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Various preparations of Oak Ridge radioiodide were diluted with glass-distilled water and applied to filter paper strips for chromatography. Spontaneous drying of the applied samples for 10–20 min before the papers were placed in the chromatography jar resulted in variable, and sometimes very marked (up to 70%), transformation of the radioiodide. At least 3 different non-iodide components were formed. These changes were largely prevented by drying in a nitrogen atmosphere, and were completely inhibited by 3 ×l0-4 M methimazole. An oxidative mechanism is therefore suggested. Addition of 3×l0-4 M carrier iodide also greatly reduced the chemical changes occurring on drying. This, coupled with the variability of the effect, suggests that a trace component of the atmosphere, whose concentration varied from day to day, was involved in the reaction. These findings raise the possibility that other labile compounds, in addition to iodide, may also undergo oxidative changes when very dilute solutions are dried on f...

To elucidate further our previous findings that patients with essential hypertension produce more sulfate conjugated 17-hydroxycorticosteroids (17-OHCS) and less glucuronide conjugated 17-OHCS than normotensive subjects, individual urinary C21 αketolic steroids were separated by paper chromatography and quantitated in 11 hypertensives and 11 normotensives. The chromatography was performed separately on free, glucuronide and sulfate conjugated steroid fractions. The results obtained revealed the following statistically significant differences between the 2 groups of subjects: 1) in the free steroid fraction, excretion of cortisol and cortisone was higher in hypertensives; 2) in the glucuronide conjugated fraction, excretion of tetrahydrocortisol, tetrahydrocortisone, tetrahydrocorticosterone and tetrahydro-11-dehydrocorticosterone was lower in the hypertensives; 3) in the sulfate fraction, excretion of the most polar steroids, 6α- and 6β-hydroxycortisol, was higher in the hypertensives. The ratio ...

Submaxillary gland slices of Wistar adult male rats were incubated in vitro with l-14C-sodium acetate under an atmosphere of 95% O2 and 5% CO2 for 5 hr. A radioactive material behaving like cholesterol was characterized by chromatographic mobility in several paper and column systems before and after acetylation, digitonide and dibromide precipitation, debromination and final recrystallization of the free compound and its derivative to constant specific activity. Labeled acetate was converted to cholesterol to the extent of 0.04–0.07%. A radioactive material behaving like pregnenolone was not sufficiently identified due to low yield. To prove with certainty that cholesterol was further metabolized, submaxillary gland homogenates of Wistar adult male rats were incubated in vitro with cholesterol-4-14C for 2 hr in the presence of NADPH generating system. Radioactive materials behaving like pregnenolone and dehydroepiandrosterone were characterized by paper chromatography mobility in several systems and recry...

The in vitro metabolism of radioactive androstenedione, pregnenolone and progesterone by rat placental homogenates has been studied. No estrogens were isolated but there was extensive ring A reduction of each compound. From the androstenedione incubations, 3α-hydroxy-5α-androstan-17-one, 3β-hydroxy- 5α-androstan-17-one, 5α-androstane-3α,17β-diol and 5α-androstane-3β,17β-diol were isolated by paper chromatography and identified by thin layer chromatography and the reverse isotope dilution technique. The metabolites of pregnenolone and progesterone were quantitatively and qualitatively similar. From these incubations 3α-hydroxy-5α-pregnan-20-one and 5α-pregnane-3α,20β-diol were isolated and identified. In the presence of a progesterone trap, progesterone- 7α-3H was found following the incubation of pregnenolone-7α-3H, demonstrating the presence of a 3β-ol-dehydrogenase Δ4–5-isomerase enzyme system. (Endocrinology 87: 151, 1970) ATTEMPTS to evaluate possible endocrine functions of the rat placenta during pre...

The separation of tritium-labeled from nonlabeled or 14C-labeled steroids on paper partition chromatograms has been described and its implications have been discussed. In addition, cortisol-1, 2-3H and cortisol-4-14C were administered simultaneously to a normal man, and labeled steroids from urine and plasma, collected at intervals during the first 24 hr, were isolated by paper chromatographic procedures. All metabolites exhibited chromatographic separation of the 3H-containing moiety from the nonlabeled or 14Clabeled compounds. However, no isotope effect was discernible for the in vivo metabolism of cortisol-1, 2-3H. When care was taken to analyze the total chromatographic peak areas, the proportions of 3H:14C in the isolated metabolites (cortisol, cortisone, 3α, 11β,17,21-tetrahydroxy-5β-pregnan-20-one, 3α,11β,17,21-tetrahydroxy-5α-pregnan-20-one,3α, 17, 21-trihydroxy-5 β-pregnane-11,20-dione and a mixture of the cortolones were identical with that in the administered steroids.

A series of in vitro experiments using 4-14C-pregnenolone and 4-14C-progesterone as precursors were performed to determine the effects of prolonged adrenocorticotrophic hormone (ACTH) administration on adrenal steroid biogenesis in rabbits. The adrenal steroids were isolated by paper chromatography and quantitated by the double isotope derivative assay method. In the control group, there was no distinction between the roles of pregnenolone and progesterone as precursors of adrenal steroids. Following the administration of 25 U of porcine ACTH for 21 days, however, 17α-hydroxylated steroids became quantitatively important. Prolonged exogenous ACTH treatment enhanced the specific activities of the 17α-hydroxycorticosteroids from both precursors, but the specific activities were enhanced more from pregnenolone than from progesterone. Under experimental conditions more than twice as much pregnenolone was metabolized via 17α-hydroxypregnenolone bypassing the progesterone step than under control conditions. Pro...

