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APPLICATION OF A HPLC METHOD OF ANALYSIS OF RETINOIDS TO METABOLIC STUDIES

In a previous publication Bhat et al. (Anal. Biochem. 102: 243-248, 1980) described a reverse phase high-pressure liquid chromatographic (HPLC) method for the separation of naturally occurring retinoids. We modified this method in the present study to achieve base line separation of 13-cisand all-trans-retinoic acid (RA) by employing reverse phase ion pair HPLC. In addition, we employed this method to investigate the interconversion of 1 3 4 s and all-trans-RA and their metabolism in normal rats. The separation of naturally occurring retinoids was achieved by reverse phase HPLC on a octadecylsilane column eluted with acetonitrile-phosphate buffer, pH 7.2 mixtures. The order of elution from a mixture of 200-250 ng of each of the following standard retinoids was mannosyl retinyl phosphate (MRP), 4OXORA, 5,6-epoxyRA, retinyl phosphate, 13-cis-RA, all-trans-RA, retinol, retinal, retinyl acetate, anhydroretinol and retinyl palmitate. All the standards were in the all-trans configuration except 13-cis-RA. The column was eluted with acetonitrile-phosphate buffer, pH 7.2 (37:63), at an initial flow rate of 2.2 ml/min for the first 30 min to elute MRP, 4-OXORA. 5,6-epoxyRA, 13-cis-RA and all-transRA and with acetonitrile-phosphate buffer, pH 7.2 (54:46), at a flow rate of 3.5 ml/min for the following 15 min to elute retinol, retinal, retinyl acetate, and anhydroretinol. The solvent composition was then altered to acetonitrile-water (98.2) and the flow rate adjusted to 1.5 mYmin for an additional 15 min to elute retinyl palmitate. Metabolic studies on retinoic acid were carried out in normal male rats (200 g) of the Sprague-Dawley strain. Each rat was injected intraperitoneally with 50 pCi of either [10-3H]-all-trans (5.4 pg] or [ll-3H]-13-cis-RA (8.8 pg) in 50 pl of ethanol and sacrificed after 0.5 and 3 hr. Blood, liver and testes were removed and lyophilized. The tissues were extracted in 99% methanol and an aliquot of the extract in absolute methanol was used for HPLC analysis. A small amount of all-trans-RA was converted 0.5 hr after injection to 13-cis-RA in all the tissues studied, the conversion being 2.4% (testes), 3.1% (liver) and 6.9% (blood) of the total recovered radioactivity. In addition, all-trans-RA was also transformed to highly polar metabolites. At 0.5 hr after injection, these constituted 17.5% for liver, 47.7% for testes and 37% for blood. At 3 hr after injection, most of the radioactivity was located in the highly polar metabolites (blood 81%, liver 87% and testes 75%). These metabolites were more polar than 5,6-epoxy-all-trans-RA