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Microsomal Enzymes (microsomal + enzyme)
Selected AbstractsCapillary electrophoretic chiral separation of hydroxychloroquine and its metabolites in the microsomal fraction of liver homogenatesELECTROPHORESIS, Issue 5-6 2006Carmem Dickow Cardoso Abstract A rapid, selective, and low-cost chiral capillary electrophoretic method was developed for the simultaneous analysis of hydroxychloroquine (HCQ) and its three chiral metabolites: desethylchloroquine (DCQ), desethylhydroxychloroquine (DHCQ), and bisdesethylchloroquine (BDCQ) in the microsomal fraction of liver homogenates. After liquid,liquid extraction using toluene as extracting solvent, the drug and metabolites were resolved on a fused-silica capillary (50,,m ID, 50,cm total length, and 42,cm effective length), using 100,mmol/L of Tris/phosphate buffer, pH,9.0 containing 1% w/v sulfated-,-CD and 30,mg/mL hydroxypropyl-,-CD. Detection was carried out at 220,nm. The extraction procedure was efficient in removing endogenous interferents, and low values (,15%) for CVs and deviation from theoretical values were demonstrated for both within-day and between-day assays. The quantitation limit was 125,ng/mL with linear response over the 125,2000,ng/mL of concentration range for all metabolites. After validation, the method was used for an in vitro metabolism study of HCQ. The major HCQ metabolite formed by microsomal enzymes was (,)-(R)-DHCQ. [source] Comparative hepatic activity of xenobiotic-metabolizing enzymes and concentrations of organohalogens and their hydroxylated analogues in captive greenland sledge dogs (Canis familiaris),ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 1 2009Jonathan Verreault Abstract A captive study was performed with Greenland sledge dogs (Canis familiaris) fed a naturally organohalogen-contaminated diet (Greenland minke whale [Balaenoptera acutorostrata] blubber; exposed group) or a control diet (pork fat; control group). The catalytic activity of major xenobiotic-metabolizing phase I and II hepatic microsomal enzymes was assessed. Relative to control dogs, ethoxyresorufin- O -deethylase (EROD) activity in exposed dogs was twofold higher (p = 0.001). Testosterone hydroxylation yielded 6,- and 16,-hydroxy (OH) testosterone and androstenedione, with higher rates of production (23,27%; p , 0.03) in the exposed individuals. In the exposed dogs, epoxide hydrolase (EH) activity was 31% higher (p = 0.02) relative to the control dogs, whereas uridine diphosphoglucuronosyl transferase (UDPGT) activity was not different (p = 0.62). When the exposed and control dogs were combined, the summed (,) plasma concentrations of OH-polychlorinated biphenyl (PCB) congeners were predicted by plasma ,PCB concentrations and EROD activity (p , 0.04), whereas testosterone hydroxylase, EH, and UDPGT activities were not significant predictors of these concentrations. Consistent results were found for individual OH-PCB congeners and their theoretical precursor PCBs (e.g., 4-OH-CB-187 and CB-183, and 4-OH-CB-146 and CB-146) and for EROD activity. No association was found between ,OH,polybrominated diphenyl ether (PBDE) and ,PBDE plasma concentrations, or between potential precursor-metabolite pairs, and the enzyme activities. The present results suggest that liver microsomal EROD activity and plasma PCB concentrations have a greater (e.g., relative to EH activity) predictive power for the occurrence of plasma OH-PCB residues in sledge dogs. These results also suggest that plasma OH-PBDEs likely are not products of cytochrome P450-mediated transformation but, rather, are accumulated via the diet. [source] The specificity of alcohol dehydrogenase with cis -retinoidsFEBS JOURNAL, Issue 9 2004Activity with 11- cis -retinol, localization in retina Studies in knockout mice support the involvement of alcohol dehydrogenases ADH1 and ADH4 in retinoid metabolism, although kinetics with retinoids are not known for the mouse enzymes. Moreover, a role of alcohol dehydrogenase (ADH) in the eye retinoid interconversions cannot be ascertained due to the lack of information on the kinetics with 11- cis -retinoids. We report here the kinetics of human ADH1B1, ADH1B2, ADH4, and mouse ADH1 and ADH4 with all- trans -, 7- cis -, 9- cis -, 11- cis - and 13- cis -isomers of retinol and retinal. These retinoids are substrates for all enzymes tested, except the 13- cis isomers which are not used by ADH1. In general, human and mouse ADH4 exhibit similar activity, higher than that of ADH1, while mouse ADH1 is more efficient than the homologous human enzymes. All tested ADHs use 11- cis -retinoids efficiently. ADH4 shows much higher kcat/Km values for 11- cis -retinol oxidation than for 11- cis -retinal reduction, a unique property among mammalian ADHs for any alcohol/aldehyde substrate pair. Docking simulations and the kinetic properties of the human ADH4 M141L mutant demonstrated that residue 141, in the middle region