An accurate and relatively simple radioimmunoassay method for the measurement of aldosterone in human plasma is described. One ml or less plasma with 3H aldosterone (2 pg) is extracted with dichloromethane. Two alternative purification steps are described, either a standard paper chromatography (B5) or a more rapid Sephadex LH-20 column (60 × 1 cm) in the system dichloromethane: methanol 98:2 provides adequate purification for analysis. The assay uses either an antibody of high specificity generated against aldosterone-18, 21-disuccinate, or the 3-oxime coupled to bovine albumin. The samples, or standards, are incubated for 1–2 hours at 5° C with dilute antibody and 3H aldostercne. Separation of bound from free aldosterone is affected with activated and coarse-graded Florisil. This one-day method, using the disuccinate antibody and the LH-20 column, is associated with blank values difficult to distinguish from zero (< 6 pg). Aldosterone values from adrenalectomized subjects or duplicate samples after cort...

The isolation of the C-19 and C-21 saturated urinary metabolites following cortisone administration to man have been described (Dobriner et al., 1950, 1951; Burstein et al., 1953). Recently significant quantities of hydrocortisone (17-hydroxycorticosterone) have become available and the present study reports the isolation from urine, utilizing paper chromatography, of the major α-ketolic and 17-ketosteroid metabolites isolated following the oral administration of hydrocortisone to a man. The isolated steroids were identified by chromatographic mobility on paper, melting points and infra-red spectrometry. The significance of the present study lies in the demonstration of: 1) The in vivo conversion of hydrocortisone into cortisone, 2) the products of reduction of the Δ4-3-keto system, and 3) the pattern of the 11-oxygenated 17-ketosteroid metabolites arising from hydrocortisone.

The 17-ketosteroid content of urine has been examined by 4 methods: a clinical “total 17-ketosteroid” determination after acid hydrolysis; a total ketosteroid determination on the ketonic fraction from a micro-Girard procedure; a simple column chromatographic separation of the C19O2 and C19O3 steroids of an extract of urine subjected to enzyme hydrolysis and solvolysis; and a 2-stage paper chromatographic separation of androsterone, etiocholanolone, dehydroepiandrosterone and the C19O3 steroids of a similar urine extract. Paper chromatographic analysis revealed that up to 90 per cent of the clinical “total 17-ketosteroid” value may consist of nonspecific chromogens, thus largely invalidating the quantitative significance of such measurements. Girard fractionation achieved satisfactory purification with some loss of steroids. The column chromatographic method proved more practical, yielding 2 biologically significant ketosteroid fractions which correlated well with the results of paper chromatography.

The present study describes the isolation from human urine and definitive chemical identification of 4 members of a new class of acidic metabolites of corticol, characterized by a 21-oic acid group: 3α, 17, 20α-trihydroxy-11-oxopregnan-21-oic acid; 3α,17,20β-trihydroxy-11-oxopregnan-21-oic acid; 3α,11β,17,20α-tetrahydroxypregnan-21-oic acid; 3α,11β,17,20β-tetrahydroxypregnan-21-oic acid; The trivial name “cortoic acids” is suggested for this group of metabolites; the individual compounds, named after the corresponding 21-hydroxy metabolites, would be: “cortolonic acid,” “β-cortolonic acid,” “cortolic acid” and “β-cortolic acid,” respectively. Together these metabolites constitute 5–25% (mean 12%) of the administered cortisol. The most abundant by far is “cortolonic acid” which is one of the 4 most abundant metabolites of cortisol. Transformation of cortisol to these metabolites involves an intramolecular shift of a hydrogen atom from C-21 to C-20 as well as oxidative removal of the other hydrogen...

A simplified, rapid, and highly reproducible technique is described for measuring 5 alpha-reductase activity (5 alpha RA) in small skin biopsies. Human genital skin was obtained from 23 nonhirsute and 20 hirsute premenopausal women (HW) and 5 normal men. Skin samples were minced at 4 C and incubated with RPMI-1640 in the presence of 95% O2-5% CO2 and 4.15 nmol [14C]testosterone ([14C]T) for 2 h at 37 C. Steroids were extracted with diethyl ether and separated by Celite and paper chromatography. Radioactivity in specific eluates was quantified, and the mass of each steroid was measured by RIA. The separate formation of 5 alpha-androstane-17 beta-ol-3-one (DHT), 5 alpha-androstane-3 alpha, 17B diol (3 alpha diol), androstenedione, and androsterone from [14C]T was measured. In separate experiments it was demonstrated that an incubation time of 2 h was optimum and that the addition of cofactors was unnecessary. Radiochemical purity was confirmed after chromatography. The mean +/- SE conversion ratio (CR) of T to DHT (in 2 h) in HW was higher than that in normal women (16.80 +/- 1.62% vs. 4.48 +/- 0.36%; P less than 0.01). In men, the CR of T to DHT averaged 31.60 +/- 3.96%. Individual values for the CR of T to DHT in HW and normal women did not overlap. The CR of T to 3 alpha diol was significantly higher in HW (9.66 +/- 0.86%) and men (15.98 +/- 2.0%) compared to that in normal women (2.96 +/- 0.32%; P less than 0.05). The CR of T to androstenedione was significantly greater in HW and men (6.18 +/- 0.42 and 7.28 +/- 1.92%) compared to that in normal women (2.64 +/- 0.64%; P less than 0.05). The CR of T to androsterone was very low and was similar in the three groups. The production of DHT in HW (4.50 +/- 1.0 pmol/mg X 2 h) was significantly greater than that in normal women (0.48 +/- 0.08; P less than 0.01) and was similar to the production in men (6.18 +/- 1.94 pmol/mg X 2 h). There was a significant correlation between the CR of T to DHT and DHT production, and the CR of T to 3 alpha diol and 3 alpha diol production as well as between the CRs of T to DHT and T to 3 alpha diol. These data suggest that measurements of DHT formation are best suited for the assessment of 5 alpha RA and that the measurement of 5 alpha RA in vitro from small skin biopsies is suitable for the clinical evaluation of hirsutism.

