Search

Background: Many hormones display distinct circadian rhythms, driven by central regulators, hormonal bioavailability, and half-life. A set of 11-oxygenated C19 steroids (11-oxyandrogens) and pregnenolone sulfate (PregS) are elevated in congenital adrenal hyperplasia and other disorders, but their circadian patterns have not been characterized., Participants and Methods: Peripheral blood was collected every 2 h over 24 h from healthy volunteer men (10 young, 18-30 years, and 10 older, 60-80 years). We used mass spectrometry to quantify 15 steroids, including androstenedione (A4), testosterone (T), 11β-hydroxy- and 11-ketotestosterone (11OHT, 11KT),11β-hydroxy- and 11-ketoandrostenedione (11OHA4, 11KA4), and 4 ∆5-steroid sulfates. Diurnal models including mesor (rhythm adjusted median), peak, and nadir concentrations, acrophase, and amplitude were computed., Results: 11OHA4 followed a rhythm similar to cortisol: acrophase 8:00 h, nadir 21:00 h and were similar in young and old men. 11KT had similar diurnal patterns, but the peak was lower in older than in young men, as was the case for A4. All four steroid sulfates were higher in young vs older men. PregS and 17-hydroxypregnenolone sulfate (17OHPregS) showed sustained elevations between 8:00 and 18:00 h, and nadirs around midnight, while DHEAS and AdiolS displayed minimal diurnal variations. All 4 11-oxyandrogens correlated tightly with cortisol (r from 0.54 for 11OHT to 0.81 for 11OHA4, P < 0.0001 for all), but very weakly with T, supporting their adrenal origin and ACTH governance., Conclusions: 11-Oxyandrogens, PregS, and 17OHPregS display distinct circadian and age variations, which should be accounted for when used as clinical biomarkers.

