Two-Dimensional Thin Layer Chromatography of Melatonin and Related Compounds.

Two-dimensional thin layer chromatography has been used by workers in the field to separate radiolabeled serotonin derivatives from complex mixtures of culture media and homogenates of glands. The compounds resolved include N-acetylserotonin, melatonin, hydroxytryptophol, methoxytryptophol, hydroxyindole acetic acid, and methoxyindole acetic acid. The method requires either radiolabeled tryptophan or serotonin, if an investigator wants to study conversion. It is also useful in the chemical synthesis of serotonin metabolites because it is relatively fast. It pointed to the enzyme that converts serotonin to N-acetylserotonin as being key in controlling the nocturnal increase in vertebrate melatonin production. This enzyme, arylalkylamine N-acetyltransferase (E.C. 2.3.1.87), has been the focus of hundreds of papers which probed its biology, biochemistry, molecular biology, structural biology, neural regulation, development, evolution, and genetics.

Anti-VEGF Signalling Mechanism in HUVECs by Melatonin, Serotonin, Hydroxytyrosol and Other Bioactive Compounds.

Angiogenesis drives evolution and destabilisation of atherosclerotic plaques and the growth and expansion of tumour cells. Vascular endothelial growth factor (VEGF) is the main endogenous pro-angiogenic factor in humans. The aim was to provide insight into the anti-VEGF activity of bioactive compounds derived from aromatic amino acids (serotonin, melatonin, 3-indoleacetic acid, 5-hydroxytryptophol and hydroxytyrosol). Experiments involved endothelial cell migration (wound-healing assay), the molecular mechanisms (ELISA assay) and the downstream effects (phospholipase C gamma 1 (PLCγ1), protein kinase B (Akt) and endothelial nitric oxide synthase (eNOS) by Western blot) on human umbilical vein endothelial cells (HUVECs). The data suggest for the first time that hydroxytyrosol interacts with surface components of the endothelial cell membrane (, preventing VEGF from activating its receptor. Serotonin and 5-hydroxytryptophol significantly inhibited HUVEC migration (98% and 50%, respectively) following the same mechanism. Conversely to other bioactive compounds, the anti-angiogenic effect of melatonin, serotonin, 3-indoleacetic acid and 5-hydroxytryptophol is not mediated via PLCγ1. However, hydroxytyrosol inhibits PLCγ1 phosphorylation. Additionally, melatonin and serotonin maintained eNOS phosphorylation and hydroxytyrosol significantly activated eNOS-all via Akt. These data provide new evidence supporting the interest in melatonin, serotonin, 3-indoleacetic acid, 5-hydroxytryptophol and hydroxytyrosol for their further exploitation as anti-VEGF ingredients in food.

Concerted derivatization and concentration method with dispersive liquid-liquid microextraction for liquid chromatographic analysis of 5-hydroxyindoles in human serum.

We developed a concerted derivatization and concentration method based on dispersive liquid-liquid microextraction (DLLME) for the liquid chromatography (LC) determination of 5-hydroxyindoles (5-HIs; serotonin, 5-hydroxyindole-3-acetic acid, N-acetylserotonin, and 5-hydroxytryptohol). Concerted derivatization and concentration could be affected by adding a mixture of an ionic liquid (1-hexyl-3-methylimidazolium hexafluorophosphate, extraction solvent), methanol (disperser), and water containing fluorescence derivatization reagents [benzylamine and potassium hexacyanoferrate(III)] into the sample. The resulting sedimented phase was injected into a reversed-phase LC column using a mixture of acetonitrile and 250 mM acetate buffer (pH 4.3) as the mobile phase for gradient elution, and the derivatives obtained were fluorometrically detected at excitation and emission wavelengths of 345 nm and 452 nm, respectively. The derivatization (reagent concentrations and pH) and extraction (extraction and disperser solvent type) conditions were optimized simultaneously. The limits of detection of the 5-HIs were in the range of 0.08-0.33 nM. The method was validated for 10 and 50 pmol/mL human serum levels, and the recovery of 5-HIs was between 66% and 98%, within a relative standard deviation of 9.5%. The proposed method is well suited for the highly sensitive analysis of trace amounts of 5-HIs in human serum samples.

Acyclovir inhibits rat liver tryptophan-2,3-dioxygenase and induces a concomitant rise in brain serotonin and 5-hydroxyindole acetic acid levels.

Viral diseases of the brain may induce changes in neurotransmitter synthesis and metabolism. In experimental herpes simplex encephalitis, brain serotonin is reduced, whilst it's major metabolite, 5-hydroxyindole acetic acid and turnover is increased. It is well established that reduced levels of brain monoamines, serotonin and norepinephrine may contribute to the symptoms of clinical depression, which raises the possibility that this condition is prevalent in herpes simplex encephalitis. An inverse relationship exists between liver tryptophan-2,3-dioxygenase activity and brain serotonin levels and there is an interdependency between serotonin and norepinephrine levels. The aim of this study is to determine the effect of acyclovir, an antiviral used in the treatment of herpes simplex encephalitis, on rat liver tryptophan-2,3-dioxygenase activity in vitro and in vivo as well as on rat forebrain serotonin, 5-hydroxyindole acetic acid and norepinephrine levels. The results show that acyclovir inhibits tryptophan-2,3-dioxygenase activity in vitro and in vivo, with a concomitant rise in serotonin and 5-hydroxyindole acetic acid levels. However, acyclovir reduces the turnover of serotonin to 5-hydroxyindole acetic acid, without any effect on norepinephrine levels. It appears that acyclovir may have the potential to reduce the clinical symptoms of depression in herpes simplex encephalitis. However, a greater turnover of serotonin to 5-hydroxyindole acetic acid could possibly be masked by conversion of serotonin to 5-hydroxytryptophol, which needs to be investigated further.

