Normal Rat Liver Microsomes Research Articles

Metabolic study of ginsenoside Rg3 and glimepiride in type 2 diabetic rats by liquid chromatography coupled with quadrupole‐Orbitrap mass spectrometry

RationaleGinsenoside Rg3 and glimepiride have been applied to treat type 2 diabetes (T2DM) because of their good hypoglycemic effects. In this study, the effects of ginsenoside Rg3 acting synergistically with glimepiride were investigated in liver microsomes from rats with type 2 diabetes.MethodsAn in vitro incubation system with normal rat liver microsomes (RLM) and type 2 diabetic rat liver microsomes (TRLM) was developed. The system also included two experimental groups consisting of RLM and TRLM pretreated with ginsenoside Rg3 and glimepiride (named the RLMR and TRLMR groups, respectively). The metabolism in the different groups was analyzed by ultra‐performance liquid chromatography coupled with quadrupole‐orbitrap mass spectrometry (UPLC/Q‐Orbitrap MS).ResultsThe results showed that the concentration of glimepiride increased in RLM and TRLM after treatment with ginsenoside Rg3. Five metabolites (M1–M5) of glimepiride were found, and they were named 3N‐hydroxyglimepiride, hydroxyglimepiride, 1,2‐epoxy ether‐3‐hydroxyglimepiride, 1N‐hydroxyglimepiride and 1N,2C,S,O,O‐epoxy ether‐3‐hydroxyglimepiride. The metabolite of ginsenoside Rg3 was ginsenoside Rh2.ConclusionsAn in vitro incubation system with RLM and TRLM was developed. The system revealed pathways that produce glimepiride metabolites. Ginsenoside Rg3 may inhibit the activity of cytochrome P450 enzymes in vitro. The present study showed that ginsenoside Rg3 and glimepiride may be combined for the treatment of T2DM.

Off-line two-dimensional liquid chromatography coupled with diode array detection and quadrupole-time of flight mass spectrometry for the biotransformation kinetics of Ginkgo biloba leaves extract by diabetic rat liver microsomes

Ginkgo biloba leaves extract (GBE), one of the most widely used traditional Chinese medicines worldwide, can be used for the treatment of diabetes mellitus (DM). However, its biotransformation in liver is not fully known under the state of DM. In this study, an off-line hydrophilic interaction × reversed-phase two-dimensional liquid chromatography (HILIC × RP 2D-LC) system coupled with diode array detection (DAD) and quadrupole time-of-flight mass spectrometry (q/TOF-MS) was established for the qualification and quantification of the biotransformation of GBE in normal and diabetic rat liver microsomes (RLMs). 6 metabolites were tentatively identified according to the exact molecular weights and the characteristic fragment ions provided by q/TOF-MS data. The results of metabolic stability showed that the metabolic ratio of four target compounds including quercetin, genistein, kaempferol and isorhamnetin in diabetic RLMs were significantly enhanced when comparing with normal RLMs. The results of enzyme kinetics showed that compared with normal RLMs, the Michaelis-Menten constant (Km) value of genistein was obvious increased while its maximal velocity (Vmax) and intrinsic clearance (CLint) values were significantly decreased by diabetic RLMs, and the Vmax and CLint values of kaempferol and isorhamnetin were notably enhanced while their Km values were markedly reduced. For the half-time (t1/2) values of four target compounds and the Km, Vmax and CLint values of quercetin, there were not statistically significant changes between normal and diabetic RLMs. The results suggest that the developed off-line 2D LC-DAD-q/TOF-MS method is an easy and accurate approach for the study of GBE biotransformation in RLMs and may provide the essential data for further pharmacological and clinical studies of GBE.

