Rabbit Liver Microsomes Research Articles

Synthesis of Two Metabolites of the Antiarrythmicum Amiodarone

The metabolism of the potent antiarrythmic drug amiodarone (AMI; 1) has yet not been fully investigated. Recently, in vitro experiments revealed that in rabbit-liver microsomes, AMI (1) and its main metabolite MDEA (2) were biotransformed to the hydroxylated derivatives 3′-OH-AMI (3) and 3′-OH-MDEA (4), respectively. To establish the chemical structure of 3 and 4, we developed a total synthesis of these two metabolites of AMI (1). 1H- and 13C-NMR Signal assignment from HSQC and HMBC 2D NMR data of synthesized 4 showed that the proposed structure of metabolite 4 is correct. Even the structure of 3 was found to be correct by comparing its HPLC/MS-MS/MS with the data described earlier.

Neonicotinoid insecticides: reduction and cleavage of imidacloprid nitroimine substituent by liver microsomal and cytosolic enzymes.

The major insecticide imidacloprid (IMI) is known to be metabolized by human cytochrome P450 3A4 with NADPH by imidazolidine hydroxylation and dehydrogenation to give 5-hydroxy-imidacloprid and the olefin, respectively, and by nitroimine reduction and cleavage to yield the nitrosoimine, guanidine, and urea derivatives. More extensive metabolism by human or rabbit liver microsomes with NADPH or rabbit liver cytosol without added cofactor reduces the IMI N-nitro group to an N-amino substituent, i.e., the corresponding hydrazone. A major metabolite on incubation of IMI in the human microsome-NADPH system is tentatively assigned by LC/MS as a 1,2,4-triazol-3-one derived from the hydrazone; the same product is obtained on reaction of the hydrazone with ethyl chloroformate. The hydrazone and proposed triazolone are considered here together (referred to as the hydrazone) for quantitation. Only a portion of the microsomal reduction and cleavage of the nitroimine substituent is attributable to a CYP450 enzyme. The cytosolic enzyme conversion to the hydrazone is inhibited by added cofactors (NAD > NADH > NADP > NADPH) and enhanced by an argon instead of an air atmosphere. The responsible cytosolic enzyme(s) does not appear to be DT-diaphorase (which is inhibited by several neonicotinoids), aldose reductase, aldehyde reductase, or xanthine oxidase. However, the cytosolic metabolism of IMI is inhibited by several aldo-keto-reductase inhibitors (i.e., alrestatin, EBPC, Ponalrestat, phenobarbital, and quercetin). Other neonicotinoids with nitroimine, nitrosoimine, and nitromethylene substituents are probably also metabolized by "neonicotinoid nitro reductase(s)" since they serve as competitive substrates for [(3)H]IMI metabolism.

Effect of SMP-500, a Novel Acyl-CoA:Cholesterol Acyltransferase Inhibitor, on the Cholesterol Esterification and Its Hypocholesterolemic Properties

We investigated the effects of SMP-500, a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, on ACAT activities in the liver and intestine, and in macrophages. We measured its effects on the serum cholesterol levels and hepatic cholesterol content in mice, rabbits and hamsters. SMP-500 inhibited ACAT activities in rabbit liver and small intestine microsomes with IC₅₀ values of 72 and 84 nmol/l, respectively, and acted as a competitive inhibitor of rabbit liver ACAT. SMP-500 potently inhibited cholesterol esterification in rat peritoneal macrophages (IC₅₀ = 15 nmol/l). In high-fat and high-cholesterol diet-fed mice and in high-cholesterol diet-fed rabbits, SMP-500 reduced the serum cholesterol levels and the hepatic cholesterol content. SMP-500 also reduced the serum and hepatic cholesterol in normal chow-fed hamsters in a dose-dependent manner. In all the animal models, SMP-500 reduced the hepatic free cholesterol content as well as the total and esterified cholesterol. Administered orally, SMP-500 had a direct inhibitory effect on hepatic ACAT activity. These results indicate that SMP-500 is a potent and competitive ACAT inhibitor and may have a therapeutic potential for treating hypercholesterolemia and atherosclerosis.

