HPLC-QTOFMS和MS/MS分析鉴定PF573228肝微粒体体外代谢产物

为研究PF573228的大鼠肝微粒体体外代谢特点,采用高效液相色谱-电喷雾-串联质谱(HPLC-ESI-MS/MS)的方法,结合检测的保留时间、精确质量数和特征碎片离子,鉴定了PF573228体外的3种代谢产物分子的结构,推断体外代谢产物的生物转化有3个主要途径:脱氢,N-脱烷基和氧化.     

Metabolism of fibrates by cytochrome P450s and UDP-glycosyltransferases in rat and human liver microsomes

Fibrates are widely used for the treatment of dyslipidemia.However,the contributions of the phase I and phase II metabolic pathways to the clearance of fibrates are unclear.In this study,we investigated the metabolism of gemfibrozil(Gem) ,clofibric acid(CA) ,fenofibric acid(FA) and bezafibrate(Beza) by cytochrome P450s(P450s) and UDP-glycosyltransferases(UGTs) using a substrate depletion approach.We also compared the metabolic characteristics of rat liver microsomes(RLM) and human liver microsomes(HLM) .The intrinsic clearance rates mediated by P450s,UGTs and both were 172 ± 22,643 ± 26,798 ± 103 μL min-1 mg-1,respectively,for Gem and 43 ± 11,88 ± 12,119 ± 15 μL min-1 mg-1,respectively,for CA in RLM.The fractions metabolized by P450s and UGTs in RLM were 22% and 81% for Gem,36% and 74% for CA.The P450-and UGT-mediated depletion rates for Gem were 303 and 1607 nmol min-1 mg-1 in RLM versus 86 and 243 nmol min-1 mg-1 in HLM.The corre-sponding rates for CA were 1.1 and 1.7 nmol min-1 mg-1 in RLM versus 0.025 and 0.038 nmol min-1 mg-1 in HLM.Accordingly,both P450s and UGTs substantially contribute to the clearance of Gem and CA,with UGTs playing a greater role.To avoid un-der-estimating the impact of these pathways,it is necessary to measure NADPH-and UDPGA-dependent metabolism.Although the fractions of these two pathways in RLM and HLM were similar,the depletion rate of Gem and CA in RLM was higher than that in HLM.The metabolism of FA and Beza by P450s and UGTs was too low to calculate intrinsic clearance in both RLM and HLM.These results indicate that fibrates are metabolized via similar pathways in rats and humans,and it is applicable to use RLM to predict the clearance of fibrates in human.     

Hydroxyl radicals are not involved in NADPH dependent microsomal lipid peroxidation

Summary NADPH dependent H₂O₂ formation in microsomes in the presence of chelated iron leads to formation of hydroxyl radicals. Enhancement of hydroxyl radical generation (via ferric-EDTA or sodium azide) did not result in a concomitant increase in lipid peroxidation; rather, a decrease was observed. Moreover, the hydroxyl radical scavenger DMSO did not inhibit lipid peroxidation. This comparison of hydroxyl radical formation with lipid peroxidation suggests that hydroxyl radicals do not play a part in NADPH-dependent lipid peroxidation.     

Immunoquantitation of some cytochrome P-450 isozymes in liver microsomes from streptozotocin-diabetic rats

Summary Streptozotocin-diabetes in rats leads to a decrease of cytochrome P-450 UT-A (the major form in control rats) and an increase of cytochrome P-450 PB-B (the major one induced by phenobarbital treatment) in liver microsomes. The increased benzphetamine-N-demethylase activity can be related to the induction of cytochrome P-450 PB-B.     

Ca-efflux,from direct membrane injury by CCl4, is elicited by amphiphilic vehicles in vitro

Direct membrane injury by CCl₄, in situations excluding metabolic activation, was evaluated in saponin-permeabilized hepatocytes and in microsomes by measuring immediate Ca²⁺ efflux. A good correlation appears between the Ca²⁺ efflux and the level of CCl₄ in the membrane and also the variations in fluidity. Mixtures of CCl₄ with water-soluble vehicles were used to improve the dispersion of CCl₄ in the medium. The mixtures varied in their ability to elicit the membrane effects of CCl₄. The performance of ethanol and, to a lesser degree, other alcohols, suggests the existence of a water stable structural organization between CCl₄ and these amphiphilic vehicles, facilitating the transfer of CCl₄ to the membrane.     

Differential effect of silybin on the Fe2+-ADP and t-butyl hydroperoxide-induced microsomal lipid peroxidation

Summary We have observed a differential effect of silybin dihemisuccinate on rat liver microsonal oxygen consumption and on lipid peroxidation induced by NADPH-Fe²⁺-ADP and t-butyl hydroperoxide. These results are ascribed to the antioxidant properties of the flavonoid. The differences observed in the effect of the catalysts may be a consequence of the different capacity of silybin to act as a scavenger of free radicals formed by NADPH-Fe²⁺-ADP or t-butyl hydroperoxide.This research was supported in part by grant B-2019-8412 from Dirección de Investigación y Bibliotecas, Universidad de Chile.