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Sample records for ethyl glucuronide etg

  1. A wearable biochemical sensor for monitoring alcohol consumption lifestyle through Ethyl glucuronide (EtG) detection in human sweat.

    PubMed

    Selvam, Anjan Panneer; Muthukumar, Sriram; Kamakoti, Vikramshankar; Prasad, Shalini

    2016-03-21

    We demonstrate for the first time a wearable biochemical sensor for monitoring alcohol consumption through the detection and quantification of a metabolite of ethanol, ethyl glucuronide (EtG). We designed and fabricated two co-planar sensors with gold and zinc oxide as sensing electrodes. We also designed a LED based reporting for the presence of EtG in the human sweat samples. The sensor functions on affinity based immunoassay principles whereby monoclonal antibodies for EtG were immobilized on the electrodes using thiol based chemistry. Detection of EtG from human sweat was achieved through chemiresistive sensing mechanism. In this method, an AC voltage was applied across the two coplanar electrodes and the impedance across the sensor electrodes was measured and calibrated for physiologically relevant doses of EtG in human sweat. EtG detection over a dose concentration of 0.001-100 μg/L was demonstrated on both glass and polyimide substrates. Detection sensitivity was lower at 1 μg/L with gold electrodes as compared to ZnO, which had detection sensitivity of 0.001 μg/L. Based on the detection range the wearable sensor has the ability to detect alcohol consumption of up to 11 standard drinks in the US over a period of 4 to 9 hours.

  2. A wearable biochemical sensor for monitoring alcohol consumption lifestyle through Ethyl glucuronide (EtG) detection in human sweat

    PubMed Central

    Panneer Selvam, Anjan; Muthukumar, Sriram; Kamakoti, Vikramshankar; Prasad, Shalini

    2016-01-01

    We demonstrate for the first time a wearable biochemical sensor for monitoring alcohol consumption through the detection and quantification of a metabolite of ethanol, ethyl glucuronide (EtG). We designed and fabricated two co-planar sensors with gold and zinc oxide as sensing electrodes. We also designed a LED based reporting for the presence of EtG in the human sweat samples. The sensor functions on affinity based immunoassay principles whereby monoclonal antibodies for EtG were immobilized on the electrodes using thiol based chemistry. Detection of EtG from human sweat was achieved through chemiresistive sensing mechanism. In this method, an AC voltage was applied across the two coplanar electrodes and the impedance across the sensor electrodes was measured and calibrated for physiologically relevant doses of EtG in human sweat. EtG detection over a dose concentration of 0.001–100 μg/L was demonstrated on both glass and polyimide substrates. Detection sensitivity was lower at 1 μg/L with gold electrodes as compared to ZnO, which had detection sensitivity of 0.001 μg/L. Based on the detection range the wearable sensor has the ability to detect alcohol consumption of up to 11 standard drinks in the US over a period of 4 to 9 hours. PMID:26996103

  3. Quantification of fatty acid ethyl esters (FAEE) and ethyl glucuronide (EtG) in meconium for detection of alcohol abuse during pregnancy: Correlation study between both biomarkers.

    PubMed

    Cabarcos, Pamela; Tabernero, María Jesús; Otero, José Luís; Míguez, Martha; Bermejo, Ana María; Martello, Simona; De Giovanni, Nadia; Chiarotti, Marcello

    2014-11-01

    This article presents results from 47 meconium samples, which were analyzed for fatty acid ethyl esters (FAEE) and ethyl glucuronide (EtG) for detection of gestational alcohol consumption. A validated microwave assisted extraction (MAE) method in combination with GC-MS developed in the Institute of Forensic Science (Santiago de Compostela) was used for FAEE and the cumulative concentration of ethyl myristate, ethyl palmitate and ethyl stearate with a cut-off of 600ng/g was applied for interpretation. A simple method for identification and quantification of EtG has been evaluated by ultrasonication followed solid phase extraction (SPE). Successful validation parameters were obtained for both biochemical markers of alcohol intake. FAEE and EtG concentrations in meconium ranged between values lower than LOD and 32,892ng/g or 218ng/g respectively. We have analyzed FAEE and EtG in the same meconium aliquot, enabling comparison of the efficiency of gestational ethanol exposure detection. Certain agreement between the two biomarkers was found as they are both a very specific alcohol markers, making it a useful analysis for confirmation. PMID:25137651

  4. Quantification of fatty acid ethyl esters (FAEE) and ethyl glucuronide (EtG) in meconium from newborns for detection of alcohol abuse in a maternal health evaluation study.

    PubMed

    Bakdash, Abdulsallam; Burger, Pascal; Goecke, Tamme W; Fasching, Peter A; Reulbach, Udo; Bleich, Stefan; Hastedt, Martin; Rothe, Michael; Beckmann, Matthias W; Pragst, Fritz; Kornhuber, Johannes

    2010-04-01

    Fatty acid ethyl esters (FAEE) and ethyl glucuronide (EtG) were determined in 602 meconium samples in a maternal health evaluation study for detection of gestational alcohol consumption. A validated headspace solid phase microextraction method in combination with GC-MS was used for FAEE and the cumulative concentration of ethyl palmitate, ethyl linoleate, ethyl oleate, and ethyl stearate with a cut-off of 500 ng/g was applied for interpretation. A new and simple method was developed and validated for quantification of EtG from 10-20 mg meconium with D(5)-EtG as internal standard consisting of 30 min. extraction with methanol/water (1:1, v/v), evaporation of methanol, filtration of the aqueous solution through a cellulose filter and injection into LC-MS-MS. The limits of detection and quantification for EtG were 10 and 30 ng/g, the recovery 86.6 to 106.4% and the standard deviation of the concentrations ranged from 13% at 37 ng/g to 5% at 46,700 ng/g (N = 6). FAEE above the cut-off were found in 43 cases (7.1%) with cumulative concentrations between 507 and 22,580 ng/g and with one outlier of about 150,000 ng/g (EtG not detected). EtG was detected in 97 cases (16.3%) and concentrations between LOD and 10,200 ng/g with another outlier of 82,000 ng/g (FAEE 10,500 ng/g). Optimal agreement between the two markers was obtained with a cut-off for EtG of 274 ng/g and 547 cases with both FAEE- and EtG-negative, 33 cases with both FAEE- and EtG-positive, nine cases with FAEE-positive and EtG-negative, and seven cases with FAEE-negative and EtG-positive. Differences in physical, chemical, and biochemical properties and in the pharmacokinetic behavior are discussed as reasons for the deviating cases. In none of the 602 cases, serious alcohol consumption was reported by the mothers and no evidence for gestational ethanol exposure was observed in the medical investigation of the newborns. It is concluded that the combined use of FAEE and EtG in meconium as markers for fetal

  5. Detecting alcohol abuse: traditional blood alcohol markers compared to ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs) measurement in hair.

    PubMed

    Hastedt, Martin; Büchner, Mara; Rothe, Michael; Gapert, René; Herre, Sieglinde; Krumbiegel, Franziska; Tsokos, Michael; Kienast, Thorsten; Heinz, Andreas; Hartwig, Sven

    2013-12-01

    Alcohol abuse is a common problem in society; however, the technical capabilities of evaluating individual alcohol consumption using objective biomarkers are rather limited at present. In recent years research has focused on alcohol markers using hair analysis but data on performance and reliable cut-off values are still lacking. In this study 169 candidates were tested to compare traditional biomarkers, such as carbohydrate-deficient-transferrin (CDT), gamma glutamyl transferase (GGT), aspartate amino transferase, alanine amino transferase and the mean corpuscular volume of the erythrocytes, with alcohol markers detectable in hair such as ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs). This study revealed that EtG, GGT and CDT showed the best results, demonstrating areas under the curve calculated from receiver operating characteristics of 0.941, 0.943 and 0.899 respectively. The lowest false-negative and false-positive rates were obtained by using a combined interpretation system for hair EtG and FAEEs. All markers demonstrated only low to moderate correlations. Optimum cut-off values for differentiation between social and chronic excessive drinking calculated for hair EtG and FAEEs were 28 pg/mg and 0.675 ng/mg, respectively. The critical values published in the "Consensus on Alcohol Markers 2012" by the Society of Hair Testing were confirmed.

  6. Did you drink alcohol during pregnancy? Inaccuracy and discontinuity of women's self-reports: On the way to establish meconium ethyl glucuronide (EtG) as a biomarker for alcohol consumption during pregnancy.

    PubMed

    Eichler, Anna; Grunitz, Juliane; Grimm, Jennifer; Walz, Lisa; Raabe, Eva; Goecke, Tamme W; Beckmann, Matthias W; Kratz, Oliver; Heinrich, Hartmut; Moll, Gunther H; Fasching, Peter A; Kornhuber, Johannes

    2016-08-01

    Consuming alcohol during pregnancy is one of the most verified prenatal risk factors for impaired child development. Information about the amount of alcohol consumed prenatally is needed to anticipate negative effects and to offer timely support. Women's self-reports are not reliable, often influenced by social stigmas and retrospective recall bias, causing biomarkers of intrauterine ethanol exposure to become more and more relevant. The present study compares both women's gestational and retrospective self-reports of prenatal alcohol consumption with levels of ethyl glucuronide (EtG) in meconium. Women (n = 180) gave self-reports of prenatal alcohol consumption both during their 3rd trimester (gestational self-report) and when their children were 6-8 years old (retrospective self-report). Child meconium was collected after birth and analyzed for EtG. No individual feedback of children's EtG level was given to the women. All analyses were run separately for two cut-offs: 10 ng/g (limit of detection) and 120 ng/g (established by Goecke et al., 2014). Mothers of children with EtG values above 10 ng/g (n = 42) tended to report prenatal alcohol consumption more frequently. There was no trend or significance for the EtG cut-off of 120 ng/g (n = 26) or for retrospective self-report. When focusing on women who retrospectively reported alcohol consumption during pregnancy, a claim to five or more consumed glasses per month made an EtG over the 10 ng/g and the 120 ng/g cut-off more probable. Women whose children were over the 10 ng/g EtG cut-off were the most inconsistent in their self-report behavior, whereas the consistency in the above 120 ng/g EtG group was higher than in any other group. The next step to establish EtG as a biomarker for intrauterine alcohol exposure is to correlate EtG values in meconium with child developmental impairments. PMID:27565755

  7. [Detection and application of ethyl glucuronide in forensic toxicology].

    PubMed

    Zhao, Hui; Zhuo, Xian-yi; Shen, Bao-hua

    2009-02-01

    Ethyl glucuronide is a specific metabolite of ethanol. There have been plenty of articles referring its pharmacokinetics, detection and application as a specific bio-marker of alcohol intake. This article reviews various analytical methods of EtG, relationship between EtG quantification and ethanol intake, and criteria for determining chronic alcohol abuse, and origin of ethanol found in the cadavers by EtG analysis. EtG has its potential application in forensic toxicology. PMID:19397218

  8. Influence of Gilbert's syndrome on the formation of ethyl glucuronide.

    PubMed

    Huppertz, Laura M; Gunsilius, Leonie; Lardi, Christelle; Weinmann, Wolfgang; Thierauf-Emberger, Annette

    2015-09-01

    A drinking experiment with participants suffering from Gilbert's syndrome was performed to study the possible influence of this glucuronidation disorder on the formation of ethyl glucuronide (EtG). Gilbert's syndrome is a rather common and, in most cases, asymptomatic congenital metabolic aberration with a prevalence of about 5 %. It is characterized by a reduction of the enzyme activity of the uridine diphosphate glucuronosyltransferase (UGT) isoform 1A1 up to 80 %. One of the glucuronidation products is EtG, which is formed in the organism following exposure to ethanol. EtG is used as a short-term marker for ethyl alcohol consumption to prove abstinence in various settings. After 2 days of abstinence from ethanol and giving a void urine sample, 30 study participants drank 0.1 L of sparkling wine (9 g ethanol). 3, 6, 12, and 24 h after drinking, urine samples were collected. 3 hours after drinking, an additional blood sample was taken, in which liver enzyme activities, ethanol, hematological parameters, and bilirubin were measured. EtG and ethyl sulfate (EtS), another short-term marker of ethanol consumption, were determined in the urine samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS); creatinine was measured photometrically. In all participants, EtG and EtS were detected in concentrations showing a wide range (EtG: 3 h sample 0.5-18.43 mg/L and 6 h sample 0.67-13.8 mg/L; EtS: 3 h sample 0.87-6.87 mg/L and 6 h sample 0.29-4.48 mg/L). No evidence of impaired EtG formation was found. Thus, EtG seems to be a suitable marker for ethanol consumption even in individuals with Gilbert's syndrome. PMID:25680552

  9. Detection of ethyl glucuronide in blood spotted on different surfaces.

    PubMed

    Winkler, M; Kaufmann, E; Thoma, D; Thierauf, A; Weinmann, W; Skopp, G; Alt, A

    2011-07-15

    This study aims to show that sensitive detection of ethyl glucuronide in dried blood spotted onto various surfaces after a period of 24h is feasible. At present, there is insufficient information how tightly ethyl glucuronide (EtG) binds to various materials and how easily it can be eluted. 4ml aliquots of blood samples obtained from seven volunteers after consumption of alcoholic beverages were applied to six different surfaces. After drying and a 24h-storage at 20±2°C the samples were re-dissolved in water, and EtG was subsequently analyzed by a LC-MS Paul-type ion trap. A comparison was made between dried and corresponding fluid samples. EtG was detectable in all subjects' samples following consumption of alcohol. EtG was also detectable after a storage time of four weeks at 4°C in whole blood that had been preserved with EDTA. EtG was detectable in all samples dried on different surfaces and its concentration remained relatively constant irrespective of the particular condition of the material. Detection of EtG in blood spots from the scene may indicate recent alcohol consumption in cases where collection of blood remained undone or could not be performed. PMID:21641739

  10. Testing for ethanol markers in hair: discrepancies after simultaneous quantification of ethyl glucuronide and fatty acid ethyl esters.

    PubMed

    Kintz, P; Nicholson, D

    2014-10-01

    The hair of 97 cases were analysed for ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEE, including ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate) according to the Society of Hair Testing guidelines to examine the role of both tests in documenting chronic excessive alcohol drinking, particularly when the results are in contradiction. 27 (27.8%) results were EtG negative and FAEE positive, when applying the SoHT cut-offs, probably due to the use of alcohol-containing hair products. Four cases (4.1%) were EtG positive and FAEE negative that were attributed to the use of herbal lotions containing EtG. PMID:24794020

  11. Voucher-Based Reinforcement for Alcohol Abstinence Using the Ethyl-Glucuronide Alcohol Biomarker

    ERIC Educational Resources Information Center

    McDonell, Michael G.; Howell, Donelle N,; McPherson, Sterling; Cameron, Jennifer M.; Srebnik, Debra; Roll, John M.; Ries, Richard K.

    2012-01-01

    This study assessed the effects of a contingency management (CM) intervention for alcohol consumption in 10 alcohol-dependent participants. An ABCA design was used. Vouchers were provided contingent on results of ethyl glucuronide (EtG) urine tests (an alcohol biomarker with a 2-day detection period) and alcohol breath tests during the C phase.…

  12. Practical use of ethyl glucuronide and ethyl sulfate in postmortem cases as markers of antemortem alcohol ingestion.

    PubMed

    Høiseth, Gudrun; Karinen, Ritva; Christophersen, Asbjørg; Mørland, Jørg

    2010-03-01

    In postmortem toxicology, it could be difficult to determine whether a positive blood ethanol concentration reflects antemortem ingestion or postmortem synthesis of alcohol. Measurement of the nonoxidative ethanol metabolite ethyl glucuronide (EtG) has been suggested as a marker of antemortem ingestion of alcohol, but EtG might degrade postmortem which could make interpretation difficult. So far, the published articles concern EtG only. Another nonoxidative metabolite, ethyl sulfate (EtS), which is more stable, has therefore been included in this study. We present a material of 36 deaths where postmortem formation of ethanol was suspected and where both EtG and EtS were measured in blood and urine to assist the interpretation. In 19 cases, EtG and EtS were positive in the body fluids analyzed. The median concentration of EtG and EtS in blood was 0.4 (range 0.1-23.2) and 0.9 mg/L (range 0.04-7.9), respectively. The median concentration of EtG and EtS in urine was 35.9 (range 1.0-182) and 8.5 mg/L (range 0.3-99), respectively. In another 16 cases, there was no trace of EtG or EtS in the specimens analyzed. In one case, there was inconsistency between the results of EtG and EtS; they were both positive in urine, while only EtS was positive in blood. This study showed that, out of 36 cases, antemortem ingestion of alcohol was very likely in 19 and unlikely in 16, according to EtG and EtS results. In the last case, the interpretation was more difficult. One possible explanation would be postmortem degradation of EtG in blood. PMID:19937334

  13. Examination of sex differences in fatty acid ethyl ester and ethyl glucuronide hair analysis.

    PubMed

    Gareri, Joey; Rao, Chitra; Koren, Gideon

    2014-06-01

    Clinical studies examining performance of fatty acid ethyl esters (FAEE) and ethyl glucuronide (EtG) in identifying excessive alcohol consumption have been primarily conducted in male populations. An impact of hair cosmetics in producing both false-negative EtG results and false-positive FAEE results has been demonstrated, suggesting a possible bias in female populations. This study evaluates FAEE-positive hair samples (>0.50 ng/mg) from n = 199 female and n = 73 male subjects for EtG. Higher FAEE/EtG concordance was observed amongst male over female subjects. Performance of multiple proposed EtG cut-off levels were assessed; amongst female samples, FAEE/EtG concordance was 36.2% (30 pg/mg), 36.7% (27 pg/mg), and 43.7% (20 pg/mg). Non-coloured hair demonstrated a two-fold increase in concordance (41.8 v. 20.8%) over coloured hair in the female cohort. FAEE levels did not differ between male and female subjects; however they were lower in coloured samples (p = 0.046). EtG was lower in female subjects (p = 0.019) and coloured samples (p = 0.026). A total of n = 111 female samples were discordant. Amongst discordant samples (EtG-negative), 26% had evidence of recent alcohol use including consultation histories (n = 20) and detectable cocaethylene (n = 9); 29% of discordant samples were coloured. False-negative risk with ethyl glucuronide analysis in females was mediated by cosmetic colouring. These findings suggest that combined analysis of FAEE and EtG is optimal when assessing a female population and an EtG cut-off of 20 pg/mg is warranted when using combined analysis. While concordant FAEE/EtG-positive findings constitute clear evidence, discordant FAEE/EtG findings should still be considered suggestive evidence of chronic excessive alcohol consumption. PMID:24817046

  14. Levels of ethyl glucuronide and ethyl sulfate in oral fluid, blood, and urine after use of mouthwash and ingestion of nonalcoholic wine.

    PubMed

    Høiseth, Gudrun; Yttredal, Borghild; Karinen, Ritva; Gjerde, Hallvard; Christophersen, Asbjørg

    2010-03-01

    The aim of this study is to investigate the concentrations of ethyl glucuronide (EtG) in oral fluid and both EtG and ethyl sulfate (EtS) in blood and urine following intense use of mouthwash and ingestion of nonalcoholic wine, which are proven to contain 3 mg/L EtG, 1.5 mg/L EtS, and 0.2 g/L ethanol. Twelve subjects participated in a controlled experiment. All subjects ingesting nonalcoholic wine showed urine samples negative for EtG but positive for EtS (Cmax 2.15 mg/L). All four subjects using mouthwash were negative for EtG and EtS in urine. All samples of oral fluid were negative for EtG and all samples of blood were negative for EtG and EtS. This study showed that ingestion of EtG and EtS as components of nonalcoholic wine lead to detection of urine EtS only, suggesting superior bioavailability of orally ingested EtS compared to EtG. This possibility of false-positive EtS results in urine after ingestion of nonalcoholic wine is important to remember when using EtG and EtS as relapse markers for alcohol. Finally, the study showed that a positive EtG or EtS result after accidental alcohol exposure is unlikely in blood and oral fluid. PMID:20223100

  15. Blood kinetics of ethyl glucuronide and ethyl sulphate in heavy drinkers during alcohol detoxification.

    PubMed

    Høiseth, Gudrun; Morini, Luca; Polettini, Aldo; Christophersen, Asbjørg; Mørland, Jørg

    2009-07-01

    Studies of ethyl glucuronide (EtG) blood kinetics have so far been performed on healthy volunteers with ingestion of low to moderate doses of ethanol. These data are not necessarily transferable to heavy drinkers where the consumed doses of ethanol are much higher. The aim of this study was to investigate the pharmacokinetics of EtG and ethyl sulphate (EtS) in blood in heavy drinkers after termination of alcohol ingestion. Sixteen patients from an alcohol withdrawal clinic were included directly after admission. Time of end of drinking, estimated daily intake of ethanol (EDI) and medical history were recorded. Three to five blood samples over 20-43 h were collected from each patient subsequent to admission. The median EDI was 172 g (range 60-564). The first sample was collected median 2.5 h after end of drinking (range 0.5-23.5). Two patients had levels of EtG and EtS below LOQ in all samples, the first collected 19.25 and 23.5 h after cessation of drinking, respectively. Of the remaining 14 patients, one subject, suffering from both renal and hepatic disease, showed concentrations of EtG and EtS substantially higher than the rest of the material. This patient's initial value of EtG was 17.9 mg/L and of EtS 5.9 mg/L, with terminal elimination half lives of 11.9 h for EtG and 12.5 h for EtS. Among the remaining 13 patients, the initial median values were 0.7 g/L (range 0-3.7) for ethanol, 1.7 mg/L (range 0.1-5.9) for EtG and 0.9 mg/L (range 0.1-1.9) for EtS. Elimination occurred with a median half-life of 3.3 h for EtG (range 2.6-4.3) and 3.6 h for EtS (range 2.7-5.4). In conclusion, elimination of EtG in heavy drinkers did not significantly differ from healthy volunteers, and EtS appeared to have similar elimination rate. In the present work, there was one exception to this, and we propose that this could be explained by the patient's renal disease, which would delay excretion of these conjugated metabolites. PMID:19395207

  16. Ethyl glucuronide and ethyl sulfate in meconium and hair-potential biomarkers of intrauterine exposure to ethanol.

    PubMed

    Morini, L; Marchei, E; Vagnarelli, F; Garcia Algar, O; Groppi, A; Mastrobattista, L; Pichini, S

    2010-03-20

    This study investigated ethyl glucuronide (EtG) and ethyl sulfate (EtS) concentration in meconium and in maternal and neonatal hair (HEtG and HFAEEs, respectively) as potential markers of intrauterine exposure to ethanol together with meconium fatty acid ethyl esters (FAEEs) in a cohort of 99 mother-infant dyads, 49 coming from the Arcispedale of Reggio Emilia (Italy) and 50 from the Hospital del Mar of Barcelona (Spain). FAEEs, EtG and EtS were measured in meconium samples using liquid chromatography-tandem mass spectrometry. A head space-solid phase microextraction-gas chromatography-mass spectrometry was used to test HEtG and HFAEEs in hair samples from mothers and their newborns. Eighty-two meconium samples (82.8%) tested positive for EtG, 19 (19.2%) for EtS while 22 (22.2%) showed FAEEs levels higher than 2 nmol/g, the cut-off used to differentiate daily maternal ethanol consumption during pregnancy from occasional or no use. Although EtG and EtS in meconium did not correlate with total FAEEs concentration, a good correlation between EtG, EtS and ethyl stearate was observed. Moreover, EtG correlated well with ethyl palmitoleate, while EtS with ethyl laurate, myristate and linolenate. Neither maternal nor neonatal hair appears as good predictors of gestational ethanol consumption and subsequent fetal exposure in these mother-infant dyads. In conclusion, these data show that meconium is so far the best matrix in evaluating intrauterine exposure to ethanol, with EtG and EtS being potentially good alternative biomarkers to FAEEs. PMID:20060246

  17. Influence of thermal hair straightening on ethyl glucuronide content in hair.

    PubMed

    Ettlinger, Jana; Kirchen, Luc; Yegles, Michel

    2014-06-01

    Hair analysis of ethyl glucuronide (EtG) has become a valuable marker for the detection of moderate and chronic alcohol consumption. It has been shown that bleaching and perming may decrease EtG content in hair. So far, no studies exist about the influence of thermal hair straightening on EtG content in hair. Forty-one positive EtG hair samples were treated in vitro with a hair straightener at 200°C. Duration of treatment of 1 min was chosen for this study. After washing, pulverization, incubation in ultrasonic bath, solid-phase extraction, and derivatization with heptafluorobutyric anhydride, EtG was determined by gas chromatography-mass spectrometry - negative ion chemical ionization (GC-MS-NICI). The EtG contents in straightened hair strands were then compared with those in the corresponding untreated strands. In 20 of 41 hair samples, a decrease of EtG content was found ranging from 0.7% to 79.3% (average 20%) whereas in 21 cases an increase was shown ranging from 2.0% to 50.9% (average 15%). The variation of the results seems to depend on hair colour. The decrease may be explained by thermic in vitro destruction of EtG. The increase may be explained by denaturation of the hair matrix by thermal treatment possibly causing a better extraction of EtG during incubation in ultrasonic bath. This in vitro study indicates that thermal hair straightening has an impact on the EtG content in hair. This has to be considered for a correct interpretation of EtG results in hair. However, these results should be confirmed by in vivo studies. PMID:24817051

  18. Voucher-based reinforcement for alcohol abstinence using the ethyl-glucuronide alcohol biomarker.

    PubMed

    McDonell, Michael G; Howell, Donelle N; McPherson, Sterling; Cameron, Jennifer M; Srebnik, Debra; Roll, John M; Ries, Richard K

    2012-01-01

    This study assessed the effects of a contingency management (CM) intervention for alcohol consumption in 10 alcohol-dependent participants. An ABCA design was used. Vouchers were provided contingent on results of ethyl glucuronide (EtG) urine tests (an alcohol biomarker with a 2-day detection period) and alcohol breath tests during the C phase. The percentage of negative urines was 35% during the first baseline phase, 69% during the C phase, and 20% during the return-to-baseline phase. Results suggest that EtG urine tests may be a feasible method to deliver CM to promote alcohol abstinence.

  19. Identification and preliminary characterization of UDP-glucuronosyltransferases catalyzing formation of ethyl glucuronide.

    PubMed

    Schwab, Nicole; Skopp, Gisela

    2014-04-01

    Ethyl glucuronide (EtG), a minor metabolite of ethanol, is used as a marker of alcohol consumption in a variety of clinical and forensic settings. At present there are very few studies of UDP-glucuronosyltransferases (UGT), responsible for catalyzing EtG formation, and the possible effect of nutritional components, e.g. flavonoids, which are extensively glucuronidated, on EtG formation has not been addressed at all. The following incubation conditions were optimized with regard to previously published conditions: buffer, substrate concentration, and incubation time. Isolation of EtG from the incubation mixture was also optimized. Recombinant UGT enzymes (UGT1A1, 1A3, 1A4, 1A6, 1A9, 2B7, 2B10, 2B15) were screened for their activity towards ethanol, and kinetic data were then established for all enzymes. It was decided to study the effect of the flavonoids quercetin and kaempferol on glucuronidation of ethanol. Isolation was by solid-phase extraction (SPE) to minimize matrix effects. Analysis was performed by liquid chromatography-tandem mass spectrometry (LC-MS-MS), with EtG-d5 as the internal standard. SPE was vital to avoid severe ion suppression after direct injection of the incubation solution. EtG formation was observed for all enzymes under investigation; their kinetics followed the Michaelis-Menten model, meaning the maximum reaction rate achieved at saturating substrate concentrations (V(max)) and the substrate concentration at which the reaction rate is half of V(max) (Michaelis-Menten constant, K(m)) could be calculated. The highest rate of glucuronidation was observed with UGT1A9 and 2B7. After co-incubation with both flavonoids, formation of EtG was significantly reduced for all enzymes except for UGT2B15, whose activity did not seem to be affected. Results reveal that multiple UGT isoforms are capable of catalyzing glucuronidation of ethanol; nevertheless, the effect of UGT polymorphism on glucuronidation of ethanol needs further study. Formation of EtG

  20. Influence of ethanol dose and pigmentation on the incorporation of ethyl glucuronide into rat hair.

    PubMed

    Kharbouche, Hicham; Steiner, Nadia; Morelato, Marie; Staub, Christian; Boutrel, Benjamin; Mangin, Patrice; Sporkert, Frank; Augsburger, Marc

    2010-09-01

    Ethyl glucuronide (EtG) is a minor and specific metabolite of ethanol. It is incorporated into growing hair, allowing a retrospective detection of alcohol consumption. However, the suitability of quantitative EtG measurements in hair to determine the quantity of alcohol consumed has not clearly been demonstrated yet. The purpose of this study was to evaluate the influence of ethanol dose and hair pigmentation on the incorporation of EtG into rat hair. Ethanol and EtG kinetics in blood were investigated after a single administration of ethanol. Eighteen rats were divided into four groups receiving 0 (control group), 1, 2, or 3g ethanol/kg body weight. Ethanol was administered on 4 consecutive days per week for 3 weeks by intragastric route. Twenty-eight days after the initial ethanol administration, newly grown hair was shaved. Pigmented and nonpigmented hair were analyzed separately by gas chromatography coupled to tandem mass spectrometry. Blood samples were collected within 12h after the ethanol administration. EtG and ethanol blood levels were measured by liquid chromatography coupled to tandem mass spectrometry and headspace gas chromatography-flame ionization detector, respectively. No statistically significant difference was observed in EtG concentrations between pigmented and nonpigmented hair (Spearman's rho=0.95). Thus, EtG incorporation into rat hair was not affected by hair pigmentation. Higher doses of ethanol resulted in greater blood ethanol area under the curve of concentration versus time (AUC) and in greater blood EtG AUC. A positive correlation was found between blood ethanol AUC and blood EtG AUC (Spearman's rho=0.84). Increased ethanol administration was associated with an increased EtG concentration in hair. Blood ethanol AUC was correlated with EtG concentration in hair (Pearson's r=0.89). EtG concentration in rat hair appeared to reflect the EtG concentration in blood. Ethanol was metabolized at a median rate of 0.22 g/kg/h, and the median

  1. Ethyl glucuronide in hair - A highly effective test for the monitoring of alcohol consumption.

    PubMed

    Agius, Ronald; Nadulski, Thomas; Kahl, Hans-Gerhard; Dufaux, Bertin

    2012-05-10

    In Germany drink driving offenders lose their license and must prove abstinence for one year in order to regain it. In this paper we assess the newly introduced ethyl glucuronide (EtG) tests in urine and hair in this alcohol abstinence monitoring. 20% (80 out of 386) of the 3cm long hair samples were tested positive for EtG in hair, compared to only 2% (92 out of 4248 samples) in urine in the same time period. Additionally 50% of the samples positive for EtG in hair had EtG values greater than 30pg/mg hair, indicating chronic alcohol consumption in the last three months. This study shows that four EtG tests in 3cm hair lengths reveal a significantly higher percentage of drink driving offenders who fail to be sober in the rehabilitation period, than do six random EtG tests in urine. Presumably, the hair test is more adequate to monitor long term alcohol abstinence than the urine test as defined by the new driving license re-granting medical and psychological assessment (MPA) in Germany. PMID:22019393

  2. A study of distribution of ethyl glucuronide in different keratin matrices.

    PubMed

    Pirro, V; Di Corcia, D; Pellegrino, S; Vincenti, M; Sciutteri, B; Salomone, A

    2011-07-15

    Ethyl glucuronide (EtG) is a direct metabolite of ethanol, frequently used as a biomarker of alcohol abuse. To this purpose, EtG is preferentially determined in hair samples, using a cut-off value of 30pg/mg to discriminate between social and heavy drinkers, as recently fixed by an international consensus conference. Although this cut-off value is assumed for head hair, alternative matrices, such as pubic, axillary and chest hair, are often analyzed when head hair is not available. Previous studies suggested that determination of EtG in various keratin matrices may lead to different results; growth cycle and rate, urine contamination, distribution of sebum glands and other environmental factors are likely to contribute to these differences. We analyzed more than 2700 samples (head, pubic, chest and axillary hair) to evaluate the inter- and intra-individual distribution of the EtG concentration in the different keratin matrices. The data were interpreted on a statistical basis, on the assumption that large population data-sets will level off the average alcohol consumption of each group. From both inter- and intra-individual distribution data, significant differences were observed in EtG concentrations recorded in head, axillary and pubic hair samples. It is concluded that pubic hair cannot be utilized alternatively to head hair to prove chronic alcohol abuse, nor is axillary hair, since positive and negative biases respectively affect these determinations. In contrast, for chest hair, EtG distributions similar to head hair were found, although the large discrepancy between the examined population dimensions presently prevents any definitive conclusion. Thus, chest hair represents a promising alternative to head hair for EtG determinations, deserving further investigation on samples collected from the same individuals, in order to establish a clear correlation between their respective EtG concentrations. PMID:21511419

  3. Combined use of fatty acid ethyl esters and ethyl glucuronide in hair for diagnosis of alcohol abuse: interpretation and advantages.

    PubMed

    Pragst, F; Rothe, M; Moench, B; Hastedt, M; Herre, S; Simmert, D

    2010-03-20

    In this study the combined use of fatty acid ethyl esters (FAEE) and ethyl glucuronide (EtG) for diagnoses of chronically excessive alcohol abuse is investigated at 174 hair samples from driving ability examination, workplace testing and child custody cases for family courts and evaluated with respect to the basics of interpretation. Using the cut-off values of 0.50 ng/mg for FAEE and 25 pg/mg for EtG, both markers were in agreement in 75% of the cases with 103 negative and 28 positive results and there were 30 cases with FAEE positive and EtG negative and 13 cases with FAEE negative and EtG positive. As the theoretical basis of interpretation, the pharmacokinetics of FAEE and EtG is reviewed for all steps between drinking of ethanol to incorporation in hair with particular attention to relationships between alcohol dose and concentrations in hair. It is shown that the concentrations of both markers are essentially determined by the area under the ethanol concentration in blood vs. time curve AUC(EtOH), despite large inter-individual variations. It is demonstrated by calculation of AUC(EtOH) on monthly basis for moderate, risky and heavy drinking that AUC(EtOH) increases very strongly in the range between 60 and 120 g ethanol per day. This specific feature which is caused by the zero-order elimination of ethanol is a favorable prerequisite for a high discrimination power of the hair testing for alcohol abuse. From the consideration of the different profiles of FAEE and EtG along the hair and in agreement with the literature survey, a standardized hair segment 0-3 cm is proposed with cut-off values of 0.5 ng/mg for FAEE and 30 pg/mg for EtG. This improves also the agreement between FAEE and EtG results in the cases of the present study. A scheme for combined interpretation of FAEE and EtG is proposed which uses the levels of abstinence and the double of the cut-off values as criteria in addition to the cut-off's. Considering the large variations in the relationship

  4. Urine tested positive for ethyl glucuronide and ethyl sulphate after the consumption of "non-alcoholic" beer.

    PubMed

    Thierauf, Annette; Gnann, Heike; Wohlfarth, Ariane; Auwärter, Volker; Perdekamp, Markus Grosse; Buttler, Klaus-Juergen; Wurst, Friedrich M; Weinmann, Wolfgang

    2010-10-10

    In abstinence maintenance programs, for reissuing the driving licence and in workplace monitoring programs abstinence from ethanol and its proof are demanded. Various monitoring programs that mainly use ethyl glucuronide (EtG) as alcohol consumption marker have been established. To abstain from ethanol, but not from the taste of alcoholic beverages, in particular non-alcoholic beer has become more and more popular. In Germany, these "alcohol-free" beverages may still have an ethanol content of up to 0.5vol.% without the duty of declaration. Due to severe negative consequences resulting from positive EtG tests, a drinking experiment with 2.5L of non-alcoholic beer per person was performed to address the question of measurable concentrations of the direct metabolites EtG and EtS (ethyl sulphate) in urine and blood. Both alcohol consumption markers - determined by LC-MS/MS - were found in high concentrations: maximum concentrations in urine found in three volunteers were EtG 0.30-0.87mg/L and EtS 0.04-0.07mg/L, i.e., above the often applied cut-off value for the proof of abstinence of 0.1mg EtG/L. In the urine samples of one further volunteer, EtG and EtS concentrations cumulated over-night and reached up to 14.1mg/L EtG and 16.1mg/L EtS in the next morning's urine. Ethanol concentrations in blood and urine samples were negative (determined by HS-GC-FID and by an ADH-based method). PMID:20457499

  5. Urine tested positive for ethyl glucuronide and ethyl sulphate after the consumption of "non-alcoholic" beer.

    PubMed

    Thierauf, Annette; Gnann, Heike; Wohlfarth, Ariane; Auwärter, Volker; Perdekamp, Markus Grosse; Buttler, Klaus-Juergen; Wurst, Friedrich M; Weinmann, Wolfgang

    2010-10-10

    In abstinence maintenance programs, for reissuing the driving licence and in workplace monitoring programs abstinence from ethanol and its proof are demanded. Various monitoring programs that mainly use ethyl glucuronide (EtG) as alcohol consumption marker have been established. To abstain from ethanol, but not from the taste of alcoholic beverages, in particular non-alcoholic beer has become more and more popular. In Germany, these "alcohol-free" beverages may still have an ethanol content of up to 0.5vol.% without the duty of declaration. Due to severe negative consequences resulting from positive EtG tests, a drinking experiment with 2.5L of non-alcoholic beer per person was performed to address the question of measurable concentrations of the direct metabolites EtG and EtS (ethyl sulphate) in urine and blood. Both alcohol consumption markers - determined by LC-MS/MS - were found in high concentrations: maximum concentrations in urine found in three volunteers were EtG 0.30-0.87mg/L and EtS 0.04-0.07mg/L, i.e., above the often applied cut-off value for the proof of abstinence of 0.1mg EtG/L. In the urine samples of one further volunteer, EtG and EtS concentrations cumulated over-night and reached up to 14.1mg/L EtG and 16.1mg/L EtS in the next morning's urine. Ethanol concentrations in blood and urine samples were negative (determined by HS-GC-FID and by an ADH-based method).

  6. Assistance of ethyl glucuronide and ethyl sulfate in the interpretation of postmortem ethanol findings.

    PubMed

    Krabseth, Hege; Mørland, Jørg; Høiseth, Gudrun

    2014-09-01

    Postmortem ethanol formation is a well-known problem in forensic toxicology. The aim of this study was to interpret findings of ethanol in blood, in a large collection of forensic autopsy cases, by use of the nonoxidative ethanol metabolites, ethyl glucuronide (EtG), and ethyl sulfate (EtS). In this study, according to previously published literature, antemortem ethanol ingestion was excluded in EtS-negative cases. Among 493 ethanol-positive forensic autopsy cases, collected during the study period, EtS was not detected in 60 (12 %) of the cases. Among cases with a blood alcohol concentration (BAC) of ≤ 0.54 g/kg, antemortem ethanol ingestion was excluded in 38 % of the cases, while among cases with a BAC of ≥ 0.55 g/kg, antemortem ethanol ingestion was excluded in 2.2 % of the cases. For all cases where ethanol was measured at a concentration >1.0 g/kg, EtS was detected. The highest blood ethanol concentration in which EtS was not detected was 1.0 g/kg. The median concentrations of EtG and EtS in blood were 9.5 μmol/L (range: not detected (n.d.) 618.1) and 9.2 μmol/L (range: n.d. 182.5), respectively. There was a statistically significant positive correlation between concentration levels of ethanol and of EtG (Spearman's rho=0.671, p<0.001) and EtS (Spearman's rho=0.670, p<0.001), respectively. In conclusion, this study showed that in a large number of ethanol-positive forensic autopsy cases, ethanol was not ingested before the time of death, particularly among cases where ethanol was present in lower blood concentrations. Routine measurement of EtG and EtS should therefore be recommended, especially in cases with BAC below 1 g/kg. PMID:24935750

  7. Determination of ethyl glucuronide in human hair by hydrophilic interaction liquid chromatography-tandem mass spectrometry.

    PubMed

    Yaldiz, Fadile; Daglioglu, Nebile; Hilal, Ahmet; Keten, Alper; Gülmen, Mete Korkut

    2013-10-01

    Ethyl glucuronide (EtG) is a direct metabolite of ethanol and has been utilized as a marker for alcohol intake. This study presents development, validation and application of a new hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method for the analysis of EtG in human hair samples. The linearity was assessed in the range of 5-2000 pg/mg hair, with a correlation coefficient of >0.99. The method was selective and sensitive, with a limit of detection (LOD) and limit of quantitation (LOQ) of 0.05 pg/mg and 0.18 pg/mg in hair, respectively. Differently from the extraction procedures in the literature, a fast and simple liquid-liquid method was used and highest recoveries and cleanest extracts were obtained. The method was successfully applied to 30 human hair samples which were taken from those who state they consume alcohol. EtG concentrations in the hair samples of alcohol users participated in this study, ranged between 1.34 and 82.73 pg/mg. From the concentration of EtG in hair strands 20 of the 30 subjects can be considered regular moderate drinkers. PMID:24112322

  8. The influence of ethanol containing cosmetics on ethyl glucuronide concentration in hair.

    PubMed

    Martins Ferreira, Liliane; Binz, Tina; Yegles, Michel

    2012-05-10

    Ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEE), non-volatile, direct metabolites of ethanol have been shown to be suitable markers for the evaluation of social and chronic excessive alcohol consumption. Previous investigations have shown that the regular use of hair-care products with high alcohol content lead to an increase of FAEE concentration and consequently gave false-positive results for the determination of FAEE in hair. In this study we investigated the influence of a long-term hair treatment with EtOH containing lotion, on the EtG concentrations in hair. In this study 7 volunteer subjects (classified as either rare, social or heavy drinkers) treated the right side of their scalp every day during a one or two month period with a commercial hair tonic (Seborin), which contains 44.0% ethanol (vol%). Collection of hair specimens from both sides of the scalp was done one day before hair treatment, one week and one month after treatment (for 5 subjects also after two months of treatment). A hair segment of 3 centimeters (cm) was cut and then washed with water and acetone, and then pulverized. EtG was quantified by GC/MS after pulverization and 2h of ultrasonication in water, extraction by solid phase extraction using Oasis MAX columns and derivatization with HFBA. Measurements were done in negative chemical ionization mode using EtG-D5 as internal standard. Comparison of EtG concentration in the treated and in the non-treated hair specimens did not show any increase at the different dates of collection for the 7 subjects. In conclusion, these results show that there is no indication for an increase of EtG after use of ethanol containing hair cosmetics.

  9. Biotransformation of ethanol to ethyl glucuronide in a rat model after a single high oral dosage.

    PubMed

    Wright, Trista H; Ferslew, Kenneth E

    2012-03-01

    Ethyl glucuronide (EtG) is a minor ethanol metabolite that confirms the absorption and metabolism of ethanol after oral or dermal exposure. Human data suggest that maximum blood EtG (BEtG) concentrations are reached between 3.5 and 5.5h after ethanol administration. This study was undertaken to determine if the Sprague-Dawley (SD) rat biotransforms ethanol to EtG after a single high oral dose of ethanol. SD rats (male, n=6) were gavaged with a single ethanol dose (4 g/kg), and urine was collected for 3 h in metabolic cages, followed by euthanization and collection of heart blood. Blood and urine were analyzed for ethanol and EtG by gas chromatography and enzyme immunoassay. Blood and urine ethanol concentrations were 195±23 and 218±19 mg/dL, whereas BEtG and urine EtG (UEtG) concentrations were 1,363±98 ng equivalents/mL and 210±0.29 mg equivalents/dL (X ± standard error of the mean [S.E.M.]). Sixty-six male SD rats were gavaged ethanol (4 g/kg) and placed in metabolic cages to determine the extent and duration of ethanol to EtG biotransformation and urinary excretion. Blood and urine were collected up to 24 h after administration for ethanol and EtG analysis. Maximum blood ethanol, urine ethanol, and UEtG were reached within 4 h, whereas maximum BEtG was reached 6 h after administration. Maximum concentrations were blood ethanol, 213±20 mg/dL; urine ethanol, 308±34 mg/dL; BEtG, 2,683±145 ng equivalents/mL; UEtG, 1.2±0.06 mg equivalents/mL (X±S.E.M.). Areas under the concentration-time curve were blood ethanol, 1,578 h*mg/dL; urine ethanol, 3,096 h*mg/dL; BEtG, 18,284 h*ng equivalents/mL; and UEtG, 850 h*mg equivalents/dL. Blood ethanol and BEtG levels were reduced to below limits of detection (LODs) within 12 and 18 h after ethanol administration. Urine ethanols were below LOD at 18 h, but UEtG was still detectable at 24h after administration. Our data prove that the SD rat biotransforms ethanol to EtG and excretes both in the urine and suggest that it

  10. Biotransformation of ethanol to ethyl glucuronide in a rat model after a single high oral dosage.

    PubMed

    Wright, Trista H; Ferslew, Kenneth E

    2012-03-01

    Ethyl glucuronide (EtG) is a minor ethanol metabolite that confirms the absorption and metabolism of ethanol after oral or dermal exposure. Human data suggest that maximum blood EtG (BEtG) concentrations are reached between 3.5 and 5.5h after ethanol administration. This study was undertaken to determine if the Sprague-Dawley (SD) rat biotransforms ethanol to EtG after a single high oral dose of ethanol. SD rats (male, n=6) were gavaged with a single ethanol dose (4 g/kg), and urine was collected for 3 h in metabolic cages, followed by euthanization and collection of heart blood. Blood and urine were analyzed for ethanol and EtG by gas chromatography and enzyme immunoassay. Blood and urine ethanol concentrations were 195±23 and 218±19 mg/dL, whereas BEtG and urine EtG (UEtG) concentrations were 1,363±98 ng equivalents/mL and 210±0.29 mg equivalents/dL (X ± standard error of the mean [S.E.M.]). Sixty-six male SD rats were gavaged ethanol (4 g/kg) and placed in metabolic cages to determine the extent and duration of ethanol to EtG biotransformation and urinary excretion. Blood and urine were collected up to 24 h after administration for ethanol and EtG analysis. Maximum blood ethanol, urine ethanol, and UEtG were reached within 4 h, whereas maximum BEtG was reached 6 h after administration. Maximum concentrations were blood ethanol, 213±20 mg/dL; urine ethanol, 308±34 mg/dL; BEtG, 2,683±145 ng equivalents/mL; UEtG, 1.2±0.06 mg equivalents/mL (X±S.E.M.). Areas under the concentration-time curve were blood ethanol, 1,578 h*mg/dL; urine ethanol, 3,096 h*mg/dL; BEtG, 18,284 h*ng equivalents/mL; and UEtG, 850 h*mg equivalents/dL. Blood ethanol and BEtG levels were reduced to below limits of detection (LODs) within 12 and 18 h after ethanol administration. Urine ethanols were below LOD at 18 h, but UEtG was still detectable at 24h after administration. Our data prove that the SD rat biotransforms ethanol to EtG and excretes both in the urine and suggest that it

  11. Ethyl glucuronide findings in hair samples from the mummies of the Capuchin Catacombs of Palermo.

    PubMed

    Musshoff, Frank; Brockmann, Christopher; Madea, Burkhard; Rosendahl, Wilfried; Piombino-Mascali, Dario

    2013-10-10

    The Capuchin Catacombs of Palermo contain over 1800 preserved bodies: friars, priests and laypeople including men, women, and children. The bodies were accessible to family members who could visit the deceased and commemorate them through prayers. The "Sicily Mummy Project" analyzed hair samples from 38 mummies to determine the presence of ethyl glucuronide (EtG) using a routine procedure in our accredited laboratory of liquid chromatography coupled with mass spectrometry. The limit of quantification was 2.3 pg/mg. The hair samples were from 1.5 to 12 cm in length. All samples were analyzed in 2 segments (seg. A 0-3 cm and seg. B the remainder). Samples <4 cm in length were cut in half. In 31 out of 76 segments positive results were obtained for EtG, with concentrations between 2.5 and 531.3 pg/mg (mean 73.8, median 13.3 pg/mg). In 14 cases positive results were obtained for both segments. In one sample a positive result was obtained for segment A but not for segment B and in a further two samples only for segment B. The results indicate that EtG analyses can be performed on mummy hair samples even several hundred years after death to identify evidence for significant alcohol consumption during life. PMID:24053883

  12. Utility of urinary ethyl glucuronide analysis in post-mortem toxicology when investigating alcohol-related deaths.

    PubMed

    Sundström, M; Jones, A W; Ojanperä, I

    2014-08-01

    Use and abuse of alcohol are common findings when unnatural deaths are investigated as evidenced by high blood- and urine- alcohol concentrations (BAC and UAC) at autopsy. Because ethanol is metabolized in the liver until the time of death, the autopsy BAC or UAC might be negative even though the deceased had consumed alcohol in the immediate ante-mortem period. Analysis of the non-oxidative metabolite of ethanol [ethyl glucuronide (EtG)] offers a more sensitive test of recent drinking. In this paper, we determined the concentrations of ethanol and EtG in urine samples from 972 consecutive forensic autopsies. In 425 cases (44%) both EtG and ethanol were positive, which supports ante-mortem drinking. In 342 cases (35%), both EtG and ethanol was negative, which speaks against any consumption of alcohol just before death. In 181 cases, ethanol was negative in urine (<0.2 g/kg), whereas EtG was positive (>0.5 mg/L), which points towards ingestion of alcohol some time before death. In these cases, mean and median concentrations of EtG were 53.2 mg/L and 23.7 mg/L, respectively, although there was no mention of alcohol on 131 of the death certificates. Alcohol was mentioned on death certificates as an underlying or immediate cause of death or a contributing factor in 435 (45%) cases, which rose to 566 (58%) cases when positive EtG results were included. This article demonstrates the usefulness of EtG analysis in routine post-mortem toxicology when ante-mortem drinking and alcohol-related deaths are investigated. PMID:24954799

  13. Ethyl glucuronide concentrations in hair: a controlled alcohol-dosing study in healthy volunteers.

    PubMed

    L Crunelle, Cleo; Cappelle, Delphine; Yegles, Michel; De Doncker, Mireille; Michielsen, Peter; Dom, Geert; van Nuijs, Alexander L N; Maudens, Kristof E; Covaci, Adrian; Neels, Hugo

    2016-03-01

    Ethyl glucuronide (EtG) is a minor phase II metabolite of alcohol that accumulates in hair. It has been established as a sensitive marker to assess the retrospective consumption of alcohol over recent months using a cut-off of ≥7 pg/mg hair to assess repeated alcohol consumption. The primary aim was to assess whether amounts of alcohol consumed correlated with EtG concentrations in hair. Additionally, we investigated whether the current applied cut-off value of 7 pg/mg hair was adequate to assess the regular consumption of low-to-moderate amounts of alcohol. A prospective controlled alcohol-dosing study in 30 healthy individuals matched on age and gender. Individuals were instructed to drink no alcohol (N = 10), 100 g alcohol per week (N = 10) or 150 g alcohol per week (N = 10) for 12 consecutive weeks, before and after which hair was collected. Throughout the study, compliance to daily alcohol consumption was assessed by analyzing urine EtG three times weekly. Participants in the non-drinking group had median EtG concentrations of 0.5 pg/mg hair (interquartile range (IQR) 1.7 pg/mg; range < 0.21-4.5 pg/mg). Participants consuming 100 and 150 g alcohol per week showed median EtG concentrations of 5.6 pg/mg hair (IQR 4.7 pg/mg; range 2.0-9.8 pg/mg) and 11.3 pg/mg hair (IQR 5.0 pg/mg; range 7.7-38.9 pg/mg), respectively. Hair EtG concentrations between the three study groups differed significantly from one another (p < 0.001). Hair EtG concentrations can be used to differentiate between repeated (low-to-moderate) amounts of alcohol consumed over a long time period. For the assessment of repeated alcohol use, we propose that the current cut-off of 7 pg/mg could be re-evaluated. PMID:26549114

  14. Immunoassay for ethyl glucuronide in vitreous humor: a new tool for postmortem diagnostics of alcohol use.

    PubMed

    Rainio, Juha; Kultti, Johanna; Kangastupa, Päivikki; Tuomi, Heidi; Ahola, Sanna; Karhunen, Pekka J; Helander, Anders; Niemelä, Onni

    2013-03-10

    Although excessive alcohol consumption plays a major role in fatal events, the role of alcohol use as a possible contributing factor at the time of death is not easy to establish due to lack of suitable biomarkers for postmortem analyses. We used an immunological approach to measure ethyl glucuronide (EtG) concentrations from vitreous humor (VH) and serum from 58 individuals representing a forensic autopsy population of cases with either a well-documented history of excessive alcohol use (n=37) or cases without such history (n=21), according to medical and police records and blood alcohol determinations (BAC). The immunoassay was based on the Microgenics DRI-EtG EIA reagents applied on an automated Abbott Architect c8000 clinical chemistry analyzer. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) determination of EtG and ethyl sulfate (EtS) was used as a reference method. At a cut-off of 0.3mg/l for VH-EtG, the immunoassay correctly identified 92% of the cases with a history of excessive alcohol use, whereas the BAC was positive (cut-off 10mg/dl) in 68% of the cases. A significant correlation emerged between VH-EtG and serum EtG (r=0.77, p<0.001) and between VH-EtG and BAC (r=0.62, p<0.001), although VH-EtG was frequently elevated also in cases with no detectable BAC. The EtG immunoassay showed a strong correlation with the LC-MS/MS reference method (r=0.94, p<0.001) and there was 100% agreement in the frequency of marker positive and negative findings between the immunoassay EtG results and the LC-MS/MS analysis of EtG and EtS. The present data indicate that the immunoassay for VH-EtG is a useful forensic tool for screening of antemortem alcohol use. PMID:23415594

  15. Ethyl sulphate and ethyl glucuronide in vitreous humor as postmortem evidence marker for ethanol consumption prior to death.

    PubMed

    Thierauf, Annette; Kempf, Jürgen; Perdekamp, Markus Grosse; Auwärter, Volker; Gnann, Heike; Wohlfarth, Ariane; Weinmann, Wolfgang

    2011-07-15

    To clarify the circumstances of death, the degree of inebriation is of importance in many cases, but for several reasons the determination of the ethanol concentration in post-mortem samples can be challenging and the synopsis of ethanol and the direct consumption markers ethyl glucuronide (EtG) and ethyl sulphate (EtS) has proved to be useful. The use of a rather stable matrix like vitreous humor offers further advantages. The aim of this study was to determine the concentrations of ethanol and the biomarkers in the robust matrix of vitreous humor and to compare them with the respective levels in peripheral venous blood and urine. Samples of urine, blood from the femoral vein and vitreous humor were taken from 26 deceased with suspected ethanol consumption prior to death and analyzed for ethanol, EtS and EtG. In the urine samples creatinine was also determined. The personal data, the circumstances of death, the post-mortem interval and the information about ethanol consumption prior to death were recorded. EtG and EtS analysis in urine was performed by LC-ESI-MS/MS, creatinine concentration was determined using the Jaffé reaction and ethanol was detected by HS-GC-FID and by an ADH-based method. In general, the highest concentrations of the analytes were found in urine and showed statistical significance. The mean concentrations of EtG were 62.8mg/L (EtG100 206.5mg/L) in urine, 4.3mg/L in blood and 2.1mg/L in vitreous humor. EtS was found in the following mean concentrations: 54.6mg/L in urine (EtS100 123.1mg/L), 1.8mg/L in blood and 0.9mg/L in vitreous humor. Ethanol was detected in more vitreous humor samples (mean concentration 2.0g/kg) than in blood and urine (mean concentration 1.6g/kg and 2.1g/kg respectively). There was no correlation between the ethanol and the marker concentrations and no statistical conclusions could be drawn between the markers and matrices.

  16. Ethyl sulphate and ethyl glucuronide in vitreous humor as postmortem evidence marker for ethanol consumption prior to death.

    PubMed

    Thierauf, Annette; Kempf, Jürgen; Perdekamp, Markus Grosse; Auwärter, Volker; Gnann, Heike; Wohlfarth, Ariane; Weinmann, Wolfgang

    2011-07-15

    To clarify the circumstances of death, the degree of inebriation is of importance in many cases, but for several reasons the determination of the ethanol concentration in post-mortem samples can be challenging and the synopsis of ethanol and the direct consumption markers ethyl glucuronide (EtG) and ethyl sulphate (EtS) has proved to be useful. The use of a rather stable matrix like vitreous humor offers further advantages. The aim of this study was to determine the concentrations of ethanol and the biomarkers in the robust matrix of vitreous humor and to compare them with the respective levels in peripheral venous blood and urine. Samples of urine, blood from the femoral vein and vitreous humor were taken from 26 deceased with suspected ethanol consumption prior to death and analyzed for ethanol, EtS and EtG. In the urine samples creatinine was also determined. The personal data, the circumstances of death, the post-mortem interval and the information about ethanol consumption prior to death were recorded. EtG and EtS analysis in urine was performed by LC-ESI-MS/MS, creatinine concentration was determined using the Jaffé reaction and ethanol was detected by HS-GC-FID and by an ADH-based method. In general, the highest concentrations of the analytes were found in urine and showed statistical significance. The mean concentrations of EtG were 62.8mg/L (EtG100 206.5mg/L) in urine, 4.3mg/L in blood and 2.1mg/L in vitreous humor. EtS was found in the following mean concentrations: 54.6mg/L in urine (EtS100 123.1mg/L), 1.8mg/L in blood and 0.9mg/L in vitreous humor. Ethanol was detected in more vitreous humor samples (mean concentration 2.0g/kg) than in blood and urine (mean concentration 1.6g/kg and 2.1g/kg respectively). There was no correlation between the ethanol and the marker concentrations and no statistical conclusions could be drawn between the markers and matrices. PMID:21367549

  17. The influence of cleansing shampoos on ethyl glucuronide concentration in hair analyzed with an optimized and validated LC-MS/MS method.

    PubMed

    Binz, Tina M; Baumgartner, Markus R; Kraemer, Thomas

    2014-11-01

    Ethyl glucuronide (EtG) is widely used as a marker for assessment of alcohol consumption behavior. In this study the influence of special cleansing shampoos on ethyl glucuronide concentrations in hair was investigated. For that purpose an optimized LC-MS/MS method was developed using a Hypercarb™ porous graphitic carbon (PGC) column and validated according to the guidelines of the German Society of Toxicological and Forensic Chemistry (GTFCh). Twenty-five hair samples of persons with known alcohol consumption behavior were investigated (21 positive samples and 4 blank samples). The hair samples were divided into two strands of hair and were analyzed after treatment with one out of four cleansing shampoos and without shampoo treatment. EtG concentrations in hair did not show any significant differences after a single application of the different cleansing shampoos. EtG was still detectable in all the positive hair samples without significant concentration change. These results clearly demonstrated that a single application of the tested cleansing shampoos did not remove EtG from hair and therefore had no influence on EtG concentration in analytical hair analysis.

  18. The influence of cleansing shampoos on ethyl glucuronide concentration in hair analyzed with an optimized and validated LC-MS/MS method.

    PubMed

    Binz, Tina M; Baumgartner, Markus R; Kraemer, Thomas

    2014-11-01

    Ethyl glucuronide (EtG) is widely used as a marker for assessment of alcohol consumption behavior. In this study the influence of special cleansing shampoos on ethyl glucuronide concentrations in hair was investigated. For that purpose an optimized LC-MS/MS method was developed using a Hypercarb™ porous graphitic carbon (PGC) column and validated according to the guidelines of the German Society of Toxicological and Forensic Chemistry (GTFCh). Twenty-five hair samples of persons with known alcohol consumption behavior were investigated (21 positive samples and 4 blank samples). The hair samples were divided into two strands of hair and were analyzed after treatment with one out of four cleansing shampoos and without shampoo treatment. EtG concentrations in hair did not show any significant differences after a single application of the different cleansing shampoos. EtG was still detectable in all the positive hair samples without significant concentration change. These results clearly demonstrated that a single application of the tested cleansing shampoos did not remove EtG from hair and therefore had no influence on EtG concentration in analytical hair analysis. PMID:25151107

  19. Ethyl glucuronide determination in meconium and hair by hydrophilic interaction liquid chromatography-tandem mass spectrometry.

    PubMed

    Tarcomnicu, Isabela; van Nuijs, Alexander L N; Aerts, Katrien; De Doncker, Mireille; Covaci, Adrian; Neels, Hugo

    2010-03-20

    Ethyl glucuronide (EtG) detection in non-conventional matrices, such as hair and meconium, can provide useful information on alcohol abuse over a long time frame, for example during pregnancy or after a withdrawal treatment. This study reports on the development, validation and application of a new hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method for the analysis of EtG in meconium and hair. For each matrix, the sample preparation and the chromatographic separation were thoroughly optimised. Additionally, experiments with reversed-phase liquid chromatography were also performed in the development stages. Analyses were carried out using a Phenomenex Luna HILIC column (150 mm x 3 mm, 5 microm) and a mobile phase composed by ammonium acetate 2mM and acetonitrile, in gradient. Different SPE cartridges (Oasis MAX, Oasis WAX, aminopropyl silica) and solvents were tested in order to obtain the highest recoveries and cleanest extracts. Optimal results were obtained for meconium with aminopropyl cartridges, while for hair an incubation of 16 h with 2 mL of water and acetonitrile (50/50, v/v) provided good results. The analytical method was validated for both matrices (meconium and hair) by assessing linearity, precision, accuracy, recovery and limit of quantification. The calibration curve concentrations ranged from 50 to 1200 pg/mg for meconium and from 20 to 1000 pg/mg for hair. Real meconium and hair samples were analyzed and results were consistent with literature. PMID:20061101

  20. Improved liquid chromatography-tandem mass spectrometric method for the determination of ethyl glucuronide concentrations in hair: applications to forensic cases.

    PubMed

    Imbert, Laurent; Gaulier, Jean-Michel; Dulaurent, Sylvain; Morichon, Julien; Bevalot, Fabien; Izac, Paul; Lachâtre, Gérard

    2014-01-01

    Ethyl glucuronide (EtG) is a direct marker of ethanol consumption, and its assay in hair is an efficient tool for chronic alcoholism diagnosis. In 2012, the Society of Hair Testing proposed a new consensus for hair concentrations interpretation, strongly advising the use of analytical methods providing a limit of quantification of less than 3 pg/mg. The present work describes the optimization and validation of a previously developed liquid chromatography-tandem mass spectrometric method in order to comply with this recommendation. The concentration range of this improved method is from 3 to 1,000 pg/mg. Some cases are then described to illustrate the usefulness of hair EtG: a forensic post-mortem case and two cases of suspension of driving licences. Finally, hair samples of some teetotallers (n = 10) have been analyzed, which allowed neither to quantitate nor to detect any trace of EtG. PMID:23824336

  1. Ethyl glucuronide in human hair after daily consumption of 16 or 32 g of ethanol for 3 months.

    PubMed

    Kronstrand, Robert; Brinkhagen, Linda; Nyström, Fredrik H

    2012-02-10

    The overall objectives of the study were to develop a sensitive method for ethyl glucuronide (EtG) determination in hair and then investigate if a low or moderate intake of ethanol could be differentiated from total abstinence. Forty-four subjects were included in the study, 12 males (7 drinkers and 5 abstinent) and 32 females (14 drinkers and 18 abstinent). The study lasted 3 months and the female drinkers consumed one glass (16 g of ethanol) and the males consumed two glasses (32 g of ethanol) of wine (13.5-14%) daily. Hair samples were collected as close as possible above the skin and the proximal 2 cm were analyzed for EtG. Hair was cut into pieces of about 0.5 cm length and washed before incubation overnight in water and then extracted on Clean Screen EtG Carbon columns. The LC/MS/MS system consisted of a Waters ACQUITY UPLC connected to an API 4000 triple quadrupole instrument. Two transitions for EtG and one for the internal standard EtG-D(5) were measured. The method was linear from 60 to 10,000 pg/sample. Imprecision studies were performed at three levels as well as with an authentic sample. Total imprecision was 16% at 200 pg/sample, 8% at 1000 pg/sample, 6% at 8000 pg/sample and 13% at 29 pg/mg in the authentic sample. Of those who drank two glasses of wine every day, four had measurable amounts of EtG in their hair (5-11 pg/mg), and in only one of the females drinking one glass of wine EtG was quantified (3 pg/mg). Among the 23 abstinent subjects two had traces of EtG in the hair. We conclude that persons who ingested 16 or 32 g of ethanol daily for 3 months presented with low concentrations of EtG in hair, well below the proposed threshold for overconsumption set at 30 pg/mg. In addition, none of those who ingested 16 g/day had concentrations over the proposed abstinence threshold of 7 pg/mg. PMID:21367545

  2. Validation of a novel method to identify in utero ethanol exposure: simultaneous meconium extraction of fatty acid ethyl esters, ethyl glucuronide, and ethyl sulfate followed by LC-MS/MS quantification

    PubMed Central

    Himes, Sarah K.; Concheiro, Marta; Scheidweiler, Karl B.

    2015-01-01

    Presence of fatty acid ethyl esters (FAEE), ethyl glucuronide (EtG), and ethyl sulfate (EtS) in meconium, the first neonatal feces, identifies maternal alcohol consumption during pregnancy. Current meconium alcohol marker assays require separate analyses for FAEE and EtG/EtS. We describe development and validation of the first quantitative liquid chromatography tandem mass spectrometry assay for 9 FAEEs, EtG, and EtS in 100 mg meconium. For the first time, these alcohol markers are analyzed in the same meconium aliquot, enabling comparison of the efficiency of gestational ethanol exposure detection. 100 mg meconium was homogenized in methanol and centrifuged. The supernatant was divided, and applied to two different solid phase extraction columns for optimized analyte recovery. Limits of quantification for ethyl laurate, myristate, linolenate, palmitoleate, arachidonate, linoleate, palmitate, oleate, and stearate ranged from 25–50 ng/g, with calibration curves to 2,500–5,000 ng/g. EtG and EtS linear dynamic ranges were 5–1,000 and 2.5–500 ng/g, respectively. Mean bias and between-day imprecision were <15 %. Extraction efficiencies were 51.2–96.5 %. Matrix effects ranged from −84.7 to 16.0 %, but were compensated for by matched deuterated internal standards when available. All analytes were stable (within ±20 % change from baseline) in 3 authentic positive specimens, analyzed in triplicate, after 3 freeze/thaw cycles (−20 °C). Authentic EtG and EtS also were stable after 12 h at room temperature and 72 h at 4 °C; some FAEE showed instability under these conditions, although there was large inter-subject variability. This novel method accurately detects multiple alcohol meconium markers and enables comparison of markers for maternal alcohol consumption. PMID:24408304

  3. Diagnosis of chronic alcohol consumption. Hair analysis for ethyl-glucuronide.

    PubMed

    Jurado, C; Soriano, T; Giménez, M P; Menéndez, M

    2004-10-29

    This paper describes a procedure for the detection and quantification of ethyl-glucuronide (EtG) in hair samples. During method development the efficacy of extraction of EtG from hair was compared in four extraction methods: (a) methanol; (b) methanol:water (1:1); (c) water; and (d) water:trifluoroacetic acid (9:1). In addition, three derivatizing agents were compared as well: N,O-bistrimethylsilyl-trifluoroacetamide (BSTFA): trimethylchlorosilane (TMCS) (99:1), pentafluoropropionic anhydride (PFPA) and heptafluorobutyric anhydride (HFBA). Water was found to be the best extracting solvent and PFPA the best derivatizing agent. Both provided the highest recoveries, with cleaner extracts and more stable derivatives. The final method is as follows: about 100mg of hair are sequentially washed with water and acetone. The decontaminated sample is finely cut with scissors, then the deuterated internal standard (EtG-d5) and 2 mL of water are added. After sonication for 2 h, the sample is maintained at room temperature overnight. Derivatization is performed with PFPA. Derivatives are injected into a GC-MS system in the electronic impact mode. The method shows linearity over the range of concentrations from 0.050 to 5 ng/mg. Detection and quantification limits are 0.025 and 0.050 ng/mg, respectively. Mean recoveries for the three studied concentrations (low, medium and high) are higher than 87%. The coefficients of variation in intra- and inter-assay precision are always lower than 7%. The method is being routinely applied in our lab for the diagnosis of chronic alcohol consumption. PMID:15451088

  4. Impact of the grinding process on the quantification of ethyl glucuronide in hair using a validated UPLC-ESI-MS-MS method.

    PubMed

    Kummer, Natalie; Wille, Sarah M R; Di Fazio, Vincent; Ramírez Fernández, Maria Del Mar; Yegles, Michel; Lambert, Willy E E; Samyn, Nele

    2015-01-01

    The Society of Hair Testing (SoHT) has provided cutoffs for the quantification of ethyl glucuronide (EtG) in hair to indicate occasional or chronic/excessive alcohol consumption. Although several sensitive methods have been reported, past proficiency test results show a lack of reproducibility. An ultra-performance liquid chromatographic mass spectrometric method (LLOQ of 10 pg EtG/mg hair) has been validated according to the international guidelines, including the successful participation in five proficiency tests. This method was subsequently used to evaluate the impact of different grinding conditions (cut, weakly or extensively pulverized hair samples) on the final measured EtG concentration. Hair from alcohol consumers (n = 2) and commercially available quality control samples (QCs) (n = 2) was used. For the QCs, extensive pulverization led to a significantly higher amount of measured EtG. In the hair samples obtained from volunteers, cut or weakly pulverized hair resulted in EtG concentrations below the LLOQ, while the mean concentrations of 14 and 40 pg EtG/mg hair were determined after extensive pulverization. Differences in sample preparation could partially explain inter-laboratory variability. As the differences in results can lead to a different interpretation even when applying the SoHT cutoffs, it is of interest to standardize sample preparation techniques in the field of EtG hair testing. PMID:25274495

  5. Degradation of the ethyl glucuronide content in hair by hydrogen peroxide and a non-destructive assay for oxidative hair treatment using infra-red spectroscopy.

    PubMed

    Ammann, Dominic; Becker, Roland; Kohl, Anka; Hänisch, Jessica; Nehls, Irene

    2014-11-01

    The assessment of quantification results of the alcohol abuse marker ethyl glucuronide (EtG) in hair in comparison to the cut-off values for the drinking behavior may be complicated by cosmetic hair bleaching. Thus, the impact of increasing exposure to hydrogen peroxide on the EtG content of hair was investigated. Simultaneously, the change of absorbance in the range of 1000-1100 cm(-1) indicative for the oxidation of cystine was investigated non-destructively by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) using pulverized portions of the respective hair samples. Hair samples treated with hydrogen peroxide consistently displayed a significantly increased absorbance at 1040 cm(-1) associated with the formation of cysteic acid. The EtG content decreased significantly if the hair was treated with alkaline hydrogen peroxide as during cosmetic bleaching. It could be shown that ATR-FTIR is capable of detecting an exposure to hydrogen peroxide when still no brightening was visible and already before the EtG content deteriorated significantly. Thus, hair samples suspected of having been exposed to oxidative treatment may be checked non-destructively by a readily available technique. This assay is also possible retrospectively after EtG extraction and using archived samples. PMID:25180828

  6. Ethyl glucuronide concentrations in oral fluid, blood, and urine after volunteers drank 0.5 and 1.0 g/kg doses of ethanol.

    PubMed

    Høiseth, Gudrun; Yttredal, Borghild; Karinen, Ritva; Gjerde, Hallvard; Mørland, Jørg; Christophersen, Asbjørg

    2010-01-01

    The aim of this study was to investigate the concentrations of ethyl glucuronide (EtG) in oral fluid, blood, and urine after healthy volunteers drank two doses of ethanol, 0.5 (n = 11) and 1.0 g/kg (n = 10), after an overnight fast. Samples of oral fluid, blood, and urine were collected before drinking started and at 1.5, 3.5, 5.5, 8.5, 11.5, and 24 h post-dosing. Following ingestion of low dose of ethanol, the Cmax for EtG was 0.36 mg/L (range 0.28-0.41 mg/L) in blood and 69.8 mg/L (range 47.1-96.5 mg/L) in urine. In oral fluid, the concentrations were < 1% of those in blood, and only three subjects exceeded the limit of quantification for EtG in oral fluid. After ingestion of the high dose of ethanol, the Cmax for EtG was 1.06 mg/L (range 0.8-1.22 mg/L) in blood, 159.9 mg/L (range 97.2-225.5 mg/L) in urine, and 0.032 mg/L (range 0.013-0.059 mg/L) in oral fluid. The median oral fluid/blood ratio was 0.029 (range 0.012-0.054) for EtG. The detection time for EtG was median 11.5 h (range 3.5-11.5 h) in oral fluid. According to this, the detection time for EtG in oral fluid is therefore only a few hours longer than for ethanol itself and represents limited additional value. PMID:20663284

  7. Validation of a headspace solid-phase microextraction-GC-MS/MS for the determination of ethyl glucuronide in hair according to forensic guidelines.

    PubMed

    Agius, Ronald; Nadulski, Thomas; Kahl, Hans-Gerhard; Schräder, Johannes; Dufaux, Bertin; Yegles, Michel; Pragst, Fritz

    2010-03-20

    The analysis of ethyl glucuronide (EtG) in hair is a powerful tool for chronic alcohol abuse control because of the typical wide detection window of the hair matrix and due to the possibility of segmentation, allowing evaluation of alcohol consumption in different periods. Additionally, EtG in hair is often the only diagnostic parameter of choice for alcohol abuse when other clinical parameters such as ALT, AST, gammaGT and CDT (asialotransferrin and disialotransferrin) are in the normal range and EtG in urine negative. In this paper, we describe the development, optimization and validation of a new method based on hair extraction with water, clean-up by solid phase extraction (SPE), derivatization with heptafluorobutyric anhydride and headspace solid-phase microextraction (HS-SPME) in combination with GC-MS/MS according to forensic guidelines. The assay linearity of EtG was confirmed over the range from 2.8 to 1000 pg/mg hair, with a coefficient of determination (r(2)) above 0.999. The LLOQ was 2.8 pg/mg and the LLOD was 0.6 pg/mg. An error profile calculated according to the "Guide to the Expression of Uncertainty in Measurement" (GUM) at 99% confidence intervals for the range 5-750 pg/mg hair did not exceed 10%. This range corresponds to more than 98% of the positive samples analysed. PMID:20061100

  8. Determination of ethyl-glucuronide in hair for heavy drinking detection using liquid chromatography-tandem mass spectrometry following solid-phase extraction.

    PubMed

    Lamoureux, Fabien; Gaulier, Jean-michel; Sauvage, François-Ludovic; Mercerolle, Magali; Vallejo, Christine; Lachâtre, Gérard

    2009-08-01

    The detection of ethyl-beta-D-6-glucuronide (EtG), a stable phase II metabolite of ethanol, is of interest in both clinical and forensic contexts with the aim of monitoring alcohol abuse. We present a liquid chromatography-electrospray ionisation-tandem mass spectrometry method for the detection and quantification of EtG in hair. Thirty milligrams of washed and cut hair were cleaned up using solid-phase extraction graphite cartridges. Separation was then performed using an Uptisphere-3SI column, and the detection was operated in the negative mode. After validation, the method was applied to hair samples taken from four fatalities (F) with documented excessive drinking habits, 12 heavy drinkers (HD) and seven social drinkers (SD). The method exhibits limits of detection and quantification of 4 and 10 pg/mg, respectively. Intra- and inter-assay standard deviation and relative bias were less than 20% over the calibrating range (10 to 3,000 pg/mg). EtG hair concentrations in SD were <10 pg/mg and >50 pg/mg for F and HD (range, 54 to 497 pg/mg). The present assay appears convenient to carry out owing to the very small quantity of hair samples required to determine an effective heavy alcohol consumption (EtG hair concentration >50 pg/mg). PMID:19517099

  9. Gas chromatographic determination of ethyl glucuronide in hair: comparison between tandem mass spectrometry and single quadrupole mass spectrometry.

    PubMed

    Cappelle, Delphine; Neels, Hugo; Yegles, Michel; Paulus, Jeff; van Nuijs, Alexander L N; Covaci, Adrian; Crunelle, Cleo L

    2015-04-01

    Ethyl glucuronide (EtG), a minor metabolite of ethanol, accumulates in hair and is currently used as a long-term marker for the detection of chronic and excessive alcohol consumption. Sensitive methods are required to differentiate teetotalers from moderate drinkers according to the established cut-off (i.e., 7 pg/mg hair). The aim of this study was to develop a sensitive method using gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) operated in the negative ion chemical ionization (NICI) mode. The validated method was applied to hair samples from teetotalers, moderate and excessive alcohol consumers, and results were compared to a previously validated GC-NICI-MS method. The developed GC-NICI-MS/MS method showed linearity over a range from 2 to 400 pg/mg hair, with a limit of detection (LOD) of 0.05 pg/mg hair and a lower limit of quantification (LLOQ) of 0.2 pg/mg hair, compared to an LOD of 0.5 pg/mg hair and LLOQ of 1.5 pg/mg hair obtained with GC-NICI-MS. Furthermore, lower background noise was observed using GC-NICI-MS/MS. Comparison of results of hair samples (n=58) obtained by GC-NICI-MS and GC-NICI-MS/MS showed no significant difference between both methods (paired-sample t-test, p>0.05; mean CV=1.0%). The differences between both methods were larger for EtG concentrations<30 mg/pg hair (mean CV=1.7%) than for EtG concentrations>30 mg/pg hair (mean CV=0.7%). This suggests a higher selectivity of GC-NICI-MS/MS at lower concentrations. In conclusion, by using GC-NICI-MS/MS, a higher analytical selectivity and an improved signal to noise ratio, can be achieved. Although GC-NICI-MS would not change the interpretation of the EtG concentrations, the present GC-NICI-MS/MS method should preferentially be used for the determination of EtG in hair, especially when differentiating between teetotalers and moderate drinkers according to the current cut-off (i.e., 7 pg/mg hair). PMID:25562794

  10. Analysis of ethyl glucuronide in hair samples by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS).

    PubMed

    Cabarcos, Pamela; Hassan, Huda M; Tabernero, María Jesús; Scott, Karen S

    2013-07-01

    Many different biomarkers can be used to evaluate ethanol intake. Ethyl glucuronide (EtG) is a direct phase II and minor metabolite of ethanol formed through the UDP-glucuronosyl transferase-catalyzed conjugation of ethanol with glucuronic acid. Its investigation is of interest in both clinical and forensic contexts because of the wide window of detection. A sensitive LC-MS/MS procedure has been developed and fully validated according to the guidelines of forensic toxicology for the analysis of EtG in hair. Sample preparation and chromatographic separation were thoroughly optimized. The analysis was performed in the multiple reaction monitoring mode using the transitions m/z 221 → 203 (for the quantification) and 221 → 85 or 75 (for the qualification) for EtG, and m/z 226 → 208 (for quantification) and 226 → 75 or 85 (for qualification) for EtG-D5, used as the internal standard. Analyses were carried out using an Inertsil ODS-3 column (100 × 3 mm i.d., 3 µm particle size) and a mobile phase composed of formic acid and acetonitrile. Various SPE cartridges and solvents were tested in order to obtain the highest recoveries and cleanest extracts. The assay linearity of EtG was confirmed over the range from 20 to 2500 pg mg(-1), with a coefficient of determination (R(2) ) above 0.99. The lower limit of quantitation (LLOQ) was 20 pg mg(-1) and the limit of detection was 10 pg mg(-1). Intra- and inter-day assays were less than 15% except at the LLOQ (20%). The analytical method was applied to 72 post-mortem hair samples. EtG concentration in the hair ranged from 0 to 653 pg mg(-1) hair. PMID:22234871

  11. Can ethyl glucuronide in hair be determined only in 3 cm hair strands?

    PubMed

    Agius, Ronald; Ferreira, Liliane Martins; Yegles, Michel

    2012-05-10

    This paper addresses the suitability of ethyl glucuronide in hair (EtGH) strands other than 3cm for alcohol consumption. This issue will be addressed (a) by statistically comparing the distribution of EtGH results for 3cm hair strands to other hair strands analysed from 4126 cases and (b) by examining the stability of EtGH in an 8cm hair strand and two 12cm hair samples of two volunteers and a post-mortem case using 1cm segmental analysis. For 3464 driving license re-granting Medical and Psychological Assessment (MPA) cases, the detection of alcohol consumption using hair lengths longer than 3cm was never significantly less than for 3cm hair lengths, even up to 12cm hair lengths analysed non-segmented. For 662 non-MPA cases, where, in contrast to MPA cases, generally no abstinence was required, an increase in the EtGH positivity rate was observed with increasing hair length analysed up to 9cm, indicating that EtG-washout effects seem to play a minor role if any. For both MPA and non-MPA hair samples less than 3cm, a drastic, significant increase in the number of positive EtGH samples were observed, compared to 3cm hair lengths, strongly supportive of EtGH incorporation from sweat after a recent alcohol consumption. Segmental studies indicated that EtG is stable in the hair matrix up to 12cm long, hence supporting the above results. Even though both the statistical and the stability studies are preliminary results which need to be confirmed by other studies, they both provide evidence for the determination of alcohol consumption using EtGH in hair lengths longer than 3cm. Amendments to the Consensus of the Society of Hair Testing, the German driving license re-granting guidelines and EWDTS hair guidelines with respect to testing for abstinence and/or alcoholism are proposed for the benefit of the donors. PMID:22019395

  12. A fully validated high-performance liquid chromatography-tandem mass spectrometry method for the determination of ethyl glucuronide in hair for the proof of strict alcohol abstinence.

