Mg Of Microsomal Protein Research Articles

A novel system to determine activity of individual uridine 5′-diphospho-glucuronosyltransferase (UGT) isoforms: Recombinant UGT-beads

Previous work demonstrated that human liver microsomes (HLM) can spontaneously bind to silica-coated magnetizable beads (HLM-beads) and that these HLM-beads retain UGT activity. However, the contributions of individual UGT isoforms is not directly assessable in this system except through use of model inhibitors. Thus, a preparation wherein rUGT microsomes bound to these same beads to form rUGT-beads of individual UGT isoforms would provide a novel system for measuring the contribution of individual UGT isoforms in a direct manner. To this end, the enzyme activities and kinetic parameter estimates of various rUGT isoforms in rUGT-beads were investigated, as well as the impact of fatty acids (FAs) on enzyme activity. The catalytic efficiencies (Vmax/Km) of the tested rUGTs were 2- to 7-fold higher in rUGT-beads compared to rUGT microsomes, except for rUGT1A6, where Vmax is the maximum product formation rate normalized to mg of microsomal protein (pmol/min/mg protein). Interestingly, in contrast to traditional rUGT preparations, the sequestration of UGT-inhibitory fatty acids (FA) using bovine serum albumin (BSA) did not alter the catalytic efficiency (Vmax/Km) of the rUGTs in rUGT-beads. Moreover, the increase in catalytic efficiency of rUGT-beads over rUGT microsomes was similar to increases in catalytic efficiency noted with rUGT microsomes (not bound to beads) incubated with BSA, suggesting the beads in some way altered the potential for FAs to inhibit activity. The rUGT-bead system may serve as a useful albumin-free tool to determine kinetic constants for UGT substrates, particularly those that exhibit high binding to albumin.

Effect of alpha-lipoic acid on rat kidney and liver microsomes subjected to oxidative stress induced by ascorbate-Fe++

Metabolic changes during kidney and liver disease can induce increased production of oxygen radicals that play an important role in the progression of kidney and liver damage and in the appearance of important comorbidities. In the study reported here, the effect of alpha-lipoic acid (ALA) on the chemiluminescence (CL) and TBARS determination of microsomes isolated from rat kidney and liver was analyzed. Oxidative stress in microsomes was induced by subjecting samples (1 mg of protein) to an ascorbate-Fe++-dependent (0.4 mM)-Fe++ (2.15 μM) prooxidant system at 37 °C for 120 min. Oxidative damage was quantified by two methods, TBARS and CL: CL was determined using a Packard 1900 TR liquid scintillation counter (Meriden CT, USA). CL was expressed as cpm (counts per minute) was read every 10 minutes to establish the course of peroxidation as a function of time. Likewise, the total value of cpm (sum of the readings) was used to compare the inhibitory effect of ALA using different concentrations corresponding to 0.05; 0.15 and 0.25 μg of ALA per mg of microsomal protein. Controls were run simultaneously without the addition of ascorbate-Fe++. It was observed that the CL was lower in the kidney and liver microsomes obtained from the ALA group than in the control group (without ALA). In addition, -ALA was found to be concentration-dependent (0.05, 0.15 and 0.25 μg of ALA per mg of solution) reduced in CL, measured as total cpm. After that it was done the determination of lipid peroxidation products (Thiobarbituric Acid Reactive Substances—TBARS). TBARS were measured in the microsomal samples. Lipoperoxidation inhibition depended on the concentration of ALA in the incubation mixture. The results show that for all the concentrations tested, a protective effect on the induced oxidative damage was found. CL and TBARS level analyzes indicate that ALA may act as an antioxidant protecting rat kidney and liver microsomes from lipoperoxidative damage.

Abundance and Associated Variations of Cytochrome P450 Drug-Metabolizing Enzymes in the Liver of East Asian Adults: A Meta-Analysis.