of the active site, is essential for such ADH4 specificity. The distinct kinetics of ADH4 with 11- cis -retinol, its wide specificity with retinol isomers and its immunolocalization in several retinal cell layers, including pigment epithelium, support a role of this enzyme in the various retinol oxidations that occur in the retina. Cytosolic ADH4 activity may complement the isomer-specific microsomal enzymes involved in photopigment regeneration and retinoic acid synthesis. [source] Induction of endogenous pathways by antiepileptics and clinical implicationsFUNDAMENTAL & CLINICAL PHARMACOLOGY, Issue 5 2005M. Strolin Benedetti Abstract The aim of this study was to review modifications of the endogenous pathways (e.g. enzyme elevations, normal body constituent depletion or higher formation/excretion of endogenous metabolites) which could be ascribed to enzyme induction by antiepileptic drugs (AEDs). Information on older (e.g. phenobarbital, phenytoin and carbamazepine) and newer drugs (where information is available) is discussed together with clinical implications. The enzymes involved in the endogenous pathways and induced by the AEDs will not be limited to the hepatic microsomal enzymes; extrahepatic enzymes and/or enzymes present in other subcellular fractions will also be discussed, if pertinent. The induction of endogenous pathways by AEDs has been taken into account in the past, but much less emphasis has been given compared with the extensive literature on induction by AEDs of the metabolism of concomitantly administered drugs, either of the same or of different classes. Not all of the endogenous pathways examined and induced by AEDs appear to result in serious clinical consequences (e.g. induction of hepatic ALP, increased excretion of d -glucaric acid or of 6, -hydroxycortisol). In some cases, induction of some pathways (e.g. increase of high-density lipoprotein cholesterol or of conjugated bilirubin) might even be a beneficial side-effect, however enzyme induction is considered rather a detrimental aspect for an AED, as induction is generally a broad and a non-specific phenomenon. The new AEDs have generally less induction potential than the older agents. Yet some (felbamate, topiramate, oxcarbazepine and lamotrigine) have the potential for inducing enzymes, whereas others (levetiracetam, gabapentin and vigabatrin) appear to be completely devoid of enzyme inducing characteristics, at least as far as the enzymes investigated are concerned. [source] CYP3A4 is a Human Microsomal Vitamin D 25-Hydroxylase,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 4 2004Ram P Gupta Abstract The human hepatic microsomal vitamin D 25-hydroxylase protein and gene have not been identified with certainty. Sixteen hepatic recombinant microsomal enzymes were screened for 25-hydroxylase activity; 11 had some 25-hydroxylase activity, but CYP3A4 had the highest activity. In characterized liver microsomes, 25-hydroxylase activity correlated significantly with CYP3A4 testosterone 6,-hydroxylase activity. Activity in pooled liver microsomes was inhibited by known inhibitors of CYP3A4 and by an antibody to CYP3A2. Thus, CYP3A4 is a hepatic microsomal vitamin D 25-hydroxylase. Introduction: Studies were performed to identify human microsomal vitamin D-25 hydroxylase. Materials and Methods: Sixteen major hepatic microsomal recombinant enzymes derived from cytochrome P450 cDNAs expressed in baculovirus-infected insect cells were screened for 25-hydroxylase activity with 1,-hydroxyvitamin D2 [1,(OH)D2], 1,-hydroxyvitamin D3 [1,(OH)D3], vitamin D2, and vitamin D3 as substrates. Activity was correlated with known biological activities of enzymes in a panel of 12 characterized human liver microsomes. The effects of known inhibitors and specific antibodies on activity also were determined. Results: CYP3A4, the most abundant cytochrome P450 enzyme in human liver and intestine, had 7-fold greater activity than that of any of the other enzymes with 1,(OH)D2 as substrate. CYP3A4 25-hydroxylase activity was four times higher with 1,(OH)D2 than with 1,(OH)D3 as substrate, was much less with vitamin D2, and was not detected with vitamin D3. 1,(OH)D2 was the substrate in subsequent experiments. In a panel of characterized human liver microsomes, 25-hydroxylase activity correlated with CYP3A4 testosterone 6,-hydroxylase activity (r = 0.93, p < 0.001) and CYP2C91 diclofenac 4,-hydroxylase activity (r = 0.65, p < 0.05), but not with activity of any of the other enzymes. Activity in recombinant CYP3A4 and pooled liver microsomes was dose-dependently inhibited by ketoconazole, troleandomycin, isoniazid, and ,-naphthoflavone, known inhibitors of CYP3A4. Activity in pooled liver microsomes was inhibited by antibodies to CYP3A2 that are known to inhibit CYP3A4 activity. Conclusion: CYP3A4 is a vitamin D 25-hydroxylase for vitamin D2 in human hepatic microsomes and hydroxylates both 1,(OH)D2 and 1,(OH)D3. [source] | |||||||||||||