ANALYTICAL BIOCHEMISTRY 89,220-224 (1978)

Calorimetric Determination of All-Trans-Retinoic Acid and 13-Cis-Retinoic Acid

CHIH-CHENG WANG, RUPERT E. HODGES, JR., AND DONALD L. HILL

Kettering-Meyer Laboratory, Southern Research Institute. Birmingham, Alabama 35205

Received January 16, 1978; accepted April 7, 1978

A method employing trifluoroacetic acid for the quantitative determination ofall- rrans-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 thera- peutic 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 con- sidered 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 in- volves 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 extinc- tion coefficient of retinoid acid-TFA complex is about 80% greater than that of retinoic acid-antimony trichloride complex (17). In preliminary

0003-2697/78/0891-0220$02.00/O Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.

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DETERMINATION OF RETINOIC ACIDS 221

experiments, we found that 13-cis-retinoic acid and all-trans-retinoic acid formed similar color complexes with TFA. These properties suggested that TFA might serve as the basis for a new, sensitive, and simple microtechnique for determining all-trans-retinoic acid and 13-cis-retinoic acid. It should be pointed out that TFA was used by Roels et al. (18) for estimation of all-trans-retinoic acid in biological materials. Results of their studies, however, have not been published.

MATERIALS AND METHODS

TFA, ethanol, chloroform, and petroleum ether (30-60°C) were reagent grade and were used without treatment. All-trans-retinoic acid and 13-cis- retinoic acid were prepared by Hoffmann-LaRoche, Inc. (Nutley, New Jersey and Basel, Switzerland) and were supplied to us by the Lung Cancer Segment of the National Cancer Institute. For oral administration, preparations (10 mg/ml) of the retinoids were dissolved in corn oil con- taining 10% alcohol. Dissolution was achieved under subdued light by shaking on a Vortex mixer.

To each 1 vol of serum (0.2-0.8 ml) in centrifuge tubes were added 0.2 vol of 1 N KOH, 1 vol of ethanol, and 2.5 vol of petroleum ether. The preparations were shaken for 1 min and centrifuged. The upper layer contained no retinoid acid and was withdrawn and discarded. The extraction with petroleum ether was repeated until the extract became free of absorbance at 365 nm, which is the absorption maximum of retinoic acid. To the alkaline residue were added 0.2 vol of 2 N HCl and 2.5 vol of petroleum ether. The preparations were again mixed and centrifuged. The upper layer was withdrawn, placed into a test tube, and evaporated to dryness with N,. The amount of retinoid acid in the residue was determined with TFA, as described below.

A Cary 14 recording spectrophotometer with l-cm quartz cells was used to measure spectra and absorbance changes as a function of time. Duplicate samples were assayed in each case. Standard solutions of all- trans-retinoic acid and 13-cis-retinoic acid were prepared in chloroform in the concentration range of 3 x lo+ to 3 x lo+ M. A portion (0.5 ml) was placed in a cuvette, and an equal volume of TFA was added. The cuvette was immediately covered, and the solution was rapidly mixed by inverting the cuvette twice. In the standard assay, absorbance was measured at 562 nm 10 set after adding the TFA. There was a linear relationship between absorbance and concentration. When standard solutions of all-trans- retinoic acid and 13-cis-retinoic acid (1.5 x lo+ to 1.5 x 10e4 M) were added to 0.2 ml of serum and the preparations were extracted as described above, the recovery was 76 to 80% for each acid.

RESULTS AND DISCUSSION

Figure 1 shows the absorption spectra of the TFA reaction with all- tmns-retinoic acid. The colorproduced has a spectrum with two peaks at

222 WANG, HODGES, AND HILL

FIG. 1. Absorption spectra of TFA reaction product of crystalline retinoic acid. (-) Spectrum at maximum color intensity (10 set). (- - -) Spectrum when color intensity at 562 nm has faded (5 min after addition of TFA). The quantity of crystalline retinoic acid in this sample was 2.9 ng. The spectrum is scanned at a rate of 10 nm per 4 set from right (higher wavelength) to left (lower wavelength).

562 and 458 nm. The absorption at 458 nm was 40% of that at the higher wavelength. The higher maximum at 562 nm is close to the 574~nm maxi- mum reported previously (16). The TFA-retinoic acid complex has a molar absorptivity at 562 nm of 53,000, which is identical to that reported (16). The color at this wavelength is of short duration; it disappears com-

Seconds

0 20 40 60 so 100 IZ Seconds

FIG. 2. Time dependence of TFA-retinoic acid absorbance at 562 nm.

DETERMINATION OF RETINOIC ACIDS 223

0 0 12 3 4 5 6 I

"OURS

HAMSTER

a 1 2 3 4 5 6 7 HOUR3

FIG. 3. Serum retinoid acid concentration at various times following oral administration of the acid to male Sprague-Dawley rats (90- 110 g), male Syrian hamsters (90- 1 IO g), and female DBAL! mice (20-23 g) at a dose of 50 mglkg. Each value is obtained from the pooled serum of three animals. (0) l3-cis-retinoic acid; and (0) retinoic acid.

pletely within 5 min after adding TFA. 13-cis-Retinoic acid has a similar spectrum (not shown); its molar absorptivity is 52,000 at 562 nm.

At 562 nm, the complex for all-trans-retinoic acid developed maximum color in about 10 set and faded rapidly (Fig. 2A). When the absorbance was plotted as a function of time on semilog paper, a straight line was obtained (Fig. 2B), indicating that the decay of the color complex fol- lowed first-order kinetics. This was observed at the substrate concentra- tions used, ranging from 1 x 10e6 to 3 x 10U5 M. Similar kinetics were ob- served for 13-cis-retinoic acid (not shown). These observations show that the absorbance value at any time can be determined from other time points by extrapolation. For both compounds, there was a linear relation- ship between absorbance and concentration with time.

Fig. 3 shows the serum concentration of all-rrans-retinoic acid and 13- cis-retinoic acid after a single po administration of the acids to rats, ham- sters, and mice. The amount of 13-cis-retinoic acid that appeared in the serum was greater than that of all-rruns-retinoic acid for all three species.

Comparison of the direct absorption spectrum, the absorption spectra of the TFA color complex, and the decay kinetics of the color complex of retinoid acid extracted from serum 1 hr after administration with those of crystalline retinoid acid showed that they were identical, a result indicating that the material being determined was unchanged retinoid acid. The assay would not, of course, distinguish between retinoic and 13-cis-retinoic acid.

ACKNOWLEDGMENTS This study was supported by Contract NOI-CP-22064, Division of Cancer Cause and

Prevention, National Cancer Institute, National Institutes of Health.

224 WANG, HODGES, AND HILL

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