Development and validation of an analytical method for the simultaneous determination of the alcohol biomarkers ethyl glucuronide, ethyl sulfate, N-acetyltaurine, and 16:0/18:1-phosphatidylethanol in human blood.

As alcohol is the most common addictive substance worldwide, it is inevitable to advance the established research. New and more substantial analytical methods can be applied to reply to complex questions in legal or forensic contexts. Therefore, an analytical method for the simultaneous determination of four different alcohol biomarkers-ethyl glucuronide, ethyl sulfate, N-acetyltaurine, and 16:0/18:1-phosphatidylethanol-in human blood was developed, validated, and verified. Despite the different chemical properties of the analytes, a specific determination via HPLC-MS/MS was achieved using a novel type of a Phenomenex Luna® Omega Sugar column. Furthermore, all criteria for a successful validation were fulfilled according to forensic guidelines. The method proved to be linear and demonstrates selectivity and sufficient sensitivity for every biomarker. LODs obtained with this method of 2.6 ng/ml (EtG), 4.7 ng/ml (EtS), 12.5 ng/ml (NAcT), and 6.9 ng/ml (PEth) were in an acceptable range for routine applications, and the stability of all analytes over a range of 12 h is given. The verification of the new developed method was performed with authentic samples. Thus, whole blood and postmortem samples were analyzed to obtain information about the drinking behavior, which can answer complex questions regarding alcohol consumption.

PK-PD Correlation of Erigeron Breviscapus Injection in the Treatment of Cerebral Ischemia-Reperfusion Injury Model Rats.

By measuring the cerebral infarction rate and neurological behavioral score of rats in a sham operation group, an MCAO model control group and an Erigeron breviscapus injection treatment group, we explored the therapeutic effects of Erigeron breviscapus injection on brain tissue and neuroethological injury in rats. Plasma samples were collected at 18 time points after intravenous injection of Erigeron breviscapus. The levels of scutellarin, 4-caffeoylquinic acid, 5-caffeoylquinic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, chlorogenic acid and isochlorogenic acid B in rat plasma at the various time points were determined by an HPLC method, and drug concentration versus time plots were constructed to estimate the pharmacokinetic parameters. Finally, a PK-PD combined model was used to analyze the relationship between the blood concentration, time and therapeutic effects of the seven active components. The results of the pharmacodynamics studies showed that the cerebral infarction rate of rats in the Erigeron breviscapus injection group decreased significantly at 5 min, 10 min, 20 min, 6 h, 8 h, 18 h, 24 h, 32 h, 40 h and 48 h after cerebral ischemia. Abnormal neurological behavior scores were significantly reduced after 4 h of cerebral ischemia. The pharmacokinetics results showed that the seven chemical constituents in Erigeron breviscapus injection reached their highest detection value after 5 min of cerebral ischemia. The lowest detection values of scutellarin and isochlorogenic acid B appeared after 6 h of cerebral ischemia but could not be detected after 8 h. The lowest detection values of 5-caffeoylquinic acid and 4,5-dicaffeoylquinic acid were found in the third hour of cerebral ischemia but not after 4 h. The lowest detection values of 4-caffeoylquinic acid, 3,5-dicaffeoylquinic acid and chlorogenic acid were found during the second hour of cerebral ischemia but not at the third hour. However, at 18 h, 24 h, 32 h and 40 h of cerebral ischemia, the cerebral infarction rates of rats in the Erigeron breviscapus injection group were significantly reduced, with decreased values of 6.22%, 11.71%, 6.92% and 4.96%, respectively, and the effects were stronger than those after 5-20 min of cerebral ischemia. The decreased values reached their highest value after 24 h of cerebral ischemia. Our results show that the effects of Erigeron breviscapus injection on reducing the cerebral infarct rate in MCAO model rats are characterized by a fast onset and long maintenance time. The 5-min blood concentration in cerebral ischemia was the highest test value, and after this time, the cerebral infarction rate of MCAO rats began to decrease. However, the peak value of the effects lagged behind that of the plasma concentration. The maximum effective time for Erigeron breviscapus injection appeared 24 h after cerebral ischemia, which provides a reference for the screening of specific drugs for ischemic stroke, optimal dosing regimens and rational clinical drug use. Graphical Abstract.

