FOCUS ON: BIOMARKERS OF FETAL ALCOHOL EXPOSURE AND FETAL ALCOHOL EFFECTS

FOCUS ON: BIOMARKERS OF FETAL maternal drinking during pregnancy. However, maternal
drinking histories are generally unreliable, and the utility
ALCOHOL EXPOSURE AND FETAL of currently available biomarkers of alcohol exposure is
ALCOHOL EFFECTS limited. Thus, one of the critical challenges for the fetal
alcohol research community is to develop better methods
for detecting drinking during pregnancy. This article will
Ludmila N. Bakhireva, M.D., Ph.D., M.P.H., examine current methods for detecting maternal and fetal
and Daniel D. Savage, Ph.D. alcohol exposure, including self­reporting and biomarkers.
The limitations of current methods, however, underscore
the critical need to develop new biomarkers with greater
One of the ongoing challenges for the accurate diagnosis sensitivity for detecting moderate levels of drinking during
and treatment of children with fetal alcohol spectrum pregnancy for longer periods of time after the last drinking
disorders (FASD) is the difficulty of confirming whether a episode. Thus future research needs are reviewed as well.
mother drank during her pregnancy. Commonly used The development of more sensitive and reliable biomarkers
screening questionnaires often are unreliable, and current for alcohol use could serve two important objectives in
established biomarkers of alcohol consumption are not clinical intervention. First, the ability to identify more
sensitive enough for use with many pregnant women. women who are drinking during pregnancy, particularly
These limitations underscore the critical need to develop early in pregnancy, would allow providers to educate such
novel biomarkers with greater sensitivity for detecting patients about the dangers of drinking during pregnancy
moderate levels of drinking during pregnancy for longer and provide counseling to reduce the risk of subsequent
periods of time after the last drinking episode. In addition, drinking episodes. Effective intervention at this point
developing reliable biomarkers of fetal alcohol effects that would benefit not only the mother and fetus but also
can identify children at risk for adverse neurobehavioral could reduce the risks of fetal alcohol damage in subse­
outcomes could lead to behavioral interventions earlier in quent pregnancies. Second, a biomarker, or perhaps a
development. The use of animal models of FASD in combination of biomarkers and other clinical measures,
biomarker development could accelerate progress in this could provide an early indication of whether a newborn
challenging field of research. KEY WORDS: Maternal alcohol child is at risk for developing behavioral and/or cognitive
exposure; prenatal alcohol exposure; pregnancy; fetal alcohol effects; problems later in life, creating opportunities for earlier
fetal alcohol spectrum disorders; prenatal diagnosis; screening and postnatal interventions that may reduce longer­term
diagnostic method; biomarkers adverse consequences. Early intervention programs, such
as targeted sociocognitive programs focused on improving
behavior and math functioning (Kable et al. 2007), language

D
espite public­awareness campaigns and warning
and literacy training (Adnams et al. 2007), and rehearsal
labels on alcoholic beverages, roughly one of every
training to address working­memory deficits (Loomes et
eight women in the United States continues to drink
al. 2008), have been shown to reduce the social and
during her pregnancy. The term fetal alcohol syndrome (FAS)
behavioral difficulties experienced by fetal alcohol–affected
was first used in the 1970s to describe a specific pattern of
children (see Paley and O’Connor, pp. 64­75, in this issue).
malformations associated with prenatal alcohol exposure
However, in the absence of reliable prognostic indicators,
(Jones et al. 1974). The continuum of clinical presentations
the adverse effects of fetal alcohol exposure may go unrec­
caused by prenatal alcohol exposure is known as fetal alcohol
ognized for years, diminishing the benefits afforded by
spectrum disorders (FASD). Attempts to accurately estimate
earlier intervention.
the prevalence of FASD have been challenging because of
An ideal biomarker for detecting alcohol use among
numerous methodological issues, including diagnostic criteria,
pregnant women should have the following attributes: (1)
passive versus active surveillance, and difficulties with ascer­
the capacity to detect low­to­moderate levels of drinking
tainment of milder cases of FASD in the absence of fetal
over extended periods of time after the last drinking episode;
alcohol–induced birth defects. The prevalence of FAS ranges
(2) a high probability of accurately detecting drinking
from 0.5 to 7.0 per 1,000 live births in the general popula­
that occurred during pregnancy (i.e., high sensitivity); and
tion and up to 9.8 per 1,000 live births in high­risk groups
(3) a low rate of false­positive test results (i.e., high speci­
(May et al. 2001, 2009). The prevalence of the entire ficity). In addition, important technical considerations
continuum of FASD might be as high as 1 to 5 percent in
young school children in the United States (Lupton et al.
2004; May et al. 2001, 2009), which is higher than the LUDMILA N. BAKHIREVA, M.D., PH.D., M.P.H., is assistant
prevalence of autism spectrum disorders. professor, Department of Pharmacy Practice and Administrative
A large majority of fetal alcohol–affected children may Sciences, College of Pharmacy; DANIEL D. SAVAGE, PH.D.,
show no physical evidence of prenatal alcohol–associated is Regents’ Professor and chair, Department of Neurosciences,
birth defects. In the absence of characteristic FAS dysmor­ School of Medicine, both at the University of New Mexico
phology, recognition of FASD requires confirmation of Health Sciences Center, Albuquerque, New Mexico.

