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Technical Briefs |
1 Division of Laboratory Medicine, Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114
aaddress correspondence to this author at: Room 235, Gray Building, Massachusetts General Hospital, Boston, MA 02114; fax 617-726-3256, e-mail mlaposata{at}partners.org
Fatty acid ethyl esters (FAEEs) are nonoxidative esterification products of ethanol metabolism found after ingestion of ethanol (1). FAEEs were proposed as toxic mediators of ethanol-induced cell injury in a study of postmortem analysis of tissues and organs (2). Subsequently, direct damage to human hepatoblastoma cells by FAEEs was reported (3), as well as in vivo pancreatic injury in rats induced by FAEE infusion (4). FAEEs in serum are both short- and long-term markers of ethanol intake (5)(6). Because serum FAEE concentrations are useful indicators of ethanol intake, test sensitivity and sample volume have become increasingly important targets for improvement.
The first use of gas chromatograph–mass spectrophotometry (GC-MS) detection systems for serum FAEE concentrations identified ethyl 16:0, ethyl 16:1, ethyl 18:0, ethyl 18:1, ethyl 18:2, and ethyl 20:4 (1). In these experiments, the FAEEs were isolated from an organic phase by thin-layer chromatography; individual species of FAEEs were then identified and quantified by GC-MS. The current procedure for isolation and quantification of FAEE using solid-phase extraction and GC-MS is based on a method developed by Bernhardt et al. (7). Improvements have increased the number of identifiable FAEEs in the current technique to include ethyl 14:0, ethyl 18:3n-3, ethyl 20:3n-6, ethyl 20:5n-3, and ethyl 22:6n-3. Preanalytical variables that affect FAEE quantification have been described in a recent report by Soderberg et al. (8).
The standard procedure for FAEE analysis uses 1 mL of serum, with the
addition of 2 mL of acetone and 50 µL of internal standard. A
chromatogram of the analysis of FAEEs from a subject with a blood
alcohol concentration of 3210 mg/L (69.8 mmol/L) is depicted in Fig. 1
. The upper tracing was obtained using a 1-mL serum sample and
extraction with 2 mL of acetone. The lower tracing was obtained using a
1-mL serum sample and extraction with 4 mL of acetone. These samples
were dried and reconstituted to the same volume and both were injected
into the GC-MS system using 1.5 µL of sample. This analysis shows
that 2 mL of acetone does not completely extract FAEEs, because there
was greater recovery of FAEEs using an extraction with 4 mL of acetone.
The ratios of abundance of various FAEEs relative to the serum and
acetone volumes used are shown in Table 1
. The abundance of the various FAEEs can be compared for the
experiment using 1 mL of serum and 2 mL of acetone vs the experiment
using 1 mL of serum and 4 mL of acetone. These are represented as 1
mL/2 mL relative to 1 mL/4 mL (Ratio 1 in Table 1
). When we used twice
the amount of acetone, 3.35- to 8.57-fold more FAEEs were extracted
into the acetone phase. The correlation between the amount obtained
using 1 mL of serum and 2 mL of acetone vs 1 mL of serum and 4 mL of
acetone was 0.980. After correction for recovery using the internal
standard, the 1 mL/2 mL ratio for total FAEEs was 29.36 nmol/mL, and
the 1 mL/4 mL ratio was 22.66 nmol/mL. The calculation of total FAEEs
is based on the area of abundance of the internal standard relative to
the area of abundance of the individual FAEEs. The total amount of
FAEEs is somewhat lower for the higher acetone volumes, most likely
because more internal standard was recovered (7.27-fold; see FAEE ethyl
17:0 in Table 1
) relative to the individual FAEEs in the serum (3.35-
to 5.63-fold, except for FAEE ethyl 18:0, for which the recovery was
8.57-fold higher) as shown in Ratio 1 of Table 1
.
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Rather than using the same amount of serum and more acetone, to try to
gain the benefit of a smaller sample size we reduced the amount of
serum, but held the acetone volume at 2 mL. The increased extraction of
FAEEs by a decreased serum-to-acetone ratio more than offset the
smaller serum sample volume. A second trial using serum with a blood
alcohol concentration of 3610 mg/L (78.5 mmol/L) was performed using 1
mL and 0.5 mL of serum and 2 mL or more of acetone. The ratios of
abundance of the individual FAEEs of the 0.5-mL samples are shown in
Table 1
(Ratios 2 and 3). Even when the sample size was reduced by 50%
(Ratio 2), the amount of FAEEs extracted was 2.11- to 5.43-fold greater
than when the standard procedure was performed using 1 mL of sample and
2 mL of acetone. When 0.5 mL of serum and 4 mL of acetone were used,
the ratio (Ratio 3) for each FAEE species increased even more. This
indicates that among the ratios tested, a 1:8 serum-to-acetone ratio
was the most efficient extraction for the FAEEs. Therefore, in the
solvent-extraction step, 0.5 mL of serum and 4 mL of acetone are
recommended.
To evaluate smaller serum sample sizes of 0.25 and 0.1 mL, a third
series of experiments was performed. The samples in these studies had a
blood alcohol concentration of 850 mg/L (18.5 mmol/L), which we
purposely selected because it approximates the minimum blood alcohol
concentration at which a person is considered legally intoxicated. The
serum-to-acetone volume ratios used were 1:8 and 1:20. The results are
shown in Table 1
(Ratios 4 and 5). The use of a 0.25-mL serum sample
and 2 mL of acetone (a ratio of 1:8) showed a lower FAEE abundance than
in Ratio 3 with the same sample-to-acetone ratio of 1:8. This was
presumably attributable to the reduced sample size. This suggests the
need for a minimum sample volume of 0.5 mL. The abundance ratio was <1
for Ratio 4. At a ratio of 1:20 (Ratio 5), the abundance of each FAEE
was approximately the same amount as in Ratio 4, showing that a 0.10-mL
sample size is also not recommended for analysis. The total FAEE
concentration in this sample was 4.14 µmol/L, and the highest
individual abundance of an FAEE in the sample was 799 740 for FAEE
ethyl 16:0. Although the FAEEs were in less abundance in the 0.1-mL
sample, they were still clearly detectable in a sample with a blood
ethanol concentration at the lower legal limit for intoxication. Thus
the ideal sample volume to maximize FAEE detection appears to be 0.5
mL. However, even at 850 mg/L (18.5 mmol/L) blood ethanol, a sample
size of 0.10 mL or possibly lower could be used for FAEE analysis.
In summary, the sensitivity for FAEE detection is improved by use of a smaller sample volume of 0.5 mL and a serum-to-acetone ratio of 1:8.
References
The following articles in journals at HighWire Press have cited this article:
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  D. M. Yoerger, C. A. Best, B. M. McQuillan, G. E. Supple, J. L. Guererro, J. E. Cluette-Brown, A. Hasaba, M. H. Picard, J. R. Stone, and M. Laposata Rapid Fatty Acid Ethyl Ester Synthesis by Porcine Myocardium Upon Ethanol Infusion into the Left Anterior Descending Coronary Artery Am. J. Pathol., May 1, 2006; 168(5): 1435 - 1442. [Abstract] [Full Text] [PDF]
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 M. A. Refaai, P. N. Nguyen, T. S. Steffensen, R. J. Evans, J. E. Cluette-Brown, and M. Laposata Liver and Adipose Tissue Fatty Acid Ethyl Esters Obtained at Autopsy Are Postmortem Markers for Premortem Ethanol Intake Clin. Chem., January 1, 2002; 48(1): 77 - 83. [Abstract] [Full Text] [PDF]
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