Rifampin Reduces the Plasma Concentrations of Oral and Intravenous Hydromorphone in Healthy Volunteers.

BACKGROUND: Several opioids are metabolized by the inducible cytochrome P450 (CYP) 3A isozymes. Coadministration with strong inducers of drug metabolism, such as rifampin, can dramatically reduce systemic exposure to these opioids. As the CYP metabolism of hydromorphone is of minor importance, we studied in healthy volunteers whether hydromorphone would be an effective analgesic for patients who concomitantly receive the prototypical enzyme inducer rifampin. METHODS: In this paired, randomized, crossover study, 12 participants received oral placebo or rifampin for 8 days. Oral hydromorphone (2.6 mg) was administered on day 6 followed by intravenous hydromorphone (0.02 mg/kg) on day 8. Hydromorphone and hydromorphone-3-glucuronide (HM3G) plasma concentrations were measured for 24 hours and psychomotor responses, including perceived drug effect, change in pupil diameter, and cold pressor threshold were evaluated for 6 hours. Our primary outcome was the change in the area under the concentration-time curve (AUC0-last) of oral and intravenous hydromorphone after pretreatment with rifampin or placebo. Pharmacodynamic parameters and other pharmacokinetic parameters were analyzed as secondary outcomes. RESULTS: Rifampin reduced the AUC0-last of oral and intravenous hydromorphone by 43% (ratio to control: 0.57, 90% confidence interval [CI], 0.50-0.65) and 26% (ratio to control: 0.74, 90% CI, 0.69-0.79), respectively. The maximum concentration of oral hydromorphone was reduced by 37% (ratio to control: 0.63, 90% CI, 0.55-0.72), and oral bioavailability decreased from 33% to 26% (ratio to control: 0.78, 90% CI, 0.67-0.91) in the rifampin phase compared with placebo. The HM3G-to-hydromorphone ratio increased by 50% (90% CI, 25-79) and 42% (90% CI, 29-55) after oral and intravenous hydromorphone, respectively. Rifampin did not significantly affect the pharmacodynamic parameters. CONCLUSIONS: Rifampin significantly reduces the concentrations of oral and intravenous hydromorphone. This interaction is due to an increase in the first-pass and systemic metabolism of hydromorphone, likely involving induction of uridine 5'-diphospho- glucuronosyltransferase enzymes by rifampin. The enhancement of hydromorphone elimination should be considered when managing pain of patients who are treated with strong enzyme inducers.

Evaluating the hip-flask defence using analytical data from ethanol and ethyl glucuronide. A comparison of two models.

AIM: Claimed intake of alcohol after a traffic incident, called the hip-flask defence, can be objectively assessed by different methods. One of them is the use of two consecutive ethanol concentrations in urine and the ratio between ethanol concentrations in urine and blood. Another one is the concentrations of ethyl glucuronide (EtG) and ethyl sulphate (EtS) in blood and their ratio to ethanol. The experimental basis for both these models is from single dose studies only. The aim of this study was therefore to describe the kinetics of ethanol, EtG and EtS after ingestion of two repeated doses of ethanol and to investigate the usefulness of the different models for the assessment of the hip-flask defence. METHODS: Thirty-five subjects ingested a first dose of 0.51 g of ethanol per kilo body weight, and two hours later a second dose (the hip-flask drink) of 0.25, 0.51 or 0.85 g of ethanol per kilo body weight. Ten urine and 17 blood samples were collected and analysed for ethanol, EtG and EtS using fully validated methods. It was investigated if all subjects fulfilled the criteria for recent drinking, according to the two different models, when using the samples collected 180-240 minutes after start of first dose drinking. According to the first model, increase in urinary ethanol concentrations and a ratio UAC/BAC below 1.3 indicated recent drinking. According to the second model, increase in blood EtG concentrations and a ratio ethanol (g/kg)/EtG (mg/L) above 1 indicated recent drinking. RESULTS: All subjects in the high dose group fulfilled all criteria for recent drinking. One subject in the medium dose group and nine subjects in the low dose group failed to show increasing UAC and/or a UAC/BAC ratio below 1.3. One subject in the low dose group failed to show increasing concentrations of blood EtG, but all subjects showed a ratio ethanol/EtG above 1. CONCLUSIONS: The present study showed, by the use of experimental data, that both two models used to investigate the hip-flask defence can be used, but only when the hip-flask dose is sufficiently high.

