Immunohistochemical localization and quantitation of NADPH-cytochrome P- 450 reductase in human liver

ME McManus, PD Hall, I Stupans, J Brennan, W Burgess, R Robson and DJ Birkett

An antibody raised in a goat against the human liver NADPH-cytochrome P- 450 reductase (EC 1.6.2.4.) enzyme has been used to: 1) immunoquantify the level of this enzyme in human liver microsomes, and 2) study the distribution of the reductase across the human liver acinus. Employing the Western blot procedure, anti-human reductase IgG recognized a single band in human liver microsomes which corresponded in molecular weight to the purified reductase. The content of the NADPH-cytochrome P- 450 reductase in six normal human livers varied from 87 to 121 pmol/mg of microsomal protein. NADPH-cytochrome P-450 reductase activity of the same microsomes ranged from 107 to 222 nmol of cytochrome c reduced per min per mg of protein. The correlation between reductase content and activity (r = 0.54) was not statistically significant (p greater than 0.1). The total cytochrome P-450 content (cytochrome P-450 and P-420) of the same microsomes varied from 423 to 1413 pmol/mg of microsomal protein. The average ratio of cytochrome P-450 to NADPH-cytochrome P- 450 reductase was 7.1:1 +/- 3.1 (mean +/- SD) in the human liver microsomal preparations studied. The reductase was found to be nonuniformly distributed across the human liver acinus. Although all hepatocytes stained positively for NADPH-cytochrome P-450 reductase, the staining intensity was highest in zone 3 and in some cases also in zone 1 hepatocytes. These results show that human liver contains a gross excess of cytochrome P-450 molecules to NADPH-cytochrome P-450 reductase molecules. Furthermore, the differential distribution of the reductase within the human liver acinus may lead to a better understanding of the mechanism underlining site-specific drug hepatotoxicity.

Volume 32, Issue 1, pp. 189-194, 08/01/1987
Copyright © 1987 by American Society for Pharmacology and Experimental Therapeutics

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