Apart from its role in vision, molecular function of vitamin A is not known. Problems have included the wide range of abnormalities encountered in deficiency and complicating effects of secondary inanition following onset of deficiency and loss of appetite. Hypotheses of the non-visual function of vitamin A have included a role for vitamin A in glycoprotein synthesis, or as a cofactor in enzyme activities, and in control of differentiation. The role for vitaman A in differentiation is currently the most favored hypothesis. In the present studies, the molecular basis of vitamin A in the control of liver cytochrome P-450 levels and hepatic drug metabolizing activities was examined in some detail. Tube-fed, retinoate cycled rats were used to eliminate non-specific effects associated with secondary inanition and to ensure stringent vitamin A deficiency throughout. Results obtained indicated a number of highly reproducible changes in drug metabolizing activity in vitamin A deficiency. Hexobarbital sleeping times increased from day 6 of deficiency T(,6) onwards. Radioactivity present in the plasma as native or metabolized hexobarbital in the first three hours after (14) C-hexobarbital injection indicated impaired drug transformation. Thereafter, clearance of hexobarbital or metabolites as determined by plasma t1/2 values reveled a slower rate of drug disposition. No change was observed in 24 hour renal excretion levels. Drug sensitivity based on concentration of hexobarbital and its metabolites at time of awakening was the same in deficient and control rats. Impaired drug metabolism in vitamin A deficiency was confirmed by in Vitro enzyme assays using hexobarbital, aminopyrine, benzphetamine and aniline as substrate probes. Kinetic studies during the deficiency phase (T(,0) - T(,13)) showed changes at T(,6) as for hexobarbital sleeping times. Reduction in enzyme activities intensified in the late stages of deficiency. Time course of changes in cyt P-450 levels were similar, whereas levels of cyt b(,5), another hemoprotein located in the microsomal membrance, were unaltered. Characterization of microsomal proteins by various techniques revealed numerous changes in vitamin A deficiency. A decrease in the levels of cyt P-450 was readily apparent. DEAE-Sephacel column chromatography of solubilized microsomes followed by serial SDS-PAGE showed that the molecular weight of cyt P-450 subtypes affected in vitamin A deficiency was 50K-52K. One of these subtypes was tentatively identified by high resolution two dimensional IF/SDS electrophoresis and in vitro enzyme assays as cyt P-450h. Other cyt P-450 species possibly affected included cyt P-450f, g and j. Not all proteins, however, were depressed in vitamin A deficiency

levels of some unidentified proteins increased, or were present in multiple forms. NADPH cyt P-450 reductase was found to exist in two forms. Only the minor form of molecular weight approximately 1,000 daltons less than the major form (74K) was markedly depressed in vitamin A deficiency. Decrease in concentration of this form was probably responsible for the 15% decrease in NADPH cyt P-450 reductase activity in unfractionated microsomal fractions. Cyt P-450 turnover rates determined after labelling of the heme moiety with (3)H-ALA for fast phase components increased sharply in vitamin A deficiency. Slow phase turnover rates and the ratio of fast phase to slow phase cyt P-450 were not changed. Although synthesis of cyt P-450 was depressed approximately 20% in the late stages of deficiency, total spectrally determined cyt P-450 levels decreased 50%. It seems unlikely, therefore, that altered synthesis is the primary determinant of final steady state concentrations of cyt P-450 in vitamin A deficiency, as has been proposed for fibronectin and other proteins. More probably, depressed synthesis represents a secondary response to still earlier changes in vitamin A deficiency including daily catabolic rate, the basis for which is not yet known.