Activities of human alcohol dehydrogenases in the metabolic pathways of ethanol and serotonin.
Alcohols and aldehydes in the metabolic pathways of ethanol and serotonin are substrates for alcohol dehydrogenases (ADH) of class I and II. In addition to the reversible alcohol oxidation/aldehyde reduction, these enzymes catalyse aldehyde oxidation. Class-I gammagamma ADH catalyses the dismutation of both acetaldehyde and 5-hydroxyindole-3-acetaldehyde (5-HIAL) into their corresponding alcohols and carboxylic acids. The turnover of acetaldehyde dismutation is high (kcat = 180 min-1) but saturation is reached first at high concentrations (Km = 30 mm) while dismutation of 5-HIAL is saturated at lower concentrations and is thereby more efficient (Km = 150 microm; kcat = 40 min-1). In a system where NAD+ is regenerated, the oxidation of 5-hydroxytryptophol to 5-hydroxyindole-3-acetic acid proceeds with concentration levels of the intermediary 5-HIAL expected for a two-step oxidation. Butanal and 5-HIAL oxidation is also observed for class-I ADH in the presence of NADH. The class-II enzyme is less efficient in aldehyde oxidation, and the ethanol-oxidation activity of this enzyme is competitively inhibited by acetate (Ki = 12 mm) and 5-hydroxyindole-3-acetic acid (Ki = 2 mm). Reduction of 5-HIAL is efficiently catalysed by class-I gammagamma ADH (kcat = 400 min-1; Km = 33 microm) in the presence of NADH. This indicates that the increased 5-hydroxytryptophol/5-hydroxyindole-3-acetic acid ratio observed after ethanol intake may be due to the increased NADH/NAD+ ratio on the class-I ADH. (+info)
Storage of specimens at 4 degrees C or addition of sodium fluoride (1%) prevents formation of ethanol in urine inoculated with Candida albicans.
The microbial synthesis of ethanol was investigated in urine specimens containing 0.5% or 1.0% (w/v) glucose and inoculated with the yeast Candida albicans (100 cfu/mL). Aliquots (10 mL) of urine were dispensed into plastic tubes containing enough sodium fluoride to give final concentrations of 0.1%, 0.25%, 0.5%, 0.75%, 1%, and 2% (w/v), and C. albicans was added. The tubes were tightly stoppered and allowed to stand either at room temperature (22 degrees C) or in a refrigerator (4 degrees C) for up to 34 days before concentrations of ethanol were determined by headspace gas chromatography. Urine samples stored at 22 degrees C without sodium fluoride produced 0.25 g/L ethanol after two days, and the concentration increased to 2.10 g/L and 4.50 g/L after eight days for specimens containing 0.5% (w/v) and 1% (w/v) glucose, respectively. The ratio of the serotonin metabolites 5-hydroxytryptophol/5-hydroxyindoleacetic acid (5HTOL/5HIAA) in urine remained within the reference range (< 15 pmol/nmol) despite high concentrations of ethanol being produced. Urine samples kept at 4 degrees C did not produce any ethanol (< 0.01 g/L) even without sodium fluoride present as a preservative. The production of ethanol by C. albicans was stopped completely by adding 1% or 2% (w/v) sodium fluoride but not by concentrations of 0.75% (w/v) or less. The microbial synthesis of ethanol in urine samples initially stored at room temperature without sodium fluoride was slowed down considerably by moving them into a refrigerator at 4 degrees C. In conclusion, the production of ethanol in urine by C. albicans can be prevented by storage of samples in a refrigerator at 4 degrees C or by adding sodium fluoride > or = 1% (w/v). Measuring the ratio of 5HTOL/5HIAA can help to distinguish postsampling production of ethanol from metabolism and excretion processes. (+info)
Urinary ethyl glucuronide and 5-hydroxytryptophol levels during repeated ethanol ingestion in healthy human subjects.
AIMS: This study investigated the stability and reproducibility of urinary ethyl glucuronide (EtG) and the 5-hydroxytryptophol (5-HTOL) to 5-hydroxyindole-3-acetic acid (5-HIAA) ratio, both of which are used as biochemical markers of recent alcohol consumption, after single and multiple oral doses of ethanol in healthy human subjects. METHODS: Nine females aged 19-27 years drank ethanol (8%, w/v, in juice) or placebo (juice) in random order. The intervention consisted of 0.4 g/kg (22-28 g) of ethanol or placebo twice daily (in the morning and evening) during 8 consecutive days, starting in the evening on day 1. Spot urine samples of the first morning void were collected during the 8-day drinking period and for another 3 days (days 9-11) with no intake of ethanol or placebo. Ethanol, EtG, 5-HTOL and 5-HIAA were determined in the urine samples by headspace GC, LC-MS, GC-MS and HPLC, respectively. RESULTS: The individual results during the drinking period were highly variable, both within and between subjects, ranging from 0-7.3 mmol/l for ethanol, 1.4-71.0 mg/l for EtG, 0.1-4.5 mg/mmol for the EtG/creatinine ratio, and 2-109 nmol/ micro mol for 5-HTOL/5-HIAA. The placebo group consistently showed negative values for ethanol (< 0.1 mmol/l) and 5-HTOL/5-HIAA (< 15 nmol/ micro mol), but two samples were positive for EtG (> 0.1 mg/l). In the morning of day 9 (i.e. approximately 14-15 h after the last dose), ethanol was no longer measurable in urine and the 5-HTOL/5-HIAA ratio had returned to below the reference value, but detectable levels of EtG (11.3 +/- 6.0 mg/l, mean +/- SD) and the EtG/creatinine ratio (1.0 +/- 0.3 mg/mmol) were found in all samples. CONCLUSIONS: The results confirm the increase in urinary EtG and 5-HTOL levels during acute ethanol intake, although the individual values were highly variable both within and between subjects. No significant accumulation of either compound occurred upon multiple-dose administration of 0.8 g/kg (44-57 g) ethanol per day for approximately 1 week. (+info)
Evaluation of 5-hydroxytryptophol and other endogenous serotonin (5-hydroxytryptamine) analogs as substrates for UDP-glucuronosyltransferase 1A6.
