Glucuronosyltransferase Activity Research Articles

Examining Physiologically Based Pharmacokinetic Model Assumptions for Cross-Tissue Similarity of Activity per Unit of Enzyme: The Case Example of Uridine 5′-Diphosphate Glucuronosyltransferase

The default assumption during in vitro in vivo extrapolation (IVIVE) to predict metabolic clearance in physiologically-based pharmacokinetics (PBPK) is that protein expression and activity have the same relationship in various tissues. This assumption is examined for uridine 5'-diphosphate glucuronosyltransferases (UGTs), a case example where expression and, hence, metabolic activity are distributed across various tissues. Our literature analysis presents overwhelming evidence of a greater UGT activity per unit of enzyme (higher kcat) in kidney and intestinal tissues relative to liver (greater than 200-fold for UGT2B7). This analysis is based on application of abundance values reported using similar proteomic techniques and within the same laboratory. Our findings call into question the practice of assuming similar kcat during IVIVE estimations as part of PBPK, and call for a systematic assessment of the kcat of various enzymes across different organs. The analysis focused on compiling data for probe substrates that were common for two or more of the studied tissues, to allow for reliable comparison of cross-tissue enzyme kinetics; this meant that UGT enzymes included in the study were limited to UGT1A1, 1A3, 1A6, 1A9 and 2B7. Significantly, UGT1A9 (n=24) and the liver (n=27) were each found to account for around half of the total dataset; these were found to correlate, with hepatic UGT1A9 data found in 15 of the studies, highlighting the need for more research into extrahepatic tissues and other UGT isoforms. Significance Statement During PBPK modelling (in vitro in vivo extrapolation) of drug clearance, the default assumption is that the activity per unit of enzyme (kcat) is the same in all tissues. The analysis provides preliminary evidence that this may not be the case, and that renal and intestinal tissues may have almost 250-fold greater UGT activity per unit of enzyme than liver tissues.

The possible role of ursolic acid in Covid-19: A real game changer.

Ursolic acid (UA) is a pentacyclic terpenoid is usually found in the fruit peels and stem bark as secondary metabolites. UA has antiviral, antibacterial, and antiparasitic properties. UA has a wide spectrum of pharmacological activities against different infections. Because of the greatest antiviral and anti-inflammatory properties of UA, so it could be a plausible therapeutic herbal medicine in Covid-19 treatment. Covid-19 is a recent worldwide virulent disease pandemic due to severe acute respiratory coronavirus disease 2 (SARS-CoV-2). The pathogenesis of SARS-CoV-2 infection is related to the direct cytopathic effect and exaggerated immune response by which acute lung injury (ALI) and/or acute respiratory distress syndrome might be developed in critical cases. UA may inhibit main protease of SARS-CoV-2, and inhibits the interface flanked by SARS-CoV-2 viral proteins and its entry point commonly recognized as angiotensin converting enzyme 2 (ACE2). In addition, UA attenuates SARS-CoV-2-induced inflammatory reactions and oxidative stress.Therefore, UA could avert SARS-CoV-2 infection from causing ALI. This opinion proposed that UA might be a potential candidate therapy against Covid-19 and can mitigate post-Covid-19 complications such as lung fibrosis. In this regards, forthcoming studies are reasonable to substantiate the therapeutic role of UA in Covid-19. However, taken into account that Covid-19 is yet to be investigating for further evaluations, therefore, clinical trials are recommended regarding use and dose of UA in Covid-19 treatment, as well as secondary effects.

Синдром Жильбера: термінологія, епідеміологія, генетика, патогенез (частина I)

Мета огляду — аналіз даних літератури щодо поширеності, етіології, генетики, патогенезу синдрому Жильбера. Проведений огляд наукової літератури щодо синдрому Жильбера за ключовими словами «синдром Жильбера», «гіпербілірубінемія», «уридиндифосфатглюкуронілтрансфераза» з використанням як пошукової системи PubMed. Беручи до уваги дослідження, проведені в останні 10 років, проаналізовані тези 75 статей. Більш детально вивчено результати дослідження, висвітлені у 28 статтях.

