those seen at term.113,114
In fact, changes in lipoprotein constituents are often linked to the develop-
ment and growth of specific organs. For example, TC typically declines
during gestational weeks 12 through 20 at which time the adrenal glands
increase in size nearly 10-fold.113 This period is followed by a rise in TC
through 32 weeks and is characterized by a dramatic developmental increase
in the fetal liver. From week 32, a second decline in TC appears as the fetus
approaches gestation.113 Triglyceride levels continue to increase as the fetus
develops and approaches full term.114
Some maternal factors have been shown to influence lipid and lipoprotein
concentrations in the developing fetus and neonate. Maternal hypertension
may result in fetal hypercholesterolemia as a consequence of reduced adrenal
utilization of LDL-C during the late stages of development.115 Additionally,
illness, such as respiratory distress syndrome in neonates or prematurity has
been associated with higher TC, TG, and apo A-I.116,117 Other factors such as
maternal stress at delivery, socioeconomic level, low birth weight, or seasonal
differences have been investigated and do not appear to alter lipid or lipo-
protein levels.118 In fact, at birth, healthy infants have TC in the range of
50–100 mg/dl and cholesterol is typically more evenly distributed between
HDL-C and LDL-C particles than is found at any other time of life.100,119
Early Childhood
Total cholesterol increases somewhat prior to the newborn’s first oral meal.120
Additionally, within hours of the first oral feeding, as well as over the first
few days postpartum, LDL-C and HDL-C increase regardless of diet.120,121
When infants are less than 1 year old, TC and LDL-C are typically found to
be higher in breast-fed as compared to bottle-fed (formula) infants.104,120
Other dietary influences may have a profound effect on the infant’s lipid
and lipoprotein profile. For example, lowering the intake of saturated fat
may reduce TC and LDL-C concentrations in infants and children below 5
years of age; however, this does not appear to impact HDL-C.101
Total Cholesterol and Low-Density Lipoprotein Cholesterol

With longitudinal designs, some investigators have reported TC to increase
through mid-adolescence, with the greatest rise demonstrated within the
first two years of life to levels similar to that seen during young adulthood
(~ 170–205 mg/dl).105,122–126 In a study by Rask-Nissilä et al., TC increased
progressively from ages 7 to 48 months (140.8 ± 21.8, 152.4 ± 25.4, 155.6 ±
26.9, 162.6 ± 24.3 mg/dl at 7, 13, 24, 48 months, respectively).125 Other
researchers, however, have noted decrements in TC from ages 1 to 2 years102
and through 8 years of age.127 Cross-sectional studies of young children are
more variable with respect to TC and LDL-C. Total cholesterol and LDL-C
have been shown to rise through age 8 years, with the largest difference of
increase typically seen between years 1 and 2 (~ 7 mg/dl).106,124,127 Total
cholesterol and LDL-C have also been shown to decrease103,128 or not change
when reported in cross-sectional research.129
To our knowledge no investigations have examined the influence of devel-
opment (age) during early childhood on LDL particle size. However, Stein-
beck et al. compared a group of children (~ 8 ± 1.5 years of age) with their
parents and were unable to determine differences in LDL particle size (26.7
± 0.9 versus 26.6 ± 0.8 nm, in children versus parents, respectively).130 In
addition, few investigations have examined Lp(a) in childhood, and it is
likely that Lp(a) concentrations are not related to age, at least prior to adult-
hood.131,132
Triglycerides and High-Density Lipoprotein Cholesterol

Triglycerides are less often reported in younger children and this is primarily
because of the 9–12 h fasting requirement. However, when reported, TG may
be reduced in children after age 2 years and through 8 years of age.102,127,133
Cross-sectional studies have not reported differences in TG during these
younger ages.123
HDL-C may increase with early advancing age when reported cross-sec-
tionally,103,134 but this has not been a consistent finding.103,106,127–129 Far fewer
longitudinal investigations report on HDL-C in children prior to age 8 years,
while most have not seen changes,102,127 others have found HDL-C to
increase.125,133 For example, Kaitosaari et al. reported that HDL-C increased
from 7 months through 7 years of age by greater than 40%133 and this may
be explained by a concomitant increase in lecithin cholesterol acyltransferase
(LCAT) activity observed by others in children during this period of life.135
Gender and Ethnic Differences

In contrast to older ages, TC and apo B are higher in girls than boys (begin-
ning at 7 months of age), while boys have somewhat higher HDL-C.129,136,137
Triglycerides are typically higher in young girls as compared to boys, often
by as much as 20%102,106,127 and this may be because of different LPL activity;
however, this has not been confirmed in the literature. In addition, Boulton
et al. reported that girls increased LDL-C through 8 years of age, while
LDL-C of boys remained steady from age 2 to 8 years.102
Asayama et al. reported on HDL-C and its subfractions from birth in males
and females in a cross-sectional cohort.134 At birth, boys had significantly
greater HDL3-C than girls, while HDL-C was somewhat higher. Interestingly,
in boys HDL3-C remained constant, while HDL-C and HDL2-C rose ~ 11
mg/dl from birth to ages 6–10 years and then began to decline into adulthood
(> 15 years). In girls, HDL-C and HDL2-C continued to increase beyond birth
(35.6 ± 2.3 and 17.7 ± 1.8 mg/dl, respectively) into late adolescence (15 years
of age; 51.8 ± 2.3 and 31.7 ± 2.3 mgl/dl), while HDL3-C rose immediately
after birth (16.2 ± 1.1 mg/dl) and continued to rise until 10 years of age (21.4
± 1.1 mg/dl).134 Consistently, most others do not report differences in HDL-
C between boys and girls of these ages.106,127,129
In addition, the mean LDL particle size has been reported to be larger in
young girls than boys (2–3 years),138 whereas no difference in Lp(a) has been
observed during these ages.131,139 Interestingly, it may be expected that boys
at this age would have greater LDL particle sizes than girls, since adults
with higher TG concentrations typically have smaller LDL particle sizes.159,160
However, this suggests that other factors, such as very low-density lipopro-
tein (VLDL) production and enzyme activity, may be unique at different
points in the lifespan.
With regard to ethnic differences, Freedman et al. reported that black
children 1–4 years of age had higher TC levels as compared to American
Indians, Mexican Americans or whites of the same age.124 Among Mexican
Americans and whites, TC is 2–5 mg/dl greater in girls than boys. In black
girls TC was 2–3 mg/dl lower as compared to boys during the first 4 years
of life.124 Younger black boys and girls of the Bogalusa study had TG lower
than white girls, but not boys,127 and greater HDL-C than whites,127,129 but
similar to Mexican American children especially at ages closer to 8 years.129
Mid- to Late Adolescence

As children mature, lifestyle preferences that may play a role in modeling
lipid and lipoprotein profiles are solidified, and these preferences can have
a profound impact on adult life.140 They are indicated by the fact that obesity
acquired in childhood is highly predictive of obesity in adult life,105,141 smok-
ing habits typically begin by high school,105,142 regular oral contraceptive use
in girls frequently begins by age 15 years,96,143 and dietary preferences,
including the intake of processed food, rich in saturated fats and carbohy-
drates,4,144 and patterns of physical activity in childhood may remain con-
stant into adulthood.145 Due to the overwhelming evidence that the
atherosclerotic process begins early,97,98 and in part, the fact that lifestyle can
impact risk factors such as lipid and lipoprotein concentrations, the Ameri-
can Heart Association currently makes recommendations on its primary
prevention beginning in mid- to late adolescence.140,146

The current literature suggests that TC and LDL-C decrease during adoles-
cence, continuing through puberty until 18–19 years of age. This is followed
by a steady increase in LDL-C that appears to be independent of ethnic-
ity.126,127 Table 15.2 illustrates mean TC values during these periods.19
Guo et al. reported that the concentrations of TC and LDL-C decrease
somewhat between 9 and 11 years of age through early adulthood
TABLE 15.2
Plasma Total Cholesterol (mg/dl) in the First
Two Decades of Life
Percentile
Age (years) n Mean ± S.E. 5th 95th
0–4
Male 238 154.6 ± 1.8 114 203
Female 186 156.0 ± 2.0 112 200
5–9
Male 1253 159.9 ± 0.7 121 203
Female 1118 163.7 ± 0.7 126 205
Source: Adapted from United States Department of
Health, Education, and Welfare, National Heart,
Lung, and Blood Institutes, National Institutes of
Health, Lipid Research Clinics, Vol. I. The Preva-
lence Study, Bethesda, MD, 79–1527, 1979.
(17–19 years), reaching their minimum values during pubescence in both

boys and girls, and then increasing beyond age 19 years.147 Others have
reported similar patterns whether the studies have been
longitudinal 102,127,148–151 or cross-sectional. 103,105,106,123,136,152 Reductions
observed in apo B during these periods are consistent with reductions in TC
and LDL-C.68,102,148 In addition, VLDL-C has been reported to remain constant
during these years.127,148

After a small rise in TG concentration from 8 to 11 years of age, TG levels
remain stable through aging15,102,127 whereas cross-sectional analyses indicate
a progressive rise from 10% to 40% from 11 to 15 years.106,129,136,153 With regards
to HDL-C, most prospective investigations also report increasing concentra-
tions prior to age 11 years,102 followed by stepwise decrements in HDL-C
through 18 years of age.102,147,148 These changes coincide with reductions
reported in apo A-I.68,102,148 Boulton et al. reported apo A-I concentrations
substantially lower at age 15 years as compared to 8 years in both boys (123
± 16 vs. 199 ± 58 mg/dl) and girls (127 ± 18 vs. 192 ± 55 mg/dl).102 Cross-
sectionally, Bachorik et al. reported lower apo A-I in boys,68 which may
coincide with the reduced HDL-C reported in both genders when investi-
gated by this design.103,126,129,136,152 Consistent with these findings, Ronnemaa
et al. reported that LCAT activity was lowest during these years when
compared with early adulthood.154 Contrary to these reports, others have
reported no change in HDL-C in boys and girls106,123 or increases in HDL-C
(most notably, HDL2-C) in girls of 6–10 years as compared to 11–15 years,
but not in boys.134
Gender and Ethnic Differences