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. (+info)
Comparison of serum fatty acid ethyl esters and urinary 5-hydroxytryptophol as biochemical markers of recent ethanol consumption.
AIMS: To examine the effects of an acute dose of ethanol on serum fatty acid ethyl esters (FAEEs) concentration and urinary 5-hydroxytryptophol (5-HTOL)/5-hydroxyindole-3-acetic acid (5-HIAA) ratio. METHODS: Sixteen (14 male, 2 female) heavy alcohol drinkers were tested in a single, 2-day long session. Six participants received 1.5 g/l of ethanol/l of body water (approximately 0.75 g/kg of body weight, low dose group: LD) and 10 participants received 2.0 g/l of ethanol ( approximately 1.0 g/kg of body weight, high dose group: HD) in four divided doses every 20 min. Blood, urine, and breath samples were collected repeatedly over 36 h following the ingestion of ethanol and were analyzed for the presence of FAEE, 5-HTOL/5-HIAA, and ethanol, respectively. Serum gamma-glutamyltransferase (GGT), a marker of chronic ethanol use, was also included. RESULTS: The breath ethanol level peaked approximately 1 h after the last dose, at 95 and 120 mg/dl for the LD and HD groups, respectively. The mean ratio of urinary 5-HTOL/5-HIAA was significantly elevated 5 and 9 h after ethanol administration, but returned to baseline 13 h after ethanol administration. This ratio was twice as high for the HD group compared with the LD group. Serum levels of FAEEs were significantly elevated at 5 h, but not 13 h after ethanol administration. There were no time-dependent changes in serum GGT levels. CONCLUSIONS: Measuring the levels of FAEE and 5-HTOL/5-HIAA ratio provides a convenient method to detect recent, particularly binge-type, ethanol use, but these measures may have limited applicability in detecting ethanol use in traditional clinical trial settings. (+info)
Biomarkers to disclose recent intake of alcohol: potential of 5-hydroxytryptophol glucuronide testing using new direct UPLC-tandem MS and ELISA methods.
AIMS: This study compared two new methods for direct determination of 5-hydroxytryptophol glucuronide (GTOL) in urine, a biomarker for detection of recent alcohol consumption. METHODS: Urine samples were collected from ten alcoholic patients during recovery from intoxication. A direct injection ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for measurement of the urinary GTOL to 5-hydroxyindoleacetic acid (5-HIAA) ratio, and an ELISA assay for direct measurement of GTOL, were used. Comparison was made with the urinary ethanol and ethyl glucuronide (EtG) concentrations. RESULTS: The breath ethanol concentration on admission ranged between 1.0-3.1 g/l. The UPLC-MS/MS method showed a median detection time of 39 h for an elevated urinary GTOL/5-HIAA ratio, while EtG was detected for a median of 65 h. Determination of GTOL by the ELISA assay showed 87% sensitivity in detecting positive samples at a 44% specificity, as compared with the UPLC-MS/MS method. CONCLUSIONS: The lower sensitivity of the urinary GTOL/5-HIAA ratio compared with EtG for recent drinking may be clinically useful, in cases where the EtG test provides an unwanted high sensitivity for intake of only small amounts of alcohol or unintentional ethanol exposure. (+info)
Loss of ethanol from vitreous humor in drowning death.
Two separate cases of drowning with extended periods underwater (2 and 4 weeks) are reported. The postmortem ethanol concentrations were 260 and 280 in central blood, 50 and 80 in vitreous, and 330 and 320 in urine (mg/100 mL) for cases 1 and 2, respectively. Determination of the urine 5-hydroxytryptophol/5-hydroxyindole-3-acetic acid ratios produced results of 713 and 41 pmol/nmol, respectively. The serotonin metabolite ratios support the explanation of diffusion of ethanol from the vitreous fluid into the surrounding water, rather than postmortem production of ethanol in blood, as the primary reason for the blood-vitreous ethanol differences. (+info)
Comparison between the urinary alcohol markers EtG, EtS, and GTOL/5-HIAA in a controlled drinking experiment.