Comparison between the urinary alcohol markers EtG, EtS, and GTOL/5-HIAA in a controlled drinking experiment.

AIM: Urinary ethyl glucuronide (EtG), ethyl sulfate (EtS), and the ratio between 5-hydroxytryptophol-glucuronide and 5-hydroxyindole-3-acetic acid (GTOL/5-HIAA) are all suggested as biomarkers for recent alcohol ingestion with longer detection times than measurement of ethanol itself. The aim of this controlled study was to compare the sensitivities and detection times of EtG, EtS, and GTOL/5-HIAA, after a single ingestion of ethanol. METHODS: 0.5 g ethanol/kg body weight was ingested by 10 healthy male volunteers in a fasted state. Ethanol, EtG, EtS, and GTOL/HIAA levels were measured in urine samples collected during a 45-50 h period. The total amount of ethanol excreted as EtG and EtS was also determined. RESULTS: Urinary EtG, EtS, and GTOL/5-HIAA showed 100% sensitivity as biomarkers for recent drinking. Compared to ethanol testing in urine, the detection times for GTOL/5-HIAA were approximately 5 h longer and for EtG and EtS approximately 25 h longer. The maximum EtG concentrations were higher than for EtS in all subjects, and a higher fraction of the ethanol dose was excreted as EtG (median 0.019%) compared with EtS (median 0.011%). CONCLUSIONS: This study is the first controlled experiment comparing the time-courses for ethanol, EtG, EtS, and GTOL/5-HIAA in urine. In cases where surveillance of alcohol relapse is needed, measurements of urinary EtG and EtS are sensitive and specific alternatives to ethanol testing. The GTOL/5-HIAA ratio is equally sensitive but with a much shorter window of detection.

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.

Alcohol biomarker analysis: simultaneous determination of 5-hydroxytryptophol glucuronide and 5-hydroxyindoleacetic acid by direct injection of urine using ultra-performance liquid chromatography-tandem mass spectrometry.

A direct ultra-performance liquid chromatography-tandem mass spectrometry method (UPLC-MS/MS) for simultaneous measurement of urinary 5-hydroxytryptophol glucuronide (GTOL) and 5-hydroxyindoleacetic acid (5-HIAA) was developed. The GTOL/5-HIAA ratio is used as an alcohol biomarker with clinical and forensic applications. The method involved dilution of the urine sample with deuterated analogues (internal standards), reversed-phase chromatography with gradient elution, electrospray ionisation and monitoring of two product ions per analyte in selected reaction monitoring mode. The measuring ranges were 6.7-10 000 nmol/l for GTOL and 0.07-100 micromol/l for 5-HIAA. The intra- and inter-assay imprecision, expressed as the coefficient of variation, was below 7%. Influence from ion suppression was noted for both compounds but was compensated for by the use of co-eluting internal standards. The accuracy in analytical recovery of added substance to urine samples was 96 and 98%, respectively, for GTOL and 5-HIAA. Method comparison with GC-MS for GTOL in 25 authentic patient samples confirmed the accuracy of the method with a median ratio between methods (GC-MS to UPLC-MS/MS) of 1.14 (r(2) = 0.975). The difference is explained by the fact that the GC-MS method also measures unconjugated 5-hydroxytryptophol naturally present in urine. The comparison with data for 5-HIAA obtained by an HPLC method demonstrated a median ratio of 1.05 between the methods. The UPLC-MS/MS method was capable of measuring endogenous GTOL and 5-HIAA levels in urine, which agreed with the literature data. In conclusion, a fully validated and robust direct method for the routine measurement of urinary GTOL and 5-HIAA was developed.

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.

Problem drinking in relation to treatment outcome among opiate addicts in methadone maintenance treatment.