56 Alcohol Research & Health

 FOCUS ON: BIOMARKERS OF FETAL ALCOHOL EXPOSURE AND FETAL ALCOHOL EFFECTS

to be the most sensitive screening questionnaires for iden­

Table Key Terms and Abbreviations tifying risky drinking (14 or more drinks per week) in
pregnant women, with sensitivities greater than 90 percent
FASD Fetal alcohol spectrum disorders and 70 percent, respectively (Chang et al. 1999; Russell et
FAS Fetal alcohol syndrome al. 1996). However, such questionnaires do not capture the
amount, pattern, or timing of alcohol consumption.
ARND Alcohol­related neurodevelopmental disorder
Sensitivity The proportion of truly diseased persons in The Timeline Follow­Back Procedure. A more extensive
the screened population who are identified interview technique, known as the timeline follow­back
as diseased by the screening test (TLFB) procedure (Sobell and Sobell 1992) currently is
Specificity The proportion of truly nondiseased persons considered a gold standard for assessing alcohol exposure.
who are so identified by the screening test During the TLFB interview, patients are asked to report
TLFB Timeline follow­back procedure to assess exact quantities of the specific alcoholic beverages con­
alcohol exposure sumed each day during the defined timeframe. To minimize
γ­glutamyltransferase
recall bias, questions are linked to specific days, events,
GGT
and activities during the reported period. Sensitivity and
MCV Mean corpuscular volume specificity of novel biomarkers are traditionally established
CDT Carbohydrate­deficient transferrin relative to quantities of alcohol consumption (absolute
ounces of alcohol per day and per drinking day) reported
FAEE Fatty acid ethyl esters
on the TLFB interview. A more objective gold standard
EtG Ethyl glucuronide might be the actual diagnosis of FAS, partial FAS, or
EtS Ethyl sulfate alcohol­related neurodevelopmental disorder (ARND)
in affected children; however, such a gold standard would
PEth Phosphatidylethanol
have its own methodological limitations, given that most
SOURCE: Adapted from Last, J.M. A Dictionary of Epidemiology. 4th ed. New York, NY: children are not diagnosed with FASD until they start
Oxford University Press, Inc., 2001 exhibiting cognitive and behavioral problems in school.
Furthermore, general pediatricians often lack expertise to
include (1) a biological sample easily obtained by recognize FASD (Jones et al. 2006). In addition, not all
a minimally invasive and clinically acceptable procedure children prenatally exposed to alcohol develop FASD.
that (2) requires little or no sample preparation, and (3) a
simple, relatively inexpensive analytical procedure that (4)
provides rapid results, ideally in a point­of­care setting. Laboratory Tests
Unfortunately, none of the current alcohol biomarkers ade­ Given the inherent difficulties of self­report, including
quately satisfies more than one or two of these attributes. inaccurate recall, embarrassment, and fear of stigmatization,
biomarkers of chronic and recent alcohol exposure taken
individually and in combination may provide better
Current Approaches for the Detection corroboration of maternal drinking history.
of Fetal Alcohol Exposure
Biomarkers of Alcohol­Induced Pathology
Self­Reporting Methods The most established serum biomarkers of alcohol­induced
pathology are γ­glutamyltransferase (GGT), mean corpus­
cular volume (MCV), and carbohydrate­deficient transferrin
Screening Questionnaires. Despite growing interest in (CDT). GGT is a liver enzyme elevated in blood as a
biomarkers that can objectively and accurately identify result of chronic exposure to alcohol. MCV is a measure
alcohol exposure in mothers and children, maternal self­ of average red blood cell volume, which increases with
report remains, by far, the most prevalent approach used
in clinical practice for identifying people with hazardous 1
TWEAK is a five­item scale developed to screen for risk drinking during pregnancy. It is an
or harmful alcohol consumption. Screening questionnaires acronym for the following questions (Russell, 1994): T (tolerance)—“How many drinks can you
hold?” W (worried)—“Have close friends or relatives worried or complained about your drinking in
such as TWEAK,1 the Alcohol Use Disorders Identification the past year?” E (eye­opener)—“Do you sometimes take a drink in the morning when you first get
Test (AUDIT), the Michigan Alcohol Screening Test, and up?” A (amnesia)—“Has a friend or family member ever told you about things you said or did while
CAGE2 are designed to assess a history of alcohol­related you were drinking that you could not remember?” K/Cut Down—“Do you sometimes feel the need to
cut down on your drinking?”
behaviors (Russell et al.1996). Such questionnaires are
2
brief and easy to administer in clinical settings and might The CAGE questionnaire consists of four questions: 1. Have you ever felt you should cut down on
your drinking? 2. Have people annoyed you by criticizing your drinking? 3. Have you ever felt bad or
be quite sensitive for identifying risky drinkers. Given the guilty about your drinking? 4. Eye opener: Have you ever had a drink first thing in the morning to
existing body of literature, TWEAK and AUDIT appear steady your nerves or to get rid of a hangover?