Highly sensitive quantification of key regulatory oxysterols in biological samples by LC-ESI-MS/MS

We describe a highly sensitive and specific method for the quantification of key regulatory oxysterols in biological samples. This method is based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry (LC-MS/MS). After alkaline hydrolysis of human serum (5 microl) or rat liver microsomes (1 mg protein), oxysterols were extracted, derivatized into picolinyl esters, and analyzed by LC-MS/MS using the electrospray ionization mode. The detection limits of the picolinyl esters of 4beta-hydroxycholesterol, 7alpha-hydroxycholesterol, 22R-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, and 24S,25-epoxycholesterol were 2-10 fg (5-25 amol) on-column (signal-to-noise ratio = 3). Reproducibilities and recoveries of these oxysterols were validated according to one-way layout and polynomial equation, respectively. The variances between sample preparations and between measurements by this method were calculated to be 1.8% to 12.7% and 2.9% to 11.9%, respectively. The recovery experiments were performed using rat liver microsomes spiked with 0.05 ng to 12 ng of oxysterols, and recoveries of the oxysterols ranged from 86.7% to 107.3%, with a mean recovery of 100.6%. This method provides reproducible and reliable results for the quantification of oxysterols in small amounts of biological samples.

Degradation of stearoyl-coenzyme A desaturase: endoproteolytic cleavage by an integral membrane protease.

Stearoyl-coenzyme A desaturase (SCD) is a key regulator of membrane fluidity, turns over rapidly, and represents a prototype for selective degradation of resident proteins of the endoplasmic reticulum. Using detergent-solubilized, desaturase-induced rat liver microsomes we have characterized a protease that degrades SCD. Degradation of SCD in vitro is highly selective, has a half-life of 3-4 h, and generates a 20-kDa C-terminal fragment of SCD. The N terminus of the 20-kDa fragment was identified as Phe177. The cleavage site occurs in a conserved 12-residue hydrophobic segment of SCD flanked by clusters of basic residues. The SCD protease remains associated with microsomal membranes after peripheral and lumenal proteins have been selectively removed. SCD protease is present in normal rat liver microsomes and cleaves purified SCD. We conclude that rapid turnover of SCD involves a constitutive microsomal protease with properties of an integral membrane protein.

Hematotoxicity on human bone marrow- and umbilical cord blood-derived progenitor cells and in vitro therapeutic index of methoxymorpholinyldoxorubicin and its metabolites.

MMDX [3'-deamino-3'-[2(S)-methoxy-4-morpholinyl] doxorubicin], an anthracycline derivative active in vitro and in vivo against multidrug-resistant tumors, is currently under investigation in phase I clinical trials. In vivo it is metabolically activated, resulting in more cytotoxic compounds. We determined in vitro the toxic concentration of a 1-h period of exposure to doxorubicin (DX), MMDX, and bioactivated MMDX on hematopoietic progenitors and tumor cell lines. DX and MMDX were tested on both bone marrow- (BM) and cord blood (hCB)-derived clonogenic cells, whereas the metabolites were tested on hCB only. All substances were tested on seven tumor cell lines. BM cells proved to be twice as sensitive as hCB cells to cytotoxics, and MMDX was twice as toxic as DX against hCB cells; MMDX activated with normal rat-liver microsomes and with dexamethasone-induced rat microsomes were, respectively, 70 and 230 times more toxic than MMDX. A comparison of the cytotoxic concentrations on hematopoietic progenitors and tumor cells, revealed that DX and MMDX had 5-fold stronger activity on tumor cell lines than on granulocyte/macrophage colony-forming cells (GM-CFCs), whereas bioactivated MMDX showed comparable cytotoxicity against tumor cells and hematopoietic progenitors. MMDX metabolites are very potent but display a lower degree of tumor selectivity than MMDX. Strategies to reduce MMDX metabolization should be developed to optimize the therapeutic index of this new anthracycline.