This study analyzed indicators of alcohol-related problems in opiate addicts before, during, and after leaving methadone maintenance treatment (MMT), in relation to illicit drug use and retention in treatment. The study was based on 204 patients, admitted to MMT for the first time between 1 January 1995 and 31 July 2000, and followed until 31 December 2000. Three measures were used to indicate alcohol use and alcohol-related problems; records of hospital care with an alcohol-related diagnosis, any treatment with alcohol-sensitizing drugs (disulfiram or calcium carbimide) during MMT, and results of the 5-hydroxytryptophol to 5-hydroxyindoleacetic acid ratio (5HTOL/5HIAA) in urine, a sensitive biomarker for recent drinking. Use of illicit drugs was determined by routine urine drug testing. About one third of the patients (n = 69) had a lifetime prevalence of hospital treatment for an alcohol-related diagnosis, 45 of whom had been hospitalized (mean 4.2 stays) prior to the start of MMT. There was a significant association (p<0.05) between the number of alcohol-related diagnoses prior to treatment and a positive 5HTOL/5HIAA test during MMT. The alcohol indicators first became positive on average 1.6 years after admission to treatment, compared with after about 4 months for illicit drugs. Use of cannabis or benzodiazepines was significantly associated with alcohol use. Female methadone patients with indications of alcohol-related problems relapsed more often into illicit drug use than did women without such indications (3.9 vs. 2.5 relapse periods/year; p<0.005), whereas no significant association was found for men. The results of the present study indicate that drinking problems among patients undergoing MMT is associated with an increased risk of relapse into illicit drug use and with discharge from treatment. Concurrent treatment of alcohol-related problems, including systematic monitoring of alcohol use, therefore should be recommended to reduce the risk for relapse into illicit drug use and improve overall treatment outcome in MMT.

Utilizing the urinary 5-HTOL/5-HIAA ratio to determine ethanol origin in civil aviation accident victims.

Specimens from fatal aviation accident victims are submitted to the FAA Civil Aerospace Medical Institute for toxicological analysis. During toxicological evaluations, ethanol analysis is performed on all cases. Care must be taken when interpreting a positive ethanol result due to the potential for postmortem ethanol formation. Several indicators of postmortem ethanol formation exist; however, none are completely reliable. The consumption of ethanol has been shown to alter the concentration of two major serotonin metabolites, 5-hydroxytryptophol (5-HTOL) and 5-hydroxyindole-3-acetic acid (5-HIAA). While the 5-HTOL/5-HIAA ratio is normally very low, previous studies using living subjects have demonstrated that the urinary 5-HTOL/5-HIAA ratio is significantly elevated for 11-19 h after acute ethanol ingestion. Recently, our laboratory developed and validated an analytical method for the simultaneous determination of both 5-HTOL and 5-HIAA in forensic urine samples using a simple liquid/liquid extraction and LC/MS/MS and LC/MS/MS/MS. In this previous work a 15 pmol/nmol serotonin metabolite ratio cutoff was established in postmortem urine, below which it could be conclusively determined that no recent antemortem ethanol consumption had occurred. In the current study this newly validated analytical method was applied to five ethanol-positive aviation fatalities where the origin of the ethanol present could not previously be conclusively determined. In four of the five cases examined the detected ethanol was demonstrated to be present due to postmortem microbial formation, and not consumption, even though some indication of ethanol consumption may have been present.

Detection of recent ethanol intake with new markers: comparison of fatty acid ethyl esters in serum and of ethyl glucuronide and the ratio of 5-hydroxytryptophol to 5-hydroxyindole acetic acid in urine.