Cytochrome P450 (CYP) enzymes are one of the main sources of variability in drug metabolic clearance. Information on their abundance levels is therefore crucial to optimize scaling factors for in vitro-in vivo extrapolation (IVIVE) to predict metabolic clearance. This study aims to quantify the abundance data of hepatic drug-metabolizing CYP enzymes in East Asian subjects reported from various sources in the literature using meta-analysis. We conducted a meta-analysis on the abundance of drug-metabolizingCYP enzymes in the liver of East Asian adults. Eligible reports were identified based on predefined criteria-(1) individual liver microsomal samples, and (2) absolute protein abundance data from normal tissues of East Asian adult subjects. Subgroup and sensitivity analyses were also performed. Among the 11 CYP isoforms analyzed in East Asian subjects, CYP3A5 and CYP3A4 had the highest protein levels. In particular, the number of studies and the liver sample used to quantify the abundance of CYP3A4 were the largest. Of the isoforms involved, CYP2J2 and CYP2B6 had the lowest abundance level, i.e., <5 pmol/ mg of microsomal protein. For enzymes with abundance values available in both Chinese and Japanese subjects (CYP1A2, CYP2C9, CYP3A4, and CYP3A5), the abundance level of each CYP isoform appeared to be higher in Chinese than in Japanese subjects. The most distinct difference was observed in CYP3A5 abundance. The current meta-analysis shows that the abundance levels of CYP enzymes appear to vary greatly among different East Asian individuals who have similar ethnic backgrounds and food habits. The pooled data of CYP abundance can be used as preliminary reference values along with the associated variations for the projections of pharmacokinetics through physiologically based pharmacokinetic (PBPK)approaches.

The impact of variation in scaling factors on the estimation of internal dose metrics: a case study using bromodichloromethane (BDCM)

A rate for hepatic metabolism (Vmax) determined in vitro must be scaled for in vivo use in a physiologically based pharmacokinetic (PBPK) model. This requires the use of scaling factors such as mg of microsomal protein per gram of liver (MPPGL) and liver mass (FVL). Variation in MPPGL and FVL impacts variation in Vmax, and hence PBPK model-derived estimates of internal dose used in dose response analysis. The impacts of adult human variation in MPPGL and FVL on estimates of internal dose were assessed using a human PBPK model for bromodichloromethane (BDCM), a water disinfection byproduct, for multiple internal dose metrics for two exposure scenarios (single 0.25 liter drink of water or 10 min shower) under plausible (5 μg/L) and high level (20 μg/L) water concentrations. For both concentrations, all internal dose metrics were changed less than 5% for the showering scenario (combined inhalation and dermal exposure). In contrast, a 27-fold variation in area under the curve (AUC) for BDCM in venous blood was observed at both oral exposure concentrations, whereas total amount of BDCM metabolized in liver was relatively unchanged. This analysis demonstrates that variability in the scaling factors used for in vitro to in vivo extrapolation (IVIVE) for metabolic rate parameters can have a significant route-dependent impact on estimates of internal dose under environmentally relevant exposure scenarios. This indicates the need to evaluate both uncertainty and variability for scaling factors used for IVIVE.

Functional characterization of ent-kaurene oxidase, MtKO, from Montanoa tomentosa (Zoapatle)

Kaurene oxidases are P450 proteins that catalyze the conversion of ent-kaurene into kaurenoic acid, the final enzymatic product with a wide range of pharmacological properties. We describe the functional characterization of an ent-kaurene oxidase (EC 1.14.13.78) isolated from Montanoa tomentosa after heterologous expression in Saccharomyces cerevisiae, as well as the detection of the enzymatic activity in the plant itself. In the presence of NADPH and FAD, the microsomal fraction from transformed INVSc1 cells, ent-kaurene produced ent-kaurenoic acid, which was confirmed by GC-MS analyses. The kinetic parameters for ent-kaurene using 0.5 mg of microsomal protein were Km app= 80.63±1.2 μM and V max app= 31.80±1.8 μmol-1mg-1h-1. Optimal temperature and pH were 30°C and 7.6, respectively. Similar kinetic parameters were observed when leaf microsomes from M. tomentosa were assayed under the same conditions as for yeast microsomes. This result strongly suggests that ent-kaurene oxidase activity is present in leaf microsomes. The enzymatic activity was competitively inhibited by paclobutrazol, with IC50=43.9 μM, implying that MtKO is resistant to inhibition by azolic-type compounds. This study confirmed the biochemical detection of ent-kaurene oxidase activity in the plant, and the heterologous functionality of a cDNA with an ent-kaurene oxidase identity from M. tomentosa (zoapatle).

