Identification and interspecies characterization of UDP-glucuronosyltransferase isoforms catalyzing acacetin glucuronidation using recombinant UGT enzymes and microsomes

Objective:To explore the glucuronic acid metabolism of acacetin in human liver and intestinal microsomes to better characterize human uridine 5'-diphospho (UDP)-glucuronosyltransferase (UGT) isoforms.In addition,interspecies comparisons were performed to identify the most appropriate experimental animal model for an in vivo study.Methods:Liquid chromatography tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) were used to confirm the successful biosynthesis of acacetin-7-O-glucuronide.Human isoforms of UGT and isozyme-specific chemical inhibitors were used for recombinant assays.Acacetin glucuronidation kinetics were assessed by combining acacetin with recombinant human UGT isoforms or with microsomes from humans or experimental animals.Kinetic differences between species were assessed in vitro using the same approach.Results:We identified multiple UGT isoforms that facilitated acacetin glucuronidation,and found that UGT1A1 was the major isoform that catalyzed this process.Acacetin-7-O-glucuronide formation exhibited clear substrate inhibition kinetics when combined with recombinant UGTs or with liver/intestinal microsomes derived from humans,monkeys,rats,mice,dogs,or pigs.Intrinsic metabolic clearance values of human intestinal microsomes were two-fold greater than those of human liver microsomes.Among the evaluated species,the Km value of dog microsomes (0.86 μM) was greatest in acacetin glucuronidation,while mice exhibited the highest CLint value,5.05 mL/min/mg.The CLint values of microsomes derived from monkeys and minipigs were 1.99 mL/min/mg and 2.12 mL/min/mg,respectively,exhibiting similar intrinsic metabolic clearance activity to that observed in humans.Conclusion:Monkey may represent a suitable model for experimental studies of acacetin pharmacokinetics owing to a high sequence homology of UGT1A1 and similar UGT1A1 glucuronidation activity to humans.

Glucuronidation is the dominating in-vivo metabolism pathway of herbacetin:elucidation of herbacetin pharmacokinetics after intravenous and oral administration in rats

OBJECTIVE To map a comprehensive metabolic pathway of herbacetin in rats,specifically,to elucidate the biotransformation of herbacetin in vivo and to simultaneously monitor the pharmacokinetic process of both parent drug and its major metabolites.METHODS liquid chromatography/ion trap mass spectrometry(LC/MS n) and ultra-liquid chromatography coupled with mass spectrometry(UPLC/MS) were combined in the current study for qualitative and quantitative determinations of herbacetin and its metabolites in bile,urine and feces after both oral and intravenous administration of herbacetin to rats.Enzyme kinetic studies on the intestinal and hepatic metabolism of herbacetin were further conducted to elucidate metabolic profiles of herbacetin in rat tissues and organs.Additionally,plasma concentration profiles of herbacetin and its metabolites in rats were obtained to characterize the overall pharmacokinetic behavior of herbacetin.RESULTS It was found that herbacetin was excreted primarily from rat urine in the form of glucuronide-conjugations.Subsequent in vitro enzyme kinetic studies and in vivo pharmacokinetic investigations suggested an extensive hepatic metabolism of herbacetin and the high exposure of herbacetin-glucuronides in systemic circulation.The clearance,half-life and bioavailability of herbacetin in rats were determined as(16.4±1.92)mL·kg^(-1)·min^(-1),(11.9±2.7)min,and 1.32%,respectively.On basis of these findings,a comprehensive metabolic pathway of herbacetin in rats was composed.In addition,a physiology based pharmacokinetic(PBPK) model was successfully developed with the aid of the Gastro Plus to simulate the pharmacokinetic process of herbacetin in rats.Application of the PBPK modeling can provide a useful starting point to understand and extrapolate pharmacokinetic parameters among different species,populations,and disease states.CONCLUSION After oral administration,herbacetin was subjected to colonic degradation and extensive first pass metabolism,with glucuronidation as its dominating in vivo metabolic pathway.

Glucuronidation of Two Novel Metabolites of Benproperine with Trichloroacetimidate Donor

The glucuronide metabolites of benproperine were synthesized from mono-hydroxylate metabolites of benproperine that were treated with methyl （2,3,4-tri-O-acetyl-1-O-tfichloroacetimidoyl ） -α-D-glucopyranuronate with BF3 · Eh O as the promoter followed by basic hydrolyzation with Na2 CO3. The form of basic acceptors, the order of addition, and the promoter are all important variables in this glucuronidation. The salt form of the basic acceptor was found to be better than its free form for glucuronidation with a Lewis acid as the promoter. Two mono-hydroxylated benproperines were synthesized from 2-benzylphenol in three steps.

