Microsomes separation

Ethanol also inhibits ADH-catalyzed retinol oxidation in vitro, and ethanol treatment of mouse embtyos has been demonstrated to reduce endogenous RA levels. The inhibition of cytosolic RolDH activity and stimulation of microsomal RolDH activity could explain ethanol-mediated vitamin A depletion, separate from ADH isoenzymes. Although the exact mechanism of inhibition of retinoid metabolism by ethanol is unclear, these observations are consistent with the finding that patients with alcoholic liver disease have depletedhepatic vitamin A reserves [review see [2]. 

FIGURE 2.8 Monooxygenase activities of mammals, birds, and fish, (a) Mammals and birds, (b) Mammals, birds, and fish. Activities are of hepatic microsomal monooxygenases to a range of substrates expressed in relation to body weight. Each point represents one species (males and females are sometimes entered separately) (from Walker et al. 2000). 

The researeh on dehydroepiandrosterone (DHEA) is limited beeause of the laek of radiolabeled metabolites. Robinzon et al. [126] showed that, using pig liver mierosomes, the radiolabeled metabolites of DHEA can be prepared in stable, pure form for bioehemical smdies. They utilized pig liver microsomal (PLM) fractions to prepare pH]-labeled 7a-hydroxy-DHEA (7a-OH-DHEA), 7[3-hydroxy-DHEA (7P-OH-DHEA), and 7-oxo-DHEA substrates from 50 pM [1,2,6,7-3H]DHEA. The metabolites were separated by silica gel PLC plates using ethyl aeetate-hexane-gla-eial aeetic acid (18 8 , v/v) as the mobile phase, extracted with ethyl aeetate, and dried under a stream of nitrogen. The purity of markers was determined with the use of TLC and GC/MS. 

The identification and quantification of potentially cytotoxic carbonyl compounds (e.g. aldehydes such as pentanal, hexanal, traw-2-octenal and 4-hydroxy-/mAW-2-nonenal, and ketones such as propan- and hexan-2-ones) also serves as a useful marker of the oxidative deterioration of PUFAs in isolated biological samples and chemical model systems. One method developed utilizes HPLC coupled with spectrophotometric detection and involves precolumn derivatization of peroxidized PUFA-derived aldehydes and alternative carbonyl compounds with 2,4-DNPH followed by separation of the resulting chromophoric 2,4-dinitrophenylhydrazones on a reversed-phase column and spectrophotometric detection at a wavelength of378 nm. This method has a relatively high level of sensitivity, and has been successfully applied to the analysis of such products in rat hepatocytes and rat liver microsomal suspensions stimulated with carbon tetrachloride or ADP-iron complexes (Poli etui., 1985). 

Poli, G., Dianzani, M.U., Cheeseman, K.H., Slater, T.F., Lang, J. and Esterbauer, H. (1985). Separation and characterization of the aldehydic products of lipid peroxidation stimulated by carbon tetrachloride or ADP-iron in isolated rat hepatocytes and rat liver microsomal suspensions. Biochem. J. 227, 629-638,... 

During catabolic and anabolic processes, a renovation of the molecular cellular components takes place. It should be emphasized that the catabolic and anabolic pathways are independent of each other. Be these pathways coincident and differing in the cycle direction only, the metabolism would have been side-tracked to the so-called useless, or futile, cycles. Such cycles arise in pathology, where a useless turnover of metabolites may occur. To avoid this undesirable contingency, the synthetic and degradative routes in the cell are most commonly separated in space. For example, the oxidation of fatty acids occurs in the mitochondria, while the synthesis thereof proceeds extramitochondrially, in the microsomes. 

Each entry is an average of data obtained from two separate experiments using different microsomal preparations. Enantiomeric composition was determined by CD spectral data (18) and by CSP- HPLC (19,20). 

It was recently reported that. >97% of BaP 4,5-epoxide metabolically formed from the metabolism of BaP in a reconstituted enzyme system containing purified cytochrome P-450c (P-448) is the 4S,5R enantiomer (24). The epoxide was determined by formation, separation and quantification of the diastereomeric trans-addition products of glutathione. Recently a BaP 4,5-epoxide was isolated from a metabolite mixture obtained from the metabolism of BaP by liver microsomes from 3-methylcholanthrene-treated Sprague-Dawley rats in the presence of the epoxide hydrolase inhibitor 3,3,3-trichloropropylene oxide, and was found to contain a 4S,5R/4R,5S enantiomer ratio of 94 6 (Chiu et. al., unpublished results). However, the content of the 4S,5R enantiomer was <60% when liver microsomes from untreated and phenobarbital-treated rats were used as the enzyme sources. Because BaP 4R,5S-epoxide is also hydrated predominantly to 4R,5R-dihydro-... 

