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Articles |
1
Division of Laboratory Medicine, Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114.
2
Medical Policlinic, University Hospital, Ramistrasse
100, CH-8091, Zurich, Switzerland.
a Address correspondence to this author at: Room 235, Gray Bldg., Massachusetts General Hospital, Boston, MA 02114. Fax 617-726-3256; e-mail mlaposata{at}partners.org
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Methods: For some studies, subject were recruited volunteers; in others, residual blood samples after ethanol quantification were used. FAEEs were isolated by solid-phase extraction and quantified by gas chromatography–mass spectrometry.
Results: For weight-adjusted amounts of ethanol intake, FAEE
concentrations were twofold greater for men than women
(P 
0.05). Accounting for triglycerides improved the
correlation between blood ethanol concentrations and FAEE
concentrations for both men (from r = 0.640 to
r = 0.874) and women (from r =
0.619 to r = 0.673). FAEE concentrations did not
change when samples were stored at or below 4 °C, but doubled when
stored at room temperature for 
24 h. The type of alcoholic beverage
and rate of consumption did not affect FAEE concentrations.
Conclusion: These studies advance plasma and serum FAEE measurements closer to implementation as a clinical test for ethanol intake.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The present study analyzes how gender, plasma or serum triglyceride
concentrations, specimen storage conditions, the type of alcoholic
beverage, and the rate of ethanol ingestion affect serum or plasma FAEE
concentrations. In our studies with FAEEs purified from plasma or serum
by solid-phase extraction (6) and quantified by gas
chromatography–mass spectrometry (GC-MS) (7), we determined
that (a) men have higher serum FAEE concentrations than
women for weight-adjusted amounts of ethanol ingestion (P
0.05); (b) FAEE/triglyceride correlates better with blood
ethanol than FAEE alone, improving the correlation coefficient for men
from r = 0.640 to r = 0.874 and for
women from r = 0.619 to r = 0.673;
(c) storage of plasma samples at room temperature for longer
than 1 day can lead to artifactual formation of FAEEs; (d)
the plasma FAEE concentration is not affected by the type of alcoholic
beverage consumed; and (e) the rate of ethanol ingestion
does not influence the FAEE concentrations in serum. Taken together,
these findings provide essential information to appropriately interpret
the clinical significance of an FAEE concentration in the blood of an
individual presenting for assessment of ethanol intake.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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impact of triglycerides on faee concentrations
For studies assessing the influence of triglyceride concentrations
on FAEE concentrations, 85 residual plasma or serum samples (from 64
men and 21 women) were obtained from the toxicology laboratory at the
Massachusetts General Hospital. Samples were positive for blood ethanol
and, in essentially every case, positive for FAEEs as well. The samples
were stored at 4 °C until analysis. The triglycerides were
quantified using a Hitachi 917 automated chemistry analyzer (Boehringer
Mannheim Diagnostics).
changes in faee concentrations under different storage
conditions
To evaluate the effect of time and temperature on sample storage,
frozen residual plasma from individuals in the study investigating type
of alcoholic beverage on plasma FAEE concentrations (described below)
was used. Samples were thawed, pooled, divided into five 0.5-mL
aliquots, and stored under conditions that differed in duration and
temperature. Samples were analyzed after 24 and 48 h of storage at
-80 °C, 4 °C, and room temperature (25 °C). The control
sample was thawed, processed, and analyzed immediately.
impact of type of alcoholic beverage on faee synthesis
The effect of alcoholic beverage type on FAEE concentrations was
analyzed in eight healthy volunteers (seven women, one man) who were
social drinkers. Volunteers were admitted to the General Clinical
Research Center (GCRC) on the day of the study. Subjects were required
to complete a brief food survey form, recalling the past 24 h of
dietary intake, and a drinking history survey (Khavari Alcohol Test)
(9). The researchers recorded each subject’s weight and
height. Volunteers were required to abstain from any ethanol 5 days
before and 72 h after the study. The eight participants were
equally divided into two groups, four for beer ingestion and four for
vodka intake. Alcohol type was chosen at random.
