Module 7: Lipids: Learning Objectives

MODULE 7: LIPIDS
Lipid Profile test is one of the most common requested laboratory examination. It
includes the amount determination of Total Cholesterol and Triglycerides. Lipids are
important because a clear relationship has been demonstrated between plasma lipids
and lipoproteins and atherosclerosis. Atherosclerosis is a condition of deposition of
plaques in the blood vessels that has been proven to lead to coronary artery disease. In
addition, according to the recommendations of the National Cholesterol Education
Program (NCEP), a Lipid Profile is useful in the evaluation of risk status for coronary
heart disease (CHD).
LEARNING OBJECTIVES:
At the end of the lesson, the student will be able to:
1. Identify the practical considerations in performing the triglyceride and cholesterol

methods of procedures
2. Discuss the principle associated with methods that determine cholesterol and
triglycerides
Lesson 1: Blood Lipid Determination
The most commonly employed lipid analyses are triglycerides and cholesterol
determination. An understanding of the concentration of these substances in serum and
plasma is used as an index in the diagnosis and management of certain diseases and
disorders associated with lipid metabolism.
Triglyceride determination is carried out by enzymatic method. Here, lipoprotein

lipase is required to produce glycerine and fatty acid. Glycerine will the participate in a
series of coupled reactions to yield the desired colored complex which id proportional to
the concentration of unknown triglycerides (TAG)
Modified Ferro-Ham is the most commonly used method for cholesterol

determination. This method employs the Liebermann-Burchardt reaction, which gives a
green color. This color is directly developed without prior extraction of the lipids.

Diagnostic Lipids in Human Plasma
1. Cholesterol
o Two-thirds of plasma cholesterol is esterified and one-third of plasma

cholesterol is unesterified. The greater part of the cholesterol of the body
arises by synthesis (about one gram per day), whereas only about 0.3
gram per day is provided by the average diet. The microsomal and cytosol
fraction of the cell are responsible for cholesterol synthesis. Acetyl
coenzyme-A is the source of all carbon atom in cholesterol.
o Laboratory Determination
a. Chemical method (Colorimetric)
b. Phenol Amino Phenazone Method: same as Van Handel and

Zilversmit method, only phenolaminophenone is used forming
yellow green at 550.
c. Reaction of hydrogen peroxide with Tylenol, forming colored

compound at 55nm.
d. Richmond modification
e. Colorimetry (Trinder’s reaction)
f. Blood Total Cholesterol (Liebermann-Burchardt method)
• Principle: Serum total cholesterol reacts with acetic

anhydride in the presence of sulphuric acid, a green solution
is formed. The serum total cholesterol concentration is
directly proportional to the absorbance reading at 600nm
after 3-5 minutes of incubation.
2. Triglyceride
o Aka: Triacylglycerol
o 95% of the pure form are stored in adipose tissues and predominant form
of glycerol esters are found in plasma. Their chief function is to serve as
the main storage form of fuel or energy in the human body. They may
generally simple and mixed triglycerides.
o Laboratory Determination:
a. Chemical Methods: Hantzch condensation method and Van Handel

& Zilversmit Method
b. Enzymatic Method
c. Nephelometric method (Stone and Thorp)
3. Phospholipid
o Phospholipids are esters of glycerol that contain two fatty acyl groups and
phosphatic acid.
o The main phospholipids are sphingomyelin, lecithin, and cephalins.
o Phospholipids constitute about 25% of low density lipoprotein (LDL) mass

(Lecithin:Sphingomyelin ratio of 2:1) and about 30% of high density
lipoprotein (HDL) mass (Lecitihin:Sphingomyelin ratio of 5:1).
o Total phospholipid determination is significant in the evaluation of the lipid

metabolism although findings may not be very significant.
4. Lipoprotein
o Lipoproteins (LPPs) are large, complex molecules which contain various

properties, water, insoluble lipids, and specific proteins (apoproteins).
These proteins, by their “detergent properties’ render the lipids soluble in
plasma and water. The lipid component causes LPPs to have densities
less than the densities of albumin and the globulins. The differences in
relative proportions of lipids and proteins among the several LPPs give
them varying densities that permit their separation in ultracentrifugation.
The LPPs do differ in the density of electrical charges. This property, in

combination with differences in size, permits separation of the LPPs
electrophoresis.
Practical Considerations:
Cholesterol Determination
- In specimen collection, a fasting period of 8-12 hours is necessary.
- Serum/plasma (EDTA) should be promptly separated from red blood cells.