Overexpression of CYP3A5 attenuates inducibility and activity of CYP3A4 in HepG2 cells.

There have been conflicting reports regarding the catalytic role of cytochrome P450 (CYP)3A5, which range from deeming it irrelevant to suggesting it is equally as important as CYP3A4, the most potent and abundant catalytic cytochrome enzyme in the human liver. This was partially attributed to the fact that CYP3A5 is highly polymorphic. However the importance of other underlying mechanisms remain unclear. The aim of the present study was to investigate the interaction between these enzymes. A human HepG2 hepatocellular line stably overexpressing CYP3A5 was constructed. The results suggested that CYP3A5 does not affect CYP3A4 expression directly. However, overexpression of CYP3A5 attenuated the inducibility of CYP3A4 in response to dexamethasone. A luciferase reporter assay indicated that this attenuation was due to a decrease in CYP3A4 promoter activity. Furthermore, a pharmacokinetic assay using quinidine and amlodipine showed that CYP3A4 enzyme activity per mg of microsomal protein was also decreased in the group overexpressing CYP3A5 compared with the dexamethasone‑induced control group. In conclusion, the current study demonstrated that CYP3A5 may affect CYP3A4 at the transcriptional level and may thus modify CYP3A4 expression and activity in the presence of substrates and inducers. The results indicate that CYPs may interact with each other under certain conditions and that this interaction may be a novel mechanism by which drug‑drug interactions are mediated.

Heme oxygenase activity (HOA) in rat liver: Response to severe hemorrhage in an inbred rat strain

In efforts to identify cellular mechanisms involved in differences among inbred rat strains in survival time after severe hemorrhage (STaH) (Klemcke et al. Physiol Genomics 43: 758, 2011), we evaluated HOA, a cytoprotective enzyme that catalyzes conversion of cellular heme into biliverdin, free iron (Fe+2), and carbon monoxide. We adapted a HOA procedure used with purified HO (Reed et al, DMD 38: 2060, 2010) for use in tissues (liver microsomal fraction); the assay measures the appearance of Fe+2 using hemin substrate and NADPH as cofactor. HOA had an apparent Km of 7.8 μM of hemin; Vmax= 170 nM Fe+2 /mg protein/min; time period of initial velocity of 0 to 40 min; and a linearity of Fe+2 formation for 0.25 to 3 mg of microsomal protein. Dark Agouti (DA) rats (longest STaH) were assigned to one of three treatments (n=5/ treatment): non‐operated controls, catheterized (CATH) only, or CATH plus hemorrhage (49% of blood volume) 24 h post‐surgery. Rats were euthanized 30 min after handling or after the start of the hemorrhage. HOA increased 32% 24 hrs after CATH alone (4.69 vs 3.54 nmol Fe+2/mg protein/30 min; P = 0.05). HOA increased 36% further after hemorrhage to 6.37 nmol Fe+2/mg protein/30 min (P = 0.01). Hence in liver of DA rats, HOA is increased and may be exerting a cytoprotective effect after stressors such as surgery and hemorrhage. Moreover, this assay allows for an easier, more direct procedure for measuring HOA in tissues.

Arachidonic Acid-metabolizing Cytochrome P450 Enzymes Are Targets of ω-3 Fatty Acids*