Cloning of UGT1A9 cDNA from liver tissues and its expression in CHL cells

AIM: To clone the cDNA of UGT1 A9 from a Chinese human liver and establish the Chinese hamster lung (CHL) cell line expressing human UGTI A9.METHODS: cDNA of UGT1A9 was transcripted from mRNA by reverse transcriptase-ploymerase chain reaction, and was cloned into the pGEM-T vector which was amplified in the host bacteric E. CoIl DH5α. The inserted fragment, verified by DNA sequencing, wes subcloned into the Hind III/Not I site of a mammalian expression vector pREP9 to construct the plasmid termed pREP9-UGT1A9. CHL cells were transfected with the resultant recombinants, pREP9- UGT1A9, and selected by G418 (400 mg. L-1) for one month. The surviving clone (CHL-UGT1A9) was harvested as a pool and sub-cultured in medium containing G418 to obtain samples for UGT1 A9 assays. The enzyme activity of CHL-UGT1A9 towards propranolol in S9 protein of the cell was determined by HPLC.RESULTS: The sequence of the cDNA segment cloned,which was 1666 bp in length, was identical to that released by Gene Bank (GenBank accession number: AF056188) in coding region. The recombinant constructed, pREP9- UGT1 A9, contains the entire coding region, along with 18bp of the 5' and 55 bp of the 3′ untranslated region of the UGT1A9 cDNA, respectively. The cell lines established expressed the protein of UGT1 A9, and the enzyme activity towards propranolol in S9 protein was found to be 101 ± 24pmol.min-1 .mg-1 protein (n = 3), but was not detectable in parental CHL cells.CONCLUSION: The cDNA of UGT1A9 was successfully cloned from a Chinese human liver and transfected into CHL ceils. The CHL-UGT1A9 cell lines established efficiently expressed the protein of UGT1A9 for the further enzyme study of drug glucuronidation.

Androgen-independent growth in LNCaP cell lines and steroid uridine diphosphate-glucuronosyltransferase expression

<abstract>Aim: To investigate the mechanism of androgen-independent growth of prostate cancer after androgen ablation in LNCaP cells and the effect of glucuronidation activity. Methods: To establish androgen-independent growth in prostate cancer LNCaP-SF, continuous passage was performed in androgen-stripped medium and the cells were evaluated for glucuronidation activity. The expression vector of antisense uridine diphosphate glucuronosyltransferase (UGT) 2B15 cDNA was also constructed and evaluated. Results: LNCaP-SF lead to a higher expression in UGT2B15 and their glucuronidation activity is 2.5 times higher than that of LNCaP cells. Significantly fewer LNCaP and LNCaP-SF than control were transfected with the antisense UGT2B15 cDNA, suggesting that UGT2B15 plays an important part in the glucuronidation activity of androgens in both cells. Conclusion: The alteration of UGT2B15 expression in LNCaP-SF cells is proposed as a biological characteristic involved in the growth of hormonerefractory prostate cancer.

Enzymatic treatment of estrogens and estrogen glucuronide

Natural and synthetic estrogens from sewage treatment systems are suspected to influence the reproductive health of the animals in the rivers. In this article, we investigated the enzymatic treatment of three estrogens （estrone, 17β-estradiol, and 17α-ethynyletstradiol） by a fungal laccase which oxidize phenolic compounds with dissolved oxygen. The elimination of the estrogenic activities by enzymatic oxidation was demonstrated by medaka vitellogenin assay. In addition, we developed an enzymatic treatment system comprised of β-D-glucuronidase and the laccase for 17β-estradiol 3-（β-D-glucuronide） degradation. The two enzymes eliminated 17β-estradiol 3-（β- D-glucuronide） and the intermediate, 17β-estradiol, efficiently.