6-1) ALTERATION OF AFLATOXIN B<SUB>1</SUB> METABOLIC PROFILES AND REDUCTION OF AFLATOXIN B<SUB>1</SUB> MUTAGENICITY BY HEPATIC MICROSOMES OF RATS FED BUTYLATED HYDROXYANISOLE

Effect of administering butylated hydroxyanisole (BHA) on the metabolism of aflatoxin B1 (AFB1) and production of mutagenic metabolites have been compared with those of phenobarbital (PB) and 3-methylcholanthrene (MC) administration in rat liver microsomes. Male Sprague-Dawley rats were treated with these inducers and liver microsomes were isolated. These microsomes were used to metabolize AFB1 and to produce mutagenic metabolites. Results showed that normal rat liver were able to metabolize AFB1 quite actively and produced large amounts of AFB-8,9-epoxide (appearing as the AFB-8,9-dihydrodiol-Tris complex). Upon incubations of normal rat liver microsomes with increasing concentrations of AFB1, a steep dose-related increases of mutagenicity was observed in the Ames test. The PB-microsomes had an increased ability to metabolize AFB1 and particularly the rate for the production of the weakly mutagenic AFQ1 metabolite was markedly increased. Conversely, PB-microsomes had a moderate decrease in its ability to form the strongly mutagenic of AFB-8,9-epoxide metabolite. However, the ability of PB-microsomes to form mutagenic metabolites from AFB1 was somewhat greater than that of the control-microsomes. The MC-microsomes had an increased ability to metabolize AFB1 also. However, instead of the weakly mutagenic AFQ1 metabolite seen with the PB-microsomes, large amounts of the strongly mutagenic AFM1 metabolite was formed. Although AFM1 is not known to be a direct mutagen, it was highly mutagenic upon activation with microsomes. The very steep dose-related increases of mutagenicity and appearance of bacterial toxicity at relatively lower doses of AFB1 may have been caused by the secondary metabolic activation. The ability of BHA-microsomes to metabolize AFB1 was decreased. Among the metabolites produced by the BHA-microsomes, the non-mutagenic AFB2a was formed in significantly increased amounts but the toxic AFB-8,9-epoxide was produced only in much reduced amounts. The AFB2a was not mutagenic even after metabolic activation with microsomes. When increasing concentrations of AFB1 was incubated with BHA-microsomes, a very mild dose-related increases of mutagenicity was observed and the occurence of toxic effects on bacterial growth appeared only at high doses of AFB1. This may have been due both to the reduced rate of overall AFB1 metabolism and to the decreased formation of the highly mutagenic AFB-8,9-epoxide but an increased formation of the non-mutagenic AFB2a metabolite by the BHA-microsomes.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of various steroids on the biosynthesis of arachidonic acid in isolated hepatocytes and HTC cells.

The effect of various steroids on the incorporation and desaturation of eicosa-8,11,14-trienoic acid in normal hepatocytes and HTC cells was investigated. After 3 hr incubation with 11-deoxycorticosterone, both kinds of cells showed an increase in the incorporation of eicosatrienoic acid. In contrast, progesterone, cortexolone, 17-beta-estradiol, testosterone, estriol, aldosterone, corticosterone, dexamethasone, dehydroepiandrosterone, 11-beta-hydroxyandrosterone, 11-ketoaetiocholanolone, epiaetiocholanolone and 5-beta-pregnane-3 alpha,20 alpha-diol, provoked no significant changes in the uptake of the exogenous acid. Of all the steroids tested, only 11-deoxycorticosterone, dexamethasone and 17-beta-estradiol evoked a significant inhibition on the arachidonate biosynthesis in both kinds of cells. Testosterone, estriol, aldosterone and corticosterone provoked a significant inhibition of delta 5-desaturase in HTC cells. In dexamethasone, this effect was dose-dependent (0 to 10(-4) M). Simultaneous incubation with 17-beta-estradiol or 11-deoxycorticosterone with dexamethasone led to an extent of inhibition on arachidonate biosynthesis that did not surpass the effect of each drug. Pretreatment of isolated hepatocytes with the antiglucocorticoid, cortexolone, prevented the dexamethasone-induced inhibition of arachidonate biosynthesis. Normal rat liver microsomes preincubated in vitro with dexamethasone, 11-deoxycorticosterone, 17-beta-estradiol, corticosterone or estriol (10(-6) or 10(-4) M concentration), showed no significant changes in the delta 5-desaturase activity. The results obtained suggest that the effect of the steroids on arachidonic acid biosynthesis in normal hepatocytes and HTC cells requires receptor occupancy and probably is mediated through a common biochemical mechanism.