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) protect against cardiovascular disease by largely unknown mechanisms. We tested the hypothesis that EPA and DHA may compete with arachidonic acid (AA) for the conversion by cytochrome P450 (CYP) enzymes, resulting in the formation of alternative, physiologically active, metabolites. Renal and hepatic microsomes, as well as various CYP isoforms, displayed equal or elevated activities when metabolizing EPA or DHA instead of AA. CYP2C/2J isoforms converting AA to epoxyeicosatrienoic acids (EETs) preferentially epoxidized the ω-3 double bond and thereby produced 17,18-epoxyeicosatetraenoic (17,18-EEQ) and 19,20-epoxydocosapentaenoic acid (19,20-EDP) from EPA and DHA. We found that these ω-3 epoxides are highly active as antiarrhythmic agents, suppressing the Ca(2+)-induced increased rate of spontaneous beating of neonatal rat cardiomyocytes, at low nanomolar concentrations. CYP4A/4F isoforms ω-hydroxylating AA were less regioselective toward EPA and DHA, catalyzing predominantly ω- and ω minus 1 hydroxylation. Rats given dietary EPA/DHA supplementation exhibited substantial replacement of AA by EPA and DHA in membrane phospholipids in plasma, heart, kidney, liver, lung, and pancreas, with less pronounced changes in the brain. The changes in fatty acids were accompanied by concomitant changes in endogenous CYP metabolite profiles (e.g. altering the EET/EEQ/EDP ratio from 87:0:13 to 27:18:55 in the heart). These results demonstrate that CYP enzymes efficiently convert EPA and DHA to novel epoxy and hydroxy metabolites that could mediate some of the beneficial cardiovascular effects of dietary ω-3 fatty acids.

Biochemical Conservation and Evolution of Germacrene A Oxidase in Asteraceae

Sesquiterpene lactones are characteristic natural products in Asteraceae, which constitutes approximately 8% of all plant species. Despite their physiological and pharmaceutical importance, the biochemistry and evolution of sesquiterpene lactones remain unexplored. Here we show that germacrene A oxidase (GAO), evolutionarily conserved in all major subfamilies of Asteraceae, catalyzes three consecutive oxidations of germacrene A to yield germacrene A acid. Furthermore, it is also capable of oxidizing non-natural substrate amorphadiene. Co-expression of lettuce GAO with germacrene synthase in engineered yeast synthesized aberrant products, costic acids and ilicic acid, in an acidic condition. However, cultivation in a neutral condition allowed the de novo synthesis of a single novel compound that was identified as germacrene A acid by gas and liquid chromatography and NMR analyses. To trace the evolutionary lineage of GAO in Asteraceae, homologous genes were further isolated from the representative species of three major subfamilies of Asteraceae (sunflower, chicory, and costus from Asteroideae, Cichorioideae, and Carduoideae, respectively) and also from the phylogenetically basal species, Barnadesia spinosa, from Barnadesioideae. The recombinant GAOs from these genes clearly showed germacrene A oxidase activities, suggesting that GAO activity is widely conserved in Asteraceae including the basal lineage. All GAOs could catalyze the three-step oxidation of non-natural substrate amorphadiene to artemisinic acid, whereas amorphadiene oxidase diverged from GAO displayed negligible activity for germacrene A oxidation. The observed amorphadiene oxidase activity in GAOs suggests that the catalytic plasticity is embedded in ancestral GAO enzymes that may contribute to the chemical and catalytic diversity in nature.

Microsomal Triglyceride Transfer Protein Enhances Cellular Cholesteryl Esterification by Relieving Product Inhibition

Cholesteryl ester synthesis by the acyl-CoA:cholesterol acyltransferase enzymes ACAT1 and ACAT2 is, in part, a cellular homeostatic mechanism to avoid toxicity associated with high free cholesterol levels. In hepatocytes and enterocytes, cholesteryl esters are secreted as part of apoB lipoproteins, the assembly of which is critically dependent on microsomal triglyceride transfer protein (MTP). Conditional genetic ablation of MTP reduces cholesteryl esters and enhances free cholesterol in the liver and intestine without diminishing ACAT1 and ACAT2 mRNA levels. As expected, increases in hepatic free cholesterol are associated with decreases in 3-hydroxy-3-methylglutaryl-CoA reductase and increases in ATP-binding cassette transporter 1 mRNA levels. Chemical inhibition of MTP also decreases esterification of cholesterol in Caco-2 and HepG2 cells. Conversely, coexpression of MTP and apoB in AC29 cells stably transfected with ACAT1 and ACAT2 increases cholesteryl ester synthesis. Liver and enterocyte microsomes from MTP-deficient animals synthesize lesser amounts of cholesteryl esters in vitro, but addition of purified MTP and low density lipoprotein corrects this deficiency. Enrichment of microsomes with cholesteryl esters also inhibits cholesterol ester synthesis. Thus, MTP enhances cellular cholesterol esterification by removing cholesteryl esters from their site of synthesis and depositing them into nascent apoB lipoproteins. Therefore, MTP plays a novel role in regulating cholesteryl ester biosynthesis in cells that produce lipoproteins. We speculate that non-lipoprotein-producing cells may use different mechanisms to alleviate product inhibition and modulate cholesteryl ester biosynthesis.