Production of carbon monoxide by cytochrome P450 during iron-dependent lipid peroxidation

Carbon monoxide (CO) formation was studied in the process of lipid peroxidation in phenobarbital-induced rabbit liver microsomes. The reaction was NADPH-dependent and required Fe 2+, which occurs in microsomes as being protein bound and is not a consequence of heme destruction. Zn-protoporphyrin IX, an inhibitor of the heme oxygenase activity, proved to have no effect on CO production, suggesting that heme oxygenase is not involved into the CO generation reaction. At the same time, the addition of cytochrome P450 typical inhibitors SKF 525A and metyrapone to the reaction mixture had an inhibitory effect on the CO formation rate. Antioxidants such as α-tocopherol and desferal inhibited lipid peroxidation in phenobarbital-induced rabbit liver microsomes, and in this case the CO production was not registered. Thus, on the basis of the results presented here it is possible to assert that the process of NADPH, Fe 2+-dependent carbon monoxide formation in microsomes is a result of lipid peroxidation with cytochrome P450 2B4 participation.

Species Differences in the Metabolism of (+)- and (−)-Limonenes and their Metabolites, Carveols and Carvones, by Cytochrome P450 Enzymes in Liver Microsomes of Mice, Rats, Guinea Pigs, Rabbits, Dogs, Monkeys, and Humans

(+)-Limonene is shown to cause renal toxicity in male rats, but not in female rats and other species of animals including mice, guinea pigs, rabbits, and dogs. We have previously shown that male-specific rat CYP2C11 (but not female-specific CYP2C12) is able to convert limonenes to carveols and perillyl alcohols (M. Miyazawa, M. Shindo, and T. Shimada: Chem. Res. Toxicol., 15, 15-20, 2002). Here, we investigated whether (+)- and (-)-limonene enantiomers are differentially metabolized by P450 enzymes in liver microsomes of mice, rats, guinea pigs, rabbits, dogs, monkeys, and humans. Limonene enantiomers were converted to respective carveols, perillyl alcohols, and carvones (oxidative metabolites of carveols) by liver microsomes of dogs, rabbits, and guinea pigs. Mice, rats, monkeys, and humans produced carveols and perilly alcohols, but not carvones. Reconstituted monooxygenase systems containing purified rabbit CYP1A2 and 2B4 and NADPH-P450 reductase were found to catalyze (+)-limonene to (+)-carveol, (+)-carvone, and (+)-perillyl alcohol, being more active with CYP2B4. When (+)-carveol and (+)-carvone were used as substrates, dogs, rabbits, and guinea pigs metabolized them to (+)-carvone and (+)-carveol, respectively. Again humans, monkeys, rats, and mice did not convert (+)-carveol to (+)-carvone, but metabolized (+)-carvone to (+)-carveol, with male rats having the highest rates. CYP2C enzymes were suggested to play major roles in metabolizing (+)-carveol to (+)-carvone and (+)-carvone to (+)-carveol by liver microsomes, since the activities were inhibited significantly by anti-human CYP2C9 antibodies in these animal species. Studies with recombinant P450 enzymes suggested that CYP2C9 and 2C19 in humans and CYP2C11 in untreated male rats were the major enzymes in metabolizing (+)-carvone. These results suggest that there are species-related differences in the metabolism of limonenes by P450 enzymes, particularly in the way from (+)-carveol to (+)-carvone. However, it remains unclear whether these differences in limonene metabolism by these animal species explain species-related differences in limonene-induced renal toxicity.