The role of ethanol in fatalities in Jeddah, Saudi Arabia.

In Saudi Arabia, alcohol consumption is prohibited by law, but interpreting postmortem ethanol can be complicated by its postmortem production. This study developed and validated a method using headspace gas chromatography with flame ionization detection and liquid chromatography tandem mass spectroscopy to detect ethanol and its polar metabolites (ethyl glucuronide [EtG] and ethyl sulfate [EtS]) in postmortem blood and urine specimens, respectively. All calibration curves were linear with coefficients of determination greater than 0.999. The limits of detection ranged 4.5-5.0mg/dL for ethanol and 0.05-0.06mg/L for EtG and EtS. The limits of quantification were 10.0mg/dL for ethanol and 0.075mg/L for EtG and EtS. Within-run precision was less than 11% for all analytes of interest. Matrix effects for EtG and EtS ranged 3-47%. After excluding matrix effects, analytical recoveries ranged 72-100%. This validated method was then used for routine postmortem forensic toxicology analyses in 592 routine postmortem cases to distinguish between antemortem ethanol consumption and its postmortem microbial formation. Among them, 98 blood samples (17%) were positive for ethanol or its polar metabolites. Thirty-two of these cases (33%) were positive for EtG and EtS and therefore due to antemortem ethanol consumption. The remaining 66 (67%) cases were negative for both EtG and EtS and therefore due to postmortem ethanol synthesis. Because this is the first study to report the problem of alcohol consumption in Saudi Arabia, further studies are essential for validating these findings.

The Relationship Between Ingested Dose of Ethanol and Amount of Ethyl Glucuronide Formed in Blood.

A positive non-linear relation between the dose of ethanol ingested and the area under the curve (AUC) for ethyl glucuronide (EtG) in urine is previously observed. The relation between both doses and AUC of ethanol and the AUC for EtG in blood is not previously published, and this study aimed to investigate this relationship. After an overnight fast, 10 healthy volunteers ingested 0.5-g ethanol per kilo body weight (low dose) in one occasion and 1.0-g ethanol per kilo body weight (high dose) in the next occasion. Results showed that there was a significant higher median ratio between blood AUC for EtG and dose of ethanol in the high-dose (8.99; range 7.37-10.94) group compared to the low-dose (5.02; range 4.25-6.15) group (P = 0.005). The median ratio between the AUC for EtG and AUC for ethanol was actually significantly higher in the low-dose (1.77; range 1.51-2.24) group compared to the high-dose (1.67; range 1.30-2.02) group (P = 0.005), although values are quite similar. This study therefore showed that the ratio between the AUC for EtG in blood and dose of ethanol is higher after intake of 1.0 g/kg than 0.5 g/kg. This pattern is however not seen when AUC for EtG is compared to AUC for ethanol. Results therefore support that the percentage of ethanol converted to EtG is not increasing when the doses increase. An explanation for the positive non-linear relation previously observed between the dose of ethanol ingested and amount of EtG formed may be a relative higher first-pass metabolism of ethanol at lower doses.