    PubMed

    Albermann, Maria Elena; Musshoff, F; Madea, B

    2010-04-01

    Hair analysis has become a powerful tool for the detection of chronic and past drug consumption. For several years, it has been possible to determine even the intake of ethanol in hair samples by detecting the ethanol metabolites ethyl glucuronide or fatty acid ethyl esters. Recently, new requirements were published for the use of EtG as an abstinence test (c(EtG) < 7 pg/mg) as well as for heavy-drinking detection (c(EtG) > 30 pg/mg). In order to perform abstinence tests, a sensitive LC-MS/MS procedure has been developed and fully validated according to the guidelines of forensic toxicology. The nine-point calibration curve showed linearity over the range of concentrations from 2-1,000 pg/mg. Detection and quantification limits were 1 and 4 pg/mg respectively. The intra- and inter-day precision and accuracy were always better than 20%. The validated procedure has successfully been applied to perform abstinence tests and to analyze hair samples from persons in withdrawal treatment. Concentrations between EtG concentration is close to a cut-off value. PMID:20130844

  13. Glucuronic acid and the ethanol metabolite ethyl-glucuronide cause Toll-like receptor 4 activation and enhanced pain

    PubMed Central

    Lewis, Susannah S.; Hutchinson, Mark R.; Zhang, Yingning; Hund, Dana K.; Maier, Steven F.; Rice, Kenner C.; Watkins, Linda R.

    2013-01-01

    We have previously observed that the non-opioid morphine metabolite, morphine-3-glucuronide, enhances pain via a toll-like receptor 4 (TLR4) dependent mechanism. The present studies were undertaken to determine whether TLR4-dependent pain enhancement generalizes to other classes of glucuronide metabolites. In silico modeling predicted that glucuronic acid alone and ethyl glucuronide, a minor but long-lasting ethanol metabolite, would dock to the same MD-2 portion of the TLR4 receptor complex previously characterized as the docking site for morphine-3-glucuronide. Glucuronic acid, ethyl glucuronide and ethanol all caused an increase in TLR4-dependent reporter protein expression in a cell line transfected with TLR4 and associated co-signaling molecules. Glucuronic acid-, ethyl glucuronide-, and ethanol-induced increases in TLR4 signaling were blocked by the TLR4 antagonists LPS-RS and (+)-naloxone. Glucuronic acid and ethyl glucuronide both caused allodynia following intrathecal injection in rats, which was blocked by intrathecal co-administration of the TLR4 antagonist LPS-RS. The finding that ethyl glucuronide can cause TLR4-dependent pain could have implications for human conditions such as hangover headache and alcohol withdrawal hyperalgesia, as well as suggesting that other classes of glucuronide metabolites could have similar effects. PMID:23348028

  14. Determination of ethyl glucuronide in hair samples of Chinese people by protein precipitation (PPT) and large volume injection-gas chromatography-tandem mass spectrometry (LVI-GC/MS/MS).

    PubMed

    Shi, Yan; Shen, Baohua; Xiang, Ping; Yan, Hui; Shen, Min

    2010-11-15

    Ethyl glucuronide (EtG) has been shown to be a suitable marker of excessive alcohol consumption. Determination of EtG in hair samples may help to differentiate social drinkers from alcoholics, and this testing can be widely used in forensic science, treatment programs, workplaces, military bases as well as driving ability test to provide legal proof of drinking. A method for determination of EtG in hair samples using large volume injection-gas chromatography-tandem mass spectrometry (LVI-GC/MS/MS) was developed and validated. Hair samples (in 1 mL deionized water) were ultrasonicated for 1h and incubated overnight; these samples were then deproteinated to remove impurities and derivatisated with 15 μL of pyridine and 30 μL of BSTFA. EtG was detected using GC/MS/MS in multiple-reaction monitoring mode. This method exhibited good linearity: y=0.0036 x+0.0437, R²=0.9993, the limit of detection and the limit of quantification were 5 pg/mg and 10 pg/mg, respectively. The extraction recoveries were more than 60%, and the inter-day and intra-day relative standard deviations (RSD) were less than 15%. This method has been applied to the analysis of EtG in hair samples from 21 Chinese subjects. The results for samples obtained from all of those who were teetotallers were negative, and the results for the other 15 samples ranged from 10 to 78 pg/mg, except for one negative sample. These data are the basis for interpretation of alcohol abuse. PMID:20977979

  15. Chemometric evaluation of nine alcohol biomarkers in a large population of clinically-classified subjects: pre-eminence of ethyl glucuronide concentration in hair for confirmatory classification.

    PubMed

    Pirro, Valentina; Valente, Valeria; Oliveri, Paolo; De Bernardis, Angela; Salomone, Alberto; Vincenti, Marco

    2011-10-01

    An important goal of forensic and clinical toxicology is to identify biological markers of ethanol consumption that allow an objective diagnosis of chronic alcohol misuse. Blood and head hair samples were collected from 175 subjects-objectively classified as non-drinkers (N=65), social drinkers (N=51) and active heavy drinkers (N=59)-and analyzed to determine eight traditional indirect biomarkers of ethanol consumption [aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyltransferase (γ-GT), alkaline phosphatase (ALP), mean corpuscular volume (MCV), carbohydrate-deficient transferrin (CDT), and cholesterol and triglycerides in blood] and one direct biomarker [ethyl glucuronide (EtG) in head hair]. The experimental values obtained from these determinations were submitted to statistical evaluations. In particular, Kruskal-Wallis, Mann-Whitney and ROC curve analyses, together with principal component analysis (PCA), allowed the diagnostic performances of the various biomarkers to be evaluated and compared consistently. From these evaluations, it was possible to deduce that EtG measured in head hair is the only biomarker that can conclusively discriminate active heavy drinkers from social and non-drinkers, using a cut-off value of 30 pg/mg. In contrast, a few indirect biomarkers such as ALP, cholesterol, and triglycerides showed extremely low diagnostic abilities and may convey misleading information. AST and ALT proved to be highly correlated and exhibited quite low sensitivity and specificity. Consequently, either of these parameters can be discarded without compromising the classification efficiency. Among the indirect biomarkers, γ-GT provided the highest diagnostic accuracy, while CDT and MCV yielded high specificity but low sensitivity. It was therefore concluded that EtG in head hair is the only biomarker capable of supporting a confirmatory diagnosis of chronic alcohol abuse in both forensic and clinical practice, while it was found

  16. Positive EtG findings in hair as a result of a cosmetic treatment.

    PubMed

    Sporkert, Frank; Kharbouche, Hicham; Augsburger, Marc P; Klemm, Clementine; Baumgartner, Markus R

    2012-05-10

    In a case of a driving ability assessment, hair analysis for ethyl glucuronide (EtG) was requested by the authorities. The person concerned denied alcohol consumption and did not present any clinical sign of alcoholism. However, EtG was found in concentrations of up to 910pg/mg in hair from different sampling dates suggesting an excessive drinking behavior. The person declared to use a hair lotion on a regularly base. To evaluate a possible effect of the hair lotion, prospective blood and urine controls as well as hair sampling of scalp and pubic hair were performed. The traditional clinical biomarkers of ethanol consumption, CDT and GGT, were inconspicuous in three blood samples taken. EtG was not detected in all collected urine samples. The hair lotion was transmitted to our laboratory. The ethanol concentration in this lotion was determined with 35g/L. The EtG immunoassay gave a positive result indicating EtG, which could be confirmed by GC-MS/MS-NCI. In a follow-up experiment the lotion was applied to the hair of a volunteer over a period of six weeks. After this treatment, EtG could be measured in the hair at a concentration of 72pg/mg suggesting chronic and excessive alcohol consumption. Overnight incubation of EtG free hair in the lotion yielded an EtG concentration of 140pg/mg. In the present case, the positive EtG hair findings could be interpreted as the result of an EtG containing hair care product. To our knowledge, the existence of such a product has not yet been reported, and it is exceptionally unusual to find EtG in cosmetics. Therefore, external sources for hair contamination should always be taken into account when unusual cosmetic treatment is mentioned. In those cases, it is recommended to analyze the hair product for a possible contamination with EtG. The analysis of body hair can help to reveal problems occurring from cosmetic treatment of head hair. As a consequence, the assessment of drinking behavior should be based on more than one

  17. A novel, simultaneous extraction of FAEE and EtG from meconium and analysis by LC-MS/MS.

    PubMed

    Vaiano, Fabio; Favretto, Donata; Palumbo, Diego; Cooper, Gail; Mactier, Helen; Busardò, Francesco P; Mari, Francesco; Bertol, Elisabetta

    2016-04-01

    Fatty acid ethyl esters (FAEEs) and ethyl-glucuronide (EtG) in meconium have been widely studied as biomarkers of maternal alcohol consumption during pregnancy. Many analytical approaches have been proposed for their analysis, mostly consisting of separated extraction procedures requiring the use of two meconium aliquots. This study aimed to validate a new analytical procedure for the simultaneous extraction of FAEEs and EtG from a meconium aliquot through a single solid-phase extraction (SPE) applied to 242 anonymized samples of meconium. Targeted FAEEs were: ethyl-myristate (Myr), ethyl-palmitate (Pal), ethyl-oleate (Ole) and ethyl-stearate (Ste). Two hundred milligrams of meconium was sonicated with acetonitrile, and a single SPE performed by means of aminopropyl columns. FAEEs were eluted with hexane, followed by EtG elution with water. Both the mixtures were dried, recovered, and analyzed by liquid chromatography-tandem mass spectrometry using C8 (FAEEs) and C18 (EtG) columns. Transitions were: m/z 257 → 57,88, Myr; m/z 262 → 57,88, Myr-d5; m/z 285 → 57, 72, Pal; m/z 290 → 57,258, Pal-d5; m/z 311 → 72,114, Ole; m/z 316 → 72,265, Ole-d5; m/z 257 → 57,72 Ste; m/z 318 → 57,286, Ste-d5; m/z 221 → 75,85, EtG; m/z 226 → 75,85, EtG-d5. Lower limit of quantification range was 10-15 ng/g for FAEEs and 10 ng/g for EtG. Linearity was evaluated for different concentration ranges; the mean coefficients of determination (R (2)) were above 0.9961. Precision and accuracy for FAEEs and EtG were consistently ≤20 % and ±20 %, respectively. Ion suppression was observed for all the analytes. Matrix effect did not significantly affect the analyses. Recovery efficiency was 93 % for EtG and 75-85 % for FAEEs. PMID:26873204

  18. Involvement of UDP-glucuronosyltransferases UGT1A9 and UGT2B7 in ethanol glucuronidation, and interactions with common drugs of abuse.

    PubMed

    Al Saabi, Alaa; Allorge, Delphine; Sauvage, François-Ludovic; Tournel, Gilles; Gaulier, Jean-Michel; Marquet, Pierre; Picard, Nicolas

    2013-03-01

    Ethyl glucuronide (EtG) determination is increasingly used in clinical and forensic toxicology to document ethanol consumption. The enzymes involved in EtG production, as well as potential interactions with common drugs of abuse, have not been extensively studied. Activities of human liver (HLM), kidney (HKM), and intestinal (HIM) microsomes, as well as of 12 major human recombinant UDP-glucuronosyltransferases (UGTs), toward ethanol (50 and 500 mM) were evaluated in vitro using liquid chromatography-tandem mass spectrometry. Enzyme kinetic parameters were determined for pooled microsomes and recombinant UGTs with significant activity. Individual contributions of UGTs were estimated using the relative activity factor approach, proposed for scaling activities obtained with cDNA-expressed enzymes to HLM. Interaction of morphine, codeine, lorazepam, oxazepam, nicotine, cotinine, cannabinol, and cannabidiol (5, 10, 15 mg/l) with ethanol (1.15, 4.6, 11.5 g/l; i.e., 25, 100, 250 mM) glucuronidation was assessed using pooled HLM. Ethanol glucuronidation intrinsic clearance (Cl(int)) was 4 and 12.7 times higher for HLM than for HKM and HIM, respectively. All recombinant UGTs, except UGT1A1, 1A6, and 1A10, produced EtG in detectable amounts. UGT1A9 and 2B7 were the most active enzymes, each accounting for 17 and 33% of HLM Cl(int), respectively. Only cannabinol and cannabidiol significantly affected ethanol glucuronidation. Cannabinol increased ethanol glucuronidation in a concentration-dependent manner, whereas cannabidiol significantly inhibited EtG formation in a noncompetitive manner (IC(50) = 1.17 mg/l; inhibition constant (K(i)) = 3.1 mg/l). UGT1A9 and 2B7 are the main enzymes involved in ethanol glucuronidation. In addition, our results suggest that cannabinol and cannabidiol could significantly alter ethanol glucuronidation. PMID:23230132

  19. Ethyl glucuronide concentration in hair for detecting heavy drinking and/or abstinence: a meta-analysis.

    PubMed

    Boscolo-Berto, Rafael; Viel, Guido; Montisci, Massimo; Terranova, Claudio; Favretto, Donata; Ferrara, Santo Davide

    2013-05-01

    In both clinical and forensic settings, hair analysis for ethyl glucuronide (HEtG) has been increasingly employed for diagnosing chronic excessive drinking and, more recently, for monitoring abstinence. This paper aims at meta-analysing published data on HEtG concentrations in teetotallers, social drinkers and heavy drinkers in order to evaluate the use of this marker in hair for identifying chronic excessive drinking and for monitoring abstinence. In May 2012, a systematic multi-database search retrieved 366 records related to HEtG and further screened for relevant publications in the field. Fifteen (4.1 %) records matched the selection criteria and were included in the meta-analysis. The mean and 95 % confidence intervals (CI) of HEtG concentrations in social drinkers (mean 7.5 pg/mg; 95 % CI 4.7-10.2 pg/mg; p < 0.001), heavy drinkers (mean 142.7 pg/mg; 95 % CI 99.9-185.5 pg/mg; p < 0.001) and deceased subjects with a known history of chronic excessive drinking (mean 586.1 pg/mg; 95 % CI 177.2-995.0 pg/mg; p < 0.01) were calculated. The ranges of mean values and 95 % confidence intervals for single studies involving teetotallers/social or social/heavy drinkers showed a partial overlap with a down-trespassing of both the 7 and 30 pg/mg thresholds for social and heavy drinkers, respectively. Although larger and well-designed population studies are required to draw any definitive conclusion, our data show that the cut-off of 30 pg/mg limits the false-negative effect in differentiating heavy from social drinkers, whereas the recently proposed 7 pg/mg cut-off value might only be used for suspecting an active alcohol use, and not for proving complete abstinence. PMID:23250386

  20. Serum/whole blood concentration ratio for ethylglucuronide and ethyl sulfate.

    PubMed

    Høiseth, Gudrun; Morini, Luca; Polettini, Aldo; Christophersen, Asbjørg S; Johnsen, Lene; Karinen, Ritva; Mørland, Jørg

    2009-05-01

    Serum/blood (S/B) concentration ratios for ethyl glucuronide (EtG) and ethyl sulfate (EtS) are missing from the literature, and the aim of this study was to determine these ratios in samples from patients at admission to an alcohol rehabilitation clinic. Two blood samples were collected simultaneously, and EtG and EtS were analyzed in whole blood and serum, respectively, using a liquid chromatography-mass spectrometry method. Separate calibration standards were prepared in both whole blood and serum for the calculation of whole blood and serum concentrations, respectively. Thirteen pairs of serum and whole blood were analyzed. The median S/B value for EtG was 1.69, and the range was 1.33-1.90. For EtS, the median S/B ratio was 1.30, and the range was 1.08-1.47. The S/B ratio was significantly lower for EtS than for EtG (p < 0.001). The higher concentrations of EtG and EtS in serum than in whole blood have to be considered when whole blood results obtained from forensic toxicology are compared to serum or plasma results from clinical laboratories. PMID:19470223

  1. Computer assisted modeling of ethyl sulfate pharmacokinetics.

    PubMed

    Schmitt, Georg; Halter, Claudia C; Aderjan, Rolf; Auwaerter, Volker; Weinmann, Wolfgang

    2010-01-30

    For 12 volunteers of a drinking experiment the concentration-time-courses of ethyl sulfate (EtS) and ethanol were simulated and fitted to the experimental data. The concentration-time-courses were described with the same mathematical model as previously used for ethyl glucuronide (EtG). The kinetic model based on the following assumptions and simplifications: a velocity constant k(form) for the first order formation of ethyl sulfate from ethanol and an exponential elimination constant k(el). The mean values (and standard deviations) obtained for k(form) and k(el) were 0.00052 h(-1) (0.00014) and 0.561 h(-1) (0.131), respectively. Using the ranges of these parameters it is possible to calculate minimum and maximum serum concentrations of EtS based on stated ethanol doses and drinking times. The comparison of calculated and measured concentrations can prove the plausibility of alleged ethanol consumption and add evidence to the retrospective calculation of ethanol concentrations based on EtG concentrations. PMID:19913378

  2. Comparison of ethyl glucuronide in hair with carbohydrate-deficient transferrin in serum as markers of chronic high levels of alcohol consumption.

    PubMed

    Morini, Luca; Politi, Lucia; Acito, Silvia; Groppi, Angelo; Polettini, Aldo

    2009-07-01

    This study was designed with the aim to compare sensitivity and specificity of ethyl glucuronide in hair (HEtG) and carbohydrate-deficient transferrin (CDT) in serum as markers of heavy drinking. Eighty-six volunteers, including teetotalers, social, and heavy drinkers, were interviewed to evaluate their ethanol daily intake (EDI) during the last 2-week and 3-month periods. HEtG determination was performed by a fully validated LC-MS-MS procedure and ranged from

  3. Ethylglucuronide and Ethyl Sulfate Assays in Clinical Trials, Interpretation and Limitations: Results of a Dose Ranging Alcohol Challenge Study and Two Clinical Trials

    PubMed Central

    Jatlow, Peter I.; Agro, Ann; Wu, Ran; Nadim, Haleh; Toll, Benjamin A.; Ralevski, Elizabeth; Nogueira, Christine; Shi, Julia; Dziura, James D.; Petrakis, Ismene L.; O'Malley, Stephanie S.

    2014-01-01

    Background The ethanol metabolites, ethyl glucuronide (EtG) and ethyl sulfate (EtS) are biomarkers of recent alcohol consumption that provide objective measures of abstinence. Our goals are to better understand the impact of cutoff concentration on test interpretation, the need for measuring both metabolites, and how best to integrate test results with self-reports in clinical trials. Methods Subjects (n=18) were administered, one week apart, 3 alcohol doses calibrated to achieve blood concentrations of 20, 80 and 120 mg/dL respectively. Urinary EtG/EtS were measured at timed intervals during a 24 hour hospitalization and twice daily thereafter. In addition, participants from 2 clinical trials provided samples for EtG/EtS and drinking histories. Cutoffs for EtG/EtS of 100/50, 200/100 and 500/250 ng/mL were evaluated. Results Twelve hours following each challenge, EtG was always positive at the 100 and 200 cutoffs, but at 24 hours sensitivity was poor at all cutoffs following the low dose, and poor after 48 hours regardless of dose or cutoff. Similarly, in the clinical trials EtG sensitivity was good for detecting any drinking during the last 24 hours at the two lowest cutoffs, but under 40% during the last 24-48 hours. Sensitivity was reduced at the 500 ng/mL cutoff. Discrepancies between EtG and EtS were few. Comparison of self- reports of abstinence and EtG confirmed abstinence indicated under-reporting of drinking. Conclusions Any drinking the night before should be detectable the following morning with EtG cutoffs of 100 or 200 ng/mL. Twenty-four hours after drinking, sensitivity is poor for light drinking, but good for heavier consumption. At 48 hours, sensitivity is low following 6 drinks or less. Increasing the cutoff to 500 ng/mL leads to substantially reduced sensitivity. Monitoring both EtG and EtS should usually be unnecessary. We recommend EtG confirmed self-reports of abstinence for evaluation of outcomes in clinical trials. PMID:24773137

  4. Gyrokinetic Simulations of ETG and ITG Turbulence

    SciTech Connect

    Dimits, A; Nevins, W; Shumaker, D; Hammett, G; Dannert, T; Jenko, F; Dorland, W; Leboeuf, J; Rhodes, T; Candy, J; Estrada-Mila, C

    2006-10-03

    Published gyrokinetic continuum-code simulations indicated levels of the electron thermal conductivity {chi}{sub e} due to electron-temperature-gradient (ETG) turbulence large enough to be significant in some tokamaks, while subsequent global particle-in-cell (PIC) simulations gave significantly lower values. We have carried out an investigation of this discrepancy. We have reproduced the key features of the aforementioned PIC simulations using the flux-tube gyrokinetic PIC code, PG3EQ, thereby eliminating global effects and as the cause of the discrepancy. We show that the late-time low-transport state in both of these sets of PIC simulations is a result of discrete particle noise, which is a numerical artifact. Thus, the low value of {chi}{sub e} along with conclusions about anomalous transport drawn from these particular PIC simulations are unjustified. In our attempts to benchmark PIC and continuum codes for ETG turbulence at the plasma parameters used above, both produce very large intermittent transport. We have therefore undertaken benchmarks at an alternate reference point, magnetic shear s=0.1 instead of s=0.796, and have found that PIC and continuum codes reproduce the same transport levels. Scans in the magnetic shear show an abrupt transition to a high-{chi}{sub e} state as the shear is increased above s=0.4. When nonadiabatic ions are used, this abrupt transition is absent, and {chi}{sub e} increases gradually reaching values consistent with transport analyses of DIII-D, JET, and JT60-U discharges. New results on the balances of zonal-flow driving and damping terms in late-time quasi-steady ITG turbulence and on real-geometry gyrokinetic simulations of shaped DIII-D discharges are also reported.

  5. Select steroid hormone glucuronide metabolites can cause Toll-like receptor 4 activation and enhanced pain

    PubMed Central

    Lewis, Susannah S.; Hutchinson, Mark R.; Frick, Morin M.; Zhang, Yingning; Maier, Steven F.; Sammakia, Tarek; Rice, Kenner C.; Watkins, Linda R.

    2014-01-01

    We have recently shown that several classes of glucuronide metabolites, including the morphine metabolite morphine-3-glucuronide and the ethanol metabolite ethyl glucuronide, cause toll like receptor 4 (TLR4)-dependent signalling in vitro and enhanced pain in vivo. Steroid hormones, including estrogens and corticosterone, are also metabolized through glucuronidation. Here we demonstrate that in silico docking predicts that corticosterone, corticosterone-21-glucuronide, estradiol, estradiol-3-glucuronide and estradiol-17-glucuronide all dock with the MD-2 component of the TLR4 receptor complex. In addition to each docking with MD-2, the docking of each was altered by pre-docking with (+)-naloxone, a TLR4 signaling inhibitor. As agonist versus antagonist activity cannot be determined from these in silico interactions, an in vitro study was undertaken to clarify which of these compounds can act in an agonist fashion. Studies using a cell line transfected with TLR4, necessary co-signaling molecules, and a reporter gene revealed that only estradiol-3-glucuronide and estradiol-17-glucuronide increased reporter gene product, indicative of TLR4 agonism. Finally, in in vivo studies, each of the 5 drugs was injected intrathecally at equimolar doses. In keeping with the in vitro results, only estradiol-3-glucuronide and estradiol-17-glucuronide caused enhanced pain. For both compounds, pain enhancement was blocked by the TLR4 antagonist lipopolysaccharide from Rhodobacter sphaeroides, evidence for the involvement in TLR4 in the resultant pain enhancement. These findings have implications for several chronic pain conditions, including migraine and tempromandibular joint disorder, in which pain episodes are more likely in cycling females when estradiol is decreasing and estradiol metabolites are at their highest. PMID:25218902

  6. The UV window on counter rotating ETGs: insight from SPH simulations with chemo-photometric implementation

    NASA Astrophysics Data System (ADS)

    Bettoni, D.; Mazzei, P.; Rampazzo, R.; Marino, A.; Galletta, G.; Buson, L. M.

    2014-11-01

    The Galaxy Evolution Explorer ( GALEX) detected ultraviolet emission in about 50 % of multi-spin early-type galaxies (ETGs), suggesting the occurrence of a recent rejuvenation episode connected to the formation of these kinematical features. With the aim at investigating the complex evolutionary scenario leading to the formation of counter rotating ETGs (CR-ETGs) we use our Smooth Particle Hydrodynamic (SPH) code with chemo-photometric implementation. We discuss here the UV evolutionary path of two CR-ETGs, NGC 3593 and NGC 5173, concurrently best fitting their global observed properties, i.e., morphology, dynamics, as well as their total B-band absolute magnitude and spectral energy distribution (SED) extended over three orders of magnitude in wavelength. These simulations correspond to our predictions about the target evolution which we follow in the color-magnitude diagram (CMD), near-UV (NUV) versus r-band absolute magnitude, as a powerful diagnostic tool to emphasize rejuvenation episodes.

  7. Autism and Phthalate Metabolite Glucuronidation

    ERIC Educational Resources Information Center

    Stein, T. Peter; Schluter, Margaret D.; Steer, Robert A.; Ming, Xue

    2013-01-01

    Exposure to environmental chemicals may precipitate autism spectrum disorders (ASD) in genetically susceptible children. Differences in the efficiency of the glucuronidation process may substantially modulate substrate concentrations and effects. To determine whether the efficiency of this pathway is compromised in children with ASD, we measured…

  8. Different Electrostatic Potentials Define ETGE and DLG Motifs as Hinge and Latch in Oxidative Stress Response▿

    PubMed Central

    Tong, Kit I.; Padmanabhan, Balasundaram; Kobayashi, Akira; Shang, Chengwei; Hirotsu, Yosuke; Yokoyama, Shigeyuki; Yamamoto, Masayuki

    2007-01-01

    Nrf2 is the regulator of the oxidative/electrophilic stress response. Its turnover is maintained by Keap1-mediated proteasomal degradation via a two-site substrate recognition mechanism in which two Nrf2-Keap1 binding sites form a hinge and latch. The E3 ligase adaptor Keap1 recognizes Nrf2 through its conserved ETGE and DLG motifs. In this study, we examined how the ETGE and DLG motifs bind to Keap1 in a very similar fashion but with different binding affinities by comparing the crystal complex of a Keap1-DC domain-DLG peptide with that of a Keap1-DC domain-ETGE peptide. We found that these two motifs interact with the same basic surface of either Keap1-DC domain of the Keap1 homodimer. The DLG motif works to correctly position the lysines within the Nrf2 Neh2 domain for efficient ubiquitination. Together with the results from calorimetric and functional studies, we conclude that different electrostatic potentials primarily define the ETGE and DLG motifs as a hinge and latch that senses the oxidative/electrophilic stress. PMID:17785452

  9. Scaling of Electron Thermal Conductivity during the Transition between Slab and Mixed Slab-Toroidal ETG Mode

    NASA Astrophysics Data System (ADS)

    Sokolov, Vladimir; Balbaky, Abed; Sen, Amiya K.

    2015-11-01

    Transition from the slab to the toroidal branch of the electron temperature gradient (ETG) mode has been successfully achieved in a basic experiment in Columbia Linear Machine CLM. We found a modest increase in saturated ETG potential fluctuations (~ 2 ×) and a substantial increase in the power density of individual mode peaks (~ 4 - 5 ×) with increased levels of curvature. We have obtained a set of experimental scalings for electron thermal conductivity χ⊥e as a function of the inverse radius of curvature Rc-1 for different fluctuation levels of the initial slab ETG mode. We found that thermal conductivity for pure slab modes is larger than it is for mixed slab-toroidal ETG modes with the same level of mode fluctuation. This effective reduction in diffusive transport can be partly explained by the flute nature of the toroidal ETG mode. This research was supported by the Department of Electrical Engineering of Columbia University.

  10. Icosapent Ethyl

    MedlinePlus

    ... pharmacist if you are allergic to icosapent ethyl; fish, including shellfish (clams, scallops, shrimp, lobster, crayfish, crab, ... and ticlopidine (Ticlid); aspirin or aspirin-containing products; beta-blockers such as atenolol (Tenormin), labetalol (Normodyne), metoprolol ( ...

  11. Ethyl carbamate

    Integrated Risk Information System (IRIS)

    Ethyl carbamate ; CASRN 51 - 79 - 6 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Ef

  12. Ethyl ether

    Integrated Risk Information System (IRIS)

    Ethyl ether ; CASRN 60 - 29 - 7 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Effect

  13. Ethyl acetate

    Integrated Risk Information System (IRIS)

    Ethyl acetate ; CASRN 141 - 78 - 6 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Eff

  14. Ethyl chloride

    Integrated Risk Information System (IRIS)

    Ethyl chloride ; CASRN 75 - 00 - 3 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Eff

  15. The MCM-binding protein ETG1 aids sister chromatid cohesion required for postreplicative homologous recombination repair.

    PubMed

    Takahashi, Naoki; Quimbaya, Mauricio; Schubert, Veit; Lammens, Tim; Vandepoele, Klaas; Schubert, Ingo; Matsui, Minami; Inzé, Dirk; Berx, Geert; De Veylder, Lieven

    2010-01-01

    The DNA replication process represents a source of DNA stress that causes potentially spontaneous genome damage. This effect might be strengthened by mutations in crucial replication factors, requiring the activation of DNA damage checkpoints to enable DNA repair before anaphase onset. Here, we demonstrate that depletion of the evolutionarily conserved minichromosome maintenance helicase-binding protein ETG1 of Arabidopsis thaliana resulted in a stringent late G2 cell cycle arrest. This arrest correlated with a partial loss of sister chromatid cohesion. The lack-of-cohesion phenotype was intensified in plants without functional CTF18, a replication fork factor needed for cohesion establishment. The synergistic effect of the etg1 and ctf18 mutants on sister chromatid cohesion strengthened the impact on plant growth of the replication stress caused by ETG1 deficiency because of inefficient DNA repair. We conclude that the ETG1 replication factor is required for efficient cohesion and that cohesion establishment is essential for proper development of plants suffering from endogenous DNA stress. Cohesion defects observed upon knockdown of its human counterpart suggest an equally important developmental role for the orthologous mammalian ETG1 protein. PMID:20090939

  16. Lithocholate glucuronide is a cholestatic agent

    SciTech Connect

    Oelberg, D.G.; Chari, M.V.; Little, J.M.; Adcock, E.W.; Lester, R.

    1984-06-01

    Lithocholic acid and its taurine, glycine, and sulfate derivatives are potent cholestatic agents. (3 beta-/sup 3/H)lithocholate 3-O-beta-D-glucuronide was synthesized, and chemical and radiochemical purity were established. The aqueous solubility of lithocholate glucuronide was determined and found to be greater than that of lithocholic acid or several of its derivatives. In the range of concentrations examined, calcium ions precipitated lithocholate glucuronide stoichiometrically. The material was administered to rats prepared with an external biliary fistula. When 17-25 micrograms quantities were administered, 89.1 +/- 4.5% (mean +/- SEM) of the radiolabel was secreted in bile within the first 20 h after administration, the major fraction being secreted in less than 20 min. Four-fifths of the radiolabeled material in bile was the administered unaltered parent compound, while a minor fraction consisted of a more polar derivative(s). We showed that increasing biliary concentrations of more polar derivatives were observed with milligram doses of (3H)lithocholate glucuronide, and with time after the administration of these loading doses. Milligram doses of (3H)lithocholate glucuronide resulted in partial or complete cholestasis. When induced cholestasis was partial, secretion in bile remained the primary excretory route (82.5-105.6% recovery in bile), while, when complete cholestasis was induced, wide tissue distribution of radiolabel was observed. Cholestasis developed rapidly during infusion of (3H)lithocholate glucuronide. Bile flow was diminished within 10-20 min of the start of an infusion of 0.05 mumol, 100 g-1 body weight, minute-1, administered concomitantly with an equimolar infusion of taurocholate. The results establish that lithocholate glucuronide exerts cholestatic effects comparable to those exerted by unconjugated lithocholic acid.

  17. AGN feedback and star formation in ETGs: negative and positive feedback

    NASA Astrophysics Data System (ADS)

    Ciotti, Luca; Ostriker, Jeremiah P.; Novak, Greg; Negri, Andrea; Pellegrini, Silvia; Posacki, Silvia

    2015-08-01

    AGN feedback from supermassive black holes at the center of Early Type Galaxies is commonly invoked as the explanation for the quenching of star formation in these systems, that after this phase are considered “red and dead”. The situation is by far more complicated, due to the significant amount of mass injected in the galaxy by the evolving stellar population over cosmological times. In absence of feedback, this mass would lead to unobserved galactic cooling flows, and to central black holes two orders of magnitude more massive than observed. I will present the results of state-of-the-art hydrodynamical simulations with radiative transport and star formation of the “passive” evolution of ETGs, focusing in particular on highly structured spatial and temporal nature of the intermittent AGN feedback, that is not only negative (shutting down the cooling episodes of the ISM), but also positive, inducing star formation in the inner regions of the host galaxy.

  18. Determination of propofol glucuronide from hair sample by using mixed mode anion exchange cartridge and liquid chromatography tandem mass spectrometry.

    PubMed

    Kwak, Jae-Hwan; Kim, Hye Kyung; Choe, Sanggil; In, Sangwhan; Pyo, Jae Sung

    2016-03-15

    The main objective of this study was to develop and validate a simpler and less time consuming analytical method for determination of propofol glucuronide from hair sample, by using mixed mode anion exchange cartridge and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The study uses propofol glucuronide, a major metabolite of propofol, as a marker for propofol abuse. The hair sample was digested in sodium hydroxide solution and loaded in mixed-mode anion cartridge for solid phase extraction. Water and ethyl acetate were used as washing solvents to remove interfering substances from the hair sample. Consequently, 2% formic acid in ethyl acetate was employed to elute propofol glucuronide from the sorbent of mixed-mode anion cartridge, and analyzed by LC-MS/MS. The method validation parameters such as selectivity, specificity, LOD, LLOQ, accuracy, precision, recovery, and matrix effect were also tested. The linearity of calibration curves showed good correlation, with correlation coefficient 0.998. The LOD and LLOQ of the propofol glucuronide were 0.2 pg/mg and 0.5 pg/mg, respectively. The intra and inter-day precision and accuracy were acceptable within 15%. The mean values of recovery and matrix effect were in the range of 91.7-98.7% and 87.5-90.3%, respectively, signifying that the sample preparation, washing and extraction procedure were efficient, and there was low significant hair matrix effect for the extraction of propofol glucuronide from hair sample on the mixed mode anion cartridge. To evaluate the suitability of method, the hair of propofol administered rat was successfully analyzed with this method.

  19. IFU Spectroscopy of 10 ETG nuclei: Properties of the circumnuclear gas emission

    NASA Astrophysics Data System (ADS)

    Ricci, T. V.

    2015-09-01

    LINERs are galactic nuclei containing spectra with prominence of low-ionization lines. Several ionization sources are able to produce a LINER-like spectra (e.g. shocks, low-luminosity AGNs, pAGB stars). In this work, we will present an analysis performed on a sample of 10 massive ETGs with distances up to 30 Mpc. This sample was observed with the GMOS-IFU installed on the Gemini-South Telescope. The data cubes of the galaxies have a FOV of 3.5 - 5 arcsec2 and a spatial resolution of about 0.6 -- 1.0 arcsec. After subtracting the stellar component of the galaxies by means of spectral synthesis, we studied the emission lines all over the FOV of the data cubes. In galaxies where an AGN was clearly detected, we found ionized gaseous discs and also a low-velocity extended emission perpendicular to the gas discs. We concluded that only ionizing photons emerging from the AGNs are not enough to photoionize the gaseous discs of the galaxies. On the other hand, it seems to be responsible for the photoionization of the low-velocity extended gas perpendicular to the discs. We propose a scheme where some collimating agent, somehow aligned to the gaseous discs, may be present in some LINER-like AGNs in the local universe.

  20. Acyl glucuronides: the good, the bad and the ugly.

    PubMed

    Regan, Sophie L; Maggs, James L; Hammond, Thomas G; Lambert, Craig; Williams, Dominic P; Park, B Kevin

    2010-10-01

    Acyl glucuronidation is the major metabolic conjugation reaction of most carboxylic acid drugs in mammals. The physiological consequences of this biotransformation have been investigated incompletely but include effects on drug metabolism, protein binding, distribution and clearance that impact upon pharmacological and toxicological outcomes. In marked contrast, the exceptional but widely disparate chemical reactivity of acyl glucuronides has attracted far greater attention. Specifically, the complex transacylation and glycation reactions with proteins have provoked much inconclusive debate over the safety of drugs metabolised to acyl glucuronides. It has been hypothesised that these covalent modifications could initiate idiosyncratic adverse drug reactions. However, despite a large body of in vitro data on the reactions of acyl glucuronides with protein, evidence for adduct formation from acyl glucuronides in vivo is limited and potentially ambiguous. The causal connection of protein adduction to adverse drug reactions remains uncertain. This review has assessed the intrinsic reactivity, metabolic stability and pharmacokinetic properties of acyl glucuronides in the context of physiological, pharmacological and toxicological perspectives. Although numerous experiments have characterised the reactions of acyl glucuronides with proteins, these might be attenuated substantially in vivo by rapid clearance of the conjugates. Consequently, to delineate a relationship between acyl glucuronide formation and toxicological phenomena, detailed pharmacokinetic analysis of systemic exposure to the acyl glucuronide should be undertaken adjacent to determining protein adduct concentrations in vivo. Further investigation is required to ascertain whether acyl glucuronide clearance is sufficient to prevent covalent modification of endogenous proteins and consequentially a potential immunological response. PMID:20830700

  1. Non-linear Dynamics in ETG Mode Saturation and Beam-Plasma Instabilities

    NASA Astrophysics Data System (ADS)

    Tokluoglu, Erinc K.

    Non-linear mechanisms arise frequently in plasmas and beam-plasma systems resulting in dynamics not predicted by linear theory. The non-linear mechanisms can influence the time evolution of plasma instabilities and can be used to describe their saturation. Furthermore time and space averaged non-linear fields generated by instabilities can lead to collisionless transport and plasma heating. In the case of beam-plasma systems counter-intuitive beam defocusing and scaling behavior which are interesting areas of study for both Low-Temperature and High Energy Density physics. The non-linear mode interactions in form of phase coupling can describe energy transfer to other modes and can be used to describe the saturation of plasma instabilities. In the first part of this thesis, a theoretical model was formulated to explain the saturation mechanism of Slab Electron Temperature Gradient (ETG) mode observed in the Columbia Linear Machine (CLM), based on experimental time-series data collected through probe diagnostics [1]. ETG modes are considered to be a major player in the unexplained high levels of electron transport observed in tokamak fusion experiments and the saturation mechanism of these modes is still an active area of investigation. The data in the frequency space indicated phase coupling between 3 modes, through a higher order spectral correlation coefficient known as bicoherence. The resulting model is similar to [2], which was a treatment for ITG modes observed in the CLM and correctly predicts the observed saturation level of the ETG turbulence. The scenario is further supported by the fact that the observed mode frequencies are in close alignment with those predicted theoretical dispersion relations. Non-linear effects arise frequently in beam-plasma systems and can be important for both low temperature plasma devices commonly used for material processing as well as High Energy Density applications relevant to inertial fusion. The non-linear time averaged

  2. 21 CFR 584.200 - Ethyl alcohol containing ethyl acetate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... Ethyl alcohol containing ethyl acetate. The feed additive ethyl alcohol containing ethyl acetate meets the requirement of 27 CFR 21.62, being not less than 92.5 percent ethyl alcohol, each 100 gallons... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Ethyl alcohol containing ethyl acetate....

  3. 21 CFR 584.200 - Ethyl alcohol containing ethyl acetate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... Ethyl alcohol containing ethyl acetate. The feed additive ethyl alcohol containing ethyl acetate meets the requirement of 27 CFR 21.62, being not less than 92.5 percent ethyl alcohol, each 100 gallons... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Ethyl alcohol containing ethyl acetate....

  4. 21 CFR 584.200 - Ethyl alcohol containing ethyl acetate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... Ethyl alcohol containing ethyl acetate. The feed additive ethyl alcohol containing ethyl acetate meets the requirement of 27 CFR 21.62, being not less than 92.5 percent ethyl alcohol, each 100 gallons... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Ethyl alcohol containing ethyl acetate....

  5. 21 CFR 584.200 - Ethyl alcohol containing ethyl acetate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Ethyl alcohol containing ethyl acetate. The feed additive ethyl alcohol containing ethyl acetate meets the requirement of 27 CFR 21.62, being not less than 92.5 percent ethyl alcohol, each 100 gallons... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Ethyl alcohol containing ethyl acetate....

  6. 21 CFR 584.200 - Ethyl alcohol containing ethyl acetate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Ethyl alcohol containing ethyl acetate. The feed additive ethyl alcohol containing ethyl acetate meets the requirement of 27 CFR 21.62, being not less than 92.5 percent ethyl alcohol, each 100 gallons... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Ethyl alcohol containing ethyl acetate....

  7. Conjugation position of quercetin glucuronides and effect on biological activity.

    PubMed

    Day, A J; Bao, Y; Morgan, M R; Williamson, G

    2000-12-15

    Quercetin glycosides are common dietary antioxidants. In general, however, potential biological effects of the circulating plasma metabolites (e.g., glucuronide conjugates) have not been measured. We have determined the rate of glucuronidation of quercetin at each position on the polyphenol ring by human liver cell-free extracts containing UDP-glucuronosyltransferases. The apparent affinity of UDP-glucuronosyltransferase followed the order 4'- > 3'- > 7- > 3, although the apparent maximum rate of formation was for the 7-position. The 5-position did not appear to be a site for conjugation. After isolation of individual glucuronides, the inhibition of xanthine oxidase and lipoxygenase were assessed. The K(i) for the inhibition of xanthine oxidase by quercetin glucuronides followed the order 4'- > 3'- > 7- > 3-, with quercetin-4'-glucuronide a particularly potent inhibitor (K(i) = 0. 25 microM). The glucuronides, with the exception of quercetin-3-glucuronide, were also inhibitors of lipoxygenase. Quercetin glucuronides are metabolites of quercetin in humans, and these compounds can retain some biological activity depending on conjugation position at expected plasma concentrations. PMID:11118813

  8. A specific immunoassay for the determination of morphine and its glucuronides in human blood.

    PubMed

    Beike, J; Blaschke, G; Mertz, A; Köhler, H; Brinkmann, B

    1998-01-01

    The development of specific antisera for immunochemical determination of morphine, morphine-3-glucuronide and morphine-6-glucuronide is described. Morphine was N-demethylated to normorphine and N-alkylated to give N-aminopropyl-normorphine as hapten for antisera against morphine. As haptens for antisera against morphine-3-glucuronide and morphine-6-glucuronide, N-aminopropyl-nor-morphine was glucuronidated in position 3 or 6 respectively. Each of these three haptens were coupled to BSA employing the glutaraldehyde method to obtain three different immunogens. Immunisation of rabbits with these conjugates gave anti-morphine, anti-morphine-3-glucuronide and anti-morphine-6-glucuronide antisera, which were tested in a competitive, heterogeneous radioimmunoassay. Tracers for this radioimmunoassay procedure were synthesised by substitution of morphine and morphine-6-glucuronide in position 2 with 125I and indirect iodination of the morphine-3-glucuronide hapten according to the method of Bolton and Hunter. The resulting antisera show very specific reactions with morphine, morphine-3-glucuronide and morphine-6-glucuronide. Cross reactivities of each antiserum with structurally related opiates and opioides are very low. The cross reactivities of the anti-morphine antiserum against morphine-3-glucuronide, morphine-6-glucuronide, codeine, codeine-6-glucuronide or dihydrocodeine were less than 0.3%, the anti-morphine-3-glucuronide antiserum against morphine, morphine-6-glucuronide, codeine, codeine-6-glucuronide or dihydrocodeine less than 0.1% and the anti-morphine-6-glucuronide antiserum against morphine, morphine-3-glucuronide, codeine or dihydrocodeine less than 0.1%, against codeine-6-glucuronide less than 2.3%. The determination of morphine, morphine-3-glucuronide and morphine-6-glucuronide in blood samples (limit of detection= 3, 1, 0.5 ng/g) of nine cases of fatal heroin overdose with this radioimmunoassay method and the comparison with a GC/MS method is described.

  9. Detection of pentachlorophenol and its glucuronide and sulfate conjugates in fish bile and exposure water

    SciTech Connect

    Stehly, G.R.; Hayton, W.L.

    1988-08-01

    The glucuronide and sulfate conjugates of pentachlorophenol (PCP) that were present in the bile and exposure water of goldfish (Carassius auratus) were used to develop methodology to quantify PCP and its metabolites. Reverse phase HPLC with radioactivity detection separated PCP and its metabolites, and was used to verify a method of quantification that used differential extraction and scintillation counting. Extractions of aqueous phase at pH 2 or 8, with butanol, ethyl acetate, or ether indicated that ether at pH 8 best separated PCP from its metabolites. The sulfate conjugate of PCP was the major metabolite produced when goldfish were exposed to 125 micrograms UC-PCP/l. It was present primarily in the exposure water, but also appeared in the bile.

  10. Determination of major UDP-glucuronosyltransferase enzymes and their genotypes responsible for 20-HETE glucuronidation[S

    PubMed Central

    Jarrar, Yazun Bashir; Cha, Eun-Young; Seo, Kyung-Ah; Ghim, Jong-Lyul; Kim, Hyo-Ji; Kim, Dong-Hyun; Lee, Su-Jun; Shin, Jae-Gook

    2014-01-01

    The compound 20-HETE is involved in numerous physiological functions, including blood pressure and platelet aggregation. Glucuronidation of 20-HETE by UDP-glucuronosyltransferases (UGTs) is thought to be a primary pathway of 20-HETE elimination in humans. The present study identified major UGT enzymes responsible for 20-HETE glucuronidation and investigated their genetic influence on the glucuronidation reaction using human livers (n = 44). Twelve recombinant UGTs were screened to identify major contributors to 20-HETE glucuronidation. Based on these results, UGT2B7, UGT1A9, and UGT1A3 exhibited as major contributors to 20-HETE glucuronidation. The Km values of 20-HETE glucuronidation by UGT1A3, UGT1A9, and UGT2B7 were 78.4, 22.2, and 14.8 μM, respectively, while Vmax values were 1.33, 1.78, and 1.62 nmol/min/mg protein, respectively. Protein expression levels and genetic variants of UGT1A3, UGT1A9, and UGT2B7 were analyzed in human livers using Western blotting and genotyping, respectively. Glucuronidation of 20-HETE was significantly correlated with the protein levels of UGT2B7 (r2 = 0.33, P < 0.001) and UGT1A9 (r2 = 0.31, P < 0.001), but not UGT1A3 (r2 = 0.02, P > 0.05). A correlation between genotype and 20-HETE glucuronidation revealed that UGT2B7 802C>T, UGT1A9 −118T9>T10, and UGT1A9 1399T>C significantly altered 20-HETE glucuronide formation (P < 0.05–0.001). Increased levels of 20-HETE comprise a risk factor for cardiovascular diseases, and the present data may increase our understanding of 20-HETE metabolism and cardiovascular complications. PMID:25249502

  11. Synthesis, hydrolysis and stability of psilocin glucuronide.

    PubMed

    Martin, Rafaela; Schürenkamp, Jennifer; Pfeiffer, Heidi; Lehr, Matthias; Köhler, Helga

    2014-04-01

    A two-step synthesis of psilocin glucuronide (PCG), the main metabolite of psilocin, with methyl 2,3,4-tri-O-isobutyryl-1-O-trichloroacetimidoyl-α-d-glucopyranuronate is reported. With the synthesized PCG, hydrolysis conditions in serum and urine were optimized. Escherichia coli proved to be a better enzyme source for β-glucuronidase than Helix pomatia. It was essential to add ascorbic acid to serum samples to protect psilocin during incubation. Furthermore the stability of PCG and psilocin was compared as stability data are the basis for forensic interpretation of measurements. PCG showed a greater long-term stability after six months in deep frozen serum and urine samples than psilocin. The short-term stability of PCG for one week in whole blood at room temperature and in deep frozen samples was also better than that of psilocin. Therefore, PCG can be considered to be more stable than the labile psilocin and should always be included if psilocin is analyzed in samples. PMID:24513688

  12. Synthesis, hydrolysis and stability of psilocin glucuronide.

    PubMed

    Martin, Rafaela; Schürenkamp, Jennifer; Pfeiffer, Heidi; Lehr, Matthias; Köhler, Helga

    2014-04-01

    A two-step synthesis of psilocin glucuronide (PCG), the main metabolite of psilocin, with methyl 2,3,4-tri-O-isobutyryl-1-O-trichloroacetimidoyl-α-d-glucopyranuronate is reported. With the synthesized PCG, hydrolysis conditions in serum and urine were optimized. Escherichia coli proved to be a better enzyme source for β-glucuronidase than Helix pomatia. It was essential to add ascorbic acid to serum samples to protect psilocin during incubation. Furthermore the stability of PCG and psilocin was compared as stability data are the basis for forensic interpretation of measurements. PCG showed a greater long-term stability after six months in deep frozen serum and urine samples than psilocin. The short-term stability of PCG for one week in whole blood at room temperature and in deep frozen samples was also better than that of psilocin. Therefore, PCG can be considered to be more stable than the labile psilocin and should always be included if psilocin is analyzed in samples.

  13. Hepatocyte cotransport of taurocholate and bilirubin glucuronides: Role of microtubules

    SciTech Connect

    Crawford, J.M.; Gollan, J.L. )

    1988-07-01

    Modulation of bile pigment excretion by bile salts has been attributed to modification of canalicular membrane transport or a physical interaction in bile. Based on the observation that a microtubule-dependent pathway is involved in the hepatocellular transport of bile salts, the authors investigated the possibility that bilirubin glucuronides are associated with bile salts during intracellular transport. Experiments were conducted in intact rats (basal) or after overnight biliary diversion and intravenous reinfusion of taurocholate (depleted/reinfused). All rats were pretreated with intravenous low-dose colchicine or its inactive isomer lumicolchicine. Biliary excretion of radiolabeled bilirubin glucuronides derived from tracer ({sup 14}C)bilirubin-({sup 3}H)bilirubin monoglucuronide (coinjected iv) was unchanged in basal rats but was consistently delayed in depleted/reinfused rats. This was accompanied by a significant shift toward bilirubin diglucuronide formation from both substrates. In basal Gunn rats, with deficient bilirubin glucuronidation, biliary excretion of intravenous ({sup 14}C)bilirubin monoglucuronide-({sup 3}H)bilirubin diglucuronide was unaffected by colchicine but was retarded in depleted/reinfused Gunn rats. Colchicine had no effect on the rate of bilirubin glucuronidation in vitro in rat liver microsomes. They conclude that a portion of the bilirubin glucuronides generated endogenously in hepatocytes or taken up directly from plasma may be cotransported with bile salts to the bile canalicular membrane via a microtubule-dependent mechanism.

  14. Microvascular protective activity of flavonoid glucuronides fraction from Tulipa gesneriana.

    PubMed

    Budzianowski, J; Korzeniowska, K; Chmara, E; Mrozikiewicz, A

    1999-03-01

    A mixture of flavonoid glucuronides, consisting of 7-O-glucuronides of kaempferol and quercetin 3-O-rutinosides, 3-O-gentiobiosides and 3-O-glucosides, was isolated from the perianths of Tulipa gesneriana L. var. 'Paradae'. It showed protective activity against the increased (both chloroform and histamine) skin vascular permeability in rabbits. The protective effect, measured as the reduction in leakage of Evans blue, was 59.8% after peritoneal treatment at a dose of 25 mg/kg, while that of troxerutin was 45.5%.

  15. Quantitation of Buprenorphine, Norbuprenorphine, Buprenorphine Glucuronide, Norbuprenorphine Glucuronide, and Naloxone in Urine by LC-MS/MS.

    PubMed

    Marin, Stephanie J; McMillin, Gwendolyn A

    2016-01-01

    Buprenorphine is an opioid drug that has been used to treat opioid dependence on an outpatient basis, and is also prescribed for managing moderate to severe pain. Some formulations of buprenorphine also contain naloxone to discourage misuse. The major metabolite of buprenorphine is norbuprenorphine. Both compounds are pharmacologically active and both are extensively metabolized to their glucuronide conjugates, which are also active metabolites. Direct quantitation of the glucuronide conjugates in conjunction with free buprenorphine, norbuprenorphine, and naloxone in urine can distinguish compliance with prescribed therapy from specimen adulteration intended to mimic compliance with prescribed buprenorphine. This chapter quantitates buprenorphine, norbuprenorphine, their glucuronide conjugates and naloxone directly in urine by liquid chromatography tandem mass spectrometry (LC-MS/MS). Urine is pretreated with formic acid and undergoes solid phase extraction (SPE) prior to analysis by LC-MS/MS. PMID:26660175

  16. Diethylstilbestrol can effectively accelerate estradiol-17-O-glucuronidation, while potently inhibiting estradiol-3-O-glucuronidation

    SciTech Connect

    Zhu, Liangliang; Xiao, Ling; Xia, Yangliu; Zhou, Kun; Wang, Huili; Huang, Minyi; Ge, Guangbo; Wu, Yan; Wu, Ganlin; Yang, Ling

    2015-03-01

    This in vitro study investigates the effects of diethylstilbestrol (DES), a widely used toxic synthetic estrogen, on estradiol-3- and 17-O- (E2-3/17-O) glucuronidation, via culturing human liver microsomes (HLMs) or recombinant UDP-glucuronosyltransferases (UGTs) with DES and E2. DES can potently inhibit E2-3-O-glucuronidation in HLM, a probe reaction for UGT1A1. Kinetic assays indicate that the inhibition follows a competitive inhibition mechanism, with the Ki value of 2.1 ± 0.3 μM, which is less than the possible in vivo level. In contrast to the inhibition on E2-3-O-glucuronidation, the acceleration is observed on E2-17-O-glucuronidation in HLM, in which cholestatic E2-17-O-glucuronide is generated. In the presence of DES (0–6.25 μM), K{sub m} values for E2-17-O-glucuronidation are located in the range of 7.2–7.4 μM, while V{sub max} values range from 0.38 to 1.54 nmol/min/mg. The mechanism behind the activation in HLM is further demonstrated by the fact that DES can efficiently elevate the activity of UGT1A4 in catalyzing E2-17-O-glucuronidation. The presence of DES (2 μM) can elevate V{sub max} from 0.016 to 0.81 nmol/min/mg, while lifting K{sub m} in a much lesser extent from 4.4 to 11 μM. Activation of E2-17-O-glucuronidation is well described by a two binding site model, with K{sub A}, α, and β values of 0.077 ± 0.18 μM, 3.3 ± 1.1 and 104 ± 56, respectively. However, diverse effects of DES towards E2-3/17-O-glucuronidation are not observed in liver microsomes from several common experimental animals. In summary, this study issues new potential toxic mechanisms for DES: potently inhibiting the activity of UGT1A1 and powerfully accelerating the formation of cholestatic E2-17-O-glucuronide by UGT1A4. - Highlights: • E2-3-O-glucuronidation in HLM is inhibited when co-incubated with DES. • E2-17-O-glucuronidation in HLM is stimulated when co-incubated with DES. • Acceleration of E2-17-O-glucuronidationin in HLM by DES is via activating the

  17. PRACTICAL PREPARATION OF RESVERATROL 3-O-β-D-GLUCURONIDE

    PubMed Central

    Jungong, Christian S.; Novikov, Alexei V.

    2012-01-01

    A practical synthesis of resveratrol 3-O-β-D-glucuronide, suitable for preparation of large quantities, was developed using selective deacetylation of resveratrol triacetate with ammonium acetate. A simplified procedure for large scale preparation of resveratrol is also reported. PMID:22919115

  18. Fate of glucuronide conjugated estradiol in the environment

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The fate and transport of conjugated reproductive hormones, which are polar compared to parent hormones, are little understood. Laboratory bench-scale soil (Hamar; Sandy, mixed, frigid typic Endoaquolls) sorption studies were conducted using [14C] 17ß-estradiol-3-glucuronide for a range of concentra...

  19. Ethyl alcohol production

    SciTech Connect

    Hofman, V.; Hauck, D.

    1980-11-01

    Recent price increases and temporary shortages of petroleum products have caused farmers to search for alternate sources of fuel. The production of ethyl alcohol from grain is described and the processes involved include saccharification, fermentation and distillation. The resulting stillage has potential as a livestock feed.

  20. Methyl ethyl ketone (MEK)

    Integrated Risk Information System (IRIS)

    Methyl ethyl ketone ( MEK ) ( CASRN 78 - 93 - 3 ) Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Nonc

  1. Chlorimuron-ethyl

    Integrated Risk Information System (IRIS)

    Chlorimuron - ethyl ; CASRN 90982 - 32 - 4 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinog

  2. Analysis of glucuronide and sulfate steroids in urine by ultra-high-performance supercritical-fluid chromatography hyphenated tandem mass spectrometry.

    PubMed

    Doué, Mickael; Dervilly-Pinel, Gaud; Pouponneau, Karinne; Monteau, Fabrice; Le Bizec, Bruno

    2015-06-01

    Profiling conjugated urinary steroids to detect anabolic-steroid misuse is recognized as an efficient analytical strategy in both chemical-food-safety and anti-doping fields. The relevance and robustness of such profiling rely on the analysis of glucuronide and sulfate steroids, which is expected to have properties including accuracy, specificity, sensitivity, and, if possible, rapidity. In this context, the ability of ultra-high-performance supercritical-fluid chromatography (UHPSFC) hyphenated tandem mass spectrometry (MS-MS) to provide reliable and accurate phase II analysis of steroids was assessed. Four stationary phases with sub-2 μm particles (BEH, BEH 2-ethyl-pyridine, HSS C18 SB, and CSH fluorophenyl) were screened for their capacity to separate several conjugated steroid isomers. Analytical conditions including stationary phase, modifier composition and percentage, back pressure, column temperature, and composition and flow rate of make-up solvent were investigated to improve the separation and/or the sensitivity. Thus, an analytical procedure enabling the analysis of eight glucuronide and 12 sulfate steroids by two different methods in 12 and 15 min, respectively, was optimized. The two procedures were evaluated, and UHPSFC-MS-MS analysis revealed its ability to provide sensitive (limits of quantification: 0.1 ng mL(-1) and 0.5 ng mL(-1) for sulfate and glucuronide steroids, respectively) and reliable quantitative performance (R(2) > 0.995, RSD < 20%, and bias < 30%) through the use of suitable labeled internal standards. Comparison with UHPLC-MS-MS was performed, and UHPSFC-MS-MS obtained better performance in terms of sensitivity. Finally, as a proof of concept, this so-called green technology was used in a chemical-food-safety context to profile steroid conjugates in urine samples from bovines treated with estradiol. Graphical Abstract Glucuronide and sulfate steroids analysis in urine by ultra-high performance supercritical fluid

  3. Simultaneous quantification of buprenorphine, norbuprenorphine, buprenorphine glucuronide, and norbuprenorphine glucuronide in human placenta by liquid chromatography mass spectrometry

    PubMed Central

    Concheiro-Guisan, Marta; Shakleya, Diaa M.; Huestis, Marilyn A.

    2011-01-01

    A LCMS method was developed and validated for the determination of buprenorphine (BUP), norbuprenorphine (NBUP), buprenorphine glucuronide (BUP-Gluc), and norbuprenorphine glucuronide (NBUP-Gluc) in placenta. Quantification was achieved by selected ion monitoring of m/z 468.4 (BUP), 414.3 (NBUP), 644.4 (BUP-Gluc), and 590 (NBUP-Gluc). BUP and NBUP were identified monitoring MS2 fragments m/z 396, 414 and 426 for BUP, and 340, 364 and 382 for NBUP, and glucuronide conjugates monitoring MS3 fragments m/z 396 and 414 for BUP-Gluc, and 340 and 382 for NBUP-Gluc. Linearity was 1–50 ng/g. Intra-day, inter-day and total assay imprecision (% RSD) were <13.4%, and analytical recoveries were 96.2–113.1%. Extraction efficiencies ranged from 40.7–68%, process efficiencies 38.8–70.5%, and matrix effect 1.3–15.4%. Limits of detection were 0.8 ng/g for all compounds. An authentic placenta from an opioid-dependent pregnant woman receiving BUP pharmacotherapy was analyzed. BUP was not detected but metabolite concentrations were NBUP-Gluc 46.6, NBUP 15.7 and BUP-Gluc 3.2 ng/g. PMID:19247639

  4. Enzyme-assisted synthesis and structural characterization of the 3-, 8-, and 15-glucuronides of deoxynivalenol.

    PubMed

    Uhlig, Silvio; Ivanova, Lada; Fæste, Christiane Kruse

    2013-02-27

    4-Deoxynivalenol is one of the most prevalent mycotoxins in grain-based food and feed products worldwide. Conjugation of deoxynivalenol to glucuronic acid and elimination via the urine appears to be the major metabolism pathway, although with differing efficiency in different species. In order to make pure deoxynivalenol glucuronides for analytical methodologies available we intended to enzymatically synthesize glucuronides of deoxynivalenol using rat and human liver microsomes supplemented with uridine 5'-diphosphoglucuronic acid and alamethicin as detergent. Three glucuronides were isolated and purified using solid-phase extraction of microsomal incubations and subsequent semipreparative hydrophilic interaction chromatography. NMR spectra were obtained for all three compounds from solutions in methanol, showing that deoxynivalenol 3-O-β-D-glucuronide and deoxynivalenol 15-O-β-D-glucuronide were the major products from incubations of deoxynivalenol with rat and human liver microsomes, respectively. The NMR spectra of a third glucuronide showed replacement of the C-8 carbonyl by a ketal carbon. This glucuronide was finally identified as deoxynivalenol 8-O-β-D-glucuronide. The present study provides unequivocal structural evidence for three glucuronides of deoxynivalenol formed by liver enzymes.

  5. Organochlorines inhibit acetaminophen glucuronidation by redirecting UDP-glucuronic acid towards the D-glucuronate pathway

    SciTech Connect

    Chan, Tom S. Wilson, John X.; Selliah, Subajini; Bilodeau, Marc; Zwingmann, Claudia; Poon, Raymond; O'Brien, Peter J.

    2008-11-01

    Industry-derived organochlorines are persistent environmental pollutants that are a continuing health concern. The effects of these compounds on drug metabolism are not well understood. In the current study we present evidence that the inhibition of acetaminophen (APAP) glucuronidation by minute concentrations of organochlorines correlates well with their ability to stimulate the D-glucuronate pathway leading to ascorbate synthesis. A set of 6 arylated organochlorines, including 5 PCB (polychlorinated biphenyl) congeners, were assessed for their effects on APAP glucuronidation in isolated hepatocytes from male Sprague-Dawley rats. The capacity of each organochlorine to inhibit APAP glucuronidation was found to be directly proportional to its capacity to stimulate ascorbate synthesis. PCB153, PCB28 and bis-(4-chlorophenyl sulfone) (BCPS) in increasing order were the most effective organochlorines for inhibiting APAP glucuronidation and stimulating the D-glucuronate pathway. None of the 3 inhibitors of APAP glucuronidation were able to alter the expression of UGT1A6, UGT1A7 and UGT1A8 (the major isoforms responsible for APAP glucuronidation in the rat), however, their efficacy at inhibiting APAP glucuronidation was proportional to their capacity to deplete UDP-glucuronic acid (UDPGA). BCPS-mediated inhibition of APAP glucuronidation in isolated hepatocytes had non-competitive characteristics and was insensitive to the inactivation of cytochrome P450. The effective organochlorines were also able to selectively stimulate the hydrolysis of UDPGA to UDP and glucuronate in isolated microsomes, but could not inhibit APAP glucuronidation in microsomes when UDPGA was in excess. We conclude that organochlorines are able to inhibit APAP glucuronidation in hepatocytes by depleting UDPGA via redirecting UDPGA towards the D-glucuronate pathway. Because the inhibition is non-competitive, low concentrations of these compounds could have long term inhibitory effects on the

  6. In vitro glucuronidation of five rhubarb anthraquinones by intestinal and liver microsomes from humans and rats.

    PubMed

    Wu, Wenjin; Hu, Nan; Zhang, Qingwen; Li, Yaping; Li, Peng; Yan, Ru; Wang, Yitao

    2014-08-01

    Anthraquinones naturally distribute in many plants including rhubarb and have widespread applications throughout industry and medicine. Recent studies provided new insights in potential applications of these traditional laxative constituents. Glucuronidation was the main metabolic pathway of rhubarb anthraquinones in vivo. This study examined the activity and regioselectivity of glucuronidation of rhubarb anthraquinones (aloe-emodin, emodin, chrysophanol, physcion, rhein) in liver and intestinal microsomes from rats and humans, by comparing with the core structure danthron. All anthraquinones formed mono-glucuronides and, except for rhein, the conjugation sites of the main metabolites were unambiguously identified. Two minor glucuronides of emodin were first reported together with the dominant emodin-3-O-β-D-glucuronide. The substitution on the anthraquinone ring was crucial to the activity and regioselectivity of glucuronidation. In general, the activity was decreased greatly with a β-COOH (rhein), while enhanced dramatically with a β-OH (emodin). Glucuronidation showed an absolute preference towards β-OH, followed by α-OH and β-alcoholic OH. The glucuronidation activity and regioselectivity also varied slightly with organs and species. All glucuronides of aloe-emodin, emodin, chrysophanol and physcion were formed by multiple human UGT isoforms with 1A9 being the most prominent in most cases. The UGT2B subfamily (2B7 and 2B15) only showed high activity towards a β-OH. In conclusion, the substitution at the anthraquinone ring was crucial to the rate and preference of glucuronidation. The high glucuronidation activity of UGT1A9 towards anthraquinones highlighted potential drug interactions.

  7. Multiplexed Targeted Quantitative Proteomics Predicts Hepatic Glucuronidation Potential

    PubMed Central

    Margaillan, Guillaume; Rouleau, Michèle; Klein, Kathrin; Fallon, John K.; Caron, Patrick; Villeneuve, Lyne; Smith, Philip C.; Zanger, Ulrich M.

    2015-01-01

    Phase II metabolism is prominently governed by UDP-glucuronosyltransferases (UGTs) in humans. These enzymes regulate the bioactivity of many drugs and endogenous small molecules in many organs, including the liver, a major site of regulation by the glucuronidation pathway. This study determined the expression of hepatic UGTs by targeted proteomics in 48 liver samples and by measuring the glucuronidation activity using probe substrates. It demonstrates the sensitivity and accuracy of nano-ultra-performance liquid chromatography with tandem mass spectrometry to establish the complex expression profiles of 14 hepatic UGTs in a single analysis. UGT2B7 is the most abundant UGT in our collection of livers, expressed at 69 pmol/mg microsomal proteins, whereas UGT1A1, UGT1A4, UGT2B4, and UGT2B15 are similarly abundant, averaging 30–34 pmol/mg proteins. The average relative abundance of these five UGTs represents 81% of the measured hepatic UGTs. Our data further highlight the strong relationships in the expression of several UGTs. Most notably, UGT1A4 correlates with most measured UGTs, and the expression levels of UGT2B4/UGT2B7 displayed the strongest correlation. However, significant interindividual variability is observed for all UGTs, both at the level of enzyme concentrations and activity (coefficient of variation: 45%–184%). The reliability of targeted proteomics quantification is supported by the high correlation between UGT concentration and activity. Collectively, these findings expand our understanding of hepatic UGT profiles by establishing absolute hepatic concentrations of 14 UGTs and further suggest coregulated expression between most abundant hepatic UGTs. Data support the value of multiplexed targeted quantitative proteomics to accurately assess specific UGT concentrations in liver samples and hepatic glucuronidation potential. PMID:26076694

  8. Disposition of acetone, methyl ethyl ketone and cyclohexanone in acute poisoning.

    PubMed

    Sakata, M; Kikuchi, J; Haga, M; Ishiyama, N; Maeda, T; Ise, T; Hikita, N

    1989-01-01

    A case of coma due to the drinking of a liquid cement for polyvinyl chloride resin, containing acetone, methyl ethyl ketone, cyclohexanone and polyvinyl chloride is described. The patient also simultaneously ingested the alcoholic beverage, sake. After gastric lavage, plasma exchanges and direct hemoperfusions, the patient recovered. The concentrations of these chemicals in plasma and urine were analyzed at various time intervals to estimate the clearance. The elimination half lives for acetone and methyl ethyl ketone were 18 hours and 10 hours, respectively. Although cyclohexanone made up the largest component in the solvents, the blood level was extremely low and a large amount of cyclohexanol, a metabolite of cyclohexanone was detected in the blood and urine. The glucuronide metabolite of cyclohexanol was also estimated after the hydrolysis with beta-glucuronidase. Since the conversion of cyclohexanone to cyclohexanol is known to be catalyzed by alcohol dehydrogenase, possible interactions between sake ingestion and cyclohexanone metabolism is proposed.

  9. UDP-glucuronosyltransferases 1A6 and 1A10 catalyze reduced menadione glucuronidation

    SciTech Connect

    Nishiyama, Takahito; Ohnuma, Tomokazu; Inoue, Yuu; Kishi, Takehiko; Ogura, Kenichiro; Hiratsuka, Akira

    2008-06-27

    Menadione (2-methyl-1,4-naphthoquine), also known as vitamin K3, has been widely used as a model compound in the field of oxidative stress-related research. The metabolism of menadione has been studied, and it is known that menadione undergoes a two-electron reduction by NAD(P)H:Quinone oxidoreductase 1 (NQO1) after which the reduced form of menadione (2-methyl-1,4-naphthalenediol, menadiol) is glucuronidated and excreted in urine. To investigate which human UDP-glucuronosyltransferase (UGT) isoforms participate in the glucuronidation of menadiol reduced by NQO1 from menadione, we first constructed heterologously expressed NQO1 in Sf9 cells and tested the menadiol glucuronidating activity of 16 human recombinant UGT isoforms. Of the 16 UGT isoforms, UGTs 1A6, 1A7, 1A8, 1A9, and 1A10 catalyzed menadiol glucuronidation, and, of these, UGTs 1A6 and 1A10 catalyzed menadiol glucuronidation at much higher rates than the other UGTs. Menadiol was regioselectively glucuronidated in the manner of 4-position > 1-position by UGTs 1A7, 1A8, 1A9, and 1A10. In contrast to these UGTs, only UGT1A6 exhibited 1-menadiol-preferential glucuronidating activity. The results suggest possible detoxification pathways for quinones via NQO1 reduction followed by UGT glucuronidation.

  10. Biosynthesis of Drug Glucuronide Metabolites in the Budding Yeast Saccharomyces cerevisiae.

    PubMed

    Ikushiro, Shinichi; Nishikawa, Miyu; Masuyama, Yuuka; Shouji, Tadashi; Fujii, Miharu; Hamada, Masahiro; Nakajima, Noriyuki; Finel, Moshe; Yasuda, Kaori; Kamakura, Masaki; Sakaki, Toshiyuki

    2016-07-01

    Glucuronidation is one of the most common pathways in mammals for detoxification and elimination of hydrophobic xenobiotic compounds, including many drugs. Metabolites, however, can form active or toxic compounds, such as acyl glucuronides, and their safety assessment is often needed. The absence of efficient means for in vitro synthesis of correct glucuronide metabolites frequently limits such toxicological analyses. To overcome this hurdle we have developed a new approach, the essence of which is a coexpression system containing a human, or another mammalian UDP-glucuronosyltransferases (UGTs), as well as UDP-glucose-6-dehydrogenase (UGDH), within the budding yeast, Saccharomyces cerevisiae. The system was first tested using resting yeast cells coexpressing UGDH and human UGT1A6, 7-hydroxycoumarin as the substrate, in a reaction medium containing 8% glucose, serving as a source of UDP-glucuronic acid. Glucuronides were readily formed and recovered from the medium. Subsequently, by selecting suitable mammalian UGT enzyme for the coexpression system we could obtain the desired glucuronides of various compounds, including molecules with multiple conjugation sites and acyl glucuronides of several carboxylic acid containing drugs, namely, mefenamic acid, flufenamic acid, and zomepirac. In conclusion, a new and flexible yeast system with mammalian UGTs has been developed that exhibits a capacity for efficient production of various glucuronides, including acyl glucuronides. PMID:27241161

  11. A fluorescent assay amenable to measuring production of beta-D-glucuronides produced from recombinant UDP-glycosyl transferase enzymes.

    PubMed

    Trubetskoy, O V; Shaw, P M

    1999-05-01

    Beta-glucuronidase cleavage of 4-methylumbelliferyl beta-D-glucuronide generates the highly fluorescent compound, 4-methylumbelliferone. We show that other beta-D-glucuronide compounds act as competitors in this assay. The 4-methylumbelliferyl beta-D-glucuronide cleavage assay can easily be adapted to high throughput formats to detect the presence of beta-D glucuronides generated using recombinant glycosyl transferase preparations.

  12. High-throughput screening technologies for drug glucuronidation profiling.

    PubMed

    Trubetskoy, Olga; Finel, Moshe; Trubetskoy, Vladimir

    2008-08-01

    A significant number of endogenous and exogenous compounds, including many therapeutic agents, are metabolized in humans via glucuronidation, catalysed by uridine diphosphoglucuronosyltransferases (UGTs). The study of the UGTs is a growing field of research, with constantly accumulated and updated information regarding UGT structure, purification, substrate specificity and inhibition, including clinically relevant drug interactions. Development of reliable UGT assays for the assessment of individual isoform substrate specificity and for the discovery of novel isoform-specific substrates and inhibitors is crucial for understanding the function and regulation of the UGT enzyme family and its clinical and pharmacological relevance. High-throughput screening (HTS) is a powerful technology used to search for novel substrates and inhibitors for a wide variety of targets. However, application of HTS in the context of UGTs is complicated because of the poor stability, low levels of expression, low affinity and broad substrate specificity of the enzymes, combined with difficulties in obtaining individual UGT isoforms in purified format, and insufficient information regarding isoform-specific substrates and inhibitors. This review examines the current status of HTS assays used in the search for novel UGT substrates and inhibitors, emphasizing advancements and challenges in HTS technologies for drug glucuronidation profiling, and discusses possible avenues for future advancement of the field.

  13. Species difference in glucuronidation formation kinetics with a selective mTOR inhibitor.

    PubMed

    Berry, Loren M; Liu, Jingzhou; Colletti, Adria; Krolikowski, Paul; Zhao, Zhiyang; Teffera, Yohannes

    2014-04-01

    The mammalian target of rapamycin (mTOR) is a protein kinase that shows key involvement in age-related disease and promises to be a target for treatment of cancer. In the present study, the elimination of potent ATP-competitive mTOR inhibitor 3-(6-amino-2-methylpyrimidin-4-yl)-N-(1H-pyrazol-3-yl)imidazo[1,2-b]pyridazin-2-amine (compound 1) is studied in bile duct-cannulated rats, and the metabolism of compound 1 in liver microsomes is compared across species. Compound 1 was shown to undergo extensive N-glucuronidation in bile duct-catheterized rats. N-glucuronides were detected on positions N1 (M2) and N2 (M1) of the pyrazole moiety as well as on the primary amine (M3). All three N-glucuronide metabolites were detected in liver microsomes of the rat, dog, and human, while primary amine glucuronidation was not detected in cynomolgus monkey. In addition, N1- and N2-glucuronidation showed strong species selectivity in vitro, with rat, dog, and human favoring N2-glucuronidation and monkey favoring N1-glucuronide formation. Formation of M1 in monkey liver microsomes also followed sigmoidal kinetics, singling out monkey as unique among the species with regard to compound 1 N-glucuronidation. In this respect, monkeys might not always be the best animal model for N-glucuronidation of uridine diphosphate glucuronosyltransferase (UGT) 1A9 or UGT1A1 substrates in humans. The impact of N-glucuronidation of compound 1 could be more pronounced in higher species such as monkey and human, leading to high clearance in these species. While compound 1 shows promise as a candidate for investigating the impact of pan-mTOR inhibition in vivo, opportunities may exist through medicinal chemistry efforts to reduce metabolic liability with the goal of improving systemic exposure. PMID:24423753

  14. Trans-stilbene oxide administration increased hepatic glucuronidation of morphine but decreased biliary excretion of morphine glucuronide in rats

    SciTech Connect

    Fuhrman-Lane, C.; Fujimoto, J.M.

    1982-09-01

    The effect of the inducing agent trans-stilbene oxide (TSO) on the metabolism and biliary excretion of (/sup 14/C)morphine was studied in the isolated in situ perfused rat liver. After administration of morphine by intraportal injection or by the segmented retrograde intrabiliary injection technique, the TSO-treated group showed a marked decrease in the biliary recovery of morphine as its glucuronide conjugate (morphine-3-glucuronide (MG)). However, recovery of MG in the venous outflow of the single pass perfusate was greatly increased. These findings suggested that TSO treatment enhanced the formation of MG from morphine and changed the primary route of hepatic elimination of MG. TSO treatment also decreased the excretion of morphine (as MG) in the bile of anesthetized renal-ligated rats. This decreased biliary function required several days to develop and appeared closely associated with the inductive effect of TSO. After i.v. administration of (/sup 14/C)MG itself, biliary recovery was also markedly decreased in TSO-treated rats. It is postulated that the effect of the TSO treatment led to either a decrease in canalicular transport of MG into bile or an increase in the efficiency of transfer of MG to the blood at the sinusoidal side of the hepatocyte. Regardless of the mechanism, the results indicate the need to study compartmentalization of drug transport and metabolism functions.

  15. HT-2 toxin 4-glucuronide as new T-2 toxin metabolite: enzymatic synthesis, analysis, and species specific formation of T-2 and HT-2 toxin glucuronides by rat, mouse, pig, and human liver microsomes.

    PubMed

    Welsch, Tanja; Humpf, Hans-Ulrich

    2012-10-10

    Glucuronides of the mycotoxin T-2 toxin and its phase I metabolite HT-2 toxin are important phase II metabolites under in vivo and in vitro conditions. Since standard substances are essential for the direct quantitation of these glucuronides, a method for the enzymatic synthesis of T-2 and HT-2 toxin glucuronides employing liver microsomes was optimized. Structure elucidation by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry revealed that besides T-2 toxin glucuronide and HT-2 toxin 3-glucuronide also the newly identified isomer HT-2 toxin 4-glucuronide was formed. Glucuronidation of T-2 and HT-2 toxin in liver microsomes of rat, mouse, pig, and human was compared and metabolites were analyzed directly by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). A distinct, species specific pattern of glucuronidation of T-2 and HT-2 toxin was observed with interesting interindividual differences. Until recently, glucuronides have frequently been analyzed indirectly by quantitation of the aglycone after enzymatic cleavage of the glucuronides by β-glucuronidase. Therefore, the hydrolysis efficiencies of T-2 and HT-2 toxin glucuronides using β-glucuronidases from Helix pomatia, bovine liver, and Escherichia coli were compared. PMID:22967261

  16. Species Differences in Microsomal Oxidation and Glucuronidation of 4-Ipomeanol: Relationship to Target Organ Toxicity.