Effect of microsome–liposome fusion on the rotational mobility of cytochrome P450IIB4 in rabbit liver microsomes

Membrane fusion of microsomes with soybean phospholipid vesicles was performed at pH 6.5 to investigate the effect of lipid-enrichment in the membrane on the rotational mobility of cytochrome P450. Rotational diffusion of cytochrome P450 in the microsomal membrane of phenobarbital-induced rabbit liver was measured by detecting the decay of absorption anisotropy after photolysis of the heme CO complex by a vertically polarized laser flash. The fusion procedures yielded three separate fractions upon sucrose density gradient centrifugation with lipid-to-protein ratio in weight (L/P) as follows: 1.5 in the bottom fraction, 2.2 in the middle fraction, and 3.9 in the top fraction. In each fraction, co-existence of mobile and immobile cytochrome P450 was observed. The percentage of rotationally mobile P450 (with the mean rotational relaxation time of φ=505–828 μs) in each of the different bands was found to be 59% in the bottom fraction, 61% in the middle fraction, and 68% in the top fraction. This increase in mobile population of P450 due to lipid-enrichment indicates that aggregated proteins in microsomal membranes dissociate with increasing L/P which is inversely proportional to the protein concentration in the membrane. With freeze-fracture electron microscopy, it was shown that the average distance increased between intramembrane particles by lipid-enrichment. Thus, the significant immobile population (32%) of P450 in microsomal membranes can be explained by nonspecific protein aggregation which is a consequence of the low L/P of 0.8. The decrease in the mobile population in the bottom fraction compared with intact microsomes was shown to be due to the pH 6.5 incubation used for fusion.

Metabolism of amiodarone (Part III): identification of rabbit cytochrome P450 isoforms involved in the hydroxylation of mono-N-desethylamiodarone

1. Amiodarone (AMI) is a potent anti-arrhythmic drug and mono-N-desethylamiodarone (MDEA) is its only known metabolite. It was found recently that in rabbit liver microsomes MDEA was biotransformed to n-3-hydroxybutyl-MDEA (3OH-MDEA). 2. In liver microsomes isolated from the untreated rabbit, the formation of 3OHMDEA obeyed Michaelis-Menten enzyme kinetics with Km = 6.39 ± 1.07 μm and Vmax = 0.56 ± 0.21 nmol min−1 mg−1 protein. 3. Furthermore, (1) among chemicals usually used as inhibitors of cytochrome P450, only midazolam (MDZ), cyclosporin A and ketoconazole inhibited the MDEA hydroxylase activity significantly (> 60% inhibition), (2) MDZ, a substrate of CYP3A, inhibited the 3OH-MDEA formation competitively (Ki = 10 ± 5 μm), (3) the formation rates of 3OH-MDEA correlated positively with those of 1′OH-MDZ (r = 0.81; n = 6), and (4) MDEA hydroxylase activity of microsomes isolated from rabbit rifampicininduced cultured hepatocytes was 4-fold more active than the control. 4. Since CYP3A6 is mainly induced by rifampicin in rabbit-cultured hepatocytes, the data suggest that this isoform is involved in the biotransformation of MDEA to 3OHMDEA. 5. Since α-naphthoflavone, cimetidine and quinidine also partially inhibited the MDEA hydroxylase activity, it is possible that other CYPs, such as 1A, 2C and 2D, may also be active in the metabolism of amiodarone.

Stimulation of aniline, p-nitrophenol and N-nitrosodimethylamine metabolism in kidney by pyridine pretreatment of rabbits.

Pyridine has been shown to cause liver and kidney damage in animals and in humans. In a previous study we examined the effects of pyridine on rabbit liver and lung microsomal drug-metabolizing enzymes. In this study, in vivo i.p. administration of pyridine to rabbits caused a significant 3.4-fold increase in kidney N-nitrosodimethylamine (NDMA) N-demethylase activity as compared to the activity in control rabbits. The same treatment also significantly stimulated the activity of other cytochrome P4502E1-associated enzymes. The activities of p-nitrophenol hydroxylase and aniline 4-hydroxylase in kidney microsomes were increased 4.9-and 4.5-fold, respectively. Pyridine treatment increased the P450 content of the kidney 1.6-fold (P<0.05). SDS-PAGE of both kidney and liver microsomes of pyridine-treated rabbits showed a protein band of enhanced intensity at 51,000 Mr migrating in the region of cytochrome P4502E1. p-Aminophenol, a 4-hydroxylation product of aniline, has been shown to be nephrotoxic and NDMA, a procarcinogen, has been shown to be carcinogenic following bioactivation by NDMA N-demethylase in a number of tissues including the kidney. Since pyridine was shown to be nephrotoxic, it is expected that pyridine potentiates the toxic and/or carcinogenic effects of aniline, p-nitrophenol and NDMA through induction of their metabolism by the cytochrome P450-dependent drug-metabolizing enzymes.

