Microsomes products

Recent studies suggest that many factors may affect hydroxyl radical generation by microsomes. Reinke et al. [34] demonstrated that the hydroxyl radical-mediated oxidation of ethanol in rat liver microsomes depended on phosphate or Tris buffer. Cytochrome bs can also participate in the microsomal production of hydroxyl radicals catalyzed by NADH-cytochrome bs reductase [35,36]. Considering the numerous demonstrations of hydroxyl radical formation in microsomes, it becomes obvious that this is not a genuine enzymatic process because it depends on the presence or absence of free iron. Consequently, in vitro experiments in buffers containing iron ions can significantly differ from real biological systems. 

Inducer(s) (agents employed to alter microsomal production and/or enzyme activity)... 

Corbett D, Baden DG, Chipko BR (1979) The non-microsomal production of N-(4-chloro-phenyl)glycolhydroxamic acid from 4-chloronitrosobenzene by rat liver homogenates. Bioorg Chem 8 227-235... 

Fig. 9. Cotranslational glycosylalion in the presence of PDl depleted-microsomes. Products of translation were immunoprecipitated with a specific antibody for IKN-y and were resolved by SDS—PAGE. The gel was loaded as follows lane 1, molecular-weight markers lane 2, translation products in the absence of microsomes lane 3, translation products in the presence of native microsomes lane 4, as lane 3 but treated posttranslationally with N-glycanase to remove oligosaccharide side chains lane 5, translation products synthesized in the presence of PDI-depleted microsomes (saponin washed). The inferred products of translation are labeled as follows x, product arising from incorrect initiation of translation core, unglycosylated core polypeptide singly and doubly glycosylated products as indicated.

Acetaminophen-induced hepatic toxicity (see Chapter 26) has been associated with alcoholic cirrhosis as a result of alcohol-induced increases in microsomal production of toxic acetaminophen metabolites. Hepatitis C appears to be an important cofactor in the development of end-stage... 

Literature reports iadicate that sodium sorbate causes weak genotoxic effects such as chromosomal aberrations and mutations ia mammalian cells (172,173). This effect is thought to be caused by oxidative products of sodium sorbate ia stored solutions (173—175). The main oxidation product of sodium sorbate, 4,5-oxohexenoate, is mutagenic ia a Salmonella mammahan-microsome test (176). Sorbic acid and potassium sorbate were not genotoxic under the same test procedures (167,172,174—177). 

Medicinal chemistry has frequently drawn inspiration and important new leads from the examination of natural products, and this was proven to be the case once more. In 1992, researchers at Merck and Glaxo announced, almost simultaneously, the independent discovery of the same new class of natural products from two different fungi. As a consequence, the same family of natural products has two names - the zaragozic acids (Merck)4 or the squalestatins (Glaxo).5 A typical member of the family, zaragozic acid A (squa-lestatin SI) (1) was shown to have a tremendous affinity for squalene synthase (K, = 79 pM for rat microsomal squalene synthase) and could even lower serum cholesterol levels in vivo in a population of marmosets.6... 

Ethanol is oxidized by alcohol dehydrogenase (in the presence of nicotinamide adenine dinucleotide [NAD]) or the microsomal ethanol oxidizing system (MEOS) (in the presence of reduced nicotinamide adenine dinucleotide phosphate [NADPH]). Acetaldehyde, the first product in ethanol oxidation, is metabolized to acetic acid by aldehyde dehydrogenase in the presence of NAD. Acetic acid is broken down through the citric acid cycle to carbon dioxide (CO2) and water (H2O). Impairment of the metabolism of acetaldehyde to acetic acid is the major mechanism of action of disulfiram for the treatment of alcoholism. 

Figure 5.55 MS" spectra from rntz 329, the (M + H)+ ion of an hydroxy metabolite of Praziquantel, and the base peaks in subsequent product-ion spectra. Reprinted from J. Chromatogr., B, 708, Lerch, C. and Blaschke, G., Investigation of the stereoselective metabolism of Praziquantel after incubation with rat liver microsomes by capillary electrophoresis and liquid chromatography-mass spectrometry , 267-275, Copyright (1998), with permission from Elsevier Science.