Identification of Human UDP-Glucuronosyltransferase and Sulfotransferase as Responsible for the Metabolism of Dotinurad, a Novel Selective Urate Reabsorption Inhibitor.

Dotinurad, a novel selective urate reabsorption inhibitor, is used to treat hyperuricemia. In humans, orally administered dotinurad is excreted mainly as glucuronide and sulfate conjugates in urine. To identify the isoforms of UDP-glucuronosyltransferase (UGT) and sulfotransferase (SULT) involved in dotinurad glucuronidation and sulfation, microsome and cytosol fractions of liver, intestine, kidney, and lung tissues (cytosol only) were analyzed along with recombinant human UGT and SULT isoforms. Dotinurad was mainly metabolized to its glucuronide conjugate by human liver microsomes (HLMs), and the glucuronidation followed the two-enzyme Michaelis-Menten equation. Among the recombinant human UGT isoforms expressed in the liver, UGT1A1, UGT1A3, UGT1A9, and UGT2B7 catalyzed dotinurad glucuronidation. Based on inhibition analysis using HLMs, bilirubin, imipramine, and diflunisal decreased glucuronosyltransferase activities by 45.5%, 22.3%, and 22.2%, respectively. Diflunisal and 3'-azido-3'-deoxythymidine, in the presence of 1% bovine serum albumin, decreased glucuronosyltransferase activities by 21.1% and 13.4%, respectively. Dotinurad was metabolized to its sulfate conjugate by human liver cytosol (HLC) and human intestinal cytosol (HIC) samples, with the sulfation reaction in HLC samples following the two-enzyme Michaelis-Menten equation and that in HIC samples following the Michaelis-Menten equation. All eight recombinant human SULT isoforms used herein catalyzed dotinurad sulfation. Gavestinel decreased sulfotransferase activity by 15.3% in HLC samples, and salbutamol decreased sulfotransferase activity by 68.4% in HIC samples. These results suggest that dotinurad glucuronidation is catalyzed mainly by UGT1A1, UGT1A3, UGT1A9, and UGT2B7, whereas its sulfation is catalyzed by many SULT isoforms, including SULT1B1 and SULT1A3. SIGNIFICANCE STATEMENT: The identification of enzymes involved in drug metabolism is important to predicting drug-drug interactions (DDIs) and interindividual variability for safe drug use. The present study revealed that dotinurad glucuronidation is catalyzed mainly by UGT1A1, UGT1A3, UGT1A9, and UGT2B7 and that its sulfation is catalyzed by many SULT isoforms, including SULT1B1 and SULT1A3. Therefore, dotinurad, a selective urate reabsorption inhibitor, is considered safe for use with a small risk of DDIs and low interindividual variability.

Gene mutation pattern of Gilbert's syndrome combined with viral hepatitis and its relationship with the exploration of clinical data