ChemInform Abstract: New Selective Inhibitors of Cytochrome P450 2B4 and an Activator of Cytochrome P450 3A6 in Rabbit Liver Microsomes.

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Inhibitory effect of azole antifungal agents on the glucuronidation of lorazepam using rabbit liver microsomes in vitro.

Azole antifungal agents (azoles) have inhibitory effects on the cytochrome P450. However, the effect of azoles on conjugative metabolism has not been given much attention. Lorazepam (LZP), a benzodiazepine sedative agent, is known to be metabolized by uridine 5'-diphosphate (UDP)-glucuronyltransferase. Herein we report investigation of the effect of azoles on the enzyme-kinetics of glucuronidation of lorazepam using rabbit liver microsomes in vitro. The Km and Vmax for LZP glucuronidation were determined to be 0.26+/-0.08 mM and 1.25+/-0.21 nmol/min/mg protein, respectively, when evaluated in the presence of a detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) (0.8 mg/mg protein). Azoles fluconazole, miconazole, and ketoconazole competitively inhibited the glucuronidation of LZP, with Ki values of 7.17+/-4.78 mM, 0.17+/-0.08 mM, and 0.092+/-0.026 mM, respectively. These results are comparable to the previously reported Ki values of azoles with zidovudine (AZT) glucuronidation (1.4, 0.18, and 0.08 mM for fluconazole, miconazole, and ketoconazole, respectively) [Sampol et al., Br. J. Clin. Pharmacol., 40, 83-86, 1995]. Therefore, in order to avoid possible side effects of LZP, the concomitant administration of LZP and azoles should be carefully evaluated.

New Selective Inhibitors of Cytochrome P450 2B4 and an Activator of Cytochrome P450 3A6 in Rabbit Liver Microsomes

We investigated interactions of adamantane, diamantane and their two substituted derivatives, 2-isopropenyl-2-methyladamantane (2-PMADA,1) and 3-isopropenyl-3-methyldiamantane (3-PMDIA,2), with various isoforms of rabbit cytochrome P450 (CYP). The data of spectroscopic experiments showed that all the substances are bound to the substrate binding site of rabbit CYP2B4 and CYP3A6.1and2are compounds having higher affinities to these CYP isoforms than adamantane and diamantane. All compounds inhibit CYP2B4 specific enzyme activity (the 7-pentoxyresorufinO-depentylase activity). The 50% inhibition of CYP2B4 was due to 3.82, 0.61, 0.66 and 0.37 μM adamantane, diamantane,1and2, respectively. The products formed during the CYP2B4-mediated metabolism of studied substances are less effective inhibitors than parent compounds. An opposite effect of1on CYP3A6 was determined. The specific enzyme activity of CYP3A6 increased to 138% of control when1was used in the presence of 40 μM erythromycin as a substrate. Here, we report the finding of a new activator of CYP3A6 having the structure quite different from that of CYP3A6 activators known to date.

Methionine S-Oxidation in Human and Rabbit Liver Microsomes: Evidence for a High-Affinity Methionine S-Oxidase Activity That Is Distinct from Flavin-Containing Monooxygenase 3

