A PROCEDURE FOR CONCOMITANT PHOTOMETRIC ESTIMATION OF PREGNANEDIOL AND ESTROGENS IN HUMAN URINE*†

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Abstract
BECAUSE of the intimate interrelationship of estrogen and progesterone metabolism to human reproductive physiology, there is need for a procedure which provides concomitant colorimetric pregnanediol and estrogen determinations in single twenty-four-hour urine specimens. Furthermore, since the sulfuric acid color reaction by which pregnanediol usually is determined is not specific for this steroid, incorporation of procedural modifications which contribute to more adequate characterization of the final pregnanediol sulfuric acid color product is highly desirable. This has been accomplished by the utilization of a liquid chromatogram technique which fractionates dihydroxysteroids from mono- and trihydroxysteroids, and by special attention to the spectral character of the pregnanediol sulfuric acid color reaction. In our preparation of urinary phenol-like residues for chromatographic fractionation and photometric estimation of the estrogens (estrone, estradiol, and estriol) a neutral moiety is obtained (1). This crude neutral steroid residue may be utilized for colorimetric, i.e., concentrated sulfuric acid, pregnanediol determination in a procedure in which a liquid chromatogram technique similar to that described by us (1, 2) for the natural estrogens has been incorporated. Umberger and Curtis (3) have shown that the absorption curves obtained by heating the natural estrogens with 90 per cent sulfuric acid for twelve minutes at 100° C. are sufficiently characteristic for each to serve as a means of identification. Our somewhat similar study of a number of neutral steroids which, like pregnanediol, produce color with concentrated sulfuric acid at room temperature has shown that the absorption maximum (420 mμ) for pregnanediol is sufficiently characteristic within the group of neutral steroids investigated, to aid in the identification of pregnanediol. By our procedure, as little as 1 mg. of pregnanediol added to 1 liter of men's urine immediately after hydrolysis, or from 2 to 10 mg. of pure sodium pregnanediol glucuronidate added immediately before hydrolysis, can be recovered as pregnanediol in satisfactory yields. A description of the procedure and of some preliminary tests of its potentialities and limitations forms the subject of this report. The application of this procedure to a study of simultaneous estrogen and pregnanediol excretion throughout the normal menstrual cycle will form the subject of a future report.