    PubMed

    Parkinson, Oliver T; Teitelbaum, Aaron M; Whittington, Dale; Kelly, Edward J; Rettie, Allan E

    2016-10-01

    4-Ipomeanol (IPO) is a model pulmonary toxicant that undergoes P450-mediated metabolism to reactive electrophilic intermediates that bind to tissue macromolecules and can be trapped in vitro as the NAC/NAL adduct. Pronounced species and tissue differences in IPO toxicity are well documented, as is the enzymological component of phase I bioactivation. However, IPO also undergoes phase II glucuronidation, which may compete with bioactivation in target tissues. To better understand the organ toxicity of IPO, we synthesized IPO-glucuronide and developed a new quantitative mass spectrometry-based assay for IPO glucuronidation. Microsomal rates of glucuronidation and P450-dependent NAC/NAL adduct formation were compared in lung, kidney, and liver microsomes from seven species with different target organ toxicities to IPO. Bioactivation rates were highest in pulmonary and renal microsomes from all animal species (except dog) known to be highly susceptible to the extrahepatic toxicities induced by IPO. In a complementary fashion, pulmonary and renal IPO glucuronidation rates were uniformly low in all experimental animals and primates, but hepatic glucuronidation rates were high, as expected. Therefore, with the exception of the dog, the balance between microsomal NAC/NAL adduct and glucuronide formation correlate well with the risk for IPO-induced pulmonary, renal, and hepatic toxicities across species. PMID:27468999

  17. Species Differences in Microsomal Oxidation and Glucuronidation of 4-Ipomeanol: Relationship to Target Organ Toxicity.

    PubMed

    Parkinson, Oliver T; Teitelbaum, Aaron M; Whittington, Dale; Kelly, Edward J; Rettie, Allan E

    2016-10-01

    4-Ipomeanol (IPO) is a model pulmonary toxicant that undergoes P450-mediated metabolism to reactive electrophilic intermediates that bind to tissue macromolecules and can be trapped in vitro as the NAC/NAL adduct. Pronounced species and tissue differences in IPO toxicity are well documented, as is the enzymological component of phase I bioactivation. However, IPO also undergoes phase II glucuronidation, which may compete with bioactivation in target tissues. To better understand the organ toxicity of IPO, we synthesized IPO-glucuronide and developed a new quantitative mass spectrometry-based assay for IPO glucuronidation. Microsomal rates of glucuronidation and P450-dependent NAC/NAL adduct formation were compared in lung, kidney, and liver microsomes from seven species with different target organ toxicities to IPO. Bioactivation rates were highest in pulmonary and renal microsomes from all animal species (except dog) known to be highly susceptible to the extrahepatic toxicities induced by IPO. In a complementary fashion, pulmonary and renal IPO glucuronidation rates were uniformly low in all experimental animals and primates, but hepatic glucuronidation rates were high, as expected. Therefore, with the exception of the dog, the balance between microsomal NAC/NAL adduct and glucuronide formation correlate well with the risk for IPO-induced pulmonary, renal, and hepatic toxicities across species.

  18. An in vitro approach to estimate putative inhibition of buprenorphine and norbuprenorphine glucuronidation.

    PubMed

    Oechsler, Stephanie; Skopp, Gisela

    2010-05-01

    An in vitro inhibition study was performed to investigate potential drug-drug interactions on glucuronidation of buprenorphine (BUP) and norbuprenorphine (NBUP), which represents the major elimination pathway of the drug using cDNA-expressed uridine 5'-diphosphate glucuronosyltransferases (UGTs) and human liver microsomes (HLMs). Following identification of major UGT enzymes for BUP and NBUP glucuronidation, substrates were incubated with drugs (amitriptyline, nortriptyline, lamotrigine, oxazepam, and temazepam), which are extensively cleared by glucuronidation as well as are often used during maintenance treatment. To evaluate the inhibitory potential, the half maximal inhibitor concentration (IC(50)), the inhibition constant (K (i)), and the inhibitor concentration (K (I)) that yield half the maximum rate of inactivation and the enzyme inactivation rate constant (k (inact)) were determined, if appropriate. Amitriptyline and temazepam are inhibitors of NBUP glucuronidation (UGT1A3, HLMs), whereas BUP glucuronidation was affected by amitriptyline (HLMs), oxazepam, and temazepam (UGT2B7). Additionally, BUP inhibits NBUP glucuronidation (UGT1A1, 1A3, HLMs) and vice versa (UGT1A3). A decrease in the metabolic clearance of NBUP may increase the risk of adverse effects such as respiratory depression. Further investigations are needed to evaluate whether inhibition of BUP and NBUP glucuronidation contributes to adverse events. PMID:20111869

  19. Hydroxytyrosol glucuronides protect renal tubular epithelial cells against H(2)O(2) induced oxidative damage.

    PubMed

    Deiana, Monica; Incani, Alessandra; Rosa, Antonella; Atzeri, Angela; Loru, Debora; Cabboi, Barbara; Paola Melis, M; Lucas, Ricardo; Morales, Juan C; Assunta Dessì, M

    2011-09-30

    Hydroxytyrosol (2-(3',4'-dihydroxyphenyl)ethanol; HT), the most active ortho-diphenolic compound, present either in free or esterified form in extravirgin olive oil, is extensively metabolized in vivo mainly to O-methylated, O-sulfated and glucuronide metabolites. We investigated the capacity of three glucuronide metabolites of HT, 3'-O-β-d-glucuronide and 4'-O-β-d-glucuronide derivatives and 2-(3',4'-dihydroxyphenyl)ethanol-1-O-β-d-glucuronide, in comparison with the parent compound, to inhibit H(2)O(2) induced oxidative damage and cell death in LLC-PK1 cells, a porcine kidney epithelial cell line. H(2)O(2) treatment exerted a toxic effect inducing cell death, interacting selectively within the pro-death extracellular-signal relate kinase (ERK 1/2) and the pro-survival Akt/PKB signaling pathways. It also produced direct oxidative damage initiating the membrane lipid peroxidation process. None of the tested glucuronides exhibited any protection against the loss in renal cell viability. They also failed to prevent the changes in the phosphorylation states of ERK and Akt, probably reflecting their inability to enter the cells, while HT was highly effective. Notably, pretreatment with glucuronides exerted a protective effect at the highest concentration tested against membrane oxidative damage, comparable to that of HT: the formation of malondialdehyde, fatty acid hydroperoxides and 7-ketocholesterol was significantly inhibited.

  20. Improved detection of opioid use in chronic pain patients through monitoring of opioid glucuronides in urine.

    PubMed

    Dickerson, Jane A; Laha, Thomas J; Pagano, Monica B; O'Donnell, Brendan R; Hoofnagle, Andrew N

    2012-10-01

    When chronic pain patients are suspected of being non-compliant, their therapy can be withdrawn. Therefore, sensitive and specific confirmatory testing is important for identifying diversion and adherence. This work aimed to develop a novel liquid chromatography tandem mass spectrometry (LC-MS-MS) method to detect 14 opioids and six opioid glucuronide metabolites in urine with minimal sample preparation. Analytes included were morphine, oxymorphone, hydromorphone, oxycodone, hydrocodone, codeine, fentanyl, norfentanyl, 6-monoacetylmorphine, meperidine, normeperidine, propoxyphene, methadone, buprenorphine, morphine-3-glucuronide, morphine-6-glucuronide, oxymorphone glucuronide, hydromorphone glucuronide, codeine-6-glucuronide and norbuprenorphine glucuronide. Samples were processed by centrifugation and diluted in equal volume with a deuterated internal standard containing 14 opioids and four opioid glucuronides. The separation of all compounds was complete in nine minutes. The assay was linear between 10 and 1,000 ng/mL (fentanyl 0.25-25 ng/mL). Intra-assay imprecision (500 ng/mL, fentanyl 12.5 ng/mL) ranged from 1.0 to 8.4% coefficient of variation. Inter-assay precision ranged from 2.9 to 6.0%. Recovery was determined by spiking five patient specimens with opioid and opioid glucuronide standards at 100 ng/mL (fentanyl 2.5 ng/mL). Recoveries ranged from 82 to 107% (median 98.9%). The method correlated with our current quantitative LC-MS-MS assay for opioids, which employs different chromatography. Internal standards were not available for every analyte to critically evaluate for ion suppression. Instead, a novel approach was designed to achieve the most rigorous quality control possible, in which the recovery of each analyte was evaluated in each negative sample. PMID:22833646

  1. Glucuronidation and Sulfation Kinetics of Diflunisal in Man.

    NASA Astrophysics Data System (ADS)

    Loewen, Gordon Rapheal

    Diflunisal is a nonsteroidal anti-inflammatory drug used in the treatment of arthritis and musculoskeletal pain. Diflunisal exhibits concentration- and dose-dependent kinetics, the mechanism of which has not been determined. The purpose of this study was to determine the mechanism(s) responsible for non-linear disposition of diflunisal and to examine environmental factors which may affect the elimination of diflunisal. The metabolites of diflunisal, including a new metabolite, the sulphate conjugate, were purified by column and semi-preparative high pressure liquid chromatography. Assays for the quantitation of diflunisal and conjugates in urine and diflunisal in plasma were developed. Plasma protein binding of diflunisal in blank plasma and in plasma obtained following multiple doses of diflunisal was determined by equilibrium dialysis. Total body clearance of diflunisal decreased when dose increased from 100 to 750 mg. Total clearance increased when dose increased from 750 to 1000 mg. The percent of recovered dose eliminated as the acyl glucuronide decreased and the percent eliminated as the sulphate increased with increasing dose of diflunisal. Plasma protein binding of diflunisal was concentration dependent over a range of diflunisal plasma concentrations of 3 to 257 mug/ml. Total clearance, and to a lesser degree, unbound clearance of diflunisal were decreased following multiple dose administration of 250 and 500 mg diflunisal. Percent of recovered dose eliminated as the acyl glucuronide decreased and percent eliminated as the sulphate conjugate increased following multiple dosing. Plasma protein binding of diflunisal was similar in blank plasma and plasma obtained at steady state. Unbound clearance of diflunisal exceeded liver plasma flow. Frequency distributions of the elimination of the conjugates of diflunisal were normally distributed. Sex, smoking, and use of vitamins or oral contraceptives were identified as factors which may affect the elimination of

  2. EPA dashes ethyl`s hopes for MMT

    SciTech Connect

    Heller, K.

    1992-01-15

    Up until the Environmental Protection Agency (EPA; Washington) decided to deny Ethyl`s (Richmond, VA) petition to sell manganese-based gasoline additive MMT, many on Wall Street were bullish. Bets were that MMT sales could create an up to $200 million/year sales windfall for Ethyl with $60 million/year income, and push its near $26/share price up by at least 50 cts. But EPA ruled January 8 against MMT in unleaded gas due to its potential to increase hydrocarbon emissions. What kept analysts hoping is that octane enhancer MMT`s environmental impacts are mixed. An Ethyl spokesman says that MMT cut tailpipe emissions of nitrogen oxide by 20% and carbon monoxide by 7%. Ethyl also points out that MMT could save as much as 85,000 barrels/day of imported oil because of lower energy requirements in blending. And the product has sold for 13 years in Canada with no reported ill health effects. But, points out Smith, Barney (New York) analyst James Wilbur, Canada is not the congested Los Angeles basin, where the unknown effects of small amounts of heavy metal manganese would show up a lot faster if every car burnt MMT. For now, the financial effect of the decision is negligible, although at some point Ethyl may have to take a write-down on its Orangeburg, NC plant.

  3. Ethyl`s MMT ready to hit the road

    SciTech Connect

    Stringer, J.

    1996-01-03

    After spending two decades and about $30 million on the fight to sell the fuel octane booster methylcyclopentadienyl manganese tricarbonyl (MMT), Ethyl has started marketing the product. Ethyl president and chief operating officer Thomas Gottwald says he expects a profit from MMT from the outset. {open_quotes}MMT is a gangbuster new product,{close_quotes} says Paul Raman, an analyst with S.G. Warburg (New York), {open_quotes}and it will be very profitable for Ethyl.{close_quotes} Ethyl`s effort to bring MMT to market faced pressure from EPA and automakers. EPA says MMT should not be marketed until more research is done on health effects of the manganese-based additive. US automakers oppose MMT, fearing it will damage catalytic converters. Last October Ethyl won a federal appeals court decision compelling EPA to approve MMT use. Gottwald says the MMT fight has been well worth it: {open_quotes}We fought with our eye on the bottom line.{close_quotes}

  4. Gas chromatographic-mass spectrometric assay for 6-hydroxymelatonin sulfate and 6-hydroxymelatonin glucuronide in urine

    SciTech Connect

    Francis, P.L.; Leone, A.M.; Young, I.M.; Stovell, P.; Silman, R.E.

    1987-04-01

    Circulating melatonin is hydroxylated to 6-hydroxymelatonin and excreted in urine as the sulfate and glucuronide conjugates. We extracted these two compounds from urine by using octadecylsilane-bonded silica cartridges to eliminate most of the urea and electrolytes, and silica cartridges to separate the sulfate and glucuronide conjugates. After hydrolyzing the separated conjugates enzymically, we determined the free hydroxymelatonin by gas chromatography-mass spectrometry. Though recoveries were low and variable, we were able to quantify the analyte in the original sample by adding deuterated sulfate and glucuronide conjugates to the urines before extraction.

  5. A rapid and sensitive UPLC-MS/MS method for the simultaneous quantification of serum androsterone glucuronide, etiocholanolone glucuronide, and androstan-3α, 17β diol 17-glucuronide in postmenopausal women.

    PubMed

    Ke, Yuyong; Gonthier, Renaud; Isabelle, Maxim; Bertin, Jonathan; Simard, Jean-Nicolas; Dury, Alain Y; Labrie, Fernand

    2015-05-01

    Quantification of steroidal glucuronide conjugates by the indirect methods of immunoassay and GC-MS/MS may underestimate some conjugates since hydrolysis is needed in sample processing. In the present work, a sensitive and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous direct quantification of androsterone glucuronide, etiocholanolone glucuronide, and androstan-3α, 17β diol 17-glucuronide in postmenopausal women's serum. The quantification limits are 0.1ng/mL for 3α-diol-17G and 4ng/mL for both ADT-G and Etio-G, respectively, with an extraction from 200μL serum while the total run time is less than 6min for all three glucuronides. In this method, solid phase extraction is used for sample preparation. The assay has been validated in compliance with EndoCeutics SOPs and FDA guidelines for bioanalytical method development and validation. The recovery of glucuronides in stripped serum is consistent with that in unstripped serum, where the average difference in stripped and unstripped is less than 10%. A linear regression model fits well the standard curves of all three compounds with R≥0.99 where the weighting factor is 1/X. Interday accuracy and CV for all levels of QCs are within the range of 15% in both stripped and unstripped serum while all calibration curves are within the range of 6% except for LLOQs, which are within the range of 9%. Other parameters have also been assessed such as selectivity, matrix, lipemic and hemolysis effects as well as stabilities in solution and matrix. Incurred sample reanalysis has been performed with a result of over 93% within 20% of the original values. This reliable, sensitive and fast method is ready for large-scale clinical sample assays. PMID:25701608

  6. High-sensitivity analysis of buprenorphine, norbuprenorphine, buprenorphine glucuronide, and norbuprenorphine glucuronide in plasma and urine by liquid chromatography–mass spectrometry☆

    PubMed Central

    Regina, Karen J.; Kharasch, Evan D.

    2014-01-01

    A new method using ultra-fast liquid chromatography and tandem mass spectrometry (UFLC–MS/MS) was developed for the simultaneous determination of buprenorphine and the metabolites norbuprenorphine, buprenorphine-3β-glucuronide, and norbuprenorphine-3β-glucuronide in plasma and urine. Sample handling, sample preparation and solid-phase extraction procedures were optimized for maximum analyte recovery. All four analytes of interest were quantified by positive ion electrospray ionization tandem mass spectrometry after solid-phase microextraction. The lower limits of quantification in plasma were 1 pg/mL for buprenorphine and buprenorphine glucuronide, and 10 pg/mL for norbuprenorphine and norbuprenorphine glucuronide. The lower limits of quantitation in urine were 10 pg/mL for buprenorphine, norbuprenorphine and their glucuronides. Overall extraction recoveries ranged from 68–100% in both matrices. Interassay precision and accuracy was within 10% for all four analytes in plasma and within 15% in urine. The method was applicable to pharmacokinetic studies of low-dose buprenorphine. PMID:24095872

  7. High-sensitivity analysis of buprenorphine, norbuprenorphine, buprenorphine glucuronide, and norbuprenorphine glucuronide in plasma and urine by liquid chromatography-mass spectrometry.

    PubMed

    Regina, Karen J; Kharasch, Evan D

    2013-11-15

    A new method using ultra-fast liquid chromatography and tandem mass spectrometry (UFLC-MS/MS) was developed for the simultaneous determination of buprenorphine and the metabolites norbuprenorphine, buprenorphine-3β-glucuronide, and norbuprenorphine-3β-glucuronide in plasma and urine. Sample handling, sample preparation and solid-phase extraction procedures were optimized for maximum analyte recovery. All four analytes of interest were quantified by positive ion electrospray ionization tandem mass spectrometry after solid-phase microextraction. The lower limits of quantification in plasma were 1pg/mL for buprenorphine and buprenorphine glucuronide, and 10pg/mL for norbuprenorphine and norbuprenorphine glucuronide. The lower limits of quantitation in urine were 10pg/mL for buprenorphine, norbuprenorphine and their glucuronides. Overall extraction recoveries ranged from 68-100% in both matrices. Interassay precision and accuracy was within 10% for all four analytes in plasma and within 15% in urine. The method was applicable to pharmacokinetic studies of low-dose buprenorphine. PMID:24095872

  8. 21 CFR 173.228 - Ethyl acetate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... the specifications of the Food Chemicals Codex, 1 (Ethyl Acetate; p. 372, 3d Ed., 1981), which are... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Ethyl acetate. 173.228 Section 173.228 Food and..., Lubricants, Release Agents and Related Substances § 173.228 Ethyl acetate. Ethyl acetate (CAS Reg. No....

  9. 21 CFR 173.228 - Ethyl acetate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... the specifications of the Food Chemicals Codex, 1 (Ethyl Acetate; p. 372, 3d Ed., 1981), which are... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Ethyl acetate. 173.228 Section 173.228 Food and..., Lubricants, Release Agents and Related Substances § 173.228 Ethyl acetate. Ethyl acetate (CAS Reg. No....

  10. Chemical synthesis and growth-promoting activity of all-trans-retinyl beta-D-glucuronide.

    PubMed Central

    Barua, A B; Olson, J A

    1987-01-01

    All-trans-retinol reacts with methyl (2,3,4-tri-O-acetyl-1-bromo-1-deoxy-beta-D-glucopyran)uronate in the presence of Ag2CO3 to give the triacetate methyl ester of retinyl beta-glucuronide. Hydrolysis of this ester with sodium methylate in methanol gives retinyl beta-D-glucuronide in about 15% yield. The water-soluble retinyl beta-D-glucuronide was characterized by u.v.-visible, n.m.r. and mass spectra, by elemental analysis and by its susceptibility to hydrolysis by bacterial beta-glucuronidase. Retinyl beta-glucuronide, when administered intraperitoneally in saline (0.9% NaCl), supports well the growth of vitamin A-deficient rats. PMID:3663114

  11. UGT2B10 genotype influences nicotine glucuronidation, oxidation and consumption

    PubMed Central

    Berg, Jeannette Zinggeler; von Weymarn, Linda; Thompson, Elizabeth A.; Wickham, Katherine M.; Weisensel, Natalie A.; Hatsukami, Dorothy K.; Murphy, Sharon E.

    2010-01-01

    Background Tobacco exposure is routinely assessed by quantifying nicotine metabolites in plasma or urine. On average, 80% of nicotine undergoes C-oxidation to cotinine. However, interindividual variation in nicotine glucuronidation is substantial and glucuronidation accounts for from 0 to 40% of total nicotine metabolism. We report here the effect of a polymorphism in a UDP-glucuronsyl transferase, UGT2B10, on nicotine metabolism and consumption. Methods Nicotine, cotinine, their N-glucuronide conjugates, and total trans-3'-hydroxycotinine were quantified in the urine (n=327) and plasma (n =115) of smokers. Urinary nicotine N-oxide was quantified in 105 smokers. Nicotine equivalents, the sum of nicotine and all major metabolites, were calculated for each smoker. The relationship of the UGT2B10 Asp67Tyr allele to nicotine equivalents, N-glucuronidation, and C-oxidation was determined. Results Individuals heterozygous for the Asp67Tyr allele excreted less nicotine or cotinine as their glucuronide conjugates than wild-type, resulting in a 60% lower ratio of cotinine glucuronide:cotinine, a 50% lower ratio of nicotine glucuronide:nicotine and increased cotinine and trans-3'-hydroxycotinine. Nicotine equivalents, a robust biomarker of nicotine intake, were lower among Asp67Tyr heterozygotes compared to individuals without this allele; 58.2 nmol/ml (95% CI, 48.9 – 68.2) versus 69.2 nmol/ml (95% CI, 64.3 – 74.5). Conclusions Individuals heterozygous for UGT2B10 Asp67Tyr consume less nicotine than do wild type smokers. This striking observation suggests that variations in nicotine N-glucuronidation, as reported for nicotine C-oxidation, may influence smoking behavior. Impact UGT2B10 genotype influences nicotine metabolism and should be taken into account when characterizing the role of nicotine metabolism on smoking. PMID:20501767

  12. Glucuronidation of the aspirin metabolite salicylic acid by expressed UDP-glucuronosyltransferases and human liver microsomes.

    PubMed

    Kuehl, Gwendolyn E; Bigler, Jeannette; Potter, John D; Lampe, Johanna W

    2006-02-01

    Acetylsalicylic acid (aspirin) is a common nonsteroidal anti-inflammatory drug used for treatment of pain and arthritis. In the body, acetylsalicylic acid is rapidly deacetylated to form salicylic acid. Both compounds have been proposed as anti-inflammatory agents. Major metabolites of salicylic acid are its acyl and phenolic glucuronide conjugates. Formation of these conjugates, catalyzed by UDP-glucuronosyltransferases (UGTs), decreases the amount of pharmacologically active salicylic acid present. We aimed to identify the UGTs catalyzing the glucuronidation of salicylic acid using both heterologously expressed enzymes and pooled human liver microsomes (HLMs) and to develop a liquid chromatography-tandem mass spectrometry method to quantify glucuronidation activity of UGTs 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17 Supersomes. All UGTs tested, except 1A4, 2B15, and 2B17, catalyzed salicylic acid phenolic and acyl glucuronidation. Ratios of salicylic acid phenolic to acyl glucuronide formation varied more than 12-fold from 0.5 for UGT1A6 to 6.1 for UGT1A1. These results suggest that all UGTs except 1A4, 2B15, and 2B17 might be involved in the glucuronidation of salicylic acid in vivo. From comparisons of apparent Km values determined in pooled HLMs and in expressed UGTs, UGT2B7 was suggested as a likely catalyst of salicylic acid acyl glucuronidation, whereas multiple UGTs were suggested as catalysts of phenolic glucuronidation. The results of this UGT screening may help target future evaluation of the effects of UGT polymorphisms on response to aspirin in clinical and population-based studies.

  13. Glucuronidation of the aspirin metabolite salicylic acid by expressed UDP-glucuronosyltransferases and human liver microsomes.

    PubMed

    Kuehl, Gwendolyn E; Bigler, Jeannette; Potter, John D; Lampe, Johanna W

    2006-02-01

    Acetylsalicylic acid (aspirin) is a common nonsteroidal anti-inflammatory drug used for treatment of pain and arthritis. In the body, acetylsalicylic acid is rapidly deacetylated to form salicylic acid. Both compounds have been proposed as anti-inflammatory agents. Major metabolites of salicylic acid are its acyl and phenolic glucuronide conjugates. Formation of these conjugates, catalyzed by UDP-glucuronosyltransferases (UGTs), decreases the amount of pharmacologically active salicylic acid present. We aimed to identify the UGTs catalyzing the glucuronidation of salicylic acid using both heterologously expressed enzymes and pooled human liver microsomes (HLMs) and to develop a liquid chromatography-tandem mass spectrometry method to quantify glucuronidation activity of UGTs 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17 Supersomes. All UGTs tested, except 1A4, 2B15, and 2B17, catalyzed salicylic acid phenolic and acyl glucuronidation. Ratios of salicylic acid phenolic to acyl glucuronide formation varied more than 12-fold from 0.5 for UGT1A6 to 6.1 for UGT1A1. These results suggest that all UGTs except 1A4, 2B15, and 2B17 might be involved in the glucuronidation of salicylic acid in vivo. From comparisons of apparent Km values determined in pooled HLMs and in expressed UGTs, UGT2B7 was suggested as a likely catalyst of salicylic acid acyl glucuronidation, whereas multiple UGTs were suggested as catalysts of phenolic glucuronidation. The results of this UGT screening may help target future evaluation of the effects of UGT polymorphisms on response to aspirin in clinical and population-based studies. PMID:16258079

  14. Chemoenzymatic Synthesis, Characterization, and Scale-Up of Milk Thistle Flavonolignan Glucuronides.

    PubMed

    Gufford, Brandon T; Graf, Tyler N; Paguigan, Noemi D; Oberlies, Nicholas H; Paine, Mary F

    2015-11-01

    Plant-based therapeutics, including herbal products, continue to represent a growing facet of the contemporary health care market. Mechanistic descriptions of the pharmacokinetics and pharmacodynamics of constituents composing these products remain nascent, particularly for metabolites produced following herbal product ingestion. Generation and characterization of authentic metabolite standards are essential to improve the quantitative mechanistic understanding of herbal product disposition in both in vitro and in vivo systems. Using the model herbal product, milk thistle, the objective of this work was to biosynthesize multimilligram quantities of glucuronides of select constituents (flavonolignans) to fill multiple knowledge gaps in the understanding of herbal product disposition and action. A partnership between clinical pharmacology and natural products chemistry expertise was leveraged to optimize reaction conditions for efficient glucuronide formation and evaluate alternate enzyme and reagent sources to improve cost effectiveness. Optimized reaction conditions used at least one-fourth the amount of microsomal protein (from bovine liver) and cofactor (UDP glucuronic acid) compared with typical conditions using human-derived subcellular fractions, providing substantial cost savings. Glucuronidation was flavonolignan-dependent. Silybin A, silybin B, isosilybin A, and isosilybin B generated five, four, four, and three monoglucuronides, respectively. Large-scale synthesis (40 mg of starting material) generated three glucuronides of silybin A: silybin A-7-O-β-D-glucuronide (15.7 mg), silybin A-5-O-β-D-glucuronide (1.6 mg), and silybin A-4´´-O-β-D-glucuronide (11.1 mg). This optimized, cost-efficient method lays the foundation for a systematic approach to synthesize and characterize herbal product constituent glucuronides, enabling an improved understanding of mechanisms underlying herbal product disposition and action.

  15. In vitro glucuronidation of the antibacterial triclocarban and its oxidative metabolites.

    PubMed

    Schebb, N H; Franze, B; Maul, R; Ranganathan, A; Hammock, B D

    2012-01-01

    Triclocarban (3,4,4'-trichlorocarbanilide; TCC) is widely used as an antibacterial in bar soaps. During use of these soaps, a significant portion of TCC is absorbed by humans. For the elimination from the body, glucuronidation plays a key role in both biliary and renal clearance. To investigate this metabolic pathway, we performed microsomal incubations of TCC and its hydroxylated metabolites 2'-OH-TCC, 3'-OH-TCC, and 6-OH-TCC. Using a new liquid chromatography-UV-mass spectrometry method, we could show a rapid glucuronidation for all OH-TCCs by the uridine-5'-diphosphate-glucuronosyltransferases (UGT) present in liver microsomes of humans (HLM), cynomolgus monkeys (CLM), rats (RLM), and mice (MLM). Among the tested human UGT isoforms, UGT1A7, UGT1A8, and UGT1A9 showed the highest activity for the conjugation of hydroxylated TCC metabolites followed by UGT1A1, UGT1A3, and UGT1A10. Due to this broad pattern of active UGTs, OH-TCCs can be efficiently glucuronidated in various tissues, as shown for microsomes from human kidney (HKM) and intestine (HIM). The major renal metabolites in humans, TCC-N-glucuronide and TCC-N'-glucuronide, were formed at very low conversion rates (<1%) by microsomal incubations. Low amounts of N-glucuronides were generated by HLM, HIM, and HKM, as well as by MLM and CLM, but not by RLM, according to the observed species specificity of this metabolic pathway. Among the human UGT isoforms, only UGT1A9 had activity for the N-glucuronidation of TCC. These results present an anomaly where in vivo the predominant urinary metabolites of TCC are N and N'-glucuronides, but these compounds are slowly produced in vitro. PMID:21953915

  16. In vitro antioxidative activity of (-)-epicatechin glucuronide metabolites present in human and rat plasma.

    PubMed

    Natsume, Midori; Osakabe, Naomi; Yasuda, Akiko; Baba, Seigo; Tokunaga, Takashi; Kondo, Kazuo; Osawa, Toshihiko; Terao, Junji

    2004-12-01

    Recently we identified four conjugated glucuronide metabolites of epicatechin, (-)-epicatechin-3'-O-glucuronide (E3'G), 4'-O-methyl-(-)-epicatechin-3'-O-glucuronide (4'ME3'G), (-)-epicatechin-7-O-glucuronide (E7G) and 3'-O-methyl-(-)-epicatechin-7-O-glucuronide (3'ME7G) from plasma and urine. E3'G and 4'ME3'G were isolated from human urine, while E7G and 3'ME7G were isolated from rats that had received oral administration of (-)-epicatechin (Natsume et al. (2003), Free Radic. Biol. Med. 34,840-849). It has been suggested that these metabolites possess considerable in vivo activity, and therefore we carried out a study to compare the antioxidant activities of the metabolites with that of the parent compound. This was achieved by measuring superoxide scavenging activity, reduction of plasma TBARS production and reduced susceptibility of low-density-lipoprotein (LDL) to oxidation. (-)-Epicatechin was found to have more potent antioxidant activity than the conjugated glucuronide metabolites. Both (-)-epicatechin and E7G had marked antioxidative properties with respect to superoxide radical scavenging activity, plasma oxidation induced by 2,2'-azobis-(2-aminopropane) dihydrochloride (AAPH) and LDL oxidation induced by copper ions or 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN). In contrast, the other metabolites had light antioxidative activities over the range of physiological concentrations found in plasma.

  17. Separation of substrates and closely related glucuronide metabolites using various chromatographic modes.

    PubMed

    Romand, Stéphanie; Rudaz, Serge; Guillarme, Davy

    2016-02-26

    The aim of this study was to assess the retention and selectivity of a cocktail of 10 substrates of uridine diphosphate glucuronosyltransferase enzymes (UGTs) and their respective glucuronides using four chromatographic approaches. For this purpose, seven different stationary phases were employed in reversed phase liquid chromatography (RPLC), two in hydrophilic interaction liquid chromatography (HILIC), one in aqueous normal phase chromatography (ANPC) and four in subcritical fluid chromatography (SFC). Highly orthogonal separations were achieved with these chromatographic modes. Hydrophobic interactions mainly governed the retention of the substrates and their polar glucuronides in RPLC despite the use of different chemical stationary phase bonding, involving additional possible interactions. In ANPC, atypical separations and poor peak shapes were observed with the selected compounds. In HILIC and SFC conditions, the metabolites were more retained than the substrates because of the polarity increase related to the glucuronic acid moiety. For the latter, a very high proportion of organic solvent (up to 80%) was required to elute the glucuronides that often displayed poor peak shapes. Finally, the selectivity of nine chromatographic systems was compared for the separation of isomeric and diastereoisomeric compounds. The stationary phases used in RPLC mode were more selective towards the two positional isomers of morphine glucuronides since they possess distinct lipophilicity. HILIC and SFC columns were found to be promising for the separation of a critical diastereoisomers pair, namely epitestosterone-glucuronide and testosterone-glucuronide. PMID:26818236

  18. Glucuronidation of Drugs and Drug-Induced Toxicity in Humanized UDP-Glucuronosyltransferase 1 Mice

    PubMed Central

    Kutsuno, Yuki; Itoh, Tomoo; Tukey, Robert H.

    2014-01-01

    UDP-glucuronosyltransferases (UGTs) are phase II drug-metabolizing enzymes that catalyze glucuronidation of various drugs. Although experimental rodents are used in preclinical studies to predict glucuronidation and toxicity of drugs in humans, species differences in glucuronidation and drug-induced toxicity have been reported. Humanized UGT1 mice in which the original Ugt1 locus was disrupted and replaced with the human UGT1 locus (hUGT1 mice) were recently developed. In this study, acyl-glucuronidations of etodolac, diclofenac, and ibuprofen in liver microsomes of hUGT1 mice were examined and compared with those of humans and regular mice. The kinetics of etodolac, diclofenac, and ibuprofen acyl-glucuronidation in hUGT1 mice were almost comparable to those in humans, rather than in mice. We further investigated the hepatotoxicity of ibuprofen in hUGT1 mice and regular mice by measuring serum alanine amino transferase (ALT) levels. Because ALT levels were increased at 6 hours after dosing in hUGT1 mice and at 24 hours after dosing in regular mice, the onset pattern of ibuprofen-induced liver toxicity in hUGT1 mice was different from that in regular mice. These data suggest that hUGT1 mice can be valuable tools for understanding glucuronidations of drugs and drug-induced toxicity in humans. PMID:24764149

  19. In vitro glucuronidation kinetics of deoxynivalenol by human and animal microsomes and recombinant human UGT enzymes.

    PubMed

    Maul, Ronald; Warth, Benedikt; Schebb, Nils Helge; Krska, Rudolf; Koch, Matthias; Sulyok, Michael

    2015-06-01

    The mycotoxin deoxynivalenol (DON), formed by Fusarium species, is one of the most abundant mycotoxins contaminating food and feed worldwide. Upon ingestion, the majority of the toxin is excreted by humans and animal species as glucuronide conjugate. First in vitro data indicated that DON phase II metabolism is strongly species dependent. However, kinetic data on the in vitro metabolism as well as investigations on the specific enzymes responsible for DON glucuronidation in human are lacking. In the present study, the DON metabolism was investigated using human microsomal fractions and uridine-diphosphoglucuronyltransferases (UGTs) as well as liver microsomes from five animal species. Only two of the twelve tested human recombinant UGTs led to the formation of DON glucuronides with a different regiospecificity. UGT2B4 predominantly catalyzed the formation of DON-15-O-glucuronide (DON-15GlcA), while for UGT2B7 the DON-3-O-glucuronide (DON-3GlcA) metabolite prevailed. For human UGTs, liver, and intestinal microsomes, the glucuronidation activities were low. The estimated apparent intrinsic clearance (Clapp,int) for all human UGT as well as tissue homogenates was <1 mL/min mg protein. For the animal liver microsomes, moderate Clapp,int between 1.5 and 10 mL/min mg protein were calculated for carp, trout, and porcine liver. An elevated glucuronidation activity was detected for rat and bovine liver microsomes leading to Clapp,int between 20 and 80 mL/min mg protein. The obtained in vitro data points out that none of the animal models is suitable for estimating the human DON metabolism with respect to the metabolite pattern and formation rate.

  20. Human hydroxylated metabolites of BDE-47 and BDE-99 are glucuronidated and sulfated in vitro.

    PubMed

    Erratico, Claudio; Zheng, Xiaobo; Ryden, Andreas; Marsh, Goran; Maho, Walid; Covaci, Adrian

    2015-07-16

    Polybrominated diphenyl ethers (PBDEs) were used worldwide as additive flame retardants and are classified as persistent, bioaccumulable and toxic environmental pollutants. In humans, the hydroxylated metabolites of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and 2,2',4,4',5-pentabromodiphenyl ether (BDE-99) formed in vitro have also been detected in vivo. To further characterize the metabolism of BDE-47 and BDE-99 and to identify candidate markers for monitoring the human exposure to PBDEs using non-invasive approaches, glucuronidation and sulfation of hydroxylated metabolites of BDE-47 and BDE-99 were investigated using human liver microsomes and cytoplasm, respectively. The formed Phase II metabolites were analyzed by liquid chromatography-tandem mass spectrometry using a novel approach to develop analytical methods in absence of authentic standards. All available standards for hydroxylated metabolites of BDE-47 and BDE-99 were glucuronidated and sulfated, showing that glucuronidation and sulfation are part of the metabolism pathway of BDE-47 and BDE-99 in vitro. The major glucuronidated and sulfated analogs of hydroxylated metabolites of BDE-47 were (a) 2,4-DBP-Gluc and 5-Gluc-BDE-47, and (b) 2'-Sulf-BDE-28, 4-Sulf-BDE-42 and 3-Sulf-BDE-47, respectively. The major glucuronidated and sulfated analogs of hydroxylated metabolites of BDE-99 were (a) 2,4,5-TBP-Gluc and 6'-Gluc-BDE-99, and (b) 3'-Sulf-BDE-99 and 5'-Sulf-BDE-99, respectively. Apparent Km values associated with the formation of sulfated metabolites of BDE-47 and BDE-99 were ten times lower than those of the corresponding glucuronidated metabolites, suggesting that sulfated rather than glucuronidated metabolites of OH-PBDEs might be used as markers of human exposure to PBDEs using a non-invasive approach based on urine sample collection. PMID:25956475

  1. Glucuronidation of anabolic androgenic steroids by recombinant human UDP-glucuronosyltransferases.

    PubMed

    Kuuranne, Tiia; Kurkela, Mika; Thevis, Mario; Schänzer, Wilhelm; Finel, Moshe; Kostiainen, Risto

    2003-09-01

    A multidimensional study on the glucuronidation of anabolic androgenic steroids and their phase I metabolites by 11 recombinant human UDP-glucuronosyltransferases (UGTs) was carried out using liquid chromatographic-tandem mass spectrometric analyses. Large differences between the enzymes with respect to the conjugation profiles of the 11 tested aglycones were detected. Two UGTs, 1A6 and 1A7, did not exhibit measurable activity toward any of the aglycones that were examined in this study. Regioselectivity was demonstrated by UGTs 1A8, 1A9, and 2B15 that preferentially catalyzed hydroxyl glucuronidation at the 17beta-position. Most of the other enzymes glucuronidated hydroxyl groups at both the 3alpha- and the 17beta-positions. Clear stereoselectivity was observed in glucuronidation of diastereomeric nandrolone metabolites (5alpha-estran-3alpha-ol-17-one and 5beta-estran-3alpha-ol-17-one), whereas such specificity was not seen when analogous methyltestosterone metabolites were assayed. UGTs 1A1, 1A3, 1A4, 1A8, 1A9, 1A10, 2B4, 2B7, and 2B15 readily glucuronidated 5alpha-androstane-3alpha,17beta-diol, but none of them exhibited methyltestosterone glucuronidation activity. In agreement with the latter observations, we found that the methyltestosterone glucuronidation activity of human liver microsomes is extremely low, whereas in induced rat liver microsomes it was significantly higher. The homology among UGTs 1A7 to 1A10 at the level of amino acid sequence is very high, and it was thus surprising to find large differences in their activity toward this set of aglycones. Furthermore, the high activity of UGT1A8 and 1A10 toward some of the substrates indicates that extrahepatic enzymes might play a role in the metabolism of anabolic androgenic steroids. PMID:12920167

  2. Bilirubin Glucuronidation Revisited: Proper Assay Conditions to Estimate Enzyme Kinetics with Recombinant UGT1A1

    PubMed Central

    Zhou, Jin; Tracy, Timothy S.

    2010-01-01

    Bilirubin, an end product of heme catabolism, is primarily eliminated via glucuronic acid conjugation by UGT1A1. Impaired bilirubin conjugation, caused by inhibition of UGT1A1, can result in clinical consequences, including jaundice and kernicterus. Thus, evaluation of the ability of new drug candidates to inhibit UGT1A1-catalyzed bilirubin glucuronidation in vitro has become common practice. However, the instability of bilirubin and its glucuronides presents substantial technical challenges to conduct in vitro bilirubin glucuronidation assays. Furthermore, because bilirubin can be diglucuronidated through a sequential reaction, establishment of initial rate conditions can be problematic. To address these issues, a robust high-performance liquid chromatography assay to measure both bilirubin mono- and diglucuronide conjugates was developed, and the incubation conditions for bilirubin glucuronidation by human embryonic kidney 293-expressed UGT1A1 were carefully characterized. Our results indicated that bilirubin glucuronidation should be assessed at very low protein concentrations (0.05 mg/ml protein) and over a short incubation time (5 min) to assure initial rate conditions. Under these conditions, bilirubin total glucuronide formation exhibited a hyperbolic (Michaelis-Menten) kinetic profile with a Km of ∼0.2 μM. In addition, under these initial rate conditions, the relative proportions between the total monoglucuronide and the diglucuronide product were constant across the range of bilirubin concentration evaluated (0.05–2 μM), with the monoglucuronide being the predominant species (∼70%). In conclusion, establishment of appropriate incubation conditions (i.e., very low protein concentrations and short incubation times) is necessary to properly characterize the kinetics of bilirubin glucuronidation in a recombinant UGT1A1 system. PMID:20668247

  3. UDP-glucuronosyltransferase (UGT) 1A1 mainly contributes to the glucuronidation of trovafloxacin.

    PubMed

    Fujiwara, Ryoichi; Sumida, Kyohei; Kutsuno, Yuki; Sakamoto, Masaya; Itoh, Tomoo

    2015-02-01

    Identification of drug-metabolizing enzyme(s) responsible for the metabolism of drugs is an important step to understand not only interindividual variability in pharmacokinetics but also molecular mechanisms of metabolite-related toxicity. While it was reported that the major metabolic pathway of trovafloxacin, which is an antibiotic, was glucuronidation, the UDP-glucuronosyltransferase (UGT) isoform(s) responsible for the trovafloxacin glucuronidation has not been identified yet. In the present study, among the functional human UGT members, UGT1A1, UGT1A3, and UGT1A9 exhibited higher trovafloxacin acyl-glucuronidation activities. While other UGT members such as UGT1A8, UGT2B7, and UGT2B15 showed glucuronidation activity toward trovafloxacin, the metabolic velocity was extremely low. In human liver microsomes, trovafloxacin acyl-glucuronidation followed the Hill equation with S50 value of 95 μM, Vmax value of 243 pmol/min per mg, and a Hill coefficient of 2.0, while the UGT1A1-expressing system displayed Michaelis-Menten kinetics with a substrate inhibition, with Km value of 759 μM and Vmax value of 1160 pmol/min per mg. In human liver microsomes prepared from poor metabolizers (UGT1A1*28/*28), significantly reduced trovafloxacin acyl-glucuronide formation activity was observed, indicating that UGT1A1 mainly, while other UGT members such as UGT1A3 and UGT1A9 partially, contributes to the glucuronidation of trovafloxacin. PMID:25760534

  4. S-Ethyl dipropylthiocarbamate (EPTC)

    Integrated Risk Information System (IRIS)

    S - Ethyl dipropylthiocarbamate ( EPTC ) ; CASRN 759 - 94 - 4 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessme

  5. Detection of interstellar ethyl cyanide

    NASA Technical Reports Server (NTRS)

    Johnson, D. R.; Lovas, F. J.; Gottlieb, C. A.; Gottlieb, E. W.; Litvak, M. M.; Thaddeus, P.; Guelin, M.

    1977-01-01

    Twenty-four millimeter-wave emission lines of ethyl cyanide (CH3CH2CN) have been detected in the Orion Nebula (OMC-1) and seven in Sgr B2. To derive precise radial velocities from the astronomical data, a laboratory measurement of the rotational spectrum of ethyl cyanide has been made at frequencies above 41 GHz. In OMC-1, the rotational temperature of ethyl cyanide is 90 K (in good agreement with other molecules), the local-standard-of-rest radial velocity is 4.5 + or - 1.0 km/s (versus 8.5 km/s for most molecules), and the column density is 1.8 by 10 to the 14th power per sq cm (a surprisingly high figure for a complicated molecule). The high abundance of ethyl cyanide in the Orion Nebula suggests that ethane and perhaps larger saturated hydrocarbons may be common constituents of molecular clouds and have escaped detection only because they are nonpolar or only weakly polar.

  6. Direct radioimmunoassay of urinary estrogen and pregnanediol glucuronides during the menstrual cycle

    SciTech Connect

    Stanczyk, F.Z.; Miyakawa, I.; Goebelsmann, U.

    1980-06-15

    Assays measuring immunoreactive estrone glucuronide (E/sub 1/G), estradiol-3-glucuronide (E/sub 2/-3G), estradiol-17..beta..-glucuronide (E/sub 2/-17G), estriol-3-glucuronide (E/sub 3/-3G), estriol-16..cap alpha..-glucuronide (E/sub 3/-16G), and pregnanediol-3..cap alpha..-glucuronide (Pd-3G) directly in diluted urine were developed and validated. These estrogen and pregnanediol glucuronide fractions were measured in aliquots of 24-hour and overnight samples of urine collected daily from seven women for one menstrual cycle. Urinary hormone excretion was correlated with daily serum estradiol (E/sub 2/), progesterone (P), and lutenizing hormonee (LH) levels. A sharp midcycle LH peak preceded by a preovulatory rise in serum E/sub 2/ and followed by luteal phase serum P levels were noted in each of the seven apparently ovulatory cycles. Twenty-four-hour and overnight urinary excretion patterns of estrogen glucuronides were similar to those of serum E/sub 2/. Of the five estrogen glucuronide fractions tested, excretion of E/sub 2/-17G exhibited the earliest and steepest ascending slope of the preovulatory estrogen surge and correlated best with serum E/sub 2/ levels. Urinary excretion of E/sub 1/-G, E/sub 2/-3G, and E/sub 3/-16G also showed an early and steep preovulatory rise and preceded that of E/sub 3/-3G, whereas urinary excretion of E/sub 3/-3G exhibited the poorest correlation with serum E/sub 2/ concentrations. The urinary excretion of Pd-3G rose parallel to serum P levels and was markedly elevated 2 to 3 days after the midcycle LH peak in both 24-hour and overnight collections of urine. These results indicate that among the urinary estrogen conjugate fractions tested, E/sub 2/-17G is the one that most suitably predicts ovulation.

  7. Bisphenol A glucuronide/sulfate diconjugate in perfused liver of rats

    PubMed Central

    INOUE, Hiroki; KEMANAI, Shino; SANO, Chie; KATO, Seiyu; YOKOTA, Hiroshi; IWANO, Hidetomo

    2016-01-01

    In isolated hepatocytes, the environmental estrogen bisphenol A (BPA) is metabolized into a mono-glucuronide and a glucuronide/sulfate diconjugate. Little is known about the fate of the diconjugate in the liver. The present study focused on the metabolism and dispostion of BPA diconjugate in the liver using a perfusion method. In Sprague-Dawley rats, BPA (15,150 or 1,500 nmol) was applied into the liver. In male rats, the infused BPA was conjugated to both glucuronide and a diconjugate during passage through the liver. The diconjugate was observed at high-dose application of the substrate. In female rats, the chemical was conjugated almost exclusively to the glucuronide in all doses utilized in this study. In both the male and female rats, the resultant metabolites were preferentially excreted into the bile. These results suggest that BPA is conjugated primarily to mono-glucuronide in rat liver; and that in males, diconjugate production occurs under conditions of high-dose exposure to BPA. PMID:26782136

  8. Genetic and environmental factors associated with variation of human xenobiotic glucuronidation and sulfation.

    PubMed Central

    Burchell, B; Coughtrie, M W

    1997-01-01

    Glucuronidation and sulfation are phase 2 metabolic reactions catalyzed by large families of different isoenzymes in man. The textbook view that glucuronidation and sulfation lead to the production of harmless conjugates for simple excretion is not valid. Biologically active and toxic sulfates and glucuronides are produced and leed to adverse drug reactions, including immune hypersensitivity. Considerable variation in xenobiotic conjugation is observed as a result of altered expression of UDP-glucuronosyltransferases (UGTs) and sulfotransferases (STs). Recent cloning and expression of human cDNA encoding UGTs and STs has facilitated characterization of isoform substrate specificity, which has been further validated using specific antibodies and human tissue fractions. The availability of cloned/expressed human enzymes and specific antibodies has enabled the investigation of xenobiotic induction and metabolic disruption leeding to adverse responses. Genetic polymorphisms of glucuronidation and sulfation are known to exist although the characterization and assessment of the importance of these variations are hampered by appropriate ethical studies in men with suitable safe model compounds. Genetic analysis has allowed molecular identification of defects in well-known hyperbilirubinemias. However, full characterization of the specific functional roles of human UGTs and STs requires rigorous kinetic and molecular analyses of the role of each enzyme in vivo through the use of specific antibodies and inhibitors. This will leed to the better prediction of variation of xenobiotic glucuronidation and sulfation in man. PMID:9255555

  9. New Flavonol Glucuronides from the Flower Buds of Syzygium aromaticum (Clove).

    PubMed

    Ryu, Byeol; Kim, Hye Mi; Lee, Jin Su; Lee, Chan Kyu; Sezirahiga, Jurdas; Woo, Jeong-Hwa; Choi, Jung-Hye; Jang, Dae Sik

    2016-04-20

    Repeated chromatography of the EtOAc-soluble fraction from the 70% EtOH extract of the flower buds of Syzygium aromaticum (clove) led to the isolation and characterization of four new flavonol glucuronides, rhamnetin-3-O-β-d-glucuronide (1), rhamnazin-3-O-β-d-glucuronide (2), rhamnazin-3-O-β-d-glucuronide-6″-methyl ester (3), and rhamnocitrin-3-O-β-d-glucuronide-6″-methyl ester (4), together with 15 flavonoids (5-19) having previously known chemical structures. The structures of the new compounds 1-4 were determined by interpretation of spectroscopic data, particularly by 1D- and 2D-NMR studies. Six flavonoids (6, 7, 9, 14, 18, and 19) were isolated from the flower buds of S. aromaticum for the first time in this study. The flavonoids were examined for their cytotoxicity against human ovarian cancer cells (A2780) using MTT assays. Among the isolates, pachypodol (19) showed the most potent cytotoxicity on A2780 cells with an IC50 value of 8.02 μM. PMID:27045836

  10. New Flavonol Glucuronides from the Flower Buds of Syzygium aromaticum (Clove).

    PubMed

    Ryu, Byeol; Kim, Hye Mi; Lee, Jin Su; Lee, Chan Kyu; Sezirahiga, Jurdas; Woo, Jeong-Hwa; Choi, Jung-Hye; Jang, Dae Sik

    2016-04-20

    Repeated chromatography of the EtOAc-soluble fraction from the 70% EtOH extract of the flower buds of Syzygium aromaticum (clove) led to the isolation and characterization of four new flavonol glucuronides, rhamnetin-3-O-β-d-glucuronide (1), rhamnazin-3-O-β-d-glucuronide (2), rhamnazin-3-O-β-d-glucuronide-6″-methyl ester (3), and rhamnocitrin-3-O-β-d-glucuronide-6″-methyl ester (4), together with 15 flavonoids (5-19) having previously known chemical structures. The structures of the new compounds 1-4 were determined by interpretation of spectroscopic data, particularly by 1D- and 2D-NMR studies. Six flavonoids (6, 7, 9, 14, 18, and 19) were isolated from the flower buds of S. aromaticum for the first time in this study. The flavonoids were examined for their cytotoxicity against human ovarian cancer cells (A2780) using MTT assays. Among the isolates, pachypodol (19) showed the most potent cytotoxicity on A2780 cells with an IC50 value of 8.02 μM.

  11. The gusBC genes of Escherichia coli encode a glucuronide transport system.

    PubMed

    Liang, Wei-Jun; Wilson, Kate J; Xie, Hao; Knol, Jan; Suzuki, Shun'ichi; Rutherford, Nicholas G; Henderson, Peter J F; Jefferson, Richard A

    2005-04-01

    Two genes, gusB and gusC, from a natural fecal isolate of Escherichia coli are shown to encode proteins responsible for transport of beta-glucuronides with synthetic [(14)C]phenyl-1-thio-beta-d-glucuronide as the substrate. These genes are located in the gus operon downstream of the gusA gene on the E. coli genome, and their expression is induced by a variety of beta-d-glucuronides. Measurements of transport in right-side-out subcellular vesicles show the system has the characteristics of secondary active transport energized by the respiration-generated proton motive force. When the genes were cloned together downstream of the tac operator-promoter in the plasmid pTTQ18 expression vector, transport activity was increased considerably with isopropylthiogalactopyranoside as the inducer. Amplified expression of the GusB and GusC proteins enabled visualization and identification by N-terminal sequencing of both proteins, which migrated at ca. 32 kDa and 44 kDa, respectively. Separate expression of the GusB protein showed that it is essential for glucuronide transport and is located in the inner membrane, while the GusC protein does not catalyze transport but assists in an as yet unknown manner and is located in the outer membrane. The output of glucuronides as waste by mammals and uptake for nutrition by gut bacteria or reabsorption by the mammalian host is discussed. PMID:15774881

  12. Separation and Purification of Two Flavone Glucuronides from Erigeron multiradiatus (Lindl.) Benth with Macroporous Resins

    PubMed Central

    Zhang, Zhi-feng; Liu, Yuan; Luo, Pei; Zhang, Hao

    2009-01-01

    Scutellarein-7-O-β-D-glucuronide (SG) and apigenin-7-O-β-D-glucuronide (AG) are two major bioactive constituents with known pharmacological effects in Erigeron multiradiatus. In this study, a simple method for preparative separation of the two flavone glucuronides was established with macroporous resins. The performance and adsorption characteristics of eight macroporous resins including AB-8, HPD100, HPD450, HPD600, D100, D101, D141, and D160 have been evaluated. The results confirmed that D141 resin offered the best adsorption and desorption capacities and the highest desorption ratio for the two glucuronides among the tested resins. Sorption isotherms were constructed for D141 resin under optimal ethanol conditions and fitted well to the Freundlich and Langmuir models (R2 > 0.95). Dynamic adsorption and desorption tests was performed on column packed with D141 resin. After one-run treatment with D141 resin, the two-constituent content in the final product was increased from 2.14% and 1.34% in the crude extract of Erigeron multiradiatus to 24.63% and 18.42% in the final products with the recoveries of 82.5% and 85.4%, respectively. The preparative separation of SG and AG can be easily and effectively achieved via adsorption and desorption on D141 resin, and the method developed can be referenced for large-scale separation and purification of flavone glucuronides from herbal raw materials. PMID:19918373

  13. 21 CFR 172.868 - Ethyl cellulose.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Ethyl cellulose. 172.868 Section 172.868 Food and... Multipurpose Additives § 172.868 Ethyl cellulose. The food additive ethyl cellulose may be safely used in food in accordance with the following prescribed conditions: (a) The food additive is a cellulose...

  14. 21 CFR 172.868 - Ethyl cellulose.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Ethyl cellulose. 172.868 Section 172.868 Food and... Multipurpose Additives § 172.868 Ethyl cellulose. The food additive ethyl cellulose may be safely used in food in accordance with the following prescribed conditions: (a) The food additive is a cellulose...

  15. Simplified analysis of acetaminophen glucuronide for quantifying gluconeogenesis and glycogenolysis using deuterated water.

    PubMed

    Jones, J; Kahl, S; Carvalho, F; Barosa, C; Roden, M

    2015-06-15

    Measurement of acetaminophen glucuronide (AG) (2)H enrichment from deuterated water ((2)H2O) by (2)H nuclear magnetic resonance (NMR) analysis of its monoacetone glucose (MAG) derivative provides estimation of gluconeogenic and glycogenolytic contributions to endogenous glucose production (EGP). However, AG derivatization to MAG is laborious and unsuitable for high-throughput studies. An alternative derivative, 5-O-acetyl monoacetone glucuronolactone (MAGLA), was tested. Eleven healthy subjects ingested (2)H2O to 0.5% body water enrichment and 500 mg of acetaminophen. Plasma glucose and urinary glucuronide positional (2)H enrichments were measured by (2)H NMR spectroscopy of MAG and MAGLA, respectively. A Bland-Altman analysis indicated agreement at the 95% confidence level between glucose and glucuronide estimates.

  16. Characterization of retinoyl beta-glucuronide as a minor metabolite of retinoic acid in bile.

    PubMed Central

    Zile, M H; Schnoes, H K; DeLuca, H F

    1980-01-01

    Several metabolites detected in the bile of rats given radioactive retinoic acid were separated by liquid/gel partition chromatography and purified by high-pressure liquid chromatography. One of these metabolites was found to be sensitive to beta-D-glucuronidase, yielding both 13-cis- and all-trans-retinoic acid. It had the characteristic ultraviolet absorption spectrum of retinoic acid esters. Trimethylsilyl ether and acetyl derivatives of the methylated metabolite were prepared and examined by mass spectrometry. The resulting mass spectra established the structure to be retinoyl beta-glucuronide. Retinoyl glucuronide was rapidly excreted into the bile: the excretion was complete by 12 hr after the administration of retinoic acid. At this time the metabolite represented 12% of bile radioactivity (10% of dose). These observations confirm the existence of retinoyl glucuronide but demonstrate that it represents only one of several retinoic acid metabolites in bile. PMID:6932017

  17. A major glucuronidated metabolite of JWH-018 is a neutral antagonist at CB1 receptors.

    PubMed

    Seely, Kathryn A; Brents, Lisa K; Radominska-Pandya, Anna; Endres, Gregory W; Keyes, Gregory S; Moran, Jeffery H; Prather, Paul L

    2012-04-16

    Recently, hydroxylated metabolites of JWH-018, a synthetic cannabinoid found in many K2/Spice preparations, have been shown to retain affinity and activity for cannabinoid type 1 receptors (CB1Rs). The activity of glucuronidated metabolites of JWH-018 is not known; hence, this study investigated the affinity and activity of a major metabolite, JWH-018-N-(5-hydroxypentyl) β-D-glucuronide (018-gluc), for CB1Rs. The 018-gluc binds CB1Rs (K(i) = 922 nM), has no effect on G-protein activity, but antagonizes JWH-018 activity at CB1Rs. The data suggests that hydroxylation by cytochrome P450s and subsequent glucuronidation by UDP-glucuronosyltransferases produces a metabolite, 018-gluc, which possesses antagonistic activity at CB1Rs.

  18. A major glucuronidated metabolite of JWH-018 is a neutral antagonist at CB1 receptors

    PubMed Central

    Seely, Kathryn A.; Brents, Lisa K.; Radominska-Pandya, Anna; Endres, Gregory W.; Keyes, Gregory S.; Moran, Jeffery H.; Prather, Paul L.

    2014-01-01

    Recently, hydroxylated metabolites of JWH-018, a synthetic cannabinoid found in many K2/Spice preparations, have been shown to retain affinity and activity for cannabinoid type 1 receptors (CB1Rs). The activity of glucuronidated metabolites of JWH-018 is not known; hence this study investigated the affinity and activity of a major metabolite, JWH-018-N-(5-hydroxypentyl) β-D-glucuronide (018-gluc), for CB1Rs. The 018-gluc binds CB1Rs (Ki = 922 nM), has no effect on G-protein activity, but antagonizes JWH-018 activity at CB1Rs. The data suggests that hydroxylation by cytochrome P450s and subsequent glucuronidation by UDP-glucuronosyltransferases produces a metabolite, 018-gluc, which possesses antagonistic activity at CB1Rs. PMID:22404317

  19. Synthesis of 5α-androstane-3α,17β-diol 17-O-glucuronide histaminyl conjugate for immunoassays.

    PubMed

    Vinš, Petr; Černý, Ivan; Mikšátková, Petra; Drašar, Pavel

    2016-05-01

    Simple method of preparation of 5α-androstane-3α,17β-diol 17-O-glucuronide N-histaminyl amide was developed for the construction of immunoanalytical kit. Improved method of glucuronide derivative synthesis was used, followed by hydroxybenzotriazole-dicyclohexylcarbodiimide coupling with histamine. PMID:26898541

  20. Loss of exogenous androgen dependence by prostate tumor cells is associated with elevated glucuronidation potential

    PubMed Central

    Zimmer, Brenna M.; Howell, Michelle E.; Wei, Qin; Ma, Linlin; Romsdahl, Trevor; Loughman, Eileen G.; Markham, Jonathan E.; Seravalli, Javier; Barycki, Joseph J.; Simpson, Melanie A.

    2016-01-01

    Prostate epithelial cells control the potency and availability of androgen hormones in part by inactivation and elimination. UDP-glucose dehydrogenase (UGDH) catalyzes the NAD+-dependent oxidation of UDP-glucose to UDP-glucuronate, an essential precursor for androgen inactivation by the prostate glucuronidation enzymes UGT2B15 and UGT2B17. UGDH expression is androgen stimulated, which increases the production of UDP-glucuronate, and fuels UGT-catalyzed glucuronidation. In this study, we compared the glucuronidation potential and its impact on androgen-mediated gene expression in an isogenic LNCaP model for androgen dependent versus castration resistant prostate cancer. Despite significantly lower androgen-glucuronide output, LNCaP 81 castration resistant tumor cells expressed higher levels of UGDH, UGT2B15, and UGT2B17. However, the magnitude of androgen-activated UGDH and PSA expression, as well as the AR-dependent repression of UGT2B15 and UGT2B17, was blunted several-fold in these cells. Consistent with these results, the ligand-activated binding of AR to the PSA promoter and subsequent transcriptional activation were also significantly reduced in castration resistant cells. Analysis of the UDP-sugar pools and flux through pathways downstream of UDP-glucuronate production revealed that these glucuronidation precursor metabolites were channeled through proteoglycan and glycosaminoglycan biosynthetic pathways, leading to increased surface expression of Notch 1. Knockdown of UGDH diminished Notch1 and increased glucuronide output. Overall, these results support a model in which the aberrant partitioning of UDP-glucuronate and other UDP-sugars into alternative pathways during androgen deprivation contributes to the loss of prostate tumor cell androgen sensitivity by promoting altered cell surface proteoglycan expression. PMID:27307252

  1. In vitro characterization of glucuronidation of vanillin: identification of human UDP-glucuronosyltransferases and species differences.

    PubMed

    Yu, Jian; Han, Jing-Chun; Hua, Li-Min; Gao, Ya-Jie

    2013-09-01

    Vanillin is a food flavoring agent widely utilized in foods, beverages, drugs, and perfumes and has been demonstrated to exhibit multiple pharmacological activities. Given the importance of glucuronidation in the metabolism of vanillin, the UDP-glucuronosyltransferase conjugation pathway of vanillin was investigated in this study. Vanillin glucuronide was identified by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and a hydrolysis reaction catalyzed by β-glucuronidase. The kinetic study showed that vanillin glucuronidation by HLMs and HIMs followed Michaelis-Menten kinetics and the kinetic parameters were as follows: 134.9 ± 13.5 μM and 81.3 ± 11.3 μM for K(m) of HLMs and HIMs, 63.8 ± 2.0 nmol/min/mg pro and 13.4 ±2.0 nmol/min/mg pro for Vmax of HLMs and HIMs. All UDP-glucuronosyltransferase (UGT) isoforms except UGT1A4, 1A9, and 2B7 showed the capability to glucuronidate vanillin, and UGT1A6 exerted the higher V(max)/K(m) values than other UGT isoforms for the glucuronidation of vanillin when assuming expression of isoforms is similar in recombinant UGTs. Kinetic analysis using liver microsomes from six studied speices indicated that vanillin had highest affinity for the monkey liver microsomes enzyme (K(m)  = 25.6 ± 3.2 μM) and the lowest affinity for the mice liver microsomes enzyme (K(m)  = 149.1 ± 18.4 μM), and intrinsic clearance was in the following order: monkey > dog > minipig > mice > rat ~ human. These data collectively provided important information for understanding glucuronidation of vanillin.

  2. Comparison of the effects of curcumin and curcumin glucuronide in human hepatocellular carcinoma HepG2 cells.

    PubMed

    Shoji, Motomu; Nakagawa, Kiyotaka; Watanabe, Akio; Tsuduki, Tsuyoshi; Yamada, Teiko; Kuwahara, Shigefumi; Kimura, Fumiko; Miyazawa, Teruo

    2014-05-15

    Curcumin is a yellow pigment found in turmeric (Curcuma Longa L.), and is reported, in recent studies, to have several pharmacological effects, including anti-oxidant, anti-inflammatory, anti-tumour and lipid-lowering properties. However, as most curcumin is conjugated when absorbed through the intestine, free curcumin is present at extremely low levels inside the body. Therefore, curcumin metabolites have been presumed to be responsible for the curcumin bioactivity. In this study, we first confirmed that curcumin glucuronide is the major metabolite of curcumin found in the plasma after oral administration of curcumin in rats. Next, we synthesised curcumin glucuronide and compared the effects of curcumin and curcumin glucuronide on gene expression in a human hepatoma cell line (HepG2). We found that the effects of curcumin glucuronide are weaker than those of curcumin and that this difference is related to relative absorption rates of curcumin and curcumin glucuronide into HepG2 cells.

  3. Comparison of stably expressed rat UGT1.1 and UGT2B1 in the glucuronidation of opioid compounds.

    PubMed

    King, C D; Rios, G R; Green, M D; MacKenzie, P I; Tephly, T R

    1997-02-01

    Opioids are important drugs used as analgesics, antitussives, antidiarrheals, and in the therapy of myocardial infarctions, and as antagonists of opioid intoxication. The glucuronidation of these compounds, catalyzed by UDP-glucuronosyltransferases (UGTs), is well known to be a primary step in their metabolism to hydrophilic products and in their ultimate excretion. The present study was designed to compare the reactivity and relative glucuronidation efficiencies of opioid agonists, antagonists, and partial agonists with two rat UGT isoforms; UGT1.1, which is generally considered the "bilirubin UGT," and UGT2B1, which has previously been shown to catalyze the glucuronidation of testosterone, chloramphenicol, and (-)-morphine. Rat UGT2B1, stably expressed in HK293 cells, exhibited high glucuronidation rates and catalytic efficiencies for many opioids, although values for (-)-morphine and nalorphine were the highest. In contrast, these compounds were very poor substrates for expressed rat UGT1.1. Comparably high glucuronidation rates and efficiencies were found for buprenorphine and diprenorphine with both UGT isoforms. These results suggest that opioids with morphinan-based chemical structures similar to (-)-morphine interact with UGTs differently than those with oripavine-based chemical structures similar to buprenorphine. To investigate the contribution of rat UGT1.1 and UGT2B1 in the overall rate of glucuronidation of buprenorphine in the rat liver, hepatic microsomes from Gunn rats (where UGT1.1 activity is absent) and Wistar rats (where UGT1.1 activity is present) were studied. Buprenorphine glucuronidation activity in Gunn rat liver microsomes exhibit approximately 25% of rates observed in Wistar rat liver microsomes, whereas (-)-morphine, naloxone, and naltrexone glucuronidation rates were not significantly different in microsomal preparations from Gunn and Wistar rats. These data suggest that UGT2B1 is the major hepatic enzyme involved in the glucuronidation

  4. Reduction of TEM/ETG-scale Density Fluctuations in the Core and Edge of H-mode DIII-D Plasmas

    NASA Astrophysics Data System (ADS)

    Schmitz, L.

    2008-11-01

    Improved confinement during H-mode has been linked to ExB shear suppression of large-scale (kθρs<=0.3) turbulence within an edge transport barrier. While larger scale eddies are preferentially suppressed by increased shear flow in this paradigm, the effects on smaller scale (TEM/ETG-scale) turbulence are less certain. Recent results from DIII-D provide the first experimental evidence that intermediate-scale turbulence (1 < kθρs<=3) together with larger-scale electron temperature fluctuations [1] are also reduced promptly at the L-H transition. These reductions are not confined to the edge region. Intermediate-scale density fluctuations obtained via Doppler backscattering, are significantly reduced (30%-50%) over a range of normalized radii (0.5 <=r/a <=0.85) within a few ms of the L-H transition. A larger reduction (>=75%) is observed at the top of the pedestal (r/a ˜0.9) within 0.2 ms. In addition, low-k electron temperature fluctuations (kθρs<=0.3, from correlation ECE) are strongly reduced (>75%) at the L-H mode transition and during QH-mode (r/a ˜0.7). Gyrokinetic simulation results [2] predict that Te fluctuations contribute significantly to L-mode electron heat transport, hence, the observed reduction is likely an important factor in the observed improved H-mode electron heat confinement (χe^QH/χ3^L < 0.25). Doppler backscattering is also utilized to probe time-dependent shear flows (i.e. zonal flows). The results clearly indicate that zonal flow levels are anti-correlated with the amplitude of intermediate-scale density turbulence in L-mode, suggesting that zonal flows play an important role in turbulence/transport regulation. 3pt [1] L. Schmitz et al., Phys. Rev. Lett. 100, 035002 (2008).[2] A.E. White et al., Phys. Plasmas 15, 056116 (2008).

  5. Age-related increases in F344 rat intestine microsomal quercetin glucuronidation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to establish the extent age modifies intestinal quercetin glucuronidation capacity. Pooled microsomal fractions of three equidistant small intestine (SI) segments from 4, 12, 18, and 28 mo male F344 rats (n=8/group) were employed to model the enzyme kinetics of UDP-gl...

  6. Incomplete recovery of prescription opioids in urine using enzymatic hydrolysis of glucuronide metabolites.

    PubMed

    Wang, Ping; Stone, Judith A; Chen, Katherine H; Gross, Susan F; Haller, Christine A; Wu, Alan H B

    2006-10-01

    Confirmation of opioids in urine samples of clinical patients requires liberation of opioids from their glucuronide conjugates. Both acid hydrolysis and enzyme hydrolysis using beta-glucuronidase from various sources have been reported, with the latter approach prevailing in most clinical toxicology laboratories. The goal of this study was to compare the efficiency of acid versus different enzyme hydrolysis methods in recovering morphine and common semisynthetic opioids from glucuronide standards and 78 patient urine samples that were screened positive for opioids as a class. Specimens were analyzed with a validated gas chromatography-mass spectrometry (GC-MS) procedure. With the exception of oxycodone, the results indicated that the majority of opioids tested were extensively glucuronide-conjugated in urine. Significantly, acid hydrolysis liberated > 90% of morphine and hydromorphone from their glucuronide standards but enzyme hydrolysis had lower and variable efficiency, depending on the opiate type and the enzyme source. In patient specimens, much higher concentrations of free codeine, morphine, hydromorphone, and oxymorphone were obtained with acid hydrolysis than with various enzyme methods. Incomplete hydrolysis using beta-glucuronidase could lead to false-negative results for many opioids when urine is tested for drugs of abuse. We conclude that acid hydrolysis is the method of choice for GC-MS confirmation of urine opioids.

  7. DEVELOPMENT OF A CLASS-SELECTIVE ENZYME IMMUNOASSAY FOR URINARY PHENOLIC GLUCURONIDES. (R825433)

    EPA Science Inventory

    Class-selective immunoassays for the measurement of glucuronides in human urine can aid evaluation of human exposure to complex mixtures of xenobiotics. Therefore, an enzyme immunoassay (EIA) for the group-selective detection of phenolic Profiling serum bile acid glucuronides in humans: gender divergences, genetic determinants and response to fenofibrate

    PubMed Central

    Trottier, Jocelyn; Perreault, Martin; Rudkowska, Iwona; Levy, Cynthia; Dallaire-Theroux, Amélie; Verreault, Mélanie; Caron, Patrick; Staels, Bart; Vohl, Marie-Claude; Straka, Robert J.; Barbier, Olivier

    2014-01-01

    Glucuronidation, catalyzed by UDP-glucuronosyltransferase (UGT) enzymes detoxifies cholestatic bile acids (BAs). We aimed at i) characterizing the circulating BA-glucuronide (-G) pool composition in humans, ii) evaluating how sex and UGT polymorphisms influence this composition, and iii) analyzing the effects of lipid-lowering drug fenofibrate on the circulating BA-G profile in 300 volunteers and 5 cholestatic patients. Eleven BA-Gs were determined in pre- and post-fenofibrate samples. Men exhibited higher BA-G concentrations, and various genotype/BA-G associations were discovered in relevant UGT genes. The chenodeoxycholic acid-3G concentration was associated with the UGT2B7 802C>T polymorphism. Glucuronidation assays confirmed the predominant role of UGT2B7 and UGT1A4 in CDCA-3G formation. Fenofibrate exposure increased the serum levels of 5 BA-G species, including CDCA-3G, and up-regulated expression of UGT1A4, but not UGT2B7, in hepatic cells. This study demonstrates that fenofibrate stimulates BA glucuronidation in humans, and thus reduces bile acid toxicity in the liver. PMID:23756370

  8. Urinary excretion of bile acid glucosides and glucuronides in extrahepatic cholestasis.

    PubMed

    Wietholtz, H; Marschall, H U; Reuschenbach, R; Matern, H; Matern, S

    1991-04-01

    Recently the formation of bile acid glucosides has been described as a novel conjugation mechanism in vitro and in vivo. In 10 patients with extrahepatic cholestasis caused by carcinoma of the head of the pancreas we investigated excretion rates and profiles of urinary bile acid glucosides. Urinary bile acid glucosides and, for comparison, bile acid glucuronides were extracted and characterized according to established methods. In controls total urinary bile acid glucoside excretion was 0.22 +/- 0.03 mumol/24 hr (mean +/- S.E.M.)-in the range of bile acid glucuronide excretion (0.41 +/- 0.06 mumol/24 hr; mean +/- S.E.M.). A gas chromatography-mass spectrometry-characterized trihydroxy bile acid glucoside of still-unknown hydroxyl positions accounted for 65% of total urinary bile acid glucosides. In extrahepatic cholestasis total urinary bile acid glucoside excretion was 0.52 +/- 0.13 mumol/24 hr (mean +/- SEM), yet significantly lower than bile acid glucuronide excretion (1.53 +/- 0.13 mumol/24 hr; mean +/- SEM; p less than 0.001). In cholestasis the primary bile acid derivatives cholic and chenodeoxycholic acid glucosides amounted to 90%, whereas the trihydroxy bile acid glucoside had decreased to 5% of total bile acid glucoside excretion, indicating its alteration during enterohepatic circulation. The data establish the composition and quantity of urinary bile acid glucosides in healthy controls and cholestasis and constitute a quantitative comparison with another glycosidic conjugation reaction, bile acid glucuronidation.

  9. A validated method for simultaneous determination of codeine, codeine-6-glucuronide, norcodeine, morphine, morphine-3-glucuronide and morphine-6-glucuronide in post-mortem blood, vitreous fluid, muscle, fat and brain tissue by LC-MS.

    PubMed

    Frost, Joachim; Løkken, Trine N; Brede, Wenche R; Hegstad, Solfrid; Nordrum, Ivar S; Slørdal, Lars

    2015-04-01

    The toxicodynamics and, to a lesser degree, toxicokinetics of the widely used opiate codeine remain a matter of controversy. To address this issue, analytical methods capable of providing reliable quantification of codeine metabolites alongside codeine concentrations are required. This article presents a validated method for simultaneous determination of codeine, codeine metabolites codeine-6-glucuronide (C6G), norcodeine and morphine, and morphine metabolites morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) in post-mortem whole blood, vitreous fluid, muscle, fat and brain tissue by high-performance liquid chromatography mass spectrometry. Samples were prepared by solid-phase extraction. The validated ranges were 1.5-300 ng/mL for codeine, norcodeine and morphine, and 23-4,600 ng/mL for C6G, M3G and M6G, with exceptions for norcodeine in muscle (3-300 ng/mL), morphine in muscle, fat and brain (3-300 ng/mL) and M6G in fat (46-4,600 ng/mL). Within-run and between-run accuracy (88.1-114.1%) and precision (CV 0.6-12.7%), matrix effects (CV 0.3-13.5%) and recovery (57.8-94.1%) were validated at two concentration levels; 3 and 150 ng/mL for codeine, norcodeine and morphine, and 46 and 2,300 ng/mL for C6G, M3G and M6G. Freeze-thaw and long-term stability (6 months at -80°C) was assessed, showing no significant changes in analyte concentrations (-12 to +8%). The method was applied in two authentic forensic autopsy cases implicating codeine in both therapeutic and presumably lethal concentration levels.

  10. Glucuronidation by UGT1A1 Is the Dominant Pathway of the Metabolic Disposition of Belinostat in Liver Cancer Patients

    PubMed Central

    Wang, Ling-Zhi; Ramírez, Jacqueline; Yeo, Winnie; Chan, Mei-Yi Michelle; Thuya, Win-Lwin; Lau, Jie-Ying Amelia; Wan, Seow-Ching; Wong, Andrea Li-Ann; Zee, Ying-Kiat; Lim, Robert; Lee, Soo-Chin; Ho, Paul C.; Lee, How-Sung; Chan, Anthony; Ansher, Sherry; Ratain, Mark J.; Goh, Boon-Cher

    2013-01-01

    Belinostat is a hydroxamate class HDAC inhibitor that has demonstrated activity in peripheral T-cell lymphoma and is undergoing clinical trials for non-hematologic malignancies. We studied the pharmacokinetics of belinostat in hepatocellular carcinoma patients to determine the main pathway of metabolism of belinostat. The pharmacokinetics of belinostat in liver cancer patients were characterized by rapid plasma clearance of belinostat with extensive metabolism with more than 4-fold greater relative systemic exposure of major metabolite, belinostat glucuronide than that of belinostat. There was significant interindividual variability of belinostat glucuronidation. The major pathway of metabolism involves UGT1A1-mediated glucuronidation and a good correlation has been identified between belinostat glucuronide formation and glucuronidation of known UGT1A1 substrates. In addition, liver microsomes harboring UGT1A1*28 alleles have lower glucuronidation activity for belinostat compared to those with wildtype UGT1A1. The main metabolic pathway of belinostat is through glucuronidation mediated primarily by UGT1A1, a highly polymorphic enzyme. The clinical significance of this finding remains to be determined. Trial Registration ClinicalTrials.gov NCT00321594 PMID:23382909

  11. Biotransformation of bisphenol AF to its major glucuronide metabolite reduces estrogenic activity.

    PubMed

    Li, Ming; Yang, Yunjia; Yang, Yi; Yin, Jie; Zhang, Jing; Feng, Yixing; Shao, Bing

    2013-01-01

    Bisphenol AF (BPAF), an endocrine disrupting chemical, can induce estrogenic activity through binding to estrogen receptor (ER). However, the metabolism of BPAF in vivo and the estrogenic activity of its metabolites remain unknown. In the present study, we identified four metabolites including BPAF diglucuronide, BPAF glucuronide (BPAF-G), BPAF glucuronide dehydrated and BPAF sulfate in the urine of Sprague-Dawley (SD) rats. BPAF-G was further characterized by nuclear magnetic resonance (NMR). After treatment with a single dose of BPAF, BPAF was metabolized rapidly to BPAF-G, as detected in the plasma of SD rats. Biotransformation of BPAF to BPAF-G was confirmed with human liver microsomes (HLM), and Vmax of glucuronidation for HLM was 11.6 nmol/min/mg. We also found that BPAF glucuronidation could be mediated through several human recombinant UDP-glucuronosyltransferases (UGTs) including UGT1A1, UGT1A3, UGT1A8, UGT1A9, UGT2B4, UGT2B7, UGT2B15 and UGT2B17, among which UGT2B7 showed the highest efficiency of glucuronidation. To explain the biological function of BPAF biotransformation, the estrogenic activities of BPAF and BPAF-G were evaluated in ER-positive breast cancer T47D and MCF7 cells. BPAF significantly stimulates ER-regulated gene expression and cell proliferation at the dose of 100 nM and 1 μM in breast cancer cells. However, BPAF-G did not show any induction of estrogenic activity at the same dosages, implying that formation of BPAF-G is a potential host defense mechanism against BPAF. Based on our study, biotransformation of BPAF to BPAF-G can eliminate BPAF-induced estrogenic activity, which is therefore considered as reducing the potential threat to human beings. PMID:24349450

  12. Luminal accumulation of newly synthesized morphine-3-glucuronide in rat liver microsomal vesicles.

    PubMed

    Révész, Katalin; Tóth, Blanka; Staines, Adam G; Coughtrie, Michael W H; Mandl, József; Csala, Miklós

    2013-01-01

    Morphine is converted to morphine 3-β-D-glucuronide (M3G) by the UDP-glucuronosyltransferase Ugt2b1 in the endoplasmic reticulum (ER) of rat liver. Because of its luminal localization, UGT activity requires UDP-glucuronate import and glucuronide export across the ER membrane. The former transport is generally considered to be rate limiting and to explain the latency of UGT activities in intact microsomal vesicles. However, some observations indicate that the release of bulky glucuronides, such as M3G, might also be rate limiting for glucuronidation. This assumption was tested by characterizing the transport of M3G and its distribution between the intra- and extravesicular spaces during synthesis in rat liver microsomes. The amount of vesicle-associated M3G was measured using rapid filtration and LC-MS measurement. Our results reveal a remarkable accumulation of newly synthesized M3G in the microsomal lumen above the equilibrium. The transport showed a linear concentration-dependence in a wide range (5-200 μM). Therefore, the build-up of high (about 20 μM) luminal M3G concentration could adjust the rate of release to that of synthesis (44.85 ± 4.08 pmol/min/mg protein) during the conjugation of 100 μM morphine. These data can explain earlier findings indicative of separate intracellular pools of M3G in rat liver. Accumulation of bulky glucuronides in the ER lumen might also play an important role in their targeting and in the control of biliary excretion.