Vol. 34, No. 1, 2011 57

 FOCUS ON: BIOMARKERS OF FETAL ALCOHOL EXPOSURE AND FETAL ALCOHOL EFFECTS

chronic alcohol consumption over a 1­ to 3­month period. lenging in a clinical setting for the following reasons:(1)
GGT is reported to be a better marker of heavy alcohol the limited window for specimen collection (usually the
consumption in pregnant women because MCV is physi­ first 48 hours after birth); (2) the unavailability of the
ologically elevated during the second half of pregnancy sample in up to 10 percent of newborns, especially
(Barrison et al. 1982; Halmesmaki et al. 1992; Ylikorkala preterm infants; and (3) the need to freeze the specimen
et al. 1987). However, although MCV has limited sensi­ at ultralow temperatures within the first 12 hours (later
tivity during the second and third trimesters of pregnancy, freezing is acceptable, but it can diminish FAEE levels).
it is a more specific biomarker than GGT (Sarkola et al. Finally, the validity of meconium FAEEs in identifying
2000; Ylikorkala et al. 1987). The half­life for detecting risky and binge drinkers who discontinued or substantially
elevated GGT is 2 to 3 weeks compared with 17 weeks decreased alcohol use later in pregnancy is unknown.
for MCV, making GGT a better marker to detect recent
changes in drinking patterns. Ethyl Glucuronide and Ethyl Sulfate. Ethyl glucuronide
CDT is a modified form of the iron­transporting protein (EtG) is a metabolite of alcohol produced through a reac­
transferrin and is a more specific measure of alcohol con­ tion with glucuronic acid in the liver. EtG can be detected
sumption than GGT or MCV. CDT is a reliable marker in urine for up to 5 days after a drinking episode (Wurst
of current heavy drinking that is usually elevated after an et al. 2005). Studies evaluating the validity of EtG for
intake of 50 to 80 grams of alcohol (4 to 6 standard drinks) the identification of alcohol abuse and/or social drinking
per day during the previous 1 to 3 weeks (Allen et al. among pregnant women are lacking. Results of a pilot
1994; Helander and Jones 2000). However, because CDT study (Wurst et al. 2008b) in Sweden indicate that screening
has a relatively short half­life, its levels usually normalize for EtG and FAEEs can identify more potential alcohol
after 4 weeks of abstinence (Allen et al. 1994). In pregnant consumers among pregnant women than the AUDIT
women, total transferrin increases with advanced gestational questionnaire alone, but the sensitivity of this test in pregnant
age; therefore, CDT currently is measured as a percentage women has not been established. Among nonpregnant
of total transferrin (percentage CDT). patients, EtG reached a sensitivity of 83.5 percent for the
identification of recent (within 4 days) alcohol consumption
(Wurst et al. 2004b). One drawback of EtG’s high sensi­
Biomarkers of Alcohol Metabolism tivity is the potential for false­positive results, which can
The most sensitive and specific measures for detecting be produced as a result of exposure to alcohol­containing
drinking are the presence of alcohol in body fluids or hand sanitizers, mouthwashes, cough syrups, and some
exhaled breath or the presence of alcohol’s most common beauty products (Kissack et al. 2008). In addition, false­
metabolite, acetaldehyde, in blood. However, the short negative results also can result from the hydrolysis of EtG
half­lives of alcohol and acetaldehyde limit their utility in by E. coli in patients with urinary tract infections, which
clinical practice, especially among pregnant women who are particularly common among pregnant women.
may have more intermittent episodes of alcohol consump­ Ethyl sulfate (EtS) is another alcohol metabolite produced
tion. The following sections review the potential use of by a reaction in the liver (Helander and Beck 2005). Although
other alcohol metabolites as biomarkers. it is not sensitive to bacterial hydrolysis (Helander and
Dahl 2005), it has a shorter detection period in urine (up
Fatty Acid Ethyl Esters. Fatty acid ethyl esters (FAEEs) are to 30 hours after alcohol ingestion) compared with EtG
nonoxidative metabolites that are formed when alcohol (Kissack et al. 2008). Both EtG and EtS concentrations
conjugates to endogenous free fatty acids and fatty acyl­CoA can be significantly affected by water­induced diuresis;
(Laposata 1998). The sensitivity of FAEEs extracted from thus, they need to be expressed relative to the creatinine
meconium, the first stool of a newborn, for the identification value to compensate for urine dilution (Helander and Beck
of prenatal alcohol exposure varies from 26.9 percent to 2005). Given the limitations of each test, a combined
100 percent across studies (Bearer et al. 1999, 2003; EtG/EtS battery can improve the validity of each individual
Chan et al. 2003; Derauf et al. 2003; Moore et al. 2003; biomarker for identification of recent drinking. The validity
Ostrea et al. 2006). These variations in sensitivity may be of these tests in pregnant women is largely unknown, however.
attributed to difficulties in the extraction of FAEEs from In a pilot study conducted among pregnant women in
complex media, differences in the study populations, and Sweden, only one woman tested positive for urine EtG,
potential confounders, such as diet. However, the advan­ and none were positive for EtS among nine women who
tages of meconium FAEE analysis include the ability to stated that they consumed alcohol during their ongoing
identify moderate drinking (Bearer et al. 1999) and a greater pregnancy (Wurst et al. 2008b). Thus, despite the high
sensitivity for identifying binge­drinking patterns rather sensitivity of EtG and EtS, their major limitation is the
than average consumption per week (Bearer et al. 2003). short detection period.
In addition, the presence of FAEEs in meconium has been
reported to be predictive of subsequent mental and psy­ Phosphatidylethanol. Phosphatidylethanol (PEth) is a
chomotor delays in children (Peterson et al. 2008). Despite phospholipid formed in the presence of alcohol that is
these advantages, the collection of meconium can be chal­ detectable in blood up to 3 weeks after sustained alcohol