Objective: To study whether gene mutation pattern of Gilbert's syndrome (GS) is combined with viral hepatitis and its relationship with relevant clinical data. Methods: Clinical data of GS patients combined with viral hepatitis who was admitted to the Department of Infectious Diseases of Henan Provincial People's Hospital from August 2013 to December 2018 was retrospectively analyzed. The relationship between gene mutation pattern, general data (age, gender, etc.) and liver biochemical indexes was analyzed. The differences of the above data in patients with or without combined viral hepatitis were analyzed. The measurement data were compared by t-test. The categorical data was compared by the χ (2) test. The median and interquartile range of non-normally distributed data was used to indicate the central and discrete tendency. Results: A total of 107 GS eligible cases data were collected. The male to female ratio was 4.94:1 (89:18). The average age of onset was (36.36 ± 12.51) years. Alanine aminotransferase and total bilirubin levels were normal or slightly elevated, while aspartate aminotransferase, alkaline phosphatase, and γ-glutamyltransferase were all within the normal range. There were 49 cases in the combined viral hepatitis group (36 cases with HBV and 13 cases with HCV), and 58 cases in the GS alone group. Total bilirubin level in GS alone group was higher than the combined viral hepatitis group (z = 0.035, P < 0.05), and there were no statistically significant differences in gender, age, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and gamma glutamyltransferase (P > 0.05). Uridine diphosphate glucuronide transferase 1A1 (UGT1A1), specifically encoded by GS was detected in all 107 cases. Mutations was mainly occurred in the upstream promoter PBREM-3263 (-3279) (86 cases) and TATA box TA insertion mutation (71 cases), and GGA-AGA Gly71Arg (57 cases) mutation in EXON1 of the coding region. All mutation forms had manifestations of homozygous and heterozygous abnormalities. The combined incidence of main mutation forms in the genetic testing data were sequenced as: A2 + B2 + C2 (17 cases, 25.23%), A1 + B1 (17 cases, 15.89%), A2 (11 cases, 10.28%), C2 (10 Cases, 9.34%), A2 + B2 (7 cases, 6.54%), A1 + B2 (7 cases, 6.54%), C1 (7 cases, 6.54%), and there was no statistically significant difference between different mutation combinations in patients with or without hepatitis (P > 0.05). The results of total data analysis showed that the total bilirubin level in the single-site mutation group was higher than the multi-site mutation group (Z=2.019, P = 0.043), and other biochemical indicators had no effect (P > 0.05) and the differences were not statistically significant. Further analysis showed that the total bilirubin level of the single-site mutation subgroup in the GS alone group was higher than the multi-site mutation subgroup (Z = 1.999, P = 0.046), and the statistical difference was similar to the combined viral hepatitis group (P > 0.05). Different mutation combinations had no effect on biochemical indexes, and had no relationship with combined viral hepatitis (P > 0.05). Conclusion: GS is common in patients with combined viral hepatitis, and there is no significant difference between the incidence of gene mutation, mutation forms, biochemical indexes, and non-hepatitis group. The increase in the number of GS mutation sites does not aggravate the deterioration of bilirubin levels due to the decrease in the content and activity of uridine diphosphate glucuronosyltransferase, and the combination of different mutation sites does not affect the changes of various biochemical indexes, and at the same time it is not related to hepatitis.

SLC35B1 significantly contributes to the uptake of UDPGA into the endoplasmic reticulum for glucuronidation catalyzed by UDP-glucuronosyltransferases

The transport of UDP-glucuronic acid (UDPGA), a co-substrate of UDP-glucuronosyltransferase (UGT), to the intraluminal side of the endoplasmic reticulum (ER) is an essential step in the glucuronidation of exogenous and endogenous compounds. According to a previous study, the expression of recombinant SLC35B1, SLC35B4, or SLC35D1, nucleotide sugar transporters, in V79 cells has the potential to transport UDPGA into the lumen of microsomes. The purpose of this study is to examine whether the transport of UDPGA by these transporters substantially affects UGT activity. Since the knockdown of UDP-glucose 6-dehydrogenase, a synthetase of UDPGA, in HEK293 cells stably expressing UGT1A1 (HEK/UGT1A1 cells) resulted in a significant decrease in 4-methylumbelliferone (4-MU) glucuronosyltransferase activity, supplementation of a sufficient amount of UDPGA is required for UGT activity. By performing qRT-PCR using cDNA samples from 21 human liver samples, we observed levels of the SLC35B1 and SLC35D1 mRNAs that were 15- and 14-fold higher, respectively, than the levels of the SLC35B4 mRNA, and SLC35B1 showed the largest (37-fold) interindividual variability. Interestingly, 4-MU glucuronosyltransferase activity was significantly decreased upon the knockdown of SLC35B1 in HEK/UGT1A1 cells, and this phenomenon was also observed in HepaRG cells. Using siRNAs targeting 23 different SLC35 subfamilies, the knockdown of SLC35B1 and SLC35E3 decreased 4-MU glucuronosyltransferase activity in HEK/UGT1A1 cells. However, the 4-MU glucuronosyltransferase activity was not altered by SLC35E3 knockdown in HepaRG cells, suggesting that SLC35B1 was the main transporter of UDPGA into the ER in the human liver. In conclusion, SLC35B1 is a key modulator of UGT activity by transporting UDPGA to the intraluminal side of the ER.

