Interlaboratory comparison of the assessment of P450 activities in human hepatic microsomal samples.
1. Although the importance of in vitro technology in supporting drug development is widely accepted, there is no real consensus about which approaches should be taken, which substrates should be used, or on the reliability and application of in vitro data. Consequently, as part of a collaborative project to characterize human liver with respect to the major forms of cytochrome P450, an interlaboratory comparison of the analysis of samples for form-specific activities was undertaken. 2. Microsomal fractions were isolated from five different human liver samples taken from the liver bank maintained at the Royal Postgraduate Medical School (RPMS). Aliquots from the five samples were sent to the 11 collaborating laboratories for characterization using their in-house, form-specific assays for cytochrome P450 activities. Although each laboratory assayed protein concentration, total cytochrome P450 content and enzyme activities were calculated using the protein estimation generated by RPMS to eliminate this possible source of variability. 3. With the exception of one laboratory, all estimates of protein concentration were similar (coefficient of variation, CoV, 9-13%) and the rank-order of the five samples was consistent across the laboratories. There was greater variability in the estimates of total cytochrome P450 content (CoV 28-43%), although again rank order of the samples across laboratories was fairly consistent. 4. The various laboratories used a number of different probe substrates, together with a range of conditions (substrate concentration, time of incubation, amount of protein), to assay for activity of CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4. However, apart from the occasional outlier, the five samples were ranked for activity of all these forms of cytochrome P450 with a high degree of consistency by the various laboratories and the choice of substrate had no appreciable effect on the ranking of the samples. 5. While this interlaboratory comparison has shown that greater consistency in the approach to in vivo determination of drug-metabolizing activity is desirable, there was little indication that any particular approach or substrate was superior to the others.
Boobis
A R
AR
Clinical Pharmacology Section, Imperial College School of Medicine, London, UK.
McKillop
D
D
Robinson
D T
DT
Adams
D A
DA
McCormick
D J
DJ
D001189:Aryl Hydrocarbon Hydroxylases; D019388:Cytochrome P-450 CYP1A2 / Q000378:metabolism; D065729:Cytochrome P-450 CYP2C9; D019389:Cytochrome P-450 CYP2D6 / Q000378:metabolism; D019392:Cytochrome P-450 CYP2E1 / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000032:analysis / Q000378:metabolism*; D007753:Laboratories / Q000592:standards; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000378:metabolism; D011785:Quality Assurance, Health Care; D015203:Reproducibility of Results; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000378:metabolism; D013379:Substrate Specificity; D015262:Xenobiotics / Q000493:pharmacokinetics
D007527:Isoenzymes; D015262:Xenobiotics; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D013250:Steroid Hydroxylases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D019392:Cytochrome P-450 CYP2E1; D001189:Aryl Hydrocarbon Hydroxylases; C504573:CYP1A2 protein, human; C104464:CYP3A protein, human; D019388:Cytochrome P-450 CYP1A2; D019389:Cytochrome P-450 CYP2D6; D051544:Cytochrome P-450 CYP3A; D039181:Steroid 16-alpha-Hydroxylase; C510163:CYP3A4 protein, human