Rat liver microsomes

L14. Lucier, G. W., McDaniel, O. S., and Matthews, H. B., Microsomal rat liver UDP glucuronyltransferase. Effects of piperonyl butoxide and other factors on enzyme activity. Arch. Biochem. Biophys. 145, 520-530 (1971). 

It also appears that tiaprofenic acid, an NSAID that also undergoes inversion in rats, is not a substrate for purified microsomal rat liver long-chain acyl-CoA synthetase for which R-ibuprofen is a substrate [25]. This data may suggest that metabolic pathways involved in the inversion of tiaprofenic acid and possibly other 2-APA NSAIDs are different from those known for R-ibuprofen. It has been recently reported that in both an in vitro cell-free system and in rat liver homogenates the chiral inversion of ibuprofen was apparent when both CoA and ATP were present however, the NSAID KE-748 was not inverted [26]. To induce hepatic microsomal and outer mitochondrial long-chain fatty acid CoA ligase, rats were treated with clofibric acid [27]. Whereas chiral inversion of ibuprofen was enhanced significantly compared to controls, this was not the case for R(—)-KE-748. 

Both reductive steps in the conversion of HMG-CoA (3) into mevalonic acid (5) catalysed by microsomal rat liver HMG-CoA reductase occur by direct hydrogen transfer from the 4R position of NADPH this stereochemistry agrees with that previously reported for the corresponding yeast enzyme. The hemithioacetal addition compound (13) of 3R mevaldic add and coenzyme A... 

Squalene epoxidase, like most enzymes responsible for the later steps of sterol biosynthesis [43, 51], is membrane-bound which makes its purification in native form challenging. The purification is additionally complicated by the presence of a large number of cytochrome P450 and other enzymes that have similar hydro-phobicity and size as squalene epoxidase and are hence difficult to remove [52]. Most studies have been carried out with rat liver microsome squalene epoxidase either partially purified or as a homogenate of the cell membrane fraction. In vitro reconstitution of squalene epoxidase activity is absolutely dependent on molecular oxygen, NADPH, FAD, and NADPH-cytochrome c reductase [52, 53]. In this respect, squalene epoxidase resembles the cytochrome P450 enzymes described... 

Dexamethasone, the macrolide antibiotic triacetylo-leandromycin, and phenobarbital are all well established inducers of the CYP3A subfamily, and can increase microsomal 4-hydroxylation of RA in rat liver. To what extent this is also the case for humans is not completely clear. 

Figure 5.54 Structures of Praziquantel and its metabolites, cis- and fraw5-4-hydroxy-praziquantel. Reprinted from 7. 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.

Kitada, M., Igarashi, K., Hirose, S. Kitagawa, H. (1979). Inhibition by polyamines of lipid peroxide formation in rat liver microsomes. Biochemical Biophysical Research Communications, 87, 388-92. 

Tyagi SR, Singh Y, Sriram K, et al. 1985. Quality and quantity of dietary protein and acute endosulfan metabolic toxicity in rat liver microsomes. Indian J Med Res 81 480-487. 

Types of Carboxylesterase Isolated from Rat Liver Microsomes... 

Polyphenols and flavanoids in rat liver microsomal fractions have been demonstrated to inhibit glucuronidation of estrone and estradiol in vitro (Zhu et al, 1998). In addition, flavonoids have also been found to induce phase I and II enzymes in rats including UDP-glucuronosyl transferase (Seiss et al, 1996). However, the effects of phytoestrogens have not been evaluated for either their inhibition or induction of glucuronosyl transferase activity. 

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. 

Since the results of our experiments with isolated rat liver fractions supported a reaction sequence Initiated by microsomal oxidation of the nitrosamine leading to formation of a carbonium ion, the results of the animal experiment suggested that in the intact hepatocyte, one of the earlier electrophilic intermediates (II, III or V, Figure 1) is intercepted by nucleophilic sites in DNA (exemplified here by the N7 position of guanine) before a carbocation is formed. 

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). 

NPYR Rat Liver microsomes HPLC assay for a-hydroxylation 15... 

NPYR Rat Liver microsomes and postmitochondrial supernatant identification of 1,4-butanediol, and 4-hydroxybutyric acid 12... 

NPYR Rat Liver and lung microsomes and postmitochondrial supernatant a-hydroxylation but not p-hydroxy-lation in both tissues 13... 

NNN Rat Liver microsomes assay for a-hydroxylation and the effect of a-deuterium substitution 33... 

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

NPIP Rat Liver microsomes identification of 5-hydroxypenta-nal from a-hydroxylation 46... 

NHEX Rat Liver microsomes and post-mitochondrial supernatant identification of 3-hydroxyNHEX and 4-hydroxyNHEX 53... 

Table III. Rates of Formation of NNN Metabolites by F-344 Rat Liver Microsomes ...

The effects of deuterium substitution on the rates of a-hydroxylation of NNN have been measured. The results obtained in vitro, with rat liver microsomes, showed only a small deuterium isotope effect of 1.2 for 2 -hydroxylation, whereas a significant effect of 2.4-2.7 was observed for 5 -hydroxylation (33). Analogous results were obtained 2n vivo when the urinary metabolites... 

Only limited metabolic studies have been carried out on NPIP. It undergoes a-hydroxylation by rat liver microsomes to give 5-hydroxypentanal, a process analogous to the formation of... 

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. 

It appears that considerable metabolism of nitrosamines takes place in the liver, from the finding that most of them are activated by rat liver microsomes to bacterial mutagens. However, relatively few nitrosamines induce liver tumors in rats. The most common target is the esophagus and a wide variety of nitrosamines induce tumors in this organ of rats, some only tumors of the esophagus, others tumors of other organs also. 

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