Isolation of Testosterone-(17β→1β-oside)-d-glucopyranosuronic Acid from Human Blood1

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Abstract
To a 71-yr-old woman with an arrhenoblastoma, 50 μ of 4-14C-testosterone was administered and 135 ml of blood was withdrawn 15 min later. The blood extract was separated into 4 different compartments by chromatography on alumina. The glucuronide compartment was separated by countercurrent distribution into 4 fractions, 3 of which were identified: testosterone-(17β → 1β-oside)-d-glucopyranosuronic acid (TG); 5β-androstan-(3α→lβ-oside)-d-glucopyranosuronic acid-17-one (EG); and 5α-androstan-(3α→lβ-oside)-d-glucopyranosuronic acid-17-one(AG). TG was identified upon conversion to methyl [testosterone-(17β→lβ-oside)-d-2,3,4-tri-0-acetyl-glucopyranosuronate] (TGAc3M) not separable from cold synthetic compound by repeated crystallization. The steroid portion was identified as testosterone after enzymatic hydrolysis of TG, addition of nonradioactive steroid, and the maintenance of constant specific activity after repeated crystallizations. These procedures identified the steroid and its glucuronic conjugate. This is the first time a steroid 17β→lβ-d-glucuronide has been isolated from human blood.