Standing at room temperature may alter free-cholesterol ratio in the specimen. It
may also affect hydrolysis of cholesterol esters, and enzyme reaction of
cholesterol.
- Avoid using hemolyzed serum samples, especially if colorimetric procedures are

to be employed. This may cause false elevated values due to haemoglobin
extrachromogens present in red blood cells.
- Use clean, dry test tube or container during the process because the presence of
water causes sudden color changes, especially in Libermann-Burchardt reaction
and results in falsely decreased values.
- Serum protein produces turbidity and should be extracted using an organic

solvent.
- Posture variation can be a source of variation in findings.
- Interference in different method is observed wiith turbid, lipemic, icteric, or

hemolyzed samples.
Triglyceride Determination
- Fasting for 8-14 hrs is recommended in order to avoid the presence of

chylomicrons which appear in serum 2-6hrs after meal. In the presence of
hyperlipidemias, chylomicrons will appear in the blood even after overnight
fasting.
- Plasma triglycerides are higher than that of serum due to entrapment of

chylomicrions and lipoproteins (LPPs) during the clopping process and due to
lipolysis.
- Heparinized or EDTA-treated plasma can be used but fluorides and oxalates can
cause interference.
- Glucose and phospholipids presents interference in the non-enzymatic methods

for TAG assay because oxidation step-off glycerol to formaldehyde is not specific
for glycerol alone.
Materials Needed:
Apparatus
• Spectrophotometer and accessories
• Centrifuge
Glassware and equipment
• Test tubes
• Pipette
• Water bath set at 37 degrees celsius
• Beaker
Chemicals
• Triglyceride reagents and standards (Profame)
• Cholesterol reagents and standards (Profame)
Procedures:
A. Cholesterol Determination
1. Prepare the reagent mixture
2. Mix reagent A and reagent B in the ratio 1:1; placing 1500 µL (reagent A) + 1500 µL
(reagent B) in a test tube
3. Prepare the following:
Blank Standard Sample
Test Sample - - 25 µL
Standard - 25 µL -
Cholesterol working
1500 µL 1500 µL 1500 µL
reagent
4. Mix well and read the absorbance at 600 nm after 3-5 minutes
5. Concentration of unknown = Optical density of unknown/control X Concentration of

Standard
Optical density of standard
Note: To convert mg% to mmol/L, multiply mg% by 0.0258
Reference Value: 110-210 mg/dL
B. Blood Triglycerides Determination (Triglyceride-Enzymatic, Glycerol

Phosphate Oxidase)
1. Prepare the following:
Blank Standard Sample
Test sample - - 0.025 mL
Standard - 0.025 mL -
Distilled water 0.025 mL - -
Working agent 2.5 mL 2.5 mL 2.5 mL
2. Mix and incubate for 10 minutes at 25 degrees celsius.
3. Measure the absorbance of the sample and the standard against reagent blank within
an hour.

Concentration of unknown = Optical density of unknown/control X Concentration of

Standard
Optical density of standard
Normal Range: 36-165 mg/dL (0.4-1.86 mmol/L)

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MODULE 7: LIPIDS

At the end of the lesson, the student will be able to:

1. Identify the practical considerations in performing the triglyceride and cholesterol

Lesson 1: Blood Lipid Determination

Triglyceride determination is carried out by enzymatic method. Here, lipoprotein

Modified Ferro-Ham is the most commonly used method for cholesterol

Diagnostic Lipids in Human Plasma

o Two-thirds of plasma cholesterol is esterified and one-third of plasma

a. Chemical method (Colorimetric)

b. Phenol Amino Phenazone Method: same as Van Handel and

c. Reaction of hydrogen peroxide with Tylenol, forming colored

e. Colorimetry (Trinder’s reaction)

f. Blood Total Cholesterol (Liebermann-Burchardt method)

• Principle: Serum total cholesterol reacts with acetic

a. Chemical Methods: Hantzch condensation method and Van Handel

c. Nephelometric method (Stone and Thorp)

o The main phospholipids are sphingomyelin, lecithin, and cephalins.

o Phospholipids constitute about 25% of low density lipoprotein (LDL) mass

o Total phospholipid determination is significant in the evaluation of the lipid

o Lipoproteins (LPPs) are large, complex molecules which contain various

The LPPs do differ in the density of electrical charges. This property, in

- In specimen collection, a fasting period of 8-12 hours is necessary.

- Serum/plasma (EDTA) should be promptly separated from red blood cells.

- Avoid using hemolyzed serum samples, especially if colorimetric procedures are

- Serum protein produces turbidity and should be extracted using an organic

- Posture variation can be a source of variation in findings.

- Interference in different method is observed wiith turbid, lipemic, icteric, or

- Fasting for 8-14 hrs is recommended in order to avoid the presence of

- Plasma triglycerides are higher than that of serum due to entrapment of

- Glucose and phospholipids presents interference in the non-enzymatic methods

• Spectrophotometer and accessories

Glassware and equipment

• Water bath set at 37 degrees celsius

• Triglyceride reagents and standards (Profame)

• Cholesterol reagents and standards (Profame)

1. Prepare the reagent mixture

3. Prepare the following:

Blank Standard Sample

5. Concentration of unknown = Optical density of unknown/control X Concentration of

Note: To convert mg% to mmol/L, multiply mg% by 0.0258

Reference Value: 110-210 mg/dL

B. Blood Triglycerides Determination (Triglyceride-Enzymatic, Glycerol

1. Prepare the following:

Blank Standard Sample

Test sample - - 0.025 mL

Distilled water 0.025 mL - -

Working agent 2.5 mL 2.5 mL 2.5 mL

2. Mix and incubate for 10 minutes at 25 degrees celsius.

Concentration of unknown = Optical density of unknown/control X Concentration of

Normal Range: 36-165 mg/dL (0.4-1.86 mmol/L)

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