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.

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.

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.

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.

Specific antagonism of enteric neural serotonin receptors by dipeptides of 5-hydroxytryptophan: evidence that serotonin is a mediator of slow synaptic excitation in the myenteric plexus.

Research on the role of serotonin (5-hydroxytryptamine, 5-HT) in the function of the enteric nervous system has been impeded by the lack of specific inhibitors of the enteric neural actions of 5-HT. Saturable, reversible, high affinity enteric binding sites for 3H-5-HT have recently been characterized and radioautographically located. Affinity for the 3H-5-HT binding site requires an indole ring substituted with a free hydroxyl group. These 3H-5-HT binding sites have been proposed to be enteric neural 5-HT receptors. This hypothesis was tested in the current study by comparing the ability of compounds to inhibit the binding of 3H-5-HT with their electrophysiologically determined actions on myenteric neurons. 5-Methoxytryptamine did not inhibit the binding of 3H-5-HT to enteric membranes and neither mimicked nor antagonized the effects of 5-HT on the membrane potential of myenteric neurons. Two dipeptides of 5-hydroxytryptophan, N-acetyl- and N-hexanoyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP and N-hex-5-HTP-DP) inhibited the binding of 3H-5-HT (K1 = 0.25 microM for 5-HTP-DP and 1.19 microM for N-hex-5-HTP-DP). 5-HTP-DP applied by pressure microejection or superfusion (10 microM) antagonized the slow postsynaptic depolarization of myenteric neurons evoked by microejection of 5-HT. 5-HTP-DP also blocked the 5-HT-induced presynaptic reduction in amplitude of nicotinic fast synaptic potentials; however, 5-HTP-DP itself did not affect these responses. Moreover, 5-HTP-DP also failed to affect responses of myenteric neurons to microejected substance P, their muscarinic response to acetylcholine, or antidromic action potentials. In contrast, both dipeptides blocked the slow synaptic potentials seen in type II/AH neurons following stimulation of fiber tracts in interganglionic connectives. These data support the hypotheses that enteric 3H-5-HT binding sites are enteric neural 5-HT receptors, that dipeptides of 5-hydroxytryptophan are specific antagonists at these receptors, and that 5-HT is one of the mediators of slow synaptic potentials in the myenteric plexus.

The influence of GABA on the synthesis of N-acetylserotonin, melatonin, O-acetyl-5-hydroxytryptophol and O-acetyl-5-methoxytryptophol in the pineal gland of the male Wistar rat.

The influence of GABA on the synthesis of N-acetylserotonin, melatonin, O-acetyl-5-hydroxytryptophol and O-acetyl-5-methoxytryptophol has been investigated using different experimental procedures. It was demonstrated that when GABA and an acetyl donor were added to the incubation medium together, a significant increase in synthesis of the N-acetylated products occurred during the night. Moreover there was a large increase in N-acetylserotonin synthesis at 15(00) hrs although none was observed in the control experiments. However, when GABA was added 20 min before the acetyl donor, synthesis of the N-acetylated products was significantly less. The opposite effect was observed for the O-acetylated indoles. These results confirm the proposal by Ebadi et al. (1982) that GABA, like norepinephrine, may be a regulator of melatonin synthesis. As melatonin is implicated in the regulation of reproduction it may be that GABA is equally significant in this regulatory effect.