BACKGROUND: At present, recent ethanol consumption can be routinely detected with certainty only by direct measurement of ethanol concentration in blood or urine. Because ethanol is rapidly eliminated from the circulation, however, the time span for this detection is in the range of hours. Several new markers have been proposed to extend the detection interval, but their characteristics have not yet justified their use in routine clinical practice. We therefore investigated three new markers and compared their kinetics and sensitivities: (1) fatty acid ethyl esters (FAEEs) in serum, (2) ethyl glucuronide (EtG) in urine, and (3) the ratio of 5-hydroxytryptophol to 5-hydroxyindole acetic acid (5-HTOL/5-HIAA) in urine. METHODS: Seventeen healthy men participated in a drinking experiment. Blood and urine samples were collected twice daily on three consecutive days and once daily on days 4 and 5. Ethanol concentration was determined by gas chromatography, FAEE levels, by gas chromatography with mass spectrometry, EtG concentration, by liquid chromatography-tandem mass spectrometry, and 5-HTOL/5-HIAA ratio, by high-performance liquid chromatography. RESULTS: The peak serum ethanol concentrations of the subjects ranged from 5.4 to 44.7 mmol/liter (mean +/- SD, 30.1 +/- 9.1 mmol/liter). In the case of the serum ethanol determination, 100% sensitivity was reached only immediately after the end of the drinking experiment, and in the case of FAEE levels and 5-HTOL/5-HIAA ratio, it tested for 6.7 hr after the end of the ethanol intake. Thereafter, these latter parameters declined until 15.3 hr (FAEEs) and 29.4 hr (5-HTOL/5-HIAA), subsequently remaining in a stable range until 78.5 hr without further decrease. In contrast, EtG concentration showed 100% sensitivity until 39.3 hr and thereafter decreased, falling to below the limit of quantification of 0.1 mg/liter at 102.5 hr. CONCLUSION: After moderate drinking, EtG in the urine proved to be a superior marker of recent ethanol consumption in healthy subjects. This is because EtG is a direct ethanol metabolite, it occurs in the urine only when ethanol has been consumed, and its sensitivity remains at the level of 100% for 39.3 hr.

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.

Accurate assignment of ethanol origin in postmortem urine: liquid chromatographic-mass spectrometric determination of serotonin metabolites.

Toxicological examination of fatal aviation accident victims routinely includes analysis of ethanol levels. However, distinguishing between antemortem ingestion and postmortem microbial formation complicates all positive ethanol results. 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 h after acute ingestion. The liquid-liquid extracts of postmortem urine samples were subjected to liquid chromatography-mass spectrometry (LC-MS) for the simultaneous quantitation of these two analytes, yielding detection limits of 0.1 ng/ml for each. Examination of the 5-HTOL/5-HIAA ratio was undertaken for 44 urine samples known to be antemortem ethanol-positive or antemortem ethanol-negative. Recent ethanol ingestion was conveniently and accurately separated using a 5-HTOL/5-HIAA ratio of 15 pmol/nmol, a value previously suggested using human volunteers. All 21 ethanol-negative postmortem samples were below this cutoff, while all 23 ethanol-positive postmortem samples were above this cutoff. Thus, we recommend the employment of this cutoff value, established using this straightforward LC-MS procedure, to confirm or deny recent antemortem ethanol ingestion in postmortem urine samples.

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.

[5-HTOL--a new biochemical alcohol marker with forensic applications]. / 5-HTOL--ny biokemisk alkoholmarkör med rättsmedicinska tillämpningar.

The concentration of ethanol in blood and urine provides important evidence in criminal and civil litigation when alcohol-related crimes are investigated (e.g., drunk driving). The determination of ethanol in body fluids is a routine procedure at forensic chemistry and toxicology laboratories and when gas chromatographic methods are used accurate and precise results are obtained. However, the risk for artifactual formation of ethanol, especially in postmortem specimens, always needs to be considered. The ratio of 5-HTOL/5-HIAA in urine provides a useful way to distinguish between ethanol produced after death, or generated in vitro after sampling, from the ethanol consumed. This article describes the application of the 5-HTOL/5-HIAA ratio as a biochemical marker for acute alcohol intake in various forensic situations. Examples include suspected drunk drivers, rape victims, and medico-legal autopsies where forensic ethanol analysis is requested.

Urinary excretion patterns of 5-hydroxyindole-3-acetic acid and 5-hydroxytryptophol in various animal species: implications for studies on serotonin metabolism and turnover rate.