Assessment of recent alcohol consumption by detecting ethyl glucuronide and ethyl sulphate level among traffic accident patients.

Objective: This study aims to explain whether EtG and EtS, which are two non-oxidative ethanol metabolites secreted by the liver and used as markers of alcohol intake related to an incident, were also present in whole blood samples of patients with traffic accident injuries.Methods: EtG and EtS concentrations in all of the whole blood samples were sent to the Forensic Medicine Department of Cukurova University for testing Blood Alcohol Concentrations (BAC) via validated liquid chromatography-tandem mass spectrometer method.Results: The scope of the study covers 200 patients that were admitted to medical services within 12 h following the incident. Of all patients 16.5% were found to be BAC positive, while 35.5 and 23.5 were positive for EtG and EtS, respectively. All samples with a positive BAC result were also positive for EtG and EtS.Conclusion: Detection of EtG and EtS, minor metabolites along with ethanol in the blood can serve as an objective tool to provide further analysis regarding alcohol-related traffic accidents at emergency services.

Ethyl glucuronide, a marker of alcohol consumption, correlates with metabolic markers of oxidant stress but not with hemolysis in stored red blood cells from healthy blood donors.

BACKGROUND: Red blood cell (RBC) storage in the blood bank is associated with the progressive accumulation of oxidant stress. While the mature erythrocyte is well equipped to cope with such stress, recreative habits like alcohol consumption may further exacerbate the basal level of oxidant stress and contribute to the progress of the storage lesion. STUDY DESIGN AND METHODS: RBC levels of ethyl glucuronide, a marker of alcohol consumption, were measured via ultra-high-pressure liquid chromatography coupled with high-resolution mass spectrometry. Analyses were performed on 599 samples from the recalled donor population at Storage Days 10, 23, and 42 (n = 250), as part of the REDS-III RBC-Omics (Recipient Epidemiology Donor Evaluation Study III Red Blood Cell-Omics) study. This cohort consisted of the 5th and 95th percentile of donors with extreme hemolytic propensity out of the original cohort of 13,403 subjects enrolled in the REDS-III RBC Omics study. Ehtyl glucuronide levels were thus correlated to global metabolomics and lipidomics analyses and RBC hemolytic propensity. RESULTS: Ethyl glucuronide levels were positively associated with oxidant stress markers, including glutathione consumption and turnover, methionine oxidation, S-adenosylhomocysteine accumulation, purine oxidation, and transamination markers. Decreases in glycolysis and energy metabolism, the pentose phosphate pathway and ascorbate system were observed in those subjects with the highest levels of ethyl glucuronide, though hemolysis values were comparable between groups. CONCLUSION: Though preliminary, this study is suggestive that markers of alcohol consumption are associated with increases in oxidant stress and decreases in energy metabolism with no significant impact on hemolytic parameters in stored RBCs from healthy donor volunteers.

Performance of PEth Compared With Other Alcohol Biomarkers in Subjects Presenting For Occupational and Pre-Employment Medical Examination.

AIMS: To compare the performance of short- and long-term alcohol biomarkers for the evaluation of alcohol drinking in employment-related health controls. METHODS: The 519 blood samples originated from 509 patients (80% men) presenting at occupational health units and medical centers at employment agencies for the evaluation of risky drinking. The laboratory investigation comprised the measurement of phosphatidylethanol (PEth 16:0/18:1), carbohydrate-deficient transferrin (CDT; % disialotransferrin), gamma-glutamyl transferase (GGT), mean corpuscular volume (MCV), ethanol and ethyl glucuronide (EtG). RESULTS: Many samples tested positive for acute (57%) and chronic (69%) alcohol biomarkers. PEth was the single most positive biomarker (64%; cut-off 0.05 µmol/l or 35 µg/l) and the only positive chronic biomarker in 100 cases. The highest PEth concentrations were seen in samples positive for all chronic biomarkers, followed by those also being CDT positive (cut-off 2.0%). All 126 CDT-positive samples were positive for PEth using the lower reporting limit (≥0.05 µmol/l) and for 114 cases (90%) also using the higher limit (≥0.30 µmol/l or 210 µg/l). In the CDT-positive cases, the PEth median concentration was 1.71 µmol/l, compared with 0.45 µmol/l for the CDT-negative cases (P < 0.0001). PEth and CDT values were correlated significantly (r = 0.63, P < 0.0001). Among the EtG-positive cases (≥1.0 ng/ml), 95% were also PEth positive, and all ethanol-positive cases (≥0.10 g/l) were also PEth positive. CONCLUSIONS: For optimal detection of drinking habits, using a combination of short- and long-term alcohol biomarkers provided best information. PEth was the single most positive alcohol biomarker, whereas GGT and MCV offered little additional value over PEth and CDT.

