ABSTRACT
Serotonin is a specific in vitro substrate for human UDP-glucuronosyltransferase (UGT) 1A6. In this study, the contribution of UGT1A6 to the glucuronidation of endogenous structural analogs of serotonin, including 5-hydroxytryptophol, N-acetylserotonin, and 6-hydroxymelatonin, was evaluated using available recombinant human UGT isoforms, human liver microsomes, and liver microsomes from animals that do not express functional UGT1A6 (Gunn rats and cats). Only UGT1A6 and UGT1A9 were found to glucuronidate 5-hydroxytryptophol at a concentration of 2 mM, although the glucuronidation rate with UGT1A6 was over 10 times that of UGT1A9. K(m) values for human liver microsomes (156, 141, and 134 microM) were most similar to that of expressed UGT1A6 (135 microM) but vastly different from that of UGT1A9 (3674 microM). 5-Hydroxytryptophol glucuronidation by human liver microsomes (n = 54) correlated well with serotonin glucuronidation (R(s) = 0.83) and UGT1A6 protein content (R(s) = 0.85). 5-Hydroxytryptophol also competitively inhibited serotonin glucuronidation by human liver microsomes (K(i) = 291 microM) and UGT1A6 (K(i) = 200 microM). N-acetylserotonin was glucuronidated most extensively by UGT1A6, although UGT1A9 and UGT1A10 showed moderate catalysis. 6-Hydroxymelatonin was glucuronidated largely by UGT1A9 and UGT1A10 but not at all by UGT1A6. Gunn rat liver glucuronidation rates for serotonin, 5-hydroxytryptophol, N-acetylserotonin, and 6-hydroxymelatonin were 11, 5, 32, and 3%, respectively, of that of normal rat liver. Cat liver microsomes did not glucuronidate serotonin, whereas relatively low activities were observed for the other indole substrates. In conclusion, these results indicate that human UGT1A6 plays a predominant role in the glucuronidation of 5-hydroxytryptophol and N-acetylserotonin, whereas 6-hydroxymelatonin is not a substrate for this enzyme.
Subject(s)
Glucuronosyltransferase/metabolism , Hydroxytryptophol/metabolism , Serotonin/analogs & derivatives , Serotonin/metabolism , Adolescent , Adult , Aged , Animals , Cats , Child , Child, Preschool , Drug Evaluation, Preclinical/methods , Female , Humans , Hydroxytryptophol/chemistry , Male , Middle Aged , Rats , Rats, Gunn , Rats, Wistar , Substrate Specificity/physiologyABSTRACT
HYPOTHESIS: The unknown prevalence of alcohol use and misuse among aviation pilots, crewmembers and associated support personnel call for continuous improvement of methods for detecting recent alcohol use. Early detection is essential to proper treatment and prevention of potentially catastrophic mishaps. Urinary 5-hydroxytryptophol (5HTOL), a serotonin (5HT) metabolite, has shown promise in the clinical setting as a noninvasive marker of recent alcohol consumption. METHODS: The urinary 5HTOL concentrations of 11 male and female subjects were followed for approximately 24 h following dosing with ethanol 0.6 g.kg-1. Concentrations were reported as a ratio of 5HTOL to 5-hydroxy-indoleacetic acid (5HIAA), 5HTOL/5HIAA (pmol/nmol), to compensate for urinary dilution and elevated 5HTOL levels due to dietary intake. Data from one male subject was excluded after he admitted to continued alcohol consumption subsequent to dosing and missing several urine samples. RESULTS: 5HTOL/5HIAA ratios remained above the 15 pmol/nmol cutoff for recent alcohol use for approximately 11-16 h in all except one subject. Calculations based on body weight and administered alcohol dose suggest that measurable blood alcohol levels would exist for only 5-7 h post ingestion. CONCLUSION: This study confirmed the extended elevation of 5HTOL/5HIAA ratios observed in earlier studies, even at the relatively low alcohol dose used herein. 5HTOL appears to be a marker for acute alcohol consumption worthy of further investigation by military and civilian authorities. Potential aviation applications of 5HTOL include validation of measurable blood alcohol concentrations, investigation of poor performance due to hangover effects, and as a forensic toxicology tool in aircraft accident investigations to distinguish between actual alcohol ingestion and post-mortem alcohol synthesis.
Subject(s)
Alcoholic Intoxication/urine , Aviation , Hydroxytryptophol/urine , Substance Abuse Detection/methods , Acute Disease , Adult , Alcoholic Intoxication/metabolism , Biomarkers/urine , Body Weight , Female , Humans , Hydroxytryptophol/chemistry , Male , Middle Aged , Military Personnel , Occupational Health , Reproducibility of Results , Time FactorsABSTRACT
A quantitative gas chromatographic-mass spectometric method using a deuterated analogue as internal standard was developed for the analysis of 5-hydroxytryptophol in cerebrospinal fluid. The analytical procedure involves the addition of the internal standard and 5-hydroxyindole to the cerebrospinal fluid followed by extraction into chloroform and derivatization with pentafluoropropionic anhydride. 5-Hydroxytryptophol levels in the CSF of male alcoholics and a control group were examined. During ethanol intoxication the mean level, 10.4 +/- 4.4 pmoles/ml, was significantly (P < 0.001) higher than in the controls, 3.31 +/- 0.94 pmoles/ml. The following morning the mean had decreased (P < 0.01) to 4.46 +/- 1.81 pmoles/ml. The level after 8 days was 4.70 +/- 2.47 pmoles/ml, which is lower than during intoxication (P <0.01). The levels found during the recovery from ethanol intoxication was not statistically different from the levels in the control group. These results indicate that serotonin metabolism in the central nervous system is affected by ethanol consumption.