The metabolism of iv-administered 14C-cortisol (F) by pregnant baboons (107, 124 and 150 days gestation) was compared with that previously reported for nonpregnant animals and with that of animals examined 6-18 h after spontaneous vaginal delivery (178 plus or minus 4 days). Unconjugated, glucuronoside (beta-glucuronidase) and sulfate (H2SO4-ethyl acetate) fractions were extracted with ethyl acetate from urine containing more than 80% of injected 14C. Metabolites of interest were isolated by paper partition chromatography and purified by crystallization and derivative formation. Compared with nonpregnant animals, the following changes (P less than 0.05) were observed in pregnancy: (1) an increase in the percent urinary 14C in the unconjugated fraction and a decrease in the proportion of 14C appearing in the glucuronoside fraction; (2) an increase in excretion of metabolites more polar than the cortols; (3) a decrease in excretion of metabolites less polar than cortisone in the glucuronoside fraction; (4) an increase in unconjugated F excretion. Production rate of F (11.9 plus or minus 0.7 mg/day) estimated by isotope dilution and percent urinary 14C in tetrahydrocortisol and tetrahydrocortisone from the glucuronoside fraction were as in nonpregnant animals. With the exception of an increase in F production (22.7 plus or minus 0.8 mg/day), presumably the result of the stress of labor, F metabolism in the immediate postpartum period was strikingly similar to timals indicates that changes in the mother alone can account for the altered metabolic disposition of F in pregnancy and suggests that the fetus takes little part in metabolism of maternal circulating F.

The increase of plasma deoxycorticosterone (DOC) levels after administration of spironolactone is a real effect and not due to cross-interference of the drug or its metabolites with the DOC-assay. This is proved by in vitro and in vivo results. Of all the metabolites only canrenone interferes to some extent with DOC after liquid-liquid-extraction and paper chromatography. The antiserum, however, is so highly specific, that the final cross-interference of the total procedure amounts to less than 2% of the DOC plasma levels. Moreover, in patients with Addison's disease only a very small increase of DOC plasma levels can be observed after administration of spironolactone in contrast to normal subjects.

We have determined the structures of high mannose (Man) oligosaccharide units at individual glycosylation sites of mouse TSH. Mouse thyrotropic tumor tissue was incubated with D-[2-3H]Man with or without [14C]tyrosine ([14C] Tyr) for 2, 3, or 6 h, and for a 3-h pulse followed by a 2-h chase. TSH heterodimers or free alpha-subunits were obtained from homogenates using specific antisera. After reduction and alkylation, subunits were treated with trypsin. The tryptic fragments were then loaded on a reverse phase HPLC column to separate tryptic fragments bearing labeled oligosaccharides. The N-linked oligosaccharides were released with endoglycosidase-H and analyzed by paper chromatography. Man9GlcNac2 and Man8GlcNac2 units predominated at each time point and at each specific glycosylation site, but the processing of high Man oligosaccharides differed at each glycosylation site. The processing at Asn23 of TSH beta-subunits was slower than that at Asn56 or Asn82 of alpha-subunits. The processing at Asn82 was slightly faster than that at Asn56 for both alpha-subunits of TSH heterodimers and free alpha-subunits. The present study demonstrates that the early processing of oligosaccharides differs at the individual glycosylation sites of TSH and free alpha-subunits, perhaps because of local conformational differences.

Testicular homogenates from rhesus monkeys 1.0, 2.0, 2.5, 2.7, 2.8, 3.0, 7, 8, 8.5 and 9 years of age and those from 33- and 100-day-old rats were incubated with 3H-progesterone, 3H-testosterone and 14C-pregnenolone in the presence of co-factors. In addition, homogenates of testes obtained from patients 3, 4, 5, 7, 15 and 83 years of age at the time of orehiectomy were incubated with 3H-progesterone and 14C-pregnenolone. After incubation, radioactive products were separated and identified by column and paper chromatography with derivative formation and recrystallization to constant specific activity. Testes from monkeys and humans of different ages converted significant amounts (up to 70%) of progesterone and pregnenolone to 17α-hydroxyprogesterone, androstenedione, testosterone, 20α-hydroxypregn- 4-en-3-one and/or 16α-hydroxyprogesterone. Testes of monkeys 1.0–9 years of age converted 1–30% of testosterone to androstenedione. However, no or very small amounts (less than 0.4% of each product) of 5α-reduce...

A shortened approach to the measurement of 17α-hydroxyprogesterone (17-OHP) is described. The extracted plasma sample with 3H indicator is purified by only one paper chromatography. No acetylation step or further chromatography is necessary. Values obtained in males and females are the same as reported by Strott et al. Both LH and FSH increased plasma 17-OHP in men. Measurable, but low, plasma values were observed in prepubertal children. Further evidence for adrenal secretion was provided by values from children and castrates, and by failure to suppress 17-OHP during the follicular phase with estrogen.

A method is described for the determination of unconjugated androstenediol (3β, 17β-dihydroxy-5-androstene) in peripheral plasma based on a competitive protein-binding technique. The procedure includes ether extraction and paper chromatography in the Bush A2 system. Sex steroid binding globulin is derived from patients under estrogen therapy suffering from a carcinoma of the prostate. Separation of bound and unbound androstenediol is carried out by ammonium sulfate precipitation. The precision of the method, evaluated by determining the coefficient of variation of analyses of pools of plasma, was 8.5% for plasma of men and 12.3% for plasma of women. Accuracy was satisfactory, the specificity was proved by mass spectrometry. The mean plasma androstenediol concentration in 19 normal men was 109.9 ± 42.6 (sd) ng/100 ml. In 11 normal women the mean was 61.5 ± 22.7 (sd) ng/100 ml. The difference between male and female values was significant. After stimulation with hCG, plasma androstenediol increased...