The samples of l,6-T2-DBpD and l,6-T2-2,3,7,8-Cl4-DBpD are useful in metabolism and mode of action studies. For example, when incubated with rabbit liver microsomes, l,6-T.>-DBpD is extensively metabolized to polar product(s) but only when these preparations are fortified with reduced nicotinamide-adenine dinucleotide phosphate. Under the same conditions l,6-T2-2,3,7,8-Cl4-DBpD is completely resistant to metabolic attack. In some types of studies, a higher specific activity possibly is desirable i.e., >1 Ci/mmole), and this can be achieved, with the methodology already developed, by using larger amounts of tritium gas or working on a larger synthetic scale so that it is not necessary to add unlabeled materials to assist in crystallization steps where a certain minimum amount of compound is necessary. 

Figure 1. Reaction scheme for production of alkylating agent following microsomal metabolism of S-nitrosodialkylamine (3),...

Analysis of reaction mixtures for 1-propanol and 2-propanol following incubation of NDPA with various rat liver fractions in the presence of an NADPH-generating system is shown in Table I ( ). Presence of microsomes leads to production of both alcohols, but there was no propanol formed with either the soluble enzyme fraction or with microsomes incubated with SKF-525A (an inhibitor of cytochrome P450-dependent oxidations). The combined yield of propanols from 280 ymoles of NDPA was 6.1 ymoles and 28.5 ymoles for the microsomal pellet and the 9000 g supernatant respectively. The difference in the ratio of 1- to 2-propanol in the two rat liver fractions may be due to differences in the chemical composition of the reaction mixtures (2) Subsequent experiments have shown that these ratios are quite reproducible. For comparison, Table I also shows formation of propanols following base catalyzed decomposition of N-propyl-N-nitrosourea. As expected (10,11), both propanol isomers were formed, the total yield in this case being almost quantitative. 

Further experiments on the metabolism of NDPA by rat liver fractions have also provided support for the 3-oxidation mechanism of Kruger. In addition to the products of a-oxidation, we have isolated and characterized NHPPA as a major product of the microsomal oxidation of NDPA (18). We have also shown that NHPPA is further oxidized to NOPPA by microsomal preparations from rat liver (18), Finally, with NOPPA as substrate, we have shown that metabolism takes place principally by reduction with the microsomal or soluble fraction of rat liver to yield NHPPA, although microsomal a-oxidation also takes place to some extent (19). 

NNN Rat Liver microsomes identification of products of p-hydroxylation and pyridine-N-oxidation 36... 

Analysis of the products formed from NHEX in vitro using rat liver microsomes and postmitochondrial supernatant resulted in the identification of 3-hydroxyNHEX from p-hydroxylation and 4-hydroxyNHEX from y-hydroxylation. The ratio of 4-hydroxyNHEX to 3-hydroxyNHEX was 3 to 1. Both conformeric forms of each of these metabolites were detected (53). From ix vitro data which are available so far for NPYR, NNN, NPIP, and NHEX, it does appear that the rates of a- and y-hydroxylation (when possible), exceed those of p-hydroxylation. 

Salgo, M.G. et al.. Beta carotene and its oxidation products have different effects on microsome mediated binding of benzo[a]pyrene to DNA, Free Rad Biol. Med, 26, 162, 1999. 

A full understanding of the role of pectin in plant development requires elucidation of the mechanisms that regulate p>ectin biosynthesis (6). Our strategy for studying the biosynthesis of HGA was to 1) establish a PGA-GalAT assay that would allow detection of synthesized HGA, 2) characterize the enzyme in microsomal membranes, 3) characterize the product synthesized by the enzyme in microsomal membranes, and 4) solubilize the enzyme and characterize the solubilized enzyme and its product. 

CHARACTERIZATION OF THE PRODUCTS SYNTHESIZED BY PGA-GALAT IN MICROSOMAL MEMBRANES... 

Second-derivative spectrophotometry has been used to monitor the time-dependent production of cis,tmns-(Xmax 242 nm) and trans, tram- (Xmax 232 nm) diene conjugates of microsomal PUFAs following the exposure of rats to carbon tetrachloride (CCU) (Corongui et al., 1986). These signals have been postulated to be derived from mixtures of peroxidized substrates. Previous studies using chemical model systems have established that autoxidation of linolenic or arachidonic acid results in the production of cis, trans- and tmns, trawr-conjugated diene... 

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,... 

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