A Gunn rat model of preterm hyperbilirubinemia.

BackgroundThe impact of bilirubin in preterm infants is poorly understood. An animal model would assist in improving understanding. The Gunn rat lacks uridine diphosphate-glucuronylsyl transferase 1 and can be made acutely hyperbilirubinemic by injection of sulfodimethoxine (sulfa), a drug that displaces bilirubin from albumin and thus increases free bilirubin.MethodsOn postnatal day (P) 5, Gunn rats either heterozygous (Nj) or homozygous (jj) for glucuronosyltransferase activity were injected with either saline or sulfa. Behavior and cerebellar weight were measured.ResultsPups did not show any signs of acute bilirubin encephalopathy. Pup weight dropped significantly on P8 only in the jj-sulfa group. Behavior was affected only in the jj-sulfa group. Cerebellar weight was significantly less in the jj-sulfa group.DiscussionThe Gunn rat pup model may be a good model to study hyperbilirubinemia in preterm infants.

Effect of feed restriction on hepatic estradiol metabolism and liver function in cows

In this study, the effects of restriction feeding on the liver function, hepatic uridine diphosphate glucuronosyltransferase (UGT) activity, hepatic insulin-like growth factor (IGF)-1 mRNA expression and response to high-dose estradiol-17β (E2) administration were investigated in non-lactating cows. Cows were assigned to either restricted feeding (30% of total digestible nutrient requirement) or ad libitum feeding of a dent corn-based concentrate and roughage for a 2-week feeding trial (Day 1=day of beginning the feeding trial). On day 14, a high-dose E2 administration study was carried out to examine plasma E2 levels as an indicator of hepatic E2 metabolism. Plasma E2 concentration in the restricted feeding group was consistently higher after high-dose E2 administration than in the control group. In addition, indocyanine green half-life value was prolonged by restricted feeding for 13 days, and increased liver triglyceride concentration and decreased liver UGT activity were caused by this restriction over 14 days. Restricted feeding did not affect plasma IGF-1 concentration or hepatic IGF-1 mRNA expression. These results suggest that two weeks of restriction feeding led to accumulation of triglyceride, decreased liver blood flow, and slightly impaired liver function, which in turn slowed down the hepatic metabolism of E2 without significantly impacting hepatic IGF-1 production.

Upregulation of UDP-Glucuronosyltransferases 1a1 and 1a7 Are Involved in Altered Puerarin Pharmacokinetics in Type II Diabetic Rats.

Puerarin is an isoflavonoid extracted from Pueraria lobata roots, and displays a broad range of pharmacological activities, including antidiabetic activity. However, information about the pharmacokinetics of puerarin in diabetics is scarce. This study was conducted to investigate the difference in pharmacokinetic effects of puerarin in normal rats and rats with diabetes mellitus (DM), and the mechanism involved. DM was induced by a combined high-fat diet (HFD) and streptozotocin (STZ) injection. Plasma concentrations of puerarin in DM, HFD, and control rats were determined after intravenous (20 mg/kg) and oral administration (500 mg/kg) of puerarin, and pharmacokinetic parameters were estimated. The messenger RNA (mRNA) and protein expression levels of Ugt1a1 and Ugt1a7 in rat livers and intestines were measured using qRT-PCR and western blot, respectively. The area under the concentration–time curve and the clearance of puerarin in the DM rats statistically differed from those in the control rats (p <0.05) with both administration routes. The hepatic and intestinal gene and protein expressions of Ugt1a1 and Ugt1a7 were significantly increased in the DM rats (p <0.05). Therefore, the metabolic changes in diabetes could alter the pharmacokinetics of puerarin. This change could be caused by upregulated uridine diphosphate (UDP)-glucuronosyltransferase activity, which may enhance puerarin clearance, and alter its therapeutic effects.