  13. Glucuronidation of Monohydroxylated Warfarin Metabolites by Human Liver Microsomes and Human Recombinant UDP-Glucuronosyltransferases

    PubMed Central

    Zielinska, Agnieszka; Lichti, Cheryl F.; Bratton, Stacie; Mitchell, Neil C.; Gallus-Zawada, Anna; Le, Vi-Huyen; Finel, Moshe; Miller, Grover P.; Radominska-Pandya, Anna; Moran, Jeffery H.

    2008-01-01

    Our understanding of human phase II metabolic pathways which facilitate detoxification and excretion of warfarin (Coumadin) is limited. The goal of this study was to test the hypothesis that there are specific human hepatic and extrahepatic UDP-glucuronosyltransferase (UGT) isozymes, which are responsible for conjugating warfarin and hydroxylated metabolites of warfarin. Glucuronidation activity of human liver microsomes (HLMs) and eight human recombinant UGTs toward (R)- and (S)-warfarin, racemic warfarin, and major cytochrome P450 metabolites of warfarin (4′-, 6-, 7-, 8-, and 10-hydroxywarfarin) has been assessed. HLMs, UGT1A1, 1A8, 1A9, and 1A10 showed glucuronidation activity toward 4′-, 6-, 7-, and/or 8-hydroxywarfarin with Km values ranging from 59 to 480 μM and Vmax values ranging from 0.03 to 0.78 μM/min/mg protein. Tandem mass spectrometry studies and structure comparisons suggested glucuronidation was occurring at the C4′-,C6-, C7-, and C8-positions. Of the hepatic UGT isozymes tested, UGT1A9 exclusively metabolized 8-hydroxywarfarin, whereas UGT1A1 metabolized 6-, 7-, and 8-hydroxywarfarin. Studies with extrahepatic UGT isoforms showed that UGT1A8 metabolized 7- and 8-hydroxywarfarin and that UGT1A10 glucuronidated 4′-, 6-, 7-, and 8-hydroxywarfarin. UGT1A4, 1A6, 1A7, and 2B7 did not have activity with any substrate, and none of the UGT isozymes evaluated catalyzed reactions with (R)- and (S)-warfarin, racemic warfarin, or 10-hydroxywarfarin. This is the first study identifying and characterizing specific human UGT isozymes, which glucuronidate major cytochrome P450 metabolites of warfarin with similar metabolic rates known to be associated with warfarin metabolism. Continued characterization of these pathways may enhance our ability to reduce life-threatening and costly complications associated with warfarin therapy. PMID:17921187

  14. 21 CFR 184.1293 - Ethyl alcohol.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies are available from the National Academy Press, Box... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Ethyl alcohol. 184.1293 Section 184.1293 Food and....1293 Ethyl alcohol. (a) Ethyl alcohol (ethanol) is the chemical C2H5OH. (b) The ingredient meets...

  15. 21 CFR 184.1293 - Ethyl alcohol.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... is incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies are... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Ethyl alcohol. 184.1293 Section 184.1293 Food and... Substances Affirmed as GRAS § 184.1293 Ethyl alcohol. (a) Ethyl alcohol (ethanol) is the chemical C2H5OH....

  16. 21 CFR 184.1293 - Ethyl alcohol.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... is incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies are... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Ethyl alcohol. 184.1293 Section 184.1293 Food and... Substances Affirmed as GRAS § 184.1293 Ethyl alcohol. (a) Ethyl alcohol (ethanol) is the chemical C2H5OH....

  17. 21 CFR 184.1293 - Ethyl alcohol.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... is incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies are... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Ethyl alcohol. 184.1293 Section 184.1293 Food and... Substances Affirmed as GRAS § 184.1293 Ethyl alcohol. (a) Ethyl alcohol (ethanol) is the chemical C2H5OH....

  18. 21 CFR 184.1293 - Ethyl alcohol.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... is incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies are... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Ethyl alcohol. 184.1293 Section 184.1293 Food and... Substances Affirmed as GRAS § 184.1293 Ethyl alcohol. (a) Ethyl alcohol (ethanol) is the chemical C2H5OH....

  19. The Human UGT1A3 Enzyme Conjugates Norursodeoxycholic Acid into a C23-ester Glucuronide in the Liver*

    PubMed Central

    Trottier, Jocelyn; El Husseini, Diala; Perreault, Martin; Pâquet, Sophie; Caron, Patrick; Bourassa, Sylvie; Verreault, Mélanie; Inaba, Ted T.; Poirier, Guy G.; Bélanger, Alain; Guillemette, Chantal; Trauner, Michael; Barbier, Olivier

    2010-01-01

    Norursodeoxycholic acid (norUDCA) exhibits efficient anti-cholestatic properties in an animal model of sclerosing cholangitis. norUDCA is eliminated as a C23-ester glucuronide (norUDCA-23G) in humans. The present study aimed at identifying the human UDP-glucuronosyltransferase (UGT) enzyme(s) involved in hepatic norUDCA glucuronidation and at evaluating the consequences of single nucleotide polymorphisms in the coding region of UGT genes on norUDCA-23G formation. The effects of norUDCA on the formation of the cholestatic lithocholic acid-glucuronide derivative and of rifampicin on hepatic norUDCA glucuronidation were also explored. In vitro glucuronidation assays were performed with microsomes from human tissues (liver and intestine) and HEK293 cells expressing human UGT enzymes and variant allozymes. UGT1A3 was identified as the major hepatic UGT enzyme catalyzing the formation of norUDCA-23G. Correlation studies using samples from a human liver bank (n = 16) indicated that the level of UGT1A3 protein is a strong determinant of in vitro norUDCA glucuronidation. Analyses of the norUDCA-conjugating activity by 11 UGT1A3 variant allozymes identified three phenotypes with high, low, and intermediate capacity. norUDCA is also identified as a competitive inhibitor for the hepatic formation of the pro-cholestatic lithocholic acid-glucuronide derivative, whereas norUDCA glucuronidation is weakly stimulated by rifampicin. This study identifies human UGT1A3 as the major enzyme for the hepatic norUDCA glucuronidation and supports that some coding polymorphisms affecting the conjugating activity of UGT1A3 in vitro may alter the pharmacokinetic properties of norUDCA in cholestasis treatment. PMID:19889628

  1. The human UGT1A3 enzyme conjugates norursodeoxycholic acid into a C23-ester glucuronide in the liver.

    PubMed

    Trottier, Jocelyn; El Husseini, Diala; Perreault, Martin; Pâquet, Sophie; Caron, Patrick; Bourassa, Sylvie; Verreault, Mélanie; Inaba, Ted T; Poirier, Guy G; Bélanger, Alain; Guillemette, Chantal; Trauner, Michael; Barbier, Olivier

    2010-01-01

    Norursodeoxycholic acid (norUDCA) exhibits efficient anti-cholestatic properties in an animal model of sclerosing cholangitis. norUDCA is eliminated as a C(23)-ester glucuronide (norUDCA-23G) in humans. The present study aimed at identifying the human UDP-glucuronosyltransferase (UGT) enzyme(s) involved in hepatic norUDCA glucuronidation and at evaluating the consequences of single nucleotide polymorphisms in the coding region of UGT genes on norUDCA-23G formation. The effects of norUDCA on the formation of the cholestatic lithocholic acid-glucuronide derivative and of rifampicin on hepatic norUDCA glucuronidation were also explored. In vitro glucuronidation assays were performed with microsomes from human tissues (liver and intestine) and HEK293 cells expressing human UGT enzymes and variant allozymes. UGT1A3 was identified as the major hepatic UGT enzyme catalyzing the formation of norUDCA-23G. Correlation studies using samples from a human liver bank (n = 16) indicated that the level of UGT1A3 protein is a strong determinant of in vitro norUDCA glucuronidation. Analyses of the norUDCA-conjugating activity by 11 UGT1A3 variant allozymes identified three phenotypes with high, low, and intermediate capacity. norUDCA is also identified as a competitive inhibitor for the hepatic formation of the pro-cholestatic lithocholic acid-glucuronide derivative, whereas norUDCA glucuronidation is weakly stimulated by rifampicin. This study identifies human UGT1A3 as the major enzyme for the hepatic norUDCA glucuronidation and supports that some coding polymorphisms affecting the conjugating activity of UGT1A3 in vitro may alter the pharmacokinetic properties of norUDCA in cholestasis treatment.

  2. Simultaneous determination of sulfation and glucuronidation of flavones in FVB mouse intestine in vitro and in vivo.

    PubMed

    Fan, Yanfang; Tang, Lan; Zhou, Juan; Feng, Qian; Xia, Bijun; Liu, Zhongqiu

    2013-04-01

    Glucuronidation and sulfation are the two major phase II metabolic pathways for flavones, natural compounds that hold great potential for improving human health. We investigated the positional preference for sulfation and glucuronidation of seven structurally similar flavones in vitro and in situ. An FVB mouse intestinal perfusion model was used in addition to three small intestine S9 fractions catalyzing sulfation only (Sult enzymes), glucuronidation only (Ugt enzymes) or both (Sult and Ugt enzymes). In both the single and co-reaction S9 systems, flavones containing 7-OH groups were conjugated only at 7-OH despite the presence of other hydroxyl groups, and 7-OH glucuronidation was faster than sulfation (P <0.05). The sulfation rate was enhanced in the Sult-Ugt co-reaction system, while glucuronidation was usually unchanged by the presence of Sult. In the intestinal perfusate, sulfation patterns were the same in the small intestine and colon, and the excretion rate of 7-O-sulfate was the fastest or second fastest. The excretion of 7-O-glucuronidates was faster in small intestine (P < 0.05) than in colon. The S9-mediated sulfation rates of the different flavones were significantly correlated with the excretion rates of the same flavones from perfused intestine. In conclusion, flavone glucuronidation and sulfation rates were sensitive to minor changes in molecular structure. In intestinal S9 fractions, both Ugts and Sults preferentially catalyzed reactions at 7-OH. The sulfation rate was significantly enhanced by simultaneous glucuronidation, but glucuronidation was unaltered by sulfation. Sulfation rates in mouse S9 fractions correlated with sulfation rates in perfused intestine.

  3. Alternative sampling strategies for the assessment of alcohol intake of living persons.

    PubMed

    Kummer, Natalie; Lambert, Willy E E; Samyn, Nele; Stove, Christophe P

    2016-09-01

    Monitoring of alcohol consumption by living persons takes place in various contexts, amongst which workplace drug testing, driving under the influence of alcohol, driving licence regranting programs, alcohol withdrawal treatment, diagnosis of acute intoxication or fetal alcohol ingestion. The matrices that are mostly used today include blood, breath and urine. The aim of this review is to present alternative sampling strategies that allow monitoring of the alcohol consumption in living subjects. Ethanol itself, indirect (carbohydrate deficient transferrin, CDT%) as well as direct biomarkers (ethyl glucuronide, EtG; ethyl sulphate, EtS; fatty acid ethyl esters, FAEEs and phosphatidylethanol species, PEths) of ethanol consumption will be considered. This review covers dried blood spots (CDT%, EtG/EtS, PEths), dried urine spots (EtG/EtS), sweat and skin surface lipids (ethanol, EtG, FAEEs), oral fluid (ethanol, EtG), exhaled breath (PEths), hair (EtG, FAEEs), nail (EtG), meconium (EtG/EtS, FAEEs), umbilical cord and placenta (EtG/EtS and PEth 16:0/18:1). Main results, issues and considerations specific to each matrix are reported. Details about sample preparation and analytical methods are not within the scope of this review.

  4. Alternative sampling strategies for the assessment of alcohol intake of living persons.

    PubMed

    Kummer, Natalie; Lambert, Willy E E; Samyn, Nele; Stove, Christophe P

    2016-09-01

    Monitoring of alcohol consumption by living persons takes place in various contexts, amongst which workplace drug testing, driving under the influence of alcohol, driving licence regranting programs, alcohol withdrawal treatment, diagnosis of acute intoxication or fetal alcohol ingestion. The matrices that are mostly used today include blood, breath and urine. The aim of this review is to present alternative sampling strategies that allow monitoring of the alcohol consumption in living subjects. Ethanol itself, indirect (carbohydrate deficient transferrin, CDT%) as well as direct biomarkers (ethyl glucuronide, EtG; ethyl sulphate, EtS; fatty acid ethyl esters, FAEEs and phosphatidylethanol species, PEths) of ethanol consumption will be considered. This review covers dried blood spots (CDT%, EtG/EtS, PEths), dried urine spots (EtG/EtS), sweat and skin surface lipids (ethanol, EtG, FAEEs), oral fluid (ethanol, EtG), exhaled breath (PEths), hair (EtG, FAEEs), nail (EtG), meconium (EtG/EtS, FAEEs), umbilical cord and placenta (EtG/EtS and PEth 16:0/18:1). Main results, issues and considerations specific to each matrix are reported. Details about sample preparation and analytical methods are not within the scope of this review. PMID:27208822

  5. Simultaneous Quantification of Buprenorphine, Norbuprenorphine, Buprenorphine-Glucuronide and Norbuprenorphine-Glucuronide in Human Umbilical Cord by Liquid Chromatography Tandem Mass Spectrometry

    PubMed Central

    Concheiro, Marta; Shakleya, Diaa M.; Huestis, Marilyn A.

    2009-01-01

    A LCMS method was developed and validated for the simultaneous determination of buprenorphine (BUP), norbuprenorphine (NBUP), buprenorphine glucuronide (BUP-Gluc) and norbuprenorphine glucuronide (NBUP-Gluc) in human umbilical cord. Quantification was achieved by selected ion monitoring of precursor ions m/z 468.4 for BUP; 414.3 for NBUP; 644.4 for BUP-Gluc and 590 for NBUP-Gluc. BUP and NBUP were identified by MS2, with m/z 396, 414 and 426 for BUP, and m/z 340, 364 and 382 for NBUP. Glucuronide conjugates were identified by MS3 with m/z 396 and 414 for BUP-Gluc and m/z 340 and 382 for NBUP-Gluc. The assay was linear 1–50 ng/g. Intra, inter-day and total assay imprecision (%RSD) were <14.5%, and analytical recovery ranged from 94.1% to 112.3% for all analytes. Extraction efficiencies were >66.3%, and process efficiency >73.4%. Matrix effect ranged, in absolute value, from 3.7% to 27.4% (CV<21.8%, n=8). The method was selective with no endogenous or exogenous interferences from 41 compounds evaluated. Sensitivity was high with limits of detection of 0.8 ng/g. In order to prove method applicability, an authentic umbilical cord obtained from an opioid-dependent pregnant woman receiving BUP pharmacotherapy was analyzed. Interestingly, BUP was not detected but concentrations of the other metabolites were NBUP-Gluc 13.4 ng/g, BUP-Gluc 3.5 ng/g and NBUP 1.2 ng/g. PMID:19406593

  6. Post-mortem levels and tissue distribution of codeine, codeine-6-glucuronide, norcodeine, morphine and morphine glucuronides in a series of codeine-related deaths.

    PubMed

    Frost, Joachim; Løkken, Trine Nordgård; Helland, Arne; Nordrum, Ivar Skjåk; Slørdal, Lars

    2016-05-01

    This article presents levels and tissue distribution of codeine, codeine-6-glucuronide (C6G), norcodeine, morphine and the morphine metabolites morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) in post-mortem blood (peripheral and heart blood), vitreous fluid, muscle, fat and brain tissue in a series of 23 codeine-related fatalities. CYP2D6 genotype is also determined and taken into account. Quantification of codeine, C6G, norcodeine, morphine, M3G and M6G was performed with a validated solid phase extraction LC-MS method. The series comprise 19 deaths (83%) attributed to mixed drug intoxication, 4 deaths (17%) attributed to other causes of death, and no cases of unambiguous monointoxication with codeine. The typical peripheral blood concentration pattern in individual cases was C6G≫codeine≫norcodeine>morphine, and M3G>M6G>morphine. In matrices other than blood, the concentration pattern was similar, although in a less systematic fashion. Measured concentrations were generally lower in matrices other than blood, especially in brain and fat, and in particular for the glucuronides (C6G, M3G and M6G) and, to some extent, morphine. In brain tissue, the presumed active moieties morphine and M6G were both below the LLOQ (0.0080mg/L and 0.058mg/L, respectively) in a majority of cases. In general, there was a large variability in both measured concentrations and calculated blood/tissue concentration ratios. There was also a large variability in calculated ratios of morphine to codeine, C6G to codeine and norcodeine to codeine in all matrices, and CYP2D6 genotype was not a reliable predictor of these ratios. The different blood/tissue concentration ratios showed no systematic relationship with the post-mortem interval. No coherent degradation or formation patterns for codeine, morphine, M3G and M6G were observed upon reanalysis in peripheral blood after storage.

  7. 27 CFR 21.108 - Ethyl ether.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2012-04-01 2012-04-01 false Ethyl ether. 21.108 Section 21.108 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specifications for Denaturants § 21.108 Ethyl ether. (a) Odor. Characteristic odor....

  8. 27 CFR 21.108 - Ethyl ether.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2014-04-01 2014-04-01 false Ethyl ether. 21.108 Section 21.108 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY ALCOHOL FORMULAS FOR DENATURED ALCOHOL AND RUM Specifications for Denaturants § 21.108 Ethyl ether. (a) Odor. Characteristic odor....

  9. 27 CFR 21.108 - Ethyl ether.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2013-04-01 2013-04-01 false Ethyl ether. 21.108 Section 21.108 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY ALCOHOL FORMULAS FOR DENATURED ALCOHOL AND RUM Specifications for Denaturants § 21.108 Ethyl ether. (a) Odor. Characteristic odor....

  10. 21 CFR 573.420 - Ethyl cellulose.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ..., FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.420 Ethyl cellulose. The food additive ethyl cellulose may be safely used in animal feed in accordance with the following prescribed conditions: (a) The food additive is a cellulose ether...

  11. 21 CFR 573.420 - Ethyl cellulose.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ..., FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.420 Ethyl cellulose. The food additive ethyl cellulose may be safely used in animal feed in accordance with the following prescribed conditions: (a) The food additive is a cellulose ether...

  12. 21 CFR 573.420 - Ethyl cellulose.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ..., FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.420 Ethyl cellulose. The food additive ethyl cellulose may be safely used in animal feed in accordance with the following prescribed conditions: (a) The food additive is a cellulose ether...

  13. 21 CFR 573.420 - Ethyl cellulose.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ..., FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.420 Ethyl cellulose. The food additive ethyl cellulose may be safely used in animal feed in accordance with the following prescribed conditions: (a) The food additive is a cellulose ether...

  14. 21 CFR 573.420 - Ethyl cellulose.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ..., FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.420 Ethyl cellulose. The food additive ethyl cellulose may be safely used in animal feed in accordance with the following prescribed conditions: (a) The food additive is a cellulose ether...

  15. 27 CFR 21.108 - Ethyl ether.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Ethyl ether. 21.108 Section 21.108 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT....108 Ethyl ether. (a) Odor. Characteristic odor. (b) Specific gravity at 15.56 °/15.56 °C. Not...

  16. 27 CFR 21.108 - Ethyl ether.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Ethyl ether. 21.108 Section 21.108 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT....108 Ethyl ether. (a) Odor. Characteristic odor. (b) Specific gravity at 15.56 °/15.56 °C. Not...

  17. Glucuronidation of estrone and 16α-hydroxyestrone by human UGT enzymes: The key roles of UGT1A10 and UGT2B7.

    PubMed

    Kallionpää, Roope A; Järvinen, Erkka; Finel, Moshe

    2015-11-01

    The glucuronidation of estrone and 16α-hydroxyestrone by recombinant human UDP-glucuronosyltransferase enzymes (UGTs) of subfamilies 1A, 2A and 2B was studied. Microsomes from human liver and small intestine were also tested for the glucuronidation of these two estrogens. The results revealed that UGT1A10 is by far the most active enzyme in estrone glucuronidation. UGT1A10 also exhibited high rate of 16α-hydroxyestrone conjugation at the 3-OH, whereas UGT2B7 catalyzed its glucuronidation at high rates at the 16-OH. Human liver microsomes exhibited high rates of 16α-hydroxyestrone-16-glucuronide formation, but very low formation rates of either 16α-hydroxyestrone-3-glucuronide or estrone glucuronide. On the other hand, human intestine microsomes catalyzed the formation of all these 3 different glucuronides at high rates. Kinetic analyses revealed very low Km value for 16α-hydroxyestrone glucuronidation by UGT2B7, below 4 μM, suggesting higher affinity than commonly found among UGTs and their substrates. In further studies with UGT1A10, mutant F93G exhibited increased glucuronidation rates of 16α-hydroxyestrone, but not estrone, whereas mutations in F90 did not reveal any activity with either estrogen. Taken together, the results of this study significantly expand our understanding on the metabolism of estrogens and their interactions with the human UGTs. PMID:26220143

  18. Identification and characterization of oxymetazoline glucuronidation in human liver microsomes: evidence for the involvement of UGT1A9.

    PubMed

    Mahajan, Mukesh K; Uttamsingh, Vinita; Gan, Liang-Shang; Leduc, Barbara; Williams, David A

    2011-02-01

    The incubation of oxymetazoline, a nonprescription nasal decongestant, with human liver microsomes (HLMs) supplemented with uridine-5-diphosphoglucuronic acid (UDPGA) generated glucuronide metabolite as observed by LC/MS/MS. The uridine glucuronosyltransferases (UGTs) responsible for the O-glucuronidation of oxymetazoline remain thus far unidentified. The glucuronide formed in HLMs was identified by LC/MS/MS and characterized by one- and two-dimensional NMR to be the β-O-glucuronide of oxymetazoline. UGT screening with expressed UGTs identified UGT1A9 as the single UGT isoform catalyzing O-glucuronidation of oxymetazoline. Oxymetazoline O-glucuronidation by using HLMs was best fitted to the allosteric sigmoidal model. The derived S(50) and V(max) values were 2.42 ± 0.40 mM and 8.69 ± 0.58 pmole/(min mg of protein), respectively, and maximum clearance (CL(max)) was 3.61 L/min/mg. Oxymetazoline O-glucuronidation by using expressed UGT1A9 was best fitted to the substrate inhibition model. The derived K(m) and V(max) values were 2.53 ± 1.03 mM and 54.18 ± 16.92 pmole/(min mg of protein), respectively, and intrinsic clearance (CL(int)) was 21.41 L/(min mg). Our studies indicate that oxymetazoline is not glucuronidated at its nanomolar intranasal dose and thus is eliminated unchanged, because UGT1A9 would only contribute to its elimination at the toxic plasma concentrations.

  19. Hormonal monitoring of early pregnancy by a direct radioimmunoassay of steroid glucuronides in first morning urine

    SciTech Connect

    Mendizabal, A.F.; Quiroga, S.; Farinati, Z.; Lahoz, M.; Nagle, C.

    1984-11-01

    The usefulness of the direct 4-hour radioimmunoassay of estriol-16-glucuronide (E/sub 3/G) and pregnanediol-3-glucuronide (P/sub 2/G) in first morning urine (FMU) for establishing a prognosis of the early pregnancy outcome was evaluated in 106 patients that became pregnant. Microaliquots of FMU were serially assayed from day 3 of the conception cycle until day 80 of pregnancy. The E/sub 3/G and P/sub 2/G profiles of 19 pregnancies which terminated in spontaneous abortion with either a diagnosis of the blighted ovum syndrome (n = 11) or presumption of a corpus luteum/trophoblast failure (n = 8) have been compared with those of clinically normal pregnancies (n = 87). Normal pregnancies displayed typical patterns of E/sub 3/G and P/sub 2/G development, while variations were observed in abortive events that reflected changes of the fetoplacental unit.

  20. Soy isoflavone metabolism in cats compared with other species: urinary metabolite concentrations and glucuronidation by liver microsomes.

    PubMed

    Redmon, Joanna M; Shrestha, Binu; Cerundolo, Rosario; Court, Michael H

    2016-01-01

    1. Soybean is a common source of protein in many pet foods. Slow glucuronidation of soy-derived isoflavones in cats has been hypothesized to result in accumulation with adverse health consequences. Here, we evaluated species' differences in soy isoflavone glucuronidation using urine samples from cats and dogs fed a soy-based diet and liver microsomes from cats compared with microsomes from 12 other species. 2. Significant concentrations of conjugated (but not unconjugated) genistein, daidzein and glycitein, and the gut microbiome metabolites, dihydrogenistein and dihydrodaidzein, were found in cat and dog urine samples. Substantial amounts of conjugated equol were also found in cat urine but not in dog urine. 3. β-Glucuronidase treatment showed that all these compounds were significantly glucuronidated in dog urine while only daidzein (11%) and glycitein (37%) showed any glucuronidation in cat urine suggesting that alternate metabolic pathways including sulfation predominate in cats. 4. Glucuronidation rates of genistein, daidzein and equol by cat livers were consistently ranked within the lowest 3 out of 13 species' livers evaluated. Ferret and mongoose livers were also ranked in the lowest four species. 5. Our results demonstrate that glucuronidation is a minor pathway for soy isoflavone metabolism in cats compared with most other species.

  1. Soy isoflavone metabolism in cats compared with other species: urinary metabolite concentrations and glucuronidation by liver microsomes.

    PubMed

    Redmon, Joanna M; Shrestha, Binu; Cerundolo, Rosario; Court, Michael H

    2016-01-01

    1. Soybean is a common source of protein in many pet foods. Slow glucuronidation of soy-derived isoflavones in cats has been hypothesized to result in accumulation with adverse health consequences. Here, we evaluated species' differences in soy isoflavone glucuronidation using urine samples from cats and dogs fed a soy-based diet and liver microsomes from cats compared with microsomes from 12 other species. 2. Significant concentrations of conjugated (but not unconjugated) genistein, daidzein and glycitein, and the gut microbiome metabolites, dihydrogenistein and dihydrodaidzein, were found in cat and dog urine samples. Substantial amounts of conjugated equol were also found in cat urine but not in dog urine. 3. β-Glucuronidase treatment showed that all these compounds were significantly glucuronidated in dog urine while only daidzein (11%) and glycitein (37%) showed any glucuronidation in cat urine suggesting that alternate metabolic pathways including sulfation predominate in cats. 4. Glucuronidation rates of genistein, daidzein and equol by cat livers were consistently ranked within the lowest 3 out of 13 species' livers evaluated. Ferret and mongoose livers were also ranked in the lowest four species. 5. Our results demonstrate that glucuronidation is a minor pathway for soy isoflavone metabolism in cats compared with most other species. PMID:26366946

  2. Synthesis and Evaluation of the Anti-Oxidant Capacity of Curcumin Glucuronides, the Major Curcumin Metabolites.

    PubMed

    Choudhury, Ambar K; Raja, Suganya; Mahapatra, Sanjata; Nagabhushanam, Kalyanam; Majeed, Muhammed

    2015-01-01

    Curcumin metabolites namely curcumin monoglucuronide and curcumin diglucuronide were synthesized using an alternative synthetic approach. The anti-oxidant potential of these curcumin glucuronides was compared with that of curcumin using DPPH scavenging method and Oxygen Radical Absorbance Capacity (ORAC) assay. The results show that curcumin monoglucuronide exhibits 10 fold less anti-oxidant activity (DPPH method) and the anti-oxidant capacity of curcumin diglucuronide is highly attenuated compared to the anti-oxidant activity of curcumin. PMID:26783957

  3. Sequestered endoplasmic reticulum space for sequential metabolism of salicylamide. Coupling of hydroxylation and glucuronidation.

    PubMed

    Tirona, R G; Pang, K S

    1996-08-01

    The metabolic disposition of simultaneously delivered [14C]salicylamide (SAM) (100 microM) and a tracer concentration of its hydroxylated metabolite [3H]gentisamide (GAM) was studied with single-pass followed by recirculating rat liver perfusion (10 ml/min). The use of dual radiolabeling of precursor-product pairs in single-pass and recirculating perfusions allowed for characterization of the differential metabolism of preformed [3H]GAM and formed [14C]GAM, which arose in situ in the liver with [14C]SAM single-pass perfusion, and the behavior of circulating [14C]GAM, which behaved as a preformed species in recirculation. In both modes of perfusion, [14C]SAM was mainly sequentially metabolized to [14C]GAM-5-glucuronide, whereas [3H]GAM predominantly formed [3H]GAM-5-sulfate. The steady-state and time-averaged clearances of SAM were identical and approached the value of flow, yielding a high hepatic extraction ratio (E = 0.98). The apparent extraction ratio of formed GAM [E(mi) = 0.96] was greater than that of the preformed species [E(pmi) approximately 0.7]. Because the coupling of (SAM) oxidation and (GAM) glucuronidation was a plausible explanation for the observation, a novel physiological pharmacokinetic model was developed to interpret the data. In this model, the liver was divided into three zonal units, within which acinar distribution of enzymatic activities was considered, namely periportal sulfation, evenly distributed glucuronidation, and perivenous hydroxylation. Each zonal region was subdivided into extracellular, cytosolic, and endoplasmic reticulum compartments, with cytosolic (sulfotransferases) and microsomal (cytochromes P-450 and UDP-glucuronosyltransferase) enzymes being segregated intracellularly into the cytosolic compartment and endoplasmic reticulum compartment, respectively. The simulations provided a good prediction of the present experimental data as well as previously obtained data with increasing SAM concentration and retrograde flow and

  4. Synthesis and Evaluation of the Anti-Oxidant Capacity of Curcumin Glucuronides, the Major Curcumin Metabolites

    PubMed Central

    Choudhury, Ambar K.; Raja, Suganya; Mahapatra, Sanjata; Nagabhushanam, Kalyanam; Majeed, Muhammed

    2015-01-01

    Curcumin metabolites namely curcumin monoglucuronide and curcumin diglucuronide were synthesized using an alternative synthetic approach. The anti-oxidant potential of these curcumin glucuronides was compared with that of curcumin using DPPH scavenging method and Oxygen Radical Absorbance Capacity (ORAC) assay. The results show that curcumin monoglucuronide exhibits 10 fold less anti-oxidant activity (DPPH method) and the anti-oxidant capacity of curcumin diglucuronide is highly attenuated compared to the anti-oxidant activity of curcumin. PMID:26783957

  5. IN VITRO GLUCURONIDATION OF APREPITANT: A MODERATE INHIBITOR OF UGT2B7

    PubMed Central

    House, Larry; Ramirez, Jacqueline; Seminerio, Michael; Mirkov, Snezana; Ratain, Mark J.

    2016-01-01

    Aprepitant, an oral antiemetic, commonly used in the prevention of chemotherapy-induced nausea and vomiting, is primarily metabolized by CYP3A4. Aprepitant glucuronidation has yet to be evaluated in humans. The contribution of human UDP-glucuronosyltransferase (UGT) isoforms to the metabolism of aprepitant was investigated by performing kinetic studies, inhibition studies, and correlation analyses. In addition, aprepitant was evaluated as an inhibitor of UGTs.Glucuronidation of aprepitant was catalyzed by UGT1A4 (82%), UGT1A3 (12%), and UGT1A8 (6%) and Kms were 161.6 ± 15.6 µM, 69.4 ± 1.9 µM, and 197.1 ± 28.2 µM, respectively. Aprepitant glucuronidation was significantly correlated with both UGT1A4 substrates anastrazole and imipramine (rs = 0.77, P < 0.0001 for both substrates; n = 44), and with the UGT1A3 substrate thyroxine (rs = 0.58, P < 0.0001; n = 44).We found aprepitant to be a moderate inhibitor of UGT2B7 with a Ki of ~10 µM for 4-MU, morphine, and zidovudine. Our results suggest aprepitant can alter clearance of drugs primarily eliminated by UGT2B7. Given the likelihood for first-pass metabolism by intestinal UGT2B7, this is of particular concern for oral aprepitant co-administered with oral substrates of UGT2B7, such as zidovudine and morphine. PMID:26053558

  6. Identification of a hydroxylamine glucuronide metabolite of an oral hypoglycemic agent.

    PubMed

    Miller, Randall R; Doss, George A; Stearns, Ralph A

    2004-02-01

    Glucuronides of piperazine hydroxylamines are rarely reported in the literature, and even more rarely are their structures unambiguously identified. One major metabolite was detected by liquid chromatography/mass spectrometry-radioactivity in urine from monkeys treated with the aryl piperazine oral hypoglycemic agent 9-[(1S,2R)-2-fluoro-1-methylpropyl]-2-methoxy-6-(1-piperazinyl) purine hydrochloride (1). The mass spectrum of this metabolite indicated that it was both monooxygenated and glucuronidated on the piperazine ring. Possible structures included the N- or O-glucuronic acid conjugates of a carbinolamine, hydroxylamine, or N-oxide. Treatment with beta-glucuronidase gave a monooxygenated derivative of the parent compound. 1H NMR analysis of either the glucuronic acid conjugate or the monooxygenated product provided insufficient evidence to unambiguously determine their structures. Incubation of 1 with pig liver microsomes resulted in formation of the same monooxygenated derivative derived from beta-glucuronidase treatment of the glucuronide metabolite. This in vitro system was used to generate sufficient material for analysis by 13C NMR, and the metabolite was identified as a hydroxylamine derivative 2. Incubation of the hydroxylamine with monkey liver microsomes and uridine diphospho-5'-glucuronic acid gave the same glucuronic acid conjugate as that observed in monkey urine. 13C NMR analysis of this biosynthetic product led to its unequivocal structure assignment as the O-glucuronic acid conjugate of the hydroxylamine 3.

  7. Characterization of oligomeric procyanidins and identification of quercetin glucuronide from lotus ( Nelumbo nucifera Gaertn.) seedpod.

    PubMed

    Xiao, Jun-Song; Xie, Bi-Jun; Cao, Yan-Ping; Wu, Hua; Sun, Zhi-Da; Xiao, Di

    2012-03-21

    Procyanidins are a class of polyphenols in the plant kingdom. Lotus ( Nelumbo nucifera Gaertn.) seedpods, the inedible part of lotus and a byproduct during the production of lotus seeds, were found to be a new source rich in procyanidins. Detailed information about oligomeric procyanidins in lotus seedpods remains unknown. In this study, lotus seedpods were extracted using 60% aqueous methanol and characterized with phloroglucinolysis and liquid chromatography (mass spectrometry with an electrospray ionization source). The results indicate that the oligomeric and polymeric fraction had a mean degree of polymerization of 3.2 and 15.4, respectively, and consisted of (+)-catechin (m/z 289), gallocatechin or epigallocatechin (m/z 305), quercetin glycoside (m/z 463), quercetin glucuronide (m/z 477), procyanidin dimers (m/z 577.1), proanthocyanidin dimer gallate (m/z 593.3), prodelphinidin dimers (m/z 609.1), procyanidin trimers (m/z 865.1), etc. Quercetin glucuronide was further purified using flash chromatography and identified as quercetin-3-O-β-glucuronide by determining its exact mass using ion-trap time-of-flight mass spectrometry and ¹H and ¹³C nuclear magnetic resonance, ¹H-detected heteronuclear single-quantum coherence, and ¹H-detected heteronuclear multiple-bond correlation analyses.

  8. Simultaneous Quantification of Free and Glucuronidated Cannabinoids in Human Urine by Liquid Chromatography-Tandem Mass Spectrometry

    PubMed Central

    Scheidweiler, Karl B.; Desrosiers, Nathalie A.; Huestis, Marilyn A.

    2012-01-01

    Background Cannabis is the most commonly abused drug of abuse and is commonly quantified during urine drug testing. We conducted a controlled drug administration studies investigating efficacy of urinary cannabinoid glucuronide metabolites for documenting recency of cannabis intake and for determining stability of urinary cannabinoids. Methods A liquid chromatography tandem mass spectrometry method was developed and validated quantifying Δ9-tetrahydrocannabinol (THC), 11-hydroxy-THC (11-OH-THC), 11-nor-9-carboxy-THC (THCCOOH), cannabidiol, cannabinol, THC-glucuronide and THCCOOH-glucuronide in 0.5 ml human urine via supported-liquid extraction. Chromatography was performed on an Ultra Biphenyl column with a gradient of 10 mmol/l ammonium acetate, pH 6.15 and 15% methanol in acetonitrile at 0. 4ml/min. Analytes were monitored by positive and negative mode electrospray ionization and multiple reaction monitoring mass spectrometry. Results Linear ranges were 0.5–50 ng/ml for THC-glucuronide, 1–100 ng/ml for THCCOOH, 11-OH-THC and cannabidiol, 2–100 ng/ml for THC and cannabinol, and 5–500 ng/ml for THCCOOH-glucuronide (R2>0.99). Mean extraction efficiencies were 34–73% with analytical recovery (bias) 80.5–118.0% and total imprecision 3.0–10.2% coefficient of variation. Conclusion This method simultaneously quantifies urinary cannabinoids and phase II glucuronide metabolites, and enables evaluation of urinary cannabinoid glucuronides for documenting recency of cannabis intake and cannabinoid stability. The assay is applicable for routine urine cannabinoid testing. PMID:22771478

  9. Process for the preparation of ethyl benzene

    DOEpatents

    Smith, L.A. Jr.; Arganbright, R.P.; Hearn, D.

    1995-12-19

    Ethyl benzene is produced in a catalyst bed under 0.25 to 50 atmospheres of pressure and at temperatures in the range of 50 C to 300 C, using as the catalyst a mole sieve characterized as acidic by feeding ethylene to the catalyst bed while benzene is conveniently added through the reflux to result in a molar excess present in the reactor to that required to react with ethylene, thereby reacting substantially all of the ethylene and recovering benzene as the principal overhead and ethyl benzene and diethyl benzene in the bottoms. The bottoms are fractionated, the ethyl benzene recovered and the bottoms are contacted with benzene in the liquid phase in a fixed bed straight pass reactor under conditions to transalkylate the benzene thereby converting most of the diethyl benzene to ethyl benzene which is again separated and recovered. 2 figs.

  10. Process for the preparation of ethyl benzene

    DOEpatents

    Smith, Jr., Lawrence A.; Arganbright, Robert P.; Hearn, Dennis

    1995-01-01

    Ethyl benzene is produced in a catalyst bed under 0.25 to 50 atmospheres of pressure and at temperatures in the range of 50.degree. C. to 300.degree. C., using as the catalyst a mole sieve characterized as acidic by feeding ethylene to the catalyst bed while benzene is conveniently added through the reflux to result in a molar excess present in the reactor to that required to react with ethylene, thereby reacting substantially all of the ethylene and recovering benzene as the principal overhead and ethyl benzene and diethyl benzene in the bottoms. The bottoms are fractionated, the ethyl benzene recovered and the bottoms are contacted with benzene in the liquid phase in a fixed bed straight pass reactor under conditions to transalkylate the benzene thereby converting most of the diethyl benzene to ethyl benzene which is again separated and recovered.

  11. Day-to-day variations during clinical drug monitoring of morphine, morphine-3-glucuronide and morphine-6-glucuronide serum concentrations in cancer patients. A prospective observational study

    PubMed Central

    Klepstad, Pål; Hilton, Priscilla; Moen, Jorunn; Kaasa, Stein; Borchgrevink, Petter C; Zahlsen, Kolbjørn; Dale, Ola

    2004-01-01

    Background The feasibility of drug monitoring of serum concentrations of morphine, morphine-6-glucuronide (M6G) and morphine-3-glucuronide (M3G) during chronic morphine therapy is not established. One important factor relevant to drug monitoring is to what extent morphine, M6G and M3G serum concentrations fluctuate during stable morphine treatment. Methods We included twenty-nine patients admitted to a palliative care unit receiving oral morphine (n = 19) or continuous subcutaneous (sc) morphine infusions (n = 10). Serum concentrations of morphine, M6G and M3G were obtained at the same time on four consecutive days. If readmitted, the patients were followed for another trial period. Day-to-day variations in serum concentrations and ratios were determined by estimating the percent coefficient of variation (CV = (mean/SD) ×100). Results The patients' median morphine doses were 90 (range; 20–1460) mg/24 h and 135 (range; 30–440) mg/24 h during oral and sc administration, respectively. Intraindividual fluctuations of serum concentrations estimated by median coefficients of day-to-day variation were in the oral group for morphine 46%, for M6G 25% and for M3G 18%. The median coefficients of variation were lower in patients receiving continuous sc morphine infusions (morphine 10%, M6G 13%, M3G 9%). Conclusion These findings indicate that serum concentrations of morphine and morphine metabolites fluctuate. The fluctuations found in our study are not explained by changes in morphine doses, administration of other drugs or by time for collection of blood samples. As expected the day-to-day variation was lower in patients receiving continuous sc morphine infusions compared with patients receiving oral morphine. PMID:15461818

  12. Anti-inflammatory, antiviral and quantitative study of quercetin-3-O-β-D-glucuronide in Polygonum perfoliatum L.

    PubMed

    Fan, Dongsheng; Zhou, Xin; Zhao, Chao; Chen, Huaguo; Zhao, Yang; Gong, Xiaojian

    2011-09-01

    Quercetin-3-O-β-D-glucuronide, isolated from Polygonum perfoliatum L., was evaluated by antiviral efficacy against influenza A virus and anti-inflammatory activity in vivo in mouse, and it was used for quality evaluation of P. perfoliatum L.. In vivo study, oral administration of quercetin-3-O-β-D-glucuronide significantly suppressed ear edema induced by dimethyl benzene and peritoneal permeability induced by acetic acid in mice, and quercetin-3-O-β-D-glucuronide also showed to possess inhibitory activity against influenza A virus (FLUAV). In the present study, additionally, a rapid, simple and sensitive method for quantitative analysis of quercetin-3-O-β-D-glucuronide in P. perfoliatum L. was developed using high performance liquid chromatography (HPLC) coupled with photodiode array detection. The separation was carried out on a Lichrosher-C18 column (250 mm × 4.6mm, 5 μm) together with a C18 guard column at isocratic elution systems of methanol (A) and 0.05% aqueous phosphoric acid (B) (43:57, v/v) with detection wavelength at 258 nm and column temperature at 30°C. The method was validated for linearity, repeatability, limit of quantification (LOQ), precision and robustness. The contents of quercetin-3-O-β-D-glucuronide in 28 samples from different regions of China were between 0.06% and 2.09%. The developed analytical method was applied to investigate P. perfoliatum L. and for quality control of the herb.

  13. Oral coadministration of β-glucuronidase to increase exposure of extensively glucuronidated drugs that undergo enterohepatic recirculation.

    PubMed

    Eichenbaum, Gary; Hsu, C-P; Subrahmanyam, Vangala; Chen, Jing; Scicinski, Jan; Galemmo, Robert A; Tuman, Robert W; Johnson, Dana L

    2012-07-01

    Extensive first-pass metabolism can significantly limit a drug's oral exposure levels. In this work, we introduce an innovative approach for increasing the oral bioavailability of a drug that undergoes extensive reversible glucuronidation and enterohepatic recirculation through intraduodenal coadministration of the deconjugating enzyme β-glucuronidase. Intraduodenal administration of JNJ-10198409 (10 mg/kg) with β-glucuronidase (34,000-140,000 units/kg) to catheterized rats resulted in a significant increase (p < 0.005) in the mean area under the plasma concentration versus time curve (AUC; approx. threefold) and maximum plasma concentration (C(max); approx. twofold) of JNJ-10198409. The AUC and C(max) were 60 ± 18 ng h/mL and 76 ± 29 ng/mL, respectively, with no enzyme and 177 ± 55 ng h/mL and 129 ± 41 ng/mL, respectively, with β-glucuronidase coadministered. Moreover, the AUC of the primary glucuronide metabolite increased approximately sevenfold from 1173 ± 361 (ng h)/mL with no enzyme coadministered to 8723 ± 2133 ng h/mL with coadministered enzyme. These pharmacokinetic data support the hypothesis that when the primary glucuronide is secreted into the duodenum via the bile duct, the glucuronide is converted by β-glucuronidase back to the parent compound. The parent compound is then reabsorbed and reconjugated, resulting in elevated systemic exposures to both parent and glucuronide. Potential clinical and preclinical applications and considerations for this approach are discussed.

  14. The detection and quantification of lorazepam and its 3-O-glucuronide in fingerprint deposits by LC-MS/MS.

    PubMed

    Goucher, Edward; Kicman, Andrew; Smith, Norman; Jickells, Sue

    2009-07-01

    The use of fingerprints as an alternative biological matrix to test for the presence of drugs and/or their metabolites is a novel area of research in analytical toxicology. This investigation describes quantitative analysis for the benzodiazepine lorazepam and its 3-O-glucuronide conjugate in fingerprints following the oral administration of a single 2 mg dose of lorazepam to five volunteers. Creatinine was also measured to investigate whether the amount of drug relative to that of creatinine would help to account for the variable amount of secretory material deposited. Fingerprints were deposited on glass cover slips and extracted by dissolving them in a solution of dichloromethane/methanol, containing tetradeuterated lorazepam as an internal standard. The samples were evaporated, reconstituted with mobile phase and analysed by LC-MS/MS. Chromatography was achieved using an RP (C18) column for the analysis of lorazapem and its glucuronide, and a hydrophilic interaction column (HILIC) for the analysis of creatinine. Lorazepam and its glucuronide were only detected where ten prints had been combined, up to 12 h following drug administration. In every case, the amount of lorazepam glucuronide exceeded that of lorazepam, the peak amounts being 210 and 11 pg, respectively. Adjusting for creatinine smoothed the elimination profile. To our knowledge, this represents the first time a drug glucuronide has been detected in deposited fingerprints.

  15. Involvement of the inhibition of intestinal glucuronidation in enhancing the oral bioavailability of resveratrol by labrasol containing nanoemulsions.

    PubMed

    Zhou, Jing; Zhou, Man; Yang, Fei-Fei; Liu, Chun-Yu; Pan, Rui-Le; Chang, Qi; Liu, Xin-Min; Liao, Yong-Hong

    2015-04-01

    Nanoemulsions have been developed for the oral delivery of poorly bioavailable phenolic compounds that are sensitive to intestinal glucuronidation. However, little is known about the contribution of UDP-glucuronosyltransferase (UGT) inhibitory excipients in nanoemulsions toward the inhibition of intestinal glucuronidation and the consequent enhanced bioavailability. In this study, Labrasol but not poloxamer 188 (F68) was found to inhibit the glucuronidation of resveratrol (RES), a model phenolic compound, in an inhibition assay with rat microsomes. Subsequently, two nanoemulsions, Lab-N and F68-N, were prepared with similar particle size distribution, zeta potentials, and entrapment efficiency by coemulsifying with Labrasol or F68, respectively. Although Lab-N exhibited inferior or comparable profiles of in vitro release, cellular uptake in Caco-2 cells, and lymphatic transport in rats to F68-N, the in vitro absorption study with everted sacs suggested that Labrasol containing formulations significantly and dose-dependently increased the transport of RES relative to free RES or F68-N by decreasing the amount of permeated metabolite, RES-3-glucuronide (RES-G). The in vivo pharmacokinetic experiments indicated that Lab-N exhibited increments in the maximum plasma concentration and the bioavailability of RES by 1098% and 560%, respectively, and significant decreases in those of RES-G, compared to F68-N. The overall results demonstrated that the improved oral bioavailability of RES by Lab-N was mainly attributable to the inhibition of intestinal glucuronidation by the presence of UGT inhibitory excipient. PMID:25723098

  16. Methylation, Glucuronidation, and Sulfonation of Daphnetin in Human Hepatic Preparations In Vitro: Metabolic Profiling, Pathway Comparison, and Bioactivity Analysis.

    PubMed

    Liang, Si-Cheng; Xia, Yang-Liu; Hou, Jie; Ge, Guang-Bo; Zhang, Jiang-Wei; He, Yu-Qi; Wang, Jia-Yue; Qi, Xiao-Yi; Yang, Ling

    2016-02-01

    Our previous study demonstrated that daphnetin is subject to glucuronidation in vitro. However, daphnetin metabolism is still poorly documented. This study aimed to investigate daphnetin metabolism and its consequent effect on the bioactivity. Metabolic profiles obtained by human liver S9 fractions and human hepatocytes showed that daphnetin was metabolized by glucuronidation, sulfonation, and methylation to form 6 conjugates which were synthesized and identified as 7-O-glucuronide, 8-O-glucuronide, 7-O-sulfate and 8-O-sulfate, 8-O-methylate, and 7-O-suflo-8-O-methylate. Regioselective 8-O-methylation of daphnetin was investigated using in silico docking calculations, and the results suggested that a close proximity (2.03 Å) of 8-OH to the critical residue Lysine 144 might be the responsible mechanism. Compared with glucuronidation and sulfonation pathways, the methylation of daphnetin had a high clearance rate (470 μL/min/mg) in human liver S9 fractions and contributed to a large amount (37.3%) of the methyl-derived metabolites in human hepatocyte. Reaction phenotyping studies showed the major role of SULT1A1, -1A2, and -1A3 in daphnetin sulfonation, and soluble COMT in daphnetin 8-O-methylation. Of the metabolites, only 8-O-methyldaphnetin exhibited an inhibitory activity on lymphocyte proliferation comparable to that of daphnetin. In conclusion, methylation is a crucial pathway for daphnetin clearance and might be involved in pharmacologic actions of daphnetin in humans. PMID:26869431

  17. Glucuronidated Quercetin Lowers Blood Pressure in Spontaneously Hypertensive Rats via Deconjugation

    PubMed Central

    Galindo, Pilar; Rodriguez-Gómez, Isabel; González-Manzano, Susana; Dueñas, Montserrat; Jiménez, Rosario; Menéndez, Carmen; Vargas, Félix; Tamargo, Juan; Santos-Buelga, Celestino; Pérez-Vizcaíno, Francisco; Duarte, Juan

    2012-01-01

    Background Chronic oral quercetin reduces blood pressure and restores endothelial dysfunction in hypertensive animals. However, quercetin (aglycone) is usually not present in plasma, because it is rapidly metabolized into conjugated, mostly inactive, metabolites. The aim of the study is to analyze whether deconjugation of these metabolites is involved in the blood pressure lowering effect of quercetin. Methodology/Principal Findings We have analyzed the effects on blood pressure and vascular function in vitro of the conjugated metabolites of quercetin (quercetin-3-glucuronide, Q3GA; isorhamnetin-3-glucuronide, I3GA; and quercetin-3′-sulfate, Q3'S) in spontaneously hypertensive rats (SHR). Q3GA and I3GA (1 mg/kg i.v.), but not Q3'S, progressively reduced mean blood pressure (MBP), measured in conscious SHR. The hypotensive effect of Q3GA was abolished in SHR treated with the specific inhibitor of β-glucuronidase, saccharic acid 1,4-lactone (SAL, 10 mg/ml). In mesenteric arteries, unlike quercetin, Q3GA had no inhibitory effect in the contractile response to phenylephrine after 30 min of incubation. However, after 1 hour of incubation Q3GA strongly reduced this contractile response and this effect was prevented by SAL. Oral administration of quercetin (10 mg/Kg) induced a progressive decrease in MBP, which was also suppressed by SAL. Conclusions Conjugated metabolites are involved in the in vivo antihypertensive effect of quercetin, acting as molecules for the plasmatic transport of quercetin to the target tissues. Quercetin released from its glucuronidated metabolites could be responsible for its vasorelaxant and hypotensive effect. PMID:22427863

  18. Milk Thistle Constituents Inhibit Raloxifene Intestinal Glucuronidation: A Potential Clinically Relevant Natural Product-Drug Interaction.

    PubMed

    Gufford, Brandon T; Chen, Gang; Vergara, Ana G; Lazarus, Philip; Oberlies, Nicholas H; Paine, Mary F

    2015-09-01

    Women at high risk of developing breast cancer are prescribed selective estrogen response modulators, including raloxifene, as chemoprevention. Patients often seek complementary and alternative treatment modalities, including herbal products, to supplement prescribed medications. Milk thistle preparations, including silibinin and silymarin, are top-selling herbal products that may be consumed by women taking raloxifene, which undergoes extensive first-pass glucuronidation in the intestine. Key constituents in milk thistle, flavonolignans, were previously shown to be potent inhibitors of intestinal UDP-glucuronosyl transferases (UGTs), with IC50s ≤ 10 μM. Taken together, milk thistle preparations may perpetrate unwanted interactions with raloxifene. The objective of this work was to evaluate the inhibitory effects of individual milk thistle constituents on the intestinal glucuronidation of raloxifene using human intestinal microsomes and human embryonic kidney cell lysates overexpressing UGT1A1, UGT1A8, and UGT1A10, isoforms highly expressed in the intestine that are critical to raloxifene clearance. The flavonolignans silybin A and silybin B were potent inhibitors of both raloxifene 4'- and 6-glucuronidation in all enzyme systems. The Kis (human intestinal microsomes, 27-66 µM; UGT1A1, 3.2-8.3 µM; UGT1A8, 19-73 µM; and UGT1A10, 65-120 µM) encompassed reported intestinal tissue concentrations (20-310 µM), prompting prediction of clinical interaction risk using a mechanistic static model. Silibinin and silymarin were predicted to increase raloxifene systemic exposure by 4- to 5-fold, indicating high interaction risk that merits further evaluation. This systematic investigation of the potential interaction between a widely used herbal product and chemopreventive agent underscores the importance of understanding natural product-drug interactions in the context of cancer prevention.

  19. Milk Thistle Constituents Inhibit Raloxifene Intestinal Glucuronidation: A Potential Clinically Relevant Natural Product–Drug Interaction

    PubMed Central

    Gufford, Brandon T.; Chen, Gang; Vergara, Ana G.; Lazarus, Philip; Oberlies, Nicholas H.

    2015-01-01

    Women at high risk of developing breast cancer are prescribed selective estrogen response modulators, including raloxifene, as chemoprevention. Patients often seek complementary and alternative treatment modalities, including herbal products, to supplement prescribed medications. Milk thistle preparations, including silibinin and silymarin, are top-selling herbal products that may be consumed by women taking raloxifene, which undergoes extensive first-pass glucuronidation in the intestine. Key constituents in milk thistle, flavonolignans, were previously shown to be potent inhibitors of intestinal UDP-glucuronosyl transferases (UGTs), with IC50s ≤ 10 μM. Taken together, milk thistle preparations may perpetrate unwanted interactions with raloxifene. The objective of this work was to evaluate the inhibitory effects of individual milk thistle constituents on the intestinal glucuronidation of raloxifene using human intestinal microsomes and human embryonic kidney cell lysates overexpressing UGT1A1, UGT1A8, and UGT1A10, isoforms highly expressed in the intestine that are critical to raloxifene clearance. The flavonolignans silybin A and silybin B were potent inhibitors of both raloxifene 4′- and 6-glucuronidation in all enzyme systems. The Kis (human intestinal microsomes, 27–66 µM; UGT1A1, 3.2–8.3 µM; UGT1A8, 19–73 µM; and UGT1A10, 65–120 µM) encompassed reported intestinal tissue concentrations (20–310 µM), prompting prediction of clinical interaction risk using a mechanistic static model. Silibinin and silymarin were predicted to increase raloxifene systemic exposure by 4- to 5-fold, indicating high interaction risk that merits further evaluation. This systematic investigation of the potential interaction between a widely used herbal product and chemopreventive agent underscores the importance of understanding natural product–drug interactions in the context of cancer prevention. PMID:26070840

  20. Zomepirac Acyl Glucuronide Is Responsible for Zomepirac-Induced Acute Kidney Injury in Mice.

    PubMed

    Iwamura, Atsushi; Watanabe, Katsuhito; Akai, Sho; Nishinosono, Tsubasa; Tsuneyama, Koichi; Oda, Shingo; Kume, Toshiyuki; Yokoi, Tsuyoshi

    2016-07-01

    Glucuronidation, an important phase II metabolic route, is generally considered to be a detoxification pathway. However, acyl glucuronides (AGs) have been implicated in the toxicity of carboxylic acid drugs due to their electrophilic reactivity. Zomepirac (ZP) was withdrawn from the market because of adverse effects such as renal toxicity. Although ZP is mainly metabolized to acyl glucuronide (ZP-AG) by UDP-glucuronosyltransferase, the role of ZP-AG in renal toxicity is unknown. In this study, we established a ZP-induced kidney injury mouse model by pretreatment with tri-o-tolyl phosphate (TOTP), a nonselective esterase inhibitor, and l-buthionine-(S,R)-sulfoximine (BSO), a glutathione synthesis inhibitor. The role of ZP-AG in renal toxicity was investigated using this model. The model showed significant increases in blood urea nitrogen (BUN) and creatinine (CRE), but not alanine aminotransferase. The ZP-AG concentrations were elevated by cotreatment with TOTP in the plasma and liver and especially in the kidney. The ZP-AG concentrations in the kidney correlated with values for BUN and CRE. Upon histopathological examination, vacuoles and infiltration of mononuclear cells were observed in the model mouse. In addition to immune-related responses, oxidative stress markers, such as the glutathione/disulfide glutathione ratio and malondialdehyde levels, were different in the mouse model. The suppression of ZP-induced kidney injury by tempol, an antioxidant agent, suggested the involvement of oxidative stress in ZP-induced kidney injury. This is the first study to demonstrate that AG accumulation in the kidney by TOTP and BSO treatment could explain renal toxicity and to show the in vivo toxicological potential of AGs. PMID:27112166

  1. Bilirubin glucuronidation by intact Gunn rat fibroblasts expressing bilirubin UDP-glucuronosyltransferase.

    PubMed Central

    Seppen, J; Tada, K; Hellwig, S; Bakker, C T; Prasad, V R; Roy Chowdhury, N; Roy Chowdhury, J; Bosma, P J; Oude Elferink, R P

    1996-01-01

    Crigler-Najjar (CN) disease is an inherited disorder of bilirubin metabolism. The disease is caused by a deficiency of the hepatic enzyme bilirubin UDP-glucuronosyltransferase (B-UGT). Patients with CN disease have high serum levels of the toxic compound, unconjugated bilirubin. The only defect in bilirubin metabolism of CN patients is the absence of B-UGT activity. The transplantation of cells able to glucuronidate bilirubin should therefore lower serum bilirubin levels. The Gunn rat is the animal model of CN disease. Primary Gunn rat fibroblasts (GURF) were transduced with a recombinant retrovirus, capable of transferring B-UGT cDNA. A cell line was obtained expressing B-UGT at a level comparable to hepatocytes. Bilirubin added to the culture medium of these cells was glucuronidated and excreted. The B-UGT activities of transduced GURF and freshly isolated Wistar hepatocytes were compared at different bilirubin concentrations. The specific B-UGT activities of these two cell types were comparable when physiological bilirubin concentrations (5-10 microM) were present in the culture media. At higher bilirubin concentrations (20-80 microM) the hepatocytes were more active than the transduced GURF. We conclude that with the addition of only one enzyme (B-UGT) fibroblasts can perform the complete set of reactions necessary for bilirubin glucuronidation. The difference in B-UGT activity between transduced GURF and hepatocytes at 20-80 microM bilirubin can be explained by lower UDP-glucuronic acid and glutathione S-transferase levels in GURF. Our findings also indicate that these cells could be used to develop extrahepatic gene therapy for CN disease. PMID:8670060

  2. The kinetics of the urinary excretion of the N-oxide and glucuronides of methaqualone in man.

    PubMed

    Wilson, K; Burnett, D; Oram, M; Reynolds, C T

    1981-01-01

    The urinary excretion of the N-oxide and the glucuronides of five C-monohydroxy metabolites of methaqualone has been studied following the oral administration of a single dose of the drug. The apparent first order rate constants for the excretion of each metabolite (kme) were shown to be numerically smaller than the overall elimination rate constant for methaqualone (k10). The Kme values tended to be greater than or equal to the corresponding apparent first order rate constants for the formation of the metabolite (km) but corresponding kme and km values were always of the same order magnitude. The kme values for the glucuronides were much smaller than the literature kme value for paracetemol glucuronide. The rate of renal elimination of the metabolites was variably sensitive to urine flow but over a period of time of 8 hours or greater the total amount of metabolite recovered in the urine was was independent of the total urine volume.

  3. Transcriptome association analysis identifies miR-375 as a major determinant of variable acetaminophen glucuronidation by human liver.

    PubMed

    Papageorgiou, Ioannis; Freytsis, Marina; Court, Michael H

    2016-10-01

    Acetaminophen is the leading cause of acute liver failure (ALF) in many countries including the United States. Hepatic glucuronidation by UDP-glucuronosyltransferase (UGT) 1A subfamily enzymes is the major route of acetaminophen elimination. Reduced glucuronidation may predispose some individuals to acetaminophen-induced ALF, but mechanisms underlying reduced glucuronidation are poorly understood. We hypothesized that specific microRNAs (miRNAs) may reduce UGT1A activity by direct effects on the UGT1A 3'-UTR shared by all UGT1A enzyme transcripts, or by indirect effects on transcription factors regulating UGT1A expression. We performed an unbiased miRNA whole transcriptome association analysis using a bank of human livers with known acetaminophen glucuronidation activities. Of 754 miRNAs evaluated, 9 miRNAs were identified that were significantly overexpressed (p<0.05; >2-fold) in livers with low acetaminophen glucuronidation activities compared with those with high activities. miR-375 showed the highest difference (>10-fold), and was chosen for further mechanistic validation. We demonstrated using in silico analysis and luciferase reporter assays that miR-375 has a unique functional binding site in the 3'-UTR of the aryl hydrocarbon receptor (AhR) gene. Furthermore overexpression of miR-375 in LS180 cells demonstrated significant repression of endogenous AhR protein (by 40%) and mRNA (by 10%), as well as enzyme activity and/or mRNA of AhR regulated enzymes including UGT1A1, UGT1A6, and CYP1A2, without affecting UGT2B7, which is not regulated by AhR. Thus miR-375 is identified as a novel repressor of UGT1A-mediated hepatic acetaminophen glucuronidation through reduced AhR expression, which could predispose some individuals to increased risk for acetaminophen-induced ALF. PMID:27531059

  4. Inhibition of lung cancer cell growth by quercetin glucuronides via G2/M arrest and induction of apoptosis.

    PubMed

    Yang, Jen-Hung; Hsia, Te-Chun; Kuo, Hsiu-Maan; Chao, Pei-Dawn Lee; Chou, Chi-Chung; Wei, Yau-Huei; Chung, Jing-Gung

    2006-02-01

    Lung cancer is the leading cause of cancer death in many developed countries, including Taiwan. Quercetin, a widely distributed bioflavonoid, is well known to induce growth inhibition in a variety of human cancer cells. Quercetin glucuronides are the main circulating metabolites after dietary supplements with quercetin in humans. However, there is little information available as to how quercetin glucuronides affect human cancer cells. We investigated the effects of quercetin glucuronides in a human lung cancer cell line NCI-H209. We checked the cell viability, cell cycle checkpoint proteins, pro- and antiapoptotic proteins, caspase-3 activity, and gene expression by flow cytometry and Western blot. The viability of cells decreased in a dose- and time-dependent manner. Cell cycle analysis revealed a significant increase of the proportion of cells in G2/M phase and subG0/G1 phase (corresponding to apoptotic cells). Moreover, quercetin glucuronides increased the expressions of cyclin B, Cdc25c-ser-216-p, and Wee1 proteins, indicating the G2/M arrest. We also demonstrated a concurrent decrease of the mitochondrial membrane potential, release of cytochrome c, up-regulation of Bax, down-regulation of Bcl-2, and activation of caspase-3, and subsequently, cleavage of poly(ADP-ribose) polymerase. In addition, quercetin glucuronide-induced apoptosis was totally blocked by the broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone. Taken together, we demonstrated that quercetin glucuronides inhibited proliferation through G2/M arrest of the cell cycle and induced apoptosis via caspase-3 cascade in the human lung cancer cell line NCI-H209. Delineation of the biological effects of specific major quercetin metabolites on chemotherapeutic potential or chemoprevention of human cancers warrants further investigation. PMID:16280456

  5. 2D QSAR Study for Gemfibrozil Glucuronide as the Mechanism-based Inhibitor of CYP2C8

    PubMed Central

    Taxak, N.; Bharatam, P. V.

    2013-01-01

    Mechanism-based inhibition of cytochrome P450 involves the bioactivation of the drug to a reactive metabolite, which leads to cytochrome inhibition via various mechanisms. This is generally seen in the Phase I of drug metabolism. However, gemfibrozil (hypolipidemic drug) leads to mechanism-based inhibition after generating glucuronide conjugate (gemfibrozil acyl-β-glucuronide) in the Phase II metabolism reaction. The mechanism involves the covalent binding of the benzyl radical (generated from the oxidation of aromatic methyl group in conjugate) to the heme of CYP2C8. This article deals with the development of a 2D QSAR model based on the inhibitory potential of gemfibrozil, its analogues and corresponding glucuronide conjugates in inhibiting the CYP2C8-catalysed amodiaquine N-deethylation. The 2D QSAR model was developed using multiple linear regression analysis in Accelrys Discovery Studio 2.5 and helps in identifying the descriptors, which are actually contributing to the inhibitory potency of the molecules studied. The built model was further validated using leave one out method. The best quantitative structure activity relationship model was selected having a correlation coefficient (r) of 0.814 and cross-validated correlation coefficient (q2) of 0.799. 2D QSAR revealed the importance of volume descriptor (Mor15v), shape descriptor (SP09) and 3D matrix-based descriptor (SpMax_RG) in defining the activity for this series of molecules. It was observed that volume and 3D matrix-based descriptors were crucial in imparting higher potency to gemfibrozil glucuronide conjugate, as compared with other molecules. The results obtained from the present study may be useful in predicting the inhibitory potential (IC50 for CYP2C8 inhibition) of the glucuronide conjugates of new molecules and compare with the standard gemfibrozil acyl-β-glucuronide (in terms of pIC50 values) in early stages of drug discovery and development. PMID:24591743

  6. Carrier-mediated mechanism for the biliary excretion of the quinolone antibiotic grepafloxacin and its glucuronide in rats.

    PubMed

    Sasabe, H; Tsuji, A; Sugiyama, Y

    1998-03-01

    Grepafloxacin (GPFX) has a comparatively greater hepatobiliary transport than other quinolone antibiotics. The biliary excretion mechanism of GPFX was investigated in a series of in vivo and in vitro studies with Sprague-Dawley rats and the mutant strain Eisai-hyperbilirubinemia rats (EHBR), which have a hereditary defect in their bile canalicular multispecific organic anion transport system (cMOAT). The biliary excretion of the parent drug in EHBR was 38% of that in normal rats, whereas the 3-glucuronide, a main metabolite of GPFX, was scarcely excreted into the bile in EHBR. To clarify the biliary excretion mechanism of GPFX, studies of uptake by bile canalicular membrane vesicle (CMV) were performed. ATP dependence was observed in the uptake of GPFX by CMV, although the extent was not very marked, whereas no ATP-dependent uptake was observed by CMV prepared from EHBR. An inhibition study of the ATP-dependent uptake of the glutathione conjugate, 2,4-dinitrophenyl-S-glutathione (DNP-SG), a typical substrate for cMOAT, was performed in order to differentiate among the affinities of six quinolone antibiotics for this transporter. All quinolone antibiotics inhibited the ATP-dependent uptake of DNP-SG with different half-inhibition concentrations (IC50), and GPFX had the lowest IC50 value. The uptake of GPFX-glucuronide by CMV from normal rats showed a marked ATP dependence, whereas there was little ATP-dependent uptake in EHBR. The K(m) value (7.2 microM) for the higher-affinity component of the glucuronide uptake was comparable to the Ki value (9.2 microM) of the glucuronide in terms of inhibition of the ATP-dependent uptake of DNP-SG, which indicates that DNP-SG and the glucuronide may share the same transporter, cMOAT. The Ki value of the glucuronide observed in this inhibition was less than 1/200 that of the parent, which suggests that the glucuronide had a much higher affinity than the parent drug. These results lead us to conclude that at least a part of the

  7. Identification of Human UDP-Glucuronosyltransferase 1A4 as the Major Isozyme Responsible for the Glucuronidation of 20(S)-Protopanaxadiol in Human Liver Microsomes

    PubMed Central

    Li, Jia; He, Chunyong; Fang, Lianxiang; Yang, Li; Wang, Zhengtao

    2016-01-01

    20(S)-protopanaxadiol (PPD), one of the representative aglycones of ginsenosides, has a broad spectrum of pharmacological activities. Although phase I metabolism has been investigated extensively, information regarding phase II metabolism of this compound remains to be elucidated. Here, a glucuronidated metabolite of PPD in human liver microsomes (HLMs) and rat liver microsomes (RLMs) was unambiguously identified as PPD-3-O-β-d-glucuronide by nuclear magnetic resonance spectroscopy and high resolution mass spectrometry. The chemical inhibition and recombinant human UDP-Glucuronosyltransferase (UGT) isoforms assay showed that the PPD glucuronidation was mainly catalyzed by UGT1A4 in HLM, whereas UGT1A3 showed weak catalytic activity. In conclusion, PPD-3-O-β-d-glucuronide was first identified as the principal glucuronidation metabolite of PPD in HLMs, which was catalyzed by UGT1A4. PMID:27005621

  8. Antimicrobial and demelanizing activity of Ganoderma lucidum extract, p-hydroxybenzoic and cinnamic acids and their synthetic acetylated glucuronide methyl esters.

    PubMed

    Heleno, Sandrina A; Ferreira, Isabel C F R; Esteves, Ana P; Ćirić, Ana; Glamočlija, Jasmina; Martins, Anabela; Soković, Marina; Queiroz, Maria João R P

    2013-08-01

    Mushroom extracts or isolated compounds may be useful in the search of new potent antimicrobial agents. Herein, it is described the synthesis of protected (acetylated) glucuronide derivatives of p-hydroxybenzoic and cinnamic acids, two compounds identified in the medicinal mushroom Ganoderma lucidum. Their antimicrobial and demelanizing activities were evaluated and compared to the parent acids and G. lucidum extract. p-Hydroxybenzoic and cinnamic acids, as also their protected glucuronide derivatives revealed high antimicrobial (antibacterial and antifungal) activity, even better than the one showed by commercial standards. Despite the variation in the order of parent acids and the protected glucuronide derivatives, their antimicrobial activity was always higher than the one revealed by the extract. Nevertheless, the extract was the only one with demelanizing activity against Aspergillus niger. The acetylated glucuronide derivatives could be deprotected to obtain glucuronide metabolites, which circulate in the human organism as products of the metabolism of the parent compounds.

  9. Identification of a new metabolite of GHB: gamma-hydroxybutyric acid glucuronide.

    PubMed

    Petersen, Ida Nymann; Tortzen, Christian; Kristensen, Jesper Langgaard; Pedersen, Daniel Sejer; Breindahl, Torben

    2013-06-01

    Gamma-hydroxybutyric acid (GHB) is an important analyte in clinical and forensic toxicology with a narrow detection window of 3-6 h. In the search of improved detection methods, the existence in vivo of a glucuronated GHB metabolite (GHB-GLUC) was hypothesized. Chemically pure standards of GHB-GLUC and a deuterated analogue for chromatography were synthesized. Liquid chromatography and tandem mass spectrometry were used for targeted analysis in anonymous clinical urine samples (n = 50). GHB-GLUC was found in concentrations ranging from 0.11 to 5.0 µg/mL (mean: 1.3 ± 1.2 µg/mL). Thus far, this is the first report of a GHB glucuronide detected in biological samples. Given that glucuronides generally have longer half-life values than their corresponding free drugs, GHB-GLUC should theoretically be a biomarker of GHB intoxication. It is also proposed that the hitherto unexplained reports of elevated GHB concentrations in some biological samples, which has caused the setting of a relatively high cutoff value (10 µg/mL), represent total GHB measurements (sum of free GHB and actively chemically hydrolyzed GHB-GLUC). To address these challenges, the present study must be followed by comprehensive pharmacokinetic and stability studies after the controlled administration of GHB.

  10. CM2 antigen, a potential novel molecule participating in glucuronide transport on rat hepatocyte canalicular membrane.

    PubMed

    Wang, L; Wang, J; Zhou, X; Li, J; Shi, Y; Han, Z; Wang, X; Li, S; Yang, Z; Wang, R; Fan, D; Han, Y

    2012-06-29

    The polarized molecules predominately distributing at hepatocyte canalicular surface play a vital role in disclosing the process of bile formation and etiopathogenisis of cholestatic live diseases. Therefore, it is important to find novel polarized molecules on hepatocyte canalicular membrane. In the present study, canalicular membrane vesicles (CMVs) isolated from rat hepatocyte by density gradient centrifugation were used as immunogens to produce hybridoma and 46 strains of monoclonal antibodies (mAb) against CMVs were obtained. With a series of morphological assay methods, including immunohistochemistry, immunofluorescence and immuno-electron microscope, the antigens recognized by canalicular mAb1 (CM1) and canalicular mAb2 (CM2) were confirmed to predominately distribute at hepatocyte canalicular membrane. Transport activity assay revealed that CM2 could inhibit ATP-dependent E217βG uptake of rat hepatocyte CMVs. Meanwhile, Western blotting analysis showed that the molecular mass of CM2 antigen was approximately 110kDa, which was much less than Mr 180kDa of multidrug resistance-associated protein 2 (MRP2) involved in glucuronide transport. These data indicated that CM2 antigen might be a potential novel molecule participating in glucuronide transport on the hepatocyte canalicular membrane.

  11. UGT1A1*28 polymorphism influences glucuronidation of bazedoxifene.

    PubMed

    Lušin, T Trdan; Mrhar, A; Trontelj, J

    2015-02-01

    Bazedoxifene is used for the prevention and treatment of osteoporosis. After peroral application, bazedoxifene is metabolized by UDP-glucuronosyltransferases (UGTs) to bazedoxifene-4'-glucuronide (M4) and bazedoxifene-5-glucuronide (M5). It has already been shown that a relatively common UGT1A1*28 polymorphism can considerably affect raloxifene pharmacokinetics and pharmacodynamics. As pharmacokinetics of bazedoxifene and raloxifene are very similar, the influence of UGT1A1*28 polymorphism on metabolism of bazedoxifene was investigated by genotyped microsomes. Our results indicate an influence of UGT1A1*28 allele on the formation clearance of both bazedoxifene metabolites. The decreased metabolic clearance was most pronounced in microsomes from polymorphic homozygote (*28/*28) where a 7 to 10-fold lower metabolic clearance was observed for both metabolites compared to other genotypes. In conclusion, the significant UGT1A1*28 genotype effect on bazedoxifene intrinsic metabolic clearance indicates that this subject is worth further exploration in vivo and provides valuable information research in this field. PMID:25997248

  12. Chemical reactivity of the naproxen acyl glucuronide and the naproxen coenzyme A thioester towards bionucleophiles.

    PubMed

    Olsen, Jørgen; Bjørnsdottir, Inga; Tjørnelund, Jette; Honoré Hansen, Steen

    2002-06-20

    Drugs may be metabolised to reactive electrophilic species that spontaneously react with proteins. The presence of such drug-protein adducts has been associated with drug toxicity. In this study, the reactivity of the major metabolite of naproxen--the 1-beta-O-glucuronide (Nap-GlcU)--was compared to the corresponding naproxen coenzyme A (Nap-CoA) thioester. The reactivity of the two metabolites was assessed in vitro in a phosphate buffer (pH 7.4; 0.1 M) at 37 degrees C towards the model bionucleophiles glutathione and human serum albumin (HSA). The reaction between the electrophilic species (Nap-GlcU and Nap-CoA) and glutathione forming the Nap-glutathione conjugate was monitored using LC-MS-MS and LC-UV, respectively. It was shown that Nap-CoA resulted in an approximate 100-fold higher formation of Nap-glutathione conjugate than Nap-GlcU. The presence of Nap-CoA also resulted in acylated HSA with a rate and a yield that was significantly higher than reported for Nap-GlcU. In summary, the data suggest that CoA metabolites may be more reactive species than acyl glucuronides that previously have been associated with severe drug related side effects in vivo.

  13. Isolation and determination of benzo(a)pyrene glucuronide and sulfate conjugates in soybean leaves

    SciTech Connect

    Negishi, T.; Nakano, M.; Kobayashi, S.; Kim, C.H.

    1987-08-01

    BaP is metabolized in mammalian systems by the mixed function oxidase system of liver microsomes. This system catalyzes the oxidation of BaP via epoxide intermediate to phenol, diol and quinone metabolites. One of these 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro-BaP is thought to act as the ultimate carcinogen by binding covalently to cellular DNA. It is also known that Cunninghamella elegans oxidized BaP to its phenol, diol and quinone metabolites. In addition, the alcohols were detected as glucuronide and sulfate conjugates. These metabolites are remarkably similar to those observed in higher organisms. On the other hand, some investigators have demonstrated that plants take up BaP and anthracene from soil or culture medium containing these compounds. This paper reports the finding that soybeans grown in BaP polluted soil take it up and metabolize to its phenol, diol and the glucuronide and sulfate conjugates of the alcohols.

  14. 40 CFR 721.10244 - Phosphonic acid, P-[2-[bis(2-hydroxyethyl)amino]ethyl]-, 2-[bis(2- chloroethoxy)phosphinyl]ethyl...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Phosphonic acid, P- ethyl]-, 2- ethyl... New Uses for Specific Chemical Substances § 721.10244 Phosphonic acid, P- ethyl]-, 2- ethyl 2... substance identified as phosphonic acid, P- ethyl]-, 2- ethyl 2-chloroethyl ester (PMN P-09-195; CAS...

  15. 40 CFR 721.10244 - Phosphonic acid, P-[2-[bis(2-hydroxyethyl)amino]ethyl]-, 2-[bis(2- chloroethoxy)phosphinyl]ethyl...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Phosphonic acid, P- ethyl]-, 2- ethyl... New Uses for Specific Chemical Substances § 721.10244 Phosphonic acid, P- ethyl]-, 2- ethyl 2... substance identified as phosphonic acid, P- ethyl]-, 2- ethyl 2-chloroethyl ester (PMN P-09-195; CAS...

  16. 40 CFR 721.10244 - Phosphonic acid, P-[2-[bis(2-hydroxyethyl)amino]ethyl]-, 2-[bis(2- chloroethoxy)phosphinyl]ethyl...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Phosphonic acid, P- ethyl]-, 2- ethyl... New Uses for Specific Chemical Substances § 721.10244 Phosphonic acid, P- ethyl]-, 2- ethyl 2... substance identified as phosphonic acid, P- ethyl]-, 2- ethyl 2-chloroethyl ester (PMN P-09-195; CAS...

  17. Elastic electron scattering by ethyl vinyl ether

    NASA Astrophysics Data System (ADS)

    Khakoo, M. A.; Hong, L.; Kim, B.; Winstead, C.; McKoy, V.