Minced adrenal tissue from a woman with Cushing's syndrome was incubated with progesterone-4-14C. After addition of carrier steroids and their reisolation by gradient elution partition chromatography and paper chromatography, radiochemical purity was established by repeated crystallization, derivative formation and countercurrent distribution. The following metabolic products were identified: Δ4-androstene-3,17-dione (2.3%),11β-hydroxy-Δ4-androstene-3,17-dione (0.3%), 17,21-dihydroxy-Δ4-pregnene-3,20-dione (1.2%), cortisol (2.5%), deoxycorticosterone (6.2%), 16αhydroxy-Δ4-pregnene-3,20-dione (24.8%), 2 unidentified compounds (18.1%) and unreacted progesterone (9.1%).

INTRODUCTION ALTHOUGH the quantitative and qualitative pattern of neutral 17-ketosteroids in the urine of normal individuals has been fairly well established, until recently, little has been known of these substances in the human blood. Gardner (1) has described a method for their determination in human plasma. He reported that adult males have levels of from 40 to 130 μg. per 100 ml. of plasma. Migeon and Plager (2), by means of paper chromatography, have isolated and identified dehydroepiandrosterone from human plasma. Another compound in smaller amounts was isolated, which was thought to be androsterone and/or etiocholanolone. The study to be reported was undertaken in an attempt to define more clearly the pattern of neutral 17-ketosteroids in human plasma. A large volume of plasma was extracted so that there would be enough steroidal material for fractionation by means of column chromatography and identification by definitive means. This communication deals with a description and the results of such a...

Previous work showed a distribution of labeled cortisol in rat liver from 30 sec to 40 min after intravenous administration. The ultracentrifuged supernatant fraction (cytosol), containing about 2'3 of the label in the liver, has now been separated into a sharp, Sephadexchromatographed peak containing most of the supernatant label. Various findings indicate that this material is associated in a complex; this may be of molecular weight 5000 or a little less, a complex that could possibly be composed of steroid, polynucleotide and polypeptide. Further column and paper chromatographic procedures separated this material into 2 bands, each labeled and possessing 260 mμ absorption characteristics. Studies with corticosterone gave similar results. (Endocrinology 87: 486, 1970)

The response of the guinea pig adrenal to intraperitoneal injections of ACTH is reflected by a four to eightfold increase in urinary corticoid and a one to twofold increase in urinary 17-ketosteroids. All the identified corticoids and 17-ketosteroids are eliminated in sufficient amounts to make their detection, isolation and quantitation possible by means of paper chromatography and conventional colorimetric assays. The corticoids and the 17-ketosteroid 6β,11β- dihydroxy-Δ4-androstene-3,17-dione are eliminated in the urine as free compounds, while the ring A saturated 17-ketosteroids are eliminated as their water-soluble conjugated forms. Cortisone has been isolated for the first time from guinea pig urine. THE isolation and identification of corticoids from the urine of normal and ACTH treated guinea pigs has been reported (1, 2, 3), whereas the quantitative estimation of tKese substances has only been partly determined by Burstein et al. (2).

THE application of paper chromatography and infra-red spectrometry has permitted the isolation and identification of the major C-21 and C-19 metabolites excreted in urine, following the administration of adrenosterone (Savard et al., 1953) hydro cortisone and 21-desoxycortisone (Burstein et al., 1953). It has been shown that the C-21 steroids are reduced in the human body to the pregnane (5β) and transformed to the corresponding C-19 etiocholane (5β) derivatives. On the other hand, the Δ4-3 keto system in adrenosterone is converted predominantly into derivatives of the androstane (5α) series, which indicates that the ethyl side chain in position 17 of these steroids appears to direct the reduction of the Δ4,5 double bond to the 5β form in vivo. The in vivo metabolism of cortisone has been described by Dobriner et al. (1950, 1951) and Lieberman et al. (1951). However, no details were given as to the relative quantities of the metabolites excreted. In view of the importance of cortisone in endocrinological ...

A method for measuring cortisol, corticosterone, testosterone, progesterone, and 17-OH progesterone simultaneously in 2 ml of plasma has been developed especially for episodic secretion studies in which many frequent assays are required. Steroids were extracted from plasma, separated into 4 fractions by paper chromatography, and individual steroids in each fraction were measured by radioimmunoassay. The individual steroid radioimmunoassays were demonstrated to be sensitive, specific, accurate, and precise. Base line 8 AM plasma steroid levels were measured in normal males and menstruating females both before and after ovulation. The values obtained were in good agreement with those previously reported by other investigators using different methods. In order to establish the normal diurnal variation in both sexes, plasma steroid levels were measured at 20-min intervals throughout the day in a normal man and a preovulatory woman. When plasma steroid levels were plotted against time frequent intermittent pea...

11β-Hydroxyandrostenedione, cortisone, 20α- and 20β-dihydrocortisol and cortisol were identified, by means of paper and thin layer chromatography and reverse isotope dilution technique, as transformation products of cortisol-4-14C in rheumatoid synovial membrane. Non-enzymatic conversion of cortisol to 11β-hydroxyandrostenedione and other unknown compounds has been shown in control experiments. The major metabolic pathways of cortisol in human rheumatoid synovial membrane involve oxidation of the 11β-hydroxyl to 11-ketone, reduction of the 20-ketone to 20α-ol and 20β-ol. No ring A reduced metabolites could be detected.

Plasma levels of 2-hydroxyestradiol (2-OHE2) were measured using a new RIA procedure. Values were below the detection limit of the assay (less than 10 pg/ml), except in the third trimester of pregnancy, when they rose to approximately 15 pg/ml. The infusion of 130 microgram/h purified 2-OHE2 elevated its plasma concentration to 155 pg/ml, consistent with a plasma MCR (MCRp) of approximately 20,000 liters/day. The infusion of [3H] 2-OHE2 to equilibrium and chromatographic separation of the extracted plasma metabolites yielded an MCRp of about 13,000 liters/day; the major plasma metabolite comigrated with 2-methoxyestradiol, and [3H] xi-methoxyestrone was also formed. The MCRp, of 2-OHE2 is approximately half that of 2-hydroxyestrone (2-OHE1), but much higher than those of other steroids. As is true for 2-OHE1, the clearance of 2-OHE2 must occur primarily in the blood compartment. Together, the measured MCRp values and estrogen receptor affinities of 2-OHE2 and 2-OHE1 predict a relative potency for effects upon gonadotropin secretion which is close to that observed in vivo.