This study describes a validated LC-MS/MS method for assaying 23 steroids within a single run from 150 μl of human plasma, serum or prostatic tissue homogenate. Isotope-labeled steroids were used as internal standards. Samples were extracted with toluene, and ketosteroids were derivatized with hydroxylamine prior to LC-MS/MS analysis. The steroids were separated on a C18 column and methanol was used as an organic solvent with the addition of 0.2 mM ammonium fluoride to improve underivatized estradiol (E2) ionization. Certified reference serums as well as plasma samples, and homogenates of prostate tissue were utilized in the method validation. The specificity of the method was inspected with a total of 27 steroids. The validation proved that the method was suitable for the quantitative analysis of a wide panel of androgens (testosterone, T (3.3 pM-13 nM); androstenedione, A4 (3.3 pM-13 nM); 5α-androstanedione, DHA4 (13 pM-13 nM); dehydroepiandrosterone, DHEA (67 pM-133 nM); dihydrotestosterone, DHT (33 pM-33 nM); 11-ketodihydrotestosterone, 11KDHT (13 pM-13nM); 11-ketotestosterone, 11KT (33 pM-6.7 nM); 11β-hydroxyandrostenedione, 11bOHA4 (33 pM-13 nM); 11β-hydroxytestosterone, 11OHT (13 pM-33 nM)), as well as estrogens (estrone, E1 (3.3 pM-13 nM)), progestagens (17α-hydroxypregnenolone, 17OHP5 (32 pM-127 nM); 17α-hydroxyprogesterone, 17OHP4 (67 pM-133 nM); progesterone, P4 (3.3 pM-13 nM); pregnenolone, P5 (6.6 pM-13 nM)), and glucocorticoids (cortisol, F (33 pM-134 nM); cortisone E (66 pM-131 nM); corticosterone, B (33 pM-67 nM); 11-deoxycortisol, S (33 pM-66 nM); 21-hydroxyprogesterone, 21OHP4 (32 pM-13 nM)). Furthermore, E2 (335 pM-134 nM) and 11α-hydroxyandrostenedione, 11aOHA4 (33 pM-33 nM) could be analyzed if the concentration in the sample was high enough. In addition, aldosterone, A (128 pM-64 nM) and 11-ketoandrostenedione, 11KA4 (33 pM-13 nM) could be analyzed semiquantitatively. The limits of quantification for all compounds ranged from 0.9 to 91 pg/ml, and from 0.009 to 0.9 pg/mg tissue. Compared to our previous method, this new method also permits the analysis of the more challenging steroids, like DHT, DHEA and P5, and a panel of 11-ketosteroids., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Objective: To comprehensively characterize androgens and androgen precursors in classic 21-hydroxylase deficiency (21OHD) and to gain insights into the mechanisms of their formation., Design: Serum samples were obtained from 38 patients (19 men) with classic 21OHD, aged 3-59, and 38 sex- and age-matched controls; 3 patients with 11β-hydroxylase deficiency; 4 patients with adrenal insufficiency; and 16 patients (8 men) undergoing adrenal vein sampling. Paraffin-embedded normal (n = 5) and 21OHD adrenal tissues (n = 3) were used for immunohistochemical studies., Methods: We measured 11 steroids in all sera by liquid chromatography-tandem mass spectrometry. Immunofluroescence localized 3β-hydroxysteroid dehydrogenase type 2 (HSD3B2) and cytochrome b5 (CYB5A) within the normal and 21OHD adrenals., Results: Four 11-oxygenated 19-carbon (11oxC19) steroids were significantly higher in male and female 21OHD patients than in controls: 11β-hydroxyandrostenedione, 11-ketoandrostenedione 11β-hydroxytestosterone, and 11-ketotestosterone (3-4-fold, P < 0.0001). For 21OHD patients, testosterone and 11-ketotestosterone were positively correlated in females, but inversely correlated in males. All 11oxC19 steroids were higher in the adrenal vein than in the inferior vena cava samples from men and women and rose with cosyntropin stimulation. Only trace amounts of 11oxC19 steroids were found in the sera of patients with 11β-hydroxylase deficiency and adrenal insufficiency, confirming their adrenal origin. HSD3B2 and CYB5A immunoreactivities were sharply segregated in the normal adrenal glands, whereas areas of overlapping expression were identified in the 21OHD adrenals., Conclusions: All four 11oxC19 steroids are elevated in both men and women with classic 21OHD. Our data suggest that 11oxC19 steroids are specific biomarkers of adrenal-derived androgen excess., (© 2016 European Society of Endocrinology.)

Objectives: Cerebrotendinous xanthomatosis (CTX) is a rare genetic disorder of bile acid (BA) synthesis that can cause progressive neurological damage and premature death. Blood (normally serum or plasma) testing for CTX is performed by a small number of specialized laboratories, routinely by gas chromatography-mass spectrometry (GC-MS) measurement of elevated 5α-cholestanol. We report here on a more sensitive biochemical approach to test for CTX particularly useful for confirmation of CTX in the case of a challenging diagnostic sample with 5α-cholestanol that, although elevated, was below the cut-off used for diagnosis of CTX (10 μg/mL or 1.0 mg/dL)., Design and Methods: We have previously described liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) methodology utilizing keto derivatization to enable the sensitive quantification of plasma ketosterol BA precursors that accumulate in CTX. We have expanded this methodology to perform isotope dilution LC-ESI-MS/MS quantification of a panel of plasma ketosterol BA precursors, with internal standards readily generated using isotopically-enriched derivatization reagent., Results: Quantification of plasma ketosterol BA precursors (7α-hydroxy-4-cholesten-3-one, 7α,12α-dihydroxy-4-cholesten-3-one and 7α,12α-dihydroxy-5β-cholestan-3-one) in a single LC-ESI/MS/MS test provided better discrimination between a CTX-positive and negative samples analyzed (n=20) than measurement of 5α-cholestanol alone., Conclusions: Quantification of plasma ketosterol BA precursors provides a more sensitive biochemical approach to discriminate between CTX negative and positive samples. A multiplexed LC-ESI-MS/MS test quantifying a panel of plasma ketosterols, with simple sample preparation, rapid analysis time and readily available internal standards, can be performed by most clinical laboratories. Wider availability of testing will benefit those affected with CTX., (Copyright © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