    2010-02-01

    We report measured and calculated results for elastic scattering of low-energy electrons by ethyl vinyl ether (ethoxyethene), a prototype system for studying indirect dissociative attachment processes that may play a role in DNA damage. The integral cross section displays the expected π* shape resonance. The agreement between the calculated and measured cross sections is generally good.

  18. Manufacturing Ethyl Acetate From Fermentation Ethanol

    NASA Technical Reports Server (NTRS)

    Rohatgi, Naresh K.; Ingham, John D.

    1991-01-01

    Conceptual process uses dilute product of fermentation instead of concentrated ethanol. Low-concentration ethanol, extracted by vacuum from fermentation tank, and acetic acid constitutes feedstock for catalytic reaction. Product of reaction goes through steps that increases ethyl acetate content to 93 percent by weight. To conserve energy, heat exchangers recycle waste heat to preheat process streams at various points.

  19. Ethyl p-nitrophenyl phenylphosphorothioate (EPN)

    Integrated Risk Information System (IRIS)

    Ethyl p - nitrophenyl phenylphosphorothioate ( EPN ) ; CASRN 2104 - 64 - 5 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Ha

  20. Human and Rat ABC Transporter Efflux of Bisphenol A and Bisphenol A Glucuronide: Interspecies Comparison and Implications for Pharmacokinetic Assessment

    EPA Science Inventory

    Significant interspecies differences exist between human and rodent with respect to absorption, distribution, and excretion of bisphenol A (BPA) and its primary metabolite, BPA-glucuronide (BPA-G). ATP-Binding Cassette (ABC) transporter enzymes play important roles in these physi...

  1. Differences in the glucuronidation of bisphenols F and S between two homologous human UGT enzymes, 1A9 and 1A10.

    PubMed

    Gramec Skledar, Darja; Troberg, Johanna; Lavdas, Jason; Peterlin Mašič, Lucija; Finel, Moshe

    2015-01-01

    1. Bisphenol S (BPS) and bisphenol F (BPF) are bisphenol A (BPA) analogues commonly used in the manufacturing of industrial and consumer products. 2. Bisphenols are often detoxified through conjugation with glucuronic acid or sulfate. In this work, we have examined the glucuronidation of BPS and BPF by recombinant human UDP-glucuronosyltransferase (UGT) enzymes. In addition, we have reexamined BPA glucuronidation, using extra-hepatic UGTs that were not tested previously. 3. The results revealed that UGT1A9, primarily a hepatic enzyme, is mainly responsible for BPS glucuronidation, whereas UGT1A10, an intestine enzyme that is highly homologous to UGT1A9 at the protein level, is by far the most active UGT in BPF glucuronidation. In contrast to the latter two UGTs that display significant specificity in the glucuronidation of BPS and BPF, UGT2A1 that is mainly expressed in the airways, exhibited high activity toward all the tested bisphenols, BPS, BPF and BPA. UGT1A10 exhibited somewhat higher BPA glucuronidation activity than UGT1A9, but it was lower than UGT2A1 and UGT2B15. 4. The new findings demonstrate interesting differences in the glucuronidation patterns of bisphenols and provide new insights into the role of extra-hepatic tissues in their detoxification.

  2. Quantitative Profiling of Human Renal UDP-glucuronosyltransferases and Glucuronidation Activity: A Comparison of Normal and Tumoral Kidney Tissues

    PubMed Central

    Margaillan, Guillaume; Rouleau, Michèle; Fallon, John K.; Caron, Patrick; Villeneuve, Lyne; Turcotte, Véronique; Smith, Philip C.; Joy, Melanie S.

    2015-01-01

    Renal metabolism by UDP-glucuronosyltransferase (UGT) enzymes is central to the clearance of many drugs. However, significant discrepancies about the relative abundance and activity of individual UGT enzymes in the normal kidney prevail among reports, whereas glucuronidation in tumoral kidney has not been examined. In this study, we performed an extensive profiling of glucuronidation metabolism in normal (n = 12) and tumor (n = 14) kidneys using targeted mass spectrometry quantification of human UGTs. We then correlated UGT protein concentrations with mRNA levels assessed by quantitative polymerase chain reaction and with conjugation activity for the major renal UGTs. Beyond the wide interindividual variability in expression levels observed among kidney samples, UGT1A9, UGT2B7, and UGT1A6 are the most abundant renal UGTs in both normal and tumoral tissues based on protein quantification. In normal kidney tissues, only UGT1A9 protein levels correlated with mRNA levels, whereas UGT1A6, UGT1A9, and UGT2B7 quantification correlated significantly with their mRNA levels in tumor kidneys. Data support that posttranscriptional regulation of UGT2B7 and UGT1A6 expression is modulating glucuronidation in the kidney. Importantly, our study reveals a significant decreased glucuronidation capacity of neoplastic kidneys versus normal kidneys that is paralleled by drastically reduced UGT1A9 and UGT2B7 mRNA and protein expression. UGT2B7 activity is the most repressed in tumors relative to normal tissues, with a 96-fold decrease in zidovudine metabolism, whereas propofol and sorafenib glucuronidation is decreased by 7.6- and 5.2-fold, respectively. Findings demonstrate that renal drug metabolism is predominantly mediated by UGT1A9 and UGT2B7 and is greatly reduced in kidney tumors. PMID:25650382

  3. Focused ultrasound-assisted acceleration of enzymatic hydrolysis of alkylphenols and 17β-oestradiol glucuronide in fish bile.

    PubMed

    Vallejo, Asier; Usobiaga, Aresatz; Ortiz-Zarragoitia, Maren; Cajaraville, Miren P; Fernández, Luis A; Zuloaga, Olatz

    2010-11-01

    According to the European Water Framework Directive (WFD), alkylphenols, such as octylphenols and nonylphenols, and 17β-oestradiol are considered as priority or emerging pollutants, respectively, mainly due to their possible properties as endocrine-disrupting compounds (EDCs). EDCs are accumulated in liver, fat, kidney and bile in the glucuronide form. In order to determine the concentration of these compounds in bile, an enzymatic hydrolysis step is necessary. This step is usually long (~16 h), and in this sense, ultrasound probes were studied as a possible alternative energy source to accelerate this process. Enzymatic hydrolysis was reduced to 20 min using an ultrasound probe at one cycle and 10% of amplitude. For validation of analytical procedure, nonylphenol glucuronide (4NP-G), 4-tert-octylphenol glucuronide (4tOP-G) and 4-n-octylphenol glucuronide (4nOP-G) were synthesised while 17β-oestradiol glucuronide (E2-G) was commercially available. Bile from thick-lip grey mullets (Chelon labrosus) was spiked with known amounts of 4NP-G, 4tOP-G, 4nOP-G and E2-G and submitted to the optimised procedure. Good recoveries (77-122%), precision in the 5% to 12% range and limits of detection, ranging from the low nanogramme per gramme level for 4tOP, 4nOP and E2 to the low microgramme per gramme level for nonylphenols, were obtained. The optimised method was applied for the determination of alkylphenol in the bile of thick-lip grey mullets fish bile from the Urdaibai estuary (UNESCO reserve of the Biosphere, Bay of Biscay), and high concentrations (2.3-14.2 μg/g), such as those obtained in polluted areas, were measured. E2 was determined in the bile of thick-lip grey mullets, intraperitoneally injected with E2.

  4. 21 CFR 172.872 - Methyl ethyl cellulose.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Methyl ethyl cellulose. 172.872 Section 172.872... Methyl ethyl cellulose. The food additive methyl ethyl cellulose may be safely used in food in accordance with the following prescribed conditions. (a) The additive is a cellulose ether having the...

  5. 21 CFR 177.1320 - Ethylene-ethyl acrylate copolymers.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... prescribed for polyethylene in § 177.1520. (1) Specifications—(i) Infrared identification. Ethylene-ethyl acrylate copolymers can be identified by their characteristic infrared spectra. (ii) Quantitative determination of ethyl acrylate content. The ethyl acrylate can be determined by the infrared spectra. Prepare...

  6. 21 CFR 177.1320 - Ethylene-ethyl acrylate copolymers.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... prescribed for polyethylene in § 177.1520. (1) Specifications—(i) Infrared identification. Ethylene-ethyl acrylate copolymers can be identified by their characteristic infrared spectra. (ii) Quantitative determination of ethyl acrylate content. The ethyl acrylate can be determined by the infrared spectra. Prepare...

  7. Photoinduced covalent binding of frusemide and frusemide glucuronide to human serum albumin

    PubMed Central

    Mizuma, Takashi; McDonagh, Antony F; Lin, Emil T; Benet, Leslie Z

    1999-01-01

    Aims To study reaction of photoactivated frusemide (F) and F glucuronide (Fgnd metabolite) with human serum albumin in order to find a clue to clarify a mechanism of phototoxic blisters from high frusemide dosage. Methods F was exposed to light in the presence of human serum albumin (HSA). HSA treated with this method (TR-HSA) was characterized by fluorescence spectroscopic experiment, alkali treatment and reversible binding experiment. Results Less 4-hydroxyl-N-furfuryl-5-sulphamoylanthranilic acid (4HFSA, a photodegradation product of F) was formed in the presence of HSA than in the absence of HSA. A new fluorescence spectrum excited at 320 nm was observed for TR-HSA. Alkali treatment of TR-HSA released 4HFSA. Quenching of the fluorescence due to the lone tryptophan near the warfarin-binding site of HSA was observed in TR-HSA. The reversible binding of F or naproxen to the warfarin-binding site of TR-HSA was less than to that of native HSA. These results indicate the photoactivated F was covalently bound to the warfarin-binding site of HSA. The covalent binding of Fgnd, which is also reversibly bound to the wafarin-binding site of HSA, was also induced by exposure to sunlight. Fgnd was more photoactive than F, indicating that F could be activated by glucuronidation to become a more photoactive compound. Conclusions The reactivity of photoactivated F and Fgnd to HSA and/or to other endogenous compounds may cause the phototoxic blisters that result at high F dosage. PMID:10383564

  8. Inhibitory effect of ciprofloxacin on β-glucuronidase-mediated deconjugation of mycophenolic acid glucuronide.

    PubMed

    Kodawara, Takaaki; Masuda, Satohiro; Yano, Yoshitaka; Matsubara, Kazuo; Nakamura, Toshiaki; Masada, Mikio

    2014-07-01

    The interaction between mycophenolate (MPA) and quinolone antibiotics such as ciprofloxacin is considered to reduce the enterohepatic recycling of MPA, which is biotransformed in the intestine from MPA glucuronide (MPAG) conjugate excreted via the biliary system; however, the molecular mechanism underlying this biotransformation of MPA is still unclear. In this study, an in vitro system was established to evaluate β-glucuronidase-mediated deconjugation and to examine the influence of ciprofloxacin on the enzymatic deconjugation of MPAG and MPA resynthesis. Resynthesis of MPA via deconjugation of MPAG increased in a time-dependent manner from 5 to 60 min in the presence of β-glucuronidase. Ciprofloxacin and phenolphthalein-β-d-glucuronide (PhePG), a typical β-glucuronidase substrate, significantly decreased the production of MPA from MPAG in the β-glucuronidase-mediated deconjugation system. In addition, enoxacin significantly inhibited the production of MPA from MPAG, while levofloxacin and ofloxacin had no inhibitory effect on MPA synthesis. Pharmacokinetic analysis revealed that ciprofloxacin showed a dose-dependent inhibitory effect on MPA production from MPAG via β-glucuronidase with a half-maximal inhibitory concentration (IC50 ) value of 30.4 µm. While PhePG inhibited the β-glucuronidase-mediated production of MPA from MPAG in a competitive manner, ciprofloxacin inhibited MPA synthesis via noncompetitive inhibition. These findings suggest that the reduction in the serum MPA concentration during the co-administration of ciprofloxacin is at least in part due to the decreased enterohepatic circulation of MPA because of noncompetitive inhibition of deconjugation of MPAG by intestinal β-glucuronidase.

  9. Evaluation of Clopidogrel Conjugation Metabolism: PK Studies in Man and Mice of Clopidogrel Acyl Glucuronide.

    PubMed

    Savu, Simona Nicoleta; Silvestro, Luigi; Surmeian, Mariana; Remis, Lina; Rasit, Yuksel; Savu, Simona Rizea; Mircioiu, Constantin

    2016-09-01

    The existence of a glucuronide conjugate of the major circulating clopidogrel metabolites, called clopidogrel acyl glucuronide (CAG), is already known. However, information regarding its pharmacokinetics (PK), metabolism, and clearance are modest. We investigated in vivo the potential CAG trans-esterification to clopidogrel (reaction occurring in vitro in particular conditions) by administering the metabolite to mice. Experiments were then carried out on men, clopidogrel administered alone or followed by activated charcoal intake (intestinal reabsorption blockade). Study objectives included: PK comparison of CAG, clopidogrel carboxylic acid (CCA), and clopidogrel in plasma, determination of their elimination patterns in urine and feces, and tracking of charcoal-induced changes in PK and/or urinary excretion that would indicate relevant enterohepatic recycling of CAG. In mice, CAG was rapidly hydrolyzed to CCA after oral administration, whereas by intravenous route metabolic conversion to CCA was delayed. No levels of clopidogrel were detected in mice plasma, excluding any potential trans-esterification or other form of back-conversion in vivo. PK experiments in man showed that CAG is hydrolyzed in the gastrointestinal tract (very low concentrations in feces), but there is no evidence of enterohepatic recirculation. Quantitation of the three moieties in stool samples accounted for only 1.2% of an administered dose, suggesting that other yet unknown metabolites/degradation products formed through metabolic processes and/or the activity of local microflora are mainly excreted by this route. In man CAG was confirmed as one of the major terminal metabolites of clopidogrel, with a PK behavior similar to CCA. PMID:27402727

  10. Identifying and applying a highly selective probe to simultaneously determine the O-glucuronidation activity of human UGT1A3 and UGT1A4.

    PubMed

    Jiang, Li; Liang, Si-Cheng; Wang, Chao; Ge, Guang-Bo; Huo, Xiao-Kui; Qi, Xiao-Yi; Deng, Sa; Liu, Ke-Xin; Ma, Xiao-Chi

    2015-01-01

    Glucuronidation mediated by uridine 5'-diphospho (UDP)-glucuronosyltransferase is an important detoxification pathway. However, identifying a selective probe of UDP- glucuronosyltransferase is complicated because of the significant overlapping substrate specificity displayed by the enzyme. In this paper, desacetylcinobufagin (DACB) 3-O- and 16-O-glucuronidation were found to be isoform-specific probe reactions for UGT1A4 and UGT1A3, respectively. DACB was well characterized as a probe for simultaneously determining the catalytic activities of O-glucuronidation mediated by UGT1A3 and UGT1A4 from various enzyme sources, through a sensitive analysis method. PMID:25884245

  11. 40 CFR 180.595 - Flufenpyr-ethyl; tolerances for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... residues of the herbicide, flufenpyr-ethyl; acetic acid, -phenoxy]-ethyl ester], in or on the following...) Tolerances are established for residues of the herbicide flufenpyr-ethyl; acetic acid, -phenoxy]-ethyl...

  12. 40 CFR 180.595 - Flufenpyr-ethyl; tolerances for residues.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... residues of the herbicide, flufenpyr-ethyl; acetic acid, -phenoxy]-ethyl ester], in or on the following...) Tolerances are established for residues of the herbicide flufenpyr-ethyl; acetic acid, -phenoxy]-ethyl...

  13. 40 CFR 180.595 - Flufenpyr-ethyl; tolerances for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... residues of the herbicide, flufenpyr-ethyl; acetic acid, -phenoxy]-ethyl ester], in or on the following...) Tolerances are established for residues of the herbicide flufenpyr-ethyl; acetic acid, -phenoxy]-ethyl...

  14. 40 CFR 180.595 - Flufenpyr-ethyl; tolerances for residues.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... residues of the herbicide, flufenpyr-ethyl; acetic acid, -phenoxy]-ethyl ester], in or on the following...) Tolerances are established for residues of the herbicide flufenpyr-ethyl; acetic acid, -phenoxy]-ethyl...

  15. Identification of the Position of Mono-O-Glucuronide of Flavones and Flavonols by Analyzing Shift in Online UV Spectrum (λmax) Generated from an Online Diode-arrayed Detector

    PubMed Central

    Singh, Rashim; Wu, Baojian; Tang, Lan; Liu, Zhongqiu; Hu, Ming

    2012-01-01

    The beneficial pharmacological effects of flavonoids such as chemo-prevention against cancer, aging and heart diseases are severely limited due to their extensive in vivo glucuronidation by UGTs. UGTs showed regiospecificity (i.e. position preference) in the glucuronidation of the flavonoids based on substrate’s chemical structure. In this paper, glucuronide(s) of 36 flavones and flavonols were generated using an in vitro glucuronidation reaction. UPLC/MS/MS was used to confirm the degree (mono- or di-) of glucuronidation in flavonoids with up to four hydroxyl group. UV spectra of flavonoids and their respective mono-O-glucuronides were generated using UPLC with an online diode-arrayed detector. Analysis of the extent of shift in spectra of glucuronides in Band I and Band II regions as reflected by changes in λmax value was used to identify the position of glucuronidation. The data showed that glucuronidation of 3- and 4’-hydroxyl resulted in Band I λmax hypsochromic shift (or blue shift) of 13–30 nm and 5–10 nm, respectively. And glucuronidation of 5-hydroxyl group caused Band II λmax hypsochromic shift of 5–10 nm. In contrast, glucuronidation of 7-hydroxyl group did not cause any λmax change in Band I or II λmax whereas glucuronidation of 6-hydroxyl group did not cause predictable changes in λmax values. The paper demonstrated for the first time that a rapid and robust analysis method using λmax changes in online UV spectra can be used to pinpoint region-specific glucuronidation of flavones and flavonols with hydroxyl groups at 4’, 3, 5, and/or 7 position(s). PMID:20687611

  16. Extrapolation of diclofenac clearance from in vitro microsomal metabolism data: role of acyl glucuronidation and sequential oxidative metabolism of the acyl glucuronide.

    PubMed

    Kumar, Sanjeev; Samuel, Koppara; Subramanian, Ramaswamy; Braun, Matthew P; Stearns, Ralph A; Chiu, Shuet-Hing Lee; Evans, David C; Baillie, Thomas A

    2002-12-01

    Diclofenac is eliminated predominantly (approximately 50%) as its 4'-hydroxylated metabolite in humans, whereas the acyl glucuronide (AG) pathway appears more important in rats (approximately 50%) and dogs (>80-90%). However, previous studies of diclofenac oxidative metabolism in human liver microsomes (HLMs) have yielded pronounced underprediction of human in vivo clearance. We determined the relative quantitative importance of 4'-hydroxy and AG pathways of diclofenac metabolism in rat, dog, and human liver microsomes. Microsomal intrinsic clearance values (CL(int) = V(max)/K(m)) were determined and used to extrapolate the in vivo blood clearance of diclofenac in these species. Clearance of diclofenac was accurately predicted from microsomal data only when both the AG and the 4'-hydroxy pathways were considered. However, the fact that the AG pathway in HLMs accounted for ~75% of the estimated hepatic CL(int) of diclofenac is apparently inconsistent with the 4'-hydroxy diclofenac excretion data in humans. Interestingly, upon incubation with HLMs, significant oxidative metabolism of diclofenac AG, directly to 4'-hydroxy diclofenac AG, was observed. The estimated hepatic CL(int) of this pathway suggested that a significant fraction of the intrahepatically formed diclofenac AG may be converted to its 4'-hydroxy derivative in vivo. Further experiments indicated that this novel oxidative reaction was catalyzed by CYP2C8, as opposed to CYP2C9-catalyzed 4'-hydroxylation of diclofenac. These findings may have general implications in the use of total (free + conjugated) oxidative metabolite excretion for determining primary routes of drug clearance and may question the utility of diclofenac as a probe for phenotyping human CYP2C9 activity in vivo via measurement of its pharmacokinetics and total 4'-hydroxy diclofenac urinary excretion.

  17. Synthesis of Ethyl Salicylate Using Household Chemicals

    NASA Astrophysics Data System (ADS)

    Solomon, Sally; Hur, Chinhyu; Lee, Alan; Smith, Kurt

    1996-02-01

    Ethyl salicylate is synthesized, isolated, and characterized in a three-step process using simple equipment and household chemicals. First, acetylsalicylic acid is extracted from aspirin tablets with isopropyl alcohol, then hydrolyzed to salicylic acid with muriatic acid, and finally, the salicylic acid is esterified using ethanol and a boric acid catalyst. The experiment can be directed towards high school or university level students who have sufficient background in organic chemistry to recognize the structures and reactions that are involved.

  18. Production of ethyl alcohol from bananas

    SciTech Connect

    Jones, R.L.; Towns, T.

    1983-12-01

    The production of ethyl alcohol from waste bananas presents many special problems. During cooking, matting of the latex fibers from the banana peel recongeal when cooled and left untreated. This problem has been addressed by Alfaro by the use of CaC1/sub 2/. Separation of solids prior to distillation of the mashes in an economical fashion and use of the by product are also of concern to banana processors.

  19. Chemical and thermochemical aspects of the ozonolysis of ethyl oleate: decomposition enthalpy of ethyl oleate ozonide.

    PubMed

    Cataldo, Franco

    2013-01-01

    Neat ethyl oleate was ozonized in a bubble reactor and the progress of the ozonolysis was followed by infrared (FT-IR) spectroscopy and by the differential scanning calorimetry (DSC). The ozonolysis was conducted till a molar ratio O3/C=C≈1 when the exothermal reaction spontaneously went to completion. A specific thermochemical calculation on ethyl oleate ozonation has been made to determine the theoretical heat of the ozonization reaction using the group increment approach. A linear relationship was found both in the integrated absorptivity of the ozonide infrared band at 1110 cm(-1) and the ozonolysis time as well as the thermal decomposition enthalpy of the ozonides and peroxides formed as a result of the ozonation. The DSC decomposition temperature of ozonated ethyl oleate occurs with an exothermal peak at about 150-155 °C with a decomposition enthalpy of 243.0 kJ/mol at molar ratio O3/C=C≈1. It is shown that the decomposition enthalpy of ozonized ethyl oleate is a constant value (≈243 kJ/mol) at any stage of the O3/C=C once an adequate normalization of the decomposition enthalpy for the amount of the adsorbed ozone is taken into consideration. The decomposition enthalpy of ozonized ethyl oleate was also calculated using a simplified thermochemical model, obtaining a result in reasonable agreement with the experimental value.

  20. Chemical and thermochemical aspects of the ozonolysis of ethyl oleate: decomposition enthalpy of ethyl oleate ozonide.

    PubMed

    Cataldo, Franco

    2013-01-01

    Neat ethyl oleate was ozonized in a bubble reactor and the progress of the ozonolysis was followed by infrared (FT-IR) spectroscopy and by the differential scanning calorimetry (DSC). The ozonolysis was conducted till a molar ratio O3/C=C≈1 when the exothermal reaction spontaneously went to completion. A specific thermochemical calculation on ethyl oleate ozonation has been made to determine the theoretical heat of the ozonization reaction using the group increment approach. A linear relationship was found both in the integrated absorptivity of the ozonide infrared band at 1110 cm(-1) and the ozonolysis time as well as the thermal decomposition enthalpy of the ozonides and peroxides formed as a result of the ozonation. The DSC decomposition temperature of ozonated ethyl oleate occurs with an exothermal peak at about 150-155 °C with a decomposition enthalpy of 243.0 kJ/mol at molar ratio O3/C=C≈1. It is shown that the decomposition enthalpy of ozonized ethyl oleate is a constant value (≈243 kJ/mol) at any stage of the O3/C=C once an adequate normalization of the decomposition enthalpy for the amount of the adsorbed ozone is taken into consideration. The decomposition enthalpy of ozonized ethyl oleate was also calculated using a simplified thermochemical model, obtaining a result in reasonable agreement with the experimental value. PMID:23969233

  1. 40 CFR 721.3152 - Ethanaminium, N-ethyl-2-hydroxy-N,N-bis(2-hydroxyethyl)-, diester with C12-18 fatty acids, ethyl...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...-bis(2-hydroxyethyl)-, diester with C12-18 fatty acids, ethyl sulfates (salts). 721.3152 Section 721... Ethanaminium, N-ethyl-2-hydroxy-N,N-bis(2-hydroxyethyl)-, diester with C12-18 fatty acids, ethyl sulfates... ethanaminium, N-ethyl-2-hydroxy-N,N-bis(2-hydroxyethyl)-, diester with C12-18 fatty acids, ethyl...

  2. Estimation of measurement uncertainty for the quantification of 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid and its glucuronide in urine using liquid chromatography-tandem mass spectrometry.

    PubMed

    Kim, Jin Young; Kwon, Woonyong; Kim, Hee Seung; Suh, Sungill; In, Moon Kyo

    2014-04-01

    Recently, the estimation of the measurement uncertainty has become a significant issue in the quality control of forensic drug testing. In the present study, the uncertainty of the measurement was calculated for the quantification of 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) and its glucuronide conjugate (THC-COOH-glu) in urine using liquid chromatography-tandem mass spectrometry. The procedure was based on liquid-liquid extraction of a volume of urine (800 µL) with ethyl acetate. The sources of uncertainty were identified and classified into four major categories as follows: standard preparation, calibration curve, method precision and bias. The overall contribution of combined standard uncertainty on THC-COOH increased in the order of standard preparation (0.9%), method precision (10.4%), calibration curve (30.3%) and bias (58.4%) and, while calibration curve (53.0%) and bias (40.4%) gave the bigger contributions to the combined standard uncertainty for THC-COOH-glu than method precision and standard preparation, which accounted for 6.3 and 0.3%, respectively. The reliability of a measurement was expressed by stating the expanded uncertainty of the measurement result at 95% confidence level. The concentrations of THC-COOH and THC-COOH-glu in the urine sample with their expanded uncertainties were 10.20 ± 1.14 ng/mL and 25.42 ± 5.01 ng/mL, respectively.

  3. Development, validation and application of a comprehensive stereoselective LC/MS-MS assay for bupropion and oxidative, reductive, and glucuronide metabolites in human urine.

    PubMed

    Teitelbaum, Aaron M; Flaker, Alicia M; Kharasch, Evan D

    2016-08-01

    A stereoselective assay was developed for the quantification of bupropion and oxidative, reductive, and glucuronide metabolites (16 analytes total) in human urine. Initially, authentic glucuronide standards obtained from commercial sources were found to be incorrectly labeled with regard to stereochemistry; the correct stereochemistry was unequivocally reassigned. A trifurcated urine sample preparation and analysis procedure was employed for the stereoselective analysis of bupropion, hydroxybupropion, erythrohydrobupropion, and threohydrobupropion enantiomers, and hydroxybupropion, erythrohydrobupropion and threohydrobupropion β-d-glucuronide diastereomers in urine. Method 1 stereoselectively analyzed bupropion (R and S), and unconjugated free hydroxybupropion (R,R and S,S), erythrohydrobupropion (1R,2S and 1S,2R), and threohydrobupropion (1R,2R and 1S,2S) using chiral chromatography with an α1-acid glycoprotein column. Because no hydroxybupropion β-d-glucuronide standards were commercially available, method 2 stereoselectively analyzed total hydroxybupropion aglycones (R,R and S,S-hydroxybupropion) after urine hydrolysis by β-glucuronidase. Hydroxybupropion β-d-glucuronide (R,R and S,S) urine concentrations were calculated as the difference between total and free hydroxybupropion (R,R and S,S) concentrations. Due to incomplete β-glucuronidase hydrolysis of erythrohydrobupropion and threohydrobupropion β-d-glucuronide diastereomers, method 3 stereoselectively analyzed intact erythrohydrobupropion and threohydrobupropion β-d-glucuronide diastereomers using C18 column chromatography. All analytes were quantified by positive ion electrospray tandem mass spectrometry. The assay was fully validated over analyte-specific concentrations. Intra- and inter assay precision were within 15% for each analyte. The limits of quantification for bupropion (R and S), hydroxybupropion (R,R and S,S), threohydrobupropion (1S,2S and 1R,2R), erythrohydrobupropion (1R,2S and 1S,2R

  4. Development, validation and application of a comprehensive stereoselective LC/MS-MS assay for bupropion and oxidative, reductive, and glucuronide metabolites in human urine.

    PubMed

    Teitelbaum, Aaron M; Flaker, Alicia M; Kharasch, Evan D

    2016-08-01

    A stereoselective assay was developed for the quantification of bupropion and oxidative, reductive, and glucuronide metabolites (16 analytes total) in human urine. Initially, authentic glucuronide standards obtained from commercial sources were found to be incorrectly labeled with regard to stereochemistry; the correct stereochemistry was unequivocally reassigned. A trifurcated urine sample preparation and analysis procedure was employed for the stereoselective analysis of bupropion, hydroxybupropion, erythrohydrobupropion, and threohydrobupropion enantiomers, and hydroxybupropion, erythrohydrobupropion and threohydrobupropion β-d-glucuronide diastereomers in urine. Method 1 stereoselectively analyzed bupropion (R and S), and unconjugated free hydroxybupropion (R,R and S,S), erythrohydrobupropion (1R,2S and 1S,2R), and threohydrobupropion (1R,2R and 1S,2S) using chiral chromatography with an α1-acid glycoprotein column. Because no hydroxybupropion β-d-glucuronide standards were commercially available, method 2 stereoselectively analyzed total hydroxybupropion aglycones (R,R and S,S-hydroxybupropion) after urine hydrolysis by β-glucuronidase. Hydroxybupropion β-d-glucuronide (R,R and S,S) urine concentrations were calculated as the difference between total and free hydroxybupropion (R,R and S,S) concentrations. Due to incomplete β-glucuronidase hydrolysis of erythrohydrobupropion and threohydrobupropion β-d-glucuronide diastereomers, method 3 stereoselectively analyzed intact erythrohydrobupropion and threohydrobupropion β-d-glucuronide diastereomers using C18 column chromatography. All analytes were quantified by positive ion electrospray tandem mass spectrometry. The assay was fully validated over analyte-specific concentrations. Intra- and inter assay precision were within 15% for each analyte. The limits of quantification for bupropion (R and S), hydroxybupropion (R,R and S,S), threohydrobupropion (1S,2S and 1R,2R), erythrohydrobupropion (1R,2S and 1S,2R

  5. Theoretical study of the decomposition of ethyl and ethyl 3-phenyl glycidate.

    PubMed

    Josa, Daniela; Peña-Gallego, Angeles; Rodríguez-Otero, Jesús; Cabaleiro-Lago, Enrique M

    2013-01-01

    The mechanism of the decomposition of ethyl and ethyl 3-phenyl glycidate in gas phase was studied by density functional theory (DFT) and MP2 methods. A proposed mechanism for the reaction indicates that the ethyl side of the ester is eliminated as ethylene through a concerted six-membered cyclic transition state, and the unstable intermediate glycidic acid decarboxylates rapidly to give the corresponding aldehyde. Two possible pathways for glycidic acid decarboxylation were studied: one via a five-membered cyclic transition state, and the other via a four-membered cyclic transition state. The results of the calculations indicate that the decarboxylation reaction occurs via a mechanism with five-membered cyclic transition state.

  6. Evidence for differences in regioselective and stereoselective glucuronidation of silybin diastereomers from milk thistle (Silybum marianum) by human UDP-glucuronosyltransferases.

    PubMed

    Jančová, Petra; Siller, Michal; Anzenbacherová, Eva; Křen, Vladimír; Anzenbacher, Pavel; Simánek, Vilím

    2011-09-01

    The flavonolignan silybin, the main component of silymarin, extract from the seeds of Silybum marianum, is used mostly as a hepatoprotectant. Silybin is almost 1:1 mixture of two diastereomers A and B. The individual UDP-glucuronosyltransferases (UGTs) contributing to the metabolism of silybin diastereomers have not been identified yet. In this study, the contribution of UGTs to silybin metabolism was examined. The potential silybin metabolites were formed in vitro by incubating silybin (i) with the human liver microsomal fraction, (ii) with human hepatocytes and finally (iii) with 12 recombinant UGTs (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15 and 2B17). High-performance liquid chromatographic (HPLC) techniques with UV detection and additionally MS detection were used for metabolite identification. Hepatocytes and microsomes formed silybin A-7-O-β-D-glucuronides, B-7-O-β-D-glucuronides, A-20-O-β-D-glucuronides and B-20-O-β-D-glucuronides. With recombinant UGTs, the major role of the UGT1A1, 1A3, 1A8 and 1A10 enzymes but also of the UGT1A6, 1A7, 1A9, 2B7 and 2B15 in the stereoselective reactions leading to the respective silybin glucuronides was confirmed. UGT1A4, UGT2B4 and UGT2B17 did not participate in silybin glucuronidation. The predominant formation of 7-O-β-D-glucuronides and the preferential glucuronidation of silybin B diastereomer in vitro by human UGTs were confirmed.

  7. Farnesol is glucuronidated in human liver, kidney and intestine in vitro, and is a novel substrate for UGT2B7 and UGT1A1

    PubMed Central

    2004-01-01

    Farnesol is an isoprenoid found in many aromatic plants and is also produced in humans, where it acts on numerous nuclear receptors and has received considerable attention due to its apparent anticancer properties. Although farnesol has been studied for over 30 years, its metabolism has not been well characterized. Recently, farnesol was shown to be metabolized by cytochromes P450 in rabbit; however, neither farnesol hydroxylation nor glucuronidation in humans have been reported to date. In the present paper, we show for the first time that farnesol is metabolized to farnesyl glucuronide, hydroxyfarnesol and hydroxyfarnesyl glucuronide by human tissue microsomes, and we identify the specific human UGTs (uridine diphosphoglucuronosyltransferases) involved. Farnesol metabolism was examined by a sensitive LC (liquid chromatography)–MS/MS method. Results indicate that farnesol is a good substrate for glucuronidation in human liver, kidney and intestine microsomes (values in nmol/min per mg). Initial analysis using expressed human UGTs indicated that UGTs 1A1 and 2B7 were primarily responsible for glucuronidation in vitro, with significantly lower activity for all the other UGTs tested (UGTs 1A3, 1A4, 1A6, 1A9 and 2B4). Kinetic analysis and inhibition experiments indicate that, in liver microsomes, UGT1A1 is primarily responsible for farnesol glucuronidation; however, in intestine microsomes, UGT2B7 is probably the major isoform involved, with a very-low-micromolar Km. We also show the first direct evidence that farnesol can be metabolized to hydroxyfarnesol by human liver microsomes and that hydroxyfarnesol is metabolized further to hydroxyfarnesyl glucuronide. Thus glucuronidation may modulate the physiological and/or pharmacological properties of this potent signalling molecule. PMID:15320866

  8. Effect of galactosamine-induced hepatic UDP-glucuronic acid depletion on acetaminophen elimination in rats. Dispositional differences between hepatically and extrahepatically formed glucuronides of acetaminophen and other chemicals.

    PubMed

    Gregus, Z; Madhu, C; Goon, D; Klaassen, C D

    1988-01-01

    Galactosamine (GAL) markedly depletes hepatic UDP-glucuronic acid (UDP-GA) whereas extrahepatic UDP-GA is minimally affected. This suggests that GAL predominantly inhibits hepatic glucuronidation. Therefore, the effect of GAL-induced hepatic UDP-GA depletion was examined in bile duct-cannulated rats to determine the role of hepatic glucuronidation in the disposition of acetaminophen (AA). GAL markedly altered the fate of AA-glucuronide but had little or no effect upon other AA metabolites. GAL decreased the biliary excretion of AA-glucuronide up to 92%, whereas reductions in blood levels and urinary excretion of AA-glucuronide did not exceed 50%. This suggests that AA-glucuronide excreted in bile is predominantly of hepatic origin whereas AA-glucuronide found in blood and urine is derived from both hepatic and extrahepatic tissues. Data in the present and previous studies [Gregus, Watkins, Thompson, Klaassen: J. Pharmacol. Exp. Ther. 225, 256, (1983)] indicate that GAL greatly reduced the biliary excretion of AA- and valproic acid-glucuronide whereas the biliary excretion of the glucuronides of phenolphthalein, iopanoic acid, bilirubin, and diethylstilbestrol was only partially decreased. This difference appears to be largely due to differential contributions by the liver and extrahepatic tissues in the glucuronidation of various compounds as well as the availability of glucuronides formed in extrahepatic tissues for biliary excretion. Specifically, the extrahepatically formed glucuronide conjugates of AA and valproic acid are not readily available for biliary excretion whereas the glucuronides of the other compounds are readily excreted into bile.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Pulse radiolysis of aqueous solutions of ethyl acrylate and hydroxy ethyl acrylate

    NASA Astrophysics Data System (ADS)

    Safrany, A.; Biro, A.; Wojnarovits, L.

    1993-10-01

    Ethyl- and hydroxy ethyl acrylate show high reactivities with hydrated electron and hydroxyl radical intermediates of water radiolysis. The electron adduct reversibly protonate with pK values of 5.7 and 7.3. The adducts may take part in irreversible protonation at the β carbon atom forming α-carboxyl alkyl radicals. Same type of radical forms in reaction of acrylates with OH: at low concentration the adduct mainly disappear in self termination reactions. Above 5 mmol dm -1 the signals showed the startup of oligomerization.

  10. An immunoassay for the detection of triclosan-O-glucuronide, a primary human urinary metabolite of triclosan.

    PubMed

    Ranganathan, Anupama; Gee, Shirley J; Hammock, Bruce D

    2015-09-01

    Triclosan-O-glucuronide (TCSG) is one of the primary urinary metabolites of the antibacterial compound triclosan or TCS that is found in many personal care products and consumer goods. We have developed a competitive, indirect heterologous ELISA for the detection of the target TCSG in urine. Such an ELISA for TCSG could be developed as a useful tool to measure this important biomarker of human exposure to TCS. Immunogens were prepared by conjugating TCSG to thyroglobulin, via heterobifunctional cross-linkers AEDP or 3-[(2-aminoethyl)dithio] propionic acid•hydrochloride and TFCS or N-[ε-trifluoroacetylcaproyloxy]succinimide ester. The coating antigen was prepared by the direct conjugation of TCSG to bovine serum albumin. Antibodies raised in rabbits 2619, 2621 (immunogen TCSG-AEDP-Thy), and 2623 (immunogen TCSG-TFCS-Thy), and the coating antigen were screened and characterized to determine their optimal concentrations. The optimized ELISA, developed with antibody 2621, gave an IC50 value of 2.85 ng/mL, with the linear range (IC20-IC80) determined to be 2.6-24.8 ng/mL. Selectivity of the assay was assessed by measuring cross-reactivity of antibody 2621 to related congeners such as the aglycone TCS, triclosan-O-sulfate, triclocarban, a polybrominated diphenyl ether derivative, and 3-phenoxybenzyl alcohol glucuronide. There was virtually no recognition by antibody 2621 to any of these cross-reactants. Graphical Abstract Urinary biomarker analysis of triclosan glucuronide. PMID:26255293

  11. Effect of inducers and inhibitors of glucuronidation on the biliary excretion and choleretic action of valproic acid in the rat.

    PubMed

    Watkins, J B; Klaassen, C D

    1982-02-01

    Valproic acid (VPA) induces an immediate choleresis in the rat which may be attributable to the osmotic properties of VPA-glucuronic acid conjugates in bile. The influence of inducers and inhibitors of glucuronidation of VPA on the biliary excretion and choleretic effect of VPA was studied. Hepatic UDP-glucuronyltransferase activity toward VPA was determined in vitro. Pretreatment with phenobarbital (75 mg/kg/day for 4 days) enhanced VPA glucuronidation; borneol (750 mg/kg) decreased VPA conjugation; 3-methylcholanthrene (20 mg/kg/day for 4 days) and galactosamine (600 mg/kg) had no effect on glucuronidation of VPA in vitro. Hepatic UDP-glucuronic acid content was decreased by borneol and galactosamine administration and was enhanced by phenobarbital and 3-methylcholanthrene pretreatment. The enzyme inducers increased the plasma disappearance of VPA in vivo but did not augment its biliary excretion or choleretic effect. Borneol and galactosamine, which inhibited the conjugation and plasma disappearance of VPA, decreased its biliary excretion and inhibited the VPA-induced increase in bile flow. Thus, the bile flow rate after VPA administration is closely related to the excretion of VPA-glucuronic acid. These data support the conclusion that the choleretic effect of VPA is due to the osmotic activity of VPA conjugates in bile.

  12. Dimerization of human uridine diphosphate glucuronosyltransferase allozymes 1A1 and 1A9 alters their quercetin glucuronidation activities.

    PubMed

    Liu, Yan-Qing; Yuan, Ling-Min; Gao, Zhang-Zhao; Xiao, Yong-Sheng; Sun, Hong-Ying; Yu, Lu-Shan; Zeng, Su

    2016-01-01

    Uridine diphosphate glucuronosyltransferase 1A (UGT1A) is a major phase II drug-metabolism enzyme superfamily involved in the glucuronidation of endobiotics and xenobiotics in humans. Many polymorphisms in UGT1A genes are reported to inhibit or decrease UGT1A activity. In this study, two UGT1A1 allozymes, UGT1A1 wild-type and a splice mutant, as well as UGT1A9 wild-type and its three UGT1A9 allozymes, UGT1A9*2(C3Y), UGT1A9*3(M33T), and UGT1A9*5(D256N) were single- or double-expressed in a Bac-to-Bac expression system. Dimerization of UGT1A1 or UGT1A9 allozymes was observed via fluorescence resonance energy transfer (FRET) and co-immunoprecipitation analysis. SNPs of UGT1A altered the ability of protein-protein interaction, resulting in differential FRET efficiencies and donor-acceptor r distances. Dimerization changed the chemical regioselectivity, substrate-binding affinity, and enzymatic activity of UGT1A1 and UGT1A9 in glucuronidation of quercetin. These findings provide molecular insights into the consequences of homozygous and heterozygous UGT1A1 and UGT1A9 allozymes expression on quercetin glucuronidation. PMID:27025983

  13. An immunoassay for the detection of triclosan-O-glucuronide, a primary human urinary metabolite of triclosan.

    PubMed

    Ranganathan, Anupama; Gee, Shirley J; Hammock, Bruce D

    2015-09-01

    Triclosan-O-glucuronide (TCSG) is one of the primary urinary metabolites of the antibacterial compound triclosan or TCS that is found in many personal care products and consumer goods. We have developed a competitive, indirect heterologous ELISA for the detection of the target TCSG in urine. Such an ELISA for TCSG could be developed as a useful tool to measure this important biomarker of human exposure to TCS. Immunogens were prepared by conjugating TCSG to thyroglobulin, via heterobifunctional cross-linkers AEDP or 3-[(2-aminoethyl)dithio] propionic acid•hydrochloride and TFCS or N-[ε-trifluoroacetylcaproyloxy]succinimide ester. The coating antigen was prepared by the direct conjugation of TCSG to bovine serum albumin. Antibodies raised in rabbits 2619, 2621 (immunogen TCSG-AEDP-Thy), and 2623 (immunogen TCSG-TFCS-Thy), and the coating antigen were screened and characterized to determine their optimal concentrations. The optimized ELISA, developed with antibody 2621, gave an IC50 value of 2.85 ng/mL, with the linear range (IC20-IC80) determined to be 2.6-24.8 ng/mL. Selectivity of the assay was assessed by measuring cross-reactivity of antibody 2621 to related congeners such as the aglycone TCS, triclosan-O-sulfate, triclocarban, a polybrominated diphenyl ether derivative, and 3-phenoxybenzyl alcohol glucuronide. There was virtually no recognition by antibody 2621 to any of these cross-reactants. Graphical Abstract Urinary biomarker analysis of triclosan glucuronide.

  14. Dimerization of human uridine diphosphate glucuronosyltransferase allozymes 1A1 and 1A9 alters their quercetin glucuronidation activities

    PubMed Central

    Liu, Yan-Qing; Yuan, Ling-Min; Gao, Zhang-Zhao; Xiao, Yong-Sheng; Sun, Hong-Ying; Yu, Lu-Shan; Zeng, Su

    2016-01-01

    Uridine diphosphate glucuronosyltransferase 1A (UGT1A) is a major phase II drug-metabolism enzyme superfamily involved in the glucuronidation of endobiotics and xenobiotics in humans. Many polymorphisms in UGT1A genes are reported to inhibit or decrease UGT1A activity. In this study, two UGT1A1 allozymes, UGT1A1 wild-type and a splice mutant, as well as UGT1A9 wild-type and its three UGT1A9 allozymes, UGT1A9*2(C3Y), UGT1A9*3(M33T), and UGT1A9*5(D256N) were single- or double-expressed in a Bac-to-Bac expression system. Dimerization of UGT1A1 or UGT1A9 allozymes was observed via fluorescence resonance energy transfer (FRET) and co-immunoprecipitation analysis. SNPs of UGT1A altered the ability of protein-protein interaction, resulting in differential FRET efficiencies and donor-acceptor r distances. Dimerization changed the chemical regioselectivity, substrate-binding affinity, and enzymatic activity of UGT1A1 and UGT1A9 in glucuronidation of quercetin. These findings provide molecular insights into the consequences of homozygous and heterozygous UGT1A1 and UGT1A9 allozymes expression on quercetin glucuronidation. PMID:27025983

  15. Bioanalytical LC-MS Method for the Quantification of Plasma Androgens and Androgen Glucuronides in Breast Cancer.

    PubMed

    Kalogera, Eleni; Pistos, Constantinos; Provatopoulou, Xeni; Christophi, Costas A; Zografos, George C; Stefanidou, Maria; Spiliopoulou, Chara; Athanaselis, Sotirios; Gounaris, Antonia

    2016-04-01

    The physiological and pathological development of the breast is strongly affected by the hormonal milieu consisting of steroid hormones. Mass spectrometry (MS) technologies of high sensitivity and specificity enable the quantification of androgens and consequently the characterization of the hormonal status. The aim of this study is the assessment of plasma androgens and androgen glucuronides, in the par excellence hormone-sensitive tissue of the breast, through the application of liquid chromatography-mass spectrometry (LC-MS). A simple and efficient fit-for-purpose method for the simultaneous identification and quantification of dehydroepiandrosterone sulfate (DHEAS), androstenedione (A4), androsterone glucuronide (ADTG) and androstane-3α, 17β-diol-17-glucuronide (3α-diol-17G) in human plasma was developed and validated. The presented method permits omission of derivatization, requires a single solid-phase extraction procedure and the chromatographic separation can be achieved on a single C18 analytical column, for all four analytes. The validated method was successfully applied for the analysis of 191 human plasma samples from postmenopausal women with benign breast disease (BBD), lobular neoplasia (LN), ductal carcinoma in situ and invasive ductal carcinoma (IDC). DHEAS plasma levels exhibited significant differences between LN, IDC and BBD patients (P < 0.05). Additionally, ADTG levels were significantly higher in patients with LN compared with those with BBD (P < 0.05). PMID:26762957

  16. Optimization of ethyl ester production assisted by ultrasonic irradiation.

    PubMed

    Noipin, K; Kumar, S

    2015-01-01

    This study presents the optimization of the continuous flow potassium hydroxide-catalyzed synthesis of ethyl ester from palm oil with ultrasonic assistance. The process was optimized by application of factorial design and response surface methodology. The independent variables considered were ethanol to oil molar ratio, catalyst concentration, reaction temperature and ultrasonic amplitude; and the response was ethyl ester yield. The results show that ethanol to oil molar ratio, catalyst concentration, and ultrasonic amplitude have positive effect on ethyl ester yield, whereas reaction temperature has negative influence on ethyl ester yield. Second-order models were developed to predict the responses analyzed as a function of these three variables, and the developed models predicts the results in the experimental ranges studied adequately. This study shows that ultrasonic irradiation improved the ethyl ester production process to achieve ethyl ester yields above 92%. PMID:25116594

  17. 21 CFR 172.872 - Methyl ethyl cellulose.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Methyl ethyl cellulose. 172.872 Section 172.872... CONSUMPTION Multipurpose Additives § 172.872 Methyl ethyl cellulose. The food additive methyl ethyl cellulose... a cellulose ether having the general formula [C6H(10 -x-y)O5(CH3)x(C2H5)y]n, where x is the...

  18. 21 CFR 172.872 - Methyl ethyl cellulose.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Methyl ethyl cellulose. 172.872 Section 172.872... CONSUMPTION Multipurpose Additives § 172.872 Methyl ethyl cellulose. The food additive methyl ethyl cellulose... a cellulose ether having the general formula [C6H(10 -x-y)O5(CH3)x(C2H5)y]n, where x is the...

  19. 21 CFR 172.872 - Methyl ethyl cellulose.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Methyl ethyl cellulose. 172.872 Section 172.872... CONSUMPTION Multipurpose Additives § 172.872 Methyl ethyl cellulose. The food additive methyl ethyl cellulose... a cellulose ether having the general formula [C6H(10 -x-y)O5(CH3)x(C2H5)y]n, where x is the...

  20. Quantitative Determination of Common Urinary Odorants and Their Glucuronide Conjugates in Human Urine

    PubMed Central

    Wagenstaller, Maria; Buettner, Andrea

    2013-01-01

    Our previous study on the identification of common odorants and their conjugates in human urine demonstrated that this substance fraction is a little-understood but nonetheless a promising medium for analysis and diagnostics in this easily accessible physiological medium. Smell as an indicator for diseases, or volatile excretion in the course of dietary processes bares high potential for a series of physiological insights. Still, little is known today about the quantitative composition of odorous or volatile targets, as well as their non-volatile conjugates, both with regard to their common occurrence in urine of healthy subjects, as well as in that of individuals suffering from diseases or other physiological misbalancing. Accordingly, the aim of our study was to develop a highly sensitive and selective approach to determine the common quantitative composition of selected odorant markers in healthy human subjects, as well as their corresponding glucuronide conjugates. We used one- and two-dimensional high resolution gas chromatography-mass spectrometry in combination with stable isotope dilution assays to quantify commonly occurring and potent odorants in human urine. The studies were carried out on both native urine and on urine that had been treated by glucuronidase assays, with analysis of the liberated odor-active compounds using the same techniques. Analytical data are discussed with regard to their potential translation as future diagnostic tool. PMID:24958143

  1. Toxicological evaluation of acyl glucuronides utilizing half-lives, peptide adducts, and immunostimulation assays.

    PubMed

    Iwamura, Atsushi; Ito, Masahito; Mitsui, Hideaki; Hasegawa, Jun; Kosaka, Keigo; Kino, Ichiro; Tsuda, Minoru; Nakajima, Miki; Yokoi, Tsuyoshi; Kume, Toshiyuki

    2015-12-25

    Chemical reactivity of acyl glucuronides (AGs) is believed to be involved in the toxicity of carboxylic acid-containing drugs. Both direct and immune-mediated toxicity have been suggested as possible mechanisms of toxicity; however, it remains unclear. In the present study, we performed assays of half-lives, peptide adducts, and immunostimulation to evaluate the potential risk of AGs of 21 drugs and analyzed the relationship to the toxic category. AGs of all withdrawn drugs tested in this study showed short half-lives and peptide adducts formation, but so did those of several safe drugs. In contrast, only AGs of withdrawn and warning drugs induced interleukin-8 (IL-8) in human peripheral blood mononuclear cells (hPBMCs). Using a DNA microarray assay, we found that zomepirac AG induced the mRNAs of 5 genes, including IL-8 in hPBMCs. In addition, withdrawn and warning drugs were distinguished from safe drugs by an integrated score of relative mRNA expression levels of 5 genes. The immunostimulation assay showed higher sensitivity, specificity, and accuracy compared with other methods. In preclinical drug development, the evaluation of the reactivity of AGs using half-lives and peptide adducts assays followed by the evaluation of immunostimulation by highly reactive AGs using hPBMCs can contribute to improved drug safety.

  2. Quercetin-3-O-glucuronide induces ABCA1 expression by LXRα activation in murine macrophages

    SciTech Connect

    Ohara, Kazuaki; Wakabayashi, Hideyuki; Taniguchi, Yoshimasa; Shindo, Kazutoshi; Yajima, Hiroaki; Yoshida, Aruto

    2013-11-29

    Highlights: •The major circulating quercetin metabolite (Q3GA) activated LXRα. •Q3GA induced ABCA1 via LXRα activation in macrophages. •Nelumbo nucifera leaf extracts contained quercetin glycosides. •N. nucifera leaf extract feeding elevated HDLC in mice. -- Abstract: Reverse cholesterol transport (RCT) removes excess cholesterol from macrophages to prevent atherosclerosis. ATP-binding cassette, subfamily A, member 1 (ABCA1) is a crucial cholesterol transporter involved in RCT to produce high density lipoprotein-cholesterol (HDLC), and is transcriptionally regulated by liver X receptor alpha (LXRα), a nuclear receptor. Quercetin is a widely distributed flavonoid in edible plants which prevented atherosclerosis in an animal model. We found that quercetin-3-O-glucuronide (Q3GA), a major quercetin metabolite after absorption from the digestive tract, enhanced ABCA1 expression, in vitro, via LXRα in macrophages. In addition, leaf extracts of a traditional Asian edible plant, Nelumbo nucifera (NNE), which contained abundant amounts of quercetin glycosides, significantly elevated plasma HDLC in mice. We are the first to present experimental evidence that Q3GA induced ABCA1 in macrophages, and to provide an alternative explanation to previous studies on arteriosclerosis prevention by quercetin.

  3. Transmembrane transport of steviol glucuronide and its potential interaction with selected drugs and natural compounds.

    PubMed

    Wang, Meiyu; Qi, Huixin; Li, Jiajun; Xu, Yunting; Zhang, Hongjian

    2015-12-01

    Steviol glucuronide (SVG) is the major metabolite derived from steviol, the aglycone of stevioside and rebaudioside A. After the ingestion of stevioside and rebaudioside A, SVG is formed and excreted into the urine in humans. In the present study, transporter mediated efflux and uptake of SVG was investigated in order to understand molecular mechanisms underlying its renal clearance. Results showed that SVG was not a substrate of efflux transporters BCRP, MRP2, MATE1 or P-gp. In contrast, OAT3 played a predominant role in the uptake of SVG in comparison to OATP1B1, OATP1B3, or OATP2B1. Quercetin, telmisartan, diclofenac, and mulberrin displayed a relatively strong inhibition against OAT3 mediated uptake of SVG with IC50 values of 1.8, 2.9, 8.0, and 10.0 μM, respectively. Because OAT3 is a major uptake transporter in the kidney, inhibition of OAT3 activity may alter SVG's renal clearance by drugs and natural compounds that are used concomitantly with stevia leaf extracts. PMID:26525112

  4. UHPLC-MS/MS quantification of buprenorphine, norbuprenorphine, methadone, and glucuronide conjugates in umbilical cord plasma.

    PubMed

    Kyle, Amy Redmond; Carmical, Jennifer; Shah, Darshan; Pryor, Jason; Brown, Stacy

    2015-10-01

    Opioid use during pregnancy can result in the newborn being physically dependent on the substance, thus experiencing drug withdrawal, termed neonatal abstinence syndrome (NAS). Buprenorphine and methadone are two drugs used to treat opioid withdrawal and are approved for use in pregnancy. Quantification of these compounds in umbilical cord plasma would help assess in utero exposure of neonates in cases of buprenorphine or methadone use during pregnancy. An LC-MS/MS method using solid-phase extraction sample preparation was developed and validated for the simultaneous quantification of methadone, buprenorphine, norbuprenorphine, and glucuronide metabolites in umbilical cord plasma. The average accuracy (percentage error) and precision (relative standard deviation) were <15% for each validated concentration. Our data establishes a 2 week maximum freezer storage window in order to achieve the most accurate cord plasma concentrations of these analytes. Additionally, we found that the umbilical cord tissue analysis was less sensitive compared with analysis with umbilical cord blood plasma, indicating that this may be a more appropriate matrix for determination of buprenorphine and metabolite concentrations. This method was successfully applied to the analysis of cord blood from women with known buprenorphine or methadone use during pregnancy. PMID:25808363

  5. Chemical and enzyme-assisted syntheses of norbuprenorphine-3-β-D-glucuronide.

    PubMed

    Fan, Jinda; Brown, Sarah M; Tu, Zhude; Kharasch, Evan D

    2011-04-20

    Norbuprenorphine-3-β-d-glucuronide (nBPN-3-β-d-G, 1) is a major phase II metabolite of buprenorphine, a pharmaceutical used for the treatment of opioid addiction. The pharmacological activity of compound 1 is not clear because investigations have been limited by the lack of chemically pure, well characterized 1 in sufficient quantities for in vitro and in vivo experiments. This work describes two concise, new methods of synthesis of 1, a chemical and an enzyme-assisted synthesis. The chemical synthesis used a strategy based on a combination of Koenig-Knorr coupling and amino-silyl protection. The enzyme-assisted synthesis used dog liver to convert the substrate norbuprenorphine (nBPN, 2) to 1. Both methods provided 1, characterized by (1)H NMR and tandem mass spectrometry, with purity >96%. The fractional yield of the enzyme-assisted synthesis was greater than that of the chemical synthesis (67% vs 5.3%), but due to larger reaction volumes, the chemical synthesis afforded greater amounts of total 1. PMID:21434652

  6. Investigation on stability of transporter protein, glucuronide transporter from Escherichia coli.

    PubMed

    Ishii, Noriyuki

    2010-06-01

    The glucuronide transporter GusB, the product of the gusB gene from Escherichia coli, is responsible for detoxification of metabolites. In this study, we successfully expressed GusB homologously in E. coli and investigated its oligomeric state in n-dodecyl-beta-D: -maltoside (DDM) detergent solution. Evidence for a pentameric state with a Stokes radius of 57 +/- 2 A for the purified GusB protein in DDM solution was obtained by analytical size-exclusion HPLC. The elution peak corresponding to pentameric GusB is commonly seen in elution profiles in the different buffer systems examined over a wide pH range. Hence, it is likely that GusB resides in the membrane as a pentamer. Stability studies with different incubation periods with the typical lipids, such as dimyristoylphosphatidylcholine, and total E. coli phospholipids, as the representatives of both phosphatidylcholine and phosphatidylethanolamine, show some clues to two-dimensional crystallization of GusB with lipids. PMID:20490474

  7. Evidence of in vitro glucuronidation and enzymatic transformation of paralytic shellfish toxins by healthy human liver microsomes fraction.

    PubMed

    García, Carlos; Rodriguez-Navarro, Alberto; Díaz, Juan Carlos; Torres, Rafael; Lagos, Néstor

    2009-02-01

    Paralytic Shellfish Toxins (PST) are endemic components found in filter bivalves in Southern Chile. Post-mortems analysis of fluid and tissue samples has shown biotransformation of PST in humans. The Gonyautoxin 3 (GTX3) and Gonyautoxin 2 (GTX2) are the major PST components in the toxin profile found in Chilean shellfish extracts, being as much as 65% of the total content of PST in filter bivalves. Therefore, they are the major accountable components of the human intoxication by shellfish consumption. The aim of this study is to show in vitro glucuronidation and biotransformation of GTX3 and GTX2 when they are incubated with microsomal fraction isolated from healthy human livers. Microsomes fractions isolated from human livers were incubated with GTX3 and GTX2 purified from contaminated mussels. After different incubation times, incubated samples were extracted and analyzed by HPLC with fluorescent on line detection and HPLC-MS analysis. The results revealed that GTX3 and GTX2, only when they were incubated with microsomal fraction and appropriated cofactors, showed to be enzymatic transformed in vitro. The glucuronidation of GTX3 and GTX2 followed typical Michaelis-Menten kinetics, resulting in apparent kinetic parameters of Km=39.4+/-0.24 microM and Vmax=6.0x10(-3) pmol/min/mg protein. In addition, the microsomes fraction also oxidized GTX3 and GTX2 into Gonyautoxin 4 (GTX 4) and Gonyautoxin 1 (GTX 1) resulting in 0.339x10(-3) pmol/min/mg protein. In conclusion, this study reports oxidation and glucuronidation of GTX3 and GTX2 when they are incubated with human liver microsomal fraction. The metabolism occurs via a glucuronidation reaction, the basis first step of biotransformation in human liver. Also it is showed that GTX4 and GTX1 came by biotransformation from GTX3 and GTX2 in humans. This data confirm human biotransformation found in human post-mortem fluid and tissue samples described previously. This data is the first evidence of in vitro glucuronidation

  8. Ethyl Lithiodiazoacetate: Extremely Unstable Intermediate Handled Efficiently in Flow.

    PubMed

    Müller, Simon T R; Hokamp, Tobias; Ehrmann, Svenja; Hellier, Paul; Wirth, Thomas

    2016-08-16

    Ethyl diazoacetate (EDA) is one of the most prominent diazo reagents. It is frequently used in metal-carbene-type reactions. However, EDA can also be used as a nucleophile under base catalysis. Whilst the addition of EDA to aldehydes can be performed using organic bases, the addition of EDA to other carbonyl electrophiles requires the use of organometallics such as lithium diisopropylamide (LDA). The generated ethyl lithiodiazoacetate is highly reactive and decomposes rapidly, even at low temperatures. Herein, we report a continuous flow protocol that overcomes the problems associated with the instantaneous decomposition of ethyl lithiodiazoacetate. The addition of ethyl lithiodiazoacetate to ketones provides direct access to tertiary diazoalcohols in good yields.

  9. Transport of estradiol-17β-glucuronide, estrone-3-sulfate and taurocholate across the endoplasmic reticulum membrane: evidence for different transport systems.

    PubMed

    Wlcek, Katrin; Hofstetter, Lia; Stieger, Bruno

    2014-03-01

    Important reactions of drug metabolism, including UGT mediated glucuronidation and steroidsulfatase mediated hydrolysis of sulfates, take place in the endoplasmic reticulum (ER) of hepatocytes. Consequently, UGT generated glucuronides, like estradiol-17β-glucuronide, have to be translocated back into the cytoplasm to reach their site of excretion. Also steroidsulfatase substrates, including estrone-3-sulfate, have to cross the ER membrane to reach their site of hydrolysis. Based on their physicochemical properties such compounds are not favored for passive diffusion and therefore likely necessitate transport system(s) to cross the ER membrane in either direction. The current study aims to investigate the transport of taurocholate, estradiol-17β-glucuronide, and estrone-3-sulfate in smooth (SER) and rough (RER) endoplasmic reticulum membrane vesicles isolated from Wistar and TR(-) rat liver. Time-dependent and bidirectional transport was demonstrated for taurocholate, showing higher uptake rates in SER than RER vesicles. For estradiol-17β-glucuronide a fast time-dependent efflux with similar efficiencies from SER and RER but no clear protein-mediated uptake was shown, indicating an asymmetric transport system for this substrate. Estrone-3-sulfate uptake was time-dependent and higher in SER than in RER vesicles. Inhibition of steroidsulfatase mediated estrone-3-sulfate hydrolysis decreased estrone-3-sulfate uptake but had no effect on taurocholate or estradiol-17β-glucuronide transport. Based on inhibition studies and transport characteristics, three different transport mechanisms are suggested to be involved in the transport of taurocholate, estrone-3-sulfate and estradiol-17β-glucuronide across the ER membrane.

  10. Transport of estradiol-17β-glucuronide, estrone-3-sulfate and taurocholate across the endoplasmic reticulum membrane: evidence for different transport systems☆

    PubMed Central

    Wlcek, Katrin; Hofstetter, Lia; Stieger, Bruno

    2014-01-01

    Important reactions of drug metabolism, including UGT mediated glucuronidation and steroidsulfatase mediated hydrolysis of sulfates, take place in the endoplasmic reticulum (ER) of hepatocytes. Consequently, UGT generated glucuronides, like estradiol-17β-glucuronide, have to be translocated back into the cytoplasm to reach their site of excretion. Also steroidsulfatase substrates, including estrone-3-sulfate, have to cross the ER membrane to reach their site of hydrolysis. Based on their physicochemical properties such compounds are not favored for passive diffusion and therefore likely necessitate transport system(s) to cross the ER membrane in either direction. The current study aims to investigate the transport of taurocholate, estradiol-17β-glucuronide, and estrone-3-sulfate in smooth (SER) and rough (RER) endoplasmic reticulum membrane vesicles isolated from Wistar and TR− rat liver. Time-dependent and bidirectional transport was demonstrated for taurocholate, showing higher uptake rates in SER than RER vesicles. For estradiol-17β-glucuronide a fast time-dependent efflux with similar efficiencies from SER and RER but no clear protein-mediated uptake was shown, indicating an asymmetric transport system for this substrate. Estrone-3-sulfate uptake was time-dependent and higher in SER than in RER vesicles. Inhibition of steroidsulfatase mediated estrone-3-sulfate hydrolysis decreased estrone-3-sulfate uptake but had no effect on taurocholate or estradiol-17β-glucuronide transport. Based on inhibition studies and transport characteristics, three different transport mechanisms are suggested to be involved in the transport of taurocholate, estrone-3-sulfate and estradiol-17β-glucuronide across the ER membrane. PMID:24406246

  11. Spironolactone and canrenone inhibit UGT2B7-catalyzed human liver and kidney microsomal aldosterone 18beta-glucuronidation: a potential drug interaction.

    PubMed

    Knights, Kathleen M; Bowalgaha, Kushari; Miners, John O

    2010-07-01

    Elevated plasma concentrations of aldosterone (ALDO) are observed in patients treated with spironolactone. Because ALDO is eliminated via UGT2B7-catalyzed 18beta-glucuronidation, this study aimed to determine whether spironolactone and its primary metabolites, canrenone and canrenoic acid, inhibit ALDO 18beta-glucuronidation by recombinant UGT2B7 and by human liver (HLM) and human kidney cortical (HKCM) microsomes. Initial experiments characterized the effects of all three compounds on 4-methylumbelliferone and ALDO glucuronidation by recombinant human UGT2B7. IC(50) values for spironolactone and canrenone ranged from 26 to 50 microM, whereas canrenoic acid was a weak inhibitor. Inhibitor constant (K(i)) values for spironolactone and canrenone inhibition of ALDO 18beta-glucuronidation were subsequently determined with HLM, HKCM, and UGT2B7 as the enzyme sources. Spironolactone and canrenone were competitive inhibitors of ALDO 18beta-glucuronidation by HLM, HKCM, and UGT2B7. Mean (+/-) K(i) values for spironolactone were 52 +/- 22 (HLM) and 34 +/- 4 microM (HKCM), and mean (+/-) K(i) values for canrenone were 41 +/- 19 (HLM) and 23 +/- 2 microM (HKCM). K(i) values for spironolactone and canrenone inhibition of ALDO 18beta-glucuronidation by recombinant UGT2B7 were 23 and 11 microM, respectively. "Actual" K(i) values for spironolactone and canrenone inhibition of ALDO 18beta-glucuronidation, which take into account the role of endogenous microsomal inhibitors, are predicted to be 3 to 5 and 2 to 4 microM, respectively. The data indicate that the elevated ALDO concentrations observed in patients treated with spironolactone may be due, at least in part, to a pharmacokinetic interaction, and spironolactone and canrenone should be considered to be potential inhibitors of the UGT2B7-mediated metabolic clearance of drugs in both liver and kidney. PMID:20304966

  12. Effect of MRP2 and MRP3 Polymorphisms on Anastrozole Glucuronidation and MRP2 and MRP3 Gene Expression in Normal Liver Samples

    PubMed Central

    Edavana, Vineetha Koroth; Penney, Rosalind B; Yao- Borengasser, Aiwei; Starlard-Davenport, Athena; Dhakal, Ishwori B; Kadlubar, Susan

    2015-01-01

    Anastrozole is an aromatase inhibitor (AI) used as adjuvant therapy for breast cancer. Anastrozole is subject to direct glucuronidation catalyzed by UDP-glucuronosyltransferase1A4 (UGT1A4). Interindividual variability in anastrozole glucuronidation may be affected by UGT1A4 SNPs. Interplay between drug metabolizing genes such as UGT1A4 and transporter genes may also be affected by genetic variability. Thus, we hypothesize that genetic variability in MRPs could influence anastrozole glucuronidation. The correlation between UGT1A4 and MRP2 or MRP3 transporter gene expressions and the correlation between MRP2 or MRP3 mRNA and anastrozole glucuronidation were analyzed in normal human liver samples. MRP2 and MRP3 mRNA levels were significantly correlated with UGT1A4 mRNA, with anastrozole glucuronidation and with each other (p<0.05). The data also demonstrated that MRP2 SNPs are positively correlated with MRP2 mRNA expression, while there was no association between MRP3 SNPs from this study and MRP3 expression. Significant correlations (p<0.05) between certain MRP2 SNPs (3972C>T, 2366C>T and −24C>T) and anastrozole glucuronidation were observed. There were no observed correlations between MRP3 SNPs and anastrozole glucuronidation. MRP2 polymorphisms have been identified as playing a role in the disposition of other drugs, and the data presented here indicate for the first time that MRP2 SNPs could influence anastrozole metabolism and contribute to interindividual variation in treatment responses. PMID:26985457

  13. Glucuronidation of bavachinin by human tissues and expressed UGT enzymes: Identification of UGT1A1 and UGT1A8 as the major contributing enzymes.

    PubMed

    Lv, Xia; Hou, Jie; Xia, Yang-Liu; Ning, Jing; He, Gui-Yuan; Wang, Ping; Ge, Guang-Bo; Xiu, Zhi-Long; Yang, Ling

    2015-10-01

    Bavachinin (BCI), a major bioactive compound in Chinese herbal Psoralea corylifolia, possesses a wide range of biological activities. In this study, the glucuronidation pathway of BCI was characterized for the first time, by using pooled human liver microsomes (HLM), pooled human intestine microsomes (HIM) and recombinant human UDP-glucosyltransferases (UGTs). One mono-glucuronide was detected in HLM in the presence of uridine-diphosphate glucuronic acid (UDPGA), and it was biosynthesized and well-characterized as BCI-4'-O-glucuronide (BCIG). Reaction phenotyping assay showed that UGT1A1, UGT1A3 and UGT1A8 were involved in BCI-4'-O-glucuronidation, while UGT1A1 and UGT1A8 displayed the higher catalytic ability among all tested UGT isoforms. Kinetic analysis demonstrated that BCI-4'-O-glucuronidation in both HLM and UGT1A1 followed sigmoidal kinetic behaviors and displayed much close Km values (12.4 μM in HLM & 9.7 μM in UGT1A1). Both chemical inhibition assays and correlation analysis demonstrated that UGT1A1 displayed a predominant role in BCI-4'-O-glucuronidation in HLM. Both HIM and UGT1A8 exhibited substrate inhibition at high concentrations, and Km values of HIM and UGT1A8 were 3.6 and 2.3 μM, respectively. Similar catalytic efficiencies were observed for HIM (199.3 μL/min/mg) and UGT1A8 (216.2 μL/min/mg). These findings suggested that UGT1A1 and UGT1A8 were the primary isoforms involved in BCI-4'-O-glucuronidation in HLM, and HIM, respectively. PMID:26320626

  14. Phase II metabolism in human skin: skin explants show full coverage for glucuronidation, sulfation, N-acetylation, catechol methylation, and glutathione conjugation.

    PubMed

    Manevski, Nenad; Swart, Piet; Balavenkatraman, Kamal Kumar; Bertschi, Barbara; Camenisch, Gian; Kretz, Olivier; Schiller, Hilmar; Walles, Markus; Ling, Barbara; Wettstein, Reto; Schaefer, Dirk J; Itin, Peter; Ashton-Chess, Joanna; Pognan, Francois; Wolf, Armin; Litherland, Karine

    2015-01-01

    Although skin is the largest organ of the human body, cutaneous drug metabolism is often overlooked, and existing experimental models are insufficiently validated. This proof-of-concept study investigated phase II biotransformation of 11 test substrates in fresh full-thickness human skin explants, a model containing all skin cell types. Results show that skin explants have significant capacity for glucuronidation, sulfation, N-acetylation, catechol methylation, and glutathione conjugation. Novel skin metabolites were identified, including acyl glucuronides of indomethacin and diclofenac, glucuronides of 17β-estradiol, N-acetylprocainamide, and methoxy derivatives of 4-nitrocatechol and 2,3-dihydroxynaphthalene. Measured activities for 10 μM substrate incubations spanned a 1000-fold: from the highest 4.758 pmol·mg skin(-1)·h(-1) for p-toluidine N-acetylation to the lowest 0.006 pmol·mg skin(-1)·h(-1) for 17β-estradiol 17-glucuronidation. Interindividual variability was 1.4- to 13.0-fold, the highest being 4-methylumbelliferone and diclofenac glucuronidation. Reaction rates were generally linear up to 4 hours, although 24-hour incubations enabled detection of metabolites in trace amounts. All reactions were unaffected by the inclusion of cosubstrates, and freezing of the fresh skin led to loss of glucuronidation activity. The predicted whole-skin intrinsic metabolic clearances were significantly lower compared with corresponding whole-liver intrinsic clearances, suggesting a relatively limited contribution of the skin to the body's total systemic phase II enzyme-mediated metabolic clearance. Nevertheless, the fresh full-thickness skin explants represent a suitable model to study cutaneous phase II metabolism not only in drug elimination but also in toxicity, as formation of acyl glucuronides and sulfate conjugates could play a role in skin adverse reactions.

  15. [Direct metabolites of ethanol as biological markers of alcohol use: basic aspects and applications].

    PubMed

    Thon, N; Weinmann, W; Yegles, M; Preuss, U; Wurst, F M

    2013-09-01

    In addition to self reports and questionnaires, biomarkers are of relevance in the diagnosis of and therapy for alcohol use disorders. Traditional biomarkers such as gamma-glutamyl transpeptidase or mean corpuscular volume are indirect biomarkers and are subject to the influence of age, gender and non-alcohol related diseases, among others. Direct metabolites of ethanol such as ethyl glucuronide (EtG), ethyl sulphate (EtS) and phosphatidylethanol (PEth) are direct metabolites of ethanol, that are positive after intake of ethyl alcohol. They represent useful diagnostic tools for identifying alcohol use even more accurately than traditional biomarkers. Each of these drinking indicators remains positive in serum and urine for a characteristic time spectrum after the cessation of ethanol intake - EtG and EtS in urine up to 7 days, EtG in hair for months after ethanol has left the body. Applications include clinical routine use, emergency room settings, proof of abstinence in alcohol rehabilitation programmes, driving under influence offenders, workplace testing, assessment of alcohol intake in the context of liver transplantation and foetal alcohol syndrome. Due to their properties, they open up new perspectives for prevention, interdisciplinary cooperation, diagnosis of and therapy for alcohol-related problems.

  16. Cognitive effects of creatine ethyl ester supplementation.

    PubMed

    Ling, Jonathan; Kritikos, Minos; Tiplady, Brian

    2009-12-01

    Supplementation with creatine-based substances as a means of enhancing athletic performance has become widespread. Until recently, however, the effects of creatine supplementation on cognitive performance has been given little attention. This study used a new form of creatine--creatine ethyl ester--to investigate whether supplementation would improve performance in five cognitive tasks, using a double-blind, placebo-controlled study. Creatine dosing led to an improvement over the placebo condition on several measures. Although creatine seems to facilitate cognition on some tasks, these results require replication using objective measures of compliance. The improvement is discussed in the context of research examining the influence of brain energy capacity on cognitive performance. PMID:19773644

  17. 21 CFR 172.872 - Methyl ethyl cellulose.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... FOR HUMAN CONSUMPTION (CONTINUED) FOOD ADDITIVES PERMITTED FOR DIRECT ADDITION TO FOOD FOR HUMAN CONSUMPTION Multipurpose Additives § 172.872 Methyl ethyl cellulose. The food additive methyl ethyl cellulose may be safely used in food in accordance with the following prescribed conditions. (a) The additive...

  18. IRIS TOXICOLOGICAL REVIEW OF METHYL ETHYL KETONE (2003 Final)

    EPA Science Inventory

    EPA is announcing the release of the final report, "Toxicological Review of Methyl Ethyl Ketone: in support of the Integrated Risk Information System (IRIS)". The updated Summary for Methyl Ethyl Ketone and accompanying Quickview have also been added to the IRIS Database.

  19. Glucuronidation of the oxidative cytochrome P450-mediated phenolic metabolites of the endocrine disruptor pesticide: methoxychlor by human hepatic UDP-glucuronosyl transferases.

    PubMed

    Hazai, Eszter; Gagne, Peter V; Kupfer, David

    2004-07-01

    Methoxychlor, a currently used pesticide, is a proestrogen exhibiting estrogenic activity in mammals in vivo. Methoxychlor undergoes oxidative metabolism by cytochromes P450, yielding 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane (mono-OH-M) and 1,1,1-trichloro-2,2-bis(4-hydroxyphenyl)ethane (bis-OH-M) as main metabolites. Since humans may be exposed to these estrogenic metabolites, which are potential substrates of UDP-glucuronosyltransferases (UGTs), their glucuronide conjugation was investigated with human liver preparations and individual UGTs. Incubation of both mono-OH-M and bis-OH-M with human liver microsomes formed monoglucuronides. The structures of the glucuronides were identified by liquid chromatography/tandem mass spectometry. Examination of cDNA-expressed recombinant human hepatic UGTs revealed that several catalyze glucuronidation of both compounds. Among the cDNA-expressed UGT1A enzymes, UGT1A9 seemed to be the main catalyst of formation of mono-OH-M-glucuronide, whereas UGT1A3 seemed to be the most active in bis-OH-M-glucuronide formation. Furthermore, the chiral selectivity of mono-OH-M glucuronidation was examined. The results of the incubation of single enantiomers generally agreed with the chiral analyses of mono-OH-M derived from the glucuronidase digestion of the glucuronides of the racemic mono-OH-M. There was a relatively slight but consistent enantioselective preference of individual UGT1A1, UGT1A3, UGT1A9, and UGT2B15 enzymes for glucuronidation of the S- over the R-mono-OH-M, whereas in human liver microsomes differences were observed among donors in generating the respective R/S-mono-OH-M ratio. Since it was previously shown that human liver microsomes demethylate methoxychlor mainly into S-mono-OH-M, the observation that UGT1A isoforms preferentially glucuronidate the S-mono-OH-M suggests a suitable mechanism for eliminating this major enantiomer. This enantiomeric preference, however, is not extended to all samples of

  20. Disruption of thyroid hormone homeostasis in Ugt1a-deficient Gunn rats by microsomal enzyme inducers is not due to enhanced thyroxine glucuronidation

    SciTech Connect

    Richardson, Terrilyn A.; Klaassen, Curtis D.

    2010-10-01

    Microsomal enzyme inducers (MEI) that increase UDP-glucuronosyltransferases (UGTs) are thought to increase glucuronidation of thyroxine (T{sub 4}), thus reducing serum T{sub 4}, and subsequently increasing thyroid stimulating hormone (TSH). Ugt1a1 and Ugt1a6 mediate T{sub 4} glucuronidation. Therefore, this experiment determined the involvement of Ugt1a enzymes in increased T{sub 4} glucuronidation, decreased serum T{sub 4}, and increased TSH after MEI treatment. Male Wistar and Ugt1a-deficient Wistar (Gunn) rats were fed a control diet or diet containing pregnenolone-16{alpha}-carbonitrile (PCN; 800 ppm), 3-methylcholanthrene (3-MC; 200 ppm), or Aroclor 1254 (PCB; 100 ppm) for 7 days. Serum T{sub 4}, triiodothyronine (T{sub 3}), and TSH concentrations, hepatic T{sub 4}/T{sub 3} glucuronidation, and thyroid histology and follicular cell proliferation were investigated. PCN, 3-MC, and PCB treatments decreased serum T{sub 4}, whereas serum T{sub 3} was maintained in both Gunn and Wistar rats (except for PCB treatment). TSH was increased in Wistar and Gunn rats after PCN (130 and 277%) or PCB treatment (72 and 60%). T{sub 4} glucuronidation in Wistar rats was increased after PCN (298%), 3-MC (85%), and PCB (450%), but was extremely low in Gunn rats, and unchanged after MEI. T{sub 3} glucuronidation was increased after PCN (121%) or PCB (58%) in Wistar rats, but only PCN increased T{sub 3} glucuronidation in Gunn rats (43%). PCN treatment induced thyroid morphological changes and increased follicular cell proliferation in both strains. These data demonstrate that T{sub 4} glucuronidation cannot be increased in Ugt1a-deficient Gunn rats. Thus, the decrease in serum T{sub 4}, increase in TSH, and increase in thyroid cell proliferation after MEI are not dependent on increased T{sub 4} glucuronidation, and cannot be attributed to Ugt1a enzymes.