Flavonoid chemical composition and antidiabetic potential of Brachychiton acerifolius leaves extract

Objective:To evaluate Brachychiton acerifolius leaf extracts as antidiabetic potential agent and to identify the main active constituents using bioactivity guided fractionation.Methods:In vitro antioxidant activity was evaluated for B.acerifolius different extracts using DPPH assay and vitamin C as control.Antidiabetic activity was then determined using STZ-induced rats treated daily with ethyl acetate and 70% ethanol leaf extracts for4 weeks at a dose of 200 g/kg body weight against gliclazide reference drug.Blood glucose,a-amylase,lipid profile,liver function enzymes and oxidative stress markers were assessed along with histopathological study for liver and pancreatic tissues.Isolation and structural elucidation of active compounds were made using Diaion and Sephadex followed by spectral analyses.Results:The results indicated that ethyl acetate and ethanol leaf extracts exhibited the strongest antioxidant activity compared to that of vitamin C(IC500.05,0.03 and 12 mg/m L,respectively).Both extracts showed potent anti-hyperglycemic activity evidenced by a significant decrease in serum glucose levels by 82.5% and 80.9% and a-amylase by45.2% and 53.6%,as compared with gliclazide 68% and 59.4%,respectively.Fractionation of ethanol extract resulted in the isolation of 9 flavonoids including apigenin-7-O-arhamnosyl(1/2)-b-D-glucuronide,apigenin-7-O-b-D-glucuronide,apigenin-7-O-b-Dglucoside and luteolin-7-O-b-D-glucuronide.Conclusions:This study highlights the potential use of B.acerifolius leaf extract enriched in flavones for the treatment of diabetes that would warrant further clinical trials investigation.

Liquid Chromatography-Tandem Mass Spectrometry Assay to Detect Ethyl Glucuronide in Human Fingernail: Comparison to Hair and Gender Differences

Over the past decade, the use of hair specimens for the long-term detection of the alcohol biomarker ethyl glucuronide has been increasing in popularity and usage. We evaluated the usefulness of fingernail clippings as a suitable alterna-tive to hair for ethyl glucuronide detection. A liquid chromatography-tandem mass spectrometry method for the detection of ethyl glucuronide in fingernail clippings was fully validated and used to analyze the hair and/or fingernail specimens of 606 college-aged study participants. The limit of detection was 2 pg/mg, the limit of quantitation was 8 pg/mg and the method was linear from 8 to 2000 pg/mg. Intra- and inter-assay imprecision studies at three different concentrations (20, 40, 200 pg/mg) were all within 7.8% and all intra- and inter-assay bias studies at these levels were within 115.1% of target concentration. Ethyl glucuronide levels in fingernail (mean = 29.1 ± 55.6 pg/mg) were higher than ethyl glucuronide levels in hair (mean = 9.48 ± 22.3 pg/mg) and a correlation of the matched pairs was observed (r = 0.552, P < 0.01, n = 529). Evaluating each gender separately revealed that the correlation of male fingernail to male hair was large and significant (r = 0.782, P < 0.01, n = 195) while female hair to female fingernail was small yet sig-nificant (r = 0.249, P < 0.01, n = 334). The study results demonstrated that fingernail may be a suitable alternative to hair for ethyl glucuronide detection and may be the preferred sample type due to the lack of a gender bias.

Biomarkers for detection of alcohol consumption in liver transplantation

Alcoholic liver disease is an established, yet controversial, indication for liver transplantation. Although an abstinence period of up to 6 mo prior to transplantation is mandatory, alcohol relapse after transplantation is a common event. In case of recurrence of heavy drinking, graft survival is significantly impaired. Guidelines on detection and surveillance of alcohol consumption in this patient cohort are lacking. This review summarizes the challenge of patient selection as well as the current knowledge on established and novel alcohol biomarkers with special focus on liver transplant candidates and recipients.

Hydrolysis of soy isoflavone conjugates using enzyme may underestimate isoflavone concentrations in tissue

Objective:To investigate the differences of using enzymatic hydrolysis and acid hydrolysis for identification and quantification of isoflavone aglycones from biomatrices. Methods: β-glucuronidase/sulfatase isolated from Helix pomatia for routine enzymatic hydrolysis or 6N HCl was used to release glucuronide and sulfate conjugates in the serum, urine and tissue samples. Profiles of soy isoflavones after enzymatic hydrolysis or acid hydrolysis in several tissues of rat fed with diets containing soy protein isolate were also compared using LC/MS and HPLC-ECD. Results: Acid hydrolysis released more aglycone than enzymatic digestion (P<0.05) in liver tissue. The total genistein, daidzein and other metabolites were 20% to 60% lower in samples from enzymatic hydrolysis than in acid hydrolysis. Conclusion: These results indicated that unknown factors in tissues reduced the enzymatic hydrolytic efficiency for releasing isoflavone aglycones even in optimized condition. This would underestimate isoflavone tissue concentrations up to 60%.