Lipopolysaccharide Induces CYP2E1 in Astrocytes through MAP Kinase Kinase-3 and C/EBPβ and -δ

Cytochrome P450 2E1 (CYP2E1) is highly inducible in a subset of astrocytes in vivo following ischemic or mechanical injury and in vitro by lipopolysaccharide (LPS) or interleukin-1beta. We have studied the mechanism of induction, and found that transcriptional activation of CYP2E1 occurred within 3 h, and CYP2E1 dependent catalytic activity was induced more than 4-fold within 5 h. The induction was sensitive to several tyrosine kinase inhibitors, and was further modulated by inhibitors of p38 MAP kinase. MAP kinase kinase-3 (MKK3) was phosphorylated in response to LPS, and expression of constitutively active MKK3, but not the MAP kinase kinases MEKK1 or MKK1, activated CYP2E1. Transcriptional activation was mediated through a C/EBPbeta and -delta binding element situated at -486/-474, and appeared to involve activation of prebound factors as well as recruitment of newly synthesized C/EBPbeta and -delta. It is thus suggested that LPS induces MKK3 activation in astrocytes, which in turn stimulates a C/EBPbeta and -delta binding element to mediate transcriptional activation of CYP2E1.

Improved expression and characterization of Ca 2+-ATPase and phospholamban in High-Five cells

The Ca 2+-ATPase accounts for the majority of Ca 2+ removed from the cytoplasm during cardiac muscle relaxation. The Ca 2+-ATPase is regulated by phospholamban, a 52 amino acid phosphoprotein, which inhibits Ca 2+-ATPase activity by decreasing the apparent affinity of the ATPase for Ca 2+. To study the physical mechanism of Ca 2+-ATPase regulation by phospholamban using spectroscopic and kinetic experiments, large amounts of both proteins are required. Therefore, we developed a Ca 2+-ATPase and phospholamban preparation based on the baculovirus-insect cell expression system using High-Five insect cells to produce large amounts of microsomal vesicles that contain either Ca 2+-ATPase expressed alone or Ca 2+-ATPase co-expressed with phospholamban. The expressed proteins were characterized using immunofluorescence spectroscopy, Ca 2+-ATPase activity assays, Ca 2+ uptake and efflux assays, and Western blotting. Our purification method yields 140 mg of microsomal protein per liter of infection (1.7 × 10 9 cells), and the Ca 2+-ATPase and phospholamban account for 16 and 1.4%, respectively, of the total microsomal protein by weight, yielding a phospholamban:Ca 2+-ATPase ratio of 1.6:1, similar to that observed in native cardiac SR vesicles. The enzymatic properties of the expressed Ca 2+-ATPase are also similar to those observed in native cardiac SR vesicles, and when co-expressed with phospholamban, the Ca 2+-ATPase is functionally coupled to phospholamban similar to that observed in cardiac SR vesicles.

Polymorphic hydroxylation of perhexiline in vitro.

The aims of this study were to examine the in vitro enzyme kinetics and CYP isoform selectivity of perhexiline monohydroxylation using human liver microsomes. Conversion of rac-perhexiline to monohydroxyperhexiline by human liver microsomes was assessed using a high-performance liquid chromatography assay with precolumn derivatization to measure the formation rate of the product. Isoform selective inhibitors were used to define the CYP isoform profile of perhexiline monohydroxylation. The rate of perhexiline monohydroxylation with microsomes from 20 livers varied 50-fold. The activity in 18 phenotypic perhexiline extensive metabolizer (PEM) livers varied about five-fold. The apparent Km was 3.3 +/- 1.5 micro m, the Vmax was 9.1 +/- 3.1 pmol min-1 mg-1 microsomal protein and the in vitro intrinsic clearance (Vmax/Km) was 2.9 +/- 0.5 micro l min-1 mg-1 microsomal protein in the extensive metabolizer livers. The corresponding values in the poor metabolizer livers were: apparent Km 124 +/- 141 micro m; Vmax 1.4 +/- 0.6 pmol min-1 mg-1 microsomal protein; and intrinsic clearance 0.026 micro l min-1 mg-1 microsomal protein. Quinidine almost completely inhibited perhexiline monohydroxylation activity, but inhibitors selective for other CYP isoforms had little effect. Perhexiline monohydroxylation is almost exclusively catalysed by CYP2D6 with activities being about 100-fold lower in CYP2D6 poor metabolizers than in extensive metabolizers. The in vitro data predict the in vivo saturable metabolism and pharmacogenetics of perhexiline.

