Ethanol Origin in Postmortem Urine: an LC/MS Determination Office of Aerospace Medicine Washington, DC 20591 of Serotonin Metabolites

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Ethanol Origin in Postmortem Urine: an LC/MS Determination Office of Aerospace Medicine Washington, DC 20591 of Serotonin Metabolites DOT/FAA/AM-03/18 Ethanol Origin in Postmortem Urine: An LC/MS Determination Office of Aerospace Medicine Washington, DC 20591 of Serotonin Metabolites Robert D. Johnson Russell J. Lewis Dennis V. Canfield Civil Aerospace Medical Institute Federal Aviation Administration Oklahoma City, OK 73125 C. LeRoy Blank Department of Chemistry and Biochemistry University of Oklahoma Norman, OK 73019 November 2003 Final Report This document is available to the public through the National Technical Information Service, Springfield, Virginia 22161. NOTICE This document is disseminated under the sponsorship of the U.S. Department of Transportation in the interest of information exchange. The United States Government assumes no liability for the contents thereof. ������������������������������������ ��������������� ����������������������������� ���������������������������� ����������������� ����������������������� ���������������� ������� ������ �� ���������� ������ �� ����� ������������� ������������ �������������� ������������ �������������� ��������������������������������� ����������������������������������������������� ��������������������������������������������� ��������������������������������������� �������������������������������������� ��������������� ��������������������������� ������������������������������ ���������������������������������������� ��������������������������� � � ����������������������������� ������������������������� ��������������������������������������� �������������������������������� ����������������� ���������������������������� ����������������������� ��������������������� ����������������� ���������������������������� 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������ ��� �� ����������������������������������������������������������������������������������������������������������� ����������������������������������������������������������������������������������������������������������� ����������� ���� ������������� ������������� �� ����������������� ��� �������������� ���� ���� ������������� ������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������� ������������������������������������������������������������������������������������������������������� ������������ ����������� ������ ������� ���� ������� �������� ����������� ���� ����������������� ������ �������� ���� �������������� ������� ������ ��� ����������� ������ �������� ���������� ���� ����������� ������� ���� ���� ����� ��������������������������������������������������������������������������������������������������������� ����������� ��������������� ���������������������������� � � � �������������������������������������� ��������������������������������������������� � ���������������������������������������������� ������������������������������������������� �������������������������������������� ���������������������������� ��������������������������������������� �������������������������������������� ������������������ ����������� � ���������������������������8 ����������������� �������� ������������������������������������������ i ETHANOL ORIGIN IN POSTMORTEM URINE: AN LC/MS DETERMINATION OF SEROTONIN METABOLITES INTRODUCTION When working with forensic specimens, postmortem The Federal Aviation Administration’s (FAA’s) Civil ethanol formation is generally avoided by storage at ap- Aerospace Medical Institute (CAMI) is responsible, under propriate temperatures and the addition of preservatives Department of Transportation (DOT) orders 8020.11A such as sodium fluoride.13 These precautions, however, do and 1100.2C, to “conduct toxicologic analysis on speci- not rule out the possibility of ethanol formation prior to mens from … aircraft accident fatalities” and “investigate sample collection and preservation.14 Furthermore, studies … general aviation and air carrier accidents and search for have demonstrated that if a specimen’s storage tempera- biomedical and clinical causes of the accidents, includ- ture is not carefully controlled, the preservative sodium ing evidence of … chemical (use).” Therefore, following fluoride may become ineffective in blocking postmortem an aviation accident, samples are collected at autopsy formation of ethanol.15, 16 Although several methods are and sent to CAMI’s Toxicology and Accident Research available to help address the postmortem ethanol issue, Laboratory, where toxicological analysis is conducted on in many cases, the source of ethanol remains elusive. various postmortem fluids and tissues. Recently, exploitation of the metabolism of serotonin Ethanol analysis is one of the most common tests per- as a biological marker for ethanol consumption has begun formed on forensic specimens. The presence of ethanol to gain interest in the field of forensic science. Serotonin in fatal aircraft accident victims constitutes an important (5-hydroxytryptamine, 5-HT) is an indoleamine com- part of aircraft accident investigations and litigation. An monly found in nature.17 In humans, 5-HT is found ethanol positive also invites legal scrutiny as to its valid- throughout the body, with substantial concentrations ity. With the accuracy of today’s analytical techniques, found in the gastrointestinal tract and blood platelets.18 there should be no doubt concerning the quantitative The metabolism of 5-HT initially involves oxidative determination of ethanol found in a biological specimen. deamination to form the intermediate aldehyde, 5-hy- However, the origin of the ethanol found in a specimen is droxyindole-3-acetaldehyde (5-HIAL). This intermediate another, and highly pertinent, variable to consider when can undergo either oxidation or reduction as shown in interpreting ethanol results. Figure 1. Oxidation of the aldehyde, catalyzed by aldehyde The microbial formation of ethanol in postmortem dehydrogenase, leads to formation of 5-hydroxyindole-3- specimens is the most significant problem encountered acetic acid (5-HIAA).19 Reduction, catalyzed by aldehyde when evaluating ethanol results. The first report deal- reductase, leads to formation of 5-hydroxytryptophol (5- ing with postmortem formation of ethanol in corpses HTOL).20 Under normal circumstances 5-HIAA is the appeared in 1936.1 But while a postmortem increase in predominant metabolite of 5-HT.18 However, ethanol ethanol was observed, the precise source of the increase consumption has been shown to significantly alter the was unknown. Today, it is known that many different normal metabolism of serotonin. microbes may be responsible for postmortem formation of An increase in 5-HTOL concentration following etha- ethanol. Investigations have been performed to identify the nol consumption was first reported in 1967.21 Since that particular species of bacteria, yeast and/or fungi primarily time it has been clearly demonstrated that the consump- responsible for ethanol production and the mechanism tion of ethanol shifts serotonin metabolism to favor forma- by which ethanol is formed.2, 3, 4, 5 Candida albicans has tion of 5-HTOL and reduces the formation of 5-HIAA.20, been identified as the microbe most often responsible for 22, 23, 24 Two primary factors contribute to this increase in postmortem production of ethanol in humans.6, 7 This the 5-HTOL/5-HIAA ratio. The first factor involves the species of yeast is commonly found in humans in vivo. oxidation of ethanol into acetaldehyde. As acetaldehyde is Although located ubiquitously throughout the body, the produced, it is rapidly oxidized by aldehyde dehydroge- highest concentrations are typically in the mouth and on nase. The oxidation of acetaldehyde results in competitive the skin.8 Glucose is the most prevalent substrate in the inhibition of aldehyde dehydrogenase and prevents its human body used by these microbes to form ethanol.9 contribution to 5-HIAA formation.25 The second factor Other endogenous compounds can also be utilized as capable of increasing the 5-HTOL/5-HIAA ratio is the substrates including, but not limited to, lactate, mannitol, higher than normal concentrations of the reduced form galactose, maltose, sucrose and lactose. 9, 10, 11, 12 of nicotinamide adenine dinucleotide (NADH), resulting 1 ��������� �� �� � � � ������������������ �� � � � � ������������������� ����������������������� ������� �� �� �� � �� � � � � ������� ������� �������������������������������������� as a byproduct from the oxidation of both
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