A complex study of the functional state of the sympathoadrenal system and adrenal cortex in 10-15-year-old children of both sexes was carried out using the indices of daily excretion of adrenaline, noradrenaline, 17-ketosteroids, and 17-hydroxycorticosteroids. A synchronism in the functional activity of the mediator component of the sympathoadrenal system as well as of the androgenic and glucocorticoid functions of the adrenal cortex was observed with age and during pubertal development of children. At the same time, heterochronic maturation was observed in the sex groups: in girls at the age of 10 and 12 years and in boys at the age of 14-15 years. The changes of different direction and intensity in the excretion of the studied hormones and hormonal metabolites were observed in the sex and age groups. A sharp increase in the daily excretion of glucocorticoid metabolites accompanied by a considerable decrease in the age index of noradrenaline secretion was observed in 14- and 15-year-old boys from beginning to end of school year; in addition, an increase in the daily excretion of sex hormones was observed at the age of 15 years. In girls, these indices varied within the age range, which points to a more sophisticated neuroendocrine control of physiological functions in girls during puberty.

Derivatization of neutral steroids for increasing sensitivity in liquid chromatography/negative atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) has been examined. Under APCI conditions, gas-phase electrons are provided by the corona discharge and captured by electron-affinitive compounds. In negative APCI-MS, therefore, ultrahigh sensitivity can be obtained by tagging neutral steroids, whose ionization efficiencies are low in the conventional APCI-MS, with electron-capturing moieties, such as a nitro group. We synthesized various boronic acid and hydrazine derivatives having electron-capturing moieties as derivatization reagents for 1,2-diol compounds and oxosteroids, respectively. Among reagents examined, those having the 2-nitro-4-trifluoromethylphenyl moiety were most effective in increasing sensitivity. That is, the detection responses of the derivatives with these reagents were increased by several to more than 200-fold over intact steroids, where limits of detection were some picograms. The developed derivatization procedures were applied to analyses of small amounts of steroids in human plasma and gave satisfactory results.

A new derivatization procedure has been developed where solid-phase catalysis is utilized to facilitate the formation of hydrazones in precolumn labeling of keto-containing compounds. This procedure has been implemented on a solid-phase enrichment and enhanced derivatization (SPEED) device, prepared from porous polyethylene that has been coated with Nafion and dansylhydrazine. The SPEED devices have been optimized using experimental design and characterized for dansylation of C-21 ketosteroids by multivariate data analysis, using progesterone as the model compound. The reaction temperature and the molar ratio between the steroid and the derivatization reagent were found to be the factors most strongly affecting the reaction. Faster reaction kinetics were achieved when the molar ratio between dansylhydrazine and the steroid was increased. Mass spectroscopic analysis showed that the four derivative peaks eluting when derivatized progesterone was separated on an octadecyl silica stationary phase were due to the syn and anti mono- and bis(hydrazones) formed in the reaction. Using optimal reaction conditions, the derivatives mainly constitute the syn and anti conformers of bis-derivatives. In contrast to solution-based acid catalysis, the SPEED device was remarkably insensitive to water in the reaction mixture. A sample volume of 400 microL was found to be the maximum, enabling sample enrichment prior derivatization. Using optimal experimental conditions, picomole amounts of ketosteroids could be derivatized in 10 min at room temperature. Analysis of spiked serum samples containing 0.4-2.0 nmol of progesterone showed overall recoveries of 52-63%. The corresponding 3delta detection limit was 1.3 pmol (n = 4, 100 microL injected), as estimated from calibration curve data.