To assess the capacity of the human fetal ovary to utilize pregnenolone sulfate as a precursor of free steroids, homogenates of fetal ovaries from 3 fetuses (88–124 days) were incubated with 3H-pregnenolone sulfate. The rate of cleavage of pregnenolone sulfate in ovaries was compared to that in adrenals and liver. The rate of cleavage was between 3 and 7 times greater in ovaries than in adrenals and liver from the same fetus. No difference in the rate of cleavage was observed in the ovaries from fetuses of different ages. The various free steroid products from the incubations of the ovaries were isolated, identified and the amounts produced in 2 hr expressed as pmoles/mg protein incubated. Pregnenolone, 17-hydroxypregnenolone, dehydroepiandrosterone and androstenedione were identified as products in each incubation of fetal ovarian tissue. Although progesterone, testosterone, estradiol and estrone were sought extensively, they were not found as products of pregnenolone sulfate metabolism.

The metabolism of pregnenolone-7α-3H has been studied in vitro in an aldosterone producing tumor and in the adrenal tissue adjacent to the tumor. The predominant products in the incubation of the tumor tissue were progesterone, and lesser amounts of deoxycorticosterone and aldosterone, with no cortisol. The adjacent adrenal tissue converted pregnenolone to androstenedione and cortisol, with no aldosterone. Although 17-oxygenated steroids were produced by both tissues, the tumor failed to convert 17-hydroxyprogesterone to 11-deoxycortisol or cortisol. Androstenedione was formed in the tumor incubation but was not converted to an 11-hydroxylated product. These findings suggest that the biosynthetic pathways in the aldosterone secreting tumor were directed both by the presence of 18-hydroxylase system and by the relative absence of alternate pathways leading to cortisol or androgen synthesis.

Two-dimensional thin-layer chromatography on aluminum oxide provides a simple, rapid, yet sensitive method for the separation and identification of individual urinary 17-ketosteroids. By employing 2 solvent systems in which the steroid compounds exhibit different mobilities, it is possible to separate not only the 11-oxygenated 17-ketosteroids but also such closely related compounds as dehydroepiandrosterone, androsterone and etiocholanolone on 1 single plate within 1 hr. The solvent systems giving the best results are 3.5 % methanol in benzene and equal volumes of ethyl ether and ethyl acetate. Good separation is obtained with pure standards as well as urine extracts. As little as 1 μg of material can readily be detected, so that 25–50 ml of urine is adequate for fractionation of the major urinary 17-ketosteroids. Recoveries of pure compounds as well as standards added to urine exceed 80%. The urinary excretion of the 6 major 17-ketosteroid metabolites in 6 normal males and 12 normal females, during a co...

Testosterone and androstenedione dynamics were studied with a constant infusion technique in adult male dogs. The metabolic clearance rate (1370 ± 660 1/day) and blood production rate (1.5 ± 0.7 mg/day) of testosterone were similar to the metabolic clearance rate (1870 ± 760 1/day) and blood production rate (1.2 ± 0.3 mg/day) of androstenedione. Plasma concentrations of testosterone and androstenedione were respectively 115 ± 46 ng/100 ml and 66 ± 15 ng/100 ml. The conversion of these two prehormones to dihydrotestosterone was studied; respectively, 8.6 ± 2.2 and 1.8 ± 1.1% of plasma testosterone and androstenedione were converted to plasma dihydrotestosterone. In addition, testosterone and dihydrotestosterone were simultaneously measured in peripheral, adrenal and spermatic vein plasma. The concentration of testosterone at these three different sites was 116 ± 18, 98 ± 41 and 6620 ± 2100 ng/100 ml while the concentration of dihydrotestosterone was 34 ± 16, 79 ± 24 and 1710 ± 780 ng/100 ml. From these dat...

The liver of a 38-year-old man was injected via the portal vein with 100 μc-1,2-3H-cortisol over a period of 1 minute. Blood samples were withdrawn via the liver vein with a heart catheter at 1-, 3-, 5-, 10- and 20-minute intervals. Metabolism of cortisol was rapid (at 5 min.). The glucuronides represented 20% of the recovered metabolites. Cortols, cortolones, tetrahydrocortisone and tetrahydrocortisol predominated in this fraction. Only the ring-A reduced metabolites were found in the conjugate fraction, indicating a limitation by the Δ4-3-keto-reducing system. There appeared to be an influence by the oxygen at C-11 on the configuration of the 20-hydroxyl. There were a considerable number of metabolites in the unconjugated fraction. Cortisol, cortisone and their 20-reduced analogues, as well as the 6-hydroxylated derivatives, were in this portion. Five 11-oxygenated-17-ketosteroids were also indicated. The sulfates appeared in such low concentration that their analysis was not attempted.

A double isotope derivative dilution procedure has been developed to measure deoxycorticosterone (DOC) in human peripheral plasma. The method involves the addition of [14C]-DOC as internal recovery indicator and [3H]-acetic anhydride for derivative formation. The method shows adequate specificity, precision and accuracy. Simultaneously, aldosterone, corticosterone, cortisol and 11-deoxycortisol have been measured with DOC. Normal plasma DOC levels were found to be 6.3 ± 1.0 ng/100 ml (mean ± se, n = 17). The corresponding levels of aldosterone, corticosterone, cortisol and 11-deoxy cortisol were 6.5 ± 1.0 ng/100 ml, 0.42 ± 0.09 μg/100 ml, 13.9 ± 1.4 μg/100 ml and 0.060 ± 0.013 μg/100 ml respectively. ACTH administered to 6 subjects produced a mean increase in plasma DOC from 4.7 to 32.4 ng/100 ml. Angiotensin II infused at subpressor doses for 2 hr into 8 subjects did not increase mean plasma DOC. Similarly, dietary sodium restriction for 5 days or postural change did not increase plasma DOC. These result...