Methionine has previously been shown to be S-oxidized by flavin-containing monooxygenase (FMO) forms 1, 2, and 3. The most efficient catalyst was FMO3, which has a Km value for methionine S-oxidation of approximately 4 mM, and exhibits high selectivity for formation of the d-diastereoisomer of methionine sulfoxide. The current studies provide evidence for an additional methionine S-oxidase activity in liver microsomes. Human and rabbit liver microsomes exhibited a biphasic response to methionine at concentrations ranging from 0.05 to 10 mM, as indicated by both Eadie–Hofstee plots and nonlinear regression. The low-affinity component of the biphasic response had Km values of approximately 3 and 5 mM for humans and rabbits, respectively, as well as high diastereoselectivity for methionine sulfoxide formation. The low-affinity activity in rabbit liver microsomes was inhibited by methimazole, S-allyl-l-cysteine, and by mild heat treatment, suggesting the activity is FMO3. The high-affinity component of the biphasic response had Km values of approximately 0.07 and 0.04 mM for humans and rabbits, respectively, as well as lower diastereoselectivity for methionine sulfoxide formation. Further characterization of the high-affinity activity in rabbit liver microsomes indicated lack of involvement of cytochrome P450s or reactive oxygen species. The high-affinity activity was inhibited 25% by potassium cyanide and greater than 50% by methimazole and S-allyl-l-cysteine. Mild heat treatment produced 85% inhibition of the low-affinity activity, but only 30% inhibition of the high-affinity activity. Both high- and low-affinity activities were decreased by 85% in flavin-depleted microsomes. Because these results suggested the additional S-oxidase activity has characteristics of an FMO, recombinant human FMO4 was evaluated as a potential catalyst of this activity. Recombinant FMO4 catalyzed S-oxidation of both methionine and S-allyl-l-cysteine, with similar diastereoselectivity to the high-affinity microsomal S-oxidase; however, the Km values for both reactions appeared to be greater than 10 mM. In summary, these studies provide evidence for two microsomal methionine S-oxidase activities. FMO3 is the predominant catalyst at millimolar concentrations of methionine. However, at micromolar methionine concentrations, there is an additional S-oxidase activity that is distinct from FMO3.

Metabolic pathways of flobufen — a new antirheumatic and antiarthritic drug. Interspecies comparison

Metabolic transformations of flobufen, [4-(2',4'-difluoro-biphenyl-4-y1)-4-oxo-2-methylbutanoic acid], a non-steroid antiinflammatory agent, were studied in vitro using the following biological models and species: rat and mouse liver homogenates and liver subcellular fractions (5 000 g and 100 000 g supernatant, mitochondria); rat, mouse, rabbit, guinea-pig and mini-pig liver microsomes; isolated rat hepatocytes; perfused rat liver and 5000 g rat muscle tissue supernatant. Reduced flobufen [4-(2',4'-difluorobiphenyl-4-yl)-4-hydroxy-2-methylbutanoic acid] is the major metabolite generated by the subcellular fractions (in the mild acidic extraction conditions during subsequent laboratory processing is converted to its lactone form). It was detected upon the incubation of flobufen with liver microsomes isolated from all the animals tested. Maximum yield of reduced flobufen in experiments with rat and mouse liver microsomes was found after anaerobic incubation with NADPH. This finding combined with the knowledge of subcellular distribution of enzymes suggest that metabolite formation depends on the activity of microsomal reductases and, probably, also on the activity of the important microsomal reductase, cytochrome P-450. Another flobufen metabolite, arylacetic acid [(2',4'-difluorobiphenyl-4-yl)ethanoic acid], is generated from the reduced metabolite by the cleavage of its side chain, and was detected in isolated hepatocytes - it was the only metabolite found in urine and faeces upon oral administration of the drug. All these metabolites were identified and quantified.

Isolation and complete covalent structure of liver microsomal paraoxonase

Paraoxonase (PON1) is a serum esterase exclusively associated with high-density lipoproteins; it might confer protection against coronary artery disease by destroying pro-inflammatory oxidized lipids in oxidized low-density lipoproteins. Here I show that rabbit liver microsomes contain a PON analogue (MsPON) and report the isolation and complete covalent structure of MsPON. In detergent-solubilized microsomes, MsPON co-purifies with the microsomal triacylglycerol transfer protein (MTP) complex. MsPON was separated from the complex and purified to homogeneity under non-denaturing conditions. Automated sequence analysis of intact MsPON and peptides obtained from enzymic and chemical cleavages led to the elucidation of the complete covalent structure of MsPON. The protein is a single polypeptide consisting of 350 residues. The sequence of rabbit liver microsomal MsPON is 60% identical with that of rabbit serum PON1, and 84% identical with the sequence predicted by a human cDNA of unknown function, designated PON3. MsPON has a hydrophobic segment at the N-terminus that might serve to anchor the protein to the microsomal membrane or to the MTP complex. Unlike in the serum enzyme, two potential N-glycan acceptor sites in MsPON are not glycosylated. An absence of N-glycans was also indicated in the rabbit liver MTP. MsPON has a single free cysteine residue at position 38 and a disulphide bond between Cys-279 and Cys-348. The microsomal enzyme lacks three residues at the N-terminus that are present in the serum protein. MsPON lacks four residues at the C-terminus that are present in the rabbit serum protein but absent from human serum PON1. On the basis of the observation that MsPON displays a high degree of similarity with serum PON1, it is proposed that MsPON might have a function related to that of PON1 in serum high-density lipoprotein complexes.