Short-term exposure of the olfactory epithelium of mature male Atlantic salmon parr to either the pesticide simazine (concentrations 1.0 and 2.0 microg l(-1)) or the pesticide atrazine (concentration 1.0 microg l(-1)) significantly reduced the olfactory response to the female priming pheromone, prostaglandin F(2alpha). In addition, the reproductive priming effect of the pheromone on the levels of expressible milt was also reduced after exposure to the individual pesticides (simazine 0.1, 0.5, 1.0 and 2.0 microg l(-1) and atrazine 0.5 and 2.0 microg l(-1)). When the olfactory epithelium was exposed to a mixture of simazine and atrazine, (concentrations of 0.5:0.5 and 1.0:1.0 microg l(-1)), there was no significant reduction in the olfactory response when compared to the single pesticides at equivalent concentrations. In addition, exposure to a mixture of simazine and atrazine had no synergistic effect on the priming response, and plasma levels of testosterone, 11-ketotestosterone and 17,20beta-dihydroxy-4-pregnen-3-one were similar in the groups of male parr exposed to the individual pesticides. Although the levels of expressible milt were reduced in all groups, there were no significant differences between the different pesticide treatments. The results of the study suggest that the two s-triazine pesticides have an additive and not a synergistic impact on olfactory-mediated endocrine function in mature male salmon parr.

A new procedure for the quantitation of C-21 ketosteroids using trifluoromethanesulfonic acid-catalyzed precolumn dansylation and coupled column liquid chromatographic separation, followed by postcolumn 1,1'-oxalyldiimidazole peroxyoxalate chemiluminescence detection is presented. In the simultaneous optimization of chromatographic resolution and chemiluminescence intensity, a coupled column chromatographic system and a stopped-flow system were used. An eluent containing 20 mM phosphate buffer at pH 6.7 accomplished an efficient separation of 3 alpha-hydroxy-5 beta-pregnan-20-one from a mixture containing 10 C-21 ketosteroids. Phosphate buffer also proved to be the most advantageous, among the six buffers tested, for sensitive detection. Experimental design and multivariate data analysis were used to characterize and optimize the postcolumn reaction chemistry in the chromatographic system. A valid full factorial design with excellent predictability showed that the flow rates for both 1,1'-oxalyldiimidazole and hydrogen peroxide were the factors most strongly affecting the sensitivity of the system. The theoretical plate numbers were above 11,000 for all 10 dansylated ketosteroids. The 3 sigma detection limit estimated from 3 alpha-hydroxy-5 beta-pregnan-20-one calibration curve data was 1.6 pmol (n = 4, 125 microL injected) and spiked serum containing 0-74 pmol of this compound showed overall recoveries of 73 +/- 9% (n = 12). Quantitation of 3 alpha-hydroxy-5 beta-pregnan-20-one was finally carried out on 45 serum samples and the results compared to those from a radioimmunoassay (RIA) method. The data acquired with the procedure described in this work compare well with the results from RIA, which confirms the reliability of the new analytical procedure.

The age changes of neural control over the function of the thyroid, adrenal cortex and testicles were examined in adult (6 months) and old (28 months) male rats. In old age, there was a weakening of adrenergic control of thyroidogenesis, alpha-adrenergic and M-cholinergic control of glucocorticoid function of the adrenal cortex and reduction of adrenergic and M-cholinergic influences in the regulation of steroidogenic function of the testicles.

The close relationship between C19O2 steroid excretion and the ratio of C19O2/C21O5 steroids indicates that it is the elevation of C17,20-lyase activity, which represents the decisive factor in the cessation of androgen-corticoid disproportion during the puberty of healthy children. In 3-6 year-old C21-hydroxylase deficient children treated with corticoids in doses only partially suppressing endogenous ACTH secretion, the excretion of total C19 steroids increased continuously parallel with a well defined elevation of 16 alpha-oxygenated-C21 steroid excretion. The patients did not show the physical signs of adrenarche before six years. This can be attributed to three factors: a) substitutional corticoid therapy; b) intraglandular control of delta 5-pathway by 16 alpha-hydroxylation of C21 steroids; c) neutralisation by 11 beta-hydroxylation of the androgen effect of C19O2 steroids.

