Formation and inhibition of ethyl glucuronide and ethyl sulfate.

Ethyl glucuronide (EtG) und ethyl sulfate (EtS) are widely accepted biomarkers in forensic and clinical settings. Even though, levels of EtG and EtS in blood and urine increase with increasing doses of alcohol, a high inter-individual variability in their production has been noticed. Therefore, we investigated the influence of dietary plant phenols on the formation of EtG and EtS and tentatively estimated the magnitude of in vivo inhibitory interactions from our in vitro results. To address these issues, formation of EtS and EtG was investigated using recombinant glucuronosyl- and sulfotransferases as well as human liver microsomes and liver cytosol. After respective kinetics had been established, inhibition experiments using quercetin, kaempferol and resveratrol were performed. These polyphenols are subject to extensive glucuronidation and/or sulfonation. EtG and EtS were determined by LC-MS/MS following solid phase extraction for EtG due to severe matrix effects and by direct injection for EtS. All enzymes investigated were involved in the conjugation of ethanol. Maximal EtG and EtS formation rates were observed with HLM and SULT1A1, respectively. All kinetics could best be described by Michaelis-Menten kinetics. Resveratrol was a competitive inhibitor of UGT1A1, UGT1A9 and HLM; quercetin and kaempferol were inhibitors of all transferases under investigation except UGT2B15. Findings for quercetin with regard to UGT2B7 and SULT2A1 and for kaempferol with regard to SULT1E1 and SULT2A1 suggested a mechanism based inhibition. Competitive inhibition of the glucuronidation and sulfonation of ethanol was estimated as weak to negligible and as moderate to weak, respectively. Beside the known polymorphisms of the transferases involved in EtG and EtS formation, prediction of the inhibitory potential indicates that polyphenols may contribute to the variable formation rate of EtG and EtS.

A methoxydiphenidine-impaired driver.

Methoxydiphenidine (MXP) was first reported in 1989 as a dissociative anesthetic but did not enter the market for pharmaceuticals. The substance re-appeared in 2013 as a new psychoactive substance. A case of driving under the influence of MXP is reported. The concentration of MXP has been determined from a serum sample (57 ng/mL) by liquid chromatography tandem mass spectrometry following liquid-liquid extraction. In addition, amphetamine, methylenedioxymethamphetamine, and its major metabolite were present in concentrations of 111, 28, and 3 ng/mL, respectively. The subject presented with amnesia, out-of-body experiences, bizarre behavior, and decreased motor abilities. At present, information on human toxicity of MXP is not available. MXP is comparable in structure as well as in action at the N-methyl-D-aspartate (NMDA) receptor to phencyclidine or ketamine. Therefore, it is likely that MXP exerts similar severe psychotropic action in man. However, there is no information on the duration and intensity of MXP's impairing effects, the interpretation of a particular concentration in the blood or serum, and its detectability in routine drug screenings. Confirmation analysis may be confined to cases where the police has specific intelligence that points to MXP use.

Identification and characterization of sulfonyltransferases catalyzing ethyl sulfate formation and their inhibition by polyphenols.

Ethyl sulfate (EtS) is a minor metabolite of ethanol, usually being present along with ethyl glucuronide in both blood and urine. At present, there have been few studies on sulfotransferases (SULTs) catalyzing EtS formation. Moreover, inhibition by nutritional components on EtS formation, e.g., polyphenols that are extensively sulfonated, has not been addressed at all. Firstly, the incubation procedure was optimized with regard to buffer, substrate concentration, and incubation time. Recombinant SULT enzymes including SULT1A1, 1A3, 1B1, 1E1, and 2A1 were screened for their activity towards ethanol; subsequently, respective kinetics was investigated. The inhibitory potential of resveratrol, quercetin, and kaempferol being abundant in beer and wine was studied thereafter. Analysis was performed by liquid chromatography/tandem mass spectrometry (LC-MS/MS) using deuterated EtS as the internal standard. All enzymes are involved in the sulfonation of ethanol; respective kinetics followed the Michaelis-Menten model. Among the five SULTs under investigation, SULT1A1 displayed the highest activity towards ethanol followed by SULT2A1. Polyphenols significantly reduced the formation of EtS. Results revealed multiple SULT isoforms being capable of catalyzing the transfer of a sulfo group to ethanol; nevertheless, the relevance of SULTs' polymorphism on the sulfonation of ethanol needs further appraisal. Nutritional components such as polyphenols effectively inhibit formation of EtS; this observation may partly serve as an explanation of the highly inter-individual variability of EtS findings in both blood and urine.

A potential new metabolite of gamma-hydroxybutyrate: sulfonated gamma-hydroxybutyric acid.

Detection of gamma-hydroxybutyric acid (GHB) became crucial in many clinical and forensic settings due to its increasing use for recreational purposes and drug-facilitated sexual assault. Its narrow window of detection of about 3-12 h in urine represents a major problem. Analogous to ethyl glucuronide, the recently identified GHB-glucuronide exhibits a longer window of detection than the parent drug. It appeared reasonable that a sulfonated metabolite of GHB (GHB-SUL) will also be formed. Due to the lack of an appropriate standard, GHB was incubated with a human liver cytosolic fraction to produce GHB-SUL. Following development of a liquid chromatography/tandem mass spectrometry (LC-MS/MS) assay to measure GHB and GHB-SUL, authentic urine samples (n = 5) were tested for GHB-SUL. These investigations revealed detectable signals of both GHB and GHB-SUL, strongly indicating that GHB is not only glucuronidated but also sulfonated. Given that sulfonated metabolites generally have longer half-life times than the corresponding free drugs, GHB-SUL may serve as a biomarker of GHB misuse along with its glucuronide.

[In vitro characterization of glucuronosyl- and sulfotransferases involved in the conjugation of ethanol]. / In vitro Charakterisierung der Glucuronidierung und Sulfatierung von Ethanol.

Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are minor metabolites of ethanol; for some years, both compounds have been used as direct biomarkers of alcohol consumption in forensic and clinical settings as well as in traffic medicine. Drinking experiments showed individual variations of the formation of EtG and EtS. At present, our knowledge on enzymes involved in the conjugation of ethanol is incomplete and partly inconsistent. The purpose of the present study was to characterize those enzymes that are capable of catalyzing glucuronidation and sulfation of ethanol including some potential inhibitors. Following optimization of incubation conditions, the formation rates of EtG and EtS from ethanol via recombinant glucuronosyltransferases (UGTs, hepatic) and sulfotransferases (SULTs, hepatic, intestinal), the kinetics and the inhibitory potential of polyphenols such as quercetin, kaempferol and resveratrol were determined. Analysis was performed following either solid phase extraction due to severe ion suppression of EtG or direct injection of the EtS-containing incubation mixture by high-pressure liquid chromatography/tandem mass spectrometry. Deuterated analogues were used as internal standards. All UGTs were capable of metabolizing ethanol through glucuronidation; UGT1A9 and UGT2B7 exhibited the highest formation rates. All SULTs showed ethanol-sulfating activity with SULT1A1 being most active. Data for all enzymes could best be described by Michaelis-Menten kinetics. All polyphenols inhibited the conjugation of ethanol except UGT2B 15. Inhibition was reversible and competitive for most enzymes; mechanism-based inhibition was evident for UGT2B7 and SULT2A1 with regard to quercetin and for SULT1E1 with regard to kaempferol. These results suggest an influence on the formation rates of EtG and EtS by common food ingredients beside known polymorphisms of UGT and SULT family members. Further studies should be conducted to achieve a better understanding of the extent and significance of this influence.