Amiodarone N‐Deethylation by CYP2C8 and its Variants, CYP2C8*3 and CYP2C8 P404A

Amiodarone is a potent Class III antiarrhythmic drug. The N-deethylation of amiodarone to desethylamiodarone is known to be catalyzed by cytochrome P450 (CYP) 2C8. In the present study, amiodarone N-deethylation by the CYP2C8s, CYP2C8*1 (wild-type), CYP2C8*3, and CYP2C8 P404A (Pro404Ala substitution in exon 8), was investigated by their transient expression in Hep G2 cells. The expression levels of CYP2C8*1 and CYP2C8*3 were similar, whereas the level of CYP2C8 P404A was 55.6% of that of CYP2C8*1. The kinetic parameters of amiodarone N-deethylation were obtained by means of Lineweaver-Burk analysis. The intrinsic clearance (Vmax/Km, per mg of microsomal protein) of amiodarone by CYP2C8 P404A but not CYP2C8*3 was significantly (48.7%) less than that of CYP2C8*1. These results suggest that CYP2C8 P404A but not CYP2C8*3 is less effective in the N-deethylation of amiodarone.

Oxidative catabolism of alpha-tocopherol in rat liver microsomes.

The goal of this study was to clarify the mechanism responsible for the catabolism of alpha-tocopherol. The vitamin, bound to albumin, was incubated with rat liver microsomes and appeared to be broken down. Optimal production of the metabolite was obtained when 1 mg of microsomal protein was incubated with 36 microM of alpha-tocopherol in the presence of 1.5 mM of NADPH. Chromatographic and mass spectrometric analyses of the metabolite led to the conclusion that it consists of an omega-acid with an opened chroman ring, although we could not perform nuclear magnetic resonance analysis to confirm this. Our data show that alpha-tocopherol is omega-oxidized to a carboxylic acid and that this process can occur in rat liver microsomes in the presence of NADPH and O2. The oxidation to the quinone structure appears to be a subsequent event that may be artifactual and/or catalyzed by a microsomal enzyme(s).

Changes in Hepatic Microsomal Triglyceride Transfer Protein and Triglyceride in Periparturient Dairy Cattle

We determined the relationship between microsomal triglyceride transfer protein (MTP) (activity, mass, and mRNA) and liver triglyceride concentration in 16 dairy cows (13 multiparous and three primiparous) from 27 d before expected calving (d –27) to 35 d postpartum (d 35), the time period when fatty liver is most likely to develop. In addition, dry matter intake, plasma nonesterified fatty acids (NEFA), and plasma glucose were monitored. There were no significant parity x time interactions. Dry matter intake, plasma NEFA, plasma glucose, and liver triglyceride were significantly affected by day of sampling. Dry matter intake was 10.7, 8.0, and 19.5 kg/d on d –27, 2, and 35, respectively. Plasma NEFA concentration was higher on d 2 (1113 μEq/L) compared with d –27 (201 μEq/L) and 35 (358 μEq/L). Plasma glucose concentration was 63.3, 54.3, and 57.8 mg/dl on d –27, 2, and 35, respectively. Hepatic triglyceride (TG) concentration increased from 1.8 to 11.8% liver TG (DM basis) on d –27 and 2, respectively. There was no difference between hepatic triglyceride concentration on d 2 and 35. There was a significant effect of day of sampling on hepatic MTP activity and mRNA. Hepatic MTP activity decreased from 2.08 to 1.79 nmole triolein transferred/h per mg of microsomal protein on d –27 and 2, respectively, and increased from 1.79 to 2.17 nmole triolein transferred/h per mg of microsomal protein on d 2 and 35, respectively. Hepatic MTP mRNA increased from d –27 to 2 and remained elevated from d 2 to 35.There was no effect of day of sampling on MTP mass. There were no significant correlations between hepatic MTP activity, mass, or mRNA with either liver TG or plasma NEFA on any of the sampling days. The cause of a decrease in hepatic MTP activity and increase in mRNA on d 2 is unknown. However, the lack of correlation between MTPactivity, mass, or mRNAwith either liver TG or plasma NEFA on d 2 postpartum suggests that MTP probably does not play a role in the etiology of fatty liver that occurs in dairy cows at calving.