The amount of ethanol that each person received was based on his or her body weight and was intended to increase the blood alcohol concentration to at least 21.7 mmol/L (100 mg/dL). The total ethanol dose was divided into nine aliquots and consumed over a 90-min period. Five blood samples were drawn from each subject. One vial (10 mL) of blood was collected before ingestion of ethanol to establish a baseline; the second and third samples were drawn 15 and 30 min after drinking was completed (105 and 120 min after the onset of drinking, respectively); and the fourth and fifth samples were collected 24 and 72 h after the beginning of ethanol ingestion. Food was provided during the study by staff dieticians immediately after the 120-min time point.
Subjects remained in the GCRC until they could be safely discharged, as determined by the blood alcohol concentration and subjective sense of sobriety. Subjects later returned to the GCRC for the 24- and 72-h blood collections.
At each time point, blood was collected in 10-mL Vacutainer Tubes containing 0.117 mL of 150 g/L potassium EDTA solution, and the tubes were placed immediately on ice. Tubes were centrifuged at 3420g for 20 min at 4 °C, and the plasma was isolated. An aliquot of plasma was reserved for ethanol analysis, and another aliquot was used for subsequent FAEE isolation and quantification.
Plasma ethanol concentrations were determined by GC. Briefly, the plasma sample was mixed with an internal standard, 1-propanol, and a 1-µL sample was injected into a Hewlett Packard 5890 GC equipped with a 5% Carbowax 20 M 60/80 Carbopack B column. The oven program was isothermal at 100 °C, and the ethanol peak was identified by comparison with a known standard.
influence of rate of ethanol consumption on faee synthesis
In studies to determine whether the rate of ethanol intake affects
FAEE pharmacokinetics in the blood, six young healthy male Caucasian
volunteers were studied. The mean ± SE age was 24.2 ± 0.7 years; the
mean ± SE body weight was 73.4 ± 1.7 kg the mean ± SE body mass
index was 22.3 ± 0.3 kg/m2; and the mean ± SE lean body
mass was 85.8% ± 1.4% total body weight. All were nonsmokers, and
their habitual ethanol intake, based on a representative 1-week dietary
recall, was 46 ± 22 g/week. All men had negative clinical
histories, and serum aminotransferase, alkaline phosphatase, bilirubin,
and albumin concentrations within the appropriate reference intervals,
and none had any serological evidence of viral hepatitis.
The subjects fasted for at least 12 h before the study. At the initiation of the study, subjects were placed on a bed in a semirecumbent position, and an indwelling catheter (kept open with normal saline) was inserted into an antecubital vein for blood sampling. After 1 h of baseline measurements (three blood drawings spaced exactly 20 min apart), the subjects were given 31.9 ± 0.6 g of ethanol (0.43 ± 0.004 g/kg body weight) to ingest in a 2-min oral bolus. The subjects ingested the ethanol as a 10% volume solution (diluted with tap water only), containing 2 drops of a concentrated flavored extract used for baking. Venous blood was then sampled at 20-min intervals over a total period of 5 h. Blood ethanol concentrations were determined by GC, using head space injection. Serum was isolated from blood by centrifugation at 2800g for 10 min at 4 °C and frozen immediately at -70 °C for subsequent measurement of FAEEs. The rates of FAEE increase and decrease in the blood from this study were compared with those reported previously by our laboratory from a study in which subjects consumed ethanol to intoxication over 90 min (8).
faee isolation and quantification
In the above experiments, serum or plasma samples were thawed, and
a 1-mL aliquot was removed for analysis. There was no statistically
significant difference in FAEE concentrations quantified from serum vs
plasma (data not shown). Ethyl heptadecanoate (E17:0; 1 nmol) was added
as an internal standard. Using a method we have described previously
(4), samples were extracted with acetone-hexane (2:8, by
volume) and dried under nitrogen vapors to an ~300 µL volume, and
FAEEs were isolated by solid-phase extraction using Bond Elut-LRC
aminopropyl columns. A complete analysis of FAEE isolation by
solid-phase extraction was performed in our laboratory, and the results
of this analysis have been published previously (6). Columns
were prewashed with dichloromethane followed by hexane; the sample was
then applied to the column and eluted by successive washes of hexane
and dichloromethane. Combined eluates were concentrated, and FAEEs were
then quantified by GC-MS using a Hewlett Packard 5890 Series II gas
chromatograph equipped with a Supelcowax SP-2330 capillary column
coupled to an HP-5971 mass spectrometer. The injector and detector were
maintained at 260 °C and 280 °C, respectively. The oven program
was initially maintained at 130 °C for 2 min, then ramped to
160 °C at 5 °C/min, ramped again at 2 °C/min to 180 °C,
held for 7 min, and finally, ramped to 230 °C at 15 °C/min and
maintained for 2 min. Carrier gas flow rate was maintained at a
constant 0.8 mL/min throughout. Single-ion monitoring was performed,
quantifying appropriate base ions for individual FAEE species [i.e.,
ions m/z 67, 88, and 101 for ethyl palmitate (E16:0), ethyl
heptadecanoate (E17:0), ethyl stearate (E18:0), ethyl oleate (E18:1),
and ethyl linoleate (E18:2); and ions m/z 79 and 91 for
ethyl arachidonate (E20:4), ethyl eicosapentaenoate (E20:5), and ethyl
docosahexaenoate (E22:6)]. FAEE quantification was determined by
interpolation of the slope generated from individually prepared
calibration curves comparing areas of varying concentrations of
E16:0–E22:6 to fixed concentrations of the internal standard (E17:0).