A radioimmunoassay of the 15-keto- metabolite of prostaglandin E2 has been developed. The details of the assay are described. Using unextracted plasma, serial measurements of 15-keto-PGE2 have been carried out every 15 minutes for 3 hours in 9 women ingesting 0.5 mg PGE2 oral tablets used for the induction of labour.

17beta-Hydroxyandrost-4-ene-3,11-dione was linked via its 3-(O-carboxymethyl) oxime to bovine serum albumin to give a conjugate which was used to generate antiserum in rabbits. The antiserum, at an overall dilution of 1 in 16,000, together with [1,2-3H] 17beta-hydroxyandrost-4-ene-3,11-dione synthesized from [1,2-3H] cortisone have been used to develop a radioimmunoassay for the parent steroid. The assay incorporates a purification step in which serum or plasma extracts are chromatographed on silica gel layers bound to plastic or aluminum sheets and the steroid containing zones cut out and eluted directly with assay buffer. The cross-reactivities of several steroids with the antiserum and the specificity, sensitivity, accuracy and precision of the assay are described. Blood sera from immature male rainbow trout contain ca 0.2-0.4 mature, serum levels rise sharply to reach values of 2 to greater than 9 microng/100 ml of 17beta-hydroxyandrost-4-ene-3,11 dione. As male fish mature, serum levels rise sharply to reach values of 2 to greater than 9 microng/100 ml. Levels in immature females rarely exceeded the assay sensitivity but serum from three ripe females showed low but detectable levels (ca 0.2 microng/100 ml) of steroid. The assay has found application in sexing live fish for experimental purposes.

The nature of the interaction between progesterone or testosterone and human albumin as well as the interaction between progesterone and partially purified human transcortin has been studied. Modification of lysine residues of albumin with maleic anhydride resulted in a decreased binding of the steroid as judged from equilibrium dialysis experiments. This suggested that lysine residues in albumin interact with the oxosteroids. In order to check this hypothesis, steroids labeled with 18O in their oxo function (testosterone and progesterone) were synthesized for use as probes of the interactions. However, no loss of label was noted when testosterone or progesterone specifically 18O-labeled in their oxo functions were incubated with albumin. This suggested that no covalent interaction between the steroidal oxo group and albumin took place. This was in contrast to the results obtained with 3,20-18O-labeled progesterone and partially purified transcortin, where a complete loss of 18O label in the protein-bound steroid was found. The nonbound steroid showed an almost complete retention of label. These results indicate a participation of steroid oxo groups in the binding of progesterone to transcortin. Of the possible mechanisms discussed, imine bonds between the steroid and transcortin seem most likely although other types of interactions cannot be ruled out.

A simple method is described for the simultaneous radioligand assay of four delta5-3beta-hydroxysteroids adjacent to one another on the biosynthetic pathway (pregnenolone [1], 17alpha-hydroxypregnenolone, dehydroepiandrosterone and 5-androsterone-3beta, 17beta-diol), and their four delta4-3keto products (progesterone, 17alpha-hydroxyprogesterone, 4-androstene-3, 17-dione and testosterone). Two plasma aliquots are extracted and fractionated each for four steroids and individual corrections are made for losses. For fractionation, maximum use is made of the high resolution and reproducibility of celite minicolumns, using propylene glycol as stationary phase, and a discontinuous gradient of ethyl acetate in iso-octane as mobile phase. The fractions are then assayed in the appropriate radioligand end-assay system. Each assay was finally validated by demonstrating coincidence of peaks of immuno- and radioactive steroid in extracts of female plasma. Results in pre-pubertal girls and women in the follicular phase of the menstrual cycle suggest that the major change in adrenal steroid production at puberty may be an increase in 17, 20-desmolase activity. There appears to be little reversal of this change in adrenal function after ovariectomy.

