Studies on the metabolism of steroid hormones in a virilizing adrenal cortex adenoma.

Slices of an adreno-cortical adenoma which had been obtained at operation from an 11-year-old girl with clinical signs of virilism were incubated with each of the following steroids: [1,2-3H]progesterone, [4-14C]pregnenolone, [1,2-3H]testosterone, [4-14C]androstenedione and [7-3H]dehydroepiandrosterone, respectively. Isolation and identification of the free radioactive metabolites were achieved by gel column chromatography on Sephadex LH-20, thin-layer chromatography, radio gas chromatography and isotope dilution. After incubation of progesterone, the following metabolites were identified: 11beta-hydroxyprogesterone, 16alpha-hydroxyprogesterone, 17alpha-hydroxyprogesterone, 21-deoxycortisol, corticosterone and cortisol. Pregnenolone was metabolized to 17alpha-hydroxypregnenolone, progesterone, dehydroepiandrosterone, androstenedione and 11beta-hydroxyandrostenedione. When testosterone was used as substrate, 11beta-hydroxytestosterone, androstenedione and 11beta-hydroxyandrostenedione were found as metabolites, whereas androstenedione was metabolized to testosterone and 11beta-hydroxyandrostenedione. After incubation of dehydroepiandrosterone, only androstenedione and 11beta-hydroxyandrostenedione were isolated and identified. From these results, it appears that cortisol was formed in the adenoma tissue via 21-deoxycortisol and corticosterone. Delta4-3oxo steroids of the C19-series arose exclusively from pregnenolone via 17alpha-hydroxypregnenolone and dehydroepiandrosterone, and not from progesterone and 17alpha-hydroxyprogesterone. Calculated on the amounts of metabolites formed, the highest enzyme activities were those of the 11beta-hydroxylase and the 17alpha-hydroxylase. It is interesting to note that only traces of testosterone were detected after incubation of androstenedione, whereas testosterone yielded large amounts of androstenedione.     

Studies on the role of intestinal bacteria in metabolism of synthetic and natural steroid hormones

Administration of antimicrobial agents to subjects taking oral contraceptives has been reported to lead to contraceptive failure and subsequent pregnancy. In women taking oral contraceptives antimicrobial agents could have an effect on both endogenous hormone levels and on the metabolism of the exogenously administered steroids. To investigate these possibilities, antimicrobial agents were administered for short periods to normal women taking various steroid drugs: Megestrol acetate (MA), medroxyprogesterone acetate (MPA), norethisterone (NET), a combination of NET and ethinylestradiol (EE) or a combination of lynestrenol and EE. During ampicillin administration the 24-h morning plasma concentrations of MA, MPA and NET were increased compared to the control values. In the MA and MPA experiments the afternoon values were determined and also found to be increased. In the subjects taking oral contraceptives plasma EE concentration showed a tendency to decrease during ampicillin administration on the third, fourth or fifth morning of ampicillin administration, but was never lower than the pretreatment values. In other experiments plasma estrone (E1) and estradiol (E2), urinary total E1, E2 and estriol (E3) and fecal unconjugated and conjugated E1, E2 or E3 were determined by RIA before, during and after administration of oxytetracycline (2 X 500 mg/day for 5 days) to 5 young male subjects. Furthermore urinary and fecal estrogens were determined in 1 male subject after administration of erythromycin for 6 days and in 2 normally menstruating women after tetracycline and trimethoprim administration, respectively. During treatment with antimicrobial drugs an increase in the excretion of fecal conjugated and, with the exception of the oxytetracycline experiments, also of unconjugated estrogens paralleled a decrease in urinary estrogen excretion, especially for E2 and E3. In both urine and feces the E1/E2 and E1 + E2/E3 ratios increased due to diminished reductive metabolism of estrogens in the gut. No significant effects on plasma unconjugated estrogen concentrations were observed. The results suggest that the intestinal bacterial flora plays a significant role in estrogen metabolism. However, further studies are necessary, because our results do not explain why administration of antibiotics may cause contraceptive failure.     

Tritium nuclear magnetic resonance spectroscopy. Part VI. Tritiated steroid hormones

The regio- and stereo-specificity of the labelling in a series of tritiated steroid hormones has been examined by ³H n.m.r., which also yields quantitative information on the distribution of the tritium between the labelled sites. Complete analysis is thus readily achieved non-destructively. Hydrogen chemical shifts for various skeletal sites are provided for the first time. The specificity of the methods of labelling steroids with tritium by catalytic reduction, catalysed exchange, and tritiodehalogenation are discussed.     

