Properties of antisera to progesterone and to 17-hydroxy-progesterone elicited by immunization with the steroids attached to protein through position 7

Antibodies to progesterone (P) and to 17-hydroxyprogesterone (17-OHP) were raised by immunization of rabbits with progesterone-7α-carboxyethyl thioether--bovine serum albumin (P-7—BSA) or with 17-OHP-7α-carboxyethyl thioether--BSA (17-OHP-7--BSA). The antisera produced were of high affinity: K_a towards the homologous hapten was 3. 7 × 10¹⁰ 1./mol for the anti-P serum and 5. 9 × 10⁹ 1/mol for the anti-17-OHP serum. The antiserum to P-7—BSA displayed little or no cross reaction (?= 2%) with the 20α-, 20β- or 5β-dihydro-derivatives of progesterone, moderate cross-reaction with pregnenolone (4%), but considerable cross-reaction with 11-deoxycorticosterone (7%), 5α-dihydro-progesterone (11%) and 17-OHP (15%). The antiserum to 17-OHP-7--BSA showed very little cross-reaction (?= 2%) with progesterone and other steroids lacking a 17α-hydroxyl group, such as pregnenolone or 11-deoxycorticosterone, but reacted significantly with 17α, 21-dihydroxy-4-pregnene-3, 20-dione (8%) and 3β, 17-dihydroxy-5-pregnen-20-one (13%). None of the sera reacted with testosterone, cortisol or estradiol-17β. It appears that conjugation of progesterone to protein through carbon-7 affords antisera comparable in specificity to those raised with 11α-conjugates and superior to those raised with 3-, 6- and 20-conjugates. The antiserum to 17-hydroxyprogesterone described is the first one that specifically recognizes this metabolite.     

Metabolism of 4-14 C-progesterone in the adult female chimpanzee.

The metabolism of 4- 14-C-progesterone was studied in two adult female chimpanzees (Pan troglodytes). After intravenous injection of 4- 14-C-progesterone, urine was collected for 5 days. The urinary conjugates were hydrolyzed with Glusulase and the extracts purified by chromatography on silica gel, paper and alumina and then crystallized to constant specific activity with or without carrier steroid. The major metabolite in both experiments was pregnanediol which accounted for 39.8% and 49.5% of the recovered dose respectively. Pregnanolone (0.6% and 2.1%) and pregnanetriol (0.1% and 1.5%) were also isolated in smaller quantities. These data suggest that the pattern of progesterone metabolism in the chimpanzee is similar to that of man in that pregnanediol is the major metabolite whereas androsterone is not.     

Specific antisera for the radioimmunoassay of estriol 3-sulfate

Antisera were raised in male guinea pigs against 6-oxoestriol 3-sulfate O-carboxymethyloxime-bovine serum albumin (BSA) conjugate. The antisera to this antigen exhibited high affinity (Ka=4.7 X 10(9)M-1) and excellent specificity for estriol 3-sulfate, showing slight cross-reactions (less than 0.43%) with other estrogen sulfates, and no cross-reactivities with free estrogens and other steroids (less than 0.01%) except cholesterol sulfate (0.22%). A standard curve using [6, 7-3H]-estriol 3-sulfate as the radioactive ligand showed high sensitivity in the range of 10-1000 pg estriol 3-sulfate.     

Isolation of 20 alpha and 20 beta 5 beta-pregnane-3 alpha,17 alpha,20,21-tetrol (hexahydro-Reichstein's compound-S) in a case of congenital adrenal hyperplasia

5β-Pregnane-3α, 17α, 20α, 21-tetrol (l) and 5β-pregnane-3α, 17α 20β, 21-tetrol (II) have been isolated and identified from the urine of a girl with congenital adrenal hyperplasia. The total 5β-pregnane-3α, 17α, 20(α+β),21-tetrol consisted of 60% of I and 40% of II. The final identity of the compounds was established by gas chromatography — mass spectrometry. The mass spectra of the two trimethylsilyl isomers were closely related to each other in contrast to the spectra of five other pairs of C₂₁-C-20(α and β)-hydroxy steroid-trimethylsilyl-ethers. The mass spectra of free I and II also exhibited many common features, but were less similar to each other than their trimethylsilyl derivatives.     