Simultaneous determination of intestinal permeability and potential drug interactions of complex mixtures using Caco-2 cells and high-resolution mass spectrometry: Studies with Rauwolfia serpentina extract

Caco-2 cells are a commonly used model for estimating the intestinal bioavailability of single chemical entity pharmaceuticals. Caco-2 cells, when induced with calcitriol, also express other biological functions such as phase I (CYP) and phase II (glucuronosyltransferases) drug metabolizing enzymes which are relevant to drug-supplement interactions. Intestinal bioavailability is an important factor in the overall safety assessment of products consumed orally. Foods, including herbal dietary supplements, are complex substances with multiple chemical components. Because of potential interactions between components of complex mixtures, more reliable safety assessments can be obtained by studying the commercial products “as consumed” rather than by testing individual chemical components one at a time. The present study evaluated the apparent intestinal permeability (Papp) of a model herbal extract, Rauwolfia serpentina, using both whole plant extracts and the individual purified Rauwolfia alkaloids. All test compounds, endpoint substrates, and their metabolites were quantified using liquid chromatography and high-resolution mass spectrometry. The Papp values for individual Rauwolfia alkaloids were comparable whether measured individually or as components of the complete extract. Both Rauwolfia extract and all individual Rauwolfia alkaloids except yohimbine inhibited CYP3A4 activity (midazolam 1′-hydroxylation). Both Rauwolfia extract and all individual Rauwolfia alkaloids except corynanthine and reserpic acid significantly increased glucuronosyltransferase activity (glucuronidation of 4-methylumbelliferone). The positive control, ketoconazole, significantly inhibited both CYP3A4 and glucuronosyltransferase activities. These findings suggest that the Caco-2 assay is capable of simultaneously identifying both bioavailability and potentially hazardous intestinal drug-supplement interactions in complex mixtures.

Hepatic Parenchymal Injury in Crigler-Najjar Type I.

Crigler-Najjar syndrome type I (CNI) arises from biallelic variants of UGT1A1 that abrogate uridine diphosphate glucuronosyltransferase (UGT1A1) activity resulting in unconjugated hyperbilirubinemia. Historically, liver parenchyma in CNI was considered structurally and histologically normal. Recent review of CNI liver explants revealed fibrosis. Our aim was to investigate the association between hepatic histology and disease phenotype in CNI. We extracted data from the medical record at the time of liver transplant from 22 patients with CNI at the Children's Hospital of Pittsburgh, and reviewed explant histology. Continuous data were normally distributed, are presented as mean (±1 SD), and analyzed using two-tailed Student t-test. Categorical data were analyzed using the Chi-square test. Both alanine transaminase (ALT; mean 87.4 IU/L) and aspartate transaminase (AST; mean 54.6 IU/L) were elevated. Nine (41%) of 22 explants had significant fibrosis. Pericentral (n = 5), periportal (n = 2), and mixed (n = 2) patterns of fibrosis occurred. A significant difference in mean age of subjects with fibrotic versus non-fibrotic livers (16.1 years vs 10.5 years; P = 0.02) was seen. There were no indices of synthetic liver dysfunction or portal hypertension. Neither a history of gallstone disease nor excess weight appeared to contribute to the development of fibrosis. For the first time, we report a 41% prevalence of clinically silent, yet histologically significant fibrosis among subjects with Crigler-Najjar type 1. Risk for fibrosis appears to accrue with time, indicating that earlier intervention may be prudent whenever considering alternative treatments such as hepatocyte transplant, auxiliary liver transplant, or viral gene therapy.