Long-Term Detection of Propofol Glucuronide in Urine Following Anesthetic Induction and Maintenance with Propofol

Propofol is the most commonly used compound for the intravenous induction and maintenance of anesthesia. Propofol addiction and abuse have become causes for concern in the healthcare community, especially among anesthesia and surgical professionals. The US Drug Enforcement Administration does not list propofol on any Schedules and most hospitals do not have inventory controls in place to prevent its misuse. Propofol is detectable in blood plasma as the parent compound for as much as 15 hours post-anesthesia. The metabolite propofol glucuronide (PPFG) has been detected in blood and urine as far out as 60 hours. Here we report the long-term renal excretion of PPFG in specimens from A) four participants following a 14-day course of orally ingested propofol dosing, and B) a female patient following anesthetic induction and 15 minutes’ maintenance with propofol. Urinary PPFG was measurable well above limits of quantitation up to 6 days following oral ingestion and 28 days post-anesthesia. We also present a third set of data evaluating the likelihood of passive exposure to aerosolized propofol in the surgical environment by analyzing the levels of urinary PPFG of healthcare workers following operating room work shifts. The results presented here demonstrate that quantitation of PPFG in urinary samples is an efficient method of long-term screening for propofol misuse and abuse.

The Detection of 1-Palmitoyl-2-oleoyl-<i>sn</i>-glycero-3-phosphoethanol and Ethyl Glucuronide in Human Umbilical Cord

In utero exposure to ethanol continues to be a significant public health issue and neonatal healthcare professionals are in need of objective means to identify exposed newborns. The aim of this study was to fully validate two methods for the detection of two direct alcohol biomarkers, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanol (POPE) and ethyl glucuronide (EtG), in umbilical cord and apply the assays to a group of authentic specimens. The limits of detections were 2 and 1 ng/g for POPE and ETG and the limits of quantitation were 4 and 3 ng/g, respectively. Inter and intra-day precision and accuracy measurements were within 15%. The assays were applied to 308 authentic specimens where we detected POPE in five (1.6%) specimens and EtG in twelve (3.9%) specimens. The mean concentrations were 11.4 ng/g ± 9.4 ng/g and 127.2 ± 227.7 ng/g for POPE and EtG, respectively. This study suggested that umbilical cord was a suitable specimen type for the identification of newborns exposed to ethanol in the womb and the prevalence of POPE and EtG detected in umbilical cord were consistent with the prevalence of self-reported binge drinking reported by the National Birth Defect Prevention Study (NBDPS) and Behavioral Risk Factor Surveillance System (BRFSS). Further studies are required to fully describe the association between the observed concentrations of POPE and EtG in umbilical cord to the level of maternal consumption of ethanol.

UDP-glucuronosyltransferases mediate coffee-associated reduction of liver fibrosis in bile duct ligated humanized transgenic UGT1A mice

Background:Coffee consumption has been shown to reduce the risk of liver fibrosis and is capable of inducing human UDP-glucuronosyltransferase(UGT)1A genes.UGT1A enzymes act as indirect antioxidants catalyzing the elimination of reactive metabolites,which in turn are potent initiators of profibrotic mechanisms.The aim of this study was to analyze the role of UGT1A genes as effectors of the protective properties of coffee in bile duct ligation(BDL)induced liver fibrosis.Methods:Fourteen days BDL with and without coffee pre-and co-treatment was performed in htgUGT1A-WT and htgUGT1A-SNP mice.Hepatic UGT1A mRNA expression levels,serum bilirubin and aminotransferase activities were determined.Liver fibrosis was assessed by collagen deposition,computational analysis of Sirius red tissue staining and expression of profibrotic marker genes.Oxidative stress was measured by hepatic peroxidase concentrations and immunofluorescence staining.Results:UGT1A transcription was differentially activated in the livers of htgUGT1A-WT mice after BDL,in contrast to a reduced or absent induction in the presence of SNPs.Co-treated(coffee+BDL)htgUGT1A-WT-mice showed significantly increased UGT1A expression and protein levels and a considerably higher induction compared to water drinking WT mice(BDL),whereas in co-treated htgUGT1A-SNP mice absolute expression levels remained below those observed in htgUGT1A-WT mice.Collagen deposition,oxidative stress and the expression of profibrotic markers inversely correlated with UGT1A expression levels in htgUGT1A-WT and SNP mice after BDL and coffee+BDL co-treatment.Conclusions:Coffee exerts hepatoprotective and antioxidative effects via activation of UGT1A enzymes.Attenuated hepatic fibrosis as a result of coffee-mediated UGT1A induction during cholestasis was detected,while the protective action of coffee was lower in a common low-function UGT1A SNP haplotype present in 10%of the Caucasian population.This study suggests that coffee consumption might constitute a potential strategy to support the conventional treatment of cholestasis-related liver diseases.