Biotransformation of pregnenolone - 7alpha-3H, progesterone - 7alpha-3H and dehydroepiandrosterone - 7alpha-3H by porcine fetal and maternal adrenal homogenate preparations.

The biotransformation of pregnenolone-7alpha-3H and of progesterone-7alpha-3H by porcine fetal and maternal adrenal homogenates at 56 and 112 days of pregnancy and of dehydroepiandrosterone-7alpha-3H by fetal adrenal homogenates has been investigated in vitro. Both pregnenolone-7alpha-3H and progesterone-7alpha-3H were metabolized extensively by maternal adrenal preparations, the principal radioactive metabolites isolated being cortisol, corticosterone, 11-deoxycortisol, deoxycorticosterone, 11beta-hydroxyprogesterone and androstenedione. In addition, 17alpha-hydroxyprogesterone, 20alpha-dihydroprogesterone and cortisone were formed from both substrates and 17alpha-hydroxypregnenolone and progesterone were formed from pregnenolone. Although essentially the same radioactive metabolites were isolated after incubation of fetal adrenal glands with pregnenolone-7alpha-3H or progesterone-7alpha-3H, a greater proportion of the radioactivity was associated with corticosteroids at 112 days of pregnancy than at 56 days. 11beta-Hydroxyandrostenedione and androstenedione were isolated and identified together with an unknown polar metabolite, after incubation of fetal adrenal tissue with dehydroepiandrosterone-7alpha-3H. These results are discussed in relation to feto-placental steroid biosynthesis and metabolism and the role of the fetal adrenal in the initiation of parturition in the pig.     

A radioimmunoassay for the measurement of 5-androstene-3β,17β-diol in plasma

A reliable radioimmunoassay for the determination of 5-androstene-3β, 17β-diol in plasma is described. Antisera were obtained by immunization of rabbits with 3β,17β-dihydroxy-5-androsten-16-one coupled to bovine serum albumin in position 16. The antiserum was characterized by titer, affinity, and specificity. Only dehydroepi-androsterone (24.3 %) and pregnenolone (2.7 %) showed a small crossreactivity. The assay method consisted of extraction with ether, thin-layer chromatography and endpoint determination.The reliability of the method was studied. The interassay variability was 7.5 % at a concentration of 1.22 μg/l. The limit of detection was 0.068 μg/l. Specificity was achieved by Chromatographic separation of the crossreacting steroids. Mass recovery experiments with 250 and 500 pg were performed, in which 99.0 ± 4.6 % of the smaller and 97.6 ± 11.3 % of the greater mass were recovered. In 45 healthy adult males plasma concentrations between 0.44 and 1.80 μg/l were found. The median was 1.06 μg/l. Stimulation of the Leydig cells with human chorionic gonadotropin (HCG) increased plasma concentrations by 93 % (average in 12 males). Therapeutic castration in 8 men caused an average decrease of 55.4 % in plasma values.     

Evaluation of adrenal function in rats by the measurement of urinary free corticosterone, free aldosterone and free 11-deoxycorticosterone.

Methods für the determination of urinary free corticosterone, free aldosterone and free 11-deoxycorticosterone (DOC) in rats are described. The free corticosteroids were measured in urine samples of 0.1–0.5 (2.0) ml by radioimmunoassay after purification by column chromatography. The validity of the methods is demonstrated by the data of the free urinary corticoids under basal conditions and after adrenal suppression and various forms of adrenal stimulation. The basal excretion of free corticosterone, free aldosterone and free DOC was 123.71 ± 15.31 (x? ± SD), 3.87 ± 1.29 and 10.61 ± 2.24 ng/day, respectively, exhibiting a decrease to 26.20 ± 5.21, 1.05 ± 0.47 and 1.35 ± 1.20 ng/day after adrenal suppression by dexamethasone. Irrespective of the mode of adrenal stimulation i.e., synthetic ACTH and systemic (cold, hunger) or neurotrophic (ether, reserpine) stress stimuli free corticosterone increased to about 450 ng/day, while free aldosterone excretion decreased during hunger and cold and was strongly enhanced after the application of reserpine. Furthermore, determination of urinary free DOC, which increased by a factor of 4, may be applied in the metyrapone test. There was a good correlation between the excretion of free corticosterone and that of free aldosterone and free DOC under basal conditions and after ACTH application, demonstrating that ACTH is responsible for the secretion of all the 3 corticoids measured. It is concluded, that the measurement of the urinary excretion of corticosterone, aldosterone and DOC is a valuable parameter of adrenal function in rats. Furthermore, in small laboratory animals like rats steroid measurements in urine are often more advantageous than Measurements in plasma.     