The concentrations of the serotonin metabolites 5-hydroxyindole-3-acetic acid (5HIAA) and 5-hydroxytryptophol (5HTOL) were determined in spot urine samples of 12 mammalian and one fish species (cat, cow, dog, ferret, golden hamster, guinea pig, horse, monkey, mouse, rabbit, rainbow trout, rat, sheep) and compared with human data. The highest urinary concentrations of 5HTOL were found in the Sprague-Dawley rat (mean 9.5 micromol/L) and NMRI mouse (8.2 micromol/L), and the lowest in rainbow trout, cynomolgus macaque, and human urine (approximately 0.1 micromol/L). The highest 5HIAA concentrations were found in hamster (89.3 micromol/L) and mouse (85.2 micromol/L), and the lowest in rainbow trout, horse and sheep (range 2.0-3.7 micromol/L). Several species showed 5HIAA concentrations similar to that normally observed in human urine (approximately 5-40 micromol/L). This study demonstrated wide inter- and intra-species variations in the urinary concentrations of 5HIAA and 5HTOL, both separately and in the sum of concentrations. The 5HTOL/5HIAA ratio, which is used as an easily accessible index of the relative importance of the reductive and oxidative pathways for serotonin metabolism, also varied considerably between different species. This observation confirms that the much higher urinary 5HTOL/5HIAA ratio in rats (mean 0.35) compared with humans (< 0.01) is due to a higher baseline formation of 5HTOL in the rat. The monkey, ferret, hamster, and rabbit most closely resembled humans in this respect, and at least the two latter species appear to be more suitable than rats as animal models for studying serotonin metabolism and turnover rate, and the metabolic interaction with ethanol.

Laboratory tests for acute alcohol consumption: results of the WHO/ISBRA Study on State and Trait Markers of Alcohol Use and Dependence.

BACKGROUND: The WHO/ISBRA Study on State and Trait Markers of Alcohol Use and Dependence aimed partly to evaluate the overall performance and cross-national validity of traditional and new biological markers of alcohol use and abuse. This article focused on the sensitivity and specificity of ethanol and methanol concentrations in plasma, and the 5-hydroxytryptophol (5HTOL) to 5-hydroxyindole-3-acetic acid (5HIAA) ratio in urine, as laboratory tests to identify acute alcohol consumption. Comparison was made with self-reported drinking levels. METHODS: Subjects were recruited in Australia, Brazil, Canada, Finland, and Japan. They were interviewed thoroughly about their alcohol consumption habits, by using the standardized WHO/ISBRA Interview Schedule, and were classified into four categories: nondrinkers, light/moderate drinkers, heavy drinkers (> or =210 g ethanol/week for men, and > or =140 g/week for women), or patients who were receiving treatment for alcohol dependence. Ethanol and methanol determinations in plasma were carried out by headspace gas chromatography. Urinary concentrations of 5HTOL and 5HIAA were determined by using gas chromatography-mass spectrometry and high-performance liquid chromatography, respectively. RESULTS: The baseline levels (in nondrinkers) for methanol and the 5HTOL/5HIAA ratio did not differ markedly between the five populations, except for a considerably higher, but probably artifactual, methanol level in the Finnish plasma samples. Moreover, there were no apparent age or sex differences. The urinary 5HTOL/5HIAA ratio was the most, and ethanol the least, sensitive indicator of recent alcohol consumption, and this was true for the different drinking categories as well as for the five study populations. The highest frequency of elevated test results was observed among those classified as heavy drinkers (e.g., 38% were positive for 5HTOL/5HIAA). However, elevated values also were obtained in nondrinkers and in drinking subjects who denied any intake of alcohol within 2 days before the interview and blood/urine sampling, which suggested a low accuracy of self-reports of alcohol consumption in certain individuals. CONCLUSIONS: The present investigation demonstrated that plasma ethanol and methanol and urinary 5HTOL/5HIAA provide useful exclusion markers for any study of biological parameters that are affected by previous acute ethanol intoxication. The major advantage of methanol and 5HTOL/5HIAA over ethanol is that they can detect recent alcohol consumption even several hours after the ethanol is no longer measurable. The results suggest that the cutoff limits to be used for these markers are not dependent on the country or population to be studied.