58 Alcohol Research & Health

 FOCUS ON: BIOMARKERS OF FETAL ALCOHOL EXPOSURE AND FETAL ALCOHOL EFFECTS

intake (Hannuksela et al. 2007). Among nonpregnant patients maternal drinking does not elevate FAEEs in neonatal
with alcohol dependence, PEth demonstrated a 94.5 per­ scalp hair. Thus, the pattern of alcohol consumption that
cent (Hartmann et al. 2007) to 99 percent (Aradottir et can be captured by FAEE analysis in neonatal hair has yet
al. 2006) sensitivity. It also appears to be sensitive for the to be determined.
identification of moderate drinking (less than 40 grams In a study of nonpregnant methadone­maintenance
per day) in nonpregnant women (Aradottir et al. 2006). patients, Wurst and colleagues (2008a) reported that EtG
A recent study (Steward et al. 2010) among women of demonstrated a relatively high correlation with AUDIT
reproductive age demonstrated that PEth was detectable scores and self­reported alcohol intake in the previous 7
in 93 percent of women averaging more than two drinks days. However, the sensitivity and specificity was not reported.
per day but in only 53 percent of women consuming up In a pilot study among pregnant women in Sweden, only
to one drink per day. In that study, the correlation between 12.5 percent of those positive for hair EtG admitted
PEth and total alcohol consumption in the past 2 weeks ongoing drinking on the AUDIT questionnaire (Wurst et
was modest but statistically significant. PEth and EtG al. 2008b). This raises questions of either severe underre­
combined were able to identify alcohol­consuming patients porting on screening questionnaires or a high false­positive
who scored below the cutoff on AUDIT in an emergency­ rate with hair EtG testing.
room setting (Kip et al. 2008). However, the validity of Although hair analysis provides important advantages over
PEth has yet to be established, particularly in pregnant other biological tissues, it also has certain limitations. As
women who consume moderate amounts of alcohol. noted above, there is no clear correlation between the dose
of alcohol consumed and concentrations of FAEEs or EtG
in hair (Pragst and Yegles 2008). In addition, differences in
Recent Developments in Biomarkers for hair growth and hair care can affect results. Furthermore,
Fetal Alcohol Exposure the technically challenging microextraction of biomarkers
from hair is not routinely performed by clinical laboratories
With the exception of measuring FAEEs in meconium, and is expensive, limiting widespread clinical use. Moreover,
most work on alcohol biomarkers to date has focused on collection of hair, especially neonate hair, can be problematic
the use of maternal blood, serum, or urine samples as the because of aesthetic reasons and cultural practices.
biological sample source. More recently, investigators have
begun examining the utility of other tissue types as sources
for alcohol biomarker detection. These include hair, fin­ Analysis of Biomarkers in Newborn Blood
gernails, umbilical cord tissue, placenta, and newborn
blood. There is some evidence to suggest that these tissue Dried blood spot (DBS) analysis, collected from a neonatal
types may serve as repositories for biomarkers of alcohol heel stick, was first introduced in 1963 to test for the
metabolism, providing detectable measures of alcohol con­ metabolic genetic disorder, phenylketonuria, in newborns
sumption over longer periods of time. Another advantage (Guthrie and Susi 1963). Since then, Guthrie card samples—
of these tissue types is their relative ease and safety in sample DBS on a filter paper—are routinely collected from more
collection. However, sample preparation and the extraction than 95 percent of newborns Nationwide and are used
of alcohol biomarkers from these tissues generally is more to detect congenital conditions, infectious disease, and
challenging, compared with preparation and extraction from DNA/RNA analyses (Clinical and Laboratory Standards
samples described above. In addition, to date, very limited Institute 2007). DBS collected for routine newborn
data are available regarding the validity of biomarker analyses screening have been used for illicit­drug testing (Alfazil
in these tissue types for identifying drinking during pregnancy. and Anderson 2008; Garcia Boy et al. 2008; Henderson
et al. 1997), and recently the United States Drug Testing
Analysis of Biomarkers in Maternal and Newborn Hair Laboratory (USDTL) developed a liquid chromatography–
mass spectrometry method for detection of PEth in DBS.
Hair FAEE analysis conducted among nonpregnant alcoholic Such analyses offer substantial advantages over existing meth­
patients in a detoxification program or among heavy ods, including (1) a minimally invasive method; (2) accuracy
drinkers demonstrated 90 to 100 percent sensitivity and similar to samples collected by drawing blood; and (3) the
90 percent specificity (Pragst and Yegles 2008; Wurst et low cost of sample collection, processing, transport, and stor­
al. 2004a). However, very low FAEE concentrations were age (McDade et al. 2007). However, the validity of alcohol
detected even among strict abstainers, possibly as a result biomarkers in DBS specimens has yet to be established.
of the use of hair cosmetics or endogenous alcohol resulting
in false­positive results (Pragst and Yegles 2008). In a guinea
pig model of fetal alcohol exposure, alcohol­exposed off­ Summary and Future Directions for
spring demonstrated a 15­fold­higher cumulative FAEE Developing Novel Biomarkers of FASD
level in hair compared with control animals (Caprara et
al. 2005a). However, researchers conducting a clinical study Despite a great deal of research to date, the goal of devel­
(Caprara et al. 2005b) reported that mild infrequent oping alcohol biomarkers that can detect lower levels of

