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Antibodies were produced in sheep against a new anti-breast cancer drug 4-hydroxyandrostenedione (4-OHA) using two hapten-ovalbumin conjugates. One of these conjugates (4-hydroxytestosterone-ovalbumin) produced an antiserum suitable for the development of a radioimmunoassay that would allow direct measurement of 4-OHA in plasma at concentrations down to 82 pmol/l, with adequate accuracy, precision and scope for further sensitivity. Although this assay would measure 4-hydroxytestosterone (4-OHT) in addition to 4-OHA, the present data suggest that the magnitude of any interference from endogenous steroids and those derived from 4-OHA could only be minimal. A comparison of solvent-extracted and unextracted samples showed that only unconjugated drug was analysed by this radioimmunoassay. A study of plasma protein binding of 4-OHA showed that at therapeutic concentrations, between 13.5 and 16.5% of plasma 4-OHA was not bound to proteins. This assay system could be a useful adjunct to the future development of 4-OHA as an anti-cancer drug.

The concentrations of dehydroepiandrosterone (DHEA), its sulfate (DHEAS), androstenedione (A-dione), testosterone (T) and dihydrotestosterone (DHT) have been measured before and after castration in men and two animal models, namely the rat and the guinea pig. In adult men, the pre-castration levels of plasma DHEAS and DHEA were measured at 1839 +/- 320 and 2.4 +/- 0.5 ng/ml, respectively, while in both animal models, the concentrations of these two steroids were below 0.3 ng/ml. Orchiectomy in men reduced plasma T and DHT levels from 2.9 +/- 0.1 and 0.60 +/- 0.10 to 0.42 +/- 0.21 and 0.05 +/- 0.01 ng/ml (P less than 0.01), respectively, while there was no significant effect observed on DHEAS, DHEA and A-dione levels. By contrast, castration in the rat reduced the low levels of circulating DHEA and A-dione below the detection of the radioimmunoassay (RIA) used. In castrated guinea pig, a small quantity of plasma A-dione (0.07 +/- 0.02 ng/ml) was measured while DHEA was undetectable. Moreover, in the rat and guinea pig, plasma T and DHT levels became undetectable. Following administration of the antiandrogen Flutamide for two weeks in the castrated rat and guinea pig, prostate weight was not further reduced, thus indicating that there is no significant androgenic activity left following castration of these two species. In fact, castration in the rat and guinea pig caused a decrease in prostatic levels of DHT from 4.24 +/- 0.351 and 9.42 +/- 1.43 ng/g, respectively, to undetectable levels. In men, on the other hand, the prostatic DHT levels were only inhibited from 5.24 +/- 0.59 to 2.70 +/