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The 7-carboxymethoximino derivative of androsterone (1) has been prepared from dehydroisoandrosterone-17-ethyleneketal by a sequence involving inversion at C-3, introduction of a carbonyl at C-7, and reduction of the double bond at C-5. The substance was condensed with BSA by the carbodiimide procedure to afford a conjugate which produced anti-androsterone antiserum in innoculated rabbits. The antiserum is sufficiently active to be useful in radioimmunoassay procedures.

Tetrahydrocortisol (THF) metabolism was studied by I.V. administration of trace amounts of 3H-THF to 3 normal men and 4 patients with miscellaneous illnesses. The principal urinary steroid was THF. This constituted about half of the “glucosiduronates” in the normal subjects, but significantly more (61–79%) in 3 of the 4 patients. Reduction of the 20-ketone of THF was quantitatively less prominent than the corresponding reaction for THE, but similarly stereoselective. Total cortols constituted 2–12% of the “glucosiduronates”, and the 111β-epimer predominated by about 15:1. The major conversion product of THF was 11β-hydroxyetiocholanolone, 12–25% of the “glucosiduronates” in normal subjects. Three of the four patients showed significantly diminished formation of this metabolite (3–5% of the “gluco-siduronate”). This suggests that diminished side-chain removal of THF may occur non-specifically in ill subjects. In one of these 3 patients, the low 11β-hydroxy-etiocholanolone formation was associated with extremely high β-cortol formation (32% of the “glucosiduronate” suggesting that β-cortol serves as one of the immediate precursors of 11β-hydroxyetiocholanolone.

Δ5-Pregnene-3α,16α,20α-triol was the principal metabolite isolated from the urine following administration of 3β, 16α-dihydroxy-Δ5-pregnen-20-one to two men. Δ5-Pregnene-3β,16α,20α-triol and 5β-pregnane-3α,16α,20α-triol were present in small amounts. The results suggest that the 16α-hydroxy group of the administered steroid alter the normal course of transformation of 3β-hydroxy-Δ5-steroids in man. Isolation of Δ5-pregnene-3α,16α,20α-triol (II) from urine of a patient with adrenocortical carcinoma was described in 1961, and was the first report of a Δ5-3α-hydroxysteroid from natural sources (1). At that time it was postulated that the probable precursor of the metabolite was 3β,16α-dihydroxy-Δ5-pregnen-20-one (I), and that the 16α-hydroxyl group interfered with the isomerization of the Δ5-3-ketosteroid sufficiently to permit reduction to the unsaturated alcohol in the favored 3α orientation. In order to explore this postulate further the in vivo transformation of 3β,16α-dihydroxy-Δ5-pregnen —20-one (I) was studied. In agreement with expectation Δ5-pregnene-3α,16α,20α-triol (II) was obtained in larger amount than the 5β-saturated trihydroxy analog (III) (2,3). It is concluded that the 16α-hydroxyl group of this substrate does in fact alter its reaction with a Δ5-3-ketosteroid isomerase.

The metabolism of pregnane-3α,17,20β-triol has been studied in man. Evidence was obtained for oxidation at C-20 and epimerization to pregnane-3α,17,20α-triol. The principal urinary product was the administered steroid. It was concluded that pregnane-3α,17,20β-triol is not on the major pathway to pregnane-3α,17,20α-triol production.

A method has been presented for the determination of the solvolyzable esters of androsterone and dehydroisoandrosterone in human plasma. The procedure involves the addition of tracer amounts of [4-14C] dehydroisoandrosterone and [3-3H] androsterone to plasma after extraction of the free steroids followed by protein precipitation, solvolysis, defatting, alumina chromatography, and thin layer chromatography. Quantitation is done by gas-liquid chromatography of the trimethylsilyl ethers. Replicate analyses of two large pools of adult human plasma gave values of 38 ± 2 μg and 47 ± 3 μg for androsterone and 147 ± 16 μg and 152 ± 10 μg for dehydroisoandrosterone as solvolyzable esters. Levels in 31 females and 24 males are reported. The procedure is applicable for routine laboratory use.

The question was studied whether the discrepancies observed in cortisol secretion rate determinations from several metabolites were due to changes in metabolic pathway of cortisol during the course of the day. Cortisol-4- 14 C was administered to two subjects in the morning at about 0800 hr. The studies were repeated in the same subjects twice in the evening at about 1800 hr and one subject received cortisol-4- 14 C at 1415 hr as well. The specific activities of 6 metabolites were determined and the apparent cortisol secretion rates were calculated. The production rates from the various metabolites were not in precise agreement at any of the times of day studied; the disagreement was apparently greatest when the tracer was given in the evening. That the time of day at which labeled cortisol is injected is not the factor responsible for the different estimates was shown in an adrenalectomized subject maintained on dexamethasone. Tracer cortisol-4- 14 C was given to this patient at 1750 hr and 36 hours later another tracer of cortisol 1,2- 3 H was given at 0900 hr. Complete urine collections were obtained from the time of administration of the first tracer until 36 hours after the second. The urine was processed as a single collection. The 3 H/ 14 C ratios of all the metabolites were identical demonstrating that the labeled cortisol followed identical metabolic pathway in the morning and in the evening. It thus appears that a diurnal variation in cortisol metabolizing enzymes is not responsible for the demonstrated discrepancies between metabolites when secretion rate is measured by the isotope dilution method from urinary metabolites.