During mid- to late adolescence lipid and lipoprotein constituents may be
different in boys and girls. Total cholesterol and LDL-C are often reported
to be greater in girls than boys in some prospective102,148 and cross-
sectional106,129,152 investigations, and this may be due to changing hormone
concentrations.153 However, greater TC and LDL-C in girls have not been
reported in all studies.123,127,136,147,153,154 Most investigations, regardless of
design, have not found HDL-C102,127,136,147,152,154 or TG concentrations to be
different between genders during these years.102,123,127,147,153,154 However,
Bachorik et al. demonstrated that apo A-I concentration was approximately
7% lower in males than females at all ages, and the male-female difference
in whites was more pronounced than in other ethnic groups (blacks or
Mexican Americans).68 Overall, apo A-I tends to remain constant with age
in boys, while being lowest in women < 20 years old.68 Furthermore, con-
centrations of Lp(a) appear to be independent of gender at a young age.102,131
Some investigations have reported LDL size to be smaller in boys aged
10–17 years than girls of the same age153–155 and this is consistent with what
is observed in younger children. However, others have reported similar LDL
sizes156 and prevalence of small, dense LDL particles between the genders.157
With regards to ethnicity during mid- to late adolescence, greater TC and
LDL-C in girls may be especially pronounced in blacks more so than
whites.126 In addition, mean levels of TC are lower in black girls by about
10 mg/dl than in white girls,127 who also tend to have a slower increase in
apo B concentrations from early ages to 19 years of age.68 The current liter-
ature also suggests that adolescent blacks have greater Lp(a) concentrations
than whites,126,131,158 while Freedman et al. identified in these ages that blacks
have larger LDL particles than whites.153 In adults, similar differences have
been observed and are explained by greater TG.159,160 However, in this inves-
tigation, despite the white children having greater TG than the black children
(boys 96 ± 51 vs. 71 ± 30 mg/dl; girls 101 ± 7 vs. 72 ± 27 mg/dl, in whites
and blacks, respectively), TG levels were only weakly correlated to LDL
particle size for the entire group of children (r = –0.21).153 Furthermore, most
reports indicate that blacks have higher HDL-C and apo A-I than
whites126,127,153 during adolescence, despite a significant drop in its concen-
tration observed from age 9 to 19 years.129 In addition, after about 12 years
of age, TG are reported to be lower in black boys and girls127 than Mexican
Americans and whites who display very similar levels.129
Middle Age
It is well recognized that the prevalence of CHD increases with age,10 and
clearly, concomitant changes in lipid and lipoprotein profiles play a signifi-
cant role in this relationship.10,161 It is generally accepted that plasma TC,
LDL-C and TG increase with advancing age in adulthood162 while HDL-C
may decrease107,163–166 or stay the same.109,111,160,162,167–76 While part of the
age-related changes are due to normal physiologic alterations associated

with aging (i.e., menopause, reduced testosterone and estrogen, reduced
muscle mass, secondary to loss of testosterone), a large part of these changes
is likely attributed to an increase in adiposity108,109 and a reduction in physical
activity.81,91,109

Early cross-sectional literature reported that changes in TC in adulthood are
extreme; from approximately 150 mg/dl at age 20 to 200 mg/dl at age 50,
thereon remaining constant to nearly 65 years of age at which time it then
begins to decline.19,167 Others, and more recent investigations, also indicate
that TC and LDL-C tend to increase with age in young or middle-aged adults
studied both cross-sectionally19,108–111,160–162,167,173,175,177–181 and prospec-
tively.107,161,163–166,176,180,182,183 Similar age-related increases during adulthood
are reported for apo B.68,111,160,173 In fact, increases in LDL-C may be explained
by reports that indicate the plasma residence time of LDL particles and that
the hepatic production of VLDL-apo B100 increase with age.112,173,184
Some investigations have also identified that the prevalence of small, dense
LDL increases160 and the mean LDL particle size decreases with age.156 How-
ever, these results are not consistent130,160,185 and potentially may be explained
by the reports of McNamara et al. and Lemieux et al., who suggest that LDL
particle size is primarily a reflection of TG concentrations, and therefore, not
necessarily age dependent.159,160
Few investigations have reported on the potential age-related changes in
Lp(a) concentrations during adulthood. While it is recognized that Lp(a)
levels are highly dependent on genetics,186,187 there is limited evidence that
they increase with age when compared cross-sectionally.132,188 Others have
not found Lp(a) to increase with age, except with menopausal status189–191
where concentrations increase unless hormonal replacement therapy (HRT)
is started.191 Furthermore, smaller apo(a) levels have also been reported to
be greatest in older individuals.132,189,190

Table 15.3 illustrates the mean fasting values of TG concentrations in adults
aged 20–59 years from a sample of men and women (not using exogenous
hormones). 1 9 Age-related increases in TG of adults are typi-
cal.108,109,160,162,173,175,176,191,192 Rifkind and Segal suggest that TG increase pro-
gressively in men at approximately 18 mg/dl per decade until age 80, but
more slowly in women to the age of 70.191 These changes are likely due to
reductions in LPL activity,192–194 but have also been correlated to increasing
visceral fat accumulation with age.109,160,181 Prospective research has also indi-
cated that TG increases with advancing age.195
Anderson et al. reported on fasting samples of 2433 individuals (men: n
= 1342; women: n = 1091) from the Framingham Study between the ages of
TABLE 15.3
Plasma Triglycerides (mg/dl) by Age in Adult
Males and Females
Percentile
Age (years) n Mean ± S.E. 5th 95th
20–24
Male 882 100.3 ± 1.9 44 201
Female 778 72.4 ± 1.3 36 131
25–29
Male 2042 115.8 ± 2.3 46 249
Female 1329 74.7 ± 1.0 37 145
30–34
Male 2444 128.3 ± 2.5 50 266
Female 1569 78.5 ± 1.0 39 151
35–39
Male 2320 144.9 ± 2.5 54 321
Female 1606 86.3 ± 1.2 40 176
40–44
Male 2428 151.4 ± 3.0 55 320
Female 1583 98.4 ± 2.1 45 191
45–49
Male 2296 151.7 ± 2.4 58 327
Female 1515 104.5 ± 1.8 46 214
50–54
Male 2138 151.8 ± 2.5 58 320
Female 1257 114.8 ± 2.0 52 233
55–59
Male 1621 141.4 ± 2.2 58 286
Female 1112 125.0 ± 2.3 55 262
60–64
Male 905 142.3 ± 3.1 58 291
Female 723 127.0 ± 3.3 56 239
65–69
Male 750 136.7 ± 5.2 57 267
Female 748 131.3 ± 4.5 60 243
70+
Male 850 129.8 ± 2.7 58 258
Female 748 132.4 ± 3.9 60 237
Triglycerides determined on plasma from fasting,
white subjects. Females are non-sex hormone users.
Source: Adapted from United States Department of
Health, Education, and Welfare, National Heart,
Lung, and Blood Institutes, National Institutes of
Health, Lipid Research Clinics, Vol. I. The Preva-
lence Study, Bethesda, MD, 79-1527, 1979.
25 and 54 years who were not using medications known to alter lipid con-
centrations.165 Comparisons were made of lipid profiles 8 years after their
first measurement and indicated that HDL-C was unchanged in men and
reduced in women.165 However, others have reported that HDL-C decreases
with age in both younger and middle-aged men and women107,163–166
although, when examined cross-sectionally, HDL-C has not been reported

to change.19,109,111,160,162,167–176 Similarly, apo A-I, apo A-II,68,175 or LCAT activity
do not appear to change with age in cross-sectional studies.196
Gender Differences
The changes that occur in lipids and lipoproteins with age in healthy indi-
viduals differ between men and women. In women, this is also dependent
on HRT. The concentrations of TC and LDL-C are significantly lower in
women than men during adulthood, especially before age 55 years19,68,162,167,196
and this is associated with higher levels of apo B.68,73,167 Heiss et al. found
that the increase in TC and LDL-C in women occurs at a much slower rate
than men.167 However, by age 55–60, the TC in women is typically equal to
that in men.167,178 In contrast, Connor et al. suggested that TC is somewhat
higher in women than in men prior to ages 20–35 years,122 and this is con-
sistent with the findings reported earlier that TC and LDL-C may be greater
in women under the age of 20 years in prospective102,129,148 and cross-sectional
studies.106,129,152
Bachorik et al. reported that apo B increased from 20 to 50 years in men,
but did not increase further during the years 50 to 69.68 In contrast, apo B
did not plateau until 60 years in women. These findings are consistent with
others.73,175 Furthermore, LDL-C may also plateau earlier in men than
women.68 Interestingly, Saito et al. reported that TC and LDL-C did not differ
between young (20–39 years) and older men (40–69 years); however, young
women (20–39 years) had lower TC and LDL-C than older women (40–69
years).175
No gender-related differences are reported to exist for Lp(a);197 however,
the literature suggests that men have smaller mean LDL particle size than
women185,198–201 regardless of menstrual status,156,201 and this may be related
in part to greater visceral adipose tissue accumulation and TG concentra-
tions.201
A large part of the discrepancy in blood lipids and lipoproteins between
men and women has to do with the use of exogenous hormones or HRT. As
reported earlier, regular oral contraceptive use frequently begins by age 15
years.96,143 Godsland reviewed literature on HRT and blood lipids and lipo-
proteins from 1974 through 2000 and surmised that, generally, postmeno-
pausal HRT use increases HDL-C and lowers LDL-C, TC, and Lp(a), while
its effects on TG depends on route of administration — oral estrogen tends
to increase TG, while transdermal estrogen has little effect.202
Estrogen has a direct influence on LDL-C by increasing LDL clearance
through an up-regulation in LDL receptor activity.203 Postmenopausal women
not receiving HRT are presumably expressing fewer LDL receptors than their
younger counterparts, and this is likely contributing to the rise in LDL-C that
occurs at menopause.180 Conversely, Bachorik et al. demonstrated that when
women were separated for use and non-use of HRT, users had significantly
greater apo B during ages 20–49 years despite having lower LDL-C.68 This
effect of HRT usage would be consistent with reports indicating an increase