ß-Glucuronidase- and OATP2B1-mediated drug interaction of scutellarin in Dengzhan Xixin Injection: A formulation aspect.

Scutellarin is the major and active constituent of Dengzhan Xixin Injection (DZXX), a traditional Chinese medicine prepared from the aqueous extract of Erigeron breviscapus and widely used for the treatment of various cerebrovascular diseases in clinic. In present study, the possible pharmacokinetic differences of scutellarin after intravenous administration of scutellarin alone or DZXX were explored. Additional, the potential roles of ß-glucuronidase (GLU) and OATP2B1 in drug-drug interaction (DDI) between scutellarin and constituents of DZXX were further evaluated in vitro. The plasma concentration, urinary and biliary excretion of scutellarin in rats after administration of DZXX, were significantly higher than those received scutellarin, while pharmacokinetic profile of Apigenin 7-O-glucuronide (AG) in rats was similar no matter AG or DZXX group. Furthermore, higher concentration in brain and plasma, however, lower level of scutellarin in intestine were observed after intravenous administration of DZXX. Finally, AG and caffeoylquinic acid esters were found to significantly inhibit GLU and OATP2B1 in vitro, which might explain, at least in part, the pharmacokinetic DDI between scutellarin and other chemical constituents in DZXX. The findings provided deep insight into the prescription-formulating principle in DZXX for treating the cerebrovascular diseases.

An LC-MS/MS method for comparing the stability of ethanol's non-oxidative metabolites in dried blood spots during 90 days.

Problems of stability were found for biomarkers of alcohol consumption: ethyl glucuronide (EtG), ethyl sulfate (EtS), phosphatidylethanols (PEths), and fatty acid ethyl esters (FAEEs) in whole blood. The purpose of this study was to establish a method for the determination of these four kinds of ethanol's non-oxidative metabolites in dried blood spots (DBS) by liquid chromatography tandem mass spectrometry (LC-MS/MS), and to evaluate their stability. In this method, 50 µL of human blood was spotted onto a filter paper for DBS analysis. Samples were extracted by methanol, reconstituted by 2-propanol, and injected into the LC-MS/MS system. Limits of detection were among 0.5-50 ng/mL, and deviations in accuracy and precision were all lower than 15% at three quality control levels. The stability of the four kinds of ethanol non-oxidative metabolites in DBS was investigated during a 90-day range under three temperatures, -20 °C, 4 °C, and 25 °C. EtG and EtS showed a high level of stability in DBS in the 90-day range, regardless of the temperature. FAEEs were unstable after three days. PEths showed stability within 15 days in postmortem DBS and 60 days in antemortem DBS, respectively, at all temperatures.

Analysis of ethyl glucuronide and ethyl sulfate in blood to determine the absorption or elimination phase of alcohol for Korean.