A comparative fluorescence polarization study of cis-parinaroyl-phosphatidylcholine and diphenylhexatriene in membranes containing different amounts of cholesterol

The steady state fluorescence anisotropy ( r s ) of 1-acryl-2- cis parinaroyl phosphatidylcholine (PnPC) was compared with that of diphenylhexatriene (DPH) in a variety of model- and biological membrane systems. The fluorescence anisotropy of both probes responded similarly to temperature changes and variations in the acyl chain composition in phosphatidylcholine (PC) liposomes. The presence of proteins and cholesterol increased r s for both DPH and PnPC in the biological membranes as compared to the isolated polar membrane lipids. Comparison of DPH and PnPC in dipalmitoyl-PC-liposomes with and without 50 mol% cholesterol, showed at temperatures above the phase transition of pure dipalmitoyl-PC the presence of cholesterol increased the r s -value for DPH strongly, whereas the r s -value for PnPC was much less affected. In the cholesterol-rich erythrocyte membrane as well as in microsomes from Morris hepatoma 7787, which have an increased cholesterol content as compared to normal rat liver microsomes, the r s of DPH was higher than that of PnPC. No large differences between the r s -values of both probes were evident in the normal cholesterol-poor rat liver microsomes. These effects are discussed in terms of structural differences between the probes and variation of cholesterol content. Alterations in the fatty acid composition of PC present in human erythrocyte membranes were introduced with the aid of a PC-specific transfer protein. Fluorescence anisotropy values of both probes hardly changed upon enrichment of the red cell membrane with either dipalmitoyl PC or 1-palmitoyl-2-arachidonyl PC.

Hydroxymethylglutaryl CoA reductase and the modulation of microsomal cholesterol content by the nonspecific lipid transfer protein.

The influence of membrane cholesterol content on 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase, EC 1.1.1.34) in rat liver microsomes was investigated. Microsomes were enriched in cholesterol by incubation with egg phosphatidylcholine-cholesterol vesicles and the nonspecific lipid transfer protein from rat liver. By this method, the microsomal cholesterol content was 2.5-fold enhanced up to final concentrations of 140 nmol cholesterol per mg microsomal protein. In another experiment, microsomes isolated from rats fed a cholesterol-rich diet were depleted of cholesterol by incubation with egg phosphatidylcholine vesicles and the transfer protein. Both cholesterol enrichment and depletion had virtually no effect on the microsomal HMG-CoA reductase activity. In another set of experiments, normal rat liver microsomes were incubated with human serum, resulting in a rise of microsomal cholesterol content. This was reflected in an increase of acyl-CoA:cholesterol acyltransferase activity but failed to have an effect on HMG-CoA reductase.

Metabolic activation of carbon tetrachloride: induction of cytochrome P-450 with phenobarbital or 3-methylcholanthrene and its effect on covalent binding

Anaerobic incubation of [ 14C]carbon tetrachloride with normal rat liver microsomes and microsomes from rats treated with the inducers phenobartital (PB) and 3-methylcholanthrene (MC) reveals distinct differences in metabolic activation. While the increase in CO-binding pigment is comparable for both inducers, metabolism of CCl 4 is enhanced only by PB-induction; MC-induced microsomes are equivalent to microsomes from untreated animals. Sodium dodecyl sulphate (SDS)-electrophoresis of microsomal proteins confirms the expected increase at 52 000 daltons (cytochrome P-450 PB) on PB-induction, at 56 000 daltons (cytochrome P-450 MC) on MC-induction; after anaerobic incubation with [ 14C]CCl 4 the electrophoretic pattern is largely unchanged. The highly reactive radical intermediates of CCl 4-metabolism should attack the closest possible partner. Most of protein-bound radioactivity is located in the mass range between 47 000 and 54 000 daltons, indicating that cytochrome P-450 PB is the isoenzyme mainly responsible for CCl 4-activation; cytochrome P-450 MC plays virtually no role in metabolic activation. The direct participation of NADPH-cytochrome P-450 reductase appears unlikely, since the specific binding to proteins in the corresponding mass range is not elevated. A significant percentage of label is attached to proteins at 120 000 daltons and above, presumably oligomers of cytochrome P-450 apoprotein.