in small, dense LDL particles;201,204 however, these results are not consis-
tent.205,206 Furthermore, spontaneous menopause,207,208 metabolically charac-
terized by low estrogen levels, elevates TG (> 10%) and reduces HDL-C
(as much as 10%) in conjunction with a reduction in LPL activity.180,209 Con-
versely, women at all ages taking oral estrogen have approximately 20–80
mg/dl higher TG than those who are not taking oral estrogen at the same
age,18,167,210 and men on exogenous estrogen also report increased TG.203 In
addition, a benefit of HRT may be that it increases HDL-C and apo A-I either
through an increase in apo A-I synthesis211 or decrease in apo A-I break-
down.212
Greater synthesis of TG-rich VLDL particles results in individuals with
larger visceral fat accumulation213 and these results are not gender depen-
dent.108,109,162 In numerous experimental conditions, it has been demonstrated
that individuals with high LPL activity have low TG and high HDL-C.27
Women typically have lower TG than men through their adult
lives.18,108,162,167,175,196 However, it is well known that men tend to accumulate
more visceral fat than women214 and investigators have demonstrated that
LPL activity is greater in women than in men,194 which may be related to fat
distribution.111,215
In addition, women have greater HDL-C, HDL2&3-C, and apo A-I than
men.68,73,216,217 However, premenopausal women have lower HDL3-C and
higher HDL2-C than the postmenopausal women who were not on HRT,
although their apo A-I and HDL-C levels are typically similar.73 Interestingly,
HRT appears to influence the age-related changes observed in HDL-C and
its subfractions. Women on HRT appear to preferentially increase HDL3-C
and decrease HDL2-C176 with age, while the opposite appears to be the case
for women who are not on HRT. Estrogens also exert their influence by
reducing HL activity,218–220 which would increase the concentration of HDL-C
by inhibiting its clearance from the plasma.27 For more detail on the topic of
HRT and lipoproteins please see chapter in this book.
While estrogen is strikingly beneficial in raising HDL-C, testosterone’s
influence on HDL-C is not well understood. Interestingly, LCAT activity is
reported to be higher in men than in women after puberty (suggesting an
influence of testosterone), despite men having lower total HDL-C.154 Cross-
sectional observations show positive relationships between HDL-C and nor-
mal physiologic levels of testosterone in adult men,221 but this has not been
confirmed by all studies.222 However, these findings are in direct contrast
with the well-documented findings of HDL-C decrease after exogenous
androgen administration of supraphysiologic levels223 or the immediate
reduction of HDL-C (by more than 10 mg/dl) in adolescent males going
through puberty when an increase in testosterone levels occurs.224 In addi-
tion, testosterone has been demonstrated to reduce apo A-I synthesis,216 and
it follows that apo A-I concentrations are approximately 5–10% lower in
males than females at all ages.68
Advanced Age
We have suggested that the prevalence of CHD increases with age;10 how-
ever, the association between cholesterol and CHD may weaken in older
individuals.225 The weakened relationship may be due, in part, to the fact
that far fewer investigations have examined the effects of older age
(≥ 65 years old) on blood lipids and lipoproteins. This section will review
what is currently known about blood lipids and lipoproteins in older indi-
viduals.

Total cholesterol and LDL-C decrease with age ≥ 65 years old in cross-
sectional18,168–172,226–229 and prospective studies.174,227,228,230,231 In fact, during
8 years of follow-up in older individuals (aged 65–79 years when initial
samples were taken) in the Framingham Study, TC declined by 0.9 mg/dl
per year.180 Other, longitudinal-design investigations have reported a
greater drop in TC of > 1.5 mg/dl per year for subjects of similar age
(Honolulu Heart Program;230 Rancho Bernardo Study;229 Zutphen Elderly
Study231). While Ericsson et al. reported that the fractional catabolic rate of
LDL apo B of older men was somewhat lower than younger men; this was
balanced by a lower production rate.112 Furthermore, Ferrara et al. suggested
that the reduced TC was not the result of survivor bias, weight loss, or use
of medications.229 It is interesting to note that even in older subjects who
gained weight, a reduction in TC was noted229,230 despite a loss of lean body
mass229 and, although no mechanisms have been proposed to explain this
phenomena, this may be related to altered nutritional intake or nutrient
absorption.174,231
The concentration of apo B is also reduced after age 69 years in men but
not in women.68 Knapp et al. reported that in older men the concentration
of Lp(a) was lower with increasing age (7th–9th decades of life).232 However,
others have reported no relationship between Lp(a) and older age.175,233,234
Moreover, to our knowledge no investigations have evaluated the relation-
ship between aging and changes in LDL particle size in this period of older
age. However, Mykkanen et al. investigated the relationship between LDL
particle size and risk of CHD in the elderly, and determined there was little
association.235

Cross-sectional analyses indicate that TG are reduced in older men and
women.101,112,172,227 However, to our knowledge no longitudinal analyses of
TG levels have been reported that have taken place over a long enough
period of time to determine the effects of aging on TG in older individuals.
One study found that 42% of its sample of older adults significantly increased
TG while the other 36% significantly decreased their TG over an 18-month
period.227
In cross-sectional investigations HDL-C does not change101,169–172 or does

apo A-I during older age.8 However, the prospective literature is equivocal
with regards to the concentration of HDL-C during older age, where inves-
tigations have reported HDL-C to decrease,68 increase230 or not change.174
Gender Differences
While few investigations have been reported in older individuals with
regards to age-related changes in lipid and lipoprotein profiles, far fewer
have reported gender differences. In older age, TC and LDL-C may be greater
in women by about 10%, with no differences in TG as compared to men.172,227
Additionally, HDL-C has been reported to increase in men, but not women
in cross-sectional studies, closing the gap between the levels reported in
prior age groups.172,175,227,229
Summary of Lipid and Lipoproteins through the Life Span in Adult

Americans
The prevalence of CHD is positively associated with the level of plasma
cholesterol throughout the life span.1,4,97,99 Plasma lipid and lipoprotein pro-
files change over the life span, and these changes begin early in development
of the growing fetus and continue into older age. It has been demonstrated
that lipid and lipoprotein measurements obtained in childhood and adoles-
cence are predictive of adult lipid and lipoprotein profiles.4,96
Many factors can influence lipid and lipoprotein profiles and this makes
it difficult to truly isolate the effects of aging. Furthermore, while changes
in lipid and lipoprotein concentrations over the life span do occur, the mech-
anisms that drive these changes are not fully understood because social and
environmental issues including education, economic status, nutrition, phys-
ical activity, obesity, tobacco use, total and abdominal obesity, and biological
issues such as activity of hepatic lipoprotein receptors, altered insulin sen-
sitivity, and hormonal changes have a profound influence over lipid and
lipoprotein levels and their metabolism. Table 15.4 illustrates some of the
major changes in plasma lipid and lipoprotein constituents in stages from
(1) gestation to birth; (2) birth to early childhood; (3) early childhood to late
adolescence; (4) late adolescence to middle age; and (5) middle age to
advanced age.
Conclusions
Lipid and lipoprotein concentrations and their constituents are highly vari-
able within and among individuals.18,20 The current literature provides
TABLE 15.4
Summary of Major Changes in Plasma Lipid and Lipoprotein
Constituents over the Lifespan
Stage I. Gestation to Birth
TC ↓ during weeks 12 through 20 of gestation

TC ↑ during weeks 20 through 32 of gestation
TC at birth between 50–100 mg/dl, however more evenly distributed
between HDL-C and LDL-C than at any other time in life
Stage II. Birth to Early Childhood
TC and LDL-C ↑↓↔

TG ↓↔
HDL-C ↑ ↔; may be explained by ↑ LCAT
TC girls > boys and HDL-C girls < boys
LDL size girls > boys
Stage III. Early Childhood to Late Adolescence
TC and LDL-C ↓
HDL-C ↑
TG ↑
TC and LDL-C girls > or = boys
TG and HDL-C = in girls and boys
LDL size girls > boys
Stage IV. Late Adolescence to Middle Age
TC and LDL-C ↑
LDL size ↓
Lp(a) may ↑
TG ↑; likely related to ↓ LPLa
HDL-C ↓ ↔
TC and LDL-C women < men
Lp(a) men = women
TG women < men; LPLa women > men
HDL-C women >men; HLa women < men
Stage V. Middle Age to Advanced Age
TC and LDL-C ↓
TG ↓
HDL-C ↑ ↓ ↔
TC and LDL-C women > men
↑, increase during this stage; ↓, decrease during this stage; ↔, does not
change during this stage; >, greater; <, lesser; LPLa, lipoprotein lipase
activity; HLa, hepatic lipase activity; TC, total cholesterol; LDL-C, low-
density lipoprotein cholesterol; TG, triglycerides; HDL-C, high-density
lipoprotein cholesterol; Lp(a), lipoprotein(a); LDL size, low-density lipo-
protein particle size; apo, apolipoprotein; LCAT, lecithin cholesterol acyl-
transferase. (Adapted from K. Watson, Plasma Lipoproteins
Concentrations in Ethnic Populations, Current Cardiol. Rep. 2003. Pub-
lished by Current Medicine.)
epidemiologic and prospective evidence to suggest that across ethnic groups

and throughout the life span, lipid and lipoprotein concentrations are asso-
ciated with CHD. The information provided in this chapter summarizes lipid
and lipoprotein differences and changes across ethnic groups in the United
States and throughout the life span. This information may be used by the
reader to recognize general trends and key age-related changes in lipid and
lipoprotein profiles that may identify individuals at greater risk for devel-
oping CHD.
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Index
A Animal studies
acylation-stimulating pathway, 162
Abbasi studies, 158 AMPK stimulation, 157
ABCA1, see Adenosine triphosphate-binding endothelial function and exercise
cassette-A1 (ABCA1) training, 89–91
ACEII Receptor 1 blockers, 86 Anitschkow, N, 67
Acetyl coenzyme (CoA), 39 Anti-atherogenic roles, HDL, 58
Acetylecholine, 65 Anti-inflammation, 101
ACS, see Acute coronary syndrome (ACS) Apolipoproteins
Acute coronary syndrome (ACS), 104 acute changes induced by exercise,
Acylation-stimulating pathway (ASP), 291–292
159–162 basics, 48
Acylation-stimulating protein (ASP), 148 concentration quantification, 123
Acylglycerides, 32, 32 exercise, impact, 268, 270–271
Adams studies, 102 L-arginine, 86
Adenosine diphosphate, 65 Arterial ischemia, pre-symptomatic, 23
Adenosine triphosphate-binding cassette-A1 Artery, anatomical structure, 63–64, 64
(ABCA1), 55 Asayama studies, 323
Adiponectin, 158–159 Asian Americans, 244, 317–318
Administration, pharmacological treatments Asian Indian Americans, see Asian Americans
bile acid-binding resins, 194, 196 ASP, see Acylation-stimulating pathway
nicotinic acid, 198 (ASP); Acylation-stimulating
statins, 191 protein (ASP)
Advanced age, 331–332 Assay linearity, 140–141
Adventitia, 64 Assay procedures, 137–138, 141
African Americans, see Black Americans Atherogenic lipid phenotype, 148
Alaskan Natives, see American Indians Atheroma, 76
Albers, Warnick and, studies, 127 Atherosclerosis
ALERT trial, 192 artery, anatomical structure, 63–64, 64
Alhassan studies, 117–142 basics, 6–7, 61–62, 62–63, 77
Almonds, 230 chronic endothelial injury hypothesis,
Alpha-adrenergic agonists, 65 66–67
Alpha-Tocopherol, Beta-Carotene Cancer endothelial dysfunction, 64–66
Prevention Study, 222 fatty streak formation, 68–71, 70–71
American Diabetes Association, 247 foam cell formation, 72
American Heart Association, 226, 247, 300, immune responsiveness, 74–75, 75
324 intracellular lipid accumulation, 72
American Indians, 318 LDL-mediated atherogenesis, 68, 69–70
Americans, adult, 320, 320–321 LDL oxidative modification, 68–71, 70–71
AMPK, see AMP-kinase (AMPK) lipid accumulation, 68, 69–70
AMP-kinase (AMPK), 156–158 lipid hypothesis, 67–68
Anderson studies, 225, 235, 327 macrophages, 72
Angelopoulos studies, 173–178 pathogenesis theories, 66, 66–68
Angiotensin II, 97 plaque formation, 75, 75–77
349
reverse cholesterol transport, 54 Callaerta-Vegh studies, 97