A monoclonal antibody recognizing an epitope shared by receptors for growth hormone, prolactin, interleukin 2 and interleukin 6.

Monoclonal antibody (MAb) termed R7B4 was generated throughout the idiotypic-anti-idiotypic network from mice immunized with human and bovine growth hormones (GH). The Ab was selected on the basis that it did not recognize human GH (hGH) neither insolubilized nor in solution but inhibited 125I-hGH binding to receptors from rat and rabbit liver and from Nb2-cell membranes. Since it inhibited Nb2-cell mitogenesis stimulated by hGH, prolactins or placental lactogens, MAb R7B4 behaved as an antagonist of lactogenic hormones. Furthermore, the Ab impaired proliferative activity of interleukin 2 (IL-2) on Nb2 cells as well as growth of 7TD1 cells, an interleukin 6 (IL-6) dependent hybridoma not expressing GH receptors. Biotin-labeled MAb R7B4 specifically bound to rat liver microsomes, and the Ab was able to recognize Nb2 and 7TD1-cell membranes as shown by flow cytometry experiments. However, MAb binding was not hampered by hGH, indicating that the Ab did not mimic GH binding site to receptors. Immunoblot assays indicated that rat and rabbit liver as well as Nb2-cells membrane antigens recognized by MAb R7B4 were similar to those revealed by a MAb directed to prolactin receptors. In addition, MAb R7B4 was able to detect two bands probably corresponding to the somatogenic receptor in rabbit liver microsomes as well as three different proteins in 7TD1-cells showing molecular weights similar to those of the IL-6 receptor complex. Results suggest that MAb R7B4 is directed to an epitope shared by receptors for lactogenic and somatogenic hormones, IL-2 and IL-6. To our knowledge, these data are the first experimental evidence of the existence of structural similarity between some of the receptors grouped in the cytokine receptor superfamily.

Studies on the Metabolic Fate of NK-104, a New Inhibitor of HMG-CoA Reductase. (5). In Vitro Metabolism and Plasma Protein Binding in Animals and Human.

NK-104 is a new and very potent competitive inhibitor of HMG-CoA reductase. NK-104 bound to plasma protein of mouse, rat, rabbit, dog and monkey, at a binding ratio of more than 96%. NK104 also highly bound to human serum albumin and a1 acid glycoprotein. However, no interaction in protein binding was found between NK-104 and selected highly protein binding drugs at their therapeutic concentrations in human plasma. The in vitro metabolism of 14C-NK 104 was investigated using the liver microsomes of rat, rabbit, guinea pig, dog, monkey and human. The radioactivity was mostly due to unchanged NK-104 except for monkey. A relatively large amount of M-13 (8-hydroxy NK-104) was observed in monkey, but not in other animal species. The kinetic study of NK 104 metabolism suggested that M-13 was formed with relatively low intrinsic clearance. Based on three different in vitro approaches, namely 1) chemical inhibition, 2) immunoinhibition and 3) metabolism by recombinant human P450, it is concluded that CYP2C9 and CYP2C8 are the enzymes responsible for the metabolism of NK-104. In addition, no inhibitory effect on CYP mediated 4-hydroxylation of tolbutamide (CYP2C9) in the presence of NK-104 was detected. In conclusion, NK-104 was highly bound to plasma protein whereas no mutual interaction in protein binding was found between NK-104 and various commonly used drugs. Furthermore, NK-104 was scarcely metabolized in the liver microsomes, and no drug/drug interaction between NK-104 and tolbutamide was noted in in vitro metabolism.