Seven patients with adrenal tumors have been studied. Two of them showed signs of feminization, 4 demonstrated only symptoms of masculinization, and 1 had a nonfunctional tumor. Peripheral and adrenal vein plasma concentrations of testosterone (T), androstenedione (Δ), dehydroepiandrosterone (D), its sulfate (DS) and glucuronide (DG) conjugates, androsterone sulfate (AS) and glucuronide (AG), and etiocholanolone sulfate (ES) and glucuronide (EG) were determined. Adrenal secretion of T, Δ, D and DS was established on the basis of differences in steroid concentrations in peripheral and adrenal vein blood. None of the other androgen conjugates (AS, AG, ES and EG) were found to be secreted by the adrenal tumors. When considering the age and sex of the patients, the concentrations in peripheral vein plasma of T, Δ, D and DS were increased in all subjects studied except for a normal level of T and D in 1 of the 2 patients with symptoms of feminization. Fractionation of urinary steroids in 3 patients wi...

The capacity of mouse testes to synthesize testosterone during fetal development, and the effects on synthesis of an inhibitor of 3- keto-4-steroid formation, 2α-cyano-4,4,17α-trimethyl- 17β-hydroxy-5-androsten-3-one (WIN) were studied. In fetal rats, WIN inhibited testicular 3 (3-hydroxy-5-steroid dehydrogenase activity and retarded development of the external genitalia (Goldman et al., Proc Soc Exp Biol Med 121: 757, 1966). Testes of fetal (14.5 and 17.5 days of gestation) and newborn mice from untreated mothers converted 4-14C-pregnenolone to 14C-testosterone during in vitro incubations. The results demonstrated testicular capacity for steroid metabolism to testosterone during the period of reproductive tract differentiation in mice. WIN, injected at doses of 1.5–7.0 mg/kg body weight into 12.5–15.5 day pregnant mice, exerted no effects on the gross development of the structures of the reproductive tract in either sex or on the conversion of 4-14C-pregnenolone to testosterone by fetal or newborn testes...

In an attempt to study the mechanism of action of minocycline and related compounds on thyroid activity, 9 chemically related compounds were administered to male and female rats fed a low or high iodine diet, to rats treated with thyroxine (T4) and to thyroidectomized, T4 maintained rats. Administration of minocycline produced a large goiter with high radioiodine uptake, the effect being more apparent in rats fed a relatively high iodine diet. Among the 9 compounds tested, minocycline, tetracycline phosphate (TC-phosphate), metacycline and doxycycline were goitrogenic in rats fed a low iodine diet, although goitrogenic activity varied greatly from compound to compound. In contrast, chlortetracycline hydrochloride (C1-TC-HC1) and demethyl chlortetracycline hydrochloride (demethyl C1-TC-HC1) reduced thyroid size under similar condition. Minocycline administration reduced thyroidal T4 and triiodothyronine (T3) and lowered plasma PBI, without affecting MIT/DIT. In sharp contrast with methylthiouracil, minocyc...

Iodide-deficient female rats were fed propylthiouracil (PTU) or dl-5-vinyl-oxazolidinethione mixed into a low-iodide diet for 10 days and then were either maintained on a low-iodide diet or given iodide supplements. Recovery of thyroid function after withdrawal of the antithyroid agents was markedly affected by the iodide supplementation. Without the iodide supplement 1) there was no regression of goiter or increase in serum PBI concentration; 2) the uptake of 131I by the thyroid was rapid and the 4-hr uptakes showed the expected ‘rebound’ phenomenon; 3) the 131I which was accumulatedby the 4th hr after administration was rapidly lost from the gland so that the 24-hr values were low and did not show the “rebound” phenomenon; 4) the ratios of radioactive DIT%MIT and T4%T3 were reversed; 5) there was a rapid loss of 131I-labeled MIT and DIT from the thyroid between the 4th and 24th hr after isotope administration, commencing 1 day after withdrawal of the drug, and a rapid loss of radioactive T4 and T3, comm...

A competitive binding assay using the cortical receptor from normal adrenal glands has been developed for ACTH. In this assay porcine, human or rabbit ACTH has been shown to compete with labeled porcine ACTH for the cortical receptor from ovine, rabbit or rat adrenal glands. One pg per assay tube reproducibly caused a significant decrease in percent bound. The mean plasma ACTH concentration was 75 pg/ml in normals at 0800 and 54 pg/ml in normals at 1530. Plasma ACTH was markedly elevated after oral metyrapone or during insulin hypoglycemia and was suppressed following dexamethasone. Addisonian patients consistently had elevated plasma ACTH concentrations prior to their maintenance hydrocortisone; ACTH concentrations were not consistently elevated in one patient with untreated Cushing's disease.

We have studied a patient with metastatic lipid cell tumor of the ovary. The tumor was responsive to HCG but not ACTH. Predominant urinary steroids were androsterone, etiocholanolone, pregnanetriol and pregnenetriol. Plasma levels of androstenedione, testosterone, 17–hydroxyprogesterone and 17-hydroxypregnenolone were increased. Most of the plasma testosterone (0.3 μg/100 ml) originated from plasma androstenedione (3 7mu;g/100 ml). From the pattern of blood and urine steroids we concluded that the important secretory product of the tumor was androstenedione. The question of the origin of this tumor was considered on the basis of its biosynthetic capacities and from a critical review of the literature. Despite many earlier statements, we conclude that this tumor has not caused Cushing's syndrome and that our studies and previous ones suggest that the origin of the tumor is the ovarian stromal cell.

