[41] ω- and (ω-1)-hydroxylation of eicosanoids and fatty acids by high-performance liquid chromatography

doi:10.1016/0076-6879(91)06112-G

Methods in Enzymology

Volume 206, 1991, Pages 432-441

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This chapter discusses the ω and (ω-1)-hydroxylation of eicosanoids and fatty acids by high-performance liquid chromatography (HPLC). Cytochromes P450 of the IVA family catalyze the hydroxylation of a number of endogenous substrates including prostaglandins (PG), leukotriene B4 (LTB4), and fatty acids at their ω and ω-1 carbon atoms. ω-Oxidation is a major route in prostaglandin metabolism, with a large proportion of prostaglandins being excreted in the urine as ω-oxidized products. Hydroxylated products are formed enzymatically by incubating fatty acids or prostaglandins with lung microsomes from pregnant rabbits, liver microsomes from diethylhexyl phthalate (DEHP)-treated rats, or purified cytochromes P450 from these tissues in reconstituted enzyme systems. The enzymatic rates of formation of products are calculated based on the percentage of radioactivity for the given metabolite relative to the total radioactivity eluting from the HPLC column.

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Microsomal protein and total CYP content were measured using methods proposed by Lowry et al. (1951) and Omura and Sato (1964), respectively, using a Hitachi U3000 dual beam spectrophotometer. Previously described chromatographic methods were also used for determination of the activities of testosterone hydroxylase and testosterone dehydrogenase (Sonderfan et al., 1987; Waxman, 1988), as well as lauric acid 11- and 12-hydroxylase (Okita et al., 1991). Ethoxyresorufin O-deethylase activity was determined by using a continual monitoring fluorimetric method.
Toxicology studies conducted with oil-soluble rosemary extracts to support authorization as a food additive (antioxidant) in the EU include an Ames test using a supercritical carbon dioxide extract (D74), a full 90-day study using D74 and an acetone extract (F62), and an investigative 90-day study with a 28-day recovery period (using D74 only). D74 was non-mutagenic in the Ames test. In the full 90-day study, where rats (20/sex/group) were either provided control diet or diets containing D74 (300, 600, or 2400 mg/kg) or F62 (3800 mg/kg), liver enlargement and hepatocellular hypertrophy were observed. To determine a mode of action and assess the reversibility of the hepatic effects, an investigative 90-day study was conducted using female rats (10/group receiving control diet or diet containing 2400 mg/kg D74). Liver enlargement was fully reversible after 28 days and microsomal enzyme analysis revealed reversible induction of cytochrome P450 enzymes (CYP2A1, CYP2A2, CYP2C11, CYP2E1, and CYP4A), demonstrating that the hepatic effects were adaptive and of no toxicological concern. Therefore, the highest dietary concentrations were established as the NOAELs. The investigative 90-day study NOAEL (providing 64 mg/kg bw/day carnosol and carnosic acid [the primary antioxidant components]) was used to establish a temporary ADI for rosemary extracts.
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It is important to investigate the induction of cytochrome P450 (CYP) enzymes by drugs. The most relevant end point is enzyme activity; however, this requires many cells and is low throughput. We have compared the CYP1A, CYP2B and CYP3A induction response to eight inducers in rat and human hepatocytes using enzyme activities (CYP1A2 (ethoxyresorufin), 2B (benzoxyresorufin for rat and bupropion for human) and CYP3A (testosterone)) and Taqman™ Low Density Array (TLDA) analysis. There was a good correlation between the induction of CYP1A2, CYP2B6 and CYP3A4 enzyme activities and mRNA expression in human hepatocytes. In contrast, BROD activities and mRNA expression in rat hepatocytes correlated poorly. However, bupropion hydroxylation correlated well with Cyp2b1 expression in rat hepatocytes. TLDA analysis of a panel of mRNAs encoding for CYPs, phase 2 enzymes, nuclear receptors and transporters revealed that the main genes induced by the 8 compounds tested were the