Inhibition of Angiogenesis by Thalidomide Requires Metabolic Activation, Which Is Species-dependent

Thalidomide has been shown to be an inhibitor of angiogenesis in a rabbit cornea micropocket model; however, it has failed to demonstrate this activity in other models. These results suggest that the anti-angiogenic effects of thalidomide may only be observed following metabolic activation of the compound. This activation process may be species specific, similar to the teratogenic properties associated with thalidomide. Using a rat aorta model and human aortic endothelial cells, we co-incubated thalidomide in the presence of either human, rabbit, or rat liver microsomes. These experiments demonstrated that thalidomide inhibited microvessel formation from rat aortas and slowed human aortic endothelial cell proliferation in the presence of human or rabbit microsomes, but not in the presence of rat microsomes. In the absence of microsomes, thalidomide had no effect on either microvessel formation or cell proliferation, thus demonstrating that a metabolite of thalidomide is responsible for its anti-angiogenic effects and that this metabolite can be formed in both humans and rabbits, but not in rodents.

Cytochrome P450-dependent desaturation of lauric acid: isoform selectivity and mechanism of formation of 11-dodecenoic acid

Cytochrome P450-catalyzed desaturation reactions have been reported infrequently in the literature. Previously, we documented the formation of the terminal olefinic metabolite of valproic acid by various members of the CYP2B and CYP4B sub-families. However, despite the extensive use of fatty acid substrates in drug metabolism studies, other examples of terminal desaturation at non-activated carbon centers are lacking. The goals of the present studies were to determine whether the archetypal P450 substrate, lauric acid (dodecanoic acid; DDA), also undergoes desaturation reactions, identify specific rabbit P450 isoforms which catalyze this reaction and examine its mechanism. A highly sensitive, capillary GC/MS assay was developed to separate and quantitate the trimethylsilyl derivatives of 11-ene-DDA, cis- and trans-10-ene-DDA and cis- and trans-9-ene-DDA. Among all of these potential olefinic metabolites, only 11-ene-DDA was formed at a significant rate by rabbit liver microsomes. The formation of 11-ene-DDA was NADPH-dependent, and was induced markedly by acetone pre-treatment, but not by phenobarbital, rifampin or Arochlor 1254. Studies with seven purified, reconstituted rabbit P450 isoforms showed that the most rapid rates of desaturation were obtained with CYP2E1, CYP4A5/7 and CYP4B1. Non-competitive, intermolecular isotope effect experiments, conducted with [12,12,12- 2H 3]DDA and [11,11- 2H 2]DDA, demonstrated further that CYP4B1-mediated terminal desaturation of DDA is initiated by removal of a hydrogen atom from the ω-1 rather than the ω position.

Rabbit liver contains one major sterol 12α-hydroxylase with broad substrate specificity

Conversion of cholesterol into cholic acid in mammalian liver requires a 12α-hydroxylation step. Results have been presented suggesting that two different enzymes are involved in this hydroxylation with different activities towards the two steroids believed to be the physiological substrates for the enzyme, 7α-hydroxy-4-cholesten-3-one and 5β-cholestane-3α,7α-diol. It is shown here that rabbit liver microsomes and partly purified sterol 12α-hydroxylase as well as COS cells transfected with a cDNA coding for this enzyme are able to catalyze 12α-hydroxylation of the two substrates at similar relative rates. Also 7α-hydroxycholesterol and 3α,7α-dihydroxy-5β-cholestanoic acid are 12α-hydroxylated by the three systems. It is concluded that rabbit liver contains one major sterol 12α-hydroxylase with a broad substrate specificity.

Isolation and structural analysis of microsomal membrane proteins.

Investigation of the endoplasmic reticulum requires a methodological background in the solubilization, purification, and structural analysis of membrane proteins. The experience of one laboratory with microsomal proteins over 30 years is summarized in this review. We focus on the isolation and structure of the major proteins of rabbit liver microsomes. The special pitfalls encountered with fragile protein complexes, hydrophobic peptides, and post-translational modifications are emphasized.