This study was conducted to understand alcohol kinetics for Koreans and to determine whether an individual is in absorption phase or elimination phase at the time of blood collection by analyzing of ethyl glucuronide and ethyl sulfate in blood. A total of 50 healthy adults was selected and assigned to drink 1g of ethanol per kg body weight of individual within 1h. Blood samples were then collected every 15min for the first 3h, 30min next 3h, and 1h last 9h. Urine samples were also collected from the individual, but not under the controlled environment. All samples were then analyzed by gas chromatography with a flame ionization detector (GC-FID) for alcohol and liquid chromatography-mass/mass spectrometry (LC-MS/MS) for EtG and EtS. The maximum BAC (Cmax) was 0.138% (g/100mL) in average under the controlled experimental condition. Alcohol elimination rates (ß) in average were 0.020% for male and 0.024% for female, respectively. It was found that the ratio of UAC and BAC was less than 1 in the absorption phase and the average ratio of UAC and BAC was 1.47 in the elimination phase. The comparison of BAC (g/L) and EtG (mg/L) absorption and elimination curves showed that the intersection time was 3.9h in average. It is shown that the ratio of EtG (mg/L)/BAC (g/L) is higher than 1, the individual would be in elimination phase of BAC. At the time of Cmax, the ratio of EtG (mg/L)/BAC (g/L) was 0.255±0.132 (SD) in average.

Identification of equol-7-glucuronide-4'-sulfate, monoglucuronides and monosulfates in human plasma of 2 equol producers after administration of kinako by LC-ESI-MS.

Equol is a product formed during the biotransformation of the naturally occurring isoflavone daidzein by intestinal bacteria. The role of equol in the prevention of several hormone-dependent diseases such as prostate cancer and osteoporosis as well as vasomotor symptoms has been extensively investigated. Equol primarily occurs in the form of major metabolites such as glucuronides and sulfates, while intact equol has been detected at only ca. 1% in human plasma. However, to date, conjugated metabolites have been evaluated by measuring the free equol obtained after selective enzymatic hydrolysis. Thus, the precise types of conjugates circulating in vivo and the position(s) of the conjugation sites on the equol skeleton have yet to be clarified. Our study describes the identification of polar equol metabolites in the plasma of 2 equol-producers obtained at 8 hours after consuming 50 g of kinako (approximately 37 mg of daidzein). The structural identification of these conjugated metabolites in plasma was performed by comparison to the LC-ESI-MS n and 1H-NMR spectral data of the corresponding chemically synthesized compounds. The results of the LC-ESI-MS/MS analysis indicated that the main conjugated metabolite in plasma was (S)-equol-7-glucuronide-4'-sulfate along with lower amounts of 7- and 4'-monoglucuronides as well as 7- and 4'-monosulfates.

Meprin ß metalloproteases associated with differential metabolite profiles in the plasma and urine of mice with type 1 diabetes and diabetic nephropathy.

BACKGROUND: Meprin metalloproteases are abundantly expressed in the brush border membranes of kidney proximal tubules and small intestines. Meprins are also expressed in podocytes and leukocytes (monocytes and macrophages). Meprins are implicated in the pathophysiology of diabetic nephropathy (DN) but underlying mechanisms are not fully understood. Single nucleotide polymophisms (SNPs) in the meprin ß gene were associated with DKD in human subjects. Furthermore, meprin α and ß double deficiency resulted in more severe kidney injury and higher mortality rates in mice with Streptozotocin (STZ)-induced type 1 diabetes. Identification of meprin substrates has provided insights on how meprins could modulate kidney injury. Meprin targets in the kidney include extracellular matrix (ECM) proteins, modulators of inflammation, and proteins involved in the protein kinase A (PKA) and PKC signaling pathways. The current study used a global metabolomics approach to determine how meprin ß expression impacts the metabolite milieu in diabetes and DKD. METHODS: Low dose STZ was used to induce type 1 diabetes in 8-week old wild-type (WT) and meprin ß knockout (ßKO) mice. Blood and urine samples were obtained at 4 and 8 weeks post-STZ injection. Assays for albumin, creatinine, neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule - 1 (KIM-1), and cystatin C were used for biochemical assessment of kidney injury. Data for biomarkers of kidney injury utilized two-way ANOVA. Metabolomics data analysis utilized UPLC-QTOF MS and multivariate statistics. RESULTS: The number of metabolites with diabetes-associated changes in levels were significantly higher in the WT mice when compared to meprin ßKO counterparts. Annotated meprin ß expression-associated metabolites with strong variable importance in projection (VIP) scores play roles in lipid metabolism (LysoPC(16:1(9Z)), taurocholic acid), amino acid metabolism (indoxyl sulfate, hippuric acid), and neurotransmitter/stress hormone synthesis (cortisol, 3-methoxy-4-hydroxyphenylethylene glycolsulfate, homovanillic acid sulfate). Metabolites that associated with meprin ß deficiency include; 3,5-dihydroxy-3',4'-dimethoxy-6,7-methylenedioxyflavone 3-glucuronide, pantothenic acid, and indoxyl glucuronide (all decreased in plasma). CONCLUSION: Taken together, the annotated metabolites suggest that meprin ß impacts complications of diabetes such as DKD by altering distinct metabolite profiles.

