A preliminary investigation on the influence of flavonoids on ethyl glucuronide formation

Abstract Aims: A large variation of the formation of ethyl glucuronide (EtG) which is a minor metabolite of ethanol has been observed in man. At present there is only a single investigation on glucuronosyltransferases (UGT) responsible for catalyzing EtG formation whereas a possible influence of nutritional components on EtG formation has not been addressed at all. Methods: Following optimization of the substrate concentration, incubation conditions such as buffer and time, as well as isolation of EtG from the incubation mixture, recombinant UGT enzymes (UGT 1A1, 1A3, 1A4, 1A6, 1A9, 2B7, 2B10, 2B15) were screened for their activity towards ethanol. Subsequently, quercetin and kaempferol were chosen to study their possible influence on the glucuronidation of ethanol for UGT1A1, 1A3 and UGT1A9. Analysis of EtG formation was performed by LC-MS/MS following solid phase extraction. EtG-d 5 was used as the internal standard. For inhibition experiments, EtG formation was determined after addition of kaempferol and quercetin in different concentrations to the incubation mixture to determine the half maximum inhibitory constants (IC 50 ). Results: Optimization of both, the incubation and isolation procedures resulted in a significant decrease of matrix effects. EtG was formed by all enzymes under investigation at variable rates ranging from 0.79 -10.41 pmol/min/mg at 50 mM ethanol and 0.95-12.9 pmol/mg/min at 200 mM ethanol. Respective kinetics followed the Michaelis-Menten model so that the Michaelis-Menten constant K m and the maximum velocity V max values could be calculated. Both flavonoids reduced formation of EtG, irrespective of the enzyme involved. The IC 50 -values ranged from 3.04 µM quercetin for UGT1A1 to 38.47 µM kaempferol for UGT1A9. Conclusions: Formation of EtG from ethanol is catalyzed by multiple UGT isoforms. In addition, co-incubation affected the glucuronidation rate, irrespective of the particular enzyme. It seems that nutritional components will influence conversion of ethanol to EtG which may partly explain its variable formation in man

Per se limits: methods of defining cut-off values for zero tolerance

Establishment of cut-off values for per se legislation

Alcohol Biomarkers in Clinical and Forensic Contexts

Background: Biomarkers of alcohol consumption are important not only in forensic contexts, e.g., in child custody proceedings or as documentation of alcohol abstinence after temporary confiscation of a driver's license. They are increasingly being used in clinical medicine as well for verification of abstinence or to rule out the harmful use of alcohol. Methods: This review is based on pertinent publications that were retrieved by a selective literature search in PubMed concerning the direct and indirect alcohol markers discussed here, as well as on the authors' experience in laboratory analysis and clinical medicine. Results: Alongside the direct demonstration of ethanol, the available markers of alcohol consumption include the classic indirect markers carbohydrate-deficient transferrin (CDT), gamma-glutamyltransferase (GGT), and mean corpuscular volume (MCV) as well as direct alcohol markers such as ethyl glucuronide (EtG) and ethyl sulfate (EtS) in serum and urine and EtG and fatty acid ethyl esters (FAEE) in hair. Phosphatidylethanol (PEth) is a promising parameter that complements the existing spectrum of tests with high specificity (48-89%) and sensitivity (88-100%). In routine clinical practice, the demonstration of positive alcohol markers often leads patients to admit previously denied alcohol use. This makes it possible to motivate the patient to undergo treatment for alcoholism. Conclusion: The available alcohol biomarkers vary in sensitivity and specificity with respect to the time period over which they indicate alcohol use and the minimum extent of alcohol use that they can detect. The appropriate marker or combination of markers should be chosen in each case according to the particular question that is to be answered by laboratory analysis

Oral Yohimbine as a New Probe Drug to Predict CYP2D6 Activity: Results of a Fixed-Sequence Phase I Trial

Objective!#!Yohimbine pharmacokinetics were determined after oral administration of a single oral dose of yohimbine 5 mg and a microdose of yohimbine 50 µg in relation to different cytochrome P450 (CYP) 2D6 genotypes. The CYP2D6 inhibitor paroxetine was used to investigate the influence on yohimbine pharmacokinetics. Microdosed midazolam was applied to evaluate a possible impact of yohimbine on CYP3A activity and the possibility of combining microdosed yohimbine and midazolam to simultaneously determine CYP2D6 and CYP3A activity.!##!Methods!#!In a fixed-sequence clinical trial, 16 healthy volunteers with a known CYP2D6 genotype [extensive (10), intermediate (2) and poor (4) metaboliser] received an oral dose of yohimbine 50 µg, yohimbine 5 mg at baseline and during paroxetine as a CYP2D6 inhibitor. Midazolam (30 µg) was co-administered to determine CYP3A activity at each occasion. Plasma concentrations of yohimbine, its main metabolite 11-OH-yohimbine, midazolam and paroxetine were quantified using validated liquid chromatography-tandem mass spectrometry assays.!##!Results!#!Pharmacokinetics of yohimbine were highly variable and a CYP2D6 genotype dependent clearance was observed. After yohimbine 5 mg, the clearance ranged from 25.3 to 15,864 mL/min and after yohimbine 50 µg, the clearance ranged from 39.6 to 38,822 mL/min. A more than fivefold reduction in clearance was caused by paroxetine in CYP2D6 extensive metabolisers, while the clearance in poor metabolisers was not affected. Yohimbine did not alter CYP3A activity as measured by microdosed midazolam.!##!Conclusions!#!The pharmacokinetics of yohimbine were highly correlated with CYP2D6, which was further supported by the clearance inhibition caused by the CYP2D6 inhibitor paroxetine. With these data, yohimbine is proposed to be a suitable probe drug to predict CYP2D6 activity. In addition, the microdose can be used in combination with microdosed midazolam to simultaneously evaluate CYP2D6 and CYP3A activity without any interaction between the probe drugs and because the microdoses exert no pharmacological effects.!##!Clinical trial registration!#!EudraCT2017-001801-34