A double isotope derivative dilution procedure is described for the measurement of aldosterone in peripheral blood of man and the sheep. 4-14C-aldosterone is used as the recovery indicator and 3H-acetic anhydride to form the radioactive derivative. The over-all nonspecific blank of the method for 76 samples was 0.22 ±0.003 mμg/20 ml when distilled water, or blood, or plasma from adrenalectomized castrated sheep or humans, was analyzed. Details of the specificity, accuracy and precision of the method are presented. The mean plasma aldosterone obtained from 25 normal recumbent humans, on a diet containing 80–120 mEq sodium/day, was 5.8 ±4.5 ±0.9 mμg/100 ml plasma. Sheep in normal sodium balance had levels below 1 mμg/100 ml blood. All values quoted have been corrected for blank. An enzymic hydrolysis procedure is described for the measurement of ring labeled tritiated steroids when 3H-acetic anhydride is used for derivative formation.

Cytoplasm from rat prostate glands binds tritiated pregnenolone and progesterone (10−8m) during incubation in vitro. The responsible binding substance, recognized by its incorporation into a radioactive steroid-“receptor” complex, depends on intact sulfhydryl groups for the binding property. The “receptor,” not present in plasma, has been detected in prostate cytoplasm using sucrose density gradients, and has a molecular weight of 45,000–50,000. The binding is not inhibited by heating the cytoplasm to 60 C, by addition of ethanol up to 7%, nor by storage up to one week at 4 C. Binding does not alter the radioactive steroids. Testosterone, estradiol-17β, deoxycorticosterone and cortisol are not bound by the “receptor”; small amounts of androstenedione and dehydro-epiandrosterone appear to be bound. After the incubation of prostatic slices with pregnenolone and progesterone (10−8m), less “bound” radioactivity appeared in the cytoplasmic fraction than was observed after incubation of the steroids and the cyt...

Groups of pregnant rats were injected with 2α-cyano-4,4,17α-trimethyl-17p-hydroxyandrost- 5-en-3-one (WIN) at varying dosage and frequency. Injections of 120-240 mg/kg body wt resulted in male fetuses with markedly reduced anogenital distances and hypospadias, but with unaffected differentiation of the reproductive ducts and of the urogenital sinus. Fetuses of rats injected with WIN or corn oil (control) at 14.5 to 16.5 days post coitum were examined on day 20.5 for testicular in vitro metabolism of 4-14C-pregnenolone and of 4-14C-dehydroepiandrosterone. Control testes converted each substrate primarily to 14C-testosterone and 14C-androstenedione. WIN-exposed testes synthesized markedly diminished amounts of 14C-testosterone and 11Candrostenedione, but accumulated Δ5-3β-hydroxysteroids: 14C-17α-hydroxypregnenolone,14Cdehydroepiandrosterone, plus residual substrate from 4-14C-pregnenolone, and 14C-androstenediol and residual substrate from 4-14C-dehydroepiandrosterone. Fetal testes of both the control and ...

Granulosa and thecal cells were isolated from 2 human ovarian follicles and the separated and recombined cells were incubated in vitro with acetate-1-14C. Radioactively labeled cholesterol, pregnenolone, progesterone, 17-hydroxyprogesterone, androstenedione and estrone were isolated in radiochemically pure form from each of the experimental situations utilizing either granulosa, theca or a mixture of the 2 cell types. In addition, labeled dehydroepiandrosterone was isolated only from the incubation with granulosa cells, while labeled estradiol-17β was isolated only from the thecal cell and recombined studies.

It has been observed that discrepancies, and often pronounced ones, exist when apparent cortisol secretion rate is measured from each of 6 individual metabolites of cortisol. This study employed adrenalectomized patients maintained on dexamethasone. Unlabeled pure cortisol was infused episodically to simulate normal adrenal secretion. Cortisol-4-14C was infused at different times in relation to the measured plasma cortisol content. Urines were collected for 3 days and the specific activities were measured for cortol, cortolone, β-cortol, β-cortolone, tetrahydrocortisol and tetrahydrocortisone. The specific activities demonstrated that the tracer was not metabolized under any circumstance in precisely the same manner as the cumulative metabolism of the unlabeled cortisol. This is a sufficient reason for the discrepancies found in secretion rate measurements and no other mechanism need be invoked. Consideration is given to the validity of approximation of the true cortisol secretion rate. It was co...

Preincubation of canine thyroid slices in KRP for 1–2 hr increased the organic binding of iodine by such slices and their response to TSH and other stimulators of iodination. TSH, dibutyryl cyclic 3′,5 ′-AMP (dbcAMP), NaF and prostaglandin Ei (PGE1) each stimulated organic binding of iodine and formation of thyroxine in preincubated slices and their effects were not additive. Anti-TSH rabbit serum blocked the effect of TSH on organisation, but not the effect of dbcAMP. Stimulation of organic binding by TSH, dbcAMP, NaF and PGE1 was still apparent in the presence of perchlorate. The presence of hydrogen peroxide in the medium enhanced organic binding of iodide and thyroxine formation in thyroid slices and this effect was not additive with the effects of TSH, dbcAMP, NaF or PGE1. Under conditions where organic binding by slices was blocked, T/M radioiodine concentration ratios after 30 min and 6 hr incubation periods were lower than controls in the presence of TSH, dbcAMP, NaF and PGE1. Adenyl cyclase activ...

In order to study the nonspecific influence of illness on the peripheral metabolism of androgens, trace amounts of 14C-testosterone were administered intravenously to 16 healthy control subjects (4 men and 4 women aged 23–35, and 5 men and 3 women aged 68–86) and to 47 patients (26 men and 21 women, aged 16–84) with a wide variety of types and severity of illness, and trace amounts of 14C-dehydroisoandrosterone were administered to 13 healthy controls (3 men and 4 women aged 21–32, and 3 men and 3 women aged 67–85) and to 15 patients (9 men and 6 women, aged 19–82) with various illnesses. It was found that both precursor hormones were converted to abnormally low proportions of androsterone and etiocholanolone in a large number of the patients, regardless of diagnosis. Mean recovery of etiocholanolone plus androsterone from 14C-testosterone in control subjects was 48% of the dose, with a coefficient of variation of 9%. Recovery in the ill patients was very variable, ranging from high normal to les...