Enantioselective liver microsomal sulphoxidation of albendazole in cattle: effect of nutritional status

1. The enantioselective liver microsomal sulphoxidation of the benzimidazole anthelmintic, albendazole (ABZ), by cattle liver microsomes has been investigated. The influence of nutritional condition on this biotransformation process was also characterized. 2. ABZ was oxidized to its sulphoxide metabolite (ABZSO) in a NADPH concentration-dependent reaction and the (+) and (-) ABZSO enantiomers formed were identified. 3. Vmax (0.27 nmol ABZSO formed per min x mg-1 microsomal protein) and Km (15.10 µM) for ABZ sulphoxidation by cattle liver microsomes were obtained. Different Vmax (0.11 and 0.16 nmol x min-1 x mg-1) and Km (9.40 and 26.70 µM) characterized the enantioselective formation of (+) and (-) ABZSO antipodes, respectively. 4. Free fatty acid (FFA) concentrations and β-hydroxybutyrate concentrations (β-OHB) in serum and liver homogenates were significantly higher in feed-restricted (poor nutritional condition) compared with control animals in an optimal nutritional status. Serum protein concentrations and liver cytosolic glucose 6-phosphate dehydrogenase (G6PD) activity were significantly lower in the feed-restricted compared with control calf. 5. Animal nutritional condition affected the pattern of ABZ sulphoxidation. A higher Km for (total) ABZSO and (+) ABZSO production was observed in the calf subjected to a period of undernutrition. 6. A nutritionally induced impairment in the affinity of microsomal mixed-function oxidases responsible of ABZ oxidation may be responsible for the observed changes in the liver microsomal sulphoxidation of ABZ in the feed-restricted calf. Furthermore, undernutrition may affect primarily the FMO-mediated formation of (+) ABZSO. These in vitro observations agree with the changes observed in vivo following the administration of ABZ to the calf subjected to a dietary restriction.

Microsomal prediction of in vivo clearance of CYP2C9 substrates in humans.

To assess the utility of human hepatic microsomes for predicting in vivo intrinsic clearance (CLint ) via the use of four cytochrome P450 2C9 substrates: phenytoin, tolbutamide (S)-ibuprofen (two pathways) and diclofenac, and to examine the role of exogenous albumin within the microsomal incubation. V max, Km and CLint (defined as V max/Km ratio) were estimated under initial rate conditions for five pathways of metabolism in a bank of 15 human hepatic microsomal samples and were scaled to in vivo units using the microsomal protein index. Non-metabolic related binding in microsomes was measured for phenytoin and tolbutamide in the presence and absence of albumin. Microsomal CLint values differed by over two orders of magnitude, with the means ranging from 0.18 (phenytoin) to 40.70 (diclofenac) microl min-1 mg-1 microsomal protein. When these data were scaled and compared with published in vivo studies a similar rank order was obtained, however, the actual CLint tended to be underpredicted. While the in vivo unbound Km for phenytoin, 1-5 micron is substantially lower than the value determined in microsomes based on total concentrations (56 micron), correction for the in vitro binding reduces this value to 20 micron and 6 micron in the absence and presence of albumin, respectively. Similar trends were seen with tolbutamide Km. An appreciation of the utility of in vitro prediction can be best achieved when the range of CLint values predicted from the individual hepatic microsomal samples are compared with the range of individual in vivo CLint values reported in the literature. The degree of underprediction is less evident using the range than the mean data and no consistent advantage in adding albumin to the incubation media is apparent.