5α-Dihydro-11-deoxycorticosterone: Effect on blood pressure in the rat

The bioactivity of 5α-reduced sex steroids such as 5α-dihydrotestosterone has increased interest in an analagous role for 5α-reduced mineralocorticoids in hypertensive syndromes. In view of its relatively high mineralocorticoid receptor affinity despite relatively low electrolyte-altering effects, 5α-dihydro-11-deoxycorticosterone, or 5αDHDOC (2) was compared to 11-deoxycorticosterone acetate (DOCA) for blood pressure-altering ability by continuous subcutaneous infusion into uninephrectomized saline-drinking Sprague-Dawley male rats, at doses selected on the basis of relative mineralocorticoid receptor affinity. After three weeks of treatment, DOCA significantly raised blood pressure, body weight, heart and kidney weight, and produced a discernible increase in fluid intake; 5αDHDOC failed to affect any of these parameters commonly influenced by mineralocorticoids. We conclude that 1) the ability of 5αDHDOC to affect blood pressure was not predicted by its relatively high affinity for the mineralocorticoid receptor, and 2) these data do not support a role for 5αDHDOC in mineralocorticoid hypertension, although differences in protein binding and clearance could affect its blood pressure-altering activity.     

In vitro metabolism studies of (1, 2, 6, 7-3-H)-cortisol in human gingiva in health and disease.

The major conversion products of incubation of 3-H-cortisol with slices of human clinically normal and inflamed gingiva were identified as 11beta-hydroxyandrostenedione and cortisone. Reduction of C-20 of cortisol to 20alpha- and 20beta-dihydro metabolites was found only after incubation of cortisol with normal gingiva and not with inflamed tissue. Thus the two major pathways of metabolism of cortisol in the gingiva appear to be the oxidative cleavage of the side chain and the oxidation of 11beta-ol while the reduction of the 20-one appear limited to the normal tissue. The mean rates of conversion of cortisol to its various metabolites in 12 normal and 12 chronically inflamed gingival tissue samples were 147 and 55.2 pmoles hr-1-mg-1 respectively. The less conversion of cortisol in inflamed tissue might explain its effectiveness as a naturally occuring antiinflammatory hormonal steroid.     

Metabolism and conjugation of [4-14C]progesterone by bovine liver and adipose tissues, in vitro

The ability of bovine liver and fat to metabolize progesterone and also to form glucuronide conjugates with these progestins $\text{in vitro}$ was investigated. Tissue supernatants were incubated with [4-¹⁴C] progesterone, UDP-glucuronic acid, and a NADPH generating system for 5 hr, at 37°C. Steroids were identified by thin-layer chromatography, high performance liquid chromatography, and recrystallization to a constant specific activity. The total original radioactivity which could not be removed by exhaustive ether extraction (presumptive conjugates) was 44.7 ± 14.2% in liver, 5.0 ± 3.6% in subcutaneous fat, and 3.7 ± 2.2% in kidney fat samples. Progestins identified in liver samples include 5β-pregnane-3α, 20α-diol (free and conjugate), 5β-pregnane-3α, 20β-diol (free and conjugate), 3α-hydroxy-5sB-pregnan-20-one (free and conjugate), 3β-hydroxy-5β-pregnan-20-one (free), 5β-pregnane-3, 20-dione (free), and progesterone (conjugate). Progestins identified in both the free and conjugate fractions of subcutaneous fat and kidney fat samples include progesterone, 3α-hydroxy-5β-pregnan-20-one, 20β-hydroxy-4-pregnen-3-one, and 20α-hydroxy-4-pregnen-3-one. Differences due to sex of bovine used were noted. These results confirm the ability of bovine liver to readily metabolize progesterone and form glucuronide conjugates of these compounds and suggest that adipose tissues take an active role in these actions in cattle.     