CYPs. AhR ligands also induced UDP-glucuronosyltransferases and glutathione S-transferases in rat and human hepatocytes. The transporters, MDR1, MDR3 and OATPA were the only transporter genes significantly up-regulated in human hepatocytes. In rat hepatocytes Bsep, Mdr2, Mrp2, Mrp3 and Oatp2 were up-regulated. We could then show a good in vivo:in vitro correlation in the induction response of isolated rat hepatocytes and ex-vivo hepatic microsomes for the drug development candidate, EMD392949. In conclusion, application of TLDA methodology to investigate the potential of compounds to induce enzymes in rat and human hepatocytes increases the throughput and information gained from one assay, without reducing the predictive capacity.
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The retention times of 6β-, 7α-, 11β- and 2α-hydroxytestosterone were approximately 10.2, 11.2, 28.5, and 29.8 min, respectively, and the retention time of testosterone was approximately 34.5 min. Lauric acid 12-hydroxylase was assayed using a modification of published methods [20,21]. A reaction mixture containing 0.2 mg/ml microsomal protein, an NADPH regenerating system and 100 μM [14C]-lauric acid (255.4 nCi/ml) in 100 mM Tris (pH 7.4), was incubated at 37 °C for 10 min.
Farnesol demonstrates antitumor activity in several animal models for human cancer and was being considered for development as a cancer chemopreventive agent. This study was performed to characterize the effects of minimally toxic doses of farnesol on the activity of phase I and II drug metabolizing enzymes. CD^® rats (20/sex/group) received daily gavage exposure to farnesol doses of 0, 500, or 1000 mg/kg/day for 28 days; 10 rats/sex/group were necropsied at the termination of farnesol exposure; remaining animals were necropsied after a 28-day recovery period. No deaths occurred during the study, and farnesol had no significant effects on body weight, food consumption, clinical signs, or hematology/coagulation parameters. Modest but statistically significant alterations in several clinical chemistry parameters were observed at the termination of farnesol exposure; all clinical pathology effects were reversed during the recovery period. At the termination of dosing, the activities of CYP1A, CYP2A1–3, CYP2B1/2, CYP2C11/12, CYP2E1, CYP3A1/2, CYP4A1–3, CYP19, glutathione reductase, NADPH/quinone oxidoreductase and UDP-glucuronosyltransferase were significantly increased in the livers of farnesol-treated rats; farnesol also increased the activity of glutathione S-transferase in the kidney. The effects of farnesol on hepatic and renal enzymes were reversed during the recovery period. At the end of the dosing period, increases in absolute and relative liver and kidney weights were seen in farnesol-treated rats. These increases may be secondary to induction of drug metabolizing enzymes, since organ weight increases were not associated with histopathologic alterations and were reversed upon discontinuation of farnesol exposure. Administration of farnesol at doses of up to 1000 mg/kg/day induced reversible increases in the activities of several hepatic and renal drug metabolizing enzymes in rats, while inducing only minimal toxicity. It is concluded that non-toxic or minimally toxic doses of farnesol could alter the metabolism, efficacy, and/or toxicity of drugs with which it is co-administered.
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A gas chromatography–mass spectrometry assay method for the analysis of lauric, myristic, and palmitic acids and their ω and ω₋₁ hydroxylated metabolites from in vitro incubations of cytochrome P450 CYP4A1, involving solid-phase extraction and trimethysilyl derivatization, was developed. The assay was linear, precise, and accurate over the range 0.5 to 50 μM for all the analytes. It has the advantages of a more rapid analysis time, an improved sensitivity, and a wider range of analytes compared with other methods. An artificial membrane system was optimized for application to purified CYP4A1 enzyme by investigating the molar ratios of cytochrome b₅ and cytochrome P450 reductase present in the incubation mixture. Using this method, the kinetics of ω and ω₋₁ oxidation of lauric, myristic, and palmitic acids by CYP4A enzymes were measured and compared in rat liver microsomes and an artificial membrane system.