risk factors, 63 Campos studies, 304
smoking, 302 Canadian Heart Health Survey, 151
smooth muscle cell immigration, 73, Canola oil, 202
73–74 Caramori studies, 104
soldier autopsies, 61–62 CARDIA study, Coronary Artery Risk
stages, 68–77 Development in Young Adults
Atherosclerotic cardiovascular disease, (CARDIA)
14–15, 14–15 Cardiovascular disease (CVD), see also Heart
Atkins diet, 235–236 disease
Atorvastatin, 192, 196, 202, 204 apolipoproteins, 291
ATP III guidelines, 20 cigarette smoking, 306–307
ATTICA Study, 232 endothelial function and exercise
training, 87, 99, 106
metabolic syndrome, 149
nutrition, 212–213
B smoking, 300–301
Cardiovascular disease (CVD), nutrition
Babies, see Early childhood effects
Bachorik studies, 325–326, 329 basics, 212–213, 246, 248, 248
Barley, 224, 230 changing macronutrient profile, 237–240
Barter studies, 57–58 DASH diet, 233
Bazzano studies, 223 dietary cholesterol, 222
Beard studies, 267 dietary fiber, 222–225
Belardinelli studies, 95 dietary patterns effect, 228–233, 229
Ben-Ezra studies, 147–163 emerging risk factors, 236–246
Bergholm studies, 96 fat type dietary intervention, 239–240
Berglund studies, 241 fiber type dietary intervention, 239–240
Bile acid-binding resins, 194–197 glycemic index/glycemic load, 225–227
Black Americans, 150–152, 244–245, 319–320 HDL particle size, 240–242
Blood-brain barrier, 308 heart disease and fiber connection,
Blood lipids, see Lipids 222–223
Blood pressure, high, see Hypertension high-fat diets, 237
BMI, see Body mass index (BMI) high-protein diets, 235–237, 239
Body mass index (BMI) LDL particle size, 236–237, 238
adiponectin, 158 Lifestyle Heart Program, 230–231
cardiovascular disease predictor, 173–174 lipid management, 224–225
metabolic syndrome, 149 lipoprotein (a), 244–246
obesity, 176 low-carbohydrate diets, 237, 239
waist circumference, 150–152 low-fat diets, 234
Bogalusa Heart Studies, 300, 324 Mediterranean Diet, 232–233
Boulton studies, 323, 325 moderate-fat diets, 234–235, 237
Brown studies, 68, 72, 224 monounsaturated fatty acids, 219–220
Butter, 219, 245 multiple dietary strategies, 239–240
NCEP recommendations, 228, 230
n-3 PUFA, 221–222
n-6 PUFA, 220–221
C nutrients effect, 213–228, 214–216
polyunsaturated fatty acids, 220–222
CACs, see Circulating angiogenic cells Portfolio diet, 230–231
(CACs) postprandial TG, 242–244
CAD, see Coronary artery disease (CAD) saturated fatty acids, 217–218
C3a-des-Arg, see Acylation-stimulating science-based dietary guidelines, 246, 247
pathway (ASP) total fat, 213, 215, 217
Calculations, 138, 141 trans fatty acids, 218–219
Index 351
weight loss effects, 233–236 atherosclerosis, 6

Cardiovascular rehabilitation (CVR), 87, bile salt conversion, 37
94–95, 98, 103 dietary, 37, 222
Cardiovascular risk assessment early childhood, 322–323
atherosclerotic cardiovascular disease, ester exchange rate, 139–141
14–15, 14–15 exercise and physical activity, 267
basics, 13–14 exercise-induced changes, 284, 290–291
diabetes, 21, 21 middle age, Americans, 327
dyslipidemia, 19–21, 20 mid- to late adolescence, 324–325, 325
hypertension, 15–18, 16–19 nutrition, 213
metabolic syndrome, 21, 21 pharmacological treatments, 183–184
multivariable risk assessment, 24–25 risk factors, 3–4
novel risk factors, 23–24 synthesis regulation, 38–41, 40–41
obesity, 22, 22 Cholesterol/cholesteryl ester transfer protein
pre-symptomatic arterial ischemia, 23 (CETP)
preventive implications, 25–26, 26 cigarette smoking, 305
risk factors, 15–23 exercise-induced changes, 292
unstable lesions, 23 indirect pathway, 57
Carter, R., ΙΙΙ, studies, 61–77 lipoprotein lipase, 155
Case studies, 102–104 post-heparin LPL activity, 273
Caucasians rate estimation, 139–141
adiponectin, 158
Cholesterol esterase, 120
apolipoproteins, 271
Cholesterol oxidase, 120
lipoprotein (a), 244
Cholestyramine, 194, 196
post-heparin LPL activity, 273
Chronic endothelial injury hypothesis, 66–67
waist circumference, 150–152
Chylomicrons, 50–51, 55, 266
CCR2, see Chemokine receptor 2 (CCR2)
Cianflone studies, 161
CD36, see Cluster differentiation 36 surface
Circulating angiogenic cells (CACs), 102
molecules (CD36) receptors
Clarkson studies, 92
Cederberg and Enderback studies, 156
Classifications, 32–37, 48–51, 49
Cell permeability, 65
Clinical applications, 274–275
Centers for Disease Control and Prevention,
148 Clinical trials
Cerivastatin, 85, 193 bile acid-binding resins, 196–197
Cessation of smoking, 306–308 fibrates, 200
CETP, see Cholesterol/cholesteryl ester fish oils, 203
transfer protein (CETP) nicotinic acid, 198
Chajek-Shaul studies, 302 statins, 194, 195
Change mechanisms, 268, 272–273, 292–293 Cluster differentiation 36 surface molecules
Changing macronutrient profile, 237–240 (CD36) receptors, 72
Chang studies, 93 CoA, see Acetyl coenzyme (CoA)
CHD, see Coronary heart disease (CHD) Coa studies, 211–248
Chelland studies, 299–310 Colesevelam, 194, 196
Chemokine receptor 2 (CCR2), 69–70 Colestipol, 194
Chemokines, 69 Connor studies, 329
Cheuvront, S., 77 Coronary artery disease (CAD)
Children, see Early childhood; Mid- to late apolipoproteins, 270
adolescence cholesterol relationship, 1–2
Chinese Americans, see Asian Americans endothelial function, 86–87, 91–98,
Chlamydia pneumoniae, 65 105–106
Chobanian studies, 25 exercise and physical activity, 268–269
Choi studies, 317 Japan incidence, 7
Cholesterol lipids and lipoprotein acceptance, 8
absorption inhibitors, 200–202 metabolic syndrome, 149
advanced age, 331 pharmacological treatments, 184–187
Coronary artery disease (CAD), exercise endothelial dysfunction, 65

training and endothelial function endothelial function and exercise
animal studies, 88–91 training, 92–93
basics, 85–86, 86, 104–106, 105 Hispanic Americans, 319
case study, 102–104 obesity, 176
dysfunction reversal, 88–91 pharmacological treatments, 187
human endothelial function, 91–97 VLDL, 5
improved endothelial dysfunction, Diabetic dyslipidemia, 176
98–102 Diacylglycerides, 26
morbidity and mortality, 87 Diacylglycerol acyltransferase (DGAT), 159
VLDL particles, 6 Diacylglycerol (DAG), 159
youth endothelial dysfunction, 97–98 Diet and Reinfarction trial, 203
Coronary Artery Risk Development in Young Dietary Approaches to Stop Hypertension
Adults (CARDIA), 319 (DASH) Diet, 229, 233
Coronary heart disease (CHD), 148 Dietary Effects on Lipoproteins and
Coronary Primary Prevention Trial, 3 Thrombogenic Activity (DELTA),
CP450, see Cytochrome P450 (CP450) 217, 241
Craig studies, 301 Dietary patterns effect, 228–233, 229
C-reactive protein, 24, 149 Digestion, lipids, 37–38, 38–39
Crouse studies, 269, 283–295 Direct pathway, 56–57
Curve development, standard, 140–141 Dreon, Krauss and, studies, 237
CVR, see Cardiovascular rehabilitation (CVR) Dreon studies, 237, 239
CY3A4 isoenzyme, 192 Drug interactions, 192, 192, 196, see also Side
CY2C0 isoenzyme, 192 effects
Cycling, postprandial lipemia, 272 Drug selection, 204
Cyclooxygenase, 42 Dumesnil studies, 237
Cyclosporine, 204 Dupont studies, 31–44
CYP3A4 system, 200, 204 Durstine and Haskell studies, 178
Cytochrome P450 (CP450), 192 Durstine studies, 177, 265–275
Cytomegaloviral infections, 65 Dysfunction reversal, 88–91
Dyslipidemias
acylation-stimulating pathway, 162
cardiovascular risk assessment, 19–21, 20
D metabolic syndrome, 148–150
obesity, 176–178
DAG, see Diacylglycerol (DAG)
Dark chocolate, 86
DASH Diet, 229, 233
Dattilo studies, 178 E
Davey studies, 239
Davis studies, 47–58, 89 Early childhood, 322–324
Decreased flow-induced vasodilation, 65 Eckel studies, 152
DELTA, see Dietary Effects on Lipoproteins EDD, see Endothelium dependent
and Thrombogenic Activity vasodilation (EDD)
(DELTA) Edwards studies, 95
DeSouza studies, 92 Eicosanoids, 41–42, 42–43
Desroches studies, 239 EID, see Endothelial-independent dilation
Developmental stages, Americans, 322 (EID)
DGAT, see Diacylglycerol acyltransferase Electrophoresis, 48, 128–131
(DGAT) ELISA techniques, 139
Diabetes, see also Insulin Elliason studies, 303
adiponectin, 158 Emerging risk factors, 236–246
American Indians, 318 Enderback, Cederberg and, studies, 156
cardiovascular disease predictor, 174–175 Endogenous pathway, 53–54
cardiovascular risk assessment, 21, 21 Endothelial dysfunction, 64–66
Index 353
Endothelial function middle age, Americans, 326–330