High performance liquid chromatography of steroid metabolites in the pregnenolone and progesterone pathways

Rapid separation of gonadal steroids in the progesterone(Δ⁴) and pregnenolone pathway (Δ⁵) has been accomplished by the use of high performance liquid chromatography (HPLC). Two HPLC systems are utilized. The first requires the use of two separate radial compression columns (C-18 and C-8), with steroids being eluted with a methanol-water gradient. The second employs a stainless steel C-18 (reversed phase) column with a 12% octadecylsilane coating. The latter system separates seven of the eight steroids in the Δ⁴ and Δ⁵ pathways in thirty-five minutes. For the quantitation of steroids directly, integration of the peak areas, using 254 nm absorption for the Δ⁴ pathway steroids (5 ng minimum limit), and 210 nm absorption for the Δ ⁵ pathway steroids (25 ng minimum limit) is used. For the quantitation of radiolabeled metabolites resulting from incubation of gonadal tissue with radiolabeled steroid precursors, either one of two methods is used: (1) the eluent can be recovered from the HPLC using a fraction collector, and counted in liquid scintillation counter or (2) the entire eluent (or a portion of it) can be counted immediately by directing the flow through a radioactivity detector.     

The conversion of testosterone to 7α-hydroxy-testosterone by incubated rat testes

Incubations of testes of adult rats with testosterone yield rather important amounts of a very polar metabolite which is identified as 7α-hydroxytestosterone. The identification of the metabolite is based on chromatography, spectrophotometry, fluorimetry, counter current distribution and NMR spectrometry.     

Synthesis of new steroid haptens for radioimmunoassay. part I. 15β-Carboxyethylmercaptotestosterone-bovine serum albumin conjugate. measurement of testosterone in male plasma without chromatography

A radioimmunoassay (RIA) procedure has been developed for measurement of testosterone in male plasma after ether:chloroform (4:1) extraction of the plasma sample without resorting to chromatography. The highly specific anti-testosterone serum was generated from both rabbits and sheep immunized with 15β-carboxyethylmercapto-testosterone-BSA conjugate. The synthesis of 15β-carboxyethylmercaptotestosterone and the preparation of its BSA conjugate are described. The high affinity (K_a = 2.38 × 10⁹ liters/mole) antiserum binds 50% of 50 picograms of tritiated testosterone at working dilutions of 1:100,000 to 1:200,000. Both 5α and 5β-dihydrotestosterone compounds exhibited less than 2% cross-reaction. The only other steroids that showed minor cross-reaction were 11β-hydroxytestosterone (3.8%), progesterone (2.1%), corticosterone (1.6%), and deoxycorticosterone (7.7%).     

The identification and quantification of three new 6alpha-hydroxylated corticosteroids in human neonatal urine

The 24 hours urines of six two days old fullterm newborn infants were investigated for polar corticosteroids. 6alpha-hydroxy-tetrahydrocortisone, 6alpha-hydroxy-20alpha-cortolone and 6alpha-hydroxy-20beta-cortolone were identified by gas chromatographic-mass spectrometric comparison of the urinary steroids to compounds synthesized previously. These 6alpha-hydroxylated corticosteroids as well as seven other polar corticosteroids were quantified by gas chromatography or mass fragmentography. It was shown that the newly identified steroids constituted a quantitatively important part of the neonatal urinary corticosteroids. The unconjugated- and glucuronic acid conjugated steroids were quantified separately. It was found that the extent of glucuronoconjugation decreased with increasing polarity of the steroid moiety.     

Influence of age on the formation of 5alpha-androstanediol and 7alpha-hydroxy-testosterone by incubated rat testes.

Testes from rats of different ages were indubated with or without tritiated testosterone. The exogenously-added or endogenously-produced testosterone is mainly metabolized to 7alpha-hydroxylated testosterone in adult animals, and to 5alpha-reduced metabolites (especially 5alpha-androstanediol) in immature animals.     