Mass relationships were obtained for each FAEE using its individual
calibration curve. Total FAEE mass was determined by the addition of
masses of individual FAEEs (E16:0–E22:6).
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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. All of the subjects drank a weight-adjusted amount of ethanol
as a mixture of vodka and fruit juice to increase their peak blood
ethanol to similar concentrations >21.7 mmol/L (100 mg/dL). As shown
by the nearly overlapping lines for ethanol and FAEEs in Fig. 1
in the
region where ethanol concentrations were <21.7 mmol/L (100 mg/dL), the
presence of FAEEs does not require that a subject has a blood ethanol
concentration that produces intoxication. Fig. 1A
illustrates that the
men in the study had a twofold higher serum FAEE concentration than the
women (P 0.05). Fig. 1B
shows the time courses for blood
ethanol concentration in the men and the women. The women showed a
modestly higher blood ethanol concentration, but only at time points
after the peak blood ethanol concentration (P 0.05) was
attained, which was the same for men and women. Thus, the higher serum
FAEE concentrations in the men were not explained by correspondingly
higher ethanol concentrations.
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impact of triglycerides on faee concentrations
The impact of serum or plasma triglyceride concentrations on FAEE
formation is shown in Fig. 2
. The results for blood ethanol vs FAEE concentration for men
and women are shown in Fig. 2A
and Fig. 2B
, respectively, and the
correlation between blood alcohol concentration and the ratio of the
FAEE concentration to triglyceride concentration for men and women is
shown in Fig. 2C
and Fig. 2D
, respectively. There was a similar degree
of correlation between ethanol and FAEEs for men (r =
0.640) and women (r = 0.619). In Fig. 2C
(for men) and
Fig. 2D
(for women), the units for the y-axis are nmol
FAEE/mg triglyceride. (The volume units were canceled in the
development of the y-axis parameter.) The inclusion of the
triglyceride concentration in the denominator greatly increased the
correlation coefficient between blood ethanol and FAEEs for men, from
0.640 to 0.874, and had a modest effect for women, increasing the value
from 0.619 to 0.673.
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changes in faee concentrations under different storage conditions
Because the currently available method for FAEE quantification
involves isolation of the lipid by solid-phase extraction and
quantification by GC-MS, samples for FAEE quantification are likely to
be sent to a limited number of laboratories for analysis. It is for
this reason that we determined the impact of temperature and time of
storage on FAEE concentrations. The sample was pooled serum or plasma
frozen within 30 min of collection from multiple patients in a clinical
trial involving ethanol ingestion, with blood alcohol concentrations in
the individual samples ranging from 21.7 to 32.6 mmol/L (100 to 150
mg/dL). The FAEEs in the pooled baseline sample were isolated
immediately after thawing and then quantified (mean ± SE,
2317 ± 458 nmol/L; n = 5). Storage of this same sample at
-80 °C for up to 2 days had no impact on the FAEE concentrations
[mean ± SE, 2812 ± 184 nmol/L (n = 5) at 24 h,
and 2940 ± 288 nmol/L (n = 5) at 48 h; Fig. 3
]. In addition, storage of the sample at 4 °C for up to 2
days did not alter the FAEE concentration [mean ± SE, 2741
± 63 nmol/L (n = 5) at 24 h, and 3014 ± 234 nmol/L
(n = 5) at 48 h]. Importantly, however, when samples were stored
at room temperature, the FAEEs increased significantly over control
concentrations [mean ± SE, 6378 ± 274 nmol/L (n = 5)
at 24 h, and 7091 ± 160 nmol/L (n = 5) at 48 h]. This
artifactual production of FAEEs may account for the limited number of
high outliers in Fig. 2
because some of the samples used for this study
may have been held at room temperature for extended periods in the
clinical laboratory before we received the specimens. There was little
difference in FAEE concentrations between 24 and 48 h at room
temperature, suggesting that most of the artifactual synthesis occurred
within the first day of storage at room temperature.