Livers from brook trout (Salvelinus fontinalis) held in water containing 3.8 p.p.m. (mg/l) Corexit and in water containing 3.8 p.p.m. Corexit + 0.2 p.p.m. polychlorinated biphenyl (PCB) were firmer in texture than livers taken from control trout held in fresh water. The testes from the PCB-treated trout were darker in color, smaller in size, contained less spermatic fluid, and appeared to be regressed when compared with testes from the control fish. Only 72% of the eggs from PCB-treated trout hatched compared to 92% for control eggs when eggs were incubated in fresh water. Fertilized eggs incubated in water containing 0.2 p.p.m. PCB + 3.8 p.p.m. Corexit resulted in less than 1% of the eggs from the untreated fish and none of the eggs from PCB-treated fish being hatched. The young fish from the control group lived only a few days in the water containing PCB. PCB greatly stimulated the in vitro 11beta-hydroxylation of testosterone by trout testicular tissue. There was no significant change in 11-ketotestosterone blood levels in maturing brook trout after treatment with 3.8 p.p.m. Corexit or 3.8 p.p.m. Corexit + 0.2 p.p.m. PCB for 21 days during the final stages of maturation. It is thought that sublethal levels of PCB can interfere with the reproduction process in this species.

5alpha-Androstenone2) has been measured in pig peripheral plasma by radioimmunoassay (RIA). In 73 mature boars values ranging from 1.2 ng to 54.1 ng per ml plasma with a mean value of 18.3 ng/ml and SD equal to 15.9 were found. In female pigs and castrated male pigs the mean concentrations were 2.3 ng/ml (n equal to 18) and 1.1 ng/ml (n equal to 19) with SD equal to 0.8 and SD equal to 0.3 respectively. Clear-cut diurnal variations in the concentration of 5alpha-androstenone in plasma samples from boars were not observed. Intravenous injection of HCG in boars caused an abrupt rise in the 5alpha-androstenone level reaching a maximum in 90 min. Twenty-eight hours after the injection a second peak of plasma 5alpha-androstenone was observed. Following castration of boars the level of 5alpha-androstenone in peripheral plasma fell within 4 days, to levels found in female pigs.

A method for the isolation of 3-ketosteroids based on the positive charge of their oximes is described. The biological extract is filtered through a column of sulphoethyl Sephadex LH-20 (H+). Steroids in the filtrate are converted into oximes and the dried reaction mixture is filtered through a column of diethylaminohydroxypropyl Sephadex LH-20 (OH-) in methanol. After evaporation of the solvent and hydroxylamine, the oximes are taken up by and separated on a column of sulphoethyl Sephadex LH-20 (H+) in methanol. Following elution of other steroids, the oximes of 3-ketosteroids are eluted as a group with methanol-pyridine (20:1, v/v), and are converted into trimethylsilyl ethers. Removal of reagents and further purification of the sample is achieved by rapid filtration through Lipidex 5000 in n-hexane-pyridine-hexamethyldisilazane-dimethoxypropane (97:1:2:10). The derivatives are then analyzed by computerized gas chromatography-mass spectrometry using open-tubular glass capillary columns. Recoveries of picogram amounts of 3H-labelled steroids carried through the entire isolation procedure are 80-90%. The purification achieved in analyses of plasma permits solid injection of the equivalent of 1-2 ml of plasma without overloading of the capillary column. The principle of the isolation procedure might be applicable to other groups of compounds that possess keto or aldehydo groups.

The iv injection of 7-ketocholesterol into rabbits, made soluble by combining with bile salts, inhibited cholesterol uptake by the aorta. However, the inhibition was not as marked or as uniform as previously demonstrated in in vitro experiments. This difference may have been the result of lower plasma concentrations of 7-ketocholesterol in the injected animals. Gastric feeding of 7-ketocholesterol failed to inhibit aortic cholesterol uptake, probably because of inadequate plasma concentrations of the inhibitory steroid. The results suggest that the mechanism of 7-ketocholesterol on aortic cholesterol uptake is through competitive inhibition.