  1. Electronic structure and normal vibrations of the 1-ethyl-3-methylimidazolium ethyl sulfate ion pair.

    PubMed

    Dhumal, Nilesh R; Kim, Hyung J; Kiefer, Johannes

    2011-04-21

    Electronic and structural properties of the ion pair 1-ethyl-3-methylimidazolium ethyl sulfate are studied using density functional methods. Three locally stable conformers of the ion pair complex are considered to analyze molecular interactions between its cation and anion. Manifestations of these interactions in the vibrational spectra are discussed and compared with experimental IR and Raman spectroscopy data. NBO analysis and difference electron density coupled with molecular electron density topography are used to interpret the frequency shifts of the normal vibrations of the ion pair, compared to the free anion and cation. Excitation energies of low-lying singlet excited states of the conformers are also studied. The density functional theory results are found to be in a reasonable agreement with experimental UV/vis absorption spectra.

  2. Bisphenol A glucuronide deconjugation is a determining factor of fetal exposure to bisphenol A.

    PubMed

    Gauderat, Glenn; Picard-Hagen, Nicole; Toutain, Pierre-Louis; Corbel, Tanguy; Viguié, Catherine; Puel, Sylvie; Lacroix, Marlène Z; Mindeguia, Pierre; Bousquet-Melou, Alain; Gayrard, Véronique

    2016-01-01

    Previous studies in experimental animals have shown that maternal exposure to bisphenol A (BPA) during late pregnancy leads to high plasma concentrations of BPA glucuronide (BPAG) in fetus compared to mother due to the inability of BPAG to cross the placental barrier. A recent in vitro study has reported that BPAG can exert adipogenic effect underlining the need for characterization of the fetal disposition of BPAG. Experiments were conducted in chronically catheterized fetal sheep to determine the contribution of BPAG hydrolysis to BPA to the elimination of BPAG from the fetal compartment and its resulting effect on the overall fetal exposure to free BPA. Serial sampling of fetal arterial blood, amniotic fluid, maternal venous blood and urine was performed following separate single doses of BPA and BPAG administered intravenously to eight fetal/maternal pairs after cesarean section, and repeated BPAG doses given to two fetal sheep. On average 67% of the BPA entering the fetal circulation was rapidly eliminated through fetal to maternal clearance, with a very short half-life (20 min), while the remaining fraction (24%) was glucuronoconjugated. BPA conjugation-deconjugation cycling was responsible for a 43% increase of the overall fetal exposure to free BPA. A very specific pattern of fetal exposure to free BPA was observed due to its highly increased persistence with a hydrolysis-dependent plasma terminal free BPA half-life of several tens of hours. These findings suggest that although the high fetal to maternal clearance of free BPA protects the fetus from transient increases in free BPA plasma concentrations associated with maternal BPA intake, low but sustained basal free BPA concentrations are maintained in the fetus through BPA conjugation-deconjugation cycling. The potential health implications of these low but sustained basal concentrations of free BPA in fetal plasma should be addressed especially when considering time-dependent effects. PMID:26540084

  3. Rapid methods to enumerate Escherichia coli in foods using 4-methylumbelliferyl-beta-D-glucuronide.

    PubMed

    Ekholm, D F; Hirshfield, I N

    2001-01-01

    Three methods to enumerate Escherichia coli in food were compared. They were based on AOAC methods using lauryl tryptose broth (LST) medium, LST-4-methylumbelliferyl-beta-D-glucuronide (MUG) medium, and a proposed method using regular LST in combination with E. coli (EC)-MUG medium. An efficacious and cost-effective method is needed that can detect E. coli and does not produce false presumptive positives. We tested 170 cheeses, 40 frozen processed seafood samples, 210 tree nuts, and 40 other samples. The method of choice for enumerating E. coli depends on the commodity itself. For a product, such as hard cheese or processed seafood, with a history of being negative for E. coli and other lactose-fermenting organisms, the proposed method using regular LST/EC-MUG is a good choice. These samples were seldom presumptive positive in the primary LST medium. If gas was produced, EC-MUG was an effective secondary medium. No false positives (fluorescence) or negatives were detected in EC-MUG medium. For a product with a history of being positive for E. coli and/or other lactose fermenting organisms, such as tree nutmeats or cheeses that are ripened by bacteria or mold, the method using LST-MUG is the method of choice. A presumptive positive in the LST-MUG medium was highly correlative with the biochemical tests that confirmed a sample contain E. coli. For samples spiked with E. coli, the results from each of these 3 methods were identical, and were consistent in enumerating E. coli. PMID:11324605

  4. Morphine-6-glucuronide: analgesic effects and receptor binding profile in rats

    SciTech Connect

    Abbott, F.V.; Palmour, R.M.

    1988-01-01

    The antinociceptive effects of morphine-6-glucuronide (M6G) were examined in two animal models of pain, the tail immersion test (reflex withdrawal to noxious heat) and the formalin test (behavioral response to minor tissue injury). In the tail immersion test, M6G produced and increase in withdrawal latency that rose rapidly between 0.01 and 0.025 ug ICV or 1 and 2 mg/kg SC. A further increase occurred at doses greater than 0.2 ug ICV or 4 mg/kg SC and was associated with marked catelepsy and cyanosis. Naloxone, 0.1 mg/kg SC, shifted the lower component of the dose-effect relation by a factor of 24. In the formalin test, 0.01 ug M6G ICV produced hyperalgesia, while between 0.05 and 0.2 ug ICV, antinociception increased rapidly without toxicity. The dose effect relations for hyperalgesia and antinociception were shifted to the right by factors of 20- and 3-fold, respectively. By comparison, ICV morphine was 60 (formalin test) to 145-200 (tail immersion test) times less potent than M6G. At sub-nanomolar concentrations, M6G enhanced the binding of (/sup 3/H)-etorphine, (/sup 3/H)-dihydromorphine and (/sup 3/H)-naloxone to rat brain membrane receptors by 20-40%. At higher concentrations, M6G displaced each ligand from binding sites, with K/sub i/ values of about 30 nM, as compared to morphine K/sub i/ values of about 3 nM.

  5. Correlation of serum 3 alpha-androstanediol glucuronide with acne and chest hair density in men.

    PubMed

    Lookingbill, D P; Egan, N; Santen, R J; Demers, L M

    1988-11-01

    Serum 3 alpha-androstanediol glucuronide (3 alpha-Adiol-G) is considered to be an indicator of peripheral tissue androgen metabolism. Precursor circulating androgens are converted in peripheral tissue to dihydrotestosterone (DHT), which is ultimately metabolized to 3 alpha-Adiol-G and secreted from the cell. Elevated serum 3 alpha-Adiol-G concentrations have been reported in women in hyperandrogenic states. We studied 44 consecutive male medical students for chest hair density, acne, and serum dehydroepiandrosterone sulfate (DHEA-S), total testosterone (total T), free and albumin-bound (bioavailable) T (bio T), and 3 alpha-Adiol-G concentrations. Although there was considerable overlap of serum 3 alpha-Adiol-G values among the groups defined by hair density or acne scores, we found statistically significant correlations between serum 3 alpha-Adiol-G and chest hairiness (P = 0.0034), acne (P = 0.0005), and a combined chest hairiness and acne score (P = 0.0018). There was no significant correlation between these clinical parameters and the levels of precursor androgens. There was, however, a strong correlation between serum 3 alpha-Adiol-G and bio T (P = 0.0005), suggesting that in men serum 3 alpha-Adiol-G levels may be dependent upon available free and albumin-bound T. The correlations in men of serum 3 alpha-Adiol-G with chest hair density, acne, and the hairiness and acne index supports the hypothesis that the serum levels of 3 alpha-Adiol-G reflect the extent of androgen action in peripheral tissues. PMID:2972739

  6. Elucidation of the Mechanisms through Which the Reactive Metabolite Diclofenac Acyl Glucuronide Can Mediate Toxicity.

    PubMed

    Scialis, Renato J; Manautou, José E

    2016-04-01

    We have previously reported that mice lacking the efflux transporter Mrp3 had significant intestinal injury after toxic diclofenac (DCF) challenge, and proposed that diclofenac acyl glucuronide (DCF-AG), as a substrate of Mrp3, played a part in mediating injury. Since both humans and mice express the uptake transporter OATP2B1 in the intestines, OATP2B1 was characterized for DCF-AG uptake. In vitro assays using human embryonic kidney (HEK)-OATP2B1 cells demonstrated that DCF-AG was a substrate with a maximal velocity (Vmax) and Km of 17.6 ± 1.5 pmol/min per milligram and 14.3 ± 0.1 μM, respectively. Another key finding from our in vitro assays was that DCF-AG was more cytotoxic compared with DCF, and toxicity occurred within 1-3 hours of exposure. We also report that 1 mM DCF-AG caused a 6-fold increase in reactive oxygen species (ROS) by 3 hours. Investigation of oxidative stress through inhibition of superoxide dismutase (SOD) revealed that DCF-AG had 100% inhibition of SOD at the highest tested dose of 1 mM. The SOD and ROS results strongly suggest DCF-AG induced oxidative stress in vitro. Lastly, DCF-AG was screened for pharmacologic activity against COX-1 and COX-2 and was found to have IC50 values of 0.620 ± 0.105 and 2.91 ± 0.36 μM, respectively, which represents a novel finding. Since cyclooxygenase (COX) inhibition can lead to intestinal ulceration, it is plausible that DCF-AG can also contribute to enteropathy via COX inhibition. Taken in context, the work presented herein demonstrated the multifactorial pathways by which DCF-AG can act as a direct contributor to toxicity following DCF administration.

  7. Bisphenol A glucuronide deconjugation is a determining factor of fetal exposure to bisphenol A.

    PubMed

    Gauderat, Glenn; Picard-Hagen, Nicole; Toutain, Pierre-Louis; Corbel, Tanguy; Viguié, Catherine; Puel, Sylvie; Lacroix, Marlène Z; Mindeguia, Pierre; Bousquet-Melou, Alain; Gayrard, Véronique

    2016-01-01

    Previous studies in experimental animals have shown that maternal exposure to bisphenol A (BPA) during late pregnancy leads to high plasma concentrations of BPA glucuronide (BPAG) in fetus compared to mother due to the inability of BPAG to cross the placental barrier. A recent in vitro study has reported that BPAG can exert adipogenic effect underlining the need for characterization of the fetal disposition of BPAG. Experiments were conducted in chronically catheterized fetal sheep to determine the contribution of BPAG hydrolysis to BPA to the elimination of BPAG from the fetal compartment and its resulting effect on the overall fetal exposure to free BPA. Serial sampling of fetal arterial blood, amniotic fluid, maternal venous blood and urine was performed following separate single doses of BPA and BPAG administered intravenously to eight fetal/maternal pairs after cesarean section, and repeated BPAG doses given to two fetal sheep. On average 67% of the BPA entering the fetal circulation was rapidly eliminated through fetal to maternal clearance, with a very short half-life (20 min), while the remaining fraction (24%) was glucuronoconjugated. BPA conjugation-deconjugation cycling was responsible for a 43% increase of the overall fetal exposure to free BPA. A very specific pattern of fetal exposure to free BPA was observed due to its highly increased persistence with a hydrolysis-dependent plasma terminal free BPA half-life of several tens of hours. These findings suggest that although the high fetal to maternal clearance of free BPA protects the fetus from transient increases in free BPA plasma concentrations associated with maternal BPA intake, low but sustained basal free BPA concentrations are maintained in the fetus through BPA conjugation-deconjugation cycling. The potential health implications of these low but sustained basal concentrations of free BPA in fetal plasma should be addressed especially when considering time-dependent effects.

  8. The liver X-receptor alpha controls hepatic expression of the human bile acid-glucuronidating UGT1A3 enzyme in human cells and transgenic mice.

    PubMed

    Verreault, Mélanie; Senekeo-Effenberger, Kathy; Trottier, Jocelyn; Bonzo, Jessica A; Bélanger, Julie; Kaeding, Jenny; Staels, Bart; Caron, Patrick; Tukey, Robert H; Barbier, Olivier

    2006-08-01

    Glucuronidation, an important bile acid detoxification pathway, is catalyzed by enzymes belonging to the UDP-glucuronosyltransferase (UGT) family. Among UGT enzymes, UGT1A3 is considered the major human enzyme for the hepatic C24-glucuronidation of the primary chenodeoxycholic (CDCA) and secondary lithocholic (LCA) bile acids. We identify UGT1A3 as a positively regulated target gene of the oxysterol-activated nuclear receptor liver X-receptor alpha (LXRalpha). In human hepatic cells and human UGT1A transgenic mice, LXRalpha activators induce UGT1A3 mRNA levels and the formation of CDCA-24glucuronide (24G) and LCA-24G. Furthermore, a functional LXR response element (LXRE) was identified in the UGT1A3 promoter by site-directed mutagenesis, electrophoretic mobility shift assays and chromatin immunoprecipitation experiment. In addition, LXRalpha is found to interact with the SRC-1alpha and NCoR cofactors to regulate the UGT1A3 gene, but not with PGC-1beta. In conclusion, these observations establish LXRalpha as a crucial regulator of bile acid glucuronidation in humans and suggest that accumulation of oxysterols in hepatocytes during cholestasis favors bile acid detoxification as glucuronide conjugates. LXR agonists may be useful for stimulating both bile acid detoxification and cholesterol removal in cholestatic or hypercholesterolemic patients, respectively. PMID:16871576

  9. Cytotoxicity of aniline mustard glucuronide alone or in a combination with glucose in Walker cells in culture and sarcoma-180 tumour bearing animals.

    PubMed

    Deliconstantinos, G; Ramantanis, G; Todorou, D K

    1983-01-01

    The effect of aniline mustard glucuronide (AMG), p-hydroxyaniline mustard (HAM), and aniline mustard (AM), on Walker ascites tumour cells in vitro showed that AM in about 80 times more toxic than its glucuronide but HAM is at least 800 times more toxic. A non toxic dose of AMG became completely lethal to Walker tumour cells in vitro, if bovine liver beta-glucuronidase was added to the incubation medium. Prior treatment of Walker tumour cells in vitro with glucose, increased the breakdown of AMG to HAM within the intact cells, while a non-toxic dose of the glucuronide became completely lethal to cells pretreated with glucose. The administration of AMG in combination with glucose to animals bearing the highly resistant to alkylating agents Sarcoma-180 tumour, increased the toxicity of the glucuronide but produced a slight effect on tumour growth. Glucose administration in Sarcoma-180 and ADJ/PC6 tumour bearing animals did not alter the tumour intracellular pH determined in vivo indirectly from the distribution of the weak non-metabolizable organic acid 5,5-dimethyl-2,4-oxazolinedione (DMO) between intra- and extra-cellular water. The present data suggest that the combination of aniline mustard glucuronide with glucose, could be effective in those tumours which have a high beta-glucuronidase activity and a lower tumour intracellular pH could be induced by glucose.

  10. 40 CFR 180.483 - O-[2-(1,1-Dimethylethyl)-5-pyrimidinyl] O-ethyl-O-(1-methyl-ethyl) phosphorothioate; tolerances...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false O- O-ethyl-O-(1-methyl-ethyl... FOOD Specific Tolerances § 180.483 O- O-ethyl-O-(1-methyl-ethyl) phosphorothioate; tolerances for residues. Time-limited tolerances are established for residues of the insecticide O-...

  11. Spectroscopy reveals that ethyl esters interact with proteins in wine.

    PubMed

    Di Gaspero, Mattia; Ruzza, Paolo; Hussain, Rohanah; Vincenzi, Simone; Biondi, Barbara; Gazzola, Diana; Siligardi, Giuliano; Curioni, Andrea

    2017-02-15

    Impairment of wine aroma after vinification is frequently associated to bentonite treatments and this can be the result of protein removal, as recently demonstrated for ethyl esters. To evaluate the existence of an interaction between wine proteins and ethyl esters, the effects induced by these fermentative aroma compounds on the secondary structure and stability of VVTL1, a Thaumatin-like protein purified from wine, was analyzed by Synchrotron Radiation Circular Dichroism (SRCD) spectroscopy. The secondary structure of wine VVTL1 was not strongly affected by the presence of selected ethyl esters. In contrast, VVTL1 stability was slightly increased by the addition of ethyl-octanoate, -decanoate and -dodecanoate, but decreased by ethyl-hexanoate. This indicates the existence of an interaction between VVTL1 and at least some aroma compounds produced during fermentation. The data suggest that proteins removal from wine by bentonite can result in indirect removal of at least some aroma compounds associated with them. PMID:27664648

  12. Mechanistic insight into alkylation of the ethyl acetoacetate anion with different ethyl halides

    NASA Astrophysics Data System (ADS)

    Marković, S.; Đurđević, J.; Vukosavljević, M.; Petrović, Z.

    2013-12-01

    The alkylation reactions of the ambident ethyl acetoacetate anion with C2H5X (X = F, Cl, Br, and I) in the O2, C3, and O4 positions of the anion were investigated at the B3LYP/6-311+G( d,p) level of theory. It was found that the ethylation reaction does not occur in the position O4, as well as with ethyl fluoride in any position of the anion, due to very high activation energies and thermodynamic instability of the hypothetic products. The activation energies for the reactions in the position O2 are lower in comparison to the position C3, but the products of the reactions in the C3 position are more stable than those in the position O4, implying that the C/O products ratio is controlled by both thermodynamic and kinetic factors, leading to the O2-product with the chloride, and C3-product with the iodide as leaving group.

  13. Parameters Affecting Ethyl Ester Production by Saccharomyces cerevisiae during Fermentation▿

    PubMed Central

    Saerens, S. M. G.; Delvaux, F.; Verstrepen, K. J.; Van Dijck, P.; Thevelein, J. M.; Delvaux, F. R.

    2008-01-01

    Volatile esters are responsible for the fruity character of fermented beverages and thus constitute a vital group of aromatic compounds in beer and wine. Many fermentation parameters are known to affect volatile ester production. In order to obtain insight into the production of ethyl esters during fermentation, we investigated the influence of several fermentation variables. A higher level of unsaturated fatty acids in the fermentation medium resulted in a general decrease in ethyl ester production. On the other hand, a higher fermentation temperature resulted in greater ethyl octanoate and decanoate production, while a higher carbon or nitrogen content of the fermentation medium resulted in only moderate changes in ethyl ester production. Analysis of the expression of the ethyl ester biosynthesis genes EEB1 and EHT1 after addition of medium-chain fatty acid precursors suggested that the expression level is not the limiting factor for ethyl ester production, as opposed to acetate ester production. Together with the previous demonstration that provision of medium-chain fatty acids, which are the substrates for ethyl ester formation, to the fermentation medium causes a strong increase in the formation of the corresponding ethyl esters, this result further supports the hypothesis that precursor availability has an important role in ethyl ester production. We concluded that, at least in our fermentation conditions and with our yeast strain, the fatty acid precursor level rather than the activity of the biosynthetic enzymes is the major limiting factor for ethyl ester production. The expression level and activity of the fatty acid biosynthetic enzymes therefore appear to be prime targets for flavor modification by alteration of process parameters or through strain selection. PMID:17993562

  14. Bioactive androgens and glucuronidated androgen metabolites are associated with subcutaneous and ectopic skeletal muscle adiposity among older black men.

    PubMed

    Miljkovic, Iva; Cauley, Jane A; Dressen, Amy S; Gordon, Christopher L; Goodpaster, Bret H; Kuller, Lewis H; Bunker, Clareann H; Patrick, Alan L; Wheeler, Victor W; Orwoll, Eric S; Zmuda, Joseph M

    2011-08-01

    Aging is associated with declining serum levels of androgenic hormones and with increased skeletal muscle fat infiltration, an emerging risk factor for type 2 diabetes mellitus (T2DM). Androgens regulate fat mass and glucose homeostasis, but the effect of androgenic hormones on skeletal muscle fat infiltration is largely unknown. Thus, the aim of the current study was to examine the association of serum androgens and their precursors and metabolites with skeletal muscle fat infiltration and T2DM in a black male population group at high risk of T2DM. Serum androgens, estrogens, and androgen precursors and metabolites were measured using mass spectrometry; and calf skeletal muscle fat distribution (subcutaneous and intermuscular fat; skeletal muscle density) was measured using quantitative computed tomography in 472 Afro-Caribbean men 65 years and older. Bioactive androgens, testosterone, free testosterone, and dihydrotestosterone were associated with less skeletal muscle fat infiltration (r = -0.14 to -0.18, P < .05) and increased skeletal muscle density (r = 0.10 to 0.14, P < .05), independent of total adiposity. In addition, glucuronidated androgen metabolites were associated with less subcutaneous fat (r = -0.11 to -0.15, P < .05). Multivariate logistic regression analysis identified an increased level of 3α-diol-3 glucuronide (odds ratio = 1.38, P < .01) and a decreased level of dihydrotestosterone (odds ratio = 0.66, P < .01) to be significantly associated with T2DM. Our findings suggest that, in elderly black men, independent of total adiposity, bioactive androgens and glucuronidated androgen metabolites may play previously unrecognized role in skeletal muscle fat distribution. Longitudinal studies are needed to further evaluate the relationship between androgens and androgen metabolites with changes in skeletal muscle fat distribution with aging and the incidence of T2DM. PMID:21353258

  15. 40 CFR 721.3152 - Ethanaminium, N-ethyl-2-hydroxy-N,N-bis(2-hydroxyethyl)-, diester with C12-18 fatty acids, ethyl...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Ethanaminium, N-ethyl-2-hydroxy-N,N... Ethanaminium, N-ethyl-2-hydroxy-N,N-bis(2-hydroxyethyl)-, diester with C12-18 fatty acids, ethyl sulfates... ethanaminium, N-ethyl-2-hydroxy-N,N-bis(2-hydroxyethyl)-, diester with C12-18 fatty acids, ethyl...

  16. 40 CFR 721.3152 - Ethanaminium, N-ethyl-2-hydroxy-N,N-bis(2-hydroxyethyl)-, diester with C12-18 fatty acids, ethyl...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Ethanaminium, N-ethyl-2-hydroxy-N,N... Ethanaminium, N-ethyl-2-hydroxy-N,N-bis(2-hydroxyethyl)-, diester with C12-18 fatty acids, ethyl sulfates... ethanaminium, N-ethyl-2-hydroxy-N,N-bis(2-hydroxyethyl)-, diester with C12-18 fatty acids, ethyl...

  17. Human UDP-Glucuronosyltransferase 1A1 is the Primary Enzyme Responsible for the N-glucuronidation of N-hydroxy-PhIP in vitro

    SciTech Connect

    Malfatti, M A; Felton, J S

    2004-04-06

    UDP-Glucuronosyltransferase 1A proteins (UGT1A) catalyze the glucuronidation of many endogenous and xenobiotic compounds including heterocyclic amines and their hydroxylated metabolites (the main topic of this study). Studies have shown that in humans UGT1A mediated glucuronidation is an important pathway in the detoxification of food-borne carcinogenic heterocyclic amines. The biotransformation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most mass abundant heterocyclic amine found in cooked meats, is highly dependent on cytochrome P4501A2 hydroxylation followed by UGT catalyzed glucuronidation of the N-hydroxy-PhIP reactive intermediate. To determine which UGT1A proteins are involved in the glucuronidation of N-hydroxy-PhIP, microsomal preparations from baculovirus infected insect cells that express all of the known functional human UGT1A isozymes (UGT1A1, -1A3, -1A4, -1A6, -1A7, -1A8, -1A9, -1A10) were exposed to N-hydroxy-PhIP and the reaction products were isolated by HPLC. All UGT1A proteins except UGT1A6 showed some degree of activity towards N-hydroxy-PhIP. The formation of both N-hydroxy-PhIP-N{sup 2}-glucuronide and N-hydroxy-PhIP-N3-glucuronide was both time and substrate concentration dependent in all the microsomal incubations that showed appreciable activity. UGT1A1 was the most efficient in converting N-hydroxy-PhIP to both conjugates producing 5 times more of the N{sup 2}-conjugate than UGT1A4, the next active UGT, and 286 times more than UGT1A7, the least active UGT. With an apparent Km of 52 {micro}M and a K{sub cat} of 114 min-1, UGT1A1 was also the most catalytically efficient in forming N-hydroxy-PhIP-N{sup 2}-glucuronide. Catalytic constants for UGT1A4, UGT1A8 and UGT1A9 were 52 min-1, 35 min{sup -1} and 3.7 min{sup -1}, respectively. The catalytic efficiency for N-hydroxy-PhIP-N3-glucuronide formation was 8, 10, and 6 times lower for UGT1A1, -1A4, and -1A8, respectively, when compared to the k{sub cat} values for N

  18. Biodegradation of pyrazosulfuron-ethyl by Acinetobacter sp. CW17.

    PubMed

    Wang, Yanhui; Du, Liangwei; Chen, Yingxi; Liu, Xiaoliang; Zhou, Xiaomao; Tan, Huihua; Bai, Lianyang; Zeng, Dongqiang

    2012-03-01

    The pyrazosulfuron-ethyl-degrading bacterium, designated as CW17, was isolated from contaminated soil near the warehouse of the factory producing pyrazosulfuron-ethyl in Changsha city, China. The strain CW17 was identified as Acinetobacter sp. based on analyses of 94 carbon source utilization or chemical sensitivity in Biolog microplates, conventional phenotypic characteristics, and 16S rRNA gene sequencing. When pyrazosulfuron-ethyl was provided as the sole carbon source, the effects of pyrazosulfuron-ethyl concentration, pH, and temperature on biodegradation were examined. The degradation rates of pyrazosulfuron-ethyl at initial concentrations of 5.0, 20.0, and 50.0 mg/L were 48.0%, 77.0%, and 32.6%, respectively, after inoculation for 7 days. The growth of the strain was inhibited at low pH buffers. The chemical degradation occurs much faster at low pH than at neutral and basic pH conditions. The degradation rate of pyrazosulfuron-ethyl at 30°C was faster than those at 20 and 37°C by CW17 strains. Two metabolites of degradation were analyzed by liquid chromatography-mass spectroscopy (LC/MS). Based on the identified products, strain CW17 seemed to be able to degrade pyrazosulfuron-ethyl by cleavage of the sulfonylurea bridge. PMID:22388979

  19. Evaluation of pharmaceutical excipients as cosolvents in 4-methyl umbelliferone glucuronidation in human liver microsomes: applications for compounds with low solubility.

    PubMed

    Argikar, Upendra A; Liang, Guiqing; Bushee, Jennifer L; Hosagrahara, Vinayak P; Lee, Wendy

    2011-01-01

    Standard incubation procedures for carrying out microsomal assays involve the use of less than 1% w/v organic solvents to minimize the potential inhibitory effects of organic solvents on metabolic activity. This presents a practical limitation for poorly soluble xenobiotics, which cannot be incubated at concentrations high enough to obtain a V(max), and therefore subsequent values for K(m) and Cl(int) cannot be calculated. Our goal was to study the application of a variety of pharmaceutical excipients to aid the solubilization of compounds in vitro in glucuronidation incubations, without affecting the reaction kinetics. In vitro glucuronidation incubations were carried out in human liver microsomes with 4-methylumbelliferone (4-MU) and the kinetics of 4-MU glucuronidation in the presence of excipients were compared to that in control incubations without any excipients. In addition, IC(75) values were calculated for each excipient. We observed that HPBCD (Hydroxypropyl-β-cyclodextrin) may be employed in in vitro glucuronidation incubations up to 0.5% w/v without affecting the Cl(int) of 4-MU. Although NMP (N-methyl-2-pyrrolidone) and DMA (N,N-dimethylacetamide); showed low IC(75) values approximately 0.1% w/v each, neither excipients altered the Cl(int) of 4-MUG (4-methylumbelliferyl-β-D-glucuronide) formation. Our studies point toward possible applications of pharmaceutical excipients to carry out in vitro glucuronidation of substrates with poor aqueous solubility, in order to estimate Cl(int) and subsequently scaled organ clearance values.

  20. A novel approach for predicting acyl glucuronide reactivity via Schiff base formation: development of rapidly formed peptide adducts for LC/MS/MS measurements.

    PubMed

    Wang, Jianyao; Davis, Margaret; Li, Fangbiao; Azam, Farooq; Scatina, JoAnn; Talaat, Rasmy

    2004-09-01

    A novel technique to study the reactivity of acyl glucuronide metabolites to protein has been developed and is described herein. Considered here are acyl glucuronide metabolites, which have undergone the rearrangement of the glucuronic acid moiety at physiological temperature and pH. The investigation of the reactivity of these electrophilic metabolites was carried out by measuring the rate of reaction of rearranged AG metabolites in forming the corresponding acyl glucuronide-peptide adduct in the presence of Lys-Phe. This differs from the parallel technique used in forming AG adducts of proteins that have been previously reported. In the study described here, the Schiff base adduct, diclofenac acyl glucuronide-Lys-Phe product, was generated and structurally elucidated by liquid chromatography tandem mass spectrometry (LC/MS/MS) analysis. The product structure was proved to be a Schiff base adduct by chemical derivatization by nucleophilic addition of HCN and chemical reduction with NaCNBH(3), followed by LC/MS/MS analysis. It is proposed here that the degree of reactivity of acyl glucuronides as measured by covalent binding to protein is proportional to the amount of its peptide adduct generated with the peptide technique described. The application of this technique to the assessment of the degree of reactivity of acyl glucuronide metabolites was validated by developing a reactivity rank of seven carboxylic acid-containing drugs. Consistency was achieved between the ranking of reactivity in the peptide technique for these seven compounds and the rankings found in the literature. In addition, a correlation (R(2) = 0.95) was revealed between the formation of a peptide adduct and the rearrangement rate of the primary acyl glucuronide of seven tested compounds. A structure effect on the degree of reactivity has demonstrated the rate order: acetic acid > propionic acid > benzoic acid derivatives. A rational explanation of this order was proposed, based on the inherent

  1. Increased estrogen sulfation of estradiol 17beta-D-glucuronide in metastatic tumor rat livers.

    PubMed

    Sun, Huadong; Liu, Lichuan; Pang, K Sandy

    2006-11-01

    Changes in the disposition of estradiol 17beta-d-glucuronide (E(2)17G), a substrate of the organic anion-transporting polypeptide family (Oatp) and multidrug resistance-associated protein 2 (Mrp2), were examined in livers of male Wag/Rij rats that were injected with CC531 cells intraportally to induce metastatic tumors (n = 5) or with phosphate-buffered saline for sham-operated controls (n = 4). Multiple indicator dilution, single-pass liver perfusions revealed extremely high influx clearances of [(3)H]E(2)17G (>190 ml/min) in both groups. In recirculating liver perfusions, [(3)H]E(2)17G decayed monoexponentially in the reservoir perfusate, and the total (9.19 +/- 1.33 versus 8.18 +/- 0.94 ml/min) and biliary (4.94 +/- 1.07 versus 4.60 +/- 0.86 ml/min) clearances were similar in both groups (P > 0.05). The metabolic clearance of E(2)17G was higher in the tumor group (4.60 +/- 0.64 versus 3.23 +/- 0.23 ml/min, P < 0.05). E(2)3S17G, the 3-sulfate metabolite, whose identity was confirmed by mass spectrometry, appeared only in bile and not perfusate. Liver microsomal incubations of E(2)3(35)S17G and [(3)H]estrone sulfate revealed similar sulfatase activities between the tumor and sham livers, albeit the activities were much lower for E(2)3(35)S17G. Oatp1a1 and Oatp1b2 protein expression in liver membrane fragments was reduced by 42% and 38%, respectively, whereas that of cytosolic estrogen sulfotransferase (Sult1e1) was significantly increased (41%) with tumor (P < 0.05). All of the observations were captured by modeling. From modeling, we showed that reduction of the high influx clearance (546 to 283 ml/min) failed to lower the total clearance of E(2)17G, whereas up-regulation of Sult1e1 increased the E(2)17G sulfation clearance (2.56 to 3.69 ml/min) in livers with metastatic tumors. PMID:16895976

  2. Ethyl Esterification for MALDI-MS Analysis of Protein Glycosylation.

    PubMed

    Reiding, Karli R; Lonardi, Emanuela; Hipgrave Ederveen, Agnes L; Wuhrer, Manfred

    2016-01-01

    Ethyl esterification is a technique for the chemical modification of sialylated glycans, leading to enhanced stability when performing matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS), as well as allowing the efficient detection of both sialylated and non-sialylated glycans in positive ion mode. In addition, the method shows specific reaction products for α2,3- and α2,6-linked sialic acids, leading to an MS distinguishable mass difference. Here, we describe the ethyl esterification protocol for 96 glycan samples, including enzymatic N-glycan release, the aforementioned ethyl esterification, glycan enrichment, MALDI target preparation, and the MS(/MS) measurement. PMID:26700047

  3. On the cause of low thermal stability of ethyl halodiazoacetates

    PubMed Central

    Mortén, Magnus; Hennum, Martin

    2016-01-01

    Summary Rates for the thermal decomposition of ethyl halodiazoacetates (halo = Cl, Br, I) have been obtained, and reported herein are their half-lives. The experimental results are supported by DFT calculations, and we provide a possible explanation for the reduced thermal stability of ethyl halodiazoacetates compared to ethyl diazoacetate and for the relative decomposition rates between the chloro, bromo and iodo analogs. We have also briefly studied the thermal, non-catalytic cyclopropanation of styrenes and compared the results to the analogous Rh(II)-catalyzed reactions. PMID:27559411

  4. A review of morphine and morphine-6-glucuronide's pharmacokinetic-pharmacodynamic relationships in experimental and clinical pain.

    PubMed

    Sverrisdóttir, Eva; Lund, Trine Meldgaard; Olesen, Anne Estrup; Drewes, Asbjørn Mohr; Christrup, Lona Louring; Kreilgaard, Mads

    2015-07-10

    Morphine is a widely used opioid for treatment of moderate to severe pain, but large interindividual variability in patient response and no clear guidance on how to optimise morphine dosage regimen complicates treatment strategy for clinicians. Population pharmacokinetic-pharmacodynamic models can be used to quantify dose-response relationships for the population as well as interindividual and interoccasion variability. Additionally, relevant covariates for population subgroups that deviate from the typical population can be identified and help clinicians in dose optimisation. This review provides a detailed overview of the published human population pharmacokinetic-pharmacodynamic studies for morphine analgesia in addition to basic drug disposition and pharmacological properties of morphine and its analgesic active metabolite, morphine-6-glucuronide, that may help identify future covariates. Furthermore, based on simulations from key pharmacokinetic-pharmacodynamic models, the contribution of morphine-6-glucuronide to the analgesic response in patients with renal insufficiency was investigated. Simulations were also used to examine the impact of effect-site equilibration half-life on time course of response. Lastly, the impact of study design on the likelihood of determining accurate pharmacodynamic parameters for morphine response was evaluated.

  5. Effects of morphine glucuronides on the function of opioid receptors in human SK-N-SH cells.

    PubMed

    Baker, L; Dye, A; Ratka, A

    2000-03-01

    Morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) are active metabolites of morphine. The effects of M3G and M6G on the opioid receptor transduction system has not yet been fully elucidated. Formation of cAMP after treatment with various doses of morphine, M3G, and M6G was studied. M6G and morphine, but not M3G, showed a dose dependent inhibition of cAMP accumulation. Naloxone blocked the inhibitory effect of M6G, M3G, and morphine. Pretreatment with M3G did not change the effects of morphine and M6G. The G-protein inhibitor PTX, prevented morphine, M3G, and M6G effects on cAMP. M3G and M6G vary in their ability to interact with the opioid receptor effector system. Inhibition of cAMP evoked by activation of opioid receptors and inhibitory G-proteins may play a role in the actions of M6G and M3G.

  6. An immunoassay for the detection of triclosan-O-glucuronide, a primary human urinary metabolite of triclosan

    PubMed Central

    Ranganathan, Anupama; Gee, Shirley J.; Hammock, Bruce D.

    2015-01-01

    Triclosan-O-glucuronide (TCSG) is one of the primary urinary metabolites of the antibacterial compound triclosan or TCS that is found in many personal care products and consumer goods. We have developed a competitive, indirect heterologous ELISA for the detection of the target TCSG in urine. Such an ELISA for TCSG could be developed as a useful tool to measure this important biomarker of human exposure to TCS. Immunogens were prepared by conjugating TCSG to thyroglobulin, via heterobifunctional cross-linkers AEDP or 3-[(2-aminoethyl)dithio] propionic acid•hydrochloride and TFCS or N-[ε-trifluoroacetylcaproyloxy]succinimide ester. The coating antigen was prepared by the direct conjugation of TCSG to bovine serum albumin. Antibodies raised in rabbits 2619, 2621 (immunogen TCSG-AEDP-Thy) and 2623 (immunogen TCSG-TFCS-Thy) and the coating antigen were screened and characterized to determine their optimal concentrations. The optimized ELISA, developed with antibody 2621, gave an IC50 value of 2.85 ng/mL, with the linear range (IC20 – IC80) determined to be 2.6 – 24.8 ng/mL. Selectivity of the assay was assessed by measuring cross-reactivity of antibody 2621 to related congeners such as the aglycone TCS, triclosan-O-sulfate, triclocarban, a polybrominated diphenyl ether derivative and 3-phenoxybenzyl alcohol glucuronide. There was virtually no recognition by antibody 2621 to any of these cross-reactants. PMID:26255293

  7. Time-Dependent Metabolism of Luteolin by Human UDP-Glucuronosyltransferases and Its Intestinal First-Pass Glucuronidation in Mice.

    PubMed

    Wu, Lili; Liu, Junjin; Han, Weichao; Zhou, Xuefeng; Yu, Xiaoming; Wei, Qiang; Liu, Shuwen; Tang, Lan

    2015-10-01

    Luteolin is a well-known flavonoid with various pharmacological properties but has low bioavailability due to glucuronidation. This study investigated the time-course of luteolin glucuronidation by 12 human UDP-glucuronosyltransferases (UGTs) and its intestinal first-pass metabolism in mice. Six metabolites, including two novel abundant diglucuronides [3',7-O-diglucuronide (diG) and 4',7-diG] and four known ones, were identified. UGT1A6 and UGT1A9 generated almost only monoglucuronides (G's). The production of 3',7-diG followed a sequential time-dependent process along with decrease of 3'-G mainly by UGT1A1, indicating that 3',7-diG was produced from 3'-G. Metabolism in mice intestine differed from that in humans. Probenecid, a nonspecific UGT inhibitor, did not affect absorption but significantly inhibited production of 7-, 4'-, and 3'-G, and enhanced the formation of another novel metabolite, 5-G, in mice. In conclusion, diglucuronide formation is time-dependent and isoform-specific. UGT1A1 preferentially generates diG, whereas UGT1A6 and UGT1A9 share a preference for G production.

  8. Alcohol Consumption during Pregnancy: Analysis of Two Direct Metabolites of Ethanol in Meconium

    PubMed Central

    Sanvisens, Arantza; Robert, Neus; Hernández, José María; Zuluaga, Paola; Farré, Magí; Coroleu, Wifredo; Serra, Montserrat; Tor, Jordi; Muga, Robert

    2016-01-01

    Alcohol consumption in young women is a widespread habit that may continue during pregnancy and induce alterations in the fetus. We aimed to characterize prevalence of alcohol consumption in parturient women and to assess fetal ethanol exposure in their newborns by analyzing two direct metabolites of ethanol in meconium. This is a cross-sectional study performed in September 2011 and March 2012 in a series of women admitted to an obstetric unit following childbirth. During admission, socio-demographic and substance use (alcohol, tobacco, cannabis, cocaine, and opiates) during pregnancy were assessed using a structured questionnaire and clinical charts. We also recorded the characteristics of pregnancy, childbirth, and neonates. The meconium analysis was performed by liquid chromatography—tandem mass spectrometry (LC-MS/MS) to detect the presence of ethyl glucuronide (EtG) and ethyl sulfate (EtS). Fifty-one parturient and 52 neonates were included and 48 meconium samples were suitable for EtG and EtS detection. The median age of women was 30 years (interquartile range (IQR): 26–34 years); EtG was present in all meconium samples and median concentration of EtG was 67.9 ng/g (IQR: 36.0–110.6 ng/g). With respect to EtS, it was undetectable (<0.01 ng/g) in the majority of samples (79.1%). Only three (6%) women reported alcohol consumption during pregnancy in face-to-face interviews. However, prevalence of fetal exposure to alcohol through the detection of EtG and EtS was 4.2% and 16.7%, respectively. Prevention of alcohol consumption during pregnancy and the detection of substance use with markers of fetal exposure are essential components of maternal and child health. PMID:27011168

  9. Alcohol Consumption during Pregnancy: Analysis of Two Direct Metabolites of Ethanol in Meconium.

    PubMed

    Sanvisens, Arantza; Robert, Neus; Hernández, José María; Zuluaga, Paola; Farré, Magí; Coroleu, Wifredo; Serra, Montserrat; Tor, Jordi; Muga, Robert

    2016-01-01

    Alcohol consumption in young women is a widespread habit that may continue during pregnancy and induce alterations in the fetus. We aimed to characterize prevalence of alcohol consumption in parturient women and to assess fetal ethanol exposure in their newborns by analyzing two direct metabolites of ethanol in meconium. This is a cross-sectional study performed in September 2011 and March 2012 in a series of women admitted to an obstetric unit following childbirth. During admission, socio-demographic and substance use (alcohol, tobacco, cannabis, cocaine, and opiates) during pregnancy were assessed using a structured questionnaire and clinical charts. We also recorded the characteristics of pregnancy, childbirth, and neonates. The meconium analysis was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect the presence of ethyl glucuronide (EtG) and ethyl sulfate (EtS). Fifty-one parturient and 52 neonates were included and 48 meconium samples were suitable for EtG and EtS detection. The median age of women was 30 years (interquartile range (IQR): 26-34 years); EtG was present in all meconium samples and median concentration of EtG was 67.9 ng/g (IQR: 36.0-110.6 ng/g). With respect to EtS, it was undetectable (<0.01 ng/g) in the majority of samples (79.1%). Only three (6%) women reported alcohol consumption during pregnancy in face-to-face interviews. However, prevalence of fetal exposure to alcohol through the detection of EtG and EtS was 4.2% and 16.7%, respectively. Prevention of alcohol consumption during pregnancy and the detection of substance use with markers of fetal exposure are essential components of maternal and child health. PMID:27011168

  10. Impact of hair-care products on FAEE hair concentrations in substance abuse monitoring.

    PubMed

    Gareri, Joey; Appenzeller, Brice; Walasek, Paula; Koren, Gideon

    2011-04-01

    Previous studies have indicated that the use of high-ethanol-content (>65%) hair-care products may elevate fatty acid ethyl ester (FAEE) concentrations in hair. In this case series, nine individuals were identified by FAEE analysis to be chronic alcohol abusers in the context of child-welfare substance abuse monitoring. Based on patient claims of moderate or no alcohol consumption, the presence of ethanol in the patients' hair-care regimens was investigated. Samples were additionally tested for the presence of ethyl glucuronide (EtG). From a total of nine patients, 12 hair samples were submitted for analysis. Patient histories were obtained as well as Material Safety Data Sheets (MSDS) listing hair-care product ethanol content. Hair samples were pre-washed to remove external contamination and analyzed for FAEE and EtG by GC-MS. According to the Society of Hair Testing consensus guidelines, FAEE levels exceeding 0.50 ng/mg and/or EtG levels exceeding 30 pg/mg indicate chronic excessive alcohol consumption. Upon initial analysis, the nine samples exhibited positive FAEE findings ranging from 0.496 to 4.984 ng/mg. MSDS review revealed the presence of ethanol from 10% to 95% by volume in at least one hair-care product used by each individual. Results of the EtG analysis ranged from 1.9 to 23.5 pg/mg. These findings indicate that regular use of products with ethanol content as low as 10% can impact FAEE results. EtG analysis should be used to confirm FAEE findings and appears to be unaffected by hair-care products, likely due to alternative mechanisms of incorporation.

  11. Impact of hair-care products on FAEE hair concentrations in substance abuse monitoring.

    PubMed

    Gareri, Joey; Appenzeller, Brice; Walasek, Paula; Koren, Gideon

    2011-04-01

    Previous studies have indicated that the use of high-ethanol-content (>65%) hair-care products may elevate fatty acid ethyl ester (FAEE) concentrations in hair. In this case series, nine individuals were identified by FAEE analysis to be chronic alcohol abusers in the context of child-welfare substance abuse monitoring. Based on patient claims of moderate or no alcohol consumption, the presence of ethanol in the patients' hair-care regimens was investigated. Samples were additionally tested for the presence of ethyl glucuronide (EtG). From a total of nine patients, 12 hair samples were submitted for analysis. Patient histories were obtained as well as Material Safety Data Sheets (MSDS) listing hair-care product ethanol content. Hair samples were pre-washed to remove external contamination and analyzed for FAEE and EtG by GC-MS. According to the Society of Hair Testing consensus guidelines, FAEE levels exceeding 0.50 ng/mg and/or EtG levels exceeding 30 pg/mg indicate chronic excessive alcohol consumption. Upon initial analysis, the nine samples exhibited positive FAEE findings ranging from 0.496 to 4.984 ng/mg. MSDS review revealed the presence of ethanol from 10% to 95% by volume in at least one hair-care product used by each individual. Results of the EtG analysis ranged from 1.9 to 23.5 pg/mg. These findings indicate that regular use of products with ethanol content as low as 10% can impact FAEE results. EtG analysis should be used to confirm FAEE findings and appears to be unaffected by hair-care products, likely due to alternative mechanisms of incorporation. PMID:21301822

  12. Characterization of dibenzo[a,l]pyrene-trans-11,12-diol (dibenzo[def,p]chrysene) glucuronidation by UDP-glucuronosyltransferases.

    PubMed

    Olson, Kristine C; Sun, Dongxiao; Chen, Gang; Sharma, Arun K; Amin, Shantu; Ropson, Ira J; Spratt, Thomas E; Lazarus, Philip

    2011-09-19

    Dibenzo[a,l]pyrene (DB[a,l]P) (dibenzo[def,p]chrysene) is a highly carcinogenic polycyclic aromatic hydrocarbon (PAH) that has been identified in tobacco smoke and is found in our environment due to incomplete combustion of organic matter. Its metabolites are known to form stable DNA adducts in bacteria and mammalian cells, and can lead to tumors in animal models. Glucuronidation of major metabolites of DB[a,l]P by the uridine-5'-diphosphate glucuronosyltransferase (UGT) family of enzymes is an important route of detoxification of this pro-carcinogen. The focus of the current study was to characterize the glucuronidation of the pro-carcinogenic enantiomers DB[a,l]P-(+)-trans-11S,12S-diol and DB[a,l]P-(-)-trans-11R,12R-diol. Glucuronidation assays with HEK293 cell lines overexpressing individual human UGT enzymes demonstrated that UGTs 1A1, 1A4, 1A7, 1A8, 1A9, 1A10, and 2B7 glucuronidated one or both DB[a,l]P-trans-11,12-diol enantiomers. Three glucuronide conjugates were observed in activity assays with UGTs 1A1 and 1A10, while two glucuronides were formed by UGTs 1A7, 1A8, and 1A9, and one glucuronide was made by UGT1A4 and UGT2B7. Enzyme kinetic analysis indicated that UGT1A9 was the most efficient UGT at forming both the (+)-DB[a,l]P-11-Gluc and (-)-DB[a,l]P-11-Gluc products, while UGTs 1A1 and 1A10 were the most efficient at forming the (+)-DB[a,l]P-12-Gluc product (as determined by k(cat)/K(M)). Incubations with human liver microsomes showed the formation of three diastereomeric glucuronide products: (+)-DB[a,l]P-11-Gluc, (+)-DB[a,l]P-12-Gluc, and (-)-DB[a,l]P-11-Gluc, with an average overall ratio of 31:32:37 in four liver specimens. Human bronchus and trachea tissue homogenates demonstrated glucuronidation activity against both DB[a,l]P-trans-11,12-diol enantiomers, with both tissues producing the (+)-DB[a,l]P-11-Gluc and (+)-DB[a,l]P-12-Gluc with little or no formation of (-)-DB[a,l]P-11-Gluc. These results indicate that multiple UGTs are involved in the

  13. Characterization of Dibenzo[a,l ]pyrene-trans-11,12-diol (Dibenzo[def,p]chrysene) Glucuronidation by UDP-glucuronosyltransferases

    PubMed Central

    Olson, Kristine C.; Sun, Dongxiao; Chen, Gang; Sharma, Arun K.; Amin, Shantu; Ropson, Ira J.; Spratt, Thomas E.; Lazarus, Philip

    2011-01-01

    Dibenzo[a,l]pyrene (DB[a,l]P) (dibenzo[def,p]chrysene) is a highly carcinogenic polycyclic aromatic hydrocarbon (PAH) that has been identified in tobacco smoke and is found in our environment due to incomplete combustion of organic matter. Its metabolites are known to form stable DNA adducts in bacteria and mammalian cells, and can lead to tumors in animal models. Glucuronidation of major metabolites of DB[a,l]P by the uridine-5’-diphosphate glucuronosyltransferase (UGT) family of enzymes is an important route of detoxification of this pro-carcinogen. The focus of the current study was to characterize the glucuronidation of the pro-carcinogenic enantiomers DB[a,l]P-(+)-trans-11S,12S–diol and DB[a,l]P-(−)-trans-11R,12R–diol. Glucuronidation assays with HEK293 cell lines over-expressing individual human UGT enzymes demonstrated that UGTs 1A1, 1A4, 1A7, 1A8, 1A9, 1A10, and 2B7 glucuronidated one or both DB[a,l]P-trans-11,12-diol enantiomers. Three glucuronide conjugates were observed in activity assays with UGTs 1A1 and 1A10, while two glucuronides were formed by UGTs 1A7, 1A8, and 1A9, and one glucuronide was made by UGT1A4 and UGT2B7. Enzyme kinetic analysis indicated that UGT1A9 was the most efficient UGT at forming both the (+)-DB[a,l]P-11-Gluc and (−)-DB[a,l]P-11-Gluc products while UGTs 1A1 and 1A10 were the most efficient at forming the (+)-DB[a,l]P-12-Gluc product (as determined by the kcat/KM). Incubations with human liver microsomes showed formation of three diastereomeric glucuronide products: (+)-DB[a,l]P-11-Gluc, (+)-DB[a,l]P-12-Gluc, and (−)-DB[a,l]P-11-Gluc, with an average overall ratio of 31 : 32 : 37 in four liver specimens. Human bronchus and trachea tissue homogenates demonstrated glucuronidation activity against both DB[a,l]P-trans-11,12-diol enantiomers, with both tissues producing the (+)-DB[a,l]P-11-Gluc and (+)-DB[a,l]P-12-Gluc with little or no formation of (−)-DB[a,l]P-11-Gluc. These results indicate that multiple UGTs are

  14. 46 CFR 151.50-42 - Ethyl ether.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... shall be designed and tested to meet the rules of the American Bureau of Shipping for a head of water at... liquid. (g) Precautions shall be taken to prevent the contamination of ethyl ether by strong...

  15. Multidimensional chromatographic approach applied to the identification of novel aroma compounds in wine. Identification of ethyl cyclohexanoate, ethyl 2-hydroxy-3-methylbutyrate and ethyl 2-hydroxy-4-methylpentanoate.

    PubMed

    Campo, E; Cacho, J; Ferreira, V

    2006-12-29

    A multidimensional chromatographic strategy has been developed and optimized with the purpose of identifying different odorants potentially relevant to the aroma and flavor of aged wines from Madeira or Sherry. Different techniques of extraction and fractionation were studied in order to get clear olfactometric and spectrometric signals from the target odorants. The best results were obtained with a dynamic headspace extraction followed by a fractionation on a normal phase medium pressure liquid chromatography on a silicagel column. Large volumes (50 microl) of the concentrated fractions were further analyzed in a dual gas chromatography-mass spectrometric system (GC-MS) equipped with two olfactometric ports. The strategy made it possible to identify in wine by first time the presence of the powerful strawberry-smelling compound, ethyl cyclohexanoate, and of two other novel fruity esters, ethyl 2-hydroxy-3-methylbutyrate and ethyl 2-hydroxy-4-methylpentanoate. Some other unidentified odorants could be isolated and their mass spectra are given. PMID:17069823

  16. Residual behavior of quizalofop ethyl on onion (Allium cepa L.).

    PubMed

    Sahoo, S K; Mandal, Kousik; Singh, Gurmail; Kumar, Rajinder; Chahil, G S; Battu, R S; Singh, Balwinder

    2013-02-01

    Quizalofop ethyl, a phenoxy propionate herbicide, is used for postemergence control of annual and perennial grass weeds in broad-leaved crops in India. The experiments were designed to study the dissipation kinetics of quizalofop ethyl on onion for two seasons. A simple, rapid, and sensitive method for estimation of quizalofop ethyl residues in onion and soil was developed and validated. The recoveries of quizalofop ethyl residues from onion and soil at different spiking level range from 84.81 to 92.68 %. The limit of quantification of this method was found to be 0.01 μg g(-1). The risk assessment through consumption of the onion in comparison to its acceptable daily intake which is an important parameter for the safety of the consumer was also evaluated. Standardized methodology supported by recovery studies was adopted to estimate residues of quizalofop ethyl on onion and soil. The average initial deposits of quizalofop ethyl on onion were observed to be 0.25 and 0.33 mg kg(-1), following single application of the herbicide at 50 g active ingredient (a.i.) ha(-1) during 2009 and 2010, respectively. The half-life values (T (1/2)) of quizalofop ethyl on onion crop were worked out to be 0.85 and 0.79 days, respectively, during 2009 and 2010. At harvest time, the residues of quizalofop ethyl on onion and soil were found to be below the determination limit of 0.01 mg kg(-1) following single application of the herbicide at 50 and 100 g a.i. ha(-1) for both the periods.

  17. Atmospheric Oxidation Mechanisms for Diethyl Ether and its Oxidation Products, Ethyl Formate and Ethyl Acetate.

    NASA Astrophysics Data System (ADS)

    Orlando, J. J.; Tyndall, G. S.

    2006-12-01

    Carbon-containing compounds are present in the earth's atmosphere as the result of emissions from natural and anthropogenic sources. Their oxidation in the atmosphere, initiated by such oxidants as OH, ozone, and nitrate radicals, leads to potentially harmful secondary pollutants such as ozone, carbonyl species, organic acids and aerosols. Ethers and esters are two classes of compounds that contribute to the complex array of organic compounds found in anthropogenically-influenced air. Additional ester is present as a result of the oxidation of the ethers. In this paper, the oxidation of diethyl ether and its two main oxidation products, ethyl formate and ethyl acetate, are studied over ranges of temperature, oxygen partial pressure, and NOx concentration, using an environmental chamber / FTIR absorption technique. Major end-products (the esters from diethyl ether; organic acids and anhydrides from the esters) are quantified, and these data are interpreted in terms of the chemistry of the various alkoxy and peroxy radicals generated. Emphasis is placed on the effects of chemical activation on the behavior of the alkoxy radicals, as well as on a novel peroxy radical rearrangement that may contribute to the observed products of ether oxidation under some conditions. Finally, the data are used, in conjunction with data on similar species, to provide a general representation of ether and ester oxidation in the atmosphere.

  18. Icosapent ethyl: a review of its use in severe hypertriglyceridemia.

    PubMed

    Kim, Esther S; McCormack, Paul L

    2014-12-01

    Icosapent ethyl (Vascepa®) is a high-purity ethyl ester of eicosapentaenoic acid (EPA) that is de-esterified to EPA following oral administration. Both EPA and docosahexaenoic acid (DHA) are long-chain omega-3 fatty acids that have been associated with triglyceride (TG)-lowering. However, DHA has been associated with increased low-density lipoprotein cholesterol (LDL-C) levels. Icosapent ethyl contains ≥96 % of the EPA ethyl ester, does not contain DHA, and is approved in the USA for use as an adjunct to diet to lower TG levels in adult patients with severe (≥500 mg/dL [≥5.65 mmol/L]) hypertriglyceridemia. In a pivotal phase III trial, oral icosapent ethyl 4 g/day significantly decreased the placebo-corrected median TG levels by 33.1 %. It did not increase LDL-C, had favorable effects on other lipid parameters, and had a tolerability profile similar to that of placebo. Therefore, icosapent ethyl is an effective and well-tolerated agent for the treatment of severe hypertriglyceridemia in adults. PMID:25428605

  19. 40 CFR 180.595 - Flufenpyr-ethyl; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... residues of the herbicide, flufenpyr-ethyl; acetic acid, -phenoxy]-ethyl ester], in or on the following...) Tolerances are established for residues of the herbicide flufenpyr-ethyl; acetic acid, -phenoxy]-ethyl ester], and its metabolite, S-3153 acid-4-OH; -phenoxy]-acetic acid, free and conjugated, in or on...

  20. Is urine an alternative to cosmetically treated hair for the detection of drugs and alcohol?

    PubMed

    Agius, Ronald; Dufaux, Bertin; Kahl, Hans-Gerhard; Nadulski, Thomas

    2014-06-01

    This study attempts to assess the utility of the urine matrix as an alternative to cosmetically treated hair for the detection of drugs and alcohol for driving licence re-granting in 1026 cosmetically treated hair samples and 33 262 urine routine samples. No significant difference was observed between the percentage positive samples in cosmetically treated hair to those in urine at both the 95% and 99% significance level for amphetamines, cocaine, opiates, benzodiazepines, and methadone. Significant difference was found between the positivity rates of cannabinoids in cosmetically treated hair and that in urine indicating urine to be a better alternative to the use of the hair matrix even when cosmetically treated. The opposite was observed for the alcohol consumption marker ethyl glucuronide (EtG) for which the positivity rate in cosmetically treated hair was twice that in urine samples. Particularly for alcohol abstinence monitoring, as for the rehabilitative driving licence re-granting medical and psychological assessment (MPA) programme in Germany, it seems that ethyl glucuronide (EtG) in hair presents a much better alternative than urine testing, even when cosmetically treated hair is analyzed. Moreover, segmentation is an additional advantage of hair testing which can provide additional useful information. PMID:24817057

  1. Tentative Structural Assignment of a Glucuronide Metabolite of Methyltestosterone in Tilapia Bile by Liquid Chromatography-Quadrupole-Time-of-Flight Mass Spectrometry.

    PubMed

    Nishshanka, Upul; Chu, Pak-Sin; Evans, Eric; Reimschuessel, Renate; Hasbrouck, Nicholas; Amarasinghe, Kande; Jayasuriya, Hiranthi

    2015-06-24

    Methyltestosterone (MT), a strong androgenic steroid, is not approved for use in fish aquaculture in the United States. It is used in the U.S. under an investigational new animal drug exemption (INAD) only during the early life stages of fish. There is a possibility that farmers feed fish with MT to enhance production for economic gains. Therefore, there is a need to develop methods for the detection of MT and its metabolite residues in fish tissue for monitoring purposes. Previously, our laboratory developed a liquid chromatography-quadrupole time-of-flight (LC-QTOF) method for characterization of 17-O-glucuronide metabolite (MT-glu) in bile of tilapia dosed with MT. The system used was an Agilent 6530 Q-TOF equipped with electrospray jet stream technology, operating in positive ion mode. Retrospective analysis of the data generated in that experiment by a feature-finding algorithm, combined with a search against an in-house library of possible MT-metabolites, resulted in the discovery of a major glucuronide metabolite of MT in the bile extracts. Preliminary data indicate it to be a glucuronide of a hydroxylated MT (OHMT-glu) which persists in tilapia bile for at least 2 weeks after dosing. We present the tentative structural assignment of the OHMT-glu in tilapia bile and time course of development. This glucuronide can serve as a marker to monitor illegal use of MT in tilapia culture.

  2. Glucuronidation of the environmental oestrogen bisphenol A by an isoform of UDP-glucuronosyltransferase, UGT2B1, in the rat liver.

    PubMed Central

    Yokota, H; Iwano, H; Endo, M; Kobayashi, T; Inoue, H; Ikushiro, S; Yuasa, A

    1999-01-01

    Bisphenol A, an environmental oestrogenic chemical, was found to conjugate highly with glucuronic acid in male rat liver microsomes studied in vitro. In the various isoforms tested (1A1, 1A3, 1A5, 1A6, 1A7 and 2B1), glucuronidation of bisphenol A and of diethylstilboestrol, a synthetic crystalline compound possessing oestrogenic activity and known to be glucuronidated by liver microsomes, was catalysed by an isoform of UDP-glucuronosyltransferase (UGT), namely UGT2B1, which glucuronidates some endogenous androgens. UGT activity towards bisphenol A in liver microsomes and in UGT2B1 expressed in yeast AH22 cells (22.9 and 0.58 nmol/min per mg of microsomal proteins respectively) was higher than that towards diethylstilboestrol (75.0 and 4.66 pmol/min per mg of microsomal proteins respectively). UGT activities towards both bisphenol A and diethylstilboestrol were distributed mainly in the liver but were also observed at substantial levels in the kidney and testis. Northern blot analysis disclosed the presence of UGT2B1 solely in the liver, and about 65% of the male rat liver microsomal UGT activities towards bisphenol A were absorbed by the anti-UGT2B1 antibody. These results indicate that bisphenol A, in male rat liver, is glucuronidated by UGT2B1, an isoform of UGT. PMID:10333482

  3. Meconium Indicators of Maternal Alcohol Abuse during Pregnancy and Association with Patient Characteristics

    PubMed Central

    Goecke, Tamme W.; Burger, Pascal; Fasching, Peter A.; Bakdash, Abdulsallam; Engel, Anne; Häberle, Lothar; Voigt, Franziska; Faschingbauer, Florian; Raabe, Eva; Maass, Nicolai; Rothe, Michael; Beckmann, Matthias W.; Pragst, Fritz

    2014-01-01

    Aim. Identification of women with moderate alcohol abuse during pregnancy is difficult. We correlated self-reported alcohol consumption during pregnancy and patient characteristics with objective alcohol indicators measured in fetal meconium. Methods. A total of 557 women singleton births and available psychological tests, obstetric data and meconium samples were included in statistical analysis. Alcohol metabolites (fatty acid ethyl esters (FAEEs) and ethyl glucuronide (EtG)), were determined from meconium and correlated with patient characteristics. Results. We found that 21.2% of the 557 participants admitted low-to-moderate alcohol consumption during pregnancy. Of the parameters analyzed from meconium, only EtG showed an association with alcohol history (P < 0.01). This association was inverse in cases with EtG value above 120 ng/g. These values indicate women with most severe alcohol consumption, who obviously denied having consumed alcohol during pregnancy. No other associations between socioeconomic or psychological characteristics and the drinking status (via meconium alcohol metabolites) could be found. Conclusion. Women who drink higher doses of ethanol during pregnancy, according to metabolite measures in meconium, might be less likely to admit alcohol consumption. No profile of socioeconomic or psychological characteristics of those women positively tested via meconium could be established. PMID:24800249

  4. Expanding analytical possibilities concerning the detection of stanozolol misuse by means of high resolution/high accuracy mass spectrometric detection of stanozolol glucuronides in human sports drug testing.

    PubMed

    Schänzer, Wilhelm; Guddat, Sven; Thomas, Andreas; Opfermann, Georg; Geyer, Hans; Thevis, Mario

    2013-01-01

    Anabolic-androgenic steroids (AAS) represent one of the most frequently detected classes of prohibited substances in doping controls. Due to their long-lasting beneficial effects on athletic performance, utmost retrospectivity via urine analysis is desirable and accomplished by targeting long-term metabolites of the respective drugs. In case of stanozolol, a substantial variety of metabolites has enabled the identification of numerous adverse analytical findings in the past, and recent studies concerning complementary phase-I and phase-II metabolites has further expanded the windows of opportunity for detecting the abuse of stanozolol. In this study, the utility of liquid chromatography-high resolution/high accuracy (tandem) mass spectrometry (LC-MS/MS) for the detection of 3'-OH-stanozolol glucuronide in sports drug testing is presented and the identification of two additional and so far unreported metabolites is shown. The structures of the complementary glucuronic acid conjugates were attributed to stanozolol-N-glucuronide and 17-epistanozolol-N-glucuronide. By means of chemical synthesis, stanozolol-N-glucuronide was prepared and used to corroborate the suggested structures. The 3'-OH-stanozolol glucuronide and the newly identified target compounds were implemented into routine sports drug test assays consisting of direct injection LC-MS/MS or solid-phase extraction (SPE) followed by LC-MS/MS. A considerably expanded detection window for stanozolol abuse was demonstrated compared to the use of conventional phase-I metabolites and methodologies based on, for example, low resolution LC-MS/MS or gas chromatography-tandem mass spectrometry (GC-MS/MS). The commercial availability of 3'-OH-stanozolol glucuronide has been of great value for confirmatory purposes, and 17-epistanozolol-N-glucuronide was found to be a favourable long-term metabolite for doping controls as it was observed up to 28 days post-administration of the drug. Applying the established

  5. Investigation of a recently detected 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol isomer: Studies on the degradation of 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol glucuronide.

    PubMed

    Hanisch, Stephanie; Paulke, Alexander; Toennes, Stefan W

    2016-09-10

    An isomer of the tetrahydrocannabinol (THC) metabolite 11-nor-9-carboxy-Δ(9)-THC (THCCOOH) had been detected in blood of cannabis users. The present study was initiated to elucidate whether the labile metabolite THCCOOH-glucuronide could be the precursor. THCCOOH-glucuronide was incubated in human serum and albumin (HSA) solution at various temperatures (-18, 4.5, 22 and 37°C) and pH values (pH 7.4 and 8.3) for seven days in the presence or absence of the esterase inhibitor sodium fluoride. Analysis of incubation samples was performed using LC-MS/MS. Marked degradation of THCCOOH-glucuronide was observed at 37°C. It was found that not only THCCOOH, but also the isomer is a degradation product of THCCOOH-glucuronide and its in-vivo production is assumed. Degradation to THCCOOH and the isomer occurred at alkaline pH, in the presence of fluoride-sensitive esterases and of HSA alone. To inhibit isomer formation during sample storage, refrigeration and controlling of the pH are recommended. However, THCCOOH and the isomer exhibit similar properties during incubations in serum, but differ in their interaction with HSA. The present study confirmed the nature of the isomer as degradation product of the abundant THC metabolite THCCOOH-glucuronide. Serum albumin and esterases are obviously involved. The isomer is formed not only during storage, but also under physiological conditions, suggesting that it can be considered an in-vivo metabolite. However, the chemical structure of the isomer remains unknown and further research is necessary.

  6. Glucuronidation of the steroid enantiomers ent-17β-estradiol, ent-androsterone and ent-etiocholanolone by the human UDP-glucuronosyltransferases

    PubMed Central

    Sneitz, Nina; Krishnan, Kathiresan; Covey, Douglas F.; Finel, Moshe

    2011-01-01

    Steroids enantiomers are interesting compounds for detailed exploration of drug metabolizing enzymes, such as the UDP-glucuronosyltransferases (UGTs). We have now studied the glucuronidation of the enantiomers of estradiol, androsterone and etiocholanolone by the 19 human UGTs of subfamilies 1A, 2A and 2B. The results reveal that the pattern of human UGTs of subfamily 2B that glucuronidate ent-17β-estradiol, particularly 2B15 and 2B17, resembles the glucuronidation of epiestradiol (17α-estradiol) rather than 17β-estradiol, the main physiological estrogen. The UGTs of subfamilies 1A and 2A exhibit higher degree of regioselectivity than enantioselectivity in the conjugation of these estradiols, regardless of whether the activity is primarily toward the non-chiral site, 3-OH (UGT1A1, UGT1A3, UGT1A7, UGT1A8 and, above all, UGT1A10), or the 17-OH (UGT1A4). In the cases of etiocholanolone and androsterone, glucuronidation of the ent-androgens, like the conjugation of the natural androgens, is mainly catalyzed by UGTs of subfamilies 2A and 2B. Nevertheless, the glucuronidation of ent-etiocholanolone and ent-androsterone by both UGT2B7 and UGT2B17 differ considerably from their respective activity toward the corresponding endogenous androgens, whereas UGT2A1-catalyzed conjugation is much less affected by the stereochemistry differences. Kinetic analyses reveal that the Km value of UGT2A1 for ent-estradiol is much higher than the corresponding value in the other two high activity enzymes, UGT1A10 and UGT2B7. Taken together, the results highlight large enantioselectivity differences between individual UGTs, particularly those of subfamily 2B. PMID:21899827

  7. Investigation of a recently detected 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol isomer: Studies on the degradation of 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol glucuronide.

    PubMed

    Hanisch, Stephanie; Paulke, Alexander; Toennes, Stefan W

    2016-09-10

    An isomer of the tetrahydrocannabinol (THC) metabolite 11-nor-9-carboxy-Δ(9)-THC (THCCOOH) had been detected in blood of cannabis users. The present study was initiated to elucidate whether the labile metabolite THCCOOH-glucuronide could be the precursor. THCCOOH-glucuronide was incubated in human serum and albumin (HSA) solution at various temperatures (-18, 4.5, 22 and 37°C) and pH values (pH 7.4 and 8.3) for seven days in the presence or absence of the esterase inhibitor sodium fluoride. Analysis of incubation samples was performed using LC-MS/MS. Marked degradation of THCCOOH-glucuronide was observed at 37°C. It was found that not only THCCOOH, but also the isomer is a degradation product of THCCOOH-glucuronide and its in-vivo production is assumed. Degradation to THCCOOH and the isomer occurred at alkaline pH, in the presence of fluoride-sensitive esterases and of HSA alone. To inhibit isomer formation during sample storage, refrigeration and controlling of the pH are recommended. However, THCCOOH and the isomer exhibit similar properties during incubations in serum, but differ in their interaction with HSA. The present study confirmed the nature of the isomer as degradation product of the abundant THC metabolite THCCOOH-glucuronide. Serum albumin and esterases are obviously involved. The isomer is formed not only during storage, but also under physiological conditions, suggesting that it can be considered an in-vivo metabolite. However, the chemical structure of the isomer remains unknown and further research is necessary. PMID:27448313

  8. Synthesis and metabolism of all-trans-[11-3H]retinyl beta-glucuronide in rats in vivo.

    PubMed Central

    Barua, A B; Batres, R O; Olson, J A

    1988-01-01

    All-trans-[11-3H]retinyl beta-glucuronide (all-trans-[11-3H]ROG) was synthesized from [3H]retinol by an improved synthetic procedure. After its intraperitoneal injection into rats, ROG is initially found as the predominant labelled component in the serum, but then is distributed to the liver, intestine, kidney and other organs of the body. Esters of vitamin A, which constituted the major metabolite of ROG, were detected in the liver as well as in other tissues. Of the labelled vitamin A esters derived from tritiated ROG in the liver and intestine, about 50% contained 5,6-epoxyretinol, which was characterized by its chromatographic behaviour, formation of an acetyl ester and lack of reactivity with diazomethane. Thus ROG, although converted to retinol in vivo, might also act physiologically in an intact form. PMID:3415665

  9. Glucuronide conjugation reduces the cytotoxicity but not the mutagenicity of benzo(a)pyrene in the CHO/HGPRT assay

    SciTech Connect

    Recio, L.; Hsie, A.W.

    1984-01-01

    Benzo(a)pyrene (B(a)P) is biotransformed by the mixed-function oxidase (MFO) system to numerous metabolites some of which are cytotoxic and/or mutagenic to mammalian cells. However, conjugation of B(a)P-induced metabolites with glucuronic acid in vivo is a major pathway of detoxication and elimination. The effects of glucuronide conjugation on B(a)P-induced cytotoxicity and mutagenicity were studied using the CHO-HGPRT assay with a rat liver homogenate preparation containing MFO system cofactors (S9 mix) and uridine diphosphate ..cap alpha..-D-glucuronic acid (UDPGA). B(a)P metabolites proximate to the biologically active B(a)P quinones (B(a)P 6-OH) and to the B(a)P 7,8-diol-9,10 epoxide isomers (B(a)P 7,8-diol), were also assayed with S9 mix in the absence and presence of UDPGA. The addition of UDPGA to S9 mix reduced B(a)P-induced cytotoxicity but did not affect mutagenicity. B(a)P 6-OH-mediated cytotoxicity was also reduced in the presence of UDPGA. UDPGA had no effect on B(a)P 7,8-diol-induced cytotoxicity or mutagenicity. B(a)P phenols have been shown to be the preferred B(a)P metabolite substrates for UDP-glucuronyltransferase enzymes. Thus, the reduction of B(a)P and B(a)P 6-OH-induced cytotoxicity by glucuronide conjugation is likely due to the elimination of cytotoxic phenols and quinones. Since B(a)P 7,8-diol is a poor substrate for UDP-glucuronyltransferase enzymes, no effects on B(a)P-induced mutagenicity or B(a)P 7,8-diol-induced cytotoxicity and mutagenicity were observed. 40 references, 3 figures, 2 tables.

  10. Patterns of free (unconjugated) buprenorphine, norbuprenorphine, and their glucuronides in urine using liquid chromatography-tandem mass spectrometry.

    PubMed

    McMillin, Gwendolyn A; Davis, Rebecka; Carlisle, Heidi; Clark, Chantry; Marin, Stephanie J; Moody, David E

    2012-03-01

    Patterns of buprenorphine and metabolites were examined in 1946 positive urine samples analyzed by liquid chromatography-tandem mass spectrometry for free (unconjugated) buprenorphine and norbuprenorphine (quantitative, 2 to 1000 ng/mL) and buprenorphine-glucuronide (B3G) and norbuprenorphine-glucuronide (N3G) (semi-quantitative, 5 to 1000 ng/mL). Two distribution patterns predominated with 49.1% positive for norbuprenorphine, B3G, and N3G and 41.6% positive for buprenorphine, norbuprenorphine, B3G, and N3G. Buprenorphine, positive in 45.5% of samples, was mostly < 5 ng/mL (median 6.1 ng/mL), but 9.8% were > 1000 ng/mL. Norbuprenorphine, B3G, and N3G had semi-Gaussian distributions with medians of 64.7, 108, and 432 ng/mL, respectively. With buprenorphine < 100 ng/mL (767 samples) or ≥ 100 ng/mL (19 quantifiable samples), the respective median metabolic ratios (free norbuprenorphine/free buprenorphine) were 25.0 and 0.15. In 12 retested "> 1000 ng/mL" buprenorphine samples, free buprenorphine was 4160 to 39,400 ng/mL and free naloxone 2140 to 9560 ng/mL. In 87 subsequent samples with buprenorphine < 20 ng/mL, naloxone concentrations were < 50 ng/mL. Concentrations of buprenorphine > 100 ng/mL (particularly with low metabolite concentrations) are suspect of urine adulteration with medication (4% in the database) that can be checked in most cases by concurrent analysis for naloxone. PMID:22337776

  11. Pseudoendogenous presence of β-boldenone sulphate and glucuronide in untreated young bulls from the food chain.

    PubMed

    Chiesa, Luca; Pasquale, Elisa; Panseri, Sara; Cannizzo, Francesca T; Biolatti, Bartolomeo; Pavlovic, Radmila; Arioli, Francesco

    2015-01-01

    The administration of boldenone (bold) to bovines, either for growth promotion or therapeutic purposes, has been banned in the EU since 1981. It is, however, a pseudoendogenous hormone, thus its detection in bovine urine, in the form of α-boldenone conjugates, is considered fully compliant up to 2 ng ml(-1). Greater attention has been placed on β-boldenone, the anabolic active epimer, whose conjugated form must be absent in urine. Recently, the identification of a biomarker representing unquestionable evidence of illicit treatment with bold or its precursor androstadienedione has been a major topic in the literature regarding the detection of residues in bovine urine, and β-boldenone sulphate is a candidate molecule. In this study, we used a method previously validated according to the European Commission Decision 2002/657/EC for the determination of sulphate and glucuronide conjugates of β-boldenone. We assessed the occurrence of these molecules in young bull urine, with the aim of understanding whether they could be of endogenous origin, and to check for a possible relationship with particular environmental and stress conditions. Urine samples from 56 young bulls were collected after transport stress, under non-stressful conditions and after transport and slaughter stress. Histopathological investigation of the hormone target organs, i.e. the bulbourethral and prostate glands, was also performed. The results indicate an inverse relationship between the presence and concentration of β-boldenone sulpho- and gluco-conjugates in urine, and stress conditions, expressed by the absence of detection at the slaughterhouse. No significant macroscopic and histologic lesions were detected. Our study indicates that β-boldenone sulphate could be a biomarker of treatment only at the slaughterhouse, while at the farm, in untreated animals (i.e. after a five-month period under the control of Official Veterinarians), sulphate and glucuronide metabolites were found with a

  12. The Impact of Glucuronidation on the Bioactivation and DNA Adduction of the Cooked-Food Carcinogen 2-Amino-1-methyl-6-phenylimidazo[4,5-b] pyridine in vivo

    SciTech Connect

    Malfatti, M A; Ubick, E A; Felton, J S

    2005-03-31

    UDP-glucuronosyltransferases (UGTs) catalyze the glucuronidation of many different chemicals. Glucuronidation is especially important for detoxifying reactive intermediates from metabolic reactions, which otherwise can be biotransformed into highly reactive cytotoxic or carcinogenic species. Detoxification of certain food-borne carcinogenic heterocyclic amines (HAs) is highly dependent on UGT1A-mediated glucuronidation. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most mass abundant carcinogenic HA found in well-done cooked meat, is extensively glucuronidated by UGT1A proteins. In humans, CYP1A2 catalyzed N-hydroxylation and subsequent UGT1A-mediated glucuronidation is a dominant pathway in the metabolism of PhIP. Therefore, changes in glucuronidation rates could significantly alter PhIP metabolism. To determine the importance of UGT1A-mediated glucuronidation in the biotransformation of PhIP, UGT1A proficient Wistar and UGT1A deficient Gunn rats were exposed to a single 100 {micro}g/kg oral dose of [{sup 14}C]-PhIP. Urine was collected over 24 h and the PhIP urinary metabolite profiles were compared between the two strains. After the 24 h exposure, livers and colon were removed and analyzed for DNA adduct formation by accelerator mass spectrometry. Wistar rats produced several PhIP and N-hydroxy-PhIP glucuronides that accounted for {approx}25% of the total amount of recovered urinary metabolites. In the Gunn rats, PhIP and N-hydroxy-PhIP glucuronides were reduced by 68-92%, compared to the Wistar rats, and comprised only 4% of the total amount of recovered urinary metabolites. PhIP-DNA adduct analysis from the Gunn rats revealed a correlation between reduced PhIP and N-hydroxy-PhIP glucuronide levels in the urine and increased hepatic DNA adducts, compared to the Wistar rats. These results indicate that UGT1A-mediated glucuronidation of PhIP and N-hydroxy-PhIP is an important pathway for PhIP detoxification. Failure to form glucuronide conjugates

  13. Chemical fingerprint and metabolic profile analysis of ethyl acetate fraction of Gastrodia elata by ultra performance liquid chromatography/quadrupole-time of flight mass spectrometry.

    PubMed

    Tang, Chunlan; Wang, Li; Liu, Xinxin; Cheng, Mengchun; Xiao, Hongbin

    2016-02-01

    The chemical fingerprint and metabolic profile of traditional Chinese medicine is very complicated and has been a great challenge. In the present study, chemical fingerprint of ethyl acetate fraction of Gastrodia elata (EtAcGE) and metabolic profile of rat plasma sample after intragastric administration of EtAcGE (2.5g/kg) were investigated using ultra-high performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry (UPLC/Q-TOF MS). A total of 38 chemical constituents of EtAcGE were identified by comparing their retention time, accurate molecular mass and characteristic fragment ions with those of references, or tentatively characterized by comparing molecular formula, fragment ions with that of known compound or information available in literature. And 40 compounds were detected in dosed rat plasma sample, including 16 prototypes and 24 metabolites underwent metabolic process of glucuronidation, glucosylation, sulfation, methylation, hydroxylation, dehydrogenation or mixed modes. The metabolic "soft spots" was hydroxyl or carboxy group. This is the first research for chemical fingerprint and metabolic profile of EtAcGE, which lay a foundation for the further investigation of EtAcGE. PMID:26621783

  14. Chemical fingerprint and metabolic profile analysis of ethyl acetate fraction of Gastrodia elata by ultra performance liquid chromatography/quadrupole-time of flight mass spectrometry.

