We have investigated in rat liver whether different forms of cytochrome P-450 are alterated in hepatic disorders associated with impaired drug metabolism. Total hepatic cytochrome P-450 is decreased after either bile duct ligation or the administration of estradiol. In contrast, phenobarbital administrated alone increase hepatic content of cytochrome P-450, and when administrated with estradiol the reduction in cytochrome P-450 was prevented. Four forms of microsomal cytochrome P-450 apoproteins, ranging in molecular weight from 50 000 to 58,000, were tentatively identified in a sodium dodecyl sulfate(SDS)-6M urea polyaocrylamide gel electrophoresis system. Phenobrbital administration icreased primarily band IV(50,000 daltons). Bile duct ilgation was associated with a marked reduction in bands II and III while bands II and III were decreased with estradiol benzoate administration. Sumultaneous administration of phenobarbital and estradiol demonstrated a return of band I and an increase in density of bands III and IV. Simultaneous administration of cholic acid and estradiol demonstrated a return of band I and not altered in band III and IV. These studies support the hypothesis that multiple forms of cytochrome P-450 are present in liver microsomal membranes and that alterations in spenific apoproteins may be associated with an increase or a decrease in the functional properties of cytochrome P-450.

The effects of vitamin B complexes on both ring and N-hydroxylation of 2-ace-tylaminofluorene by rat hepatic microsomal fraction were studied.

In the presence of thiamine-Hcl during incubation, the total hydroxy-AAF was increased by 50.3%. 0.1mM and 1.0mM riboflavin inhibited only total hydroxy-AAF with 56.7% and 85.0 whereas N-hydroxy AAF was increased to some extent. 1.0mM niacin did not have much effect on the total hydroxy-AAF, but 0.1mM niacin decreased the total dydroxy-AAF by 28.6%. Presence of p-amino-benzoic acid, calcium pantothenate, pyridoxal-Hcl or vitamin B₁₂ inhibited the total hydroxy-AAF to some extent. But the radio of ring-and N-AAF hydroxylation was not changed by these vitaminn B complexes.

Our present results suggest that vitamin B complexes were not effective in vitro metabolism of 2-AAF to N-hydroxy AAF(activation step of AAF) by a cytochrome p-450 dependent mixed function oxidase system.

Our studies are concerned with cytochrome P-450 content and 2-acetylaminofluorene N-and ring-hydroxylation by thiamine deficient rat liver microsomes. Incubation medium contained 100mM HEPES buffer pH 7.8, 2mM NADPH, 100mM KF, 100nM [¹⁴C]-AAF and microsomal protein. After 30min, incubation at 37℃, ring and N-hydroxy-9-¹⁴C-AAF formations are assayed by radioactivity measurements after paper chromatography separation. The concentration of cytochrome P-450 is elevated in microsomes from rats fed the deficient diet. It appears that the thiamine deficient rat liver is capable of producing effects on the drug hydroxylation enzymes.