Verification of microbial ethanol formation in humans by examination of postmortem indoleamine metabolites.

Abstract

Toxicological examination of fatal aviation accident victims routinely includes analysis of ethanol levels. Two distinct questions immediately arise when considering such analyses. The first is, can we stop ethanol production immediately upon receipt of specimens. Previous research suggests such ethanol formation may be substantially diminished by addition of sodium fluoride to the specimen in question. This work, however, was only accomplished in antemortem blood and urine matrices. The current report clearly demonstrates the blockade of ethanol formation in postmortem tissue specimens by sodium fluoride at both 4° C and 25° C. The second question is if ethanol exists at the time of specimen receipt, can we differentiate between antemortem ingestion and postmortem microbial formation. Development of a single analytical approach to determine concentrations of 5-hydroxytryptophol (5-HTOL) and 5-hydroxyindole-3-acetic acid (5-HIAA), two well-known metabolites of serotonin, has provided a convenient, rapid and reliable solution to this problem. Antemortem ethanol leads to an elevation in the 5-HTOL/5-HIAA ratio for 11--19 hrs after acute ingestion. The liquid/liquid extracts of postmortem urine samples were derivatized and subjected to LC/MS for the simultaneous quantification of these two analytes, yielding detection limits of 0.10 ng/mL for each. Examination of the 5-HTOL/5-HIAA ratio was undertaken for 44 urine samples previously known to be antemortem ethanol-positive or antemortem ethanol-negative. A 5-HTOL/5-HIAA ratio of 15 pmol/nmol was shown to distinguish between these groups. Thus, we recommend the employment of this cutoff value to confirm or deny recent antemortem ethanol ingestion in postmortem urine samples.