    PubMed

    Tang, Chunlan; Wang, Li; Liu, Xinxin; Cheng, Mengchun; Xiao, Hongbin

    2016-02-01

    The chemical fingerprint and metabolic profile of traditional Chinese medicine is very complicated and has been a great challenge. In the present study, chemical fingerprint of ethyl acetate fraction of Gastrodia elata (EtAcGE) and metabolic profile of rat plasma sample after intragastric administration of EtAcGE (2.5g/kg) were investigated using ultra-high performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry (UPLC/Q-TOF MS). A total of 38 chemical constituents of EtAcGE were identified by comparing their retention time, accurate molecular mass and characteristic fragment ions with those of references, or tentatively characterized by comparing molecular formula, fragment ions with that of known compound or information available in literature. And 40 compounds were detected in dosed rat plasma sample, including 16 prototypes and 24 metabolites underwent metabolic process of glucuronidation, glucosylation, sulfation, methylation, hydroxylation, dehydrogenation or mixed modes. The metabolic "soft spots" was hydroxyl or carboxy group. This is the first research for chemical fingerprint and metabolic profile of EtAcGE, which lay a foundation for the further investigation of EtAcGE.

  15. Fragrance material review on ethyl phenyl carbinyl acetate.

    PubMed

    McGinty, D; Letizia, C S; Api, A M

    2012-09-01

    A toxicologic and dermatologic review of ethyl phenyl carbinyl acetate when used as a fragrance ingredient is presented. Ethyl phenyl carbinyl acetate is a member of the fragrance structural group Aryl Alkyl Alcohol Simple Acid Esters (AAASAE). The AAASAE fragrance ingredients are prepared by reacting an aryl alkyl alcohol with a simple carboxylic acid (a chain of 1-4 carbons) to generate formate, acetate, propionate, butyrate, isobutyrate and carbonate esters. This review contains a detailed summary of all available toxicology and dermatology papers that are related to this individual fragrance ingredient and is not intended as a stand-alone document. Available data for ethyl phenyl carbinyl acetate were evaluated, then summarized, and includes: physical properties; acute toxicity; skin irritation; and skin sensitization data. A safety assessment of the entire AAASAE will be published simultaneously with this document; please refer to Belsito et al. (2012) for an overall assessment of the safe use of this material and all AAASAE in fragrances.

  16. Fragrance material review on 2-(p-tolyloxy)ethyl acetate.

    PubMed

    McGinty, D; Letizia, C S; Api, A M

    2012-09-01

    A toxicologic and dermatologic review of 2-(p-tolyloxy)ethyl acetate when used as a fragrance ingredient is presented. 2-(p-tolyloxy)ethyl acetate is a member of the fragrance structural group aryl alkyl alcohol simple acid esters (AAASAE). The AAASAE fragrance ingredients are prepared by reacting an aryl alkyl alcohol with a simple carboxylic acid (a chain of 1-4 carbons) to generate formate, acetate, propionate, butyrate, isobutyrate and carbonate esters. This review contains a detailed summary of all available toxicology and dermatology papers that are related to this individual fragrance ingredient and is not intended as a stand-alone document. Available data for 2-(p-tolyloxy)ethyl acetate were evaluated, then summarized, and includes physical properties data. A safety assessment of the entire AAASAE will be published simultaneously with this document. Please refer to Belsito et al. (2012) for an overall assessment of the safe use of this material and all AAASAE in fragrances.

  17. Fragrance material review on 2-(p-tolyloxy)ethyl acetate.

    PubMed

    McGinty, D; Letizia, C S; Api, A M

    2012-09-01

    A toxicologic and dermatologic review of 2-(p-tolyloxy)ethyl acetate when used as a fragrance ingredient is presented. 2-(p-tolyloxy)ethyl acetate is a member of the fragrance structural group aryl alkyl alcohol simple acid esters (AAASAE). The AAASAE fragrance ingredients are prepared by reacting an aryl alkyl alcohol with a simple carboxylic acid (a chain of 1-4 carbons) to generate formate, acetate, propionate, butyrate, isobutyrate and carbonate esters. This review contains a detailed summary of all available toxicology and dermatology papers that are related to this individual fragrance ingredient and is not intended as a stand-alone document. Available data for 2-(p-tolyloxy)ethyl acetate were evaluated, then summarized, and includes physical properties data. A safety assessment of the entire AAASAE will be published simultaneously with this document. Please refer to Belsito et al. (2012) for an overall assessment of the safe use of this material and all AAASAE in fragrances. PMID:22414652

  18. NEW GROUND-STATE MEASUREMENTS OF ETHYL CYANIDE

    SciTech Connect

    Brauer, Carolyn S.; Pearson, John C.; Drouin, Brian J.; Yu, Shanshan

    2009-09-01

    The spectrum of ethyl cyanide, or propionitrile (CH{sub 3}CH{sub 2}CN), has been repeatedly observed in the interstellar medium with large column densities and surprisingly high temperatures in hot core sources. The construction of new, more sensitive, observatories accessing higher frequencies such as Herschel, ALMA, and SOFIA have made it important to extend the laboratory data for ethyl cyanide to coincide with the capabilities of the new instruments. We report extensions of the laboratory measurements of the rotational spectrum of ethyl cyanide in its ground vibrational state to 1.6 THz. A global analysis of the ground state, which includes all of the previous data and 3356 newly assigned transitions, has been fitted to within experimental error to J = 132, K = 36, using both Watson A-reduced and Watson S-reduced Hamiltonians.

  19. Nitrosamine-induced carcinogenesis. The alkylation of N-7 of guanine of nucleic acids of the rat by diethylnitrosamine, N-ethyl-N-nitrosourea and ethyl methanesulphonate

    PubMed Central

    Swann, P. F.; Magee, P. N.

    1971-01-01

    1. The extent of ethylation of N-7 of guanine in the nucleic acids of rat tissue in vivo by diethylnitrosamine, N-ethyl-N-nitrosourea and ethyl methanesulphonate was measured. 2. All compounds produced measurable amounts of 7-ethyl-guanine. 3. A single dose of diethylnitrosamine or N-ethyl-N-nitrosourea produced tumours of the kidney in the rat. Three doses of ethyl methanesulphonate produced kidney tumours, but a single dose did not. 4. A single dose of diethylnitrosamine produced twice as much ethylation of N-7 of guanine in DNA of kidney as did N-ethyl-N-nitrosourea. A single dose of both compounds induced kidney tumours, although of a different histological type. 5. A single dose of ethyl methanesulphonate produced ten times as much ethylation of N-7 of guanine in kidney DNA as did N-ethyl-N-nitrosourea without producing tumours. 6. The relevance of these findings to the hypothesis that alkylation of a cellular component is the mechanism of induction of tumours by nitroso compounds is discussed. PMID:5145908

  20. Decreased Expression of Multidrug Resistance-Associated Protein 4 (MRP4/ABCC4) Leads to Reduced Glucuronidation of Flavonoids in UGT1A1-Overexpressing HeLa Cells: The Role of Futile Recycling.

    PubMed

    Sun, Hua; Zhou, Xiaotong; Zhang, Xingwang; Wu, Baojian

    2015-07-01

    In this study, the role of futile recycling (or deglucuronidation) in the disposition of two flavonoids (i.e., genistein and apigenin) was explored using UGT1A1-overexpressing HeLa cells (or HeLa1A1 cells). Glucuronidation of the flavonoids by HeLa1A1 cell lysate followed the substrate inhibition kinetics (Vmax = 0.10 nmol/min/mg, Km = 0.54 μM, and Ksi = 2.0 μM for genistein; Vmax = 0.19 nmol/min/mg, Km = 0.56 μM, and Ksi = 3.7 μM for apigenin). Glucuronide was efficiently generated and excreted after incubation of the cells with the aglycone (at doses of 1.25-20 nmol). The excretion rates were 0.40-0.69 and 0.84-1.1 nmol/min/mg protein for genistein glucuronide (GG) and apigenin glucuronide (AG), respectively. Furthermore, glucuronide excretion and total glucuronidation were significantly reduced in MRP4 knocked-down as compared to control cells. The alterations were well characterized by a two-compartment pharmacokinetic model incorporating the process of futile recycling (defined by a first-order rate constant, Kde). The derived Kde values were 15 and 25 h(-1) for GG and AG, respectively. This was well consistent with the in vitro observation that AG was subjected to more efficient futile recycling compared to GG. In conclusion, futile recycling was involved in cellular glucuronidation, accounting for transporter-dependent glucuronidation of flavonoids.

  1. Enantiomer selective glucuronidation of the non-steroidal pure anti-androgen bicalutamide by human liver and kidney: role of the human UDP-glucuronosyltransferase (UGT)1A9 enzyme

    PubMed Central

    Grosse, Laurent; Campeau, Anne-Sophie; Caron, Sarah; Morin, Frédéric-Alexandre; Meunier, Kim; Trottier, Jocelyn; Caron, Patrick; Verreault, Mélanie; Barbier, Olivier

    2013-01-01

    Bicalutamide (Casodex®) is a non-steroidal pure anti-androgen used in the treatment of localized prostate cancer. It is a racemate drug and its activity resides in the (R)-enantiomer, with little in the (S)-enantiomer. A major metabolic pathway for bicalutamide is glucuronidation catalyzed by UDP-glucuronosyltransferase (UGT) enzymes. While (S)bicalutamide is directly glucuronidated, (R)bicalutamide requires hydroxylation prior to glucuronidation. The contribution of human tissues and UGT isoforms in the metabolism of these enantiomers has not been extensively investigated. In this study, both (R) and/or (S)bicalutamide were converted into glucuronide (-G) derivatives following incubation of pure and racemic solutions with microsomal extracts from human liver and kidney. Intestinal microsomes exhibited only low reactivity with these substrates. Km values of liver and kidney samples for (S)bicalutamide glucuronidation were similar, and lower than values obtained with the (R)-enantiomer. Among the 16 human UGTs tested, UGT1A8 and UGT1A9 were able to form both (S) and (R)bicalutamide-G from pure or racemic substrates. UGT2B7 was also able to form (R)bicalutamide-G. Kinetic parameters of the recombinant UGT2B7, UGT1A8 and UGT1A9 enzymes support a predominant role of the UGT1A9 isoform in bicalutamide metabolism. Accordingly, (S)bicalutamide inhibited the ability of human liver and kidney microsomes to glucuronidate the UGT1A9 probe substrate, propofol. In conclusion, the present study provides the first comprehensive analysis of in vitro bicalutamide glucuronidation by human tissues and UGTs, and identifies UGT1A9 as a major contributor for (R) and (S) glucuronidation in the human liver and kidney. PMID:23527766

  2. Utility of coloured hair for the detection of drugs and alcohol.

    PubMed

    Agius, Ronald

    2014-06-01

    The aim of this paper is to assess the utility of coloured hair for the detection of drugs and alcohol in a large statistically significant population. The positivity rate, the 1st, 5th, 50th, 95th, and 99th percentiles of five amphetamines, cannabinoids, cocaine, four opiates, methadone, buprenorphine, seven benzodiazepines, and ethyl glucuronide (EtG) in 9488 non-treated and 1026 cosmetically treated (dyed or bleached) authentic hair samples was compared. Analytical methods used were accredited for forensic purposes at the cut-offs defined by the German driving licence re-granting medical and psychological assessment (MPA) guidelines. Considering only the drug classes for which at least 10 positive samples were detected, the positivity rate in non-treated hair was highest for alcohol (4.50%; measured using EtG at concentrations ≥ 7 pg/mg hair), followed by THC (2.00%), cocaine (1.75%), and amphetamine (0.59%). While the 1st to 99th percentile range was significantly lower for drugs in treated, compared to non-treated hair, no significant change was observed for EtG. Additionally, no significant difference in the positivity rate was observed between treated hair and non-treated hair for both drugs and EtG. This study is the first attempt to evaluate the influence of cosmetic treatment, mainly dying, on the positivity rate for both drugs and EtG in hair samples submitted routinely for abstinence testing and the first to indicate that dyed and eventually bleached hair is not necessarily useless in detecting drugs and/or alcohol consumption, thus making coloured hair analysis still useful, often being the only possibility to prove such misuse. PMID:24817056

  3. Development of a fast screening and confirmatory method by liquid chromatography-quadrupole-time-of-flight mass spectrometry for glucuronide-conjugated methyltestosterone metabolite in tilapia.

    PubMed

    Amarasinghe, Kande; Chu, Pak-Sin; Evans, Eric; Reimschuessel, Renate; Hasbrouck, Nicholas; Jayasuriya, Hiranthi

    2012-05-23

    This paper describes the development of a fast method to screen and confirm methyltestosterone 17-O-glucuronide (MT-glu) in tilapia bile. The method consists of solid-phase extraction (SPE) followed by high-performance liquid chromatography-mass spectrometry. The system used was an Agilent 6530 Q-TOF with an Agilent Jet stream electrospray ionization interface. The glucuronide detected in the bile was characterized as MT-glu by comparison with a chemically synthesized standard. MT-glu was detected in bile for up to 7 days after dosing. Semiquantification was done with matrix-matched calibration curves, because MT-glu showed signal suppression due to matrix effects. This method provides a suitable tool to monitor the illegal use of methyltestosterone in tilapia culture.

  4. An in vitro experiment on the interaction of charcoal or wheat bran with 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol and its glucuronide.

    PubMed

    Skopp, Gisela; Mikus, Gerd

    2013-11-01

    The rather long yet variable terminal half-lives and detection times since last use of urinary cannabinoids may partly be attributed to their enterohepatic circulation which generally can be interrupted or restricted by chemical adsorbents. Therefore, an in vitro experiment was performed to study the adsorption/binding of 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) and its glucuronide to activated charcoal and wheat bran; remaining concentrations were determined by liquid chromatography/tandem mass spectrometry. Adsorption/binding of 1,000 ng/mL of free or conjugated THC-COOH was complete using as little as 5 mg of charcoal whereas adsorption/binding to wheat bran increased with increasing amounts. Taking of remedies affecting enterohepatic recycling of THC-COOH and its glucuronide may challenge interpretation of cannabinoid concentrations used to detect or assess frequency of drug use or the time since last drug consumption. PMID:24077855

  5. 77 FR 12740 - Trinexapac-ethyl; Pesticide Tolerances

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-03-02

    ... plant growth regulator, trinexapac-ethyl and its primary metabolite CGA-179500, in or on grass, forage... Tolerance In the Federal Register of August 4, 2010, (75 FR 46925) (FRL-8834- 9), EPA issued a notice... Order 12866, entitled Regulatory Planning and Review (58 FR 51735, October 4, 1993). Because this...

  6. Higher permeability for water than for ethyl alcohol in Nitella.

    PubMed

    OSTERHOUT, W J V

    1950-03-01

    If we apply water at one end of a Nitella cell, A, and place at the other end, B, a solution of a substance which does not penetrate, such as sucrose, water enters the cell at A, passes along inside the cell, and escapes at B. But if in place of sucrose we use a substance which penetrates such as ethyl alcohol the flow of water is lessened and this fact makes it possible to measure the amount of alcohol which enters. (An increase in the size of cells placed in solutions of alcohol does not necessarily indicate that the number of mols of alcohol entering is greater than the number of mols of water leaving the cell.) The permeability for water is more than 18 times as great as for ethyl alcohol. The behavior of the 2 substances was compared in the same individual cell with a driving force which at the start was the same for both substances. The number of mols entering per second per cm.(2) of surface with a driving force of 1 atmosphere at 25 degrees C. is 0.772 (10(-6)) for water and 0.042 (10(-6)) for ethyl alcohol. The experiments indicate that the non-aqueous substance at the surface of the protoplasm has a higher partition coefficient for water than for ethyl alcohol, although the protoplasmic surface is composed of materials not miscible with water.

  7. Reactivity of 2-ethyl-1-hexanol in the atmosphere.

    PubMed

    Gallego-Iniesta García, María Paz; Moreno Sanroma, Alberto; Martín Porrero, María Pilar; Tapia Valle, Araceli; Cabañas Galán, Beatriz; Salgado Muñoz, María Sagrario

    2010-04-01

    Rate coefficients at room temperature for the reaction of 2-ethyl-1-hexanol with OH and NO(3) radicals and with Cl atoms have been determined in a 150 L PTFE chamber using GC-FID/SPME and FTIR as detection systems. The rate coefficients k (in units of cm(3) molecule(-1) s(-1)) obtained were: (1.13 +/- 0.31) 10(-11) for the OH reaction, (2.93 +/- 0.92) 10(-15) for the NO(3) reaction and (1.88 +/- 0.25) 10(-10) for the Cl reaction. Despite the high concentrations of 2-ethyl-1-hexanol, especially in indoor air, this is the first kinetic study carried out to date for these reactions. The results are consistent with the expected reactivity given the chemical structure of 2-ethyl-1-hexanol. Calculated atmospheric lifetimes reveal that the dominant loss process for 2-ethyl-1-hexanol is clearly the daytime reaction with the hydroxyl radical. PMID:20237722

  8. 76 FR 31479 - Pyraflufen-ethyl; Pesticide Tolerances

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-06-01

    ... Federal Register of June 23, 2010 (75 FR 35801) (FRL-8831- 3), EPA issued a notice pursuant to section 408...- ethyl. C. Revisions to Petitioned-for Tolerances In the Federal Register of December 8, 2010 (75 FR..., entitled Regulatory Planning and Review (58 FR 51735, ] October 4, 1993). Because this final rule has...

  9. 40 CFR 180.429 - Chlorimuron ethyl; tolerances for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Chlorimuron ethyl; tolerances for residues. 180.429 Section 180.429 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED..., field, forage 0.5 Corn, field, grain 0.01 Corn, field, stover 2.0 Grain, aspirated fractions 3.0...

  10. Enantioselective Metabolism of Quizalofop-Ethyl in Rat

    PubMed Central

    Liang, Yiran; Wang, Peng; Liu, Donghui; Shen, Zhigang; Liu, Hui; Jia, Zhixin; Zhou, Zhiqiang

    2014-01-01

    The pharmacokinetic and distribution of the enantiomers of quizalofop-ethyl and its metabolite quizalofop-acid were studied in Sprague-Dawley male rats. The two pairs of enantiomers were determined using a validated chiral high-performance liquid chromatography method. Animals were administered quizalofop-ethyl at 10 mg kg−1 orally and intravenously. It was found high concentration of quizalofop-acid in the blood and tissues by both intragastric and intravenous administration, and quizalofop-ethyl could not be detected through the whole study which indicated a quick metabolism of quizalofop-ethyl to quizalofop-acid in vivo. In almost all the samples, the concentrations of (+)-quizalofop-acid exceeded those of (−)-quizalofop-acid. Quizalofop-acid could still be detected in the samples even at 120 h except in brain due to the function of blood-brain barrier. Based on a rough calculation, about 8.77% and 2.16% of quizalofop-acid were excreted through urine and feces after intragastric administration. The oral bioavailability of (+)-quizalofop-acid and (−)-quizalofop-acid were 72.8% and 83.6%. PMID:24964043

  11. Kinetics of Ethyl Acetate Synthesis Catalyzed by Acidic Resins

    ERIC Educational Resources Information Center

    Antunes, Bruno M.; Cardoso, Simao P.; Silva, Carlos M.; Portugal, Ines

    2011-01-01

    A low-cost experiment to carry out the second-order reversible reaction of acetic acid esterification with ethanol to produce ethyl acetate is presented to illustrate concepts of kinetics and reactor modeling. The reaction is performed in a batch reactor, and the acetic acid concentration is measured by acid-base titration versus time. The…

  12. Dissociation of the Ethyl Radical: An Exercise in Computational Chemistry

    ERIC Educational Resources Information Center

    Nassabeh, Nahal; Tran, Mark; Fleming, Patrick E.

    2014-01-01

    A set of exercises for use in a typical physical chemistry laboratory course are described, modeling the unimolecular dissociation of the ethyl radical to form ethylene and atomic hydrogen. Students analyze the computational results both qualitatively and quantitatively. Qualitative structural changes are compared to approximate predicted values…

  13. Reactivity of 2-ethyl-1-hexanol in the atmosphere.

    PubMed

    Gallego-Iniesta García, María Paz; Moreno Sanroma, Alberto; Martín Porrero, María Pilar; Tapia Valle, Araceli; Cabañas Galán, Beatriz; Salgado Muñoz, María Sagrario

    2010-04-01

    Rate coefficients at room temperature for the reaction of 2-ethyl-1-hexanol with OH and NO(3) radicals and with Cl atoms have been determined in a 150 L PTFE chamber using GC-FID/SPME and FTIR as detection systems. The rate coefficients k (in units of cm(3) molecule(-1) s(-1)) obtained were: (1.13 +/- 0.31) 10(-11) for the OH reaction, (2.93 +/- 0.92) 10(-15) for the NO(3) reaction and (1.88 +/- 0.25) 10(-10) for the Cl reaction. Despite the high concentrations of 2-ethyl-1-hexanol, especially in indoor air, this is the first kinetic study carried out to date for these reactions. The results are consistent with the expected reactivity given the chemical structure of 2-ethyl-1-hexanol. Calculated atmospheric lifetimes reveal that the dominant loss process for 2-ethyl-1-hexanol is clearly the daytime reaction with the hydroxyl radical.

  14. 77 FR 41346 - Trinexapac-ethyl; Proposed Pesticide Tolerance

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-13

    ...-ethyl in or on barley, bran; sugarcane, molasses; and wheat, bran under the Federal Food, Drug, and... affected by this action if you are an agricultural producer, food manufacturer, or pesticide manufacturer... production (NAICS code 112). Food manufacturing (NAICS code 311). Pesticide manufacturing (NAICS code...

  15. Synthesis of Ethyl Nalidixate: A Medicinal Chemistry Experiment

    ERIC Educational Resources Information Center

    Leslie, Ray; Leeb, Elaine; Smith, Robert B.

    2012-01-01

    A series of laboratory experiments that complement a medicinal chemistry lecture course in drug design and development have been developed. The synthesis of ethyl nalidixate covers three separate experimental procedures, all of which can be completed in three, standard three-hour lab classes and incorporate aspects of green chemistry such as…

  16. 21 CFR 177.1320 - Ethylene-ethyl acrylate copolymers.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... prescribed in paragraph (c)(2) of this section, when tested by the methods prescribed for polyethylene in... identified by their characteristic infrared spectra. (ii) Quantitative determination of ethyl acrylate... less than 0.920 nor more than 0.935, as determined by ASTM method D1505-68 (Reapproved 1979),...

  17. An overview of alcohol testing and interpretation in the 21st century.

    PubMed

    Kelly, Anna T; Mozayani, Ashraf

    2012-02-01

    Ethanol analysis is the most commonly carried out drug testing in a forensic toxicology laboratory. Determination of blood alcohol concentration (BAC) is needed in a multitude of situations, including in postmortem analysis, driving under the influence (DUI) and drug-facilitated sexual assault (DFSA) cases, workplace drug monitoring, and probation investigations. These analyses are carried out by direct measurement of ethanol concentrations as well as of metabolic by-products, such as ethyl glucuronide (EtG) and ethyl sulfate (EtS). This review article will discuss pharmacokinetics, including absorption, distribution, and elimination of ethanol, methods for the detection of ethanol, the effect of ethanol on human performance, the role of alcohol in injuries and fatalities, and information regarding the interactions that may occur between alcohol and other drugs. Finally, an explanation will be given on how to interpret alcohol levels as well as the extrapolation and calculation of blood alcohol levels at times prior to sample collection. PMID:22215644

  18. Quantification of cannabinoids and their free and glucuronide metabolites in whole blood by disposable pipette extraction and liquid chromatography-tandem mass spectrometry.

    PubMed

    Scheidweiler, Karl B; Newmeyer, Matthew N; Barnes, Allan J; Huestis, Marilyn A

    2016-07-01

    Identifying recent cannabis intake is confounded by prolonged cannabinoid excretion in chronic frequent cannabis users. We previously observed detection times ≤2.1h for cannabidiol (CBD) and cannabinol (CBN) and Δ(9)-tetrahydrocannabinol (THC)-glucuronide in whole blood after smoking, suggesting their applicability for identifying recent intake. However, whole blood collection may not occur for up to 4h during driving under the influence of drugs investigations, making a recent-use marker with a 6-8h detection window helpful for improving whole blood cannabinoid interpretation. Other minor cannabinoids cannabigerol (CBG), Δ9-tetrahydrocannabivarin (THCV), and its metabolite 11-nor-9-carboxy-THCV (THCVCOOH) might also be useful. We developed and validated a sensitive and specific liquid chromatography-tandem mass spectrometry method for quantification of THC, its phase I and glucuronide phase II metabolites, and 5 five minor cannabinoids. Cannabinoids were extracted from 200μL whole blood via disposable pipette extraction, separated on a C18 column, and detected via electrospray ionization in negative mode with scheduled multiple reaction mass spectrometric monitoring. Linear ranges were 0.5-100μg/L for THC and 11-nor-9-carboxy-THC (THCCOOH); 0.5-50μg/L for 11-hydroxy-THC (11-OH-THC), CBD, CBN, and THC-glucuronide; 1-50μg/L for CBG, THCV, and THCVCOOH; and 5-500μg/L for THCCOOH-glucuronide. Inter-day accuracy and precision at low, mid and high quality control (QC) concentrations were 95.1-113% and 2.4-8.5%, respectively (n=25). Extraction recoveries and matrix effects at low and high QC concentrations were 54.0-84.4% and -25.8-30.6%, respectively. By simultaneously monitoring multiple cannabinoids and metabolites, identification of recent cannabis administration or discrimination between licit medicinal and illicit recreational cannabis use can be improved. PMID:27236483

  19. Quantification of cannabinoids and their free and glucuronide metabolites in whole blood by disposable pipette extraction and liquid chromatography-tandem mass spectrometry.

    PubMed

    Scheidweiler, Karl B; Newmeyer, Matthew N; Barnes, Allan J; Huestis, Marilyn A

    2016-07-01

    Identifying recent cannabis intake is confounded by prolonged cannabinoid excretion in chronic frequent cannabis users. We previously observed detection times ≤2.1h for cannabidiol (CBD) and cannabinol (CBN) and Δ(9)-tetrahydrocannabinol (THC)-glucuronide in whole blood after smoking, suggesting their applicability for identifying recent intake. However, whole blood collection may not occur for up to 4h during driving under the influence of drugs investigations, making a recent-use marker with a 6-8h detection window helpful for improving whole blood cannabinoid interpretation. Other minor cannabinoids cannabigerol (CBG), Δ9-tetrahydrocannabivarin (THCV), and its metabolite 11-nor-9-carboxy-THCV (THCVCOOH) might also be useful. We developed and validated a sensitive and specific liquid chromatography-tandem mass spectrometry method for quantification of THC, its phase I and glucuronide phase II metabolites, and 5 five minor cannabinoids. Cannabinoids were extracted from 200μL whole blood via disposable pipette extraction, separated on a C18 column, and detected via electrospray ionization in negative mode with scheduled multiple reaction mass spectrometric monitoring. Linear ranges were 0.5-100μg/L for THC and 11-nor-9-carboxy-THC (THCCOOH); 0.5-50μg/L for 11-hydroxy-THC (11-OH-THC), CBD, CBN, and THC-glucuronide; 1-50μg/L for CBG, THCV, and THCVCOOH; and 5-500μg/L for THCCOOH-glucuronide. Inter-day accuracy and precision at low, mid and high quality control (QC) concentrations were 95.1-113% and 2.4-8.5%, respectively (n=25). Extraction recoveries and matrix effects at low and high QC concentrations were 54.0-84.4% and -25.8-30.6%, respectively. By simultaneously monitoring multiple cannabinoids and metabolites, identification of recent cannabis administration or discrimination between licit medicinal and illicit recreational cannabis use can be improved.

  20. Diabetes Mellitus Reduces Activity of Human UDP-Glucuronosyltransferase 2B7 in Liver and Kidney Leading to Decreased Formation of Mycophenolic Acid Acyl-Glucuronide Metabolite

    PubMed Central

    Dostalek, Miroslav; Court, Michael H.; Hazarika, Suwagmani

    2011-01-01

    Mycophenolic acid (MPA) is an immunosuppressive agent commonly used after organ transplantation. Altered concentrations of MPA metabolites have been reported in diabetic kidney transplant recipients, although the reason for this difference is unknown. We aimed to compare MPA biotransformation and UDP-glucuronosyltransferase (UGT) expression and activity between liver (n = 16) and kidney (n = 8) from diabetic and nondiabetic donors. Glucuronidation of MPA, as well as the expression and probe substrate activity of UGTs primarily responsible for MPA phenol glucuronide (MPAG) formation (UGT1A1 and UGT1A9), and MPA acyl glucuronide (AcMPAG) formation (UGT2B7), was characterized. We have found that both diabetic and nondiabetic human liver microsomes and kidney microsomes formed MPAG with similar efficiency; however, AcMPAG formation was significantly lower in diabetic samples. This finding is supported by markedly lower glucuronidation of the UGT2B7 probe zidovudine, UGT2B7 protein, and UGT2B7 mRNA in diabetic tissues. UGT genetic polymorphism did not explain this difference because UGT2B7*2 or *1c genotype were not associated with altered microsomal UGT2B7 protein levels or AcMPAG formation. Furthermore, mRNA expression and probe activities for UGT1A1 or UGT1A9, both forming MPAG but not AcMPAG, were comparable between diabetic and nondiabetic tissues, suggesting the effect may be specific to UGT2B7-mediated AcMPAG formation. These findings suggest that diabetes mellitus is associated with significantly reduced UGT2B7 mRNA expression, protein level, and enzymatic activity of human liver and kidney, explaining in part the relatively low circulating concentrations of AcMPAG in diabetic patients. PMID:21123165

  1. 40 CFR 721.9514 - Ethyl silicate, reaction products with modified alkoxysilane salt (generic).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Ethyl silicate, reaction products with... Significant New Uses for Specific Chemical Substances § 721.9514 Ethyl silicate, reaction products with.... (1) The chemical substance identified generically as Ethyl silicate, reaction products with...

  2. 40 CFR 721.9514 - Ethyl silicate, reaction products with modified alkoxysilane salt (generic).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Ethyl silicate, reaction products with... Significant New Uses for Specific Chemical Substances § 721.9514 Ethyl silicate, reaction products with.... (1) The chemical substance identified generically as Ethyl silicate, reaction products with...

  3. 40 CFR 721.9514 - Ethyl silicate, reaction products with modified alkoxysilane salt (generic).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Ethyl silicate, reaction products with... Significant New Uses for Specific Chemical Substances § 721.9514 Ethyl silicate, reaction products with.... (1) The chemical substance identified generically as Ethyl silicate, reaction products with...

  4. 40 CFR 721.9514 - Ethyl silicate, reaction products with modified alkoxysilane salt (generic).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Ethyl silicate, reaction products with... Significant New Uses for Specific Chemical Substances § 721.9514 Ethyl silicate, reaction products with.... (1) The chemical substance identified generically as Ethyl silicate, reaction products with...

  5. 40 CFR 721.9514 - Ethyl silicate, reaction products with modified alkoxysilane salt (generic).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Ethyl silicate, reaction products with... Significant New Uses for Specific Chemical Substances § 721.9514 Ethyl silicate, reaction products with.... (1) The chemical substance identified generically as Ethyl silicate, reaction products with...

  6. 40 CFR 721.10064 - 2-Propenoic acid, 2-[2-(ethenyloxy)ethoxy]ethyl ester.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false 2-Propenoic acid, 2- ethyl ester. 721... Substances § 721.10064 2-Propenoic acid, 2- ethyl ester. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as 2-propenoic acid, 2- ethyl ester (PMN...

  7. 40 CFR 721.10064 - 2-Propenoic acid, 2-[2-(ethenyloxy)ethoxy]ethyl ester.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false 2-Propenoic acid, 2- ethyl ester. 721... Substances § 721.10064 2-Propenoic acid, 2- ethyl ester. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as 2-propenoic acid, 2- ethyl ester (PMN...

  8. 40 CFR 721.10064 - 2-Propenoic acid, 2-[2-(ethenyloxy)ethoxy]ethyl ester.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false 2-Propenoic acid, 2- ethyl ester. 721... Substances § 721.10064 2-Propenoic acid, 2- ethyl ester. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as 2-propenoic acid, 2- ethyl ester (PMN...

  9. 40 CFR 721.10300 - Benzeneacetic acid, .alpha.-chloro-.alpha.-phenyl-, ethyl ester.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...-.alpha.-phenyl-, ethyl ester. 721.10300 Section 721.10300 Protection of Environment ENVIRONMENTAL....-phenyl-, ethyl ester. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as benzeneacetic acid, .alpha.-chloro-.alpha.-phenyl-, ethyl ester (PMN...

  10. 40 CFR 721.10365 - Butanoic acid, 3-mercapto-2-methyl-, ethyl ester.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...-, ethyl ester. 721.10365 Section 721.10365 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.10365 Butanoic acid, 3-mercapto-2-methyl-, ethyl ester. (a) Chemical... acid, 3-mercapto-2-methyl-, ethyl ester (PMN P-10-56; CAS No. 888021-82-7) is subject to...

  11. 40 CFR 721.10365 - Butanoic acid, 3-mercapto-2-methyl-, ethyl ester.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...-, ethyl ester. 721.10365 Section 721.10365 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.10365 Butanoic acid, 3-mercapto-2-methyl-, ethyl ester. (a) Chemical... acid, 3-mercapto-2-methyl-, ethyl ester (PMN P-10-56; CAS No. 888021-82-7) is subject to...

  12. 40 CFR 721.7290 - Propanoic acid, 2-(trimethoxysilyl)-, ethyl ester.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...)-, ethyl ester. 721.7290 Section 721.7290 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.7290 Propanoic acid, 2-(trimethoxysilyl)-, ethyl ester. (a) Chemical... acid, 2-(trimethoxysilyl)-, ethyl ester (PMN P-01-22; CAS No. 137787-41-8) is subject to...

  13. 40 CFR 721.10300 - Benzeneacetic acid, .alpha.-chloro-.alpha.-phenyl-, ethyl ester.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...-.alpha.-phenyl-, ethyl ester. 721.10300 Section 721.10300 Protection of Environment ENVIRONMENTAL....-phenyl-, ethyl ester. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as benzeneacetic acid, .alpha.-chloro-.alpha.-phenyl-, ethyl ester (PMN...

  14. 40 CFR 721.7290 - Propanoic acid, 2-(trimethoxysilyl)-, ethyl ester.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...)-, ethyl ester. 721.7290 Section 721.7290 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.7290 Propanoic acid, 2-(trimethoxysilyl)-, ethyl ester. (a) Chemical... acid, 2-(trimethoxysilyl)-, ethyl ester (PMN P-01-22; CAS No. 137787-41-8) is subject to...

  15. 40 CFR 721.7290 - Propanoic acid, 2-(trimethoxysilyl)-, ethyl ester.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...)-, ethyl ester. 721.7290 Section 721.7290 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.7290 Propanoic acid, 2-(trimethoxysilyl)-, ethyl ester. (a) Chemical... acid, 2-(trimethoxysilyl)-, ethyl ester (PMN P-01-22; CAS No. 137787-41-8) is subject to...

  16. 40 CFR 721.10064 - 2-Propenoic acid, 2-[2-(ethenyloxy)ethoxy]ethyl ester.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false 2-Propenoic acid, 2- ethyl ester. 721... Substances § 721.10064 2-Propenoic acid, 2- ethyl ester. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as 2-propenoic acid, 2- ethyl ester (PMN...

  17. 40 CFR 721.10365 - Butanoic acid, 3-mercapto-2-methyl-, ethyl ester.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...-, ethyl ester. 721.10365 Section 721.10365 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.10365 Butanoic acid, 3-mercapto-2-methyl-, ethyl ester. (a) Chemical... acid, 3-mercapto-2-methyl-, ethyl ester (PMN P-10-56; CAS No. 888021-82-7) is subject to...

  18. 40 CFR 721.7290 - Propanoic acid, 2-(trimethoxysilyl)-, ethyl ester.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...)-, ethyl ester. 721.7290 Section 721.7290 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.7290 Propanoic acid, 2-(trimethoxysilyl)-, ethyl ester. (a) Chemical... acid, 2-(trimethoxysilyl)-, ethyl ester (PMN P-01-22; CAS No. 137787-41-8) is subject to...

  19. 40 CFR 721.10300 - Benzeneacetic acid, .alpha.-chloro-.alpha.-phenyl-, ethyl ester.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...-.alpha.-phenyl-, ethyl ester. 721.10300 Section 721.10300 Protection of Environment ENVIRONMENTAL....-phenyl-, ethyl ester. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as benzeneacetic acid, .alpha.-chloro-.alpha.-phenyl-, ethyl ester (PMN...

  20. 40 CFR 721.7290 - Propanoic acid, 2-(trimethoxysilyl)-, ethyl ester.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...)-, ethyl ester. 721.7290 Section 721.7290 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.7290 Propanoic acid, 2-(trimethoxysilyl)-, ethyl ester. (a) Chemical... acid, 2-(trimethoxysilyl)-, ethyl ester (PMN P-01-22; CAS No. 137787-41-8) is subject to...

  1. 40 CFR 721.10064 - 2-Propenoic acid, 2-[2-(ethenyloxy)ethoxy]ethyl ester.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false 2-Propenoic acid, 2- ethyl ester. 721... Substances § 721.10064 2-Propenoic acid, 2- ethyl ester. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as 2-propenoic acid, 2- ethyl ester (PMN...

  2. 40 CFR 721.4090 - Ethanaminium, N-[bis(diethylamino)-methylene]-N-ethyl-, bromide.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Ethanaminium, N- -N-ethyl-, bromide... Substances § 721.4090 Ethanaminium, N- -N-ethyl-, bromide. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as ethanaminium, N- -N-ethyl-, bromide (PMN...

  3. 40 CFR 721.4090 - Ethanaminium, N-[bis(diethylamino)-methylene]-N-ethyl-, bromide.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Ethanaminium, N- -N-ethyl-, bromide... Substances § 721.4090 Ethanaminium, N- -N-ethyl-, bromide. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as ethanaminium, N- -N-ethyl-, bromide (PMN...

  4. Bioavailability of curcumin and curcumin glucuronide in the central nervous system of mice after oral delivery of nano-curcumin.

    PubMed

    Szymusiak, Magdalena; Hu, Xiaoyu; Leon Plata, Paola A; Ciupinski, Paulina; Wang, Zaijie Jim; Liu, Ying

    2016-09-10

    Curcumin is a bioactive molecule extracted from Turmeric roots that has been recognized to possess a wide variety of important biological activities. Despite its great pharmacological activities, curcumin is highly hydrophobic, which results in poor bioavailability. We have formulated this hydrophobic compound into stable polymeric nanoparticles (nano-curcumin) to enhance its oral absorption. Pharmacokinetic analysis after oral delivery of nano-curcumin in mice demonstrated approximately 20-fold reduction in dose requirement when compared to unformulated curcumin to achieve comparable plasma and central nervous system (CNS) tissue concentrations. This investigation corroborated our previous study of curcumin functionality of attenuating opioid tolerance and dependence, which shows equivalent efficacy of low-dose (20mg/kg) nano-curcumin and high-dose (400mg/kg) pure curcumin in mice. Furthermore, the highly selective and validated liquid chromatography-mass spectrometry (LC-MS) method was developed to quantify curcumin glucuronide, the major metabolite of curcumin. The results suggest that the presence of curcumin in the CNS is essential for prevention and reversal of opioid tolerance and dependence. PMID:27426105

  5. Validation and Application of a Method for the Determination of Buprenorphine, Norbuprenorphine, and Their Glucuronide Conjugates in Human Meconium

    PubMed Central

    Kacinko, Sherri L.; Shakleya, Diaa M.; Huestis, Marilyn A.

    2009-01-01

    A novel liquid chromatography tandem mass spectrometry method for quantification of buprenorphine, norbuprenorphine, and glucuronidated conjugates was developed and validated. Analytes were extracted from meconium using buffer, concentrated by solid-phase extraction and quantified within 13.5 min. In order to determine free and total concentrations, specimens were analyzed with and without enzyme hydrolysis. Calibration was achieved by linear regression with a 1/x weighting factor and deuterated internal standards. All analytes were linear from 20 to 2000 ng/g with a correlation of determination of >0.98. Accuracy was ≥85.7% with intra-assay and interassay imprecision ≤13.9 and 12.4%, respectively. There was no interference from 70 licit and illicit drugs and metabolites. Buffer extraction followed by SPE yielded recoveries of ≥85.0%. There was suppression of ionization by the polar matrix; however, this did not interfere with sensitivity or analyte quantification due to inclusion of deuterated internal standards. Analytes were stable on the autosampler, at room temperature, at 4 °C, and when exposed to three freeze/thaw cycles. This sensitive and specific method can be used to monitor in utero buprenorphine exposure and to evaluate correlations, if any, between buprenorphine exposure and neonatal outcomes. PMID:18044957

  6. Transport of the coumarin metabolite 7-hydroxycoumarin glucuronide is mediated via multidrug resistance-associated proteins 3 and 4.

    PubMed

    Wittgen, Hanneke G M; van den Heuvel, Jeroen J M W; van den Broek, Petra H H; Siissalo, Sanna; Groothuis, Geny M M; de Graaf, Inge A M; Koenderink, Jan B; Russel, Frans G M

    2012-06-01

    Coumarin (1,2-benzopyrone) is a natural compound that has been used as a fragrance in the food and perfume industry and could have therapeutic usefulness in the treatment of lymphedema and different types of cancer. Several previous pharmacokinetic studies of coumarin have been performed in humans, which revealed extensive first-pass metabolism of the compound. 7-Hydroxycoumarin (7-HC) and its glucuronide (7-HC-G) are the main metabolites formed in humans, and via this route, 80 to 90% of the absorbed coumarin is excreted into urine, mainly as 7-HC-G. Active transport processes play a role in the urinary excretion of 7-HC-G; however, until now, the transporters involved remained to be elucidated. In this study, we investigated whether the efflux transporters multidrug resistance-associated proteins (MRP)1-4, breast cancer resistance protein, or P-glycoprotein play a role in 7-HC and 7-HC-G transport. For this purpose, we measured uptake of the metabolites into membrane vesicles overexpressing these transporters. Our results showed that 7-HC is not transported by any of the efflux transporters tested, whereas 7-HC-G was a substrate of MRP3 and MRP4. These results are in line with the pharmacokinetic profile of coumarin and suggest that MRP3 and MRP4 are the main transporters involved in the excretion of the coumarin metabolite 7-HC-G from liver and kidney.

  7. Bidirectional placental transfer of Bisphenol A and its main metabolite, Bisphenol A-Glucuronide, in the isolated perfused human placenta.

    PubMed

    Corbel, T; Gayrard, V; Puel, S; Lacroix, M Z; Berrebi, A; Gil, S; Viguié, C; Toutain, P-L; Picard-Hagen, N

    2014-08-01

    The widespread human exposure to Bisphenol A (BPA), an endocrine disruptor interfering with developmental processes, raises the question of the risk for human health of BPA fetal exposure. In humans, highly variable BPA concentrations have been reported in the feto-placental compartment. However the human fetal exposure to BPA still remains unclear. The aim of the study was to characterize placental exchanges of BPA and its main metabolite, Bisphenol A-Glucuronide (BPA-G) using the non-recirculating dual human placental perfusion. This high placental bidirectional permeability to the lipid soluble BPA strongly suggests a transport by passive diffusion in both materno-to-fetal and feto-to-maternal direction, leading to a calculated ratio between fetal and maternal free BPA concentrations of about 1. In contrast, BPA-G has limited placental permeability, particularly in the materno-to-fetal direction. Thus the fetal exposure to BPA conjugates could be explained mainly by its limited capacity to extrude BPA-G.

  8. Homo- and hetero-dimerization of human UDP-glucuronosyltransferase 2B7 (UGT2B7) wild type and its allelic variants affect zidovudine glucuronidation activity.

    PubMed

    Yuan, Lingmin; Qian, Sainan; Xiao, Yongsheng; Sun, Hongying; Zeng, Su

    2015-05-01

    Most human UDP-glucuronosyltransferase (UGT; EC 2.4.1.17) genes contain non-synonymous single nucleotide polymorphisms (nsSNPs) which cause amino acid substitutions. Allelic variants caused by nsSNPs may exhibit absent or reduced enzyme activity. UGT2B7 is one of the most important UGTs that glucuronidates abundant endobiotics and xenobiotics, such as estriol, morphine, and anticancer drugs. Three nsSNPs, UGT2B7*71S (211G>T), UGT2B7*2 (802C>T) and UGT2B7*5 (1192G>A) are observed in the UGT2B7 gene, and they code for allozymes UGT2B7*71S (A71S), UGT2B7*2 (H268Y), and UGT2B7*5 (D398N). UGT2B7 has been observed to form oligomers that affect its enzymatic activity and in this study, we investigated protein-protein interactions among UGT2B7 allozymes wild type (WT), A71S, H268Y and D398N, by performing a systematic quantitative fluorescence resonance energy transfer (FRET) analysis in combination with co-immunoprecipitation assay. Quantitative FRET analysis revealed that UGT2B7 allozymes formed homo- and hetero-dimers and showed distinct features in donor-acceptor distances. Both codon 71 and codon 268 in the N-terminal domain were involved in the dimeric interaction. Co-immunoprecipitation experiments also proved that UGT2B7 allozymes formed stable dimers. The glucuronidation activities of homo- and hetero-dimers were further tested with zidovudine as the substrate. An increase in activity was observed when WT hetero-dimerized with A71S compared with homo-dimers, while both H268Y and D398N impaired the activity of WT and A71S by forming hetero-dimers. In addition, zidovudine glucuronidation activity is associated with FRET distance. These findings provide insights into the consequences of amino acid substitution in UGT2B7 on zidovudine glucuronidation and the association between protein-protein interaction and glucuronidation activity. PMID:25770680

  9. [What ethanol metabolites as biological markers tell us about alcohol use].

    PubMed

    Wurst, Friedrich Martin; Thon, Natasha; Weinmann, Wolfgang; Yegles, Michel; Preuss, Ulrich

    2014-01-01

    Alcohol and tobacco related disorders are the two leading and most expensive causes of illness in central Europe. In addition to self reports and questionnaires, biomarkers are of relevance in diagnosis and therapy of alcohol use disorders. Traditional biomarkers such as gamma glutamyl transpeptidase or mean corpuscular volume are indirect biomarkers and are subject to influence of age, gender and non alcohol related diseases, among others.Direct ethanol metabolites such as ethyl glucuronide (EtG), ethyl sulphate (EtS) and phosphatidylethanol (PEth) are direct metabolites of ethanol, that are positive after intake of ethyl alcohol. They represent useful diagnostic tools for identifying alcohol use even more accurately than traditional biomarkers. Each of these drinking indicators remains positive in serum and urine for a characteristic time spectrum after the cessation of ethanol intake--EtG and EtS in urine up to 7 days, EtG in hair for months after ethanol has left the body. Applications include clinical routine use, emergency room settings, proof of abstinence in alcohol rehabilitation programs, driving under influence offenders, workplace testing, assessment of alcohol intake in the context of liver transplantation and fetal alcohol syndrome.

  10. [A study on the mechanism of reductive alkylation for preparing 3-(beta-hydroxy-ethyl-sulfonyl) N-ethyl aniline with HPLC/MS].

    PubMed

    Zhang, R; Wu, Z W; Lin, L S; Yang, H Y

    2000-11-01

    Hydrogenating 3-(beta-hydroxy-ethyl-sulfonyl)-aniline and acetaldehyde in the presence of Raney Nickel as a catalyst, 3-(beta-hydroxy-ethyl-sulfonyl)-N-ethyl-aniline was obtained with 98% conversion and 95% monoalkylation selectivity under optimum conditions. By using high performance liquid chromatography/mass selective detection technique to characterize the structures of the products, the mechanism of reductive alkylation is proposed. From the intermediates determined, it is shown that the reaction mechanism would go via an unstable N-alpha-hydroxyethylaniline derivative and Schiff base stage. After hydrogenation of Schiff base, finally the product 3-(beta-hydroxyethyl-sulfonyl)-N-ethyl aniline was formed.

  11. Elevated plasma creatinine due to creatine ethyl ester use.

    PubMed

    Velema, M S; de Ronde, W

    2011-02-01

    Creatine is a nutritional supplement widely used in sport, physical fitness training and bodybuilding. It is claimed to enhance performance. We describe a case in which serum creatinine is elevated due to the use of creatine ethyl esther. One week after withdrawal, the plasma creatinine had normalised. There are two types of creatine products available: creatine ethyl esther (CEE) and creatine monohydrate (CM). Plasma creatinine is not elevated in all creatine-using subjects. CEE , but not CM, is converted into creatinine in the gastrointestinal tract. As a result the use of CEE may be associated with elevated plasma creatinine levels. Since plasma creatinine is a widely used marker for renal function, the use of CEE may lead to a false assumption of renal failure.

  12. Effect of ethyl oleate on drying characteristics of mulberries.

    PubMed

    Doymaz, Ibrahim; Pala, Mehmet

    2003-10-01

    In this study, air-drying experiments in thin layers of mulberry grown in Istanbul, Turkey, were conducted. The effect of ethyl oleate solution on drying time of mulberry samples was investigated in a pilot air-dryer. When ethyl oleate was used as pretreatment solution, the drying time of samples was decreased. Drying curves were obtained using the Page model. The effective diffusivity varied from 2.326 x 10(-10) to 1.809 x 10(-9) m2/s the temperature range. The temperature dependence of the diffusivity coefficient was described by the Arrhenius type relationship. The activation energy for moisture diffusion was found to be 50.87 kJ/mol for treated samples and 51.85 kJ/mol for untreated samples. PMID:14609084

  13. [Influence of ethyl alcohol on diabetes pathogenesis type].

    PubMed

    Zasimowicz, Elzbieta; Wolszczak, Blanka; Zasimowicz, Barbara

    2014-03-01

    Relations between metabolism of carbohydrates and ethyl alcohol consumption became a subject of many research because they occur very frequently amongst alcoholics. One of the most often and dangerous effects of abusing ethanol is hypoglycemia. It is caused by hepatic gluconeogenesis disturbed by ethyl alcohol. Chronic result of abusing alcohol is chronic pancreas inflammation (PZT), what causes disorders of exo- and endocrine function of pancreas. Endocrine function is secretion of insulin and the glucagon what regulates metabolism of absorbed compounds. Failure of beta cells of Langerhans islets causes diabetes demanding insulin therapy. The ethanol can cause recurring diabetes resulting from damage of cells of Langerhans islets but can be also the risk factor of diabetes type 2.

  14. Structural analysis of 1-ethyl-3-methylimidazolium bifluoride melt

    NASA Astrophysics Data System (ADS)

    Matsumoto, Kazuhiko; Hagiwara, Rika; Ito, Yasuhiko; Kohara, Shinji; Suzuya, Kentaro

    2003-01-01

    The structure of 1-ethyl-3-methylimidazolium bifluoride (EMImF · HF) melt has been analyzed at 333 K by a high-energy synchrotron X-ray diffraction method. The total correlation function of the EMImF · HF melt was similar to that of the solid state, indicating that not only the short range but also the intermediate-range ordering in the solid are partially preserved in the liquid state. The intra-molecular F-F correlation in the anions clearly appears in the total correlation function of the EMImF · HF melt, whereas prominent peaks are not observed in the case of a room temperature molten salt, 1-ethyl-3-methylimidazolium fluorohydrogenate.

  15. Fragrance material review on 2-ethyl-1-butanol.

    PubMed

    McGinty, D; Letizia, C S; Api, A M

    2010-07-01

    A toxicologic and dermatologic review of 2-ethyl-1-butanol when used as a fragrance ingredient is presented. 2-Ethyl-1-butanol is a member of the fragrance structural group branched chain saturated alcohols. The common characteristic structural elements of the alcohols with saturated branched chain are one hydroxyl group per molecule, and a C(4)-C(12) carbon chain with one or several methyl side chains. This review contains a detailed summary of all available toxicology and dermatology papers that are related to this individual fragrance ingredient and is not intended as a stand-alone document. A safety assessment of the entire branched chain saturated alcohol group will be published simultaneously with this document; please refer to Belsito et al. (2010) for an overall assessment of the safe use of this material and all other branched chain saturated alcohols in fragrances.

  16. Fragrance material review on 2-ethyl-1-hexanol.

    PubMed

    McGinty, D; Scognamiglio, J; Letizia, C S; Api, A M

    2010-07-01

    A summary of the safety data available for 2-ethyl-1-hexanol when used as a fragrance ingredient is presented. 2-Ethyl-1-hexanol is a member of the fragrance structural group branched chain saturated alcohols in which the common characteristic structural element is one hydroxyl group per molecule, and a C(4) to C(12) carbon chain with one or several methyl side chains. This review contains a detailed summary of all available toxicology and dermatology papers that are related to this individual fragrance ingredient and is not intended as a stand-alone document. A safety assessment of the entire branched chain saturated alcohol group will be published simultaneously with this document; please refer to Belsito et al. (2010) for an overall assessment of the safe use of this material and all other branched chain saturated alcohols in fragrances.

  17. Identification of an antioxidant, ethyl protocatechuate, in peanut seed testa.

    PubMed

    Huang, Shiow Chyn; Yen, Gow-Chin; Chang, Lee-Wen; Yen, Wen-Jye; Duh, Pin-Der

    2003-04-01

    The antioxidant activity and identification of the antioxidant component of peanut seed testa were investigated. The antioxidant activity of peanut seed testa was studied in the linoleic acid model system by using the ferric thiocyanate method. Among the five organic solvent extracts, the ethanolic extracts of peanut seed testa (EEPST) produced higher yields and stronger antioxidant activity than other organic solvent extracts. EEPST was separated into 17 fractions on silica gel column chromatography. Fraction 17, which showed the largest yield and significant antioxidant activity, was separated by thin-layer chromatography. Four major antioxidative subfractions were present. Subfraction 17-2 was found to be effective in preventing oxidation of linoleic acid. This subfraction was further fractionated and isolated and characterized by UV, MS, IR, and (1)H NMR techniques. The active compound was identified as ethyl protocatechuate (3,4-dihydroxybenzoic acid ethyl ester).

  18. Preparation of ethyl magnesium bromide for regiospecific analysis of triacylglycerols.

    PubMed

    Ando, Yasuhiro; Tomita, Yuki; Haba, Yusuke

    2008-01-01

    This paper presents a procedure for preparation of a Grignard reagent, ethyl magnesium bromide, used for partial deacylation of triacylglycerols (TAG) in their regiospecific analysis. Magnesium turnings were reacted with ethereal solution of bromoethane in a screw-capped test tube to synthesize 2 mL of 1 M ethyl magnesium bromide. Continuously stirred with a vortex mixer, the reaction smoothly proceeded at room temperature. Regiospecific analysis of 1,3-distearoyl-2-oleoylglycerol using this product showed that fatty acid compositions of the sn-1(3) and sn-2 positions were contaminated by less than 2 mol% of fatty acids migrated from isomeric positions. The analyses of lard and cod liver/mackerel oil TAG showed typical distribution patterns of 16:0, 22:5n-3 and 22:6n-3 in pig and fish depot TAG. These results confirmed the view that the freshly prepared reagent is usable for regiospecific analysis of TAG.

  19. Fragrance material review on 2-ethyl-1-hexanol.

    PubMed

    McGinty, D; Scognamiglio, J; Letizia, C S; Api, A M

    2010-07-01

    A summary of the safety data available for 2-ethyl-1-hexanol when used as a fragrance ingredient is presented. 2-Ethyl-1-hexanol is a member of the fragrance structural group branched chain saturated alcohols in which the common characteristic structural element is one hydroxyl group per molecule, and a C(4) to C(12) carbon chain with one or several methyl side chains. This review contains a detailed summary of all available toxicology and dermatology papers that are related to this individual fragrance ingredient and is not intended as a stand-alone document. A safety assessment of the entire branched chain saturated alcohol group will be published simultaneously with this document; please refer to Belsito et al. (2010) for an overall assessment of the safe use of this material and all other branched chain saturated alcohols in fragrances. PMID:20659633

  20. Fragrance material review on 2-ethyl-1-butanol.

    PubMed

    McGinty, D; Letizia, C S; Api, A M

    2010-07-01

    A toxicologic and dermatologic review of 2-ethyl-1-butanol when used as a fragrance ingredient is presented. 2-Ethyl-1-butanol is a member of the fragrance structural group branched chain saturated alcohols. The common characteristic structural elements of the alcohols with saturated branched chain are one hydroxyl group per molecule, and a C(4)-C(12) carbon chain with one or several methyl side chains. This review contains a detailed summary of all available toxicology and dermatology papers that are related to this individual fragrance ingredient and is not intended as a stand-alone document. A safety assessment of the entire branched chain saturated alcohol group will be published simultaneously with this document; please refer to Belsito et al. (2010) for an overall assessment of the safe use of this material and all other branched chain saturated alcohols in fragrances. PMID:20659644

  1. Fatty acid ethyl esters: current facts and speculations.

    PubMed

    Laposata, M

    1999-01-01

    Increasing evidence indicates that fatty acid ethyl esters (FAEE) play a role in ethanol-induced organ damage and may serve as long-term markers of ethanol intake. This report summarizes the current knowledge on the toxicity of FAEE, the enzymes associated with FAEE synthesis, FAEE as fatty acid supplements, the in vivo degradation of orally ingested FAEE and FAEE as markers of ethanol intake. A list of major unanswered questions in each of these categories is also included.

  2. The interaction of ethyl alcohol and industrial chemicals.

    PubMed

    Hills, B W; Venable, H L

    1982-01-01

    A serious, relatively unrecognized, occupational health problem involves the interaction of ethyl alcohol and chemical agents used in industry. Workers who drink alcohol and are exposed to certain chemical agents may experience adverse health effects such as nausea, dizziness, headache, and liver damage. This report reviews the synergistic interactions of ethanol with compounds such as the thiurams, amides, oximes, halogenated hydrocarbons, and metals. Also discussed is the effect of ethanol as a cofactor with vinyl chloride in the etiology of cancer.

  3. Fragrance material review on ethyl phenyl carbinyl acetate.

    PubMed

    McGinty, D; Letizia, C S; Api, A M

    2012-09-01

    A toxicologic and dermatologic review of ethyl phenyl carbinyl acetate when used as a fragrance ingredient is presented. Ethyl phenyl carbinyl acetate is a member of the fragrance structural group Aryl Alkyl Alcohol Simple Acid Esters (AAASAE). The AAASAE fragrance ingredients are prepared by reacting an aryl alkyl alcohol with a simple carboxylic acid (a chain of 1-4 carbons) to generate formate, acetate, propionate, butyrate, isobutyrate and carbonate esters. This review contains a detailed summary of all available toxicology and dermatology papers that are related to this individual fragrance ingredient and is not intended as a stand-alone document. Available data for ethyl phenyl carbinyl acetate were evaluated, then summarized, and includes: physical properties; acute toxicity; skin irritation; and skin sensitization data. A safety assessment of the entire AAASAE will be published simultaneously with this document; please refer to Belsito et al. (2012) for an overall assessment of the safe use of this material and all AAASAE in fragrances. PMID:22433983

  4. Sensory reception of the primer pheromone ethyl oleate

    NASA Astrophysics Data System (ADS)

    Muenz, Thomas S.; Maisonnasse, Alban; Plettner, Erika; Le Conte, Yves; Rössler, Wolfgang

    2012-05-01

    Social work force distribution in honeybee colonies critically depends on subtle adjustments of an age-related polyethism. Pheromones play a crucial role in adjusting physiological and behavioral maturation of nurse bees to foragers. In addition to primer effects of brood pheromone and queen mandibular pheromone—both were shown to influence onset of foraging—direct worker-worker interactions influence adult behavioral maturation. These interactions were narrowed down to the primer pheromone ethyl oleate, which is present at high concentrations in foragers, almost absent in young bees and was shown to delay the onset of foraging. Based on chemical analyses, physiological recordings from the antenna (electroantennograms) and the antennal lobe (calcium imaging), and behavioral assays (associative conditioning of the proboscis extension response), we present evidence that ethyl oleate is most abundant on the cuticle, received by olfactory receptors on the antenna, processed in glomeruli of the antennal lobe, and learned in olfactory centers of the brain. The results are highly suggestive that the primer pheromone ethyl oleate is transmitted and perceived between individuals via olfaction at close range.

  5. Physiologically based pharmacokinetic modeling of ethyl acetate and ethanol in rodents and humans.

    PubMed

    Crowell, S R; Smith, J N; Creim, J A; Faber, W; Teeguarden, J G

    2015-10-01

    A physiologically based pharmacokinetic (PBPK) model was developed and applied to a metabolic series approach for the ethyl series (i.e., ethyl acetate, ethanol, acetaldehyde, and acetate). This approach bases toxicity information on dosimetry analyses for metabolically linked compounds using pharmacokinetic data for each compound and toxicity data for parent or individual compounds. In vivo pharmacokinetic studies of ethyl acetate and ethanol were conducted in rats following IV and inhalation exposure. Regardless of route, ethyl acetate was rapidly converted to ethanol. Blood concentrations of ethyl acetate and ethanol following both IV bolus and infusion suggested linear kinetics across blood concentrations from 0.1 to 10 mM ethyl acetate and 0.01-0.8 mM ethanol. Metabolic parameters were optimized and evaluated based on available pharmacokinetic data. The respiratory bioavailability of ethyl acetate and ethanol were estimated from closed chamber inhalation studies and measured ventilation rates. The resulting ethyl series model successfully reproduces blood ethyl acetate and ethanol kinetics following IV administration and inhalation exposure in rats, and blood ethanol kinetics following inhalation exposure to ethanol in humans. The extrapolated human model was used to derive human equivalent concentrations for the occupational setting of 257-2120 ppm ethyl acetate and 72-517 ppm ethyl acetate for continuous exposure, corresponding to rat LOAELs of 350 and 1500 ppm.

  6. Physiologically based pharmacokinetic modeling of ethyl acetate and ethanol in rodents and humans.

    PubMed

    Crowell, S R; Smith, J N; Creim, J A; Faber, W; Teeguarden, J G

    2015-10-01

    A physiologically based pharmacokinetic (PBPK) model was developed and applied to a metabolic series approach for the ethyl series (i.e., ethyl acetate, ethanol, acetaldehyde, and acetate). This approach bases toxicity information on dosimetry analyses for metabolically linked compounds using pharmacokinetic data for each compound and toxicity data for parent or individual compounds. In vivo pharmacokinetic studies of ethyl acetate and ethanol were conducted in rats following IV and inhalation exposure. Regardless of route, ethyl acetate was rapidly converted to ethanol. Blood concentrations of ethyl acetate and ethanol following both IV bolus and infusion suggested linear kinetics across blood concentrations from 0.1 to 10 mM ethyl acetate and 0.01-0.8 mM ethanol. Metabolic parameters were optimized and evaluated based on available pharmacokinetic data. The respiratory bioavailability of ethyl acetate and ethanol were estimated from closed chamber inhalation studies and measured ventilation rates. The resulting ethyl series model successfully reproduces blood ethyl acetate and ethanol kinetics following IV administration and inhalation exposure in rats, and blood ethanol kinetics following inhalation exposure to ethanol in humans. The extrapolated human model was used to derive human equivalent concentrations for the occupational setting of 257-2120 ppm ethyl acetate and 72-517 ppm ethyl acetate for continuous exposure, corresponding to rat LOAELs of 350 and 1500 ppm. PMID:26297692

  7. Impact of Association Colloids on Lipid Oxidation in Triacylglycerols and Fatty Acid Ethyl Esters.

    PubMed

    Homma, Rika; Suzuki, Karin; Cui, Leqi; McClements, David Julian; Decker, Eric A

    2015-11-25

    The impact of association colloids on lipid oxidation in triacylglycerols and fatty acid ethyl esters was investigated. Association colloids did not affect lipid oxidation of high oleic safflower and high linoleic safflower triacylglycerols, but were prooxidative in fish triacylglycerols. Association colloids retarded aldehyde formation in stripped ethyl oleate, linoleate, and fish oil ethyl esters. Interfacial tension revealed that lipid hydroperoxides were surface active in the presence of the surfactants found in association colloids. The lipid hydroperoxides from ethyl esters were less surface active than triacylglycerol hydroperoxides. Stripping decreased iron and copper concentrations in all oils, but more so in fatty acid ethyl esters. The combination of lower hydroperoxide surface activity and low metal concentrations could explain why association colloids inhibited lipid oxidation in fatty acid ethyl esters. This research suggests that association colloids could be used as an antioxidant technology in fatty acid ethyl esters.

  8. Impact of Association Colloids on Lipid Oxidation in Triacylglycerols and Fatty Acid Ethyl Esters.

    PubMed

    Homma, Rika; Suzuki, Karin; Cui, Leqi; McClements, David Julian; Decker, Eric A

    2015-11-25

    The impact of association colloids on lipid oxidation in triacylglycerols and fatty acid ethyl esters was investigated. Association colloids did not affect lipid oxidation of high oleic safflower and high linoleic safflower triacylglycerols, but were prooxidative in fish triacylglycerols. Association colloids retarded aldehyde formation in stripped ethyl oleate, linoleate, and fish oil ethyl esters. Interfacial tension revealed that lipid hydroperoxides were surface active in the presence of the surfactants found in association colloids. The lipid hydroperoxides from ethyl esters were less surface active than triacylglycerol hydroperoxides. Stripping decreased iron and copper concentrations in all oils, but more so in fatty acid ethyl esters. The combination of lower hydroperoxide surface activity and low metal concentrations could explain why association colloids inhibited lipid oxidation in fatty acid ethyl esters. This research suggests that association colloids could be used as an antioxidant technology in fatty acid ethyl esters. PMID:26506263

  9. Impact of anti-cancer drugs and other determinants on serum protein binding of morphine 6-glucuronide

    PubMed Central

    Mashayekhi, S.O.; Ghandforoush-Sattari, M.; Buss, D.C.; Routledge, P.A.; Hain, R.DW.

    2010-01-01

    Background and the purpose of the study The aim of the present study was to examine factors that may influence the protein binding of morphine 6-glucuronide (M6G), the most active metabolite of morphine. Methods An enzyme-linked immunoabsorbent assay technique was used to measure the M6G concentration in serum of 18 healthy adults, 18 neonatal and 7 children with cancer. Total and free M6G concentrations were measured following equilibrium dialysis for 3 hrs and at physiological pH at 37°C. The influence of vincristine, methotrexate, 6-mercaptopurine, morphine, human albumin, alpha-1-acid glycoprotein, palmitic acid, oleic acid and pH on M6G protein binding was examined. Results M6G was 66.87±0.73 percent free in human serum at physiological pH and temperature. The percentage free (unbound) was increased significantly by vincristine (4.33%) and methotrexate (9.68%), but 6- mercaptopurine and morphine had no significant effect on it. Free percentages of M6G was reduced by decreasing serum albumin concentration but was unaffected by the presence of alpa-1-acid glycoprotein (AAG) or changes in serum pH. Similar results were obtained in human serum albumin (HAS) solutions. Addition of palmitic acid and oleic acid reduced protein binding significantly by 6.3% and 7.4%, respectively. Major conclusion Although M6G in this study was not highly bounded, but because of its high analgesic potency, any change in its free concentration due to concurrent medication or disease caused significant changes in its effects. This dearth of evidence has been implicated in the reluctance of professionals to be cautious in prescribing them to children, particularly in the neonatal period. PMID:22615603

  10. The influence of active transport systems on morphine -6-glucuronide transport in MDCKII and MDCK-PGP cells

    PubMed Central

    Sattari, M.; Routledge, PA.; Mashayekhi, SO.

    2011-01-01

    Background and the purpose of the study Morphine-6-glucuronide (M6G) is a potent metabolite of morphine which has high penetration into the brain despite its high polarity, which could be the result of an active transport system involved in M6G transport through blood brain barrier. Examples of such transporters are p-glycoprotein (PGP), probenecid-sensitive transport mechanism, multidrug resistance related protein 1-3, the organic anion transporter family, and the organic anion transporter polypeptide family. The aim of present study was to elucidate the mechanisms involved in transporting morphine's potent metabolite, M6G. Methods M6G permeability via two cell lines; MDCKII and MDCK-PGP, was compared with that of sucrose. M6G transport was examined in different concentrations and in the presence of inhibitors of different transport systems such as cyclosporine, digoxin and probenecid. M6G concentration was measured using ELISA assay. The method was sensitive, reliable and reproducible. Results The results confirmed that M6G could cross a layer of MDCK II or MDR-PGP cells more than sucrose could. It was also observed that M6G is a PGP transporter substrate. Its permeability was increased by the use of a PGP expressed cell line, and also in the presence of a strong PGP inhibitor. Digoxin related transporters such as Oatp2 may also involved in transport of M6G. M6G seemed to be a glucose transporter 1 substrate, but was not a substrate to probenecid sensitive transporters. Major conclusion It is concluded that different transporters are responsible for M6G transports via different membrane, which could have effects on its pharmacokinetics or pharmacodynamics. PMID:23008686

  11. Quantitative determination of free and total bisphenol A in human urine using labeled BPA glucuronide and isotope dilution mass spectrometry.

    PubMed

    Kubwabo, Cariton; Kosarac, Ivana; Lalonde, Kaela; Foster, Warren G

    2014-07-01

    Bisphenol A (BPA) is a widely used industrial chemical in the manufacturing of polycarbonate plastic bottles, food and beverage can linings, thermal receipts, and dental sealants. Animal and human studies suggest that BPA may disrupt normal hormonal function and hence, potentially, have negative effects on the human health. While total BPA is frequently reported, it is recognized that free BPA is the biologically active form and is rarely reported in the literature. The objective of this study was to develop a sensitive and improved method for the measurement of free and total BPA in human urine. Use of a labeled conjugated BPA (bisphenol A-d6 β-D-glucuronide) allowed for the optimization of the enzymatic reaction and permitted an accurate determination of the conjugated BPA concentration in urine samples. In addition, a (13)C12-BPA internal standard was used to account for the analytical recoveries and performance of the isotope dilution method. Solid-phase extraction (SPE) combined with derivatization and analysis using a triple quadrupole GC-EI/MS/MS system achieved very low method detection limit of 0.027 ng/mL. BPA concentrations were measured in urine samples collected during the second and third trimesters of pregnancy in 36 Canadian women. Total maternal BPA concentrations in urine samples ranged from not detected to 9.40 ng/mL (median, 1.21 ng/mL), and free BPA concentrations ranged from not detected to 0.950 ng/mL (median, 0.185 ng/mL). Eighty-six percent of the women had detectable levels of conjugated BPA, whereas only 22 % had detectable levels of free BPA in their urine. BPA levels measured in this study agreed well with data reported internationally.

  12. Development of an enzyme immunoassay for urinary pregnanediol-3-glucuronide in a female giant panda (Ailuropoda melanoleuca).

    PubMed

    Hama, Natsuki; Kanemitsu, Hideyasu; Tanikawa, Michiyo; Shibaya, Masami; Sakamoto, Kensuke; Oyama, Yujiro; Acosta, Tomas J; Ishikawa, Osamu; Pengyan, Wang; Okuda, Kiyoshi

    2009-07-01

    In order to enable monitoring of the reproductive status of the female giant panda after observation of estrus behavior, we developed an enzyme immunoassay (EIA) system for urinary pregnanediol-3-glucuronide (PdG), a progesterone metabolite, using commercial reagents and examined the changes in the urinary concentration of PdG in a female giant panda that showed pseudopregnancy and suspicious pseudopregnancy in 6 consecutive years. The developed EIA system had good reproducibility (intra- and interassay CVs 6.1% and 16.3%, respectively), good parallelism between the standard curve and the dose response curve of serial diluted samples and positive correlation (r=0.836) with the data for PdG in the same samples measured by gas chromatography. Urinary PdG in the female panda showed two phases of increase. The first elevation was observed immediately after estrus with the levels of PdG below 100 ng/Crmg, while the second phase was characterized by a drastic elevation above 100 ng/Crmg until the level began to decrease at the end of pseudopregnancy or suspicious pseudopregnancy. The length of the second phase had wider range than that of the first phase. In the present study, a new EIA assay system for urinary PdG in the female giant panda was developed, and we found that the length of the second phase is unstable in the pseudopregnant and suspicious pseudopregnant giant panda, in contrast with the unstable length of the first phase caused by delayed implantation in the pregnant giant panda.

  13. Expression of UDP-Glucuronosyltransferase 1 (UGT1) and Glucuronidation Activity toward Endogenous Substances in Humanized UGT1 Mouse Brain

    PubMed Central

    Kutsuno, Yuki; Hirashima, Rika; Sakamoto, Masaya; Ushikubo, Hiroko; Michimae, Hirofumi; Itoh, Tomoo; Tukey, Robert H.

    2015-01-01

    Although UDP-glucuronosyltransferases (UGTs) are important phase II drug-metabolizing enzymes, they are also involved in the metabolism of endogenous compounds. Certain substrates of UGTs, such as serotonin and estradiol, play important roles in the brain. However, the expression of UGTs in the human brain has not been fully clarified. Recently, humanized UGT1 mice (hUGT1 mice) in which the original Ugt1 locus was disrupted and replaced with the human UGT1 locus have been developed. In the present study, the expression pattern of UGT1As in brains from humans and hUGT1 mice was examined. We found that UGT1A1, 1A3, 1A6, and 1A10 were expressed in human brains. The expression pattern of UGT1As in hUGT1 mouse brains was similar to that in human brains. In addition, we examined the expression of UGT1A1 and 1A6 in the cerebellum, olfactory bulbs, midbrain, hippocampus, and cerebral cortex of hUGT1 mice. UGT1A1 in all brain regions and UGT1A6 in the cerebellum and cerebral cortex of 6-month-old hUGT1 mice were expressed at a significantly higher rate than those of 2-week-old hUGT1 mice. A difference in expression levels between brain regions was also observed. Brain microsomes exhibited glucuronidation activities toward estradiol and serotonin, with mean values of 0.13 and 5.17 pmol/min/mg, respectively. In conclusion, UGT1A1 and UGT1A6 might play an important role in function regulation of endogenous compounds in a region- and age-dependent manner. Humanized UGT1 mice might be useful to study the importance of brain UGTs in vivo. PMID:25953521

  14. Glucuronidation and sulfonation, in vitro, of the major endocrine-active metabolites of methoxychlor in the channel catfish, Ictalurus punctatus, and induction following treatment with 3-methylcholanthrene

    PubMed Central

    James, Margaret O.; Stuchal, Leah D.; Nyagode, Beatrice A.

    2008-01-01

    The organochlorine pesticide, methoxychlor (MXC), is metabolized in animals to phenolic mono- and bis-demethylated metabolites (OH-MXC and HPTE respectively) that interact with estrogen receptors and may be endocrine disruptors. The phase II detoxication of these compounds will influence the duration of action of the estrogenic metabolites, but has not been investigated extensively. In this study, the glucuronidation and sulfonation of OH-MXC and HPTE were investigated in subcellular fractions of liver and intestine from untreated, MXC-treated and 3-methylcholanthrene (3-MC)-treated channel catfish, Ictalurus punctatus. MXC-treated fish were given i.p. injections of 2 mg MXC/kg daily for 6 days and sacrificed 24 hr after the last dose. The 3-MC treatment was a single 10 mg/kg i.p. dose 5 days prior to sacrifice. In hepatic microsomes from control fish, the Vmax value (mean ± S.D., n=4) for glucuronidation of OH-MXC was 270 ± 50 pmol/min/mg protein, higher than found for HPTE (110 ± 20 pmol/min/mg protein). For each substrate, the Vmax values observed in intestinal microsomes were approximately twice those found in the liver. The Km values for OH-MXC and HPTE glucuronidation in control liver were not significantly different and were 0.32 ± 0.04 mM for OH-MXC and 0.26 ± 0.06 mM for HPTE. The Km for the co-substrate, UDPGA, was higher in liver (0.28 ± 0.09 mM) than intestine (0.04 ± 0.02 mM). Treatment with 3-MC but not MXC increased the Vmax for glucuronidation in liver and intestine. Glucuronidation was a more efficient pathway than sulfonation for both substrates, in both tissues. The Vmax values for sulfonation of OH-MXC and HPTE respectively in liver cytosol were 7 ± 3 and 17 ± 4 pmol/min/mg protein and in intestinal cytosol were 13 ± 3 and 30 ± 5 pmol/min/mg protein. Treatment with 3-MC but not MXC increased rates of sulfonation of OH-MXC and HPTE and the model substrate, 3-hydroxy-benzo(a)pyrene in both intestine and liver. Comparison of the kinetics

  15. Gas chromatography-mass spectrometry of ethyl palmitate calibration and resolution with ethyl oleate as biomarker ethanol sub acute in urine application study

    NASA Astrophysics Data System (ADS)

    Suaniti, Ni Made; Manurung, Manuntun

    2016-03-01

    Gas Chromatography-Mass Spectrometry is used to separate two and more compounds and identify fragment ion specific of biomarker ethanol such as palmitic acid ethyl ester (PAEE), as one of the fatty acid ethyl esters as early detection through conyugated reaction. This study aims to calibrate ethyl palmitate and develop analysis with oleate acid. This methode can be used analysis ethanol and its chemistry biomarker in ethanol sub-acute consumption as analytical forensic toxicology. The result show that ethanol level in urine rats Wistar were 9.21 and decreased 6.59 ppm after 48 hours consumption. Calibration curve of ethyl palmitate was y = 0.2035 x + 1.0465 and R2 = 0.9886. Resolution between ethyl palmitate and oleate were >1.5 as good separation with fragment ion specific was 88 and the retention time was 18 minutes.

  16. Multiple-dose lorazepam kinetics: shuttling of lorazepam glucuronide between the circulation and the gut during day- and night-time dosing intervals in response to feeding.

    PubMed

    Chaudhary, A; Lane, R A; Woo, D; Herman, R J

    1993-12-01

    Lorazepam kinetics were examined in seven healthy males age 18 to 30 years after single- and multiple-dose lorazepam administration and in the presence and absence of neomycin and cholestyramine to block the enterohepatic circulation of the drug. Methods used a simultaneous i.v./p.o. dosing regimen with provision to measure lorazepam clearance during day- and night-time dosing intervals. The day-time steady-state clearance of free lorazepam measured 7.55 +/- 1.95 ml/min/kg (mean +/- S.D.) and was identical to that observed after single-dose administration (7.68 +/- 3.19 ml/min/kg). Neomycin and cholestyramine increased lorazepam clearances 5 to 45% (P < or = .05) as would be expected for interruption of an enterohepatic circulation and in keeping with previous observations under nonsteady-state conditions. Lorazepam clearances were the same during the day as during the night, except in the presence of neomycin and cholestyramine, where night-time clearances were significantly greater (10.16 +/- 3.52 vs. 8.77 +/- 2.43 ml/min/kg, P < or = .05). Urinary recoveries of lorazepam glucuronide, on the other hand, were greater during the day than during the night (114 +/- 11 vs. 77 +/- 15%, P < or = .05) and in all cases were greater than 100% of the administered dose for that interval. Thus, there is a diurnal variation in lorazepam elimination consistent with a fasting-induced increase in hepatic glucuronidation during the night. This, combined with the relative inactivity of the gut during this period, serves to trap the glucuronide and delay its transfer back to the systemic circulation and urine.

  17. Hesperetin and its sulfate and glucuronide metabolites inhibit TNF-α induced human aortic endothelial cell migration and decrease plasminogen activator inhibitor-1 (PAI-1) levels.