Sensitive Analysis and Pharmacokinetic Study of Berberrubine Using LC-MS/MS

Objective To develop a sensitive and reproducible liquid chromatography-tandem mass spectrometry（LC-MS/MS） method to evaluate the pharmacokinetic behavior of berberrubine（BRB） and its glucuronide（BRBG） in rats. Methods BRB, BRBG and tetrahydroberberine（THB, internal standard） were isolated by liquid-liquid extraction in rat biological samples. Chromatographic separation was achieved on an Agilent Zorbax Eclipse Plus C_（18）（2.1 mm × 50 mm, 3.5-Micron） with a gradient mobile phases primarily containing acetonitrile, water with 0.1% formic acid and 5 mm ammonium acetate. The analytes were monitored by MS/MS in positive electrospray ionization mode. Herein, the feasibility of new developed method was validated with respect to specificity, linearity, precision, accuracy, stability, extraction efficiency and matrix effect. The appropriate method was used for the pharmacokinetic study in rats.Results The new developed method could be applied to the pharmacokinetic study of BRB in rats. BRB and BRBG showed good linearity over the ranges of 2-1000 ng/mL and 5-2000 ng/mL, respectively, and precision was no more than 15%. The accuracy, specificity and stability could be acceptable. Conclusion The new method is sensitive and reproducible. In pharmacokinetic study, BRB showed nonlinear elimination property. Meanwhile, BRB was rapidly absorbed and widely distributed in various tissues with the highest exposure of BRB in kidney and liver. The absolute bioavailability of BRB was determined to be 8.2% and at the dose of 40 mg/kg, a total of 44% BRB was excreted in urine and feces.

Measurement of Free and Glucuronidated Cardamonin in Rat Plasma and Bile Using UPLC-MS/MS and Its Application to a Pharmacokinetic and Bile Excretion Study

A rapid and accurate ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry(UPLC-MS/MS)method was established and validated for the measurement of two forms of cardamonin,i.e.,free and glucuronidated,in the plasma and bile of rats.Cardamonin and an internal standard(1,8-dihydroxyanthraquinone)were extracted from plasma and bile with ethyl ether via liquid-liquid extraction.The analytes in the extracts were separated by an Agilent Zorbax Stable Bond-C18 column(2.1 mm×50 mm,1.8μm)under isocratic elution conditions[acetonitrile(A)and 0.1%ammonium formate in water(B),40:60(volume ratio)]with a flow rate of 0.4 mL/min,and mass spectrometry in negative ion MRM mode was implemented for analysis.Good linearity over the wide ranges of 0.01–5μg/mL for plasma and 0.025–10μg/mL for bile samples was acquired.Method validation was performed according to the FDA guidelines for bioanalytical methods.

Biliary excretion and enterohepatic circulation of thienorphine and its glucuronide conjugate in rats

Thienorphine(TNP)is a new partial opioid agonist currently developed as a promising drug candidate with the intended clinical indication for the treatment of opioid dependence.The pharmacokinetic profile and biliary excretion of TNP and its glucuronide conjugate(TNP-Glu)were investigated after oral administration of TNP in rats.The concentrations of TNP and TNP-Glu were simultaneously quantified using a LC-MS/MS method.A double peak phenomenon was observed in TNP plasma concentration–time curves with the secondary peak appeared at 6–8 h.A slower decline of plasma concentrations in the terminal phase was observed for TNP with T1/2 of 7.01 h.TNP-Glu was the predominant component in rat plasma and bile.Its plasma level was about 10 times higher than TNP and the 24 h accumulative bile excretion rate was 23%.Enterohepatic circulation of TNP and TNP-Glu was evaluated using a paired rat model.In bile-donor rats,no double-peak was detected and the elimination half life of TNP was significantly shortened(3.71 h)when compared to intact rats(7.01 h,P<0.05).Both TNP and TNP-Glu were detected in bile-recipient rats.Their exposures in recipient rats due to enterohepatic circulation were 15.6%and 42.6%for the parent drug and the metabolite,respectively.The deconjugation of the glucuronide conjugate and the reabsorption of free TNP were further confirmed in in situ perfused rat intestinal preparations.These results indicate that the enterohepatic circulation has a significant influence on the systemic exposure of the parent drug and its glucuronide conjugate,particularly on the terminal elimination of TNP,which may result in the prolonged retention of the drug in body.