Interlaboratory comparison of total cytochrome P-450 and protein determinations in rat liver microsomes

Rutten, Albert J.; Falke, H.E.; Catsburg, J. F.; Topp, R.J.; Blaauwboer, B.J.; Holteijn, I. van; Doorn, L. G. Van; Leeuwen, F.X.R. van

doi:10.1007/bf00324544

Cited by 199 publications

(68 citation statements)

References 8 publications

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“…The subcellular fractions were prepared according to Rutten et al [34]. Briefly, two volumes of KCL (1.15%) and EDTA (0.1mM) solution were added to a tissue sample and the mixture was homogenised in a Potter-Elvehjem apparatus with a Teflon pestle.…”

Section: Preparation Of Liver Subcellular Fractionsmentioning

confidence: 99%

Bioactivation of zearalenone by porcine hepatic biotransformation

2005

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-Zearalenone (ZEA) is a resorcylic acid lactone derivative produced by various Fusarium species that are widely found in food and feeds. The structure of zearalenone is flexible enough to allow a conformation able to bind to mammalian oestrogen receptors, where it acts as an agonist. Using oestrogen-dependent Human Breast Cancer (MCF-7) cells, the oestrogenic activity of zearalenone and its derivatives were compared using 17 β-oestradiol as a positive control. The results obtained demonstrate that the oestrogenic potency of ZEA derivatives could be ranked in the following order: α-zearalenol > α-zearalanol > zearalenone > β-zearalenol. Since pigs have been reported to be among the most sensitive animal species, biotransformation studies with pig liver subcellular fractions were conducted. These studies indicated that α-zearalenol is the main hepatic metabolite of zearalenone in pigs, and it is assumed that 3α-and 3β-hydroxysteroid dehydrogeneases are involved in the hepatic biotransformation, since the formation of α-zearalenol and β-zearalenol could be inhibited by prototypic substrates for either enzyme. The bioactivation of ZEA into the more active α-zearalenol seems to provide a possible explanation for the observed high sensitivity of pigs towards feedingstuffs contaminated with the mycotoxin. zearalenone / MCF-7 cells / oestrogenic effects / biotransformation / pigs

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Section: Preparation Of Liver Subcellular Fractionsmentioning

confidence: 99%

Bioactivation of zearalenone by porcine hepatic biotransformation

2005

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“…The CO-dierence spectrum was recorded as described by Rutten et al (1987). A lyase sample was diluted to a concentration of 5.0 nkat/ml with 0.1 M phosphate buer, pH 7.4, containing 1 mM EDTA and 20% (v/v) glycerol.…”

Section: Heme Analysesmentioning

confidence: 99%

Purification, stabilization and characterization of tomato fatty acid hydroperoxide lyase

et al. 2000

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Fatty acid hydroperoxide lyase (HPO-lyase) was puri®ed 300-fold from tomatoes. The enzymatic activity appeared to be very unstable, but addition of Triton X 100 and b-mercaptoethanol to the buer yielded an active enzyme that could be stored for several months at À808C. The enzyme was inhibited by desferoxamine mesylate (desferal), 2-methyl-1,2-di-3-pyridyl-1-propanone (metyrapone), nordihydroguaiaretic acid (NDGA), n-propyl gallate and butylated hydroxyanisole, suggesting the involvement of free radicals in the reaction mechanism and the existence of a prosthetic group in the active center. However, no heme group could be demonstrated with the methods commonly used to identify heme groups in proteins. Only 13-hydroperoxides from linoleic acid (13-HPOD) and a-linolenic acid (a-13-HPOT) were cleaved by the tomato enzyme, with a clear preference for the latter substrate. The pH-optimum was 6.5, and for concentrations lower than 300 mM a typical Michaelis±Menten curve was found with a K m of 77 mM. At higher a-13-HPOT concentrations inhibition of the enzyme was observed, which could (at least in part) be attributed to 2E-hexenal. A curve of the substrate conversion as a function of the enzyme concentration revealed that 1 nkat of enzyme activity converts 0.7 mmol a-13-HPOT before inactivation. Headspace analysis showed that tomato HPO-lyase formed hexanal from 13-HPOD and 3Z-hexenal from a-13-HPOT. A trace of the latter compound was isomerized to 2E-hexenal. In addition to the aldehydes, 12-oxo-9Z-dodecenoic acid was found by GC/MS analysis. To a small extent, isomerization to 12-oxo-10E-dodecenoic acid occurred. #