Vol. 34, No. 1, 2011 59

 FOCUS ON: BIOMARKERS OF FETAL ALCOHOL EXPOSURE AND FETAL ALCOHOL EFFECTS

drinking and for longer intervals of time after the last pregnant women, the risk of having an infant with growth
drinking episode remain elusive. As illustrated in the figure, retardation or facial features of FAS increased with an
the vast majority of biomarkers that are sensitive enough increasing number of positive test results with biological
to detect moderate and social drinking are present in body markers. To our knowledge, the validity of test batteries
fluids/tissues for relatively short periods of time after the consisting of traditional biomarkers and direct alcohol
last drinking episode. This problem is compounded by metabolites has not been established in pregnant women.
the fact that, in general, many women who continue to A third area that warrants additional development is
drink during pregnancy will cut down on either the amount biomarkers for predicting fetal alcohol­induced damage.
or frequency of drinking episodes, increasing the challenge Very little attention has been directed towards the question
of confirming a drinking history. In pregnant women, the of whether biomarkers of alcohol consumption or other
use of biomarkers to identify risky drinking early in gesta­ biomarkers have utility as prognostic indicators for newborns
tion presents a particular challenge, especially for those at risk for adverse neurobehavioral outcomes. Among
women who initiate prenatal care later in pregnancy. Thus, established alcohol biomarkers, a single study (Peterson et
more sensitive biomarkers of alcohol metabolism, such as al. 2008) has correlated increasing meconium FAEE levels
EtG/EtS, PEth, or FAEEs that can be detected in hair or with poorer mental and psychomotor development in
other tissue types for a longer period after drinking could infants up to 2 years of age. Beyond established alcohol
be very useful in clinical practice. biomarkers, a single retrospective clinical study (Robinson
In addition, the systematic assessment of a profile of et al. 1995) has examined the question of whether other
multiple biomarkers could increase the likelihood of chemical species might serve as biomarkers for FASD.
detecting drinking during pregnancy and perhaps provide These authors examined serum proteins in 12 children
better insights about drinking patterns. It has been long with FAS and 8 matched control children. Gel electrophoresis
recognized that a panel of biomarkers will detect alcohol identified eight protein spots as candidate biomarkers for
exposure more accurately than a single biomarker (Bearer FAS. No single protein spot was significantly altered in
2001; Stoler et al. 1998). However, none of the proposed all 12 FAS subjects, but the expression pattern of different
panels have been widely used in clinical practice. Stoler combinations of four of the protein spots accurately iden­
and colleagues (1998) reported that among alcohol­abusing tified all 12 FAS subjects. This study underscores the point

ʻ
ʻ
ʻ

Figure The period of time, or detection window, during which alcohol consumption can be detected and the lowest levels of alcohol consumption
detectable by current alcohol biomarkers.