Identification and characterization of chemical constituents in Dengzhan Shengmai Capsule and their metabolites in rat plasma by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

Dengzhan Shengmai Capsule (DZSMC) is a traditional Chinese medicine (TCM) formula with remarkable clinical effect in the treatment of stroke sequelae. Exploring the components of DZSMC and detecting the absorbed prototype constituents and metabolites in blood are of great significance to clarify the effective substances of this prescription. Here, a reliable method using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) was established for the comprehensive analysis of chemical constituents of DZSMC and their metabolites in rat plasma after gastric perfusion. Two acquisition modes, including MSE mode and Fast DDA mode, were performed for acquiring more precursor ions and cleaner precursor-product ions background during the study of constituents of DZSMC. As a result, a total of 125 constituents were unambiguously characterized or tentatively identified. For the first time, a total of 92 components, including 44 prototype components and 48 metabolites were unambiguously or tentatively identified in rat plasma. The metabolic pathways included phase I reactions (hydration, hydrogenation, oxidation, demethylation and hydroxylation) and phase II reactions (conjugation with glucuronide, sulfate and methyl). Furthermore, the metabolites from caffeic acid and scutellarin were characterized and validated by phase II metabolic reactions in vitro, which could be established as a simulated in vivo environment of metabolites identification and verification of TCM formula. It is the first systematic study on metabolism of DZSMC in vivo and could also provide a valid analytical strategy for characterization of the chemical compounds and metabolites of TCM formula.

A Novel Ileocolonic Release Peppermint Oil Capsule for Treatment of Irritable Bowel Syndrome: A Phase I Study in Healthy Volunteers.

INTRODUCTION: Peppermint oil (PO) has been shown to reduce abdominal pain in patients with irritable bowel syndrome (IBS). PO is assumed to induce intestinal smooth muscle relaxation and desensitization of nociceptive nerve afferents. To increase colonic PO concentration, an ileocolonic release peppermint oil (IC-PO) capsule has been developed. The aim of this study was to compare pharmacokinetic parameters of the currently available small intestinal release PO (SI-PO) and the novel IC-PO. METHODS: In this randomized, double-blind, crossover study, subjects received 182 mg of either SI-PO or IC-PO in a crossover design with a washout period of more than 14 days. Blood samples were collected to determine menthol glucuronide concentrations. RESULTS: Eight healthy volunteers (50% female, median age 22) were included. The time to reach the maximum concentration (Tmax) of IC-PO was significantly longer compared to SI-PO with a median (IQR) of 360 (360-405) versus 180 (120-180) min. The lag time (Tlag) was significantly longer with a median (IQR) of 225 (204-284) for IC-PO compared to 37 (6-65) min for SI-PO. The areas under the menthol glucuronide plasma concentration-time curves were significantly smaller with a median (IQR) of 2331 µg h/L (2006-2510) for IC-PO compared to 2623 µg h/L (2471-2920) for SI-PO. No significant differences were found in peak concentrations and elimination half-lives. CONCLUSION: IC-PO has a significantly delayed peak menthol glucuronide concentration and Tlag, both pointing to the release of PO in the more distal part of the intestine. This may enhance therapeutic efficacy as it results in increased exposure of colonic mucosal afferents to the PO. A randomized controlled trial investigating the efficacy of SI and IC-PO in IBS is currently ongoing. TRIAL REGISTRATION: ClinicalTrials.gov identifier, NCT02291445, EudraCT database 2014-004195-32.

