THE EXCRETION OF PREGNANE-3α,17α,21-TRIOL 20-ONE (TETRAHYDRO S) IN NORMAL AND PATHOLOGIC URINE

Abstract

PREGNANE-3α,17α,21-triol-20-one (tetrahydro S) has previously been identified in the urine of patients with adrenocortical tumors (1, 2) and in the urine of a man following the administration of corticotropin, by this laboratory (3). Eberlein and Bongiovanni (4) have found it in large quantities in the urine of a subject with adrenal hyperplasia. We have also identified it as the major alpha-ketolic metabolite in the urine following ingestion of Δ4-pregnene-17α,21-diol-3,20-dione (Reichstein's compound S) by a patient with anterior pituitary failure (5). The following report describes the characterization of tetrahydro S and the situations under which this steroid was or was not found in urine during a more extensive series of observations. Observations were made on pooled normal urine and on urine specimens from 2 normal subjects, 9 patients with hyperadrenocorticism, 1 with hypoadrenocorticism, and 8 with miscellaneous conditions, including pregnancy (Table 1). Methods. The methods employed and their limitations have been previously described in detail (5). Conjugates in the urine were hydrolyzed with β-glucuronidase at pH 4.5 for two days. The urine was then adjusted to pH 1 and extracted with chloroform for one hour on an oscillating shaker. Preliminary separation of fraction X (ref. 5) was made by developing the chromatograms in the toluene-propylene glycol system (6), collecting 15 cc. of effluent per ½-inch of strip (approximately ninety-six hours). Tetrahydro S migrates with cortisone in this system but is separated from hydrocortisone and other slower moving substances as well as the faster moving steroids. The zone containing the tetrahydro S and cortisone was eluted and the eluate rechromatographed in the benzene-formamide system which separated the two compounds well. After development of the chromatograms, the absorption at 245 mμ was determined directly in a Beckman DU spectrophotometer by means of an adaptor (7). Absorption at 600 mμ was similarly determined after spraying with blue tetrazolium to determine the location of alpha-ketolic compounds. Density was plotted against distance from the starting line. Semiquantitative estimates ( ± 30 per cent) of the various alpha-ketolic steroids were made by planimetric determinations of the areas of the peaks (5). Neutral reducing lipid-soluble substances were determined by the method of Heard, Sobel and Venning (8), after glucuronidase hydrolysis as previously described (5). The total neutral 17-ketosteroids were determined by a modification of the method of Holtorff and Koch (9).