Clinical Chemistry 47: 126-127, 2001;
(Clinical Chemistry. 2001;47:126-127.)
© 2001 American Association for Clinical Chemistry, Inc.
Effect of Specimen Anticoagulant and Storage on Measurement of Serum and Plasma Fatty Acid Ethyl Ester Concentrations.
Raneem O. Salem1,
Joanne E. Cluette-Brown1,
Ali Hasaba1 and
Michael Laposata1,a
1
Division of Laboratory Medicine, Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114
a Address correspondence to this author at: Room 235, Gray Bldg., Massachusetts General Hospital, Boston, MA 02114; fax 617-726-3256,
mlaposata{at}partners.org
Fatty acid ethyl esters (FAEEs) are cytotoxic nonoxidative
metabolites of ethanol that are produced by esterification of alcohol
and fatty acids (1)(2)(3). After ethanol intake, FAEEs are
found mainly in the liver, pancreas, heart, and brain, which are the
major organs adversely affected by ethanol intake
(4)(5). FAEEs are detectable in the blood for up
to 24 h after consumption of ethanol to at least 600 mg/L
(6)(7)(8), making the presence of FAEEs in blood a useful
marker for ethanol intake. This investigation presents the results of
studies on the effects of collection tube, storage time, and storage
temperature on FAEE concentrations in blood, with the goal of creating
a reliable clinical assay for serum and plasma FAEE quantification.
Four volunteers participated in this study. Each subject was given a
weight-adjusted amount of 100-proof vodka mixed with fruit juice in a
1:3 ratio (8). The vodka-juice beverage was divided into
nine equal aliquots, which were administered every 10 min over a 90-min
time period. After the last aliquot was drunk, blood samples were
collected in four different 5-mL Vacutainer Tubes from each subject to
assess the influence of the collection tube on the blood FAEE
concentration. The tubes included a red-top tube with no
anticoagulant, a purple-top tube with 150 g/L EDTA, a
green-top tube with 72 USP units of heparin, and a blue-top tube with
32 g/L sodium citrate. The tubes were kept on ice and processed within
30 min. FAEEs were then isolated and quantified as described below. To
study the effect of storage time and temperature on FAEE concentrations
in whole blood, 40 mL of blood was collected from each of the same four
subjects in red-top vacuum tubes (no anticoagulant). The 40 mL of blood
was divided into two equal aliquots that were incubated at 25 and
37 °C for 0, 2, 4, 6, 24, and 48 h. FAEEs were then isolated
from the samples. To accomplish this, serum or plasma was first
separated from the blood cells by centrifugation at 3420g at
4 °C for 20 min. FAEEs were then isolated using an acetone-hexane
(2:8 by volume) extraction followed by solid-phase extraction
(9), and measured using gas chromatography–mass
spectrometry as described previously (10). The
within-assay CV for the entire method was 17%. The CVs
for the individual stages were as follows: FAEE isolation via liquid
extraction, 
13%; solid-phase extraction, 9.7%; and
quantification by gas chromatography–mass spectrometry, 2.2%.
The effect of the collection tube on the amounts of FAEE in plasma or
serum is shown in Table 1
. For the four volunteers, the mean concentrations of FAEE in
the sodium citrate tube, the heparin tube, and the anticoagulant-free
tube were similar. However, the EDTA vacuum tube showed a lower FAEE
concentration than the others. We speculate that the extensive
chelation of calcium by EDTA contributed to this observation in some
way. The mean FAEE concentrations in plasma in the sodium citrate and
heparin tubes, and in serum in the anticoagulant-free tubes, were more
than twice the mean plasma FAEE concentration in the EDTA tubes. The
variability was least for sodium citrate tubes. This higher precision
suggests the use of 32 g/L sodium citrate as the preferred
anticoagulant.
Storage time and temperature also affected FAEE concentrations
in serum. The FAEE concentration increased over time when incubated as
whole blood at 25 and 37 °C. As shown in Fig. 1
, there was a rapid increase in FAEE concentrations at 37 °C
from 2273 ± 507 nmol/L (mean ± SE) at time 0 to 4826
± 245 nmol/L (mean ± SE) 48 h after blood collection. At
25 °C, there was no change in the FAEE concentration after up to
4 h of incubation: 1712 ± 385 nmol/L at time 0 and 1766
± 486 nmol/L at 4 h (mean ± SE for both). However, there
was an increase to 4750 ± 875 nmol/L (mean ± SE) with
storage at 25 °C for 48 h. There was less variability, in
general, for the samples stored at 37 °C, but this higher precision
is not likely to have a biological basis.
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Figure 1. Effect of time on serum FAEE concentrations at 25 and
37 °C.
Blood samples were collected from four healthy subjects and then
incubated for different times before collection of serum. The sera were
separated and processed for FAEE measurements as described in the text.
Each point represents the mean ± SE (bars); n
= 4/time point. The differences in mean values are not statistically
significant for each time point at 25 vs 37 °C. However, the values
at 25 °C at time 0 are different from those at 48 h
(P = 0.01), and they approach statistical
significance from those at 24 h (P = 0.07).
The values at 37 °C at time 0 are different from those at 24 and
48 h (P = 0.05 and P =
0.04, respectively).
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The in vitro formation of FAEEs, which is fastest at 37 °C,
is most likely the result of enzyme-mediated FAEE synthesis using the
residual ethanol in the specimen collection tubes. Ethanol remains in
the blood for up to 6 h. White bloods cells, platelets, and to a
lesser extent, red blood cells, have been shown to have FAEE synthase
activity (11). A previously published study from our
laboratory with plasma and serum samples from individuals in a clinical
trial involving ethanol ingestion showed no changes in FAEE
concentration after 2 days of storage at -4 or -80 °C
(12). Taken together, the results of the current storage
experiments and our previously reported findings indicate a need for
removal of the plasma or serum from the cells within 4 h at room
temperature, with freezing of plasma or serum at -4 °C or
-80 °C if longer periods of time are required before analysis of
the specimen. In addition, samples should not be collected for FAEE
analysis in EDTA-containing vacuum tubes.
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