Genome-wide profiling of gene expression and DNA methylation provides insight into low-altitude acclimation in Tibetan pigs

Efforts have been made to characterize the high-altitude adaption in Tibetan pigs and identified vast of genes or genomic regions undergone natural selection. Nonetheless, information concerning gene expression and DNA methylation changes response to low-altitude acclimation in Tibetan pigs is long overdue. To explore the exceptional mechanisms of gene expression and DNA methylation that are induced by low altitude environments in Tibetan pigs, we performed a comparative transcriptomic and DNA methylation analysis of skeletal muscle between indigenous Tibetan pigs that reside in high altitude regions (~4000m) and their counterparts that migrated to the geographically neighboring low-altitude regions (~500m) for nearly ten generations. Many genes that related to hypoxia response (EGLN3 and FLT1) and energy metabolism (TFB2M) were differentially expressed, but without significant DNA methylation changes. We also found genes embedded in differentially methylated regions were mainly involved in ‘Starch and sucrose metabolism’, ‘glucuronosyltransferase activity’ processes. Specifically, our results showed increased SIN3A mRNA expression, with hypomethylation status of its promoter, in longissimus dorsi muscle of low-altitude Tibetan pig. Another gene, CACNG6, showed decreasing expression level with an elevated methylation in its intron 3. These results indicated DNA-methylation-mediated expression alterations in low-altitude acclimation. We envision that this study will serve as a valuable resource for mammal acclimation research and agricultural food industry.

The effects of UDP-sugars, UDP and Mg2+on uridine diphosphate glucuronosyltransferase activity in human liver microsomes

1. The UDP-glucuronosyltransferase (UGT) enzymes are important in the metabolism, elimination and detoxification of many xenobiotics and endogenous compounds. As extrapolation of in vitro kinetics of drug metabolizing enzymes to predict in vivo clearance rates becomes more sophisticated, it is important to ensure proper optimization of enzyme assays. The luminal location of the enzyme active site (i.e. latency), and the complexity of UGT kinetics, results in consistent under-prediction of clearance of drugs metabolized by glucuronidation.2. We examined inhibition of UGT activity in alamethicin-disrupted human liver microsomes (HLM) by uridine diphosphate (UDP), a UGT reaction product, and its reversal by Mg2+ ions. We also determined whether UDP-sugars other than the co-substrate UDP-glucuronic acid (UDP-GlcA) affected glucuronidation.3. We show that other UDP-sugars do not significantly influence glucuronidation. We also demonstrate that UDP inhibits HLM UGT activity and that this is reversed by including Mg2+ in the assay. The Mg2+ effect can be offset using EDTA, and is dependent on the concentration of UDP-GlcA in the assay.4. We propose that formation of a Mg2+–UDP complex prevents UDP from affecting the enzyme. Our results suggest that 5 mM UDP-GlcA and 10 mM Mg2+ be used for UGT assays in fully disrupted HLM.

Human Biliary Amount Prediction Using Simple, Bile Flow-Rate Corrected and Uridine Diphosphate Glucuronosyltransferase Activity Corrected Allometric Methods

Aim: Retrospective scaling of human biliary amounts has been performed using allometry. Methods: Human biliary excretory data for 14 drugs were predicted using simple, bile flow-rate corrected and uridine diphosphate glucuronosyltransferase (UDPGT) activity corrected allometry methods. Allometry was performed using Y = aXb relationship with correction factors. Statistical tests consisting of fold difference (predicted/observed) and root-mean-square error (RMSE) computation were carried out. Results: UDPGT activity corrected allometry predicted higher biliary amounts in humans as compared with other methods. The RMSE values were 38, 32 and 81 for simple, bile flow-rate corrected and UDPGT activity corrected allometry, respectively. Conclusion: Although data showed the usefulness of the approaches for human biliary predictions in the decision process for nominating drug candidate(s) based on % RMSE and fold differences, for some drugs prediction appeared not satisfactory using any of the methods.

Age-related change of hepatic uridine diphosphate glucuronosyltransferase and sulfotransferase activities in male chickens and pigs.