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“…Cytochrome P450 content was determined by the DT-difference method as described by Rutten et al (1987). 7-Ethoxycoumarin deethylase and aniline-p-hydroxylase activity was determined by Greenlee & Poland (1978) and Macleod et al (1973 Warfarin and acenocoumarol enantiomers were at our disposal (Hermans & Thijssen, 1991 and inhibition mechanisms were determined by the use of Dixon plots and double reciprocal plots respectively.…”

Section: Preparation Of Microsomalfractionsmentioning

confidence: 99%

Human liver microsomal metabolism of the enantiomers of warfarin and acenocoumarol: P450 isozyme diversity determines the differences in their pharmacokinetics

Hermans

Thijssen

1993

British J Pharmacology

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1 To explain the large differences in (the stereoselectivity of) the clearances of the enantiomers of warfarin and acenocoumarol (4'-nitrowarfarin) their human liver microsomal metabolism has been studied and enzyme kinetic parameters determined. The effects of cimetidine, propafenone, sulphaphenazole, and omeprazole on their metabolism has been investigated. 2 The 4-hydroxycoumarins follow similar metabolic routes and are mainly hydroxylated at the 6-and 7-position (accounting for 63 to 99% of the metabolic clearances).3 Due to the lower Km values of R-and S-acenocoumarol and higher Vma. values of S-acenocoumarol, the overall metabolic clearances of R/S acenocoumarol exceed those of R/S warfarin 6 and 66 times respectively. 4 The metabolism of both compounds is stereoselective for the S-enantiomers, which is 10 times more pronounced in the case of acenocoumarol.5 Except for the 7-hydroxylation of the R-enantiomers (r = 0.90; P <0.025), the 6-and 7-hydroxylation rates of R/S warfarin do not correlate with those of R/S acenocoumarol. 6 Sulphaphenazole competitively inhibits the 7-and in some samples partly (up to 50%) the 6-hydroxylation of S-warfarin as well as the 7-hydroxylation of R-and S-acenocoumarol and the 6-hydroxylation of S-acenocoumarol (Kis ranging from 0.5-1.3 JAM).7 Omeprazole partly (40-80%) inhibits the 6-and 7-hydroxylation of R-warfarin (Ki = 99 and 117 pM) and of R-(Ki = 219 and 7.2 jAM) and S-acenocoumarol (Ki = 6.1 and 7.7 JiM) but not S-warfarin in a competitive manner. 8 Differences in the partial (up to 40%) inhibition of the metabolism of the enantiomers of the 4-hydroxycoumarins were also observed for the relatively weak inhibitors, propafenone and cimetidine. 9 The results suggest that the coumarin ring hydroxylations of both compounds are catalysed by different combinations of P450 isozymes. The 7-hydroxylation of R/S acenocoumarol and the 6-hydroxylation of S-acenocoumarol are at least partly conducted by (a) P450 isozyme(s) of the 2C subfamily different from P450 2C9 (the main S-warfarin 7-and 6-hydroxylase).

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Interlaboratory comparison of total cytochrome P-450 and protein determinations in rat liver microsomes

Cited by 199 publications

References 8 publications

Bioactivation of zearalenone by porcine hepatic biotransformation

Bioactivation of zearalenone by porcine hepatic biotransformation

Purification, stabilization and characterization of tomato fatty acid hydroperoxide lyase

Human liver microsomal metabolism of the enantiomers of warfarin and acenocoumarol: P450 isozyme diversity determines the differences in their pharmacokinetics

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