Dietary serotonin and alcohol combined may provoke adverse physiological symptoms due to 5-hydroxytryptophol.

The urinary excretion products of serotonin (5-hydroxytryptamine, 5HT) are 5-hydroxyindole-3-acetic acid (5HIAA) and 5-hydroxytryptophol (5HTOL), and the ratio of 5HTOL to 5HIAA is normally very low (< 0.01 ) in man. Intake of foods rich in 5HT (high amounts in banana, pineapple, and walnuts) induces a general increase in the output of 5HT metabolites, without affecting the 5HTOL/5HIAA ratio. In contrast, during metabolism of ethanol there is a shift in the catabolic pattern of 5HT, and the formation of 5HTOL increases appreciably at the expense of 5HIAA. Accordingly, the urinary 5HTOL/ 5HIAA ratio increases and does not recover to baseline levels until several hours after ethanol has been cleared from the body. When 10 healthy subjects ingested a moderate dose of ethanol (0.5 g/kg), the urinary 5HTOL/SHIAA ratio was increased approximately 70-fold on average at 4 h after intake. When the same amount of ethanol was ingested together with 3 bananas (approximately 10 mg 5HT), this ratio was increased approximately 100-fold at 4 h and still significantly higher than baseline levels at 24 h. Starting at 3-4 h after the combined intake of ethanol and banana, 7 subjects experienced one or more unpleasant symptoms (diarrhea, headache, and fatigue) which are associated with the 5HT system. The events were transient but typically lasted for several hours, and the duration correlated with the time period during which 5HTOL levels were raised. Intake of ethanol and banana separately produced much lower increases in 5HTOL output and caused no corresponding effects. This observation indicate that dietary 5HT intake together with even a moderate dose of ethanol can provoke unpleasant physiological symptoms. The symptoms may be attributed to the high concentration of 5HTOL.

Volitional ethanol consumption affects overall serotonin metabolism in Syrian golden hamsters (Mesocricetus auratus).

Methods were established for the determination of serotonin (5-HT)(1) metabolites 5-hydroxyindole-3-acetic acid (5-HIAA) and 5-hydroxytryptophol (5-HTOL) in the urine of Syrian golden hamsters (Mesocricetus auratus) and used to study the effect of volitional ethanol consumption on overall 5-HT metabolism in this ethanol-preferring rodent. The basal levels of 5-HIAA and 5-HTOL in 24-h urine of ethanol-naive hamsters were 300 +/- 101 and 4.96 +/- 1. 06 nmol (n = 8), respectively. Given free choice between water and a 15% ethanol solution, these hamsters chose to consume increasing amounts of ethanol. The increase was accompanied by a concomitant decrease in urine 5-HIAA and increase in urine 5-HTOL, indicating that volitional ethanol intake diverted part of the 5-HT metabolic flux from an oxidative into a reductive pathway. In a separate experiment, the amounts of ethanol consumed by and blood ethanol concentrations attained in ethanol-drinking golden hamsters were determined at 5 different time intervals between 6 PM and 7 AM when most feeding activities occurred. Except in the first hour after lights were turned off, ethanol was consumed at a relatively even pace throughout the night (2-3 g/kg/3 h) and blood ethanol levels were maintained at the low mM range which rarely exceeded 2 mM. These results suggest that the biochemical pathway that catalyzes 5-HT metabolism is extremely sensitive to ethanol and can play an important role in mediating the reported clinically beneficial action of a low concentration of ethanol during alcohol detoxification.

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.