Metabolism of an anionic fluorescent dye, 1-anilino-8-naphthalene sulfonate (ANS) by rat liver microsomes.

The present study was designed to examine the metabolism of 1-anilino-8-naphthalene sulfonate (ANS), an anionic compound which is transported into liver via "multispecific organic anion transporter", with rat hepatic microsomes. TLC analysis indicated that the fluorescent metabolites were not produced to a measurable extent, which made it possible to assess the ANS metabolism by measuring the fluorescence disappearance. The metabolism of ANS was remarkably inhibited by the presence of SKF-525A as well as by the substitution of O2 by CO gas. ANS metabolism by microsomes also required NADPH as a cofactor. These results indicated that the microsomal monooxygenase system might be mainly responsible for the ANS metabolism. The maximum velocity (Vmax) and Michaelis constant (K(m)) were calculated to be 4.3 +/- 0.2 nmol/min/mg protein and 42.1 +/- 2.0 microM, respectively. Assuming that 1 g of liver contains 32 mg of microsomal protein, the Vmax value was extrapolated to that per g of liver (Vmax'). The intrinsic metabolic clearance (CLint) under linear conditions calculated from this in vitro metabolic study was 3.3 ml/min/g liver, being comparable with that (3.0 ml/min/g liver) calculated by analyzing the in vivo plasma disappearance curve in a previous study. Furthermore, the effects of other organic anions on the metabolism of ANS were examined. Bromophenolblue (BPB) and rose bengal (RB) competitively inhibited the metabolism of ANS, while BSP inhibited it only slightly. The inhibition constant (Ki) of BPB (6 microM) was much smaller than that of RB (200 microM). In conclusion, the microsomal monooxygenase system plays a major role in the metabolism of ANS, and other unmetabolizable organic anions (BPB and RB) compete for this metabolism.

Effect of carbon tetrachloride-induced hepatic injury on stereoselective N-demethylation of chlorpheniramine by rat hepatic cytochrome P450 2C11 isozyme

We examined the effect of a carbon tetrachloride (CCl4)-induced hepatic injury on the stereoselective N-demethylation of RS-(+/-)-chlorpheniramine (Chp) by cytochrome P450 (CYP) 2C11 isozyme. In the non-treated rat liver microsomes, the stereoselective N-demethylation of racemic Chp was observed. However, in the CCl4-treated (0.5 ml/kg, i.p.) rat liver microsomes, the N-demethylation activities of S-(+)- and R-(-)-Chp decreased continuously up to the third day after the treatment with CCl4, and reached about 9 and 13% of control values, respectively, and the stereoselective N-demethylation of Chp was not observed. Moreover, in the liver microsomes at the 7th day after the treatment with CCl4, the N-demethylation activities of both enantiomers recovered to an original level, and the stereoselective N-demethylation of Chp was again observed. The addition of 30 microliters of the anti-rat CYP2C11 serum to the reaction mixture containing 1 mg of microsomal protein inhibited the formation of monodesmethylchlorpheniramine (DMChp) from both enantiomers to 74 and 57% of the control values for S-(+)- and R-(-)-Chp, respectively. In the liver microsomes of a male rat at the 1st day after the treatment of CCl4, the addition of the anti-rat CYP 2C11 serum (30 microliters) also caused 25% inhibition of the formation of DMChp from S-(+)-Chp, but anti-rat CYP2C11 had no inhibitory effect on the rates of microsomal N-demethylation of R-(-)-enantiomer. On the other hand, in the liver microsomes of a male rat at the 7th day after the treatment with CCl4, the anti-rat CYP2C11 serum had an inhibitory effect on the rates of microsomal N-demethylation of either S-(+)- or R-(-)-enantiomers again. Moreover, it was confirmed by Western blotting analysis that the density of the stained bands of CYP2C11 in the liver microsomes from male rats at the 1st, 2nd and 3rd days after the treatment with CCl4, was thinner than that from non-treatment male rats. These results indicated that the changes of N-demethylation activities of Chp in the CCl4-induced hepatic injury were due to the variation of microsomal CYP2C11.