Stearoyl-coenzyme A desaturase activity in the mammary gland and liver of lactating rats.

Stearoyl-CoA desaturase activity in microsomes from lactating rat mammary gland is very low (0.05-0.15 nmol/min/mg of protein) regardless of lactating time. In such microsomes, reductase activities and content of cytochrome b5 are several-fold lower than in normal rat liver microsomes. Preincubation of the mammary microsomes with purified terminal desaturase gives a 55-fold stimulation of stearoyl-CoA desaturase activity, whereas preincubation with cytochrome b5 has no effect. However, preincubation of mammary microsomes with both cytochrome b5 and terminal desaturase results in a 200-fold stimulation of overall desaturation. These observations suggest that negligible stearoyl-CoA desaturase activity in lactating rat mammary microsomes is due to a cytochrome b5 content and the absence of terminal enzyme. The hepatic stearoyl-CoA desaturase activity increases 9-fold during lactation. There is little or no change in the NADH-cytochrome c reductase activity or in the concentrations of cytochrome b5 during this period, but the activity of the terminal desaturase increases with the increase of overall desaturation. These results suggest that liver is one of the more important sources of oleic acid for milk triglycerides.

42] Fluorometric and chromatographic methods for measuring microsomal biphenyl hydroxylation

Publisher Summary This chapter describes the fluorometric and chromatographic methods for measuring microsomal biphenyl hydroxylation. This method, is the most convenient when biphenyl is used as a routine substrate for the monooxygenase. Biphenyl (I) is a useful model substrate for investigating the cytochrome P-450 (P-448)-mediated, NADPH 2 -, and oxygen-dependent microsomal aryl hydrocarbon hydroxylase. Microsomal hydroxylation of biphenyl is terminated with acid, which also ensures that the hydroxylated metabolites are not ionized and are extracted into n-heptane. The hydroxybiphenyls are then reextracted into alkali, leaving unreacted biphenyl in the organic phase. The fluorescence of the metabolites is measured at pH 5-6, achieved by adding succinic acid to the alkaline extract. Two compounds, 2-hydroxybiphenyl (II) and 4-hydroxybiphenyl (III) constitute more than 90% of the total biphenyl metabolites with liver microsomes of normal rats or hamsters. Small percentages of dihydroxybiphenyls are formed with rodent liver microsomes, and although 3-hydroxybiphenyl is isolated as a metabolite from normal rabbit urine, it is not formed in significant quantities with normal rodent liver microsomes.

Selective bioreduction of nitroxides by rat liver microsomes

It was found that control rat liver microsomes will not reduce 2-ethyl-2,4,4-trimethyl-3-oxazolidinyloxy (OXAN) even though the reduction potential for OXAN is identical to that for 2,2,6,6-tetramethylpiperidinoxyl (TEMPO) and di-tert-butyl nitroxide (DTBN) which are reduced by these microsomes. It is suggested that the conformation of OXAN prevents these control enzymes from reducing OXAN. Administration of phenobarbital for 3 days prior to sacrifice, results in a rat liver microsomal preparation which will reduce OXAN and is inhibited by very low concentrations of SKF-525A. On the other hand, rats induced with 3-methylcholanthrene did not reduce OXAN. These results suggest that phenobarbital induces the synthesis of a form of cytochrome P-450 which is not present in either normal rat liver microsomes or in 3-methylcholanthrene-induced microsomes.

Controlled trial of continuous positive airway pressure given by face mask for hyaline membrane disease--killer Pascals

The cytochrome P-450-dependent microsomal metabolism of amphetamine and N-substituted amphetamines can result in the formation of a complex which absorbs maximally at 455 nm. In normal rat liver microsomes, this complex formation is either very slow or nonexistent, depending upon the amphetamine substrate used. Phenobarbital pretreatment of rats, which increases cytochrome P-450 concentration up to 3-fold and NADPH-cytochrome <i>c</i> reductase activity up to 2-fold, dramatically enhances the rate of formation of the complex for all substrates examined, up to 30-fold for norbenzphetamine and 10-fold for <i>d</i>-amphetamine. Pretreatment of rats with 3-methylcholanthrene, although producing a 2-fold increase in total cytochrome P-450 species, decreases the rate of complex formation below that observed in normal animals. Linear regression analysis of the results from phenobarbital-pretreated rats suggests that 455-nm complex formation from amphetamines is a uniquely sensitive indication of induction by this class of inducer.