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impact of type of alcoholic beverage on faee synthesis
We also performed a controlled study in our clinical research unit
in which individuals drank either beer or vodka in weight-adjusted
intoxicating amounts of alcohol to determine whether the type of
alcoholic beverage influences the FAEE concentrations in the blood.
Fig. 4
shows that individuals drinking beer and vodka had the same
peak plasma FAEE concentrations. The peak occurred in the range of
105–120 min, and at both of these times, the subjects ingesting beer
and those drinking vodka had very similar plasma FAEE concentrations.
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influence of rate of ethanol consumption on faee synthesis
We measured the FAEE concentration in samples from a study in
which individuals were given alcohol to ingest in a 2-min oral bolus.
We previously reported the results of a study in which subjects
ingested ethanol over a 90-min period (8). We compared the
pharmacokinetic data from this investigation with those from the 2-min
bolus ingestion study. As shown in Fig. 5
, all six subjects showed a significant overlap over the time
course for blood ethanol and FAEE concentrations. These
pharmacokinetics were essentially identical to those we reported in our
earlier study in which individuals ingested ethanol over 90 min. The
time for attaining peak ethanol concentration was predictably shorter
in this study because the time of ingestion was only 2-min. In both
studies taken together, it appears that the blood ethanol peaks
~20–40 min before the FAEE concentrations. This finding is
consistent with the fact that FAEEs are metabolites of ethanol.
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There was no interference in FAEE measurements by hemoglobin 0.12
mmol/L (200 mg/dL), bilirubin 0.34 mmol/L (20 mg/dL), or cholesterol
10.3 mmol/L (400 mg/dL).
The CV for replicate analysis of samples for FAEE was <5% for specimens processed with solid-phase extraction and then quantified by GC-MS and <3% for samples quantified by GC-MS without prior extraction.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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We found in one series of experiments that there was a gender difference in serum FAEE concentrations. For a weight-adjusted amount of ethanol ingested, we observed that men had peak serum FAEE concentrations approximately twofold higher than the peak values for women. The presence of FAEEs in the serum or plasma does not necessarily indicate alcohol intake to intoxication. In a previously published clinical trial, it was shown that FAEE concentrations parallel ethanol concentrations and that FAEEs are detectable when ethanol is well below concentrations associated with intoxication (8).
It has long been known that women attain higher blood ethanol concentrations than men for equal amounts of ethanol ingested (10). In addition, women carry a higher risk for developing cirrhosis than their male counterparts (11)(12). One of the explanations that has been offered to explain this observation is that women have decreased first-pass metabolism of ethanol in the stomach, thereby permitting the transport of larger amounts of ethanol from the gastrointestinal tract into the blood (10). There are several possible explanations as to why women may have lower peak FAEE concentrations after ethanol intake. It could in part be because the women in this study were all premenopausal, with relatively high HDL-cholesterol concentrations and correspondingly low triglyceride concentrations. From the experiment concerning the effects of triglycerides on FAEE concentrations, it was found that by incorporating triglyceride concentrations into FAEE concentrations, the correlation between blood ethanol and FAEE did not improve as much for women as it did in men. Alternatively, it could be that in women there is a decreased activity of enzymes required for the synthesis of FAEEs or an increased activity of enzymes involved in the degradation of FAEEs. With in vitro (1) and in vivo (4) evidence that FAEEs are toxic, this could represent an attempt to reduce the ethanol-mediated toxicity because women already suffer greater toxic effects from ethanol ingestion than men (11)(12).
