Isolation, Identification and Quantitation of Testosterone in the Urine of Normal Adults and in Patients with Endocrine Disorders

Abstract

A procedure for the isolation of testosterone from human urine is presented. Following β-glucuronidase hydrolysis, urine was extracted with ether. The extract was chromatographed on a florisil column prior to gradient elution alumina chromatography and paper chromatography in 2 systems (Bush 1 and Bush 3). The isolated steroid has a mobility in both paper chromatographic systems similar to that of authentic testosterone. This material yielded a bright yellow fluorescent spot when subjected to the alkali-fluorescence test and a blue color with the Zimmermann reagent. Eluates from paper exhibited a UV absorption maximum at 240 mμ. The sulfuric acid and infrared absorption spectra were similar to those of authentic testosterone. The product resulting from chromic acid oxidation of the isolated steroid ran in the Bush 3 system at the same rate as authentic androstenedione. Testosterone has been isolated from the urine of normal adults, patients with untreated congenital adrenal hyperplasia, one of whom was bilaterally oophorohysterectomized, and in large amounts in a 2½-yr-old boy with a virilizing hepatoma. The daily excretion of testosterone by normal adult males was many times greater than that of normal adult females.