Storage stability study of porcine hepatic and intestinal cytochrome P450 isoenzymes by use of a newly developed and fully validated highly sensitive HPLC-MS/MS method
- 246 Downloads
Microsomes are an ideal medium to investigate cytochrome P450 (CYP450) enzyme-mediated drug metabolism. However, before microsomes are prepared, tissues can be stored for a long time. Studies about the stability of these enzymes in porcine hepatic and intestinal tissues upon storage are lacking. To be able to investigate CYP450 stability in microsomes prepared from these tissues, a highly sensitive and rapid HPLC-MS/MS method for the simultaneous determination of six CYP450 metabolites in incubation medium was developed and validated. The metabolites, paracetamol (CYP1A), 7-hydroxy-coumarin (CYP2A), 1-hydroxy-midazolam (CYP3A), 4-hydroxy-tolbutamide (CYP2C), dextrorphan (CYP2D), and 6-hydroxy-chlorzoxazone (CYP2E) were extracted with ethyl acetate at pH 1.0, followed by evaporation and separation on an Agilent Zorbax Eclipse Plus C18 column. The method was fully validated in a GLP-compliant laboratory according to European guidelines and was highly sensitive (LOQ = 0.25–2.5 ng/mL), selective, had good precision (RSD-within, 1.0–9.1%; RSD-between, 1.0–18.4%) and accuracy (within-run, 83.3–102%; between-run, 78.5–102%), and showed no relative signal suppression and enhancement. Consequently, this method was applied to study the stability of porcine hepatic and intestinal CYP450 isoenzymes when tissues were stored at − 80 °C. The results indicate that porcine CYP450 isoenzymes are stable in tissues at least up to 4 months when snap frozen and stored at − 80 °C. Moreover, the results indicate differences in porcine CYP450 stability compared to rat, rabbit, and fish CYP450, as observed by other research groups, hence stressing the importance to investigate the CYP450 stability of a specific species.
KeywordsHPLC-MS/MS Pig Cytochrome P450 Stability Liver Intestine
This work was supported by the Ghent University Special Research Fund grant BOF. DOC.2015.0075.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
Welfare of animals
All procedures followed were in accordance to the ethical standards of the ethical committee of the Faculties of Veterinary Medicine and Bioscience Engineering of Ghent University. The pigs were subject to the approval with number EC2015_113.
- 5.Yamazaki H, Inoue K, Turvy CG, Guengerich FP, Shimada T. Effects of freezing, thawing, and storage of human liver samples on the microsomal contents and activities of cytochrome p450 enzymes. Drug Metab Dispos. 1997;25(2):168–74.Google Scholar
- 6.Förlin L, Andersson T. Storage conditions of rainbow trout liver cytochrome p-450 and conjugating enzymes. Comp Biochem Physiol. 1985;80(3):569–72.Google Scholar
- 8.Tredger JM, Chhabra RS. Preservation of various microsomal drug metabolizing components in tissue preparations from the livers, lungs and small intestines of rodents. Drug Metab Dispos. 1976;4(5):451–9.Google Scholar
- 9.Paine MF, Khalighi M, Fisher JM, Shen DD, Kunze KL, Marsh CL, et al. Characterization of interintestinal and intraintestinal variations in human CYP3A-dependent metabolism 1. J Pharmacol Exp Ther. 1997;283(3):1552–62.Google Scholar
- 13.Gasthuys E, Vandecasteele T, De Bruyne P, Vande Walle J, De Backer P, Devreese M, et al. the potential use of piglets as human pediatric surrogate for preclinical pharmacokinetic and pharmacodynamic drug testing. Curr Pharm Des. 2016;22:1–17.Google Scholar
- 14.Anzenbacher P, Soucek P, Anzenbacherová E, Gut I, Hrubý K, Svoboda Z, et al. Presence and activity of cytochrome P450 isoforms in minipig liver microsomes; comparison with human liver samples. Drug Metab Dispos. 1998;26(1):56–9.Google Scholar
- 17.Anzenbacherová E, Baranová J, Zuber R, Pechová A, Anzenbacher P, Soucek P, et al. Model systems based on experimental animals for studies on drug metabolism in man: (mini)pig cytochromes P450 3A29 and 2E1. Basic Clin Pharmacol Toxicol. 2004;95:244–5.Google Scholar
- 20.Dinger J, Meyer MR, Maurer HH. Development and validation of a liquid-chromatography high-resolution tandem mass spectrometry approach for quantification of nine cytochrome P450 (CYP) model substrate metabolites in an in vitro CYP inhibition cocktail. Anal Bioanal Chem. 2014;406(18):4453–64.CrossRefGoogle Scholar
- 24.He F, Bi H, Xie Z, Zuo Z, Li J, Li X, et al. Rapid determination of six metabolites from multiple cytochrome P450 probe substrates in human liver microsome by liquid chromatography/mass spectrometry: application to high-throughput inhibition screening of terpenoids. Rapid Commun Mass Spectrom. 2007;21:635–43.CrossRefGoogle Scholar
- 32.Commision Decision of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. Off J Eur Communities. 2002;L221:8–36.Google Scholar
- 33.Knecht J, Stork G. Prozentuales und logarithmisches Verfahren zur Berechnung von Eichkurven. Fresenius’ Z Anal Chem. 1974;99(270):97–9.Google Scholar
- 35.Osselaere A, De Bock L, Eeckhaut V, De Backer P, Van Bocxlaer J, Boussery K, et al. Hepatic and intestinal CYP3A expression and activity in broilers. J Vet Pharmacol Ther. 2013;36:588–93.Google Scholar