animal studies, 88–91 mid- to late adolescence, Americans,
basics, 85–86, 86, 104–106, 105 324–326
case study, 102–104 ETICA study, 87
dysfunction reversal, 88–91 ETS, see Environmental tobacco smoke (ETS)
human endothelial function, 91–97 Exercise
improved endothelial dysfunction, apolipoproteins, 268, 270–271
98–102 basics, 265, 275
morbidity and mortality, 87 change mechanisms, 268, 272–273
youth endothelial dysfunction, 97–98 clinical application, 274–275
Endothelial-independent dilation (EID), 92, endurance training, 266–271
95 lipids, 266–268, 266–269
Endothelial-leukocyte adhesion molecules, lipoproteins, 266–269, 268–269
65 physical inactivity, 273–274
Endothelial nitric oxide synthase (eNOS), 63, postprandial lipemia, 272
88–89, 101, 105, see also Nitric resistance training, 271
oxide (NO) Exercise and obesity
Endothelial progenitor cells (EPCs), 91, basics, 173–174, 174, 178
101–102, 106 dyslipidemias, lifestyle modification,
Endothelin-1 (ET-1), 97 177–178
Endothelium dependent vasodilation (EDD), visceral adiposity, 174–177
88, 91, 94–95, 98–99, 105 Exercise-induced changes
Endurance training, 266–271 apolipoproteins, 291–292
eNOS, see Endothelial nitric oxide synthase basics, 283–284, 286–289, 294–295
(eNOS) benefits, 293–294
Environmental tobacco smoke (ETS), blood triglycerides, 285, 290
308–309, see also Smoking change mechanisms, 292–293
Enzyme analysis, intravascular, 133–141 cholesterol, 290–291
EPCs, see Endothelial progenitor cells (EPCs) HDL-C and HDL-C subfractions, 284–285
Erbs studies, 101, 105 LDL cholesterol, 290–291
Ericsson studies, 331 lipoprotein (a), 291
Essential fatty acids, 41–42, 42–43, see also Exercise training and endothelial function
Fatty acids; Fish oils; Trans fatty animal studies, 88–91
acids (TFA) basics, 85–86, 86, 104–106, 105
Estrogen, see Hormones case study, 102–104
ET-1, see Endothelin-1 (ET-1) dysfunction reversal, 88–91
Ethnic differences human endothelial function, 91–97
atherosclerosis, 62 improved endothelial dysfunction,
early childhood, 323–324 98–102
ethnicity and age, 323–324, 326 morbidity and mortality, 87
mid- to late adolescence, 326 youth endothelial dysfunction, 97–98
Ethnicity and age Exogenous pathway, 51–53
advanced age, 331–332 Exon 7 polymorphisms, 101, 105
American Indians, 318 Ezetimibe, 200, 202, 204
Americans, adult, 320, 320–321
Asian Americans, 317–318
basics, 316–321, 332, 332, 334
Black Americans, 319–320 F
developmental stages, Americans, 322
early childhood, Americans, 322–324 Familial hypercholesterolemia, 53
ethnic differences, 323–324, 326 Faraj studies, 158, 161
gender differences, 323–324, 326, 329–330, Fat, nutrition effects, 213
332 Fatty acids, see also Essential fatty acids; Fish
Hispanics, 318–319 oils; Free fatty acids (FFA); Trans
life-span, Americans, 321–332 fatty acids (TFA)
basics, 32, 33–35, 34 G

cigarette smoking, 303
lipoprotein lipase, 152 Garg studies, 220, 225
Fat-type dietary intervention, 239–240 Gebauer studies, 211–248
Fatty streak formation, 68–71, 70–71 Gel electrophoresis, 48, 128–131
Fenofibrate, 200 Gemfibrozil, 200
Gender differences
Ferrara studies, 331
advanced age, 332
Ferre studies, 157
early childhood, 323–324
FFA, see Free fatty acids (FFA)
ethnicity and age, 323–324, 326, 329–330,
Fiber, 222–225, 230, 239–240 332
Fibrates, 199–200, 204 middle age, Americans, 329–330
Fibrinogen, 23 mid- to late adolescence, 326
Filipino Americans, see Asian Americans Gerhard studies, 235
Fish oils, 202–203, 221–222, 239, 244–245, see Gestation, see Developmental stages,
also Essential fatty acids; Fatty Americans
acids GI, see Glycemic index/glycemic load
Flaxseed, 202 Gidez studies, 127
Flow-mediated dilation (FMD), 92–93, 95, 98, Gielen studies, 94, 99
100 GISSI Prevention Study, 221
5-Fluorouracil (5-FU), 91 Glide and Van Bilsen studies, 157
Fluvastatin, 191–192, 204 Glomser, Ross and, studies, 6
FMD, see Flow-mediated dilation (FMD) Glomset, Ross and, studies, 66
Foam cell formation, 72 Glucose transporters, 160
Fogarty studies, 90 Glycemic index/glycemic load, 92, 225–227
Foger studies, 292 Glycerol kinase, 120
Glycerol phosphate oxidase, 120
Folic acid, 86
Godsland studies, 329
Foster studies, 236
Gokce studies, 95
Framingham Heart Study
Goodarzi studies, 153
advanced age, 331 Goto studies, 95–96, 106
Asian Americans, 317 Graham and Rush studies, 89
cardiovascular disease, 14, 14–15, 300 Grandjean studies, 117–142
cholesterol relationship, 3 Grape juice, 86
diabetes, 21, 22 Green studies, 99
dyslipidemia, 20, 20 Griel studies, 211–248
exercise and physical activity, 274 Griffin studies, 304
hypertension, 15–17, 16–19 Grundy, Mattson and, studies, 220
metabolic syndrome, 149 Grundy and Mattson studies, 220
multivariable risk assessment, 25 Guan-Da studies, 93
obesity, 22 Guar gum, 224
pre-symptomatic arterial ischemia, 23 Guo studies, 324
preventative implications, 26
risk factor, 24
Freedman studies, 324, 326
Free fatty acids (FFA), see also Fatty acids H
acylation-stimulating pathway, 160–162
Hagberg studies, 270
cigarette smoking, 303 Halle studies, 177
lipoprotein lipase, 152, 154 Hambrecht studies, 93–94, 98, 100, 103, 105
Freeman studies, 301, 303 Hamilton studies, 274
5-FU, see 5-Fluorouracil (5-FU) Hammalainen studies, 87
Fuchsjager-Mayerl, 93 Hammond and Horn studies, 301
Fukai studies, 99 Hardman studies, 294
Future trends, 7 Harris studies, 221
Index 355
Haskell, Durstine and, studies, 178 intravascular formation, 133

Haskell studies, 91 lipid hypothesis, 67
Havel studies, 124 lipoprotein isolation, 127
Hayward studies, 91 lipoprotein lipase, 155
HDL, see High-density lipoprotein (HDL) metabolic syndrome, 148–149
HDL-C, see High-density lipoprotein (HDL- nutrition, 212
C) cholesterol and subfractions obesity, 173–178
Health impact, 8, see also Exercise; Nutrition particle size, 240–242
Health Professionals Follow-Up Study, 221 pharmacological treatments, 184–185
Heart disease, see also Cardiovascular disease reverse cholesterol transport, 55–56
(CVD) statin effects, 191
atherosclerosis, 302 High-density lipoprotein (HDL-C)
basics, 299–300, 309–310 cholesterol and subfractions
carbon monoxide, 308 advanced age, 331–332
cardiovascular disease, 300–301 basics, 51
cessation of smoking, 306–308 cigarette smoking, 303
environmental tobacco smoke, 308–309 dyslipidemia, 19–20
fiber, 222–223 early childhood, 323
HDL, 304–306 exercise and physical activity, 268–269
LDL, 304 exercise-induced changes, 284–285, 292
lipid metabolism, 302–306
foam cell formation, 72
metabolic changes, smoking cessation,
intravascular formation, 133
307–308
lipoprotein isolation, 127
nicotine, 308
triglycerides, 302–304
middle age, Americans, 327–329, 328
VLDL, 302–304
mid- to late adolescence, 325
Heart Protection Study, 26
nutrition, 213
Hedin, Thyberg and, studies, 76
pharmacological treatments, 184–185
Hegsted studies, 217
post-heparin LPL activity, 273
Heilbronn studies, 227
Heiss studies, 329 resistance training, 271
Helicobacter pylori, 65 High-fat, moderate-fat diet effects, 237
Hellerstein studies, 303 Highly sensitive C-reactive protein (hsCRP),
Henderson studies, 89 102
Heparin/manganese-chloride (MnCl2) High molecular weight (HMW) standards,
method, 127 129, 131
Hepatic (triglyceride) lipase (HL/HTGL) High-protein diets, 235–237, 239
activities, laboratory methods, 133–138 Hilpert studies, 211–248
endogenous pathway, 53 Hingorani studies, 101
exercise-induced changes, 292 Hispanic Americans
Hepatotoxicity, 193 ethnicity and age, 318–319
Herpes virus infections, 65 lipoprotein lipase, 153
He studies, 153 waist circumference, 150–152
Higashi studies, 93 Historical developments, 7
High-carbohydrate diet, 234 Homeostasis Model Assessment (HOMA) of
High-density lipoprotein (HDL) IR, 175
basics, 48, 50 Homocysteine, 24, 62, 102
cardiovascular disease, 300 Honolulu Heart Study, 222, 300, 331
cholesterol ester transfer protein, 139 Hormones, 327, 329–330
cigarette smoking, 301, 304–308 Horn, Hammond and, studies, 301
exercise-induced changes, 292 Hosakawa studies, 96
exogenous pathway, 52 Houmard studies, 153
foam cell formation, 72 Howard studies, 319
formation, 54–56 hsCRP, see Highly sensitive C-reactive
indirect pathway, 57 protein (hsCRP)
Human endothelial function, 91–97, see also J