60 Alcohol Research & Health

 FOCUS ON: BIOMARKERS OF FETAL ALCOHOL EXPOSURE AND FETAL ALCOHOL EFFECTS

that a signature pattern of changes in multiple chemical the utility that these and other high­throughput screening
species may have greater diagnostic and prognostic capability procedures could have for developing more sensitive and
than any single marker. Furthermore, high­throughput specific biomarker systems both for the diagnosis of fetal
screening of proteins, or perhaps genes or other metabolites in alcohol exposure as well as for earlier prognostic indicators
biomarker tissue samples, affords a unique opportunity for of fetal alcohol damage in exposed offspring. ■
developing more sensitive and specific biomarkers of FASD.
Finally, the development of more sensitive biomarkers
of FASD based solely on clinical research continues to be Acknowledgements
extremely difficult largely as a result of the inherent vari­
ability in patient populations, the highly variable patterns Supported by NIAAA P20AA–017068 and the Alcohol
of drinking among pregnant women, and the extended Beverage Medical Research Foundation.
time intervals between an optimal time for collecting bio­
logical samples, such as during pregnancy or at birth, and
the time at which measures of functional brain damage Financial Disclosure
and/or adverse behavioral outcomes can be assessed. One
alternative solution to this challenge is to first identify and The authors declare that they have no competing financial
validate biomarkers in established animal models of FASD interests.
and then, based on this information, pursue parallel human
studies to assess clinical utility. Despite translational research
challenges, the preclinical approach to novel biomarker
development has four important advantages. First, pre­ REFERENCES
clinical studies increase the prospects of identifying novel ADNAMS, C.M.; SOROUR, P.; KALBERG, W.O.; ET AL. Language and literacy
markers without the confounding variables associated outcomes from a pilot intervention study for children with fetal alcohol spectrum
with patient populations. Second, biomarker validation disorders in South Africa. Alcohol 41(6):403–414, 2007. PMID: 17936509
can proceed in a more systematic and controlled fashion ALFAZIL, A.A., AND ANDERSON, R.A. Stability of benzodiazepines and cocaine
over a shorter period of time and in a more cost­effective in blood spots stored on filter paper. Journal of Analytical Toxicology 32(7):
manner. Third, an animal model system provides an 511–515, 2008. PMID: 18713520
opportunity to assess more directly how a biomarker ALLEN, J.P.; LITTEN, R.Z.; ANTON, R.F.; AND CROSS, G.M. Carbohydrate­
signature may change as a function of alcohol dosing, deficient transferrin as a measure of immoderate drinking: Remaining issues.
patterns of exposure, and the influence of other interacting Alcoholism: Clinical and Experimental Research 18(4):799–812, 1994. PMID:
risk factors during pregnancy, such as nicotine, other drugs 7978088
of abuse, stress, malnutrition, or heavy­metals exposure. ARADOTTIR, S.; ASANOVSKA, G.; GJERSS, S.; ET AL. Phosphatidylethanol (PEth)
How a biosignature pattern is altered by concurrent expo­ concentrations in blood are correlated to reported alcohol intake in alcohol­
sure to other risk factors would be critically important for dependent patients. Alcohol and Alcoholism 41(4):431–437, 2006. PMID:
the interpretation of data from clinical studies. Finally, 16624837
an animal model system allows for direct correlation of BARRISON, I.G.; WRIGHT, J.T.; SAMPSON, B.; ET AL. Screening for alcohol
biomarker patterns with markers of functional damage to abuse in pregnancy. British Medical Journal (Clinical Research Edition)
the fetus and with longer­term adverse neurobehavioral 285(6351):1318, 1982. PMID: 6127136
outcomes. Such a model could, in the best­case scenario,
BEARER, C.F. Markers to detect drinking during pregnancy. Alcohol Research &
provide insights about the mechanistic basis for the ter­
Health 25(3):210–218, 2001. PMID: 11810960
atogenic damage, assessments that would be more diffi­
cult, if not impossible, to examine in a clinical study. BEARER, C.F.; JACOBSON, J.L.; JACOBSON, S.W.; ET AL. Validation of a new
To date, two studies have used rodent models of FASD biomarker of fetal exposure to alcohol. Journal of Pediatrics 143(4):463–469,
2003. PMID: 14571221
in combination with high­throughput screening procedures
to initiate a search for novel biomarker candidates. Using BEARER, C.F.; LEE, S.; SALVATOR, A.E.; ET AL. Ethyl linoleate in meconium: A
a proteomic screening procedure, Datta and colleagues biomarker for prenatal ethanol exposure. Alcoholism: Clinical and Experimental
(2008) reported a significant increase in α­fetoprotein Research 23(3):487–493, 1999. PMID: 10195823
expression in amniotic fluid taken from gestational day CAPRARA, D.L.; BRIEN, J.F.; IQBAL, U.; ET AL. A Guinea pig model for the
(GD) 17 mouse fetuses after a single high­dose alcohol identification of in utero alcohol exposure using fatty acid ethyl esters in
binge–like exposure on GD 8. Using a rat model of moderate neonatal hair. Pediatric Research 58(6):1158–1163, 2005a. PMID: 16306186
drinking throughout pregnancy that produces offspring CAPRARA, D.L.; KLEIN, J.; AND KOREN, G. Baseline measures of fatty acid ethyl
with learning deficits, Rosenberg and colleagues (2010) esters in hair of neonates born to abstaining or mild social drinking mothers.
have identified 22 placental genes whose expression at Therapeutic Drug Monitoring 27(6):811–815, 2005b. PMID: 16306859
term is significantly altered in alcohol­consuming dams. CHAN, D.; BAR­OZ, B.; PELLERIN, B.; ET AL. Population baseline of meconium
These alcohol­altered genes encode growth factors important fatty acid ethyl esters among infants of nondrinking women in Jerusalem and
for placental as well as fetal brain, vascular, endocrine, and Toronto. Therapeutic Drug Monitoring 25(3):271–278, 2003. PMID:
immune system development. These findings illustrate 12766552