    PubMed

    Giménez-Bastida, Juan Antonio; González-Sarrías, Antonio; Vallejo, Fernando; Espín, Juan Carlos; Tomás-Barberán, Francisco A

    2016-01-01

    Epidemiological, clinical and preclinical studies have reported the protection offered by citrus consumption, mainly orange, against cardiovascular diseases, which is primarily mediated by the antiatherogenic and vasculoprotective effects of the flavanone hesperetin-7-O-rutinoside (hesperidin). However, flavanone aglycones or glycosides are not present in the bloodstream but their derived phase-II metabolites could be the actual bioactive molecules. To date, only a few studies have explored the effects of circulating hesperetin-derived metabolites (glucuronides and sulfates) on endothelial cells. Herein, we describe for the first time the effects of hesperetin 3'-O-glucuronide, hesperetin 7-O-glucuronide, hesperetin 3'-O-sulfate, hesperetin 7-O-sulfate and hesperetin on human aortic endothelial cell (HAEC) migration upon pro-inflammatory stimuli as an essential step to angiogenesis. Hesperetin and its derived metabolites, at physiologically relevant concentrations (1-10 μM), significantly attenuated cell migration in the presence of the pro-inflammatory cytokine TNF-α (50 ng mL(-1)), which was accompanied and perhaps mediated by a significant decrease in the levels of the thrombogenic plasminogen activator inhibitor-1 (PAI-1). However, hesperetin metabolites did not counteract the TNF-α-induced production of pro-inflammatory interleukin-6 (IL-6) and IL-8. We also study here for the first time, the metabolism of hesperetin and its derived metabolites by HAEC with and without a pro-inflammatory stimulus. All these results reinforce the concept according to which circulating phase-II hesperetin metabolites are critical molecules contributing to the cardioprotective effects upon consumption of citrus fruits such as orange.

  18. Metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in the patas monkey: pharmacokinetics and characterization of glucuronide metabolites.

    PubMed

    Hecht, S S; Trushin, N; Reid-Quinn, C A; Burak, E S; Jones, A B; Southers, J L; Gombar, C T; Carmella, S G; Anderson, L M; Rice, J M

    1993-02-01

    The metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was examined in the patas monkey, in order to provide further information about NNK metabolic pathways in primates. Female patas monkeys were given i.v. injections of [5-3H]NNK, and metabolites in serum and urine were analyzed by HPLC. Metabolism by alpha-hydroxylation of NNK was rapid and extensive, and the products of this pathway, 4-hydroxy-4-(3-pyridyl)butyric acid and 4-oxo-4-(3-pyridyl) butyric acid, accounted for a relatively large proportion of serum and urinary metabolites at all time points. This is significant because the formation of these products is associated with modification of DNA by NNK. The other major metabolic pathway was carbonyl reduction to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which detected both unconjugated and diastereomeric O-glucuronides. One of these glucuronides had been previously identified in rat urine, but the other diastereomer, which was the more prevalent of the two in serum and urine, had not been observed in studies of NNK metabolism in rodents. It was characterized by its spectral properties, by enzymatic hydrolysis to NNAL, and by derivatization of the released NNAL enantiomer with (R)-(+)-alpha-methylbenzylisocyanate. The two NNAL glucuronides accounted for 15-20% of the urinary metabolites in monkeys given 0.1 micrograms/kg NNK, which is similar to a smoker's dose, suggesting their use as dosimeters of NNK exposure in humans. Pharmacokinetic parameters were consistent with those observed in previous studies of nitrosamines, and varied predictably with body weight of five species. The results of this study have provided new insights relevant to assessing human metabolism of NNK.

  19. Development and validation of an HPLC-MS/MS method to quantify clopidogrel acyl glucuronide, clopidogrel acid metabolite, and clopidogrel in plasma samples avoiding analyte back-conversion.

    PubMed

    Silvestro, Luigi; Gheorghe, Mihaela; Iordachescu, Adriana; Ciuca, Valentin; Tudoroniu, Ariana; Rizea Savu, Simona; Tarcomnicu, Isabela

    2011-08-01

    A new sensitive and fast quantitative analytical method for the simultaneous determination of clopidogrel, its main metabolite clopidogrel carboxylic acid, and the newly described acyl glucuronide metabolite, in human plasma samples, is presented. The analytical procedures (plasma storage, handling, and extract storage in the autosampler) were optimized in order to avoid back-conversion; a known drawback in measurements of clopidogrel. Clopidogrel acyl glucuronide was confirmed as a major source of back-conversion to the parent drug in the presence of methanol, and thorough stability experiments were carried out to find the most appropriate conditions for an accurate analysis of clopidogrel and the two metabolites. The method was validated by assessing selectivity, sensitivity, linearity, accuracy, and precision for all three analytes, in accordance to Food and Drug Administration guidelines. Spiked quality controls in plasma as well as incurred samples were used to verify back-conversion in the selected conditions, with results meeting European Medicines Agency acceptance criteria (concentrations within 80-120% of the first reading). The method was then applied to a pharmacokinetic study, and for the first time, a pharmacokinetic curve of clopidogrel acyl glucuronide in human plasma is presented. The concentrations ranged up to 1,048.684 ng/mL, with a mean of 470.268 ng/mL, while clopidogrel had a mean C(max) of 1.348 ng/mL; these orders of magnitude show how much the back-conversion of this metabolite may influence clopidogrel quantification if it is not properly controlled.

  20. Glucuronidation of thyroxine in primary monolayer cultures of rat hepatocytes: in vitro induction of UDP-glucuronosyltranferases by methylcholanthrene, clofibrate, and dexamethasone alone and in combination.

    PubMed

    Jemnitz, K; Veres, Z; Monostory, K; Vereczkey, L

    2000-01-01

    Induction of UDP-glucuronosyltransferases (UGTs) toward thyroxine (T4) and p-nitrophenol (pNP) by 3-methylcholanthrene (MC), dexamethasone (DEX), clofibrate (Cl), and MC combined with DEX or Cl was studied in rat hepatocyte culture. We have developed a sensitive method for the measurement of glucuronide conjugates of the two substrates based on HPLC analysis of culture medium. MC, Cl, or DEX increased the activity of T4 UGT. Combination of MC and Cl showed additive effect, enzyme activity was enhanced compared with either MC or Cl treatment alone (617, 441, and 217% of the control, respectively). Combination of MC and DEX did not result in higher T4 UGT activity than MC treatment alone. Both MC and DEX enhanced the pNP UGT activity (182 and 162% of the control, respectively). Combination of MC with DEX resulted in additive effect. Cl treatment did not affect pNP conjugation either alone or in combination with MC. Western blot analysis revealed that only the amount of UGT1A1 was elevated by Cl and DEX. In contrast, concentration of UGT1A6 was increased by MC. Previous studies demonstrated that UGT1A1 inducers like phenobarbital have no effect on T4 conjugation (). Our results suggest that Cl, a known inducer of UGT1A1, enhances the activity of other enzyme(s) involved in T4 glucuronidation as well. It is well documented that DEX potentiates the inductory effect of polycyclic aromatic hydrocarbon on UGT1A6 (). In our study, MC increased the rate of T4 glucuronidation, and DEX had no additional effect on this reaction, suggesting that UGT1A6 is not the only enzyme inducible by MC that can catalyze T4 conjugation. PMID:10611137

  1. Labeled 1,N6-ethenoadenosine and 3,N4-ethenocytidine in hepatic RNA of mice given[ethyl-1,2(-3)H or ethyl-1(-14)C]ethyl carbamate (urethan).

    PubMed

    Ribovich, M L; Miller, J A; Miller, E C; Timmins, L G

    1982-01-01

    Injection of a single dose of[ethyl-1,2(-3)H]or[ethyl-1(-14C]- ethyl carbamate into 12-day old male[C57BL/6 x C3H/He]F1 mice or of[ethyl-1,2(-3H]ethyl carbamate into adult male A/Jax mice resulted in the formation of labeled 1,N6-ethenoadenosine and 3,N4-ethenocytidine adducts in the hepatic RNA. These adducts were characterized by comigration on h.p.l.c. of 3H or 14C in enzymatic hydrolysates of the RNA with synthetic standards. Both the ethenoadenosine and ethenocytidine were further characterized by their conversion to acetylated products that comigrated with acetylated synthetic standards. The ethenoadenosine was also converted by anhydrous trifluoroacetic acid to a product that comigrated with synthetic 1,N6-ethenoadenine. The levels of adducts in the hepatic RNA 12 h after a single injection of 0.5-0.6 mg of ethyl carbamate/g body weight were 6-10 and 2-3 pmol/mg RNA of ethenoadenosine and ethenocytidine, respectively. No labeled ethenoadenosine or ethenocytidine could be detected in the hepatic RNA of mice given[1-14C]ethanol, an enzymatic hydrolysis product of ethyl carbamate. These data indicate that ethyl carbamate may be metabolically activated by dehydrogenation to vinyl carbamate and subsequent epoxidation of the latter compound as previously proposed. Vinyl carbamate epoxide may form etheno derivatives in a manner analogous to that demonstrated for chloroethylene oxide, an electrophilic metabolite of vinyl chloride. Vinyl carbamate has been shown to have the same spectrum of tumor induction as ethyl carbamate but to be much more active than the latter carcinogen. PMID:6178529

  2. Ocular fluorometry methodological improvements and clinical studies--with special reference to the blood-retina barrier permeability to fluorescein and fluorescein glucuronide.

    PubMed

    Larsen, M

    1993-01-01

    The measurement of fluorescence in the human eye can be made using relatively simple instruments. Fluorescence is evoked when illumination is absorbed by intrinsic fluorophores in the eye or by artificially introduced extrinsic fluorophores. Intrinsic fluorescence is evidence of important molecular characteristics of the ocular tissues, whereas the extrinsic fluorophores are used primarily in the study of the barriers between the anatomical and physiological compartments of the eye. Blood-retina barrier leakage of fluorescein can be examined after the intravenous injection of fluorescein by quantitative determination of fluorescence in plasma and in the vitreous. From these measurements of the distribution of fluorescein, the permeability of a hypothetical spherical interface between the blood and the retina can be estimated using a mathematical model of the barrier. The use of fluorescein as a tracer is problematic because of its rapid metabolic conversion to fluorescein glucuronide. This metabolite disturbs ocular fluorescence measurements because it fluoresces over the same part of the spectrum as the parent compound. Additionally, the glucuronide occurs in markedly different concentrations depending upon the patient's renal function. With the previously used fluorometry techniques it has been impossible to determine the contribution of fluorescein glucuronide to the vitreous fluorescence. The primary objective of the studies described in this thesis was to develop a method for the determination of fluorescein and fluorescein glucuronide in the human eye and in plasma, and to calculate the blood-retina barrier permeabilities of the two substances. The necessary methodological improvements included a detailed description of the geometrical optics of the eye and the optical filter properties of the lens. A new method was developed for the determination of the spatial locations of ocular fluorescence measurements and the intrinsic lens fluorescence was used to

  3. Cytotoxic constituents of ethyl acetate fraction from Dianthus superbus.

    PubMed

    Ding, Chengli; Zhang, Wu; Li, Jie; Lei, Jiachuan; Yu, Jianqing

    2013-01-01

    The ethyl acetate fraction (EE-DS) from Dianthus superbus was found to possess the cytotoxic activity against cancer cells in previous study. To investigate cytotoxic constituents, the bioassay-guided isolation of compounds from EE-DS was performed. Two dianthramides (1 and 2), three flavonoids (3-5), two coumarins (6 and 7) and three other compounds (8-10) were obtained. Structures of isolated compounds were identified by spectroscopic analysis. Cytotoxicity of the compounds against HepG2 cells was evaluated. Compound 1 showed the strongest cytotoxicity, compounds 10, 4, 3 and 5 had moderate cytotoxicity.

  4. Ethyl alcohol boiling heat transfer on multilayer meshed surfaces

    NASA Astrophysics Data System (ADS)

    Dåbek, Lidia; Kapjor, Andrej; Orman, Łukasz J.

    2016-06-01

    The paper presents the problem of heat transfer enhancement with the application of multilayer metal mesh structures during boiling of ethyl alcohol at ambient pressure. The preparation of samples involved sintering fine copper meshes with the copper base in the reduction atmosphere in order to prevent oxidation of the samples. The experiments included testing up to 4 layers of copper meshes. Significant augmentation of boiling heat transfer is possible, however, considerable number of meshes actually hinders heat transfer conditions and leads to the reduction in the heat flux transferred from the heater surface.

  5. Ethyl Pyruvate Combats Human Leukemia Cells but Spares Normal Blood Cells

    PubMed Central

    Kurz, Susanne; Bigl, Marina; Buchold, Martin; Thieme, Rene; Wichmann, Gunnar; Dehghani, Faramarz

    2016-01-01

    Ethyl pyruvate, a known ROS scavenger and anti-inflammatory drug was found to combat leukemia cells. Tumor cell killing was achieved by concerted action of necrosis/apoptosis induction, ATP depletion, and inhibition of glycolytic and para-glycolytic enzymes. Ethyl lactate was less harmful to leukemia cells but was found to arrest cell cycle in the G0/G1 phase. Both, ethyl pyruvate and ethyl lactate were identified as new inhibitors of GSK-3β. Despite the strong effect of ethyl pyruvate on leukemia cells, human cognate blood cells were only marginally affected. The data were compiled by immune blotting, flow cytometry, enzyme activity assay and gene array analysis. Our results inform new mechanisms of ethyl pyruvate-induced cell death, offering thereby a new treatment regime with a high therapeutic window for leukemic tumors. PMID:27579985

  6. Ethyl Pyruvate Combats Human Leukemia Cells but Spares Normal Blood Cells.

    PubMed

    Birkenmeier, Gerd; Hemdan, Nasr Y A; Kurz, Susanne; Bigl, Marina; Pieroh, Philipp; Debebe, Tewodros; Buchold, Martin; Thieme, Rene; Wichmann, Gunnar; Dehghani, Faramarz

    2016-01-01

    Ethyl pyruvate, a known ROS scavenger and anti-inflammatory drug was found to combat leukemia cells. Tumor cell killing was achieved by concerted action of necrosis/apoptosis induction, ATP depletion, and inhibition of glycolytic and para-glycolytic enzymes. Ethyl lactate was less harmful to leukemia cells but was found to arrest cell cycle in the G0/G1 phase. Both, ethyl pyruvate and ethyl lactate were identified as new inhibitors of GSK-3β. Despite the strong effect of ethyl pyruvate on leukemia cells, human cognate blood cells were only marginally affected. The data were compiled by immune blotting, flow cytometry, enzyme activity assay and gene array analysis. Our results inform new mechanisms of ethyl pyruvate-induced cell death, offering thereby a new treatment regime with a high therapeutic window for leukemic tumors. PMID:27579985

  7. Effects of mitragynine and 7-hydroxymitragynine (the alkaloids of Mitragyna speciosa Korth) on 4-methylumbelliferone glucuronidation in rat and human liver microsomes and recombinant human uridine 5’-diphospho-glucuronosyltransferase isoforms

    PubMed Central

    Haron, Munirah; Ismail, Sabariah

    2015-01-01

    Background: Glucuronidation catalyzed by uridine 5’- diphospho-glucuronosyltransferase (UGT) is a major phase II drug metabolism reaction which facilitates drug elimination. Inhibition of UGT activity can cause drug-drug interaction. Therefore, it is important to determine the inhibitory potentials of drugs on glucuronidation. Objective: The objective was to evaluate the inhibitory potentials of mitragynine, 7-hydroxymitragynine, ketamine and buprenorphine, respectively on 4-methylumbelliferone (4-MU) glucuronidation in rat liver microsomes, human liver microsomes and recombinant human UGT1A1 and UGT2B7 isoforms. Materials and Methods: The effects of the above four compounds on the formation of 4-MU glucuronide from 4-MU by rat liver microsomes, human liver microsomes, recombinant human UGT1A1 and UGT2B7 isoforms were determined using high-performance liquid chromatography with ultraviolet detection. Results: For rat liver microsomes, ketamine strongly inhibited 4-MU glucuronidation with an IC50 value of 6.21 ± 1.51 μM followed by buprenorphine with an IC50 value of 73.22 ± 1.63 μM. For human liver microsomes, buprenorphine strongly inhibited 4-MU glucuronidation with an IC50 value of 6.32 ± 1.39 μM. For human UGT1A1 isoform, 7-hydroxymitragynine strongly inhibited 4-MU glucuronidation with an IC50 value of 7.13 ± 1.16 μM. For human UGT2B7 isoform, buprenorphine strongly inhibited 4-MU glucuronidation followed by 7-hydroxymitragynine and ketamine with respective IC50 values of 5.14 ± 1.30, 26.44 ± 1.31, and 27.28 ± 1.18 μM. Conclusions: These data indicate the possibility of drug-drug interaction if 7-hydroxymitragynine, ketamine, and buprenorphine are co-administered with drugs that are UGT2B7 substrates since these three compounds showed significant inhibition on UGT2B7 activity. In addition, if 7-hydroxymitragynine is to be taken with other drugs that are highly metabolized by UGT1A1, there is a possibility of drug-drug interaction to occur. PMID

  8. Highly active gold-based catalyst for the reaction of benzaldehyde with ethyl diazoacetate.

    PubMed

    Fructos, Manuel R; Díaz-Requejo, M Mar; Pérez, Pedro J

    2009-09-14

    The gold complex [IPrAu(NCMe)]BF(4) catalyzes the reaction of ethyl diazoacetate with benzaldehyde to give mixtures of ethyl 3-oxo-3-phenylpropanoate and ethyl 3-hydroxy-2-phenylacrylate in the first example of a group 11 metal-based catalyst for this transformation; the catalyst activity is improved by a factor of 2500 compared to those of previously reported iron-based catalysts.

  9. Pharmacokinetic comparison between quercetin and quercetin 3-O-β-glucuronide in rats by UHPLC-MS/MS

    PubMed Central

    Yang, Le-Le; Xiao, Na; Li, Xiao-Wei; Fan, Yong; Alolga, Raphael N.; Sun, Xiao-Yue; Wang, Shi-Lei; Li, Ping; Qi, Lian-Wen

    2016-01-01

    Quercetin is a natural flavonoid widely distributed in human diet and functional foods. Quercetin 3-O-β-glucuronide (Q3G) is present in wine and some medicinal plants. Quercetin and Q3G may be metabolized from each other in vivo. While quercetin has been the subject of many studies, the pharmacokinetic profiles of quercetin and Q3G (in animals) have not yet been compared. Herein, we prepared a column-based method for rapid isolation of Q3G from Nelumbo nucifera. Then, we developed an UHPLC-MS/MS method to compare the pharmacokinetics of quercetin and Q3G. Our results showed that the plasma concentration-time curves of quercetin and Q3G show two maxima (Tmax1 ≈ 0.75 h, Tmax2 ≈ 5 h). After oral administration of 100 mg/kg quercetin or 100 mg/kg Q3G in rats, predominantly Q3G was detected in plasma with AUC at 39529.2 ± 6108.2 mg·h·L−1 or 24625.1 ± 1563.8 mg·h·L−1, 18-fold higher than quercetin with AUC at 1583.9 ± 583.3 mg·h·L−1 or 1394.6 ± 868.1 mg·h·L−1, respectively. After intravenous injection of 10 mg/kg in rats, Q3G showed extensive tissue uptake in kidney (409.2 ± 118.4 ng/g), liver (166.1 ± 52.9 ng/g), heart (97.7 ± 22.6 ng/g), and brain (5.8 ± 1.2 ng/g). In conclusion, we have shown that Q3G is a major active component in plasma and tissue for oral administration of quercetin or Q3G. PMID:27775094

  10. Transesterification process to manufacture ethyl ester of rape oil

    SciTech Connect

    Korus, R.A.; Hoffman, D.S.; Bam, N.; Peterson, C.L.; Drown, D.C.

    1993-12-31

    A process for the production of the ethyl ester of winter rape [EEWR] for use as a biodiesel fuel has been studied. The essential part of the process is the transesterification of rape oil with ethanol, in the presence of a catalyst, to yield the ethyl ester of rape oil as a product and glycerin as a by-product. Experiments have been performed to determine the optimum conditions for the preparation of EEWR. The process variables were: (1) temperature, (2) catalyst, (3) rate of agitation, (4) water content of the alcohol used, and (5) the amount of excess alcohol used. The optimum conditions were: (1) room temperature, (2) 0.5% sodium methoxide or 1% potassium hydroxide catalyst by weight of rapeseed oil, (3) extremely vigorous agitation with some splashing during the initial phase of the reaction and agitation was not necessary after the reaction mixture became homogeneous, (4) absolute ethanol was necessary for high conversion, and (5) 50% excess ethanol with NaOCH{sub 3} or 100% excess with KOH gave a maximum conversion. Viscosity, cloud point and pour point of the EEWR were measured. A preliminary break-even cost for the commercial production of EEWR was found to be $0.55/liter [$2.08/US gallon].

  11. Growth of glycine ethyl ester hydrochloride and its characterizations

    NASA Astrophysics Data System (ADS)

    Venkatesan, G.; Pari, S.

    2016-11-01

    Single crystal of glycine ethyl ester hydrochloride by slow evaporation method is reported. The grown crystal characterized by single crystal X-ray diffraction, FT-IR, UV-Vis-NIR and fluorescence spectroscopy. It is established that the crystal falls under the monoclinic system and space group P21/c with the cell parameters as: a=8.565 Å, b=12.943 Å, c=6.272 Å, α=γ=90°, β=103.630º. UV-Vis-NIR spectrum shows indirect allowed transition with a band gap of 5.21 eV and other optical properties are measured. The crystal is also shown to have a high transmittance in the visible region. The third order nonlinear property and optical limiting have been investigated using Z-Scan technique. Complex impedance spectrum measured at the dc conductivity. Dependence of dielectric constant, dielectric loss and ac conductivity on frequency at different temperature of applied ac field is analyzed. The mechanical behavior has been assessed by Vickers microhardness indenter. The thermal behavior of glycine ethyl ester hydrochloride was analyzed using TG/DTA thermal curves. From the thermal study, the material was found to possess thermal stability up to 174 °C. The predicted NLO properties, UV-Vis transmittance and Z-scan studies indicate that is an attractive material for photonics optical limiting applications.

  12. DISCOVERY OF METHYL ACETATE AND GAUCHE ETHYL FORMATE IN ORION

    SciTech Connect

    Tercero, B.; Cernicharo, J.; Lopez, A.; Caro, G. M. Munoz; Kleiner, I.; Nguyen, H. V. L. E-mail: jcernicharo@cab.inta-csic.es E-mail: munozcg@cab.inta-csic.es E-mail: nguyen@pc.rwth-aachen.de

    2013-06-10

    We report on the discovery of methyl acetate, CH{sub 3}COOCH{sub 3}, through the detection of a large number of rotational lines from each one of the spin states of the molecule: AA species (A{sub 1} or A{sub 2}), EA species (E{sub 1}), AE species (E{sub 2}), and EE species (E{sub 3} or E{sub 4}). We also report, for the first time in space, the detection of the gauche conformer of ethyl formate, CH{sub 3}CH{sub 2}OCOH, in the same source. The trans conformer is also detected for the first time outside the Galactic center source SgrB2. From the derived velocity of the emission of methyl acetate, we conclude that it arises mainly from the compact ridge region with a total column density of (4.2 {+-} 0.5) Multiplication-Sign 10{sup 15} cm{sup -2}. The derived rotational temperature is 150 K. The column density for each conformer of ethyl formate, trans and gauche, is (4.5 {+-} 1.0) Multiplication-Sign 10{sup 14} cm{sup -2}. Their abundance ratio indicates a kinetic temperature of 135 K for the emitting gas and suggests that gas-phase reactions could participate efficiently in the formation of both conformers in addition to cold ice mantle reactions on the surface of dust grains.

  13. Genotoxicity and cytotoxicity assessment of new ethyl-carbamates with ixodicidal activity.

    PubMed

    Prado-Ochoa, María Guadalupe; Muñoz-Guzmán, Marco Antonio; Vázquez-Valadez, Víctor Hugo; Velázquez-Sánchez, Ana María; Salazar, Ana María; Ramírez-Noguera, Patricia; Angeles, Enrique; Alba-Hurtado, Fernando

    2016-09-01

    The mammalian erythrocyte micronucleus test was used on the peripheral blood of Wistar rats exposed to two new ethyl-carbamates: ethyl-4-bromophenyl-carbamate (LQM 919) and ethyl-4-chlorophenyl-carbamate (LQM 996) to analyze their genotoxic potential. The mitotic index and cell proliferation kinetics in human lymphocyte cultures in the presence of these ethyl-carbamates were used to evaluate cytotoxicity and cytostaticity respectively. Exposure to greater acute doses (300mg/kg) and to all of the subchronic doses (12.5, 25 and 50mg/kg daily for 90 days) of these ethyl-carbamates induced an increased frequency (p<0.05) of micro-nucleated polychromatic erythrocytes (MN-PCE) compared with rats not exposed to the ethyl-carbamates. Increases in MN-PCE was higher in males than in females exposed to LQM 996 50mg/Kg (p<0.05). All observed changes in rats return 21days after suspending ethyl-carbamate exposure. The highest concentration (0.3mM) of both ethyl-carbamates in lymphocyte cultures increased the percentage of cells in first division metaphase and decreased the percentage of cells in third division metaphase, indicating an increase in cell cycle length or a possible cell cycle arrest in metaphase (cytostatic effect). The results of this study show that the evaluated ethyl-carbamates may induce genotoxic damage in rats and alterations in the human lymphocyte cell cycle. PMID:27542710

  14. Genotoxicity and cytotoxicity assessment of new ethyl-carbamates with ixodicidal activity.

    PubMed

    Prado-Ochoa, María Guadalupe; Muñoz-Guzmán, Marco Antonio; Vázquez-Valadez, Víctor Hugo; Velázquez-Sánchez, Ana María; Salazar, Ana María; Ramírez-Noguera, Patricia; Angeles, Enrique; Alba-Hurtado, Fernando

    2016-09-01

    The mammalian erythrocyte micronucleus test was used on the peripheral blood of Wistar rats exposed to two new ethyl-carbamates: ethyl-4-bromophenyl-carbamate (LQM 919) and ethyl-4-chlorophenyl-carbamate (LQM 996) to analyze their genotoxic potential. The mitotic index and cell proliferation kinetics in human lymphocyte cultures in the presence of these ethyl-carbamates were used to evaluate cytotoxicity and cytostaticity respectively. Exposure to greater acute doses (300mg/kg) and to all of the subchronic doses (12.5, 25 and 50mg/kg daily for 90 days) of these ethyl-carbamates induced an increased frequency (p<0.05) of micro-nucleated polychromatic erythrocytes (MN-PCE) compared with rats not exposed to the ethyl-carbamates. Increases in MN-PCE was higher in males than in females exposed to LQM 996 50mg/Kg (p<0.05). All observed changes in rats return 21days after suspending ethyl-carbamate exposure. The highest concentration (0.3mM) of both ethyl-carbamates in lymphocyte cultures increased the percentage of cells in first division metaphase and decreased the percentage of cells in third division metaphase, indicating an increase in cell cycle length or a possible cell cycle arrest in metaphase (cytostatic effect). The results of this study show that the evaluated ethyl-carbamates may induce genotoxic damage in rats and alterations in the human lymphocyte cell cycle.

  15. Determination of opiates in whole blood and vitreous humor: a study of the matrix effect and an experimental design to optimize conditions for the enzymatic hydrolysis of glucuronides.

    PubMed

    Sanches, Livia Rentas; Seulin, Saskia Carolina; Leyton, Vilma; Paranhos, Beatriz Aparecida Passos Bismara; Pasqualucci, Carlos Augusto; Muñoz, Daniel Romero; Osselton, Michael David; Yonamine, Mauricio

    2012-04-01

    Undoubtedly, whole blood and vitreous humor have been biological samples of great importance in forensic toxicology. The determination of opiates and their metabolites has been essential for better interpretation of toxicological findings. This report describes the application of experimental design and response surface methodology to optimize conditions for enzymatic hydrolysis of morphine-3-glucuronide and morphine-6-glucuronide. The analytes (free morphine, 6-acetylmorphine and codeine) were extracted from the samples using solid-phase extraction on mixed-mode cartridges, followed by derivatization to their trimethylsilyl derivatives. The extracts were analysed by gas chromatography-mass spectrometry with electron ionization and full scan mode. The method was validated for both specimens (whole blood and vitreous humor). A significant matrix effect was found by applying the F-test. Different recovery values were also found (82% on average for whole blood and 100% on average for vitreous humor). The calibration curves were linear for all analytes in the concentration range of 10-1,500 ng/mL. The limits of detection ranged from 2.0 to 5.0 ng/mL. The method was applied to a case in which a victim presented with a previous history of opiate use.

  16. Macromolecular prodrug that provides the irinotecan (CPT-11) active-metabolite SN-38 with ultralong half-life, low C(max), and low glucuronide formation.

    PubMed

    Santi, Daniel V; Schneider, Eric L; Ashley, Gary W

    2014-03-27

    We have recently reported a chemical approach for half-life extension that utilizes β-eliminative linkers to attach amine-containing drugs or prodrugs to macromolecules. The linkers release free drug or prodrug over periods ranging from a few hours to over 1 year. We adapted these linkers for use with phenol-containing drugs. Here, we prepared PEG conjugates of the irinotecan (CPT-11) active metabolite SN-38 via a phenyl ether that release the drug with predictable long half-lives. Pharmacokinetic studies in the rat indicate that, in contrast to other SN-38 prodrugs, the slowly released SN-38 shows a very low C(max), is kept above target concentrations for extended periods, and forms very little SN-38 glucuronide (the precursor of enterotoxic SN-38). The low SN-38 glucuronide is attributed to low hepatic uptake of SN-38. These macromolecular prodrugs have unique pharmacokinetic profiles that may translate to less intestinal toxicity and interpatient variability than the SN-38 prodrugs thus far studied.

  17. Chemical synthesis of all-trans-[11-3H]retinoyl beta-glucuronide and its metabolism in rats in vivo.

    PubMed Central

    Barua, A B; Olson, J A

    1989-01-01

    All-trans-[11-3H]retinoyl beta-glucuronide (RAG) was synthesized in a single step from all-trans-[11-3H]retinoyl fluoride, with a 24% yield. After its intraperitoneal injection into rats, RAG was detected in the blood, liver, intestine and kidney during the following 24 h period. Although the concentration of radiolabelled metabolites decreased with time, RAG predominated at nearly all times in nearly all tissues. Small amounts of retinoic acid (RA) were also universally present, together with unidentified polar metabolites and small amounts of non-polar esters of RA. The major excretion products of RAG in faeces and urine were RA and polar metabolites. Thus RAG, although converted in part to RA in vivo, persists as a major component in blood and tissues for at least 24 h. These observations support the concept that the retinoid beta-glucuronides might serve a physiologically significant role in the function of vitamin A. PMID:2597112

  18. Interindividual variability in hepatic drug glucuronidation: studies into the role of age, sex, enzyme inducers, and genetic polymorphism using the human liver bank as a model system.

    PubMed

    Court, Michael H

    2010-02-01

    The human liver bank has provided an invaluable model system for the study of interindividual variability in expression and activity of the major hepatic UGTs, including UGT1A1, 1A4, 1A6, 1A9, 2B7, and 2B15. Based on studies using UGT-isoform-selective probes, the rank order of activity variability is UGT 1A1>1A6>2B15>1A4 = 1A9>2B7, with coefficient of variation values ranging from 92 to 45%. Liver donor age, sex, enzyme inducers, and genetic polymorphism are factors that have been implicated as sources of this variability in UGT activity. The expression of UGTs prior to, and immediately following, birth is quite limited, explaining the susceptibility of neonates to certain drug toxicities. Old age appears to have minimal effect on UGT function. Sex differences in UGT activity are relatively small and are confined to several UGTs, including UGT2B15, which shows higher activity in males, compared with females. Enzyme inducers, including coadministered drugs, smoking, and alcohol, may increase hepatic UGT levels. Human liver bank phenotype-genotype studies, using UGT-isoform-selective probes have identified common genetic polymorphisms that are predictive of glucuronidation activity in vitro and that were subsequently verified as predictors of probe-drug clearance by glucuronidation in vivo.

  19. Mycophenolic acid glucuronide is transported by multidrug resistance-associated protein 2 and this transport is not inhibited by cyclosporine, tacrolimus or sirolimus.

    PubMed

    Patel, Chirag G; Ogasawara, Ken; Akhlaghi, Fatemeh

    2013-03-01

    1. The purpose of this study was to investigate the contribution of MRP2 to the efflux of mycophenolic acid (MPA), and its phenyl glucuronide (MPAG) and acyl glucuronide (AcMPAG) metabolites, using Madin-Darby canine kidney II cells stably transfected with human MRP2 gene (MDCKII/MRP2 cells). 2. Compared to parental MDCKII cells, MPAG was significantly translocated from basolateral (BL) to apical (AP) side in MDCKII/MRP2 cells, indicating MPAG is a substrate for MRP2. AcMPAG is highly translocated from BL to AP side in both cells, suggesting that AcMPAG is actively secreted possibly through an efflux transporter other than MRP2. Appreciable translocation of MPA was not observed in MDCKII/MRP2 cells. 3. Furthermore, using MRP2-expressing Sf9 membrane vesicles, the Michaelis-Menten constant (Km) value for MRP2-mediated MPAG transport was calculated at 224.2 ± 42.7 µM. In the vesicle system, cyclosporine, tacrolimus and sirolimus did not inhibit the uptake of MPAG via MRP2. 4. These findings indicate that only MPAG not MPA and AcMPAG is a substrate for MRP2 and that the interaction between MPAG and concomitantly administered immunosuppressive agents does not occur at MRP2 level. PMID:22934787

  20. Synthesis and application of resorufin β-D-glucuronide, a low-cost chromogenic substrate for detecting Escherichia coli in drinking water.

    PubMed

    Magro, Germinal; Bain, Robert E S; Woodall, Claire A; Matthews, Robert L; Gundry, Stephen W; Davis, Anthony P

    2014-08-19

    The development of low-cost tests for Escherichia coli is hampered by the expense and limited choice of enzyme substrates. Most chromogenic substrates are required in costly amounts, while fluorogenic substrates require an additional apparatus (e.g., an ultraviolet lamp) to be detected. Herein, we propose an alternative chromogenic substrate, resorufin β-d-glucuronide (REG), which is exceptionally sensitive and may be employed in very small amounts. We show that REG can be produced similarly to other simple glucuronides and should therefore be no more expensive. The compound is used by both healthy and injured E. coli, resulting in a pronounced color change from orange to a bright pink. Because the released dye (resorufin) has a high extinction coefficient, substantially lower amounts are needed than for commercially available substrates. The potential of this substrate is demonstrated by a presence/absence test requiring just 0.1 mg of REG/100 mL of water sample, one hundredth of the quantity needed for common chromogenic substrates, with an estimated bulk cost of ≤0.1 U.S. cents/test. REG shows promise as a chromogenic substrate for E. coli detection and should be considered in the development of new water tests, especially for low-income settings.

  1. Effects of switching from omega-3-acid ethyl esters to icosapent ethyl in a statin-treated patient with elevated triglycerides.

    PubMed

    Kedia, Anurag W; Lynch, Erin

    2015-01-01

    In patients with dyslipidemia, elevated triglyceride (TG) levels, or TG-rich lipoproteins, and cardiovascular risk may remain despite statin therapy. Prescription omega-3 fatty acid formulations containing the ethyl esters of eicosapentaenoic acid (EPA) plus docosahexaenoic acid (DHA) (omega-3-acid ethyl esters; Lovaza®) or high-purity EPA ethyl ester (icosapent ethyl; Vascepa®) are TG-lowering treatments that may be administered in addition to statins. Here we describe the effects of switching from omega-3-acid ethyl esters to icosapent ethyl in a 44-year-old obese man with dyslipidemia, hypertension, and hypothyroidism. The patient was receiving stable treatment with medications, including atorvastatin 40 mg/day and extended-release niacin 1000 mg/day. Owing to persistently elevated TG levels and other cardiovascular risk factors, the patient was initiated on omega-3-acid ethyl esters 4 g/day. After approximately 2 years on omega-3-acid ethyl esters, his total cholesterol (TC) level was 184 mg/dL, low-density lipoprotein cholesterol (LDL-C) level was 81 mg/dL, TG level was elevated at 307 mg/dL despite statin therapy, and non-high-density lipoprotein cholesterol (non-HDL-C) level was 144 mg/dL. After the switch to icosapent ethyl, TC level decreased by 34% to 121 mg/dL, LDL-C level decreased by 28% to 58 mg/dL, TG level decreased by 41% to 180 mg/dL, and non-HDL-C level decreased by 44% to 81 mg/dL. Switching from omega-3-acid ethyl esters containing both EPA and DHA to icosapent ethyl containing high-purity EPA resulted in beneficial and substantial changes in the lipid profile with a notable reduction of TG levels along with additional reductions in LDL-C levels in a statin-treated obese patient with persistently high TG levels. Treatment with icosapent ethyl was well tolerated. PMID:26453247

  2. Hydroxide as general base in the saponification of ethyl acetate.

    PubMed

    Mata-Segreda, Julio F

    2002-03-13

    The second-order rate constant for the saponification of ethyl acetate at 30.0 degrees C in H(2)O/D(2)O mixtures of deuterium atom fraction n (a proton inventory experiment) obeys the relation k(2)(n) = 0.122 s(-1) M(-1) (1 - n + 1.2n) (1 - n + 0.48n)/(1 - n + 1.4n) (1 - n + 0.68n)(3). This result is interpreted as a process where formation of the tetrahedral intermediate is the rate-determining step and the transition-state complex is formed via nucleophilic interaction of a water molecule with general-base assistance from hydroxide ion, opposite to the direct nucleophilic collision commonly accepted. This mechanistic picture agrees with previous heavy-atom kinetic isotope effect data of Marlier on the alkaline hydrolysis of methyl formate.

  3. Ethyl carbamate levels in selected fermented foods and beverages.

    PubMed

    Canas, B J; Havery, D C; Robinson, L R; Sullivan, M P; Joe, F L; Diachenko, G W

    1989-01-01

    Ethyl carbamate (EC), also known as urethane, is an animal carcinogen and a by-product of fermentation. Because EC has been found in distilled spirits and wines, a variety of fermented foods and beverages were analyzed to assess its occurrence in other products. Previously described methods using a gas chromatograph-thermal energy analyzer with a nitrogen converter were modified for each matrix and gave recoveries of greater than 80%, with a limit of detection in the 1-2 micrograms/kg (ppb) range. A total of 152 test samples were analyzed; EC levels ranged from none found to 3 ppb in 15 cheeses, 6 teas, 12 yogurts, and 8 ciders; from none found to 13 ppb in 30 breads and 69 malt beverages; and from none found to 84 ppb in 12 soy sauces. Gas chromatography/mass spectrometry/mass spectrometry was used to confirm EC identity and to quantitate EC in selected food extracts. PMID:2592308

  4. Interaction of Ethyl Alcohol Vapor with Sulfuric Acid Solutions

    NASA Technical Reports Server (NTRS)

    Leu, Ming-Taun

    2006-01-01

    We investigated the uptake of ethyl alcohol (ethanol) vapor by sulfuric acid solutions over the range approx.40 to approx.80 wt % H2SO4 and temperatures of 193-273 K. Laboratory studies used a fast flow-tube reactor coupled to an electron-impact ionization mass spectrometer for detection of ethanol and reaction products. The uptake coefficients ((gamma)) were measured and found to vary from 0.019 to 0.072, depending upon the acid composition and temperature. At concentrations greater than approx.70 wt % and in dilute solutions colder than 220 K, the values approached approx.0.07. We also determined the effective solubility constant of ethanol in approx.40 wt % H2SO4 in the temperature range 203-223 K. The potential implications to the budget of ethanol in the global troposphere are briefly discussed.

  5. Preoperative treatment of a parotid hemangioma with 100% ethyl alcohol

    PubMed Central

    Emsen, Ilteris Murat

    2008-01-01

    Hemangiomas are one of the most common childhood neoplasms, occurring in approximately 12% of infants younger than one year of age. The lesions typically appear shortly after birth, increase in size over the first year and characteristically regress over the next decade. Because hemangiomas can be visible during an important stage of a child’s social development, numerous authors have pursued alternative treatment strategies to avoid or reduce this lengthy involution process. Unfortunately, no effective medical treatment has been reported for children with large, deforming hemangiomas of the parotid gland and overlying cheek. In the present case, a patient with a large parotid hemangioma was treated preoperatively with an intralesional injection of 100% ethyl alcohol solution to reduce the size of the mass. The mass was removed 28 days later with no major postoperative complications. PMID:19949507

  6. Effects of acetone on methyl ethyl ketone peroxide runaway reaction.

    PubMed

    Lin, Yan-Fu; Tseng, Jo-Ming; Wu, Tsung-Chih; Shu, Chi-Min

    2008-05-30

    Runaway reactions by methyl ethyl ketone peroxide (MEKPO) are an important issue in Asia, due to its unstable structure and extensive heat release during upset situations. This study employed differential scanning calorimetry (DSC) to draw the experimental data for MEKPO 31 mass% and with acetone 99 mass% on three types of heating rate of 2, 4, and 10 degrees C/min; the kinetic and safety parameters were then evaluated via curve fitting. Through the reproducible tests in each condition, the results show that acetone is not a contaminant, because it could increase the activation energy (Ea) and onset temperature (To) when combined with MEKPO, which differs from the hazard information of the material safety data sheet (MSDS).

  7. Inhibition of dual-specificity phosphatase 26 by ethyl-3,4-dephostatin: Ethyl-3,4-dephostatin as a multiphosphatase inhibitor.

    PubMed

    Seo, Huiyun; Cho, Sayeon

    2016-04-01

    Protein tyrosine phosphatases (PTPs) regulate protein function by dephosphorylating phosphorylated proteins in many signaling cascades and some of them have been targets for drug development against many human diseases. There have been many reports that some chemical inhibitors could regulate particular phosphatases. However, there was no extensive study on specificity of inhibitors towardss phosphatases. We investigated the effects of ethyl-3,4-dephostatin, a potent inhibitor of five PTPs including PTP-1B and Src homology-2-containing protein tyrosine phosphatase-1 (SHP-1), on thirteen other PTPs using in vitro phosphatase assays. Of them, dual-specificity protein phosphatase 26 (DUSP26), which inhibits mitogen-activated protein kinase (MAPK) and p53 tumor suppressor and is known to be overexpressed in anaplastic thyroid carcinoma, was inhibited by ethyl-3,4-dephostatin in a concentration-dependent manner. Kinetic studies with ethyl-3,4-dephostatin and DUSP26 revealed competitive inhibition, suggesting that ethyl-3,4-dephostatin binds to the catalytic site of DUSP26 like other substrate PTPs. Moreover, ethyl-3,4-dephostatin protects DUSP26-mediated dephosphorylation of p38, a member of the MAPK family, and p53. Taken together, these results suggest that ethyl-3,4-dephostatin functions as a multiphosphatase inhibitor and is useful as a therapeutic agent for cancers overexpressing DUSP26. PMID:27209699

  8. Glucuronidation and Methylation of Procyanidin Dimers B2 and 3,3’’-Di-O-Galloyl-B2 and Corresponding Monomers Epicatechin and 3-O-Galloyl-Epicatechin in Mouse Liver

    PubMed Central

    Shrestha, Suraj P.; Thompson, John A.; Wempe, Michael F.; Gu, Mallikarjuna; Agarwal, Rajesh; Agarwal, Chapla

    2012-01-01

    Purpose The 3,3’’-di-O-galloyl ester of procyanidin B2 (B2G2) is a component of grape seed extract that inhibits growth of human prostate carcinoma cell lines. In preparation for studies in mice, its hepatic metabolism was examined in vitro and compared to B2 and the corresponding monomers, epicatechin (EC) and 3-O-galloyl-epicatechin (ECG). Methods Compounds were incubated with liver microsomes or cytosol containing cofactors for glucuronidation, sulfation or methylation, and products analyzed by liquid chromatography-mass spectrometry (LC-MS). B2G2 was administered orally to mice and plasma analyzed by LC-MS for unmodified procyanidin and metabolites. Results Glucuronides and methyl ethers of B2 and B2G2 were formed in small amounts. In contrast, EC and ECG were largely or completely converted to glucuronides, sulfates and methyl ethers under the same incubation conditions. B2G2 given orally to mice was partially absorbed intact; no significant metabolites were detected in plasma. Conclusions Glucuronidation and methylation of procyanidins B2 and B2G2 occurred but were minor processes in vitro. B2G2 was partially absorbed intact in mice after oral dosing and did not undergo significant metabolism. Unlike the flavanol monomers EC and ECG, therefore, B2G2 bioavailability should not be limited by metabolism. These results paved the way for ongoing pharmacokinetic and efficacy studies. PMID:22068277

  9. 40 CFR 721.538 - Phenol, 4-(1,1-dimethyl- ethyl)-, homopolymer.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Phenol, 4-(1,1-dimethyl- ethyl... Specific Chemical Substances § 721.538 Phenol, 4-(1,1-dimethyl- ethyl)-, homopolymer. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as phenol,...

  10. 40 CFR 721.538 - Phenol, 4-(1,1-dimethyl- ethyl)-, homopolymer.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Phenol, 4-(1,1-dimethyl- ethyl... Specific Chemical Substances § 721.538 Phenol, 4-(1,1-dimethyl- ethyl)-, homopolymer. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as phenol,...

  11. 75 FR 82069 - Ethyl Alcohol for Fuel Use: Determination of the Base Quantity of Imports

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-29

    ... COMMISSION Ethyl Alcohol for Fuel Use: Determination of the Base Quantity of Imports AGENCY: United States.... This determination is to be used to establish the ``base quantity'' of imports of fuel ethyl alcohol... CBERA-beneficiary countries. The base quantity to be used by U.S. Customs and Border Protection in...

  12. 78 FR 9938 - Ethyl Alcohol for Fuel Use: Determination of the Base Quantity of Imports

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-12

    ... recent previous determination for the 2012 amount in the Federal Register on December 30, 2011 (76 FR... COMMISSION Ethyl Alcohol for Fuel Use: Determination of the Base Quantity of Imports AGENCY: United States... is equal to 7 percent of the U.S. domestic market for fuel ethyl alcohol during the 12-month...

  13. 40 CFR 721.1085 - Benzenamine,4,4′-methylenebis[N-ethyl-N-methyl-.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Benzenamine,4,4â²-methylenebis[N-ethyl-N-methyl-. 721.1085 Section 721.1085 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.1085 Benzenamine,4,4′-methylenebis[N-ethyl-N-methyl-. (a)...

  14. 40 CFR 721.1085 - Benzenamine,4,4′-methylenebis[N-ethyl-N-methyl-.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Benzenamine,4,4â²-methylenebis[N-ethyl-N-methyl-. 721.1085 Section 721.1085 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.1085 Benzenamine,4,4′-methylenebis[N-ethyl-N-methyl-. (a)...

  15. 40 CFR 721.538 - Phenol, 4-(1,1-dimethyl- ethyl)-, homopolymer.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Phenol, 4-(1,1-dimethyl- ethyl... Specific Chemical Substances § 721.538 Phenol, 4-(1,1-dimethyl- ethyl)-, homopolymer. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as phenol,...

  16. 40 CFR 721.445 - Substituted ethyl alken-a-mide.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.445 Substituted ethyl alken-a-mide. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as a substituted ethyl...

  17. 40 CFR 721.445 - Substituted ethyl alken-a-mide.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.445 Substituted ethyl alken-a-mide. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as a substituted ethyl...

  18. Novel in vivo active anti-malarials based on a hydroxy-ethyl-amine scaffold.

    PubMed

    Ciana, Claire-Lise; Siegrist, Romain; Aissaoui, Hamed; Marx, Léo; Racine, Sophie; Meyer, Solange; Binkert, Christoph; de Kanter, Ruben; Fischli, Christoph; Wittlin, Sergio; Boss, Christoph

    2013-02-01

    A novel series of anti-malarials, based on a hydroxy-ethyl-amine scaffold, initially identified as peptidomimetic protease inhibitors is described. Combination of the hydroxy-ethyl-amine anti-malarial phramacophore with the known Mannich base pharmacophore of amodiaquine (57) resulted in promising in vivo active novel derivatives. PMID:23260352

  19. Survey of ethyl carbamate in fermented foods sold in the United Kingdom in 2004.

    PubMed

    Hasnip, Sarah; Crews, Colin; Potter, Nicholas; Christy, Julie; Chan, Danny; Bondu, Thomas; Matthews, Wendy; Walters, Barry; Patel, Krishna

    2007-04-01

    Results are presented of a survey of fermented foods and beverages sold in the United Kingdom for levels of ethyl carbamate (urethane) carried out to expand the range of food types sold in the United Kingdom for which data regarding ethyl carbamate are available. Samples were analyzed by in-house validated methods, which included measurement uncertainty estimates. The samples comprised 75 fermented liquids (beers, wines, fortified wines, spirits, liqueurs, soy sauces, and vinegars) and 25 fermented solid foods (cheeses, yogurts, soybean products, sauerkraut, yeast extract, olives, and Christmas pudding). Ethyl carbamate was not detected in the beers or the cider. Wines contained between 11 and 24 microg/kg and sake between 81 and 164 microg/kg. Fortified wines contained ethyl carbamate at levels between 14 and 60 microg/kg. Only two of five liqueurs contained ethyl carbamate. Most soy sauces and vinegars did not contain ethyl carbamate. No ethyl carbamate was detected in cheeses, yogurts, olives, or soybean-based products. Single samples of sauerkraut, yeast extract, and Christmas pudding contained low levels (29, 41, and 20 microg/kg ethyl carbamate, respectively).

  20. Biomonitoring of N-ethyl-2-pyrrolidone in automobile varnishers.

    PubMed

    Koslitz, Stephan; Meier, Swetlana; Schindler, Birgit Karin; Weiss, Tobias; Koch, Holger Martin; Brüning, Thomas; Käfferlein, Heiko Udo

    2014-12-01

    N-alkyl-2-pyrrolidones are important organic solvents for varnishes in industry. This study investigates exposure to N-ethyl-2-pyrrolidone (NEP) in varnishing of hard plastic components in an automobile plant. Two specific biomarkers of exposure, 5-hydroxy-N-ethyl-2-pyrrolidone (5-HNEP) and 2-hydroxy-N-ethylsuccinimide (2-HESI), were analyzed in urine samples of 14 workers. For this purpose, pre-shift, post-shift and next day pre-shift urine samples were collected midweek. Twelve workers performed regular work tasks (loading, wiping and packing), whereas two workers performed special work tasks including cleaning the sprayer system with organic solvents containing N-alkyl-2-pyrrolidones. Spot urine samples of nine non-exposed persons of the same plant served as controls. Median post-shift urinary levels of workers with regular work tasks (5-HNEP: 0.15 mg/L; 2-HESI: 0.19 mg/L) were ∼5-fold higher compared to the controls (0.03 mg/L each). Continuously increasing metabolite levels, from pre-shift via post-shift to pre-shift samples of the following day, were observed in particular for the two workers with the special working tasks. Maximum levels were 31.01 mg/L (5-HNEP) and 8.45 mg/L (2-HESI). No clear trend was evident for workers with regular working tasks. In summary, we were able to show that workers can be exposed to NEP during varnishing tasks in the automobile industry.

  1. Biomonitoring of N-ethyl-2-pyrrolidone in automobile varnishers.

    PubMed

    Koslitz, Stephan; Meier, Swetlana; Schindler, Birgit Karin; Weiss, Tobias; Koch, Holger Martin; Brüning, Thomas; Käfferlein, Heiko Udo

    2014-12-01

    N-alkyl-2-pyrrolidones are important organic solvents for varnishes in industry. This study investigates exposure to N-ethyl-2-pyrrolidone (NEP) in varnishing of hard plastic components in an automobile plant. Two specific biomarkers of exposure, 5-hydroxy-N-ethyl-2-pyrrolidone (5-HNEP) and 2-hydroxy-N-ethylsuccinimide (2-HESI), were analyzed in urine samples of 14 workers. For this purpose, pre-shift, post-shift and next day pre-shift urine samples were collected midweek. Twelve workers performed regular work tasks (loading, wiping and packing), whereas two workers performed special work tasks including cleaning the sprayer system with organic solvents containing N-alkyl-2-pyrrolidones. Spot urine samples of nine non-exposed persons of the same plant served as controls. Median post-shift urinary levels of workers with regular work tasks (5-HNEP: 0.15 mg/L; 2-HESI: 0.19 mg/L) were ∼5-fold higher compared to the controls (0.03 mg/L each). Continuously increasing metabolite levels, from pre-shift via post-shift to pre-shift samples of the following day, were observed in particular for the two workers with the special working tasks. Maximum levels were 31.01 mg/L (5-HNEP) and 8.45 mg/L (2-HESI). No clear trend was evident for workers with regular working tasks. In summary, we were able to show that workers can be exposed to NEP during varnishing tasks in the automobile industry. PMID:25455446

  2. UDP-Glucuronosyltransferase (UGT) 1A9-Overexpressing HeLa Cells Is an Appropriate Tool to Delineate the Kinetic Interplay between Breast Cancer Resistance Protein (BRCP) and UGT and to Rapidly Identify the Glucuronide Substrates of BCRP

    PubMed Central

    Jiang, Wen; Xu, Beibei; Wu, Baojian; Yu, Rong

    2012-01-01

    The interplay between phase II enzymes and efflux transporters leads to extensive metabolism and low bioavailability for flavonoids. To investigate the simplest interplay between one UDP-glucuronosyltransferase isoform and one efflux transporter in flavonoid disposition, engineered HeLa cells stably overexpressing UGT1A9 were developed, characterized, and further applied to investigate the metabolism of two model flavonoids (genistein and apigenin) and excretion of their glucuronides. The results indicated that the engineered HeLa cells overexpressing UGT1A9 rapidly excreted the glucuronides of genistein and apigenin. The kinetic characteristics of genistein or apigenin glucuronidation were similar with the use of UGT1A9 overexpressed in HeLa cells or the commercially available UGT1A9. Small interfering (siRNA)-mediated UGT1A9 silencing resulted in a substantial decrease in glucuronide excretion (>75%, p < 0.01). Furthermore, a potent inhibitor of breast cancer resistance protein (BCRP), 3-(6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1′,2′:1,6]pyrido[3,4-b]indol-3-yl)-propionic acid tert-butyl ester (Ko143), caused, in a dose-dependent manner, a substantial and marked reduction of the clearance (74–94%, p < 0.01), and a substantial increase in the intracellular glucuronide levels (4–8-fold, p < 0.01), resulting in a moderate decrease in glucuronide excretion (19–59%, p < 0.01). In addition, a significant, albeit moderate, reduction in the fraction of genistein metabolized (fmet) in the presence of Ko143 was observed. In contrast, leukotriene C4 and siRNA against multidrug resistance protein (MRP) 2 and MRP3 did not affect excretion of flavonoid glucuronides. In conclusion, the engineered HeLa cells overexpressing UGT1A9 is an appropriate model to study the kinetic interplay between UGT1A9 and BCRP in the phase II disposition of flavonoids. This simple cell model should also be very useful to rapidly identify whether a phase II

  3. High-yield synthesis of bioactive ethyl cinnamate by enzymatic esterification of cinnamic acid.

    PubMed

    Wang, Yun; Zhang, Dong-Hao; Zhang, Jiang-Yan; Chen, Na; Zhi, Gao-Ying

    2016-01-01

    In this paper, Lipozyme TLIM-catalyzed synthesis of ethyl cinnamate through esterification of cinnamic acid with ethanol was studied. In order to increase the yield of ethyl cinnamate, several media, including acetone, isooctane, DMSO and solvent-free medium, were investigated in this reaction. The reaction showed a high yield by using isooctane as reaction medium, which was found to be much higher than the yields reported previously. Furthermore, several parameters such as shaking rate, water activity, reaction temperature, substrate molar ratio and enzyme loading had important influences on this reaction. For instance, when temperature increased from 10 to 50 °C, the initial reaction rate increased by 18 times and the yield of ethyl cinnamate increased by 6.2 times. Under the optimum conditions, lipase-catalyzed synthesis of ethyl cinnamate gave a maximum yield of 99%, which was of general interest for developing industrial processes for the preparation of ethyl cinnamate.

  4. Distribution and organoleptic impact of ethyl 2-methylbutanoate enantiomers in wine.

    PubMed

    Lytra, Georgia; Tempere, Sophie; de Revel, Gilles; Barbe, Jean-Christophe

    2014-06-01

    The enantiomers of ethyl 2-methylbutanoate were assayed in several wines using chiral gas chromatography (β-cyclodextrin). Analyses of 37 commercial red wines from various vintages and origins revealed the almost exclusive presence of the S-enantiomeric form. The average concentration was ∼50 μg/L, but the oldest samples were found to contain higher ethyl 2-methylbutanoate levels than the youngest wines. The olfactory threshold of a racemic mixture of ethyl (2R)-2-methylbutanoate and ethyl (2S)-2-methylbutanoate (50:50, m/m) in dilute alcohol solution was 2.60 μg/L, almost twice that of the S-form, which was 1.53 μg/L. Ethyl (2S)-2-methylbutanoate and the racemic mixture of ethyl (2R)-2-methylbutanoate and ethyl (2S)-2-methylbutanoate had different aromatic nuances: the former was mainly defined by fruity descriptors, such as green apple (Granny Smith) and strawberry, whereas the latter had an unspecific, caustic, fruity, solvent odor. Sensory analysis revealed an enhancing effect of ethyl (2S)-2-methylbutanoate on the perception of fruity aromas in the matrices studied: the "olfactory threshold" of the fruity pool, consisting of esters found in red wines, in dilute alcohol solution alone was higher than that of the same mixture supplemented with 50 μg/L ethyl (2S)-2-methylbutanoate. The sensory profiles of these aromatic reconstitutions highlighted the contribution of ethyl (2S)-2-methylbutanoate to black-berry-fruit descriptors. PMID:24844693

  5. Crystal structure of (eth­oxy­ethyl­idene)di­methyl­aza­nium ethyl sulfate

    PubMed Central

    Tiritiris, Ioannis; Saur, Stefan; Kantlehner, Willi

    2015-01-01

    In the title salt, C6H14NO+·C2H5SO4 −, the C—N bond lengths in the cation are 1.2981 (14), 1.4658 (14) and 1.4707 (15) Å, indicating double- and single-bond character, respectively. The C—O bond length of 1.3157 (13) Å shows double-bond character, indicating charge delocalization within the NCO plane of the iminium ion. In the crystal, C—H⋯O hydrogen bonds between H atoms of the cations and O atoms of neighbouring ethyl sulfate anions are present, generating a three-dimensional network. PMID:26870525

  6. The mechanism of hydrothermal hydrolysis for glycyrrhizic acid into glycyrrhetinic acid and glycyrrhetinic acid 3-O-mono-β-D-glucuronide in subcritical water.

    PubMed

    Fan, Rui; Li, Nan; Xu, Honggao; Xiang, Jun; Wang, Lei; Gao, Yanxiang

    2016-01-01

    To improve the bioactivity and sweetness properties of glycyrrhizic acid (GL), the hydrothermal hydrolysis of GL into glycyrrhetinic acid (GA) and glycyrrhetinic acid 3-O-mono-β-D-glucuronide (GAMG) in subcritical water was investigated. The effects of temperature, time and their interaction on the conversion ratios were analyzed and the reactions were elaborated with kinetics and thermodynamics. The results showed that GL hydrothermal hydrolysis was significantly (P < 0.05) affected by reaction time and temperature, as well as their interaction, and could be fitted into first-order kinetics. The thermodynamic analysis indicated that the hydrolysis of GL was endergonic and non-spontaneous. The hydrolytic pathways were composed of complex consecutive and parallel reactions. It was concluded that subcritical water may be a potential medium for producing GAMG and GA.

  7. Retention of heroin and morphine-6 beta-glucuronide analgesia in a new line of mice lacking exon 1 of MOR-1.

    PubMed

    Schuller, A G; King, M A; Zhang, J; Bolan, E; Pan, Y X; Morgan, D J; Chang, A; Czick, M E; Unterwald, E M; Pasternak, G W; Pintar, J E

    1999-02-01

    Morphine produces analgesia by activating mu opioid receptors encoded by the MOR-1 gene. Although morphine-6 beta-glucuronide (M6G), heroin and 6-acetylmorphine also are considered mu opioids, recent evidence suggests that they act through a distinct receptor mechanism. We examined this question in knockout mice containing disruptions of either the first or second coding exon of MOR-1. Mice homozygous for either MOR-1 mutation were insensitive to morphine. Heroin, 6-acetylmorphine and M6G still elicited analgesia in the exon-1 MOR-1 mutant, which also showed specific M6G binding, whereas M6G and 6-acetylmorphine were inactive in the exon-2 MOR-1 mutant. These results provide genetic evidence for a unique receptor site for M6G and heroin analgesia.

  8. Slow O-demethylation of methyl triclosan to triclosan, which is rapidly glucuronidated and sulfonated in channel catfish liver and intestine.

    PubMed

    James, Margaret O; Marth, Christopher J; Rowland-Faux, Laura

    2012-11-15

    The antibacterial personal care product triclosan is discharged in municipal waste, and converted in part by bacteria in sewage sludge and soil to its more lipid-soluble methyl ether, methyl triclosan. Triclosan and methyl triclosan have been detected in water, sediment, fish and invertebrates near sewage treatment facilities. Understanding the biotransformation of methyl triclosan and triclosan in a model food fish, the channel catfish, will be of value in assessing the likelihood that these compounds will bioaccumulate in exposed fish, and therefore potentially pass up the food chain. We hypothesize that cytochrome P450 will catalyze the O-demethylation of methyl triclosan to yield triclosan, which is likely to undergo glucuronidation or sulfonation of the phenolic hydroxyl group. Conversion of methyl triclosan to triclosan was measured by LC/MS/MS following aerobic incubation of varying concentrations of methyl triclosan with NADPH and hepatic and intestinal microsomes from untreated, 3-methylcholanthrene-treated (10 mg/kg, i.p.) or PCB-126-treated (0.1 mg/kg, i.p.) channel catfish (n=4 per treatment group). The K(m) values for methyl triclosan were similar for untreated, 3-methylcholanthrene-treated and PCB-126-treated catfish liver microsomes, ranging from 80 to 250 μM. V(max) values for O-demethylation ranged from 30 to 150 pmol/min/mg protein, with no significant differences between controls, PCB-126-treated or 3-methylcholanthrene-treated fish, suggesting that methyl triclosan O-demethylation was not a CYP1-catalyzed reaction. Methyl triclosan O-demethylation activities in intestinal microsomes were similar to or lower than those found with liver microsomes. The calculated rate of O-demethylation of methyl triclosan in catfish liver at 1 μM, a concentration reported in exposed fish, and 21°C, an early summer water temperature, is 0.10 pmol/min/mg protein. This slow rate of metabolism suggests that upon continued exposure, methyl triclosan may

  9. Two-dimensional crystalline array formation of glucuronide transporter from Escherichia coli by the use of polystyrene beads for detergent removal.

    PubMed

    Ishii, Noriyuki

    2013-03-01

    n-Dodecyl-β-D-maltoside solubilized glucuronide transporter (GusB), the product of gusB gene from Escherichia coli, was treated with Bio-Beads as an agent for removing the detergent from a micellar solution under suitable combination with dimyristoylphosphatidylcholine. Optimizing conditions led to a two-dimensional crystalline array formation of GusB. The crystalline arrays appear to have a hexagonal lattice with layer group P6, the unit cell dimensions of a = b = 13.8 nm and γ = 120°. Each stain-protruding periodic unit showed approximately 11.8 ± 0.3 nm in a diameter in the inverse Fourier-filtered image to have formed with pentameric GusB (5 × 49.7 kDa). PMID:23188061

  10. Prediction of drug clearance by glucuronidation from in vitro data: use of combined cytochrome P450 and UDP-glucuronosyltransferase cofactors in alamethicin-activated human liver microsomes.

    PubMed

    Kilford, Peter J; Stringer, Rowan; Sohal, Bindi; Houston, J Brian; Galetin, Aleksandra

    2009-01-01

    Glucuronidation via UDP-glucuronosyltransferase (UGT) is an increasingly important clearance pathway. In this study intrinsic clearance (CL(int)) values for buprenorphine, carvedilol, codeine, diclofenac, gemfibrozil, ketoprofen, midazolam, naloxone, raloxifene, and zidovudine were determined in pooled human liver microsomes using the substrate depletion approach. The in vitro clearance data indicated a varying contribution of glucuronidation to the clearance of the compounds studied, ranging from 6 to 79% for midazolam and gemfibrozil, respectively. The CL(int) was obtained using either individual or combined cofactors for cytochrome P450 (P450) and UGT enzymes with alamethicin activation and in the presence and absence of 2% bovine serum albumin (BSA). In the presence of combined P450 and UGT cofactors, CL(int) ranged from 2.8 to 688 microl/min/mg for zidovudine and buprenorphine, respectively; the clearance was approximately equal to the sum of the CL(int) values obtained in the presence of individual cofactors. The unbound intrinsic clearance (CL(int, u)) was scaled to provide an in vivo predicted CL(int); the data obtained in the presence of combined cofactors resulted in 5-fold underprediction on average. Addition of 2% BSA to the incubation with both P450 and UGT cofactors reduced the bias in the clearance prediction, with 8 of 10 compounds predicted within 2-fold of in vivo values with the exception of raloxifene and gemfibrozil. The current study indicates the applicability of combined cofactor conditions in the assessment of clearance for compounds with a differential contribution of P450 and UGT enzymes to their elimination. In addition, improved predictability of microsomal data is observed in the presence of BSA, in particular for UGT2B7 substrates.

  11. Quantitative Rationalization of Gemfibrozil Drug Interactions: Consideration of Transporters-Enzyme Interplay and the Role of Circulating Metabolite Gemfibrozil 1-O-β-Glucuronide.

    PubMed

    Varma, Manthena V S; Lin, Jian; Bi, Yi-an; Kimoto, Emi; Rodrigues, A David

    2015-07-01

    Gemfibrozil has been suggested as a sensitive cytochrome P450 2C8 (CYP2C8) inhibitor for clinical investigation by the U.S. Food and Drug Administration and the European Medicines Agency. However, gemfibrozil drug-drug interactions (DDIs) are complex; its major circulating metabolite, gemfibrozil 1-O-β-glucuronide (Gem-Glu), exhibits time-dependent inhibition of CYP2C8, and both parent and metabolite also behave as moderate inhibitors of organic anion transporting polypeptide 1B1 (OATP1B1) in vitro. Additionally, parent and metabolite also inhibit renal transport mediated by OAT3. Here, in vitro inhibition data for gemfibrozil and Gem-Glu were used to assess their impact on the pharmacokinetics of several victim drugs (including rosiglitazone, pioglitazone, cerivastatin, and repaglinide) by employing both static mechanistic and dynamic physiologically based pharmacokinetic (PBPK) models. Of the 48 cases evaluated using the static models, about 75% and 98% of the DDIs were predicted within 1.5- and 2-fold of the observed values, respectively, when incorporating the interaction potential of both gemfibrozil and its 1-O-β-glucuronide. Moreover, the PBPK model was able to recover the plasma profiles of rosiglitazone, pioglitazone, cerivastatin, and repaglinide under control and gemfibrozil treatment conditions. Analyses suggest that Gem-Glu is the major contributor to the DDIs, and its exposure needed to bring about complete inactivation of CYP2C8 is only a fraction of that achieved in the clinic after a therapeutic gemfibrozil dose. Overall, the complex interactions of gemfibrozil can be quantitatively rationalized, and the learnings from this analysis can be applied in support of future predictions of gemfibrozil DDIs. PMID:25941268

  12. Design of experiments, a powerful tool for method development in forensic toxicology: application to the optimization of urinary morphine 3-glucuronide acid hydrolysis.

    PubMed

    Costa, S; Barroso, M; Castañera, A; Dias, M

    2010-04-01

    The application of the design of experiments to optimize method development in the field of forensic toxicology using the urinary morphine 3-glucuronide acid hydrolysis as an example is described. Morphine and its trideuterated analogue (used as an internal standard) were extracted from urine samples by liquid-liquid extraction (ToxiTubes A) and derivatized by silylation. Chromatographic analysis was done by gas chromatography-mass spectrometry in the selected ion monitoring mode. Using the peak area ratio (morphine-to-internal standard) as the response, we investigated the independent variables that could influence the acid hydrolysis, including temperature (range 70-130 degrees C), acid volume (range 500-1,000 microL) and time (range 15-90 min). A 2(3) full factorial design for the screening and a response surface methodology, including a central composite design for optimization, were applied. The factors which influenced the response to a greater extent were temperature and its interaction both with time and acid volume. By application of a multiple regression analysis to the experimental data, a second-order polynomial equation was obtained. The optimal predicted conditions for morphine 3-glucuronide acid hydrolysis were 115 degrees C, 38 min and 500 microL for temperature, time and acid volume, respectively. Using design of experiments, instead of the one factor at a time approach, we achieved the optimum combination of all factor values, and this allowed the best results to be obtained, simultaneously optimizing resources. In addition, time and money can be saved, since other approaches are in general more time-consuming and laborious, and do not take into account the interactions between factors.

  13. Validated LC-MS/MS method for the determination of 3-Hydroxflavone and its glucuronide in blood and bioequivalent buffers: Application to pharmacokinetic, absorption, and metabolism studies

    PubMed Central

    Xu, Beibei; Yang, Guanyi; Ge, Shufan; Yin, Taijun; Hu, Ming; Gao, Song

    2015-01-01

    The purpose of this study is to develop an UPLC-MS/MS method to quantify 3-hydroxy-flavone (3-HF) and its metabolite, 3-hydroxyflavone-glucuronide (3-HFG) from biological samples. A Waters BEH C8 column was used with acetonitrile/0.1 % formic acid in water as mobile phases. The mass analysis was performed in an API 5500 Qtrap mass spectrometer via multiple reaction monitoring (MRM) with positive scan mood. The one-step protein precipitation by acetonitrile was used to extract the analytes from plasma. The results showed that the linear response range was 0.61– 2,500.00 nM for 3-HF and 0.31– 2,500.00 nM for 3-HFG. The intra-day variance is less 16.48 % and accuracy is in 77.70–90.64 % for 3-HF and variance less than 15.86%, accuracy in 85.08–114.70 % for 3-HFG. The inter-day variance is less than 20.23 %, accuracy is in 110.58–114.2 % for 3-HF and variance less than 15.59 %, accuracy in 83.00–89.40% for 3-HFG. The analysis was done within 4.0 min. Only 10 μL of blood is needed due to the high sensitivity of this method. The validated method was successfully used to pharmacokinetic study in A/J mouse, transport study in the Caco-2 cell culture model, and glucuronidation study using mice liver and intestine microsomes. The applications revealed that this method can be used for 3-HF and 3-HFG analysis in blood as well as in bioequivalent buffers such HBSS and KPI. PMID:23973631

  14. Potential use of radiolabeled glucuronide prodrugs with auger and/or alpha emitters in combined chemo- and radio-therapy of cancer.

    PubMed

    Unak, T

    2000-07-01

    Nowadays, the scientists from different disciplines have focused their attentions to new anticancer drug design for cancer chemotherapy. An effective anticancer drug should ensure the selective drug incorporation into the targeted tumor cells without principally incorporation into the normal cells. So, the targeted tumor cells can selectively be damaged by the cytotoxic effectiveness of the drug. The basic principles of drug design have involved "prodrug" concept, which means a chemical agent which is not itself active as an anticancer drug, but it can be transformed to an active form after its administration. Prodrugs can finally be activated onto the tumor cells by some kind of enzymes. In this context, the activation of glucuronide prodrugs by b-glucuronidase have a large potential applications in cancer chemotherapy. On the other hand, combined chemo- and radio-therapy of cancer (CCRTC) concept aims to combine the cytotoxicity of an aglycone with the radiotoxicity of an appropriate radionuclide on the same prodrug. So, the cytotoxic and radiotoxic effectiveness' will be able to be concentrated into the same tumor cell to increase obviously its damage. For experimental realization of this concept an effective anticancer prodrug should be radiolabeled with a radionuclide having high level of radiotoxic effectiveness such as Auger and/or alpha-emitter radionuclides. Iodine-125 and astatine-211 are very interesting radionuclides as being effective Auger and/or alpha-emitters. Briefly, the glucuronide prodrugs radiolabeled with iodine-125 or astatine-211 promise to be designed very effective anticancer agents in the future applications of cancer chemotherapy. PMID:10903386

  15. In vitro inhibition of UDP glucuronosyltransferases by atazanavir and other HIV protease inhibitors and the relationship of this property to in vivo bilirubin glucuronidation.

    PubMed

    Zhang, Donglu; Chando, Theodore J; Everett, Donald W; Patten, Christopher J; Dehal, Shangara S; Humphreys, W Griffith

    2005-11-01

    Several human immunodeficiency virus (HIV) protease inhibitors, including atazanavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir, were tested for their potential to inhibit uridine 5'-diphospho-glucuronosyltransferase (UGT) activity. Experiments were performed with human cDNA-expressed enzymes (UGT1A1, 1A3, 1A4, 1A6, 1A9, and 2B7) as well as human liver microsomes. All of the protease inhibitors tested were inhibitors of UGT1A1, UGT1A3, and UGT1A4 with IC(50) values that ranged from 2 to 87 microM. The IC50 values found for all compounds for UGT1A6, 1A9, and 2B7 were >100 microM. The inhibition (IC50) of UGT1A1 was similar when tested against the human cDNA-expressed enzyme or human liver microsomes for atazanavir, indinavir, and saquinavir (2.4, 87, and 7.3 microM versus 2.5, 68, and 5.0 microM, respectively). By analysis of the double-reciprocal plots of bilirubin glucuronidation activities at different bilirubin concentrations in the presence of fixed concentrations of inhibitors, the UGT1A1 inhibition by atazanavir and indinavir was demonstrated to follow a linear mixed-type inhibition mechanism (Ki = 1.9 and 47.9 microM, respectively). These results suggest that a direct inhibition of UGT1A1-mediated bilirubin glucuronidation may provide a mechanism for the reversible hyperbilirubinemia associated with administration of atazanavir as well as indinavir. In vitro-in vivo scaling with [I]/Ki predicts that atazanavir and indinavir are more likely to induce hyperbilirubinemia than other HIV protease inhibitors studied when a free Cmax drug concentration was used. Our current study provides a unique example of in vitro-in vivo correlation for an endogenous UGT-mediated metabolic pathway. PMID:16118329

  16. Confirmatory analysis of buprenorphine, norbuprenorphine, and glucuronide metabolites in plasma by LCMSMS. Application to umbilical cord plasma from buprenorphine-maintained pregnant women.

    PubMed

    Concheiro, Marta; Jones, Hendreé; Johnson, Rolley E; Shakleya, Diaa M; Huestis, Marilyn A

    2010-01-01

    An LCMSMS method was developed and fully validated for the simultaneous quantification of buprenorphine (BUP), norbuprenorphine (NBUP), buprenorphine-glucuronide (BUP-Gluc), and norbuprenorphine-glucuronide (NBUP-Gluc) in 0.5mL plasma, fulfilling confirmation criteria with two transitions for each compound with acceptable relative ion intensities. Transitions monitored were 468.3>396.2 and 468.3>414.3 for BUP, 414.3>340.1 and 414.3>326.0 for NBUP, 644.3>468.1 and 644.3>396.3 for BUP-Gluc, and 590.3>414.3 and 590.3>396.2 for NBUP-Gluc. Linearity was 0.1-50ng/mL for BUP and BUP-Gluc, and 0.5-50ng/mL for NBUP and NBUP-Gluc. Intra-day, inter-day, and total assay imprecision (%RSD) were <16.8%, and analytical recoveries were 88.6-108.7%. Extraction efficiencies ranged from 71.1 to 87.1%, and process efficiencies 48.7 to 127.7%. All compounds showed ion enhancement, except BUP-Gluc that demonstrated ion suppression: variation between 10 different blank plasma specimens was <9.1%. In six umbilical cord plasma specimens from opioid-dependent pregnant women receiving 14-24mg/day BUP, NBUP-Gluc was the predominant metabolite (29.8+/-7.6ng/mL), with BUP-Gluc (4.6+/-4.8ng/mL), NBUP (1.5+/-0.8ng/mL) and BUP (0.4+/-0.2ng/mL). Although BUP biomarkers can be quantified in umbilical cord plasma in low ng/mL concentrations, the significance of these data as predictors of neonatal outcomes is currently unknown. PMID:19945361

  17. Identification of glucoside and carboxyl-linked glucuronide conjugates of mycophenolic acid in plasma of transplant recipients treated with mycophenolate mofetil

    PubMed Central

    Shipkova, Maria; Armstrong, Victor William; Wieland, Eberhard; Niedmann, Paul Dieter; Schütz, Ekkehard; Brenner-Weiß, Gerald; Voihsel, Martin; Braun, Felix; Oellerich, Michael

    1999-01-01

    Mycophenolic acid (MPA), is primarily metabolized in the liver to 7-O-MPA-β-glucuronide (MPAG). Using RP-h.p.l.c. we observed three further MPA metabolites, M-1, M-2, M-3, in plasma of transplant recipients on MMF therapy. To obtain information on the structure and source of these metabolites: (A) h.p.l.c. fractions containing either metabolite or MPA were collected and analysed by tandem mass spectrometry; (B) the metabolism of MPA was studied in human liver microsomes in the presence of UDP-glucuronic acid, UDP-glucose or NADPH; (C) hydrolysis of metabolites was investigated using β-glucosidase, β-glucuronidase or NaOH; (D) cross-reactivity of each metabolite was tested in an immunoassay for MPA (EMIT). Mass spectrometry of M-1, M-2, MPA and MPAG in the negative ion mode revealed molecular ions of m/z 481, m/z 495, m/z 319 and m/z 495 respectively. Incubation of microsomes with MPA and UDP-glucose produced M-1, with MPA and UDP-glucuronic acid MPAG and M-2 were formed, while with MPA and NADPH, M-3 was observed. β-Glucosidase hydrolysed M-1 completely. β-Glucuronidase treatment led to a complete disappearance of MPAG whereas the amount of M-2 was reduced by approximately 30%. Only M-2 was labile to alkaline treatment. M-2 and MPA but not M-1 and MPAG cross-reacted in the EMIT assay. These results suggest that: (i) M-1 is the 7-OH glucose conjugate of MPA; (ii) M-2 is the acyl glucuronide conjugate of MPA; (iii) M-3 is derived from the hepatic CYP450 system. PMID:10204993

  18. 21 CFR 176.160 - Chromium (Cr III) complex of N-ethyl-N-heptadecylfluoro-octane sulfonyl glycine.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Chromium (Cr III) complex of N-ethyl-N... § 176.160 Chromium (Cr III) complex of N-ethyl-N-heptadecylfluoro-octane sulfonyl glycine. The chromium (Cr III) complex of N-ethyl - N -heptadecylfluoro-octane sulfonyl glycine containing up to 20...

  19. 40 CFR 721.10074 - Acetic acid, 2-chloro-, 1-(3,3-dimethylcyclohexyl)ethyl ester.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...-dimethylcyclohexyl)ethyl ester. 721.10074 Section 721.10074 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.10074 Acetic acid, 2-chloro-, 1-(3,3-dimethylcyclohexyl)ethyl ester. (a... acetic acid, 2-chloro-, 1-(3,3-dimethylcyclohexyl)ethyl ester (PMN P-05-568; CAS No. 477218-59-0)...

  20. 40 CFR 721.1950 - 2-Butenedioic acid (Z), mono(2-((1-oxopropenyloxy)ethyl) ester .

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...-oxopropenyloxy)ethyl) ester . 721.1950 Section 721.1950 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.1950 2-Butenedioic acid (Z), mono(2-((1-oxopropenyloxy)ethyl) ester . (a... 2-butenedioic acid (Z), mono(2-((1-oxopropenyloxy)ethyl) ester (PMN P-85-543) is subject...