Endothelial function
Hu studies, 218 Janssen studies, 152
7α-hydroxylase, 40 Japanese Americans, see Asian Americans
Hypercholesterolemia, 65 Jogging, 272
Hyperlipidemia, 187 Jones studies, 61–77
Hypertension Judd studies, 218
cardiovascular risk assessment, 15–18,
16–19
endothelial dysfunction, 65
endothelial function and exercise K
training, 92–93
Hypothyroidism, 187 Kahn and Valdez studies, 151
Kaitosaari studies, 323
Kannel studies, 13–26
Kantor studies, 285
Kaushik, S., 77
I Kemi studies, 100
IDL, see Intermediate-density lipoprotein Keys studies, 217
(IDL) Kildsgaard studies, 161
IL-8, see Interleukin-8 (IL-8) Kingwell studies, 92
Immune responsiveness, atherosclerosis, Knapp studies, 331
74–75, 75 Kobayashi studies, 95
Kokkinos studies, 177
Improved endothelial dysfunction, 98–102
Kostner studies, 300
Indications
Kratz studies, 239
cholesterol absorption inhibitors, 202
Krauss and Dreon studies, 237
fibrates, 200
Kris-Etherton studies, 211–248
fish oils, 203
Kushnick studies, 315–334
Indirect pathway, 57
Indo-Mediterranean Diet Heart Study, 232
Infants, see Early childhood
Inflammation, 65
Insulin, see also Diabetes L
adiponectin, 158–159
Laboratory methods, lipid and lipoprotein
cardiovascular disease predictor, 174
analysis
analysis of lipoprotein, 132–133, 133–134
endothelial function and exercise apolipoprotein concentration
training, 92 quantification, 123
lipoprotein lipase, 154–155 assay linearity, 140–141
metabolic syndrome, 148 basics, 118, 142
obesity, 175–176 blood lipid concentration quantification,
VLDL, 5 120–122
Interleukin-8 (IL-8), 70 cholesterol ester exchange rate, 139–141
Intermediate-density lipoprotein (IDL), 5, gel electrophoresis, 128–130
50–51 hepatic lipase activities, 133–138
Intracellular lipid accumulation, 72 intravascular enzyme analysis, 133–141
Intrauterine development, see isolating lipoproteins, 123–133, 132
Developmental stages, lipoprotein lipase, 133–138
Americans lipoprotein precipitation, 126
Intravascular enzyme analysis, 133–141 NaBr calculation, 126
Ion chelation, 86 neutral lipids, fluorescent intensity,
IP-10, 70 140–141
Isolating lipoproteins, 123–133, 132 plasma density calculations, 125–126
Israeli population, 220 plasma preparation, 119–120
Index 357
pre-electrophoresis procedures, 130–131 apolipoprotein concentration

separating lipoprotein, 128 quantification, 123
sequential ultracentrifugation, 124–126 assay linearity, 140–141
serum preparation, 119–120 basics, 118, 142
transfer protein activity analysis, 133–141 blood lipid concentration quantification,
LaFontaine studies, 85–106 120–122
Lamarche studies, 239 cholesterol ester exchange rate, 139–141
Lamon-Fava studies, 291 gel electrophoresis, 128–130
L-arginine, 86 hepatic lipase activities, 133–138
Latinos, see Hispanic Americans intravascular enzyme analysis, 133–141
Lauer studies, 321 isolating lipoproteins, 123–133, 132
Laughlin studies, 88–90 lipoprotein lipase, 133–138
Lavrencic studies, 93 lipoprotein precipitation, 126
Layman studies, 235 NaBr calculation, 126
LCAT, see Lecithin:cholesterol acyltransferase neutral lipids, fluorescent intensity,
(LCAT) 140–141
LDL, see Low-density lipoprotein (LDL) plasma density calculations, 125–126
LDL-C, see Low-density lipoprotein (LDL-C) plasma preparation, 119–120
cholesterol pre-electrophoresis procedures, 130–131
Lecithin:cholesterol acyltransferase (LCAT) separating lipoprotein, 128
cigarette smoking, 305 sequential ultracentrifugation, 124–126
exercise-induced changes, 292 serum preparation, 119–120
foam cell formation, 72 transfer protein activity analysis, 133–141
indirect pathway, 57 Lipid and lipoprotein metabolism
post-heparin LPL activity, 273 anti-atherogenic roles, HDL, 58
reverse cholesterol transport, 56, 293 basics, 47–48, 49–50
Left internal mammary artery (LIMA), 100 classification, lipoproteins, 49, 50–51
Lemieux studies, 150–151, 327 direct pathway, 56–57
Leon studies, 270 endogenous pathway, 53–54
Leptin, 156–158, 307–308 exogenous pathway, 51–53
Lesions, unstable, 23 HDL formation, 54–56
Leukocytes indirect pathway, 57
adhesion molecules, 65 reverse cholesterol transport, 54–58, 58
atherosclerosis, 74 transport, lipids, 51–54, 52
count, unstable lesion indictor, 23 Lipid hypothesis, 67–68
Levine studies, 301 Lipid-lowering effects
Lichtenstein studies, 219 bile acid-binding resins, 196
Life-span, Americans, 321–332 cholesterol absorption inhibitors, 202
Lifestyle changes and modifications, 177–178, fibrates, 199
186–187 fish oils, 203
Lifestyle Heart Program nicotinic acid, 197
DASH diet, 233 statins, 190–191, 191
endothelial function and exercise Lipidology
training, 98 acylglycerides, 32, 32
nutrition effects, 230–231 basics, 31
summary, 229 cholesterol synthesis regulation, 38–41,
LIMA, see Left internal mammary artery 40–41
(LIMA) classes, lipids, 32–37
Linke studies, 94, 105 digestion, lipids, 37–38, 38–39
Linoleate, 41 eicosanoids, 41–42, 42–43
Linoleic acid, 41, 220, 246 essential fatty acids, 41–42, 42–43
-linolenic acid, 41, 243 fatty acids, 32, 33–35, 34
Lipid and lipoprotein analysis, laboratory peroxidation, lipids, 43–44, 44–45
methods phospholipids, 34, 36, 36
analysis of lipoprotein, 132–133, 133–134 sphingolipids, 34, 36, 36
steroids and sterols, 36–37, 37 metabolic syndrome, 152–155, 154–155

Lipid Research Clinics, 300 postprandial lipemia, 272
Lipids Lipotoxicity, 156–158
accumulation, 68, 69–70 Lithell studies, 152
atherosclerosis, 6–7
Liu studies, 223
basics, 1–2
cholesterol risk factor, 3–4 L-NMMA, 96
concentration quantification, 120–122 Lovastatin, 190–192, 231
digestion, 37–38, 38–39 Low-density lipoprotein (LDL)
exercise, impact, 266–268, 266–269 apolipoproteins, 291
fluorescent intensity, 140–141 atherosclerosis, 62, 68, 69–70, 70
future trends, 7 basics, 50–51
health impact, 8
cardiovascular disease, 300
historical development, 7
cholesterol ester transfer protein, 139
lipoproteins, 4–6
management, nutrition effects, 224–225 cigarette smoking, 301–302, 304, 307
smoking, 302–306 dyslipidemia, 19
Lipoprotein, see also High-density lipoprotein exercise-induced changes, 290–292
(HDL); Low-density lipoprotein indirect pathway, 57
(LDL) lipid hypothesis, 67–68
basics, 4–6 metabolic syndrome, 148–149
density, 48
nitric oxide, 7
exercise, impact, 266–269, 268–269
isolating, 123–133 obesity, 173–178
precipitation, 126 oxidative modification, 68–71, 70–71
separating, 128 particle size, 236–237, 238
Lipoprotein, metabolism, see also Metabolic statin effects, 190–191, 191
syndrome Low-density lipoprotein (LDL-C) cholesterol
atherosclerosis, 302 advanced age, 331
basics, 299–300, 309–310
apolipoproteins, 270
carbon monoxide, 308
dyslipidemia, 19
cardiovascular disease, 300–301
cessation of smoking, 306–308 early childhood, 322–323
endothelial dysfunction, 65 exercise and physical activity, 267–268
environmental tobacco smoke, 308–309 exercise-induced changes, 290–291
HDL, 304–306 metabolic syndrome, 149
LDL, 304 middle age, Americans, 327
lipid metabolism, 302–306 mid- to late adolescence, 324–325, 325
metabolic changes, smoking cessation,
nutrition, 212–213
307–308
pharmacological treatments, 185–186
nicotine, 308
triglycerides, 302–304 physical inactivity, 273
VLDL, 302–304 resistance training, 271
Lipoprotein (a) Low-density lipoprotein (LDL) receptor-
endogenous pathway, 54 related protein (LRP), 53
exercise and physical activity, 267 Low-density lipoprotein (LDL) receptors, 53
exercise-induced changes, 291 Low-fat, high-carbohydrate diets, 213, 215
nutrition effects, 244–246 Low-fat diets, 228, 234
Lipoprotein lipase (LPL)
LRP, see Low-density lipoprotein (LDL)
cigarette smoking, 303–304
receptor-related protein (LRP)
exercise-induced changes, 292
Ludmer studies, 65, 104
exogenous pathway, 52
laboratory methods, lipid and lipoprotein Luscombe studies, 227
analysis, 133–138 Lymphocytes, 74
lipid concentrations quantification, 120 Lyon Diet Heart Study, 232–233
Index 359
M dyslipidemia, 149–150
leptin, 156–158
MACAD, see UCLA/Cedar Sinai Mexican lipotoxicity, 156–158
American Coronary Artery LPL activity, 152–155, 154–155
Disease (MACAD) project obesity, 22
Macronutrient profile, changing, 237–240 PPARs, 156–158
Macrophage chemotactic protein 1 (MCP-1), waist circumference, 150–152, 151
69–70 Metabolism, lipid and lipoprotein
Macrophages, 70–72 anti-atherogenic roles, HDL, 58
Maeda studies, 97 basics, 47–48, 49–50
Maiorana studies, 92, 99 classifications, 49, 50–51
Margarine, 219 direct pathway, 56–57
Marine-derived fatty acids, see Fish oils endogenous pathway, 53–54
Materials, laboratory methods exogenous pathway, 51–53
apolipoprotein concentrations, 123 HDL formation, 54–56
blood lipid concentrations, 121 indirect pathway, 57
cholesterol ester exchange rate, 139 reverse cholesterol transport, 54–58, 58
gel electrophoresis, 129 transport, lipids, 51–54, 52
isolation, lipoproteins, 124 Mevalonate, 39
lipase activities, 134–135 Mexican Americans, see Hispanic Americans
lipoproteins, 124, 127 Middle age, Americans, 326–330
precipitation, 127 Mid- to late adolescence, 324–326, see also
Maternal factors, see Developmental stages, Youth
Americans Miller studies, 226
Matrix metalloproteinases (MMPs), 77 Minokoshi studies, 156
Matrix proteins, 76–77 MMPs, see Matrix metalloproteinases
Matsumoto studies, 96 (MMPs)
Mattson, Grundy and, studies, 220 MnCl2 method, see Heparin/manganese-
Mattson and Grundy studies, 220 chloride (MnCl2) method
McCall studies, 305 Moderate-fat diets, 234–235, 237
McNamara studies, 327 Moffatt studies, 1–8, 299–310
MCP-1, see Macrophage chemotactic protein Monitoring, 204–205
1 (MCP-1) Monnink studies, 104
Mechanisms of action Monoacylglycerides, 26
bile acid-binding resins, 194 Monocyte recruitment, 69
cholesterol absorption inhibitors, 202 Monounsaturated fatty acids (MUFA), 212,
fibrates, 199 217, 219–220
fish oils, 203 Morbidity and mortality, 87
nicotinic acid, 197 MRFIT, see Multiple Risk Factor Intervention
statins, 190 Trials (MRFIT)
Mediterranean Diet, 219, 228, 229, 232–233 mRNA, see Messenger RNA (mRNA)
Menopause, see Hormones MUFA, see Monounsaturated fatty acids
Mensink studies, 218 (MUFA)
Messenger RNA (mRNA), 273 Muller studies, 88
Metabolic changes, smoking cessation, Multiple dietary strategies, 239–240
307–308 Multiple Risk Factor Intervention Trials
Metabolic syndrome, see also Lipoprotein, (MRFIT)
metabolism cardiovascular disease, 300
acylation-stimulating pathway, 159–162 dyslipidemia, 19
adiponectin, 158–159 hypertension, 16
basics, 147–148, 148–149, 162–163, 163 Multivariable risk assessment, 24–25
cardiovascular risk assessment, 21, 21 Mustard and Packham studies, 302
diagnostic criteria, 4 Mykkanen studies, 331
N Nuclear magnetic resonance (NMR), 48,