Calorimetric study of microsomal membrane.

Differential scanning calorimetric measurements of normal rat liver microsomes reveal a single thermal transition at about 50 degrees. This transition is assigned to irreversible prorein denaturation. There is no evidence for a reversible lipid phase transition at any temperature above 0 degrees, indicating that the microsomal membrane is in teh fluid state under these conditions. Rats fed a fat-free diet which increases the degree of saturation of fatty acids in the membrane lipids do produce microsomal membranes exhibiting a reversible lipid phase transition. The NADH=dependent and NADPH-dependent enzymatic reductions of cytochrome c show linear Arrhenius behavior in the normal rat liver microsomes but reveal discontinuities and breaks in the Arrhenius plots at approximately the calorimetrically determined phase transition temperatures in microsomes from rats fed the fat-free diet. Hence, the fluidity of cell membranes can be altered by diet with consequent effects on membrane-supported functions. The data further show that the lipid organization of the membrane is not independent of the protein component and supports models of membrane structure where a separate class of lipids forms a boundary between the bulk phase and the proteins.

Concomitant elevations in serum sialytransferase activity and sialic acid content in rats with metastasizing mammary tumors.

Rats with transplantable spontaneously metastasizing mammary tumors have elevated levels of both serum sialoglycoconjugate and serum sialytransferase activity compared with normal female rats or rats with various nonmetastasizing mammary tumors. A direct relationship was observed between the amount of serum protein-bound sialic acid and serum sialyltransferase activity in all rats studied. Serum sialyltransferase activity in rats with a representative metastasizing mammary tumor, SMT-2A, was also correlated with tumor age. Microsomes prepared from the SMT-2A tumor have a sixfold higher sialyltransferase activity than do microsomes prepared from the nonmetastasizing mammary tumor MT-W9B. Normal rat liver microsomes have the same level of activity as microsomes prepared from livers of animals with either SMT-2A or MT-W9B tumors. The data indicate that spontaneously metastasizing mammary tumor cells have an increased production and release, perhaps through cell surface shedding, of a sialyltransferase. It is suggested that this sialyltransferase may increase the serum half-life of certain tumor-specific circulating glycoconjugates by increasing the content of protein-bound sialic acid and may thereby play a role in the immune escape mechanism of metastasizing tumor cells.

Cytochrome P-450 in the activation and inactivation of carcinogens.

The capacity of isolate mouse liver microsomes to alter the mutagenicity for bacteria of the primary carcinogen N-methyl-N'-nitro-N-nitrosoguanisine (MNNG) and the secondary one dimethylnitrosamine (DMN) was studied. Microsomal activation of DMN and inactivation of MNNG were decreased by protein- and protein-cholinedeficient diets and were increased by pretreatment with microsomal enzyme inducers. The decrease and increase paralleled the content of cytochrome P-450 present in the different microsomal preparations. With human liver microsomes of differing cytochrome P-450 contents similar correlation was obtained, whereas normal rat liver microsomes did not activate or inactivate DMN or MNNG. Oxidative demethylation of DMN by mouse liver microsomes and the activation of DMN to a mutagen followed similar kinetics. Both reactions were inhibited by carbon monoxide and the inhibition was maximally reversed by monochromatic light at 450 nm. These observations indicate that at least some carcinogens are activated or inactivated by the unspecific cytochrome P-450 dependent enzyme system, suggesting that the extent of this biotransformation may be one factor influencing human carcinogenesis.