Vol. 34, No. 1, 2011 61

 FOCUS ON: BIOMARKERS OF FETAL ALCOHOL EXPOSURE AND FETAL ALCOHOL EFFECTS

CHANG, G.; GOETZ, M.A.; WILKINS­HAUG, L.; AND BERMAN, S. Identifying KISSACK, J.C.; BISHOP, J.; AND ROPER, A.L. Ethylglucuronide as a biomarker for
prenatal alcohol use: Screening instruments versus clinical predictors. American ethanol detection. Pharmacotherapy 28(6):769–781, 2008. PMID: 18503404
Journal on Addictions 8(2):87–93, 1999. PMID: 10365188
LAPOSATA, M. Fatty acid ethyl esters: Ethanol metabolites which mediate
Clinical and Laboratory Standards Institute. Blood Collection on Filter Paper for ethanol­induced organ damage and serve as markers of ethanol intake. Progress
Newborn Screening Programs: Approved Standard, Fifth Edition. Wayne, PA: in Lipid Research 37(5):307–316, 1998. PMID: 10209651
CLSI, 2007 (CLSI Publication LA4­A5).
LAST, J.M.; SPASOFF, R.A.; AND HARRIS, S.M. A Dictionary of Epidemiology,
DATTA, S.; TURNER, D.; SINGH, R.; ET AL. Fetal alcohol syndrome (FAS) in Fourth Edition. New York: Oxford University Press, , 2001.
C57BL/6 mice detected through proteomics screening of the amniotic fluid.
Birth Defects Research. Part A, Clinical and Molecular Teratology 82(4):177–186, LOOMES, C.; RASMUSSEN, C.; PEI, J.; ET AL. The effect of rehearsal training on
working memory span of children with fetal alcohol spectrum disorder.
2008. PMID: 18240165
Research in Developmental Disabilities 29(2):113–124, 2008. PMID: 17317098
DERAUF, C.; KATZ, A.R.; AND EASA, D. Agreement between maternal self­
LUPTON, C.; BURD, L.; AND HARWOOD, R. Cost of fetal alcohol spectrum
reported ethanol intake and tobacco use during pregnancy and meconium
disorders. American Journal of Medical Genetics. Part C, Seminars in Medical
assays for fatty acid ethyl esters and cotinine. American Journal of Epidemiology
Genetics 127C(1):42–50, 2004. PMID: 15095471
158(7):705–709, 2003. PMID: 14507607
MAY, P.A., AND GOSSAGE, J.P. Estimating the prevalence of fetal alcohol
EWING, J.A. Detecting alcoholism: The CAGE questionnaire. JAMA: Journal of
syndrome: A summary. Alcohol Research & Health 25(3):159–167, 2001.
the American Medical Association, 252(14), 1905–1907, 1984. PMID: 6471323
PMID: 11810953
GARCIA BOY, R.; HENSELER, J.; MATTERN, R.; AND SKOPP, G. Determination
MAY, P.A.; GOSSAGE, J.P.; KALBERG, W.O.; ET AL. Prevalence and epidemio­
of morphine and 6­acetylmorphine in blood with use of dried blood spots.
logic characteristics of FASD from various research methods with an emphasis
Therapeutic Drug Monitoring 30(6):733–739, 2008. PMID: 18978519
on recent in­school studies. Developmental Disabilities Research Reviews
GUTHRIE, R., AND SUSI, A. A simple phenylalanine method for detecting 15(3):176–102, 2009. PMID: 19731384
phenylketonuria in large populations of newborn infants. Pediatrics 32:338– MCDADE, T.W.; WILLIAMS, S.; AND SNODGRASS, J.J. What a drop can do:
343, 1963. PMID: 14063511 Dried blood spots as a minimally invasive method for integrating biomarkers
HALMESMAKI, E.; ROINE, R.; AND SALASPURO, M. Gamma­glutamyltransferase, into population­based research. Demography 44(4):899–925, 2007. PMID:
aspartate and alanine aminotransferases and their ratio, mean cell volume and 18232218
urinary dolichol in pregnant alcohol abusers. British Journal of Obstetrics and MOORE, C.; JONES, J.; LEWIS, D.; AND BUCHI, K. Prevalence of fatty acid ethyl
Gynaecology 99(4):287–291, 1992. PMID: 1349817 esters in meconium specimens. Clinical Chemistry 49(1):133–136, 2003.
HANNUKSELA, M.L.; LIISANANTTI, M.K.; NISSINEN, A.E.; AND SAVOLAINEN, PMID: 12507969
M.J. Biochemical markers of alcoholism. Clinical Chemistry and Laboratory OSTREA, E.M., JR.; HERNANDEZ, J.D.; BIELAWSKI, D.M.; ET AL. Fatty acid
Medicine 45(8):953–961, 2007. PMID: 17579567 ethyl esters in meconium: Are they biomarkers of fetal alcohol exposure and
HARTMANN, S.; ARADOTTIR, S.; GRAF, M.; ET AL. Phosphatidylethanol as a effect? Alcoholism: Clinical and Experimental Research 30(7):1152–1159, 2006.
sensitive and specific biomarker: Comparison with gamma­glutamyl PMID: 16792562
transpeptidase, mean corpuscular volume and carbohydrate­deficient PETERSON, J.; KIRCHNER, H.L.; XUE, W.; ET AL. Fatty acid ethyl esters in
transferrin. Addiction Biology 12(1):81–84, 2007. PMID: 17407500 meconium are associated with poorer neurodevelopmental outcomes to two
HELANDER, A., AND BECK, O. Ethyl sulfate: A metabolite of ethanol in humans years of age. Journal of Pediatrics 152(6):788–792, 2008. PMID: 18492517
and a potential biomarker of acute alcohol intake. Journal of Analytical PRAGST, F., AND YEGLES, M. Determination of fatty acid ethyl esters (FAEE)
Toxicology 29(5):270–274, 2005. PMID: 16105250 and ethyl glucuronide (EtG) in hair: A promising way for retrospective detec­
HELANDER, A., AND DAHL, H. Urinary tract infection: A risk factor for false­ tion of alcohol abuse during pregnancy? Therapeutic Drug Monitoring
negative urinary ethyl glucuronide but not ethyl sulfate in the detection of 30(2):255–263, 2008. PMID: 18367991
recent alcohol consumption. Clinical Chemistry 51(9):1728–1730, 2005. ROBINSON, M.K.; MYRICK, J.E.; HENDERSON, L.O.; ET AL. Two­dimensional
PMID: 16120954 protein electrophoresis and multiple hypothesis testing to detect potential
serum protein biomarkers in children with fetal alcohol syndrome.
HENDERSON, L.O.; POWELL, M.K.; HANNON, W.H.; ET AL. An evaluation of
Electrophoresis 16(7):1176–1183, 1995. PMID: 7498162
the use of dried blood spots from newborn screening for monitoring the
prevalence of cocaine use among childbearing women. Biochemical and ROSENBERG, M.J.; WOLFF, C.R.; EL­EMAWY, A.; ET AL. Effects of moderate
Molecular Medicine 61(2):143–151, 1997. PMID: 9259979 drinking during pregnancy on placental gene expression. Alcohol
44(7–8):673–690, 2010. PMID: 20053520
JONES, K.L.; ROBINSON, L.K.; BAKHIREVA, L.N.; ET AL. Accuracy of the
diagnosis of physical features of fetal alcohol syndrome by pediatricians after RUSSELL, M. New assessment tools for drinking in pregnancy: T­ACE,
specialized training. Pediatrics 118(6):e1734–e1738, 2006. PMID: 17088402 TWEAK, and others. Alcohol Health and Research World, 18(1), 55–61, 1994.
JONES, K.L.; SMITH, D.W.; STREISSGUTH, A.P.; AND MYRIANTHOPOULOS, N.C. RUSSELL, M.; MARTIER, S.S.; SOKOL, R.J.; ET AL. Detecting risk drinking
Outcome in offspring of chronic alcoholic women. Lancet 1(7866):1076–1078, during pregnancy: A comparison of four screening questionnaires. American
1974. PMID: 4135246 Journal of Public Health 86(10):1435–1439, 1996. PMID: 9976514
KABLE, J.A.; COLES, C.D.; AND TADDEO, E. Socio­cognitive habilitation using SARKOLA, T.; ERIKSSON, C.J.; NIEMELA, O.; ET AL. Mean cell volume and
the math interactive learning experience program for alcohol­affected children. gamma­glutamyl transferase are superior to carbohydrate­deficient transferrin
Alcoholism: Clinical and Experimental Research 31(8):1425–1434, 2007. PMID: and hemoglobin­acetaldehyde adducts in the follow­up of pregnant women
17550365 with alcohol abuse. Acta Obstetricia et Gynecologica Scandinavica
79(5):359–366, 2000. PMID: 10830762
KIP, M.J.; SPIES, C.D.; NEUMANN, T.; ET AL. The usefulness of direct ethanol
metabolites in assessing alcohol intake in nonintoxicated male patients in an SOBELL, L., AND SOBELL, M. Timeline follow back: A technique for assessing
emergency room setting. Alcoholism: Clinical and Experimental Research self­reported alcohol consumption. In: Litten, R., and Allen, J., Eds. Measuring
32(7):1284–1291, 2008. PMID: 18540912 Alcohol Consumption. Totowa, NJ: Humana Press, 1992, pp. 41–72.