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Citation Excerpt :
Incubations were performed at 37 °C for 20 min and terminated by the addition of 0.1 ml of 6% acetic acid in ethanol. Samples were centrifuged and the supernatant analyzed by high performance liquid chromatography according to Okitaet al. (12) using a Radiomatic detector. As positive controls of the activity assays, freshly prepared pig liver microsomes were used and incubated with respective substrate according to the procedure described above except that NADPH-cytochrome P450 reductase was omitted.
A cytochrome P450 expressed in pig liver was cloned by polymerase chain reaction using oligonucleotide primers based on amino acid sequences of the purified taurochenodeoxycholic acid 6α-hydroxylase. This enzyme catalyzes a 6α-hydroxylation of chenodeoxycholic acid, and the product hyocholic acid is considered to be a primary bile acid specific for the pig. The cDNA encodes a protein of 504 amino acids. The primary structure of the porcine taurochenodeoxycholic acid 6α-hydroxylase, designated CYP4A21, shows about 75% identity with known members of the CYP4A subfamily in rabbit and man. Transfection of the cDNA for CYP4A21 into COS cells resulted in the synthesis of an enzyme that was recognized by antibodies raised against the purified pig liver enzyme and catalyzed 6α-hydroxylation of taurochenodeoxycholic acid. The hitherto known CYP4A enzymes catalyze hydroxylation of fatty acids and prostaglandins and have frequently been referred to as fatty acid hydroxylases. A change in substrate specificity from fatty acids or prostaglandins to a steroid nucleus among CYP4A enzymes is notable. The results of mutagenesis experiments indicate that three amino acid substitutions in a region around position 315 which is highly conserved in all previously known CYP4A and CYP4B enzymes could be involved in the altered catalytic activity of CYP4A21.AJ278474
Expressed CYP4A4 metabolism of prostaglandin E<inf>1</inf> and arachidonic acid
2001, Archives of Biochemistry and Biophysics
Cytochrome P4504A4 (CYP4A4) is a hormonally induced pulmonary cytochrome P450 which metabolizes prostaglandins and arachidonic acid (AA) to their ω-hydroxylated products. Although the physiological function of this enzyme is unknown, prostaglandins play an important role in the regulation of reproductive, vascular, intestinal, and inflammatory systems and 20-hydroxyeicosatetraenoic acid, the ω-hydroxylated product of arachidonate, is a potent vasoconstrictor. Therefore, it is important to obtain sufficient quantities of the protein for kinetic and biophysical characterization. A CYP4A4 construct was prepared and expressed in Escherichia coli. The enzyme was purified, and its activity with substrates prostaglandin E₁ (PGE₁) and AA was examined in the presence and absence of cytochrome b₅ (cyt b₅) and with a heme-depleted form of cyt b₅ (apo b₅). The stimulatory role played by cyt b₅ in this system is not dependent on electron transfer from cyt b₅ to the CYP4A4 as similar stimulation was observed with apo b₅. Rapid kinetic measurement of CYP4A4 electron transfer rates confirmed this result. Both flavin and heme reduction rates were constant in the absence and presence of cyt b₅ or apo b₅. CD spectroscopy demonstrated that a conformational change occurred in CYP4A4 protein upon binding of cyt b₅ or apo b₅. Finally, acetylenic fatty acid inhibitors 17-octadecynoic acid, 12-hydroxy-16-heptadecynoic acid, 15-hexadecynoic acid, and 10-undecynoic acid (10-UDYA) were used to probe the substrate-binding pocket of CYP4A4. The short-chain fatty acid inhibitor 10-UDYA was unable to inhibit either PGE₁ or AA metabolism. All but 10-UDYA were effective inhibitors of CYP4A4.

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[41] ω- and (ω-1)-hydroxylation of eicosanoids and fatty acids by high-performance liquid chromatography

Publisher Summary

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Pharmacol. Ther.

J. Biol. Chem.

Anal. Biochem.

Anal. Biochem.

Arch. Biochem. Biophys.