Effect of cannabis on pentobarbital-induced sleeping time and pentobarbital metabolism in the rat

The effects of two cannabis extracts with different cannabinoid compositions, as well of as pure Δ 1-tetrahydrocannabinol (THC), cannabinol (CBN) and cannabidiol (CBD), on pentobarbital metabolism were studied in the rat. Extract I, with high proportions of CBN and CBD relative to THC, when given by gavage 21·5, 40 or 63 hr before pentobarbital (30 mg/kg, i.p.), prolonged the sleeping time by 53, 42 and 21 per cent respectively. This effect was paralleled by decreases in the rate of disappearance of [ 14C]pentobarbital from the blood, and of pentobarbital metabolism by liver microsomal preparations in vitro. Extract II, with low relative proportions of CBN and CBD, did not have any significant effect on penobarbital metabolism or sleeping time. CBD alone, in the same dose as that given in Extract I, had very similar effects, while a dose of CBD equivalent to that given in Extract II had no effect. THC, CBN and CBD added to normal rat liver microsomes in vitro inhibited pentobarbital metabolism competitively, CBD being a much more potent inhibitor than THC and CBN. The CBD content may, therefore, be a significant factor in interactions between marijuana and other drugs.

Studies on the Function of Cell Membrane 7th Report: Influence of CCl4 Administration on Solubilization of the Enzymes in Electron Transport System in Rat Liver Plasma Membrane and Microsome

In liver microsomes of normal rats, both NADH-ferricyanide dehydrogenase and NADH-cytochrome c reductase were almost completely solubilized by addition of 0.5% DOC. Solubilization patterns were little affected by CCl4 injection. In membranes, solubilization of dehydrogenase and reductase was less than that in microsomes. CCl4 administration lowered the solubility of the dehydrogenase in membranes and increased three-fold the ratio of the reductase to the dehydrogenase remaining in the non-solubilized fraction. The reductase in membranes from CCl4-treated rat liver was found to be more resistant to solubilization by DOC. The possibility of accumulation of NADH-cytochrome c reductase in plasma membranes after CCl4 administration was discussed.

The conversion of pyrrolizidine alkaloids to N-oxides and to dihydropyrrolizine derivatives by rat-liver microsomes in vitro

The enzymatic conversion of pyrrolizidine alkaloids to dihydropyrrolizine derivatives (‘pyrrole derivatives’) and to N-oxides has been investigated. In rat liver both reactions were catalysed by an enzyme present in the microsomal fraction, typical of the mixed-function oxidases. Thus the conversion of alkaloids to pyrrole derivatives and to N-oxides required oxygen and reduced NADP and was activated by Mg 2+, Mn 2+ or Ca 2+ at concentrations of 5 · 10 −3 M. The liver microsomal system was less active in the female rat than in the male and was inhibited by carbon monoxide and by SKF 525A. The conversion of retrorsine to both pyrrolic metabolites and to N-oxides was significantly induced by pretreatment with phenobarbitone or DDT, but the induction of pyrrole derivatives was greater than that of N-oxide. The conversion of retrorsine to pyrrole derivatives was lower in liver microsomal fractions from rats which were starved or fed on protein-free diets. Differences between the rates of formation of pyrrole derivatives and N-oxides followed the inclusion of nicotinamide in the incubation mixtures containing microsomes from untreated rats, or substitution of a carbon monoxide-oxygen mixture for air during the incubation period. The K m values towards the formation of pyrrolic derivatives and N-oxides from retrorsine from normal rat-liver microsomes were similar. No conclusive evidence was found to establish whether N-oxides were intermediate in the formation of pyrrole derivatives. However, pyrrole derivatives could not be detected when N-oxides were added to the hepatic microsomal system in vitro. Rat-liver microsomal enzymes converted a number of alkaloids having either 1,2-dehydropyrrolizidine or saturated pyrrolizidine ring structures to pyrrole derivatives. The pyrrole derivatives from these two groups of alkaloids had different chemical properties. Only alkaloids in the first group were hepatotoxic, and in general the more toxic alkaloids in this group were those from which the greatest amounts of pyrrole derivatives were produced in vitro, although there were exceptions. The enzymatic conversion of retrorsine or monocrotaline to pyrrolic derivatives could not be demonstrated in lung microsomal fractions.