Comparison of urinary 5-hydroxytryptophol, breath ethanol, and self-report for detection of recent alcohol use during outpatient treatment: a study on methadone patients.

This study compared urinary 5-hydroxytryptophol (5HTOL) with breath-ethanol testing as objective ways to disclose recent drinking by outpatients attending a methadone maintenance treatment clinic. Information about quantity and frequency of alcohol use was obtained by confidential self-reports. Random screening was performed on Mondays-Fridays in connection with routine clinic visits for methadone dosing. An observed urine sample for monitoring of illicit drug use and determination of 5HTOL, expressed as a ratio to 5-hydroxyindole-3-acetic acid (5HIAA), was obtained from 202 patients (59 women and 143 men), 16 of whom refused to complete the self-report and/or do a breath-ethanol test. Patients taking disulfiram or calcium carbimide for alcohol detoxification were excluded. Among the 177 subjects remaining, 47 (26.6%) reported intake of any alcohol on the previous day (range, 10-230 g ethanol; median, 40). Only four of those could be identified by a positive breath-test, while 17 showed a urinary 5HTOL/5HIAA ratio above the cutoff limit. Their alcohol consumption (median, 60 g) was significantly higher compared with those showing ratios within the reference interval (median, 35 g). The sensitivity of 5HTOL/5HIAA testing for detecting self-reported drinking in excess of 50 g ethanol was 77%. An additional nine patients who claimed abstinence still showed abnormal 5HTOL/5HIAA ratios, and so did three of the patients who refused to do a breath-ethanol test and/or complete the self-report. Altogether, 59 of 190 methadone-maintained patients (31.1%) had been drinking any alcohol on the previous day (i.e. Sunday-Thursday) according to self-report and/or urinalysis data, 29 (49.2%) of whom were identified by the urinary 5HTOL/5HIAA ratio and only four (6.8%) by utilizing breathalyzer.

The urinary ratio of 5-hydroxytryptophol to 5-hydroxyindole-3-acetic acid in surgical patients with chronic alcohol misuse.

The urinary ratio of 5-hydroxytryptophol to 5-hydroxyindole-3-acetic acid was reported to be elevated for a period of up to 22 h following acute alcohol ingestion. Therefore, the ratio could detect continuous alcohol consumption, in what was considered to be a high-risk surgical group, on the evening prior to surgery. The aim of this study was to determine the preoperative ratio of 5-hydroxytryptophol to 5-hydroxyindole-3-acetic acid in patients with continuous preoperative alcohol misuse. Forty-two patients participated in this institutionally approved study, once their written informed consent had been obtained. Chronic alcoholics were defined by meeting the criteria of the Diagnostic and Statistical Manual of Mental Disorders criteria and an ethanol consumption > or =60 g/day. The urine samples were taken preoperatively and determined by means of gas chromatography-mass spectrometry and high performance liquid chromatography. The urinary ratio of 5-hydroxytryptophol to 5-hydroxyindole-3-acetic acid was significantly increased in chronic alcoholics. The ICU stay of these patients was significantly prolonged due to an increased incidence of pneumonia and sepsis. Five chronic alcoholics died, whereas no deaths occurred in the nonalcoholic group (p = 0.05). As the measurement of the urinary ratio of 5-hydroxy-tryptophol to 5-hydroxyindole-3-acetic acid could detect alcohol consumption immediately prior to operation, this marker could assist the carbohydrate-deficient transferrin in screening for patients with high-level dependency; these patients were considered to be at a high risk of developing intercurrent complications.

Changes in the concentrations of ethanol, methanol and metabolites of serotonin in two successive urinary voids from drinking drivers.