The transformation of 3α-hydroxy-5α-pregnan-20-one-4-14 C has been studied in two subjects. Only about one-third of the dose was excreted in the urine during 4 days following administration of the steroid. Considerable amounts of radioactivity, 16.4 and 29% of dose, were excreted by the fecal roote. The total identifiable urinary metabolites were very low, 7.5–10%, with 5α-pregnane-3α,20α-diol the major metabolite. The only other compound isolated was the injected steroid. The further transformation of the 5α-metabolites of progesterone may play a role in the metabolism of progesterone leading to low recovery of metabolites of this hormone.

DHA (1) has been measured in plasma by a radioimmunoassay procedure using an antiserum to DHA-7-BSA whose specificity is such that the procedure is carried out directly on diluted, unextracted plasma. The method has been used to obtain plasma DHA secretory patterns and mean concentrations and the data are in accord with those determined by related but more laborious techniques.

Details of a sensitive and specific radioimmunoassay for androsterone (1) and androsterone sulfate in plasma have been presented. Benzene extracts of plasma were chromatographed on alumina to isolate the androsterone fraction either (a) directly after extraction (A) or (b) after solvolysis (AS). Following treatment with rabbit anti-A-17-BSA, antibody bound steriod was precipitated by ammonium sulfate. Androsterone concentrations in normal male plasma averaged 57 +/- 24 (S.D.) ng/dl, range 35-135 ng/dl and for normal women, 44 +/- 21 (S.D.) ng/dl, range 18-98 ng/dl. Androsterone sulfate concentrations were: males 55 +/- 28 mug/dl (range 10-114 mug/dl); premenopausal females 52+/- 31 mug/dl (range 16-318 mug/dl).

Replicate measurements of the specific activity of urinary tetra-hydrocortisol and tetra hydrocortisone after injection of 14C-cortisol in human subjects, were made by a double isotope derivative technique employing carrier addition and recrystallization to constant isotope ratio. Twenty subjects were studied and reproducibility was found to be excellent.

Following the administration of estrone- 3 H-sulfate- 35 S to man, estrone sulfate, 2-methoxyestrone sulfate and 2-hydroxyestrone-2-sulfate were isolated from urine and purified. Their isotope content showed that estrone sulfate and 2-methoxyestrone sulfate retained 23 and 21% of the originally present 35 S, while 2-hydroxyestrone estrone-2-sulfate contained only 7%. From this it is concluded that estrone sulfate- 35 S is directly transformed to 2-hydroxyestrone-3-sulfate which is partially O-methylated to yield 2-methoxyestrone sulfate with retention of isotope and partially converted to the 2-sulfate with loss of isotope. This represents an unique example of two successive metabolic transformations at a site adjacent to an intact sulfate ester. The demonstrated direct conversion of a phenol sulfate to an O-catechol sulfate is of particular significance.

Estriol stereospecifically labelled with tritium at the 17 α position was prepared and administered together with 14 C labelled estriol to three subjects including one cirrhotic. No significant tritium was present in the plasma or urine water and hence no oxidation at C-17 had occurred. Excretion of radioactivity was very rapid particularly in the cirrhotic subjects. Analysis of the enzyme and acid hydrolysates showed that they consisted of only three compounds. The majority of the excreted radioactivity consisted of estriol which did not lose any of the tritium at 17 α . The only other compounds were 16-ketoestradiol (0.8%) and 16-epiestriol (0.2%). Both metabolites retained the major part of the tritium at C-17 α and hence could not have arisen via 17-keto intermediates.

The fate of the 4βhydrogen in dehydroisoandrosterone-4-14C, 4β-3H was studied in man. A considerable loss of tritium from the steroid nucleus was observed; approximately 30% of the tritium appeared in the body water. The saturated urinary metabolites, androsterone and etiocholanolone retained only 30% of the tritium originally present. It was demonstrated that this tritium was still present at C-4; little or no hydrogen isotope was found elsewhere in the steroid nucleus. The 3β-hydroxy-Δ5steroid metabolites retained essentially all of its tritium.

The fate of 4- 14 C-testosterone 17-N-acetyl-glucosaminide has been studied in two subjects. Following intravenous administration urinary excretion of radioactivity was rapid. Testosterone N-acetylglucosaminide was the principal metabolites; etiocholanolone and androsterone conjugated with glucuronic acid were present in a ratio of 7.3. Following oral administration only traces of metabolites conjugated with the aminosugar were present. Etiocholanolone and androsterone were obtained in ratio of 7.3. In both studies etiocholane-3α,17β-diol was the predominant non-ketonic metabolite. These results were compared with the metabolites obtained following labelled testosterone and testosterone 17-glucosiduronate. The results demonstrate that 1) the cleavage of the glycosidic linkage is almost complete by the oral route, 2) ring A is reduced prior to hydrolysis of the aminosugar and 3) there is a directive influence of the aminosugar toward 5β-reduction similar to that of a glucosiduronate group at C-17.

17-Carboxymethoximino derivatives of DHA (1), androsterone and etiocholanolone, as well as the 7-carboxymethoximino derivatives of 3β-hydroxy-androst-5-ene-7,17-dione and 7-keto-androst-5-ene-3β, 17β-diol have been prepared and conjugated to BSA for use in producing antisera to the corresponding C 19 steroids.

The metabolism of simultaneously administered adrenosterone-1,2-3H and 11β-hydroxy-Δ4-androstene-3,17-dione-4-14C has been studied in a normal and a hypothyroid subject. Conversion to the metabolites from either hormone was essentially alike demonstrating ready interconversion of the 11β-hydroxy and 11-keto groups of the administered hormones. Following a large dose of adrenosterone to a hypothyroid subject 3α-hydroxy-Δ4-and-rostene-11, 17-dione and its reaction product with urea, 3α-ureido-Δ4-and-rostene-11, 17-dione were isolated and characterized.