It has been reported that lipoprotein lipase has FAEE synthetic
activity (13)(14). This enzyme also degrades
triglycerides with release of free fatty acids from the glycerol
backbone. Another enzyme that degrades triglyceride, carboxylester
lipase, has also been shown to have FAEE synthetic activity
(15). Because these two enzymes that degrade triglycerides
have the ability to synthesize FAEEs, it has been speculated that the
rate-limiting step in FAEE synthesis is a hydrolytic event that
liberates a fatty acid from the triglyceride in the presence of ethanol
in cell water. The result could be a nonenzymatic esterification of
fatty acid and ethanol to create FAEEs. The data in Fig. 2
provide
support for this hypothesis. Fig. 2
shows that by including the
triglyceride concentration as the denominator on the y-axis,
the r value increases, especially in male subjects.
This study also gives an initial indication of specimen handling requirements for FAEE quantification. There was no FAEE degradation even when the samples were kept at refrigerator temperatures for up to 2 days. Thus, it should be quite possible to collect samples, isolate the serum or plasma, freeze or refrigerate it, and send it for analysis. There was artifactual FAEE formation in vitro when samples were maintained at room temperature for at least 1 day. This is most likely because ethanol is still present in the sample and there is some FAEE synthetic activity in the plasma sample, possibly from residual intact or disrupted white blood cells or platelets, which are known to have FAEE synthase activity (16).
It was anticipated that because FAEEs are metabolites of ethanol, the type of alcoholic beverage would not influence FAEE concentrations. This report demonstrates that this is true, at least for a comparison between beer and vodka, but most likely for all alcoholic beverages. The evidence indicates that ethanol, and not other ingredients in the alcoholic beverage, is the major component that determines the amount of FAEE synthesized.
Finally, the rate of ethanol ingestion did not impact FAEE concentrations when subjects who drank a 2-min bolus were compared with those that consumed alcohol over a 90-min period. This is consistent with the conclusion that FAEE concentrations are dependent on the amount of ethanol consumed and not the rate of consumption.
The FAEE concentrations in the individual studies in this report varied
widely for several reasons. First, as shown in Fig. 5
, there is
significant interindividual variation in FAEE production with identical
amounts of ethanol intake. Second, the samples for Fig. 2
were stored
for variable amounts of time, mostly at 4 °C, before analysis for
FAEE, whereas samples were processed immediately after collection for
the clinical studies shown in Figs. 1
, 4
, and 5
. Third, the amount of
ethanol ingested by individuals whose serum or plasma was also assayed
for FAEEs was different in the various clinical studies. In addition,
the amount of ethanol ingested in the experiment shown in Fig. 2
was
not intended to be a controlled variable, and therefore differed widely
between individuals.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The following articles in journals at HighWire Press have cited this article:
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F. M. Wurst, S. Alexson, M. Wolfersdorf, G. Bechtel, S. Forster, C. Alling, S. Aradottir, K. Jachau, P. Huber, J. P. Allen, et al. CONCENTRATION OF FATTY ACID ETHYL ESTERS IN HAIR OF ALCOHOLICS: COMPARISON TO OTHER BIOLOGICAL STATE MARKERS AND SELF REPORTED-ETHANOL INTAKE Alcohol Alcohol., January 1, 2004; 39(1): 33 - 38. [Abstract] [Full Text] [PDF]
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 W. C. Zybko, J. E. Cluette-Brown, and M. Laposata Improved Sensitivity and Reduced Sample Size in Serum Fatty Acid Ethyl Ester Analysis Clin. Chem., June 1, 2001; 47(6): 1120 - 1121. [Full Text] [PDF]
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R. O. Salem, M. A. Refaai, J. E. Cluette-Brown, J. W. Russo, and M. Laposata Fatty Acid Ethyl Esters in Liver and Adipose Tissues as Postmortem Markers for Ethanol Intake Clin. Chem., April 1, 2001; 47(4): 722 - 725. [Abstract] [Full Text] [PDF]
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R. O. Salem, J. E. Cluette-Brown, A. Hasaba, and M. Laposata Effect of Specimen Anticoagulant and Storage on Measurement of Serum and Plasma Fatty Acid Ethyl Ester Concentrations. Clin. Chem., January 1, 2001; 47(1): 126 - 127. [Full Text] [PDF]
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