BA trans-3.4-dihvdrodiol cannot be separated from BA trans-8.9-dihydrodiol in several HPLC conditions (27-29). Quantification of BA trana-3,4-dihydrodiol by HPLC can only be accomplished after converting the 3,4-dihydrodiol to its diacetate (25.26). The BA trans-3.4-dihydrodiol formed in BA metabolism by liver microsomes from pheno-barbital-treated rats was determined to have a 3R,4R/3S,4S enantiomer ratio of 69 31 (30). Recently we have determined the optical purity of the BA trans-3.4-dihvdrodiol formed in the metabolism of BA by three liver microsomes prepared from untreated rats and rats that had been pretreated with an enzyme inducer. As shown in Table II, cytochrome P-450 isozymes contained in liver microsomes from 3-methylcholanthrene- or phenobarbital-treated rats had similar stereoselectivity toward the 3,4-double bond of BA. BA trans-3.4-dihydrodiol is formed via the 3,4-epoxide intermediate (31). 

Separation into components can only be achieved by stopping the process when sedimentation of the desired component has occurred. The sediment is then resuspended in fresh solvent and centrifuged at a lower speed, when the heavier particles will sediment leaving the component in suspension. Such a method is known as differential sedimentation and is particularly useful for the fractionation of cellular components. The method outlined in Procedure 3.3 is simple and is designed to separate four main cellular fractions, namely, nuclear, mitochondrial, microsomal and soluble. 

In general, our studies with cytochrome P-450-dependent metabolism have emphasized the similarity of the hepatic MFO system in marine fish to that found in mammals. Thus, in the little skate (Raja erinaoea), a marine elasmobranch, enzyme activity is localized in the microsomal fraction, requires NADPH and molecular oxygen for maximum activity, and can be inhibited with CO (1, 2). Moreover, when hepatic microsomes from the little skate were solubilized and separated into cytochrome P-450, NADPH-cytochrome P-450 reductase, and lipid fractions, all three fractions were required for maximal MFO activity in the reconstituted system (3). We have also found, as have others, that the administration of polycyclic hydrocarbons (3-methylcholanthrene, 1,2,3,4-dibenzanthracene [DBA]), 2,3,7,8-tetrachlorodibenzo-p-dioxin... 

Initial studies designed to obtain a valid subcellular fractionation scheme for rainbow trout liver illustrated the aryl-hydrocarbon (benzo[a]pyrene] hydroxylase activity separated with glucose-6-phosphatase (35). This observation indicated that the trout hemoprotein P-450-mediated monooxygenation system was located within the endoplasmic reticulum (microsomal fraction). 

Comai, K. and Gaylor, J.L. Existence and separation of three forms of cytochrome P-450 from rat liver microsomes. J. Biol. Chem. 248 4947-4955. (1973)... 

Haugen, D.A., Van derHoeven, T.A. and Coon, M.J. Purified liver microsomal cytochrome P-450. Separation and characterization of multiple forms. J. Biol. Chem. (1975) 250 3567-3570. 

We employed various substrates to check for MFO in two bivalve species, a salt water mussel (Mytilus edulis) and a fresh water clam (Anodonta sp). Cytochrome P-450 was also studied. Organisms were exposed to 100 PPM Venezuelan crude in a stagnant system for up to one month. Enzyme assays were carried out with digestive gland 9000 g homogenates (17) and cytochrome P-450 analysis, with microsomes (21). The hydrocarbon substrates investigated included 1I+C-labelled benzo(a)pyrene, fluorene, anthracene, and naphthalene. The method used for separation of BP metabolites by thin layer radiochromatography has been described (7). The metabolite detection method for the other aromatic hydrocarbons was essentially the same except methylene chloride was used as metabolite extractant as well as TLC developer. Besides the hydrocarbon substrates, we also checked for other MFO reactions, N-dealkylase with C-imipramine (22) and 0-dealkylase with ethoxycoumarin (15). 

According to biochemical separation, location, and substrate specificity, epoxide hydrolases (EH) have been divided into a number of groups. In mammals, the insoluble microsomal epoxide hydrolases and the soluble cytosolic epoxide hydrolases are enzymes of broad and complementary substrate specificity. 

Capillary separation methods have been applied to the analysis of drugs (45-49) and of liposomes, microsomes, and viruses (34,50-53). De-... 

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