A 22-month-old Negro girl with metastatic neuroblastoma was found to be excreting markedly elevated levels of dopa, dopamine, VMA and HVA in her urine. To study the catecholamine metabolism in this patient, a solution containing both l-dopa-2,5,6-3H (167 μCi) and dopamine-2-14C (16.7 μCi) was administered intravenously, preoperatively, and serial urine specimens were collected for 48 hr. At laparotomy a 197 g tumor was removed from the region of the left adrenal gland. Dopa, dopamine, HVA, VMA and other metabolites were isolated from the urine specimens and their 3H and 14C contents and 3H/14C ratios were assayed. The urinary excretion rates of 3H and 14C were rapid and 28.5% of the injected 3H-dopa was excreted as 3H-dopamine in 2 hr, while only 5.9% of the 3H-dopa was excreted unchanged during this period. For 14C-dopamine, 11.7% was excreted unchanged and 13.7% was excreted as 14C-HVA. The 3H/14C ratios of urinary norepinephrine were strikingly high (96 to 298) and that of urinary dopamine was...

Interstitial cells were separated from seminiferous tubules in a testis from a 17-yr-old male by treatment of tissue with collagenase. The separated compartments were incubated with 3H-androstenedione and a NADPH generating system. The various products were isolated, identified and the amounts produced following a 2-hr incubation were compared in the separated tissue compartments. The conversion to testosterone was approximately the same in the 2 compartments. Seminiferous tubules effected a much greater conversion to Sa-reduced metabolites than did the interstitial cell suspension. The ratio of Sa-reduced steroids in tubules to that in interstitial cell suspension was 50 to 1. Only seminiferous tubules synthesized dihydrotestosterone, while androsterone was only identified in the interstitial cell incubation. Both 5α-androstane-3β,17β-diol and 5α-androstane-3α,17β-diol were found in equal amounts in seminiferous tubules (7% total conversion from androstenedione). The androstanediols were found o...

The metabolism of radioactive progesterone administered simultaneously intravenously and percutaneously has been studied in 4 normal subjects and 1 patient with testicular feminization syndrome. The relative contribution to urinary 5β- and 5α-pregnan-3α,20α-diol (pregnanediol and allopregnanediol) of labeled progesterone, according to its mode of administration, was determined. In normal subjects, the yield of pregnanediol originating from an intravenous dose of progesterone was 8 times higher than that of allopregnanediol. By contrast, when progesterone was administered percutaneously, the recovery of labeled allopregnanediol in urine was greater than that of pregnanediol. These data indicate that in normal subjects progesterone might be 5α-hydrogenated to a greater extent in the skin. In addition, the metabolism of progesterone administered either intravenously or percutaneously was similar in men and women; it was unchanged throughout the menstrual cycle and by estrogen treatment of men. In th...

The urinary excretion of 11β, 17α-dihydroxy-3,20-diketopregn-4-ene-21-yl sulfate (cortisol sulfate), 17α-hydroxy-3,11,20-triketopregn-4-ene-21-yl sulfate (cortisone sulfate) and of 11β-hydroxy-3,20-diketopregn-4-ene-21-yl sulfate (corticosterone sulfate) by newborn infants aged 2 and 6 days was quantified by double isotope dilution derivative assays and compared to that of known metabolites of cortisol and corticosterone. In a series of 7 newborns the sum of the urinary concentrations of cortisol and coitisone sulfates on the 2nd day of life represented represented from 43 to 114% of the amount of tetrahydrocortisone glucuronide, and on the 6th day from 8 to 155%. Corticosterone sulfate was excreted in amounts consistently much higher than tetrahydrocorticosterone glucuronide. It is concluded that the presence of these sulfate esters in urine reflects the existence of yet another pathway of corticosteioid biosynthesis and/or metabolism of particular significance during the perinatal period.

Urinary steroid excretion of 6β-OH-cortisol (6β-OH-F), tetrahydrocortisone (THE), l6α-OH-pregnenolone (16α-0H-PG) and 16a-OH-dehydroepiandrosterone (16α-OH-DHA) was determined in 23 full-term normal newborn infants, 8 normally grown premature and 8 intra-uterine growth retarded (IUGR) newborns. The method used measured free, glucuronide- and sulfate-conjugated steroids. Labeled internal standards were used for each steroid measured. The excretions were expressed in relation to urinary creatinine levels. Steroid excretion values in the normally grown full-term and premature groups were quite similar. On comparison of steroid excretion values in the IUGR and normal full-term groups by the Mann-Whitney (two-tailed) test, there were no significant differences in 6β-OH-F and THE excretions but the excretions of 16α-0H-PG and 16α-0H-DHA were significantly less (p < 0.02 and p < 0.01, respectively) in the IUGR group. The ratio 6β-OH-F + THE/16α-OH-PG + 16α-OH-DHA was significantly greater in the IUGR infants tha...

Accumulation of 35S-radioactivity by the thyroid gland was found after administration of equimolar doses of 35S-labelled thiouracil, methylthiouracil, propyl thiouracil, methimazole and carbimazole to rats. Thin-layer chromatography showed an accumulation of the parent drug in the case of methimazole, propylthiouracil, methylthiouracil and thiouracil. When carbimazole was given, an accumulation of methimazole was demonstrated in the thyroid. Propylthiouracil administration produced the largest amount of drug in the thyroid and thiouracil the least. In man both 35S-radioactivity and the parent drug accumulated in the normal thyroid after administration of methimazole and propylthiouracil. When carbimazole was given, methimazole accumulated in the thyroid. In six thyroid tumors little or no accumulation of the antithyroid drug was found.