Studies on steroid metabolism in human endometrial tissue

1. A computerized technique is described for the quantitative determination of radiometabolites from incubation studies. 2. Seven steroid substrates have been incubated with human endometrial tissue. The principal radiometabolites were identified and determined after 2hr. incubation without the addition of cofactors and after 4hr. incubation with cofactors. 3. The main products from progesterone were 20alpha-dihydroprogesterone and 5alpha-pregnanedione with lower yields of 5beta-pregnanedione and 20beta-dihydroprogesterone. There was no evidence for 17alpha-hydroxylase activity. 4. 17alpha-Hydroxyprogesterone was transformed into small yields of 17alpha,20alpha- and 17alpha,20beta-dihydroxypregn-4-en-3-one. In one incubation there was evidence for conversion into androstenedione. 5. Dehydroepiandrosterone was transformed into small amounts of androstenedione, 5alpha-androstanedione and androsterone. 6. Androstenedione and testosterone were interconvertible, the reaction favouring the formation of androstenedione. 5alpha-Androstanedione and androsterone were formed from both substrates. There was no evidence for the formation of phenolic steroids. 7. Oestrone and oestradiol-17beta were interconvertible, the reaction favouring the formation of oestrone.     

Steroid-protein interactions. Stopped flow fluorescence studies of the interaction between steroid hormones and progesterone-binding globulin.

Stopped flow fluorometry, measuring changes in the intrinsic fluorescence of progesterone-binding globulin (PBG), was used to determine the association and dissociation rates of the interaction of PBG with seven delta4-3-ketosteroids. The rates of formation and dissociation of the PBG-progesterone complex were measured as a function of concentration and temperature. At 20 degrees, kon = 8.7 X 10(7) M-1 S-1 and koff = 0.060 S-1. The association rate constants for progesterone, deoxycorticosterone, testosterone, testosterone acetate, and medrogestone were found to be the same within experimental error. The different affinities of PBG for these steroids result from the dissociation rate constants of the steroids which ranged from 0.43 S-1 for testosterone to 0.024 S-1 for medrogestone. Two corticosteroids, corticosterone and cortisol, were both bound somewhat more slowly (approximately 5 X 10(7) M-1 S-1). Reflecting their very low affinity for PBG both steroids dissociate very rapidly: corticosterone at 1.4 S-1 and cortisol at 90 S-1. The ratio of association to dissociation rate constants gave affinity constants in agreement with independently determined constants.     

Quantitative characteristics of interactions of blood lipoproteins and steroid hormones]

Human and serum lipoproteins interaction with steroid hormones (corticosterone and hydrocortisone) were studied. Methods of fluorescence quenching titration and equilibrium dialysis were used for quantitative evaluation of VLDL, LDL and HDL glucocorticoids binding ability. Association constants were found to be 0.6-2.0 x 10 M for corticosterone and 4.0-8.0 x 10 M for hydrocortisone. The number of binding sites ranged from 3 to 300 for different classes of lipoproteins. Our data suggest high specificity of serum lipoproteins binding with corticosterone and hydrocortisone.     

Studies on Bacillus stearothermophilus. Part II. Transformation of progesterone

Bacillus stearothermophilus, a thermophilic bacterium isolated from Kuwaiti desert, when incubated with exogenous progesterone for 10 days at 65 degrees C produced two new dihydroxy isomers of progesterone, and two known compounds, 5 alpha-pregnane-3,6,20-trione and 6-dehydroprogesterone, along with the earlier reported monohydroxylated metabolites and a B-Seco compound. The two new dihydroxy compounds were identified as 6 alpha,20 alpha-dihydroxyprogesterone and 6 beta,20 alpha-dihydroxyprogesterone. These metabolites were purified by TLC and HPLC followed by their identification through 1H, 13C NMR and other spectroscopic data.     

Determination of cytoplasmic receptors for specific steroid hormones. II) Androgen receptor

Cytoplasmic receptors for 5 alpha-dihydrotestosterone (3H-DHT) were determined in normal and hypertrophic human prostate using the slightly modified DCC method we previously standardized for 17beta-estradiol-receptor. Incubations were always performed at 0 degree C for 1 hr. Discrimination between 3H-DHT binding to cytoplasmic receptor and to Sex Hormone Binding Globulin (SHBG) was achieved on the basis of binding affinity, thermolability and pattern of specificity by various steroid hormones. In particular, 5 beta-DHT did not bind to cytoplasmic receptor, while it did to SHBG.