Effect of protein deficiency on the inducibility of the hepatic microsomal drug-metabolizing enzyme system—I: Effect on substrate interaction with cytochrome P-450

Abstract

Male, weanling rats divided into three groups were maintained for 15 days on a semipurified diet containing either 5% casein fed ad lib. (group 1), 20% casein pair-fed to group 1 (group 2), or 20% casein fed ad lib. (group 3). After each group was further subdivided, animals were injected i.p. on days 11, 12, 13 and 14 with either 0.9% saline or phenobarbital (80 mg/kg) in 0.9% saline. Twenty-four hr after the last injection, animals were decapitated and liver microsomes were prepared. Contents of microsomal protein, phosphatidylcholine and cytochrome P-450 were measured and used as bases of expression for spectral dissociation constants ( K s ) and maximal spectral changes ( ΔA max) associated with the binding of ethylmorphine and aniline to the cytochrome P-450 hemoprotein of microsomes. Phenobarbital administration increased microsomal protein, cytochrome P-450, and phosphatidylcholine in all three dietary groups; however, in all groups, the increase in P-450 was relatively greater than that for phosphatidylcholine. Protein deficiency (group 1 vs 2) decreased P-450 and microsomal protein, but had no effect on phosphatidylcholine contents. The effect of total food restriction (group 2 vs 3) on each of these parameters was not significant. These data suggest that a portion of the induced cytochrome P-450 binding sites may be dependent on an association with phosphatidylcholine. The fraction of such phosphatidylcholine-associated sites relative to the total sites was greater during protein deficiency and was in agreement with a greater ΔA max per nanomole P-450 for ethylmorphine. Phenobarbital induction decreases the proposed fraction of phosphatidylcholine-associated P-450 sites relative to the total P-450 sites and results in a decrease in the ΔA max per nanomole P-450 for ethylmorphine. Phenobarbital increased the ΔA max per milligram of microsomal protein for aniline, which paralleled the increase in total P-450, thus indicating that the type II site may be independent of any association of cytochrome P-450 with phosphatidylcholine. These results indicate that phosphatidylcholine may play an important role in distinguishing the effects of dietary deficiency on type I substrate binding and the corresponding capacity for induction of the rat liver microsomal enzyme system.