The hepatic activities of uridine diphosphate glucuronosyltransferase (UGT) and sulfotransferase (SULT) of male Ross 708 broiler chickens at the age of 1, 7, 14, 28, and 56days and male Camborough-29 pigs at the age of 1day and 2, 5, 10, and 20weeks were investigated. Glucuronidation and sulfation of 4-nitrophenol were used to evaluate the activities. Porcine hepatic UGT and SULT activities were low at birth, peaked at around 5-10weeks, and then declined. Both hepatic UGT and SULT activities of chickens were high at hatch and declined. Chicken hepatic UGT activity had a peak at the age of 28days. Affinity of hepatic SULT to 4-nitrophenol is similar in chickens and pigs, but the affinity of hepatic UGT in pigs was about 10 times higher than that in chickens. 4-nitrophenol was predominantly conjugated by SULT instead of UGT in chicken livers from hatch to day 56. Conversely, hepatic UGT contributed predominantly in 4-nitrophenol conjugation than the SULT in pigs from birth to 20weeks. Therefore, age has significant impact on hepatic activities of UGT and SULT, and the importance of UGT and SULT on conjugation is different in chickens and pigs.

Mechanism for Increased Expression of UGT2B in the Liver of Mice with Neuropathic Pain.

Approximately 30% of patients with cancer pain experience concurrent neuropathic pain. Since these patients are not sufficiently responsive to morphine, the development of an effective method of pain relief is urgently needed. Decreased function of the μ opioid receptor, which binds to the active metabolite of morphine M-6-G in the brain, has been proposed as a mechanism for morphine resistance. Previously, we pharmacokinetically examined morphine resistance in mice with neuropathic pain, and demonstrated that the brain morphine concentration was decreased, expression level of P-glycoprotein (P-gp) in the small intestine was increased, and expression level and activity of uridine diphosphate glucuronosyltransferase (UGT)2B in the liver were increased. In order to clarify the mechanism of the increased expression of UGT2B, we examined the phase of neuropathic pain during which UGT2B expression in the liver begins to increase, and whether this increased expression is nuclear receptor-mediated. The results of this study revealed that the increased expression of UGT2B in the liver occurred during the maintenance phase of neuropathic pain, suggesting that it may be caused by transcriptional regulation which was not accompanied by increased nuclear import of pregnane X receptor (PXR).

Differentiating Gilbert Syndrome from Crigler Najjar Syndrome Type 2 by Phenobarbitone Test

Gilbert syndrome characterized by jaundice with intermittent elevations of indirect bilirubin, in the absence of haemolysis or underlying liver disease, has both autosomal dominant and recessive inheritance. Crigler-Najjar syndrome type II (CNS2) is a hereditary disorder of bilirubin metabolism characterized by unconjugated hyperbilirubinemia due to reduced and inducible activity of hepatic bilirubin glucuronosyltransferase (GT). We report 20 children between age 5 to 15 years with unconjugated hyperbilirubenemia who were given seven days of oral phenobarbitone (5mg/kg/day) and decrease in level of bilirubin was noted. There was only 30-40% reduction of bilirubin in Crigler Najjar Syndrome Type 2 compared to Gilberts Syndrome in which bilirubin level normalised. This case series highlights the importance of simple test to differentiate these two conditions. This test is also very helpful in a place where enzyme level and mutational study cannot be done.J Nepal Paediatr Soc 2015;35(1):82-84

Toxicity of endophyte-infected ryegrass hay containing high ergovaline level in lactating ewes.