The levels of 5alpha-androstenone and testosterone in peripheral plasma and 5alpha-androstenone in fat collected from 18 boars selected according to thickness of back fat and rate of gain were evaluated. Sampling was started when the animals were about 100 days of age and samples were collected every 14 days until the boars were about 240 days old. The steroid profiles varied considerably between boars, although an increase in steroid levels was found in most animals with the onset of adolescence. The regression line for the level of 5alpha-androstenone in fat and the age of the animals in days was found to be y = 0.02x-1-31. High coefficients of correlation were found between the concentration of 5alpha-androstenone in peripheral plasma and fat (r = 0-78) and between levels of 5alpha-androstenone and testosterone in peripheral plasma (r = 0-64). Levels of 5alpha-androstenone in peripheral plasma above about 15 ng/ml were usually accompanied by a heavy accumulation of 5alpha-androstenone in adipose tissue. The use of four of the boars for mating resulted in increased steroid levels in peripheral plasma and fat. Significantly higher levels of 5alpha-androstenone and testosterone were found in the peripheral plasma of boars selected for fatness and a low rate of gain than in boars selected for low back-fat thickness and a high rate of gain.

A method for simultaneous quantitation of nine steroids in cord plasma is described which consists of Amberlite XAD-2-column chromatography at constant temperature of 45 degrees C, enzyme hydrolysis with beta-glucoronidase/aryl sulfatase, addition of five radioactive internal standards, ethyl acetate extraction, thin layer chromatography and quantitation by gas-liquid chromatography after trimethylsilyl ether derivative formation. Reliability criteria are established and the following steroid concentrations found: progesterone, 132.1 +/- 102.5 mug/100 ml; pregnenolone, 57.3 +/- 45.7 mug/100 ml; dehydroepiandrosterone, 46.5 +/- 29.4 mug/100 ml; pregnanediol, 67.5 +/- 46.6 mug/100 ml; 16-ketoandrostenediol 19.8 +/- 13.7 mug/100 ml; 16 alpha-hydroxydehydroepiandrosterone, 126.3 +/- 86.9 mug/100 ml; 16 alpha-hydroxypregnenolone, 78.2 +/- 56.5 mug/100 ml; androstenetriol, 22.2 +/- 17.5 mug/100 ml and oestriol, 127.7 +/- 116.9 mug/100.

1. [5alpha-(3)H]5alpha-Androst-16-en-3-one (5alpha-androstenone) was infused at a constant rate for 180min into the spermatic artery of a sexually mature boar. Samples of spermatic-venous blood were collected at 1min intervals for the first 10min of the infusion and thereafter at 15min intervals for the first hour, then at 64, 125, 155 and 172min. After infusion, the testis was removed and immediately cooled to -196 degrees C. 2. From both the testicular tissue and the spermatic-venous plasma, endogenous and (3)H-labelled androst-16-enes were isolated, characterized and quantitatively determined and their specific radioactivity was calculated. 3. The specific radioactivities of 5alpha-androstenore, 5alpha-androst-16-en-3alpha-ol and 5alpha-androst-16-en-3beta-ol (an-alpha and an-beta) in testicular tissue were different from those in the spermatic-venous plasma, suggesting that these compounds may be present in more than one compartment of the testis and differentially secreted into the spermatic-venous blood. 4. The ratios of the specific radioactivities of an-alpha and an-beta to their respective sulphate conjugates in the testicular tissue were less than the ratios of the same compounds in the spermatic-venous plasma. 5. The patterns of secretion of these labelled compounds in the spermatic-venous blood during the period of infusion were demonstrated. 6. The urine that accumulated during the infusion was analysed and found to contain (3)H-labelled an-beta, conjugated as both glucuronide and sulphate, the specific radioactivities of which were determined. Little or no androst-16-enes occurred as free steroids. 7. The presence of an-beta glucuronide in the urine is discussed.