50–51, 123
NaBr calculation, 126 Nuclear transcription factors, 157
National Academies, 247 Nurses' Health Study, 218, 222, 226
National Academy of Science, 223 Nutrition
National Center for Health Statistics, 148 basics, 212–213, 246, 248, 248
National Cholesterol Education Program changing macronutrient profile, 237–240
(NCEP) DASH diet, 233
cholesterol relationship, 3 dietary cholesterol, 222
exercise and physical activity, 274 dietary fiber, 222–225
lipid concentrations quantification, 122 dietary patterns effect, 228–233, 229
nutrition, 213, 228 emerging risk factors, 236–246
particle size, 240 fat type dietary intervention, 239–240
pharmacological treatments, 184 fiber type dietary intervention, 239–240
recommendations, nutrition, 228, 230 glycemic index/glycemic load, 225–227
National Heart, Lung, and Blood (NHLB) HDL particle size, 240–242
Institute, 3, 247 heart disease and fiber connection,
National Heart Institute, 300 222–223
National Institutes of Health (NIH), 3, 247 high-fat diets, 237
Navare studies, 183–205 high-protein diets, 235–237, 239
NECP, see National Cholesterol Education LDL particle size, 236–237, 238
Program (NCEP) Lifestyle Heart Program, 230–231
NEFA, see Non-esterified fatty acids (NEFA) lipid management, 224–225
Neibauer studies, 89 lipoprotein (a), 244–246
Nephrotic syndrome, 187 low-carbohydrate diets, 237, 239
Nestel studies, 245 low-fat diets, 234
Neutral lipids, fluorescent intensity, 140–141 Mediterranean Diet, 232–233
Newborns, see Early childhood moderate-fat diets, 234–235, 237
NHANES I and III studies monounsaturated fatty acids, 219–220
Hispanic Americans, 319 multiple dietary strategies, 239–240
metabolic syndrome, 147–148 NCEP recommendations, 228, 230
nutrition, 222 n-3 PUFA, 221–222
waist circumference, 150–152 n-6 PUFA, 220–221
NHLB, see National Heart, Lung, and Blood nutrients effect, 213–228, 214–216
(NHLB) Institute patterns, dietary effect, 228–233
Niacin, 163, 205 polyunsaturated fatty acids, 220–222
Niaspan, 196 Portfolio diet, 230–231
Niaura studies, 308 postprandial TG, 242–244
Nicklas studies, 178 saturated fatty acids, 217–218
Nicotinic acid, 197–198, 204 science-based dietary guidelines, 246, 247
NIH, see National Institutes of Health (NIH) total fat, 213, 215, 217
Nitric oxide (NO), see also Endothelial nitric trans fatty acids, 218–219
oxide synthase (eNOS) weight loss effects, 233–236
atherosclerosis, 7, 63 Nuts, 202
endothelial dysfunction, 65
endothelial function, 96, 99
L-NMMA, 96 O
NO, see Nitric oxide (NO)
Non-denaturing polyacrylamide gradient gel Oats, 224–225, 230, 239
electrophoresis, 48 Obesity, see also Exercise training and
Non-esterified fatty acids (NEFA), 160 endothelial function
Novel risk factors, 23–24 adiponectin, 158
N-3 PUFA, 221–222 basics, 173–174, 174, 178
N-6 PUFA, 220–221 cardiovascular risk assessment, 22, 22
Index 361
dyslipidemias, 177–178 lifestyle changes, 186–187

insulin, 156 monitoring, 204–205
leptin, 156 nicotinic acid, 197–198
lifestyle modifications, 177–178 pharmacotherapy, 187–189, 188–189
metabolic syndrome, 22 selection of drug, 204
visceral adiposity, 174–177 statins, 189–194
Oeser studies, 307 therapy goals, 185, 185–187
Okosun studies, 150, 152 treatment targets, 184–185
Olefsky studies, 157 Pharmacology
Olive oil, 219 bile acid-binding resins, 194
Omega-3 fatty acids, 202 cholesterol absorption inhibitors, 202
Oram studies, 56 fibrates, 199
Owens studies, 158 nicotinic acid, 197
Oxidation, lipoprotein, 65 statins, 189–190, 190
Oxidized low-density lipoprotein (oxLDL), Pharmacotherapy, 187–189, 188–189
67 PHLPL, see Post-heparin LPL (PHLPL)
activity
Phosphatidic acid, 34
Phosphatidylinositol 3-kinase, 159
P Phosphodiesterase 3/4 (PDE3/PDE4)-
mediated mechanisms, 159
Packham, Mustard and, studies, 302 Phospholipids, 34, 36, 36
PAGE, see Polyacrylamide gradient gel Physical activity
electrophoresis (PAGE) apolipoproteins, 268, 270–271
Panton studies, 315–334 basics, 265, 275
Paradoxical vasoconstriction, 93 change mechanisms, 268, 272–273
Particle size clinical application, 274–275
basics, 5–6 endurance training, 266–271
HDL, 240–242 lipids, 266–268, 266–269
LDL, 236–237, 238 lipoproteins, 266–269, 268–269
Pathogenesis theories, 66, 66–68 physical inactivity, 273–274
Patterns, dietary effects, 228–233 postprandial lipemia, 272
PCI, see Percutaneous intervention (PCI) resistance training, 271
PDE3/4, see Phosphodiesterase 3/4 (PDE3/ Physical inactivity, 273–274
PDE4)-mediated mechanisms Physicians' Health Study, 24, 240
PDGF, see Platelet-derived growth factor Pietinen studies, 222
(PDGF) Pima Indians, 158
Pectin, 224 Plant sterols and stanols, 230, 239
Pelkman studies, 234 Plaque formation, 75, 75–77
Percutaneous coronary angioplasty (PTCA), Plasma density calculations, 125–126
96 Plasma preparation, 119–120
Percutaneous intervention (PCI), 104 Platelet activation, 65
Permeability, 65 Platelet-derived growth factor (PDGF), 73
Peroxidase, 120 Polyacrylamide gradient gel electrophoresis
Peroxidation, lipids, 43–44, 44–45, 302 (PAGE), 129–130
Peroxisome proliferator-activated receptors Polyunsaturated fatty acids (PUFA), 217,
(PPARs), 156–158, 196 220–222
Peters and VanSlyke studies, 2 Portfolio diet, 229, 230–231
Pharmacological treatments Post-heparin LPL (PHLPL) activity, 272–273
basics, 183–184, 205 Postprandial lipemia, 272
bile acid-binding resins, 194–197 Postprandial TG, 242–244
cholesterol absorption inhibitors, 200–202 PPARs, see Peroxisome proliferator-activated
drug selection, 204 receptors (PPARs)
fibrates, 199–200 Pravastatin, 191–192, 204
fish oils, 202–203 Pre-beta1 HDL particle, 55–56
Pre-electrophoresis procedures, 130–131 Risk factors, 3–4, 15–24, 236–246

Preiss-Landl studies, 152 Rivellese studies, 239
Pre-symptomatic arterial ischemia, 23 Robbins studies, 318
Preventive implications, 25–26, 26 Robinson, S., 77
Pritikin Program, 234 Roeters van Lennep studies, 150
Procedures Roitmann studies, 85–106
apolipoprotein concentrations, 123 Ronnemaa studies, 325
blood lipid concentrations, 121–122, 122 Ross and Glomser studies, 6
cholesterol ester exchange rate, 139–141 Ross and Glomset studies, 66
gel electrophoresis, 129 Ross studies, 6, 302
lipase activities, 135–138 Rosuvastatin, 191–192, 204
lipoprotein isolation, 124, 125 Rush, Graham and, studies, 89
lipoprotein precipitation, 127–128
pre-electrophoresis, 130–131
Psota studies, 211–248
Psyllium, 224, 230 S
PTCA, see Percutaneous coronary
angioplasty (PTCA) Saito studies, 329
PUFA, see Polyunsaturated fatty acids San Antonio Heart Study, 319
(PUFA) Saturated fatty acids (SFA), 212, 217–218
Puppione, Schumaker and, studies, 124 Scavenger receptor A (SR-A), 72
Scavenger receptor class B type I (SR-BI),
56–57
Schaefer studies, 174, 176
R Schopenhauer, A., 1
Schumaker and Puppione studies, 124
Rancho Bernardo Study, 331 Science-based dietary guidelines, 246, 247
RANTES, 70 Segal, Rifkind and, studies, 327
Rask-Nissilä studies, 322 Seip and Semenkovich studies, 273
Rauscher studies, 101 Selection, drugs, 204
Reaven studies, 175–176 Selective uptake, 56
Rehman studies, 102 Semenkovich, Seip and, studies, 273
Resistance training, 271–272 Separating lipoprotein, 128
Reverse cholesterol transport Sequential ultracentrifugation, 124–126
basics, 48 Serotonin, 65
foam cell formation, 72 Seven Countries Study, 217, 219–220, 222, 232
lipid and lipoprotein metabolism, 54–58, SFA, see Saturated fatty acids (SFA)
58 Sharman studies, 239
Rhabdomyolysis, 193 SHEP trial, 15
Rifkind and Segal studies, 327 Side effects, see also Drug interactions
Risk assessment, cardiovascular bile acid-binding resins, 196
atherosclerotic cardiovascular disease, fibrates, 200
14–15, 14–15 fish oils, 203
basics, 13–14 nicotinic acid, 198
diabetes, 21, 21 statins, 192, 193–194
dyslipidemia, 19–21, 20 Simvastatin, 190, 192, 204
hypertension, 15–18, 16–19 Sjogren studies, 239
metabolic syndrome, 21, 21 SMCs, see Smooth muscle cells (SMCs)
multivariable risk assessment, 24–25 Smoking
novel risk factors, 23–24 atherosclerosis, 302
obesity, 22, 22 basics, 299–300, 309–310
pre-symptomatic arterial ischemia, 23 carbon monoxide, 308
preventive implications, 25–26, 26 cardiovascular disease, 300–301
risk factors, 15–23 cessation of smoking, 306–308
unstable lesions, 23 endothelial dysfunction, 65
Index 363
environmental tobacco smoke, 308–309 Taimela studies, 270