Objective post-mortem diagnosis of chronic alcohol abuse--a review of studies on new markers.

Identification of chronic excessive alcohol consumption in living and deceased individuals is a fundamental task in forensic pathology. Reliable methods for post-mortem diagnosis of chronic alcohol abuse are required because morphological findings are unspecific and ante-mortem data are often unreliable. In clinical practice, several biochemical markers indirectly demonstrating chronic alcohol abuse are employed, but thus far these methods have not been used in routine post-mortem investigations. We reviewed publications in which these markers have been applied to autopsy material. Based on this review, some of these biochemical parameters are useful in post-mortem diagnostics, although further systematic research is required.

HPLC determination of serotonin and its metabolites from human platelet-rich plasma; shift to 5-hydroxytryptophol formation following alcohol consumption.

A sensitive, simple, and reliable high-performance liquid chromatographic method with electrochemical detection is developed for the measurement of four natural products, the serotonin-related indols from human platelet-rich plasma (PRP) using N-methylserotonin as internal standard. Separation of serotonin (5HT), 5-hydroxytryptophan (5HTP), 5-hydroxytryptophol (5HTOL), and 5-hydroxyindole-acetic acid (5HIAA) is carried out on Supelcosil LC-18DB stationary phase. A mixture of 48 mM citric acid, 28 mM sodium phosphate dibasic, 0.027 mM Na2EDTA, and 3% methanol (pH 3.18) serves as the mobile phase. Measurements are carried out at 25 degrees C at Eox=0.65 V. The calibration curves are linear through the range of 10-200 pg/mL. Method validation is performed according to internationally accepted criteria. Blood is collected from healthy controls and schizophrenic subjects. Significantly higher PRP serotonin is measured in schizophrenics; patients with recent alcohol consumption could be characterized with significantly elevated 5HTOL/5HIAA ratio.

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.

Determination of glucuronidated 5-hydroxytryptophol (GTOL), a marker of recent alcohol intake, by ELISA technique.

OBJECTIVES: To compare markers of alcohol consumption. DESIGN AND METHODS: Measurement of urinary ethyl glucuronide, 5-hydroxytryptophol, 5-hydroxytryptophol glucuronide, 5-hydroxyindole acetic acid in 10 patients during alcohol withdrawal. RESULTS: 5-Hydroxytryptophol glucuronide was measured by ELISA with good analytical precision, its diagnostic specificity and sensitivity was better than that of 5-hydroxytryptophol and its correlation was closer to ethyl glucuronide than to 5-hydroxytryptophol. CONCLUSION: Determination of 5-hydroxytryptophol glucuronide by ELISA offers promising results in detection of previous alcohol consumption.

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.

Determination of urinary 5-hydroxytryptophol glucuronide by liquid chromatography-mass spectrometry.

5-Hydroxytryptophol glucuronide (GTOL) is the major excretion form of 5-hydroxytryptophol (5-HTOL), a minor serotonin metabolite under normal conditions. Because the concentration of 5-HTOL is markedly increased following consumption of alcohol, measurement of 5-HTOL is used as a sensitive biomarker for detection of recent alcohol intake. This study describes the development and evaluation of a liquid chromatography-electrospray ionization mass spectrometry (LC-MS) procedure for direct quantification of GTOL in human urine. Deuterium labelled GTOL (GTOL-(2)H(4)) was used as internal standard. GTOL was isolated from urine by solid-phase extraction on a C(18) cartridge prior to injection onto a gradient eluted Hypurity C(18) reversed-phase HPLC column. The detection limit of the method was 2.0 nmol/L and the measuring range 6-8500 nmol/L. The intra- and inter-assay coefficients of variation were <3.5% (n=10) and <6.0% (n=9), respectively. The new LC-MS method was highly correlated with an established GC-MS method for urinary 5-HTOL (r(2)=0.99, n=70; mean 5-HTOL/GTOL ratio=1.10). This is the first direct assay for quantification of GTOL in urine. The method is suitable for routine application.

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.

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.

Prevalence of high alcohol and benzodiazepine consumption in sleep apnea patients studied with blood and urine tests.

OBJECTIVE: To evaluate the prevalence of alcoholism and benzodiazepine abuse among patients with obstructive sleep apnea syndrome (OSAS). Such abuse may aggravate the tendency to apneas, especially in patients with OSAS. MATERIAL AND METHODS: The study included 98 consecutive OSAS patients. Two patients dropped out; blood samples could not be obtained from two other patients and a urine sample could not be obtained from one. Blood and urine samples were examined for carbohydrate-deficient transferrin (CDT) and 5-hydroxytryptophol (5-HTOL), markers of excess alcohol intake, and urine-benzodiazepines (u-Benz), a marker of drug abuse. Patients with positive screening tests were offered therapy for their abuse. RESULTS: The CDT test was positive in 8/94 patients (8.5%), the 5-HTOL test in 6/95 (6.3%) and the u-Benz test in 3/95 (3.2%). CONCLUSIONS: Our findings correlate well with current views concerning alcohol and drug abuse in Sweden, and do not indicate that the frequency of such abuse is higher among OSAS patients. It should be noted that none of the patients who screened positive in the laboratory tests admitted to being alcohol or drug abusers when they consulted their physician. We recommend screening all OSAS patients for alcohol abuse using not only a questionnaire but also a laboratory test such as the CDT test.

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.