The symbiotic association of var. (formerly named ) with perennial ryegrass () leads to the production of ergovaline (EV) and lolitrem B (LB) that are toxic for livestock. The objectives of this study were to determine the effects of feeding endophyte-infected ryegrass (SE+) hay on 16 lactating ewes (BW 80 ± 10 kg) in comparison with endophyte-free ryegrass (SE-) hay to investigate the putative mechanisms of action of EV and LB and to evaluate their persistence in milk and animal tissues. The mean EV and LB concentrations in SE+ hay were 851 and 884 μg/kg DM, respectively, whereas these alkaloids were below the limit of detection in SE- hay. No effect of SE+ was observed on animal health and skin temperature whereas prolactin decreased and significant differences between hays were observed from d 7 to 28 of the study ( < 0.03) but had no effect on milk production. Hematocrit and biochemical analyses of plasma revealed no significant difference between SE+ and SE-, whereas cortisol concentration differed significantly on d 28 ( = 0.001). Measurement of oxidative damage and antioxidant enzyme activities in plasma, liver, and kidneys revealed a slight increase in some enzyme activities involved in defense against oxidative damage in the SE+ fed ewes. Slight variations in the activities of hepatic and kidney flavin monooxygenase enzymes were observed, whereas in the kidney, glutathione -transferase activity decreased significantly ( = 0.002) in the SE+ fed ewes, whereas uridine diphosphate glucuronosyltransferase activity increased ( = 0.001). After 28 d of exposure of ewes to the SE+ hay, low EV and LB concentrations were measured in tissues. The highest concentration of EV was observed in the liver (0.68 μg/kg) whereas fat contained the highest concentration of LB (2.39 μg/kg). Both toxins were also identified at the trace level in milk.

Effect of Traumatic Brain Injury, Erythropoietin, and Anakinra on Hepatic Metabolizing Enzymes and Transporters in an Experimental Rat Model.

In contrast to considerable data demonstrating a decrease in cytochrome P450 (CYP) activity in inflammation and infection, clinically, traumatic brain injury (TBI) results in an increase in CYP and UDP glucuronosyltransferase (UGT) activity. The objective of this study was to determine the effects of TBI alone and with treatment with erythropoietin (EPO) or anakinra on the gene expression of hepatic inflammatory proteins, drug-metabolizing enzymes, and transporters in a cortical contusion impact (CCI) injury model. Microarray-based transcriptional profiling was used to determine the effect on gene expression at 24 h, 72 h, and 7 days post-CCI. Plasma cytokine and liver protein concentrations of CYP2D4, CYP3A1, EPHX1, and UGT2B7 were determined. There was no effect of TBI, TBI + EPO, or TBI + anakinra on gene expression of the inflammatory factors shown to be associated with decreased expression of hepatic metabolic enzymes in models of infection and inflammation. IL-6 plasma concentrations were increased in TBI animals and decreased with EPO and anakinra treatment. There was no significant effect of TBI and/or anakinra on gene expression of enzymes or transporters known to be involved in drug disposition. TBI + EPO treatment decreased the gene expression of Cyp2d4 at 72 h with a corresponding decrease in CYP2D4 protein at 72 h and 7 days. CYP3A1 protein was decreased at 24 h. In conclusion, EPO treatment may result in a significant decrease in the metabolism of Cyp-metabolized drugs. In contrast to clinical TBI, there was not a significant effect of experimental TBI on CYP or UGT metabolic enzymes.

Gum Arabic that is Enzymatically Modified with Arabidopsis Beta-Glucuronosyltransferase Can Make Smaller and more Stable Oil-in-Water Emulsions

A plant glycosyltransferase was utilized to modify the properties of gum arabic as an oil-in-water emulsifier. We previously reported that recombinant beta-glucuronosyltransferase (AtGlcAT14A) from Arabidopsis thaliana produced in Pichia pastoris possesses glucuronosyltransferase activity to transfer glucuronic acid (GlcA) from UDP-GlcA to beta-1,3-galactan main chain and beta-1,6-galactan side chains of type II arabinogalactan. In this paper, we report that AtGlcAT14A can also transfer GlcA from UDP-GlcA to gum arabic at the optimal pH value of 5 in the absence of dicationic ion. In the modified gum Arabic, GlcA was primarily incorporated into the beta-1,6-galactans. The oil-in-water emulsions created by the modified gum arabic were smaller, less flocculated and threefold more stable than that produced by the unmodified gum arabic. It is conceivable that the additional GlcA on the surface of gum arabic prevents flocculation by increasing surface electrostatic repulsion, which leads to more stable oil-in-water emulsions. Our study implicates the structure-function relationship and provides a potential method for the enzyme-based manipulation of gum arabic.