62 Alcohol Research & Health

 FOCUS ON: BIOMARKERS OF FETAL ALCOHOL EXPOSURE AND FETAL ALCOHOL EFFECTS

STEWARD, S.H.; LAW, T.L.; RANDALL, P.K.; AND NEWMAN, R. ethanol metabolites and self­reports. Alcoholism: Clinical and Experimental
Phosphatidylethanol and alcohol consumption in reproductive age women. Research, 32(9):1552–1557, 2008a. PMID: 18616663
Alcoholism: Clinical and Experimental Research 34(3):488–492, 2010. PMID:
20028353 WURST, F.M.; KELSO, E.; WEINMANN, W.; ET AL. Measurement of direct
ethanol metabolites suggests higher rate of alcohol use among pregnant women
STOLER, J.M.; HUNTINGTON, K.S.; PETERSON, C.M.; ET AL. The prenatal than found with the AUDIT: A pilot study in a population­based sample of
detection of significant alcohol exposure with maternal blood markers. Journal Swedish women. American Journal of Obstetrics and Gynecology198(4):407
of Pediatrics 133(3):346–352, 1998. PMID: 9738714 e1–e5, 2008b. PMID: 18221928
WURST, F.M.; ALEXSON, S.; WOLFERSDORF, M.; ET AL. Concentration of fatty WURST, F.M.; WIESBECK, G.A.; METZGER, J.W.; AND WEINMANN, W. On
acid ethyl esters in hair of alcoholics: Comparison to other biological state sensitivity, specificity, and the influence of various parameters on ethyl glu­
markers and self reported ethanol intake. Alcohol and Alcoholism 39(1):33–38, curonide levels in urine: Results from the WHO/ISBRA study. Alcoholism:
2004a. PMID: 14691072 Clinical and Experimental Research 28(8):1220–1228, 2004b. PMID:
WURST, F.M.; ALLING, C.; ARADOTTIR, S.; ET AL. Emerging biomarkers: New 15318121
directions and clinical applications. Alcoholism: Clinical and Experimental
YLIKORKALA, O.; STENMAN, U.H.; AND HALMESMAKI, E. Gamma­glutamyl
Research 29(3):465–473, 2005. PMID: 15770123
transferase and mean cell volume reveal maternal alcohol abuse and fetal alcohol
WURST, F.M.; DURSTELER­MACFARLAND, K.M.; AUWAERTER, V., ET AL. effects. American Journal of Obstetrics and Gynecology 157(2):344–348, 1987.
Assessment of alcohol use among methadone maintenance patients by direct PMID: 2887116

Now Available From NIAAA

NIAAA

The NIAAA Spectrum

Online Newsletter
The NIAAA Spectrum online newsletter features
easy­to­read summaries of the latest information from
the field of alcohol research.

Each issue includes engaging feature articles, short news

updates, interviews, and colorful graphics.

www.spectrum.niaaa.nih.gov. Also available in

To view the NIAAA Spectrum visit NIAAA’s Web site:

pdf format for easy printing.

U.S. Department
of Health and
Human Services

National Institutes
of Health

Vol. 34, No. 1, 2011 63

Copyright of Alcohol Research & Health is the property of National Institute on Alcohol Abuse & Alcoholism
and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright
holder's express written permission. However, users may print, download, or email articles for individual use.