The urine-ethanol concentration (UEC), the urine-methanol concentration (UMC) and the ratio of serotonin metabolites, 5-hydroxytryptophol (5HTOL) to 5-hydroxyindoleacetic acid (5HIAA), were determined in two successive voids from apprehended drunk drivers (n = 35). The blood-ethanol concentration (BEC) ranged from 0-3.00 g/l (mean 1.87 g/l, median 2.03 g/l) compared with 0-3.96 g/l (mean 2.48 g/l, median 2.73 g/l) in the first urinary void and 0-3.56 g/l (mean 2.24 g/l, median 2.47 g/l) in the second void. The UEC decreased significantly from 2.48 +/- 0.99 g/l to 2.24 +/- 0.95 g/l (mean +/- S.D.) between first and second voids as did the UEC/BEC ratios, changing from 1.33 +/- 0.15 to 1.20 +/- 0.10. The BEC and UEC were highly correlated; r = 0.97 +/- 0.04 (p < 0.001) for the first void and r = 0.98 +/- 0.03 (p < 0.001) for the second void. The UMC increased from 7.51 +/- 4.95 mg/l to 8.01 +/- 5.04 mg/l between the first and second voids and the mean difference of 0.50 +/- 0.78 mg/l was statistically highly significant (p < 0.001). The ratios of 5HTOL/5HIAA were 771 +/- 363 pmol/nmol and 728 +/- 377 pmol/nmol in first and second voids, respectively and the difference was not statistically significant (p > 0.05). Finding raised concentrations of methanol and a high 5HTOL/5HIAA ratio in urine specimens provides additional evidence to confirm recent drinking. These biochemical markers might prove useful whenever the integrity of blood or urine specimens is questioned, for example, owing to contamination with extraneous ethanol during collection or microbial synthesis of ethanol in vitro after sampling.

Urinary excretion of methanol and 5-hydroxytryptophol as biochemical markers of recent drinking in the hangover state.

Twenty healthy social drinkers (9 women and 11 men) drank either 50 g of ethanol (mean intake 0.75 g/kg) or 80 g (mean 1.07 g/kg) according to choice as white wine or export beer in the evening over 2 h with a meal. After the end of drinking, at bedtime, in the following morning after waking-up, and on two further occasions during the morning and early afternoon, breath-alcohol tests were performed and samples of urine were collected for analysis of ethanol and methanol and the 5-hydroxytryptophol (5-HTOL) to 5-hydroxyindol-3-ylacetic acid (5-HIAA) ratio. The participants were also asked to quantify the intensity of hangover symptoms (headache, nausea, anxiety, drowsiness, fatigue, muscle aches, vertigo) on a scale from 0 (no symptoms) to 5 (severe symptoms). The first morning urine void collected 6-11 h after bedtime as a rule contained measurable amounts of ethanol, being 0.09 +/- 0.03 g/l (mean +/- SD) after 50 g and 0.38 +/- 0.1 g/l after 80 g ethanol. The corresponding breath-alcohol concentrations were zero, except for three individuals who registered 0.01-0.09g/l. Ethanol was not measurable in urine samples collected later in the morning and early afternoon. The peak urinary methanol occurred in the first morning void, when the mean concentration after 80 g ethanol was approximately 6-fold higher than pre-drinking values. This compares with a approximately 50-fold increase for the 5-HTOL/5-HIAA ratio in the first morning void. Both methanol and the 5-HTOL/5-HIAA ratio remained elevated above pre-drinking baseline values in the second and sometimes even the third morning voids. Most subjects experienced only mild hangover symptoms after drinking 50 g ethanol (mean score 2.4 +/- 2.6), but the scores were significantly higher after drinking 80 g (7.8 +/- 7.1). The most common symptoms were headache, drowsiness, and fatigue. A highly significant correlation (r = 0.62-0.75, P <0.01) was found between the presence of headache, nausea, and vertigo and the urinary methanol concentration in the first and second morning voids, whereas 5-HTOL/5-HIAA correlated with headache and nausea. These results show that analysing urinary methanol and 5-HTOL furnishes a way to disclose recent drinking after alcohol has no longer been measurable by conventional breath-alcohol tests for at least 5-10h. The results also support the notion that methanol may be an important factor in the aetiology of hangover.