Biochemical Pharmncology, Vol. 36, No. 6, pp. 91%924, 1987. Printed in Great Britain. 1X06-2952/87 $3.00 + 0.00 Pergmon Journals Ltd. zyxwvuts THE FORMATION OF ALL-TRANS-RETINOIC ACID FROM ALL-TRANS-RETINOL IN HAIRLESS MOUSE SKIN MICHAEL J. CONNOR* and MARK H. SMIT Division of Dermatology, UCLA School of Medicine, Los Angeles, CA 90024, U.S.A. zyxwvutsrqponmlkji (Received 28 March 1986; accepted 15 August 1986) Abstract-All-tmns-retinoic acid formation from topically applied retinol has been demonstrated in the skin of skh/hrl (hairless) mice. The all-truns-retinoic acid was identified on the basis of its chro- matographic properties on HPLC at various pH values, its photoisomerization to reaction products identical to those formed from authentic all-tram-retinoic acid, and its co-chromatography with methyl retinoate after methylation with diazomethane. Topically applied retinol is about 2-fold less potent at inducing epidermal hyperplasia and 7-fold less potent at inhibiting the induction of epidermal ornithine decarboxylase by phorbol esters than all-trans-retinoic acid in this strain of mice. To elucidate the possible role all-truns-retinoic acid formation from retinol may have in these pharmacological activities, the epidermal and dermal all-trans-retinoic acid levels were compared in mice treated topically with retinol or [ll-3H]-all-trans-retinoic acid. The levels of all-truns-retinoic acid found after retinol treatment were several orders of magnitude lower than those found after [ll-3H]-all-rruns-retinoic acid treatment, and they were insufficient to account for the difference in potencies between all-truns-retinoic acid and retinol. Retinol was eliminated from the epidermis at a rate similar to that of all-trans-retinoic acid after topical administration, but the initial tissue levels achieved were lower. These results suggest that the lower potencies of retinol may simply reflect lower tissue uptake. Vitamin A is required for normal epithelial growth and differentiation. These requirements can be ful- filled by all-truns-retinoic acid (vitamin A acid) in vitamin A deficient animals, and it may be a phy- siologically active form of vitamin A in these areas [l]. All-truns-retinoic acid is a retinoid metabolite found in some tissues [2,3], and it is formed by the oxidation of retinol via the aldehyde retinal, or directly from retinal [l]. The latter oxidation step is irreversible and explains why all-truns-retinoic acid cannot completely replace retinol in the diet since it cannot be reduced back to retinal or retinol, which are required for normal vision and reproduction. Retinol is the circulating form of vitamin A and is present in the plasma in a retinal/serum retinol- binding protein-transthyretin complex, specific receptors for which are present on epidermal cells [4]. Saponified extracts of mouse epidermis con- tain about 6 nmoles retinal/g [5], although this value presumably reflects mainly the levels of retinol esters. Mouse epidermis contains enzymes which can interconvert retinol and retinal and can oxidize retinal to retinoic acid [6]. Although retinoid metab- olism in the epidermis has not been reported in depth, indirect evidence suggests that acid forms of vitamin A are physiologically important in this retinoid target tissue. The epidermis is apparently rich in cellular retinoic acid binding protein (CRABP) [7,8], a molecule implicated in the mediation of retinoid action. The therapeutic use of retinoids in dermatology currently centers on acidic species such as all-trans-retinoic acid, 13-cis-retinoic acid, and the ester and free acid forms of etretinate, although retinol shows some clinical efficacy. * Author to whom all correspondence should be addressed. Topical retinoid treatment of hairless mouse skin stimulates epidermal proliferation and turnover [9], producing a dose-dependent thickening of the prickle and granular cell layers (epidermal hyperplasia) [lo]. Retinol and retinal are about 50% as potent as all- trans-retinoic acid at inducing the hyperplasia [lo]. A retinol dose of 1.7 nmoles has been reported to inhibit the induction of ornithine decarboxylase activity in phorbol ester treated mouse epidermis by about 50% compared to a 57% inhibition after 0.17 nmole all-[runs-retinoic acid [ll]. The reasons for the lower potencies of retinol are unclear but may reflect differences in tissue uptake and loss, or a requirement for metabolic activation (such as oxi- dation to the acid). In the present study we measured the endogenous non-esterified retinol levels in hairless mouse skin, and examined the cutaneous metabolism of retinol after topical application, establishing that conversion of retinol to the acid form did occur in the skin. In an attempt to elucidate the relation, if any, of all- buns-retinoic acid formation to the mechanism of action of pharmacological doses of retinol, we com- pared the epidermal and dermal all-truns-retinoic acid levels found after topical retinol treatment to those found after topical all-truns-retinoic acid treatment. MATERIALS AND METHODS M aterials. Retinol (all-trans-retinol), retinal, reti- nyl acetate and retinyl palmitate were obtained from the Sigma Chemical Co. (St. Louis, MO). A~~-DYUW and 13-cis-methyl retinoates were synthesized from all-truns- and 13-cis-retinoic acids, respectively, using an excess of diazomethane generated by the method of Fales et al. [12]. [ll-3H]All-trans-retinoic acid 919