Synthesis of corticosteroid haptens

6α- and 6β-Hemisuccinoxy derivatives of cortisol, 11-desoxycortisol, 21-desoxycortisol and 21-desoxycortisone were synthesized. The intermediate 6-hydroxy derivatives were prepared by the auto-oxidation of 3,5-dienol 3-alkyl ethers. During this step the dihydroxyacetone side-chain of cortisol and 11-desoxycortisol was protected by the 17,21-acetonide. Following hemisuccinylation the acetonide was removed with acetic acid. The selective synthesis of 3-O-(carboxymethyl)-oximes of cortisol and 11-desoxycortisol is described.     

Antisera for radioimmunoassay of 17alpha-ethynylestradiol and mestranol

Antisera for 17α-ethynylestradiol and mestranol have been prepared by immunizing rabbits with 6-(O-carboxymethyl) oxime-bovine serum albumin conjugates prepared from 6-oxo-17α-ethynylestradiol and 6-oxomestranol, respectively. These antisera showed little cross-reaction with known metabolites of these steroids. A comparison is made between our antisera and some prepared by others, where coupling to the steroid is effected through the C-7 position.     

The identification and characterization of the c19o3 steroid metabolites of 5α-androstane-3β, 17β-diol produced by the canine prostate: 5α-androstane-3β, 6α, 17β-triol and 5α-androstane-3β, 7α, 17β-triol

This study has identified the polar metabolites of 5α-androstane-3β, 17β-diol(3β-diol) produced by the canine prostate. The major metabolite is 5α-androstane-3β, 7α, 17β-triol (7α-triol) accounting for approximately 80% of the total polar metabolites of 3β-diol. The remaining 20% is accounted for exclusively by another triol, 5α-androstane-3β, 6α, 17β-triol(6α-triol). This study has also characterized two enzymatic hydroxylases responsible for respective triol formation: 5α-androstane-3β, 17β-diol 6α-hydroxylase (6α-hydroxylase) and 5α-androstane-3β, 17β-diol 7α-hydroxylase (7α-hydroxylase). Both of these irreversible hydroxylases are located in the particulate fraction of the prostate and can utilize either NADH or NADPH as cofactor. Several $\text{in vitro}$ steroid inhibitors of these hydroxylases were identified including cholesterol, estradiol and diethylstilbestrol. Neither of the hydroxylases were found to be decreased by castration (3 months) when expressed as activity/DNA. Using a variety of C₁₉ androstane substrates, 6α- and 7α-triol were found to be major components of the total 3β-hydroxy-5α-androstane metabolites produced by the canine prostate.     

Pregnanetriolone, a normal steroid metabolite: its excretion by normal, Cushing's syndrome and congenital adrenal hyperplasia subjects.

Synthesis of 3H-pregnanetriolone permitted the estimation of pregnanetriolone in urine with a sensitivity in excess of most previous claims. A good correlation (r = +0.97) was obtained between the values from gas liquid chromatography and those of a double isotope derivative method. In contrast to previous reports, these methods indicated that pregnanetriolone is excreted by normal adults. Urinary pregnanetriolone levels were 18-59 mug/24hr for normal subjects, 35-290mug/24hr in Cushing's syndrome and 250-7000 mug/24hr with congenital adrenal hyperplasia. It is concluded that pregnanetriolone is a normal steroid metabolite and its occurrence in Cushing's syndrome does not necessary indicate an abnormal steroid biosynthetic pathway.     

Chemiluminescence enzyme immunoassay of dehydroepiandrosterone and its sulfate using peroxidase as label

We report a highly sensitive enzyme immunoassay for dehydroepiandrosterone (DHEA) and its sulfate (DHEA-S) using horseradish peroxidase as the label enzyme. Separation of free and bound DHEA-peroxidase conjugate was by insolubilized antibody, prepared by coupling purified IgG of goat anti-rabbit IgG serum with Sepharose 4B or a polystyrene tube. The enzyme activity was measured by the chemiluminescence reaction using luminol and hydrogen peroxide as substrate. The faint chemiluminescence was measured by a photon counter. The sensitivity was 25 pg/assay tube for DHEA and 100 pg/assay tube for DHEA-S. Upon comparison, results obtained by radioimmunoassay and this method showed good agreement; r = 0.86 for free DHEA, r = 0.92 for acid-hydrolyzed DHEA-S and r = 0.91 for solvolyzed DHEA-S. The present method is applicable in the routine determination of DHEA and DHEA-S in biological fluid.