The influence of caffeine on ethyl glucuronide levels in rat serum and in rat hair.

BACKGROUND: Ethanol and caffeine are the most widely used psychoactive substances in the world, with an observed steady increase in the combined consumption of alcohol and caffeine. Specific signs of ethanol-caffeine interactions have been reported both in humans and in animals. The metabolic effects of these interactions have not been fully elucidated. There are no published reports on the influence of caffeine on ethyl glucuronide (EtG) formation. EtG is a direct metabolite of ethanol and is very often used as a biomarker of alcohol consumption. Here, we investigated the influence of caffeine on the formation of EtG in rat plasma and EtG incorporation into the hair. METHODS: Studies were conducted on three male Wistar rat groups, each receiving either ethanol at 3g/kg/day, ethanol (at the same dose) with caffeine at 3mg/kg/day, or caffeine at 3mg/kg/day for four weeks. EtG and caffeine levels were evaluated in hair and in blood after the last administration. RESULTS: Blood EtG levels after the administration of ethanol together with caffeine were significantly higher than after the administration of ethanol alone. EtG levels in rat hair in the ethanol-and-caffeine group were also higher than in the ethanol-only group, but the difference was not statistically significant. CONCLUSION: This study shows the possible effect of ethanol and caffeine co-administration on EtG formation. Caffeine stimulates EtG synthesis resulting in increased blood and, possibly, hair levels of this metabolite. However, the role of these changes in estimating alcohol consumption requires further studies.

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 com - plements the existing spectrum of tests with high specificity (48-89%) and sensi - tivity (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.

Stability of Ethyl Glucuronide, Ethyl Sulfate, Phosphatidylethanols and Fatty Acid Ethyl Esters in Postmortem Human Blood.

The lack of systematic studies on the stability of ethanol's non-oxidative metabolites in postmortem specimens restricts their use in forensic cases. This study aimed to compare the stability of ethyl glucuronide (EtG), ethyl sulfate (EtS), phosphatidylethanols (PEths) and fatty acid ethyl esters (FAEEs) in postmortem human blood. Three groups were established based on the level and source of ethanol: the blank group, the ethanol-spiked group and the ethanol-positive group. Each group contained six blood samples from different corpses. The samples in each group were placed at 37, 25, 4 and -20°C. Every 24 h for 7 days, 50 µL was collected from each sample. The levels of EtG, EtS, PEths and FAEEs were determined by liquid chromatography-mass spectrometry, and their stability was evaluated. EtG was not detected in the blank group, but it was found in samples in the ethanol-spiked group placed at 37°C, and it was degraded in the ethanol-positive group at 37 and 25°C. EtS showed no change in any of the groups. PEths were not detected in the blank group, but formation was found in the ethanol-spiked group at all temperatures. In the ethanol-positive group, PEth levels fluctuated at 37°C, decreased at 25°C and increased at -20°C. FAEEs were generated in the blank group and in the ethanol-spiked group at all temperatures. In the ethanol-positive group, FAEEs were degraded at 37 and 25°C but were generated at 4 and -20°C. EtS is a reliable biomarker of ethanol consumption, and EtG could be used as a biomarker at low temperatures (4 and -20°C), but PEths and FAEEs are not appropriate biomarkers of ethanol consumption.