HDL, 304–306 Tanabe studies, 90, 270
LDL, 304 Tanaka studies, 92
lipid metabolism, 302–306 Tangier Disease, 56
metabolic changes, smoking cessation, TBARS concentrations, 304
307–308 T-786c promoter, 101
nicotine, 308 Tea, 86
triglycerides, 302–304 Testosterone, see Hormones
VLDL, 302–304 Tetrahydrobiopterin, 86
Smooth muscle cells (SMCs), see also Vascular TFA, see Trans fatty acids (TFA)
smooth muscle cells (VSMCs) TG, see Triglycerides (TG)
basics, 63 Therapeutic Lifestyle Changes, 228, 229
immigration, 73, 73–74 Therapy goals, 185, 185–187
lipid hypothesis, 67–68 Thomas studies, 242
Sniderman studies, 149, 161
Thompson studies, 183–205, 270, 293
SOD-1, see Superoxide dismutase (SOD-1)
Thrombus formation, 65
Solutions
Th1 vs. Th2 cytokine responses, 74
gel electrophoresis, 129–130
Thyberg and Hedin studies, 76
lipase activities, 135–136
TIA, see Transient ischemic attack (TIA)
lipoprotein precipitation, 127–128
Tissue-type plasminogen activator (t-PA), 99
Soybean oil, 202, 219
TNF-α, see Tumor necrosis factor alpha
Soy protein, 230, 239, 245
(TNF-α)
Sphingolipids, 34, 36, 36
Tocopherols, 44
Squalene, 40
Total cholesterol, see Cholesterol
SR-BI, see Scavenger receptor class B type I
Total fat, nutrition effects, 213, 215, 217
(SR-BI)
t-PA, see Tissue-type plasminogen activator
SREBP transcription factors, 72
(t-PA)
St-Amand studies, 270
Trans fatty acids (TFA), 34, 218–219, see also
Stamford studies, 1–8, 299–310
Essential fatty acids; Fatty acids
Standard curve development, 140–141
Transfer protein activity analysis, 133–141
Statins
Transient ischemic attack (TIA), 102
endothelial dysfunction, 65–66
Transplant patients, 192
Transport, lipids, 51–54, 52
monitoring, 205
pharmacological treatments, 189–194 Treatment targets, 184–185
Portfolio diet, 231 Triacylglycerides, 26
Steffen-Batey studies, 87 Triglycerides (TG)
Steinbeck studies, 323 advanced age, 331–332
Step I and Step II diets, 224, 228, 229, 230, 237, cigarette smoking, 303, 307
241 early childhood, 323
Steroids and sterols, 36–37, 37 exercise-induced changes, 284–285, 290,
Stewart studies, 93 293
Stiebeling studies, 31–44 levels defined, 300
Substance P, 65 middle age, Americans, 327–329, 328
Summer studies, 265–275 mid- to late adolescence, 325
Superoxide dismutase (SOD-1), 89, 99 nutrition, 212–213
Suvorava studies, 90 pharmacological treatments, 184–185, 187
smoking, 302–304
statin effects, 191
Troglitazone, 157
T Tromso Heart Study, 300
Tumor necrosis factor alpha (TNF-α), 149
Taddei studies, 92 Tunica media, 63
U VLDL-C, see Very low-density lipoprotein

cholesterol (VLDL-C)
UCLA/Cedar Sinai Mexican American Vona studies, 96
Coronary Artery Disease VSMCs, see Vascular smooth muscle cells
(MACAD) project, 153 (VSMCs)
Ultracentrifugation, sequential, 124–126
Unstable lesions, 23
U.S. Departments of Agriculture/Health and
Human Services, 247
W
Wagganer studies, 47–58
Waist circumference (WC)
V cardiovascular disease predictor, 173
metabolic syndrome, 148, 150–152, 151
Vague studies, 173 Walnuts, 86
VA-HIT trial, 26 Walsh studies, 95
Valdez, Kahn and, studies, 151 Walther studies, 100
Van Bilsen, Glide and, studies, 157 Wang studies, 88
Van Gaal studies, 178 Wannamethee studies, 87
VanSlyke, Peters and, studies, 2 Warnick and Albers studies, 127
Vascular cell adhesion molecule-1 (VCAM-1), Waters studies, 302
71, 74 Watts studies, 98
Vascular endothelial growth factor (VEGF- WC, see Waist circumference (WC)
165), 90 Weight loss effects, 233–236
Vascular smooth muscle cells (VSMCs), 76, Wei studies, 319
see also Smooth muscle cells Western diet, 219, 245
(SMCs) Western Electric Study, 222
Vasodilation, 65 Weyer studies, 158, 160
VCAM-1, see Vascular cell adhesion Wheat, 225, 239
molecule-1 (VCAM-1) Whites, see Caucasians
Vegetarian diets, 228
Williams studies, 177, 267, 269
VEGF-165, see Vascular endothelial growth
Witt studies, 87
factor (VEGF-165)
Wolk studies, 222
Very late antigen-4 (VLA-4), 71
Women's Health Study, 223
Very low-density lipoprotein cholesterol
Woodman studies, 88
(VLDL-C), 53
Woods studies, 269
Very low-density lipoprotein (VLDL)
Wood studies, 270
basics, 5, 50–51
Woolf-May studies, 177
cardiovascular disease, 300
Woo studies, 98
cholesterol ester transfer protein, 139
World Health Organization, 306
cigarette smoking, 303–304
exercise and physical activity, 266
exercise-induced changes, 292
indirect pathway, 57
lipid hypothesis, 67 X
lipoprotein lipase, 152, 155
pharmacological treatments, 184 Xanthomatosis, 1
reverse cholesterol transport, 55
smoking, 302–304
Visceral adiposity, 174–177
Vitamin C, 85 Y
Vitamin E, 85–86, 221, 304
VLA-4, see Very late antigen-4 (VLA-4) Yamashita studies, 89
VLDL, see Very low-density lipoprotein Yeast, 224
(VLDL) Ye studies, 157
Index 365
Youth, 97–98, see also Mid- to late adolescence Z

Yu-Poth studies, 228
Zeiher studies, 58
Zhang studies, 161
Zhu studies, 151–152
Zutphen Elderly Study, 331

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Lipid

Boca Raton London New York

© 2006 by Taylor & Francis Group, LLC

Library of Congress Cataloging-in-Publication Data

Visit the Taylor & Francis Web site at

Sofiya Alhassan, Ph.D. Stanford University School of Medicine, Stanford,

Theodore J. Angelopoulos, Ph.D., M.P.H. Exercise Physiology Laboratory,

Vic Ben-Ezra, Ph.D. D e p a r t m e n t o f K i n e s i o l o g y, Te x a s Wo m e n ’ s

Robert Carter III, Ph.D. Laboratory of Adaptation Physiology, Thermal

Sarah Chelland, M.S. Department of Nutrition, Food and Exercise

Yumei Coa, B.S. Department of Nutritional Sciences, Pennsylvania State

Stephen F. Crouse, Ph.D. Texas A&M University, College Station, Texas

Paul G. Davis, Ph.D. Department of Exercise and Sport Science, University

Jacqueline L. Dupont, Ph.D., Hazel K. Stiebeling Professor, Department

J. Larry Durstine, Ph.D. Department of Exercise Science, University of

Sarah Gebaur, B.S. Department of Nutritional Sciences, Pennsylvania

Peter W. Grandjean, Ph.D. Department of Health & Human Performance,

Amy E. Griel, M.Ed. Department of Nutritional Sciences, Pennsylvania

Harlan P. Jones, Ph.D. Laboratory of Psychoneuro-Immunology, Department

William B. Kannel, M.D., M.P.H., F.A.C.C. Boston University School of

Penny M. Kris-Etherton, Ph.D., R.D. Department of Nutritional Sciences,

Michael R. Kushnick, Ph.D. School of Recreation and Sport Sciences, Ohio

Tom LaFontaine, Ph.D. PREVENT Consulting Services LLC, Columbia,

Robert J. Moffatt, Ph.D., MPH, Georgia A. Stamford Professor of Exercise

Sachin M. Navare, M.D. Division of Cardiology in the Henry Low Heart

Lynn B. Panton, Ph.D. Department of Food, Nutrition and Exercise

Tricia Psota, B.S. Department of Nutritional Sciences, Pennsylvania State

Jeffrey L. Roitman, Ed.D. Research Medical Center, Kansas City, Missouri

Bryant A. Stamford, Ph.D. Professor and Chair, Department of Exercise

Andrea C. Summer, B.S. Department of Exercise Science, University of

Paul D. Thompson, M.D. Division of Cardiology in the Henry Low Heart

1 Lipids and Health: Past, Present, and Future.............................. 1

2 Cardiovascular Risk Assessment ................................................ 13

3 Basic Lipidology ........................................................................... 31

4 Lipid and Lipoprotein Metabolism ............................................ 47

5 The Vascular Biology of Atherosclerosis ................................... 61

6 Exercise Training and Endothelial Function in Patients

7 Essential Laboratory Methods for Blood Lipid and

8 Metabolic Syndrome................................................................... 147

9 Obesity, Lipoproteins, and Exercise ......................................... 173

10 Pharmacological Treatments of Lipid Abnormalities............. 183

12 Physical Activity, Exercise, Blood Lipids, and

13 Acute Changes in Lipids and Lipoprotein-Lipids

14 Smoking, Heart Disease, and Lipoprotein Metabolism......... 299

15 Lipid and Lipoprotein Concentrations in Americans:

Index ..................................................................................................... 349

Bryant A. Stamford and Robert J. Moffatt

The Cholesterol Risk Factor

while news of updated guidelines is disseminated directly to the public

phospholipids. The apolipoproteins were found to vary in size and density

that antioxidants can retard progression of lesions substantially.31 Research

Past, Present, and Future

growth factors, may lead to revision of current hypotheses and creation of

Lipids and Health

myocardial infarctions that are silent or unrecognized.2,4 Because of this

Incidence of Atherosclerotic Cardiovascular Disease

Current Status of Risk Factors — Hypertension

The primary variety of hypertension was formerly believed to be benign

increase of systolic (or 10 mmHg diastolic) throughout the entire range of