A method employing trifluoroacetic acid for the quantitative determination ofallrrans-retinoic acid and 13-cis-retinoic acid in serum is described. The method permits the detection of as little as 0.3 Kg of either compound.
Retinoic acid can serve the general tissue functions of vitamin A, except in vision and reproduction (l), and can also reverse the process of chemical carcinogenesis by eliminating preneoplastic lesions (2-8). In both man and experimental animals, however, retinoic acid, when administered in therapeutic doses, has undesirable toxic side effects (9,lO). These observations prompted the search for derivatives with similar therapeutic acitvity but lower toxicity. In animal experiments, an analog, 13-cis-retinoic acid, proved to be useful in the prevention of epithelial cancer (3,ll) and was less toxic than retinoic acid (3,4). This retinoid is presently being considered for clinical testing (5,12,13). The purpose of the present study was to develop an appropriate assay for these retinoids.
Most reported methods for the determination of retinoids in biological media involve the use of column chromatography and are cumbersome to use routinely. Two simplified methods that permit direct measurement of retinoic acid in plasma are available (14,15). One involves extraction of total retinoids with a petroleum ether-ethanol system, fractionation by thin-layer chromatography, and measurement of the absorbance of each eluted fraction by ultraviolet spectrophotometry ( 14). The other method involves the use of antimony trichloride, the Carr-Price reagent, for color estimation of retinoic acid following its separation from neutral retinoids by sequential extraction (15). A problem encountered in the use of the Carr-Price reagent is that it develops turbidity in the presence of trace amounts of moisture. In 1964, Dugan et al. ( 16) reported that trifluoroacetic acid (TFA) also forms a color complex with retinoic acid. Trifluoroacetic acid is less toxic than antimony trichloride and, unlike the latter, does not become turbid in the presence of moisture. Furthermore, the extinction coefficient of retinoid acid-TFA complex is about 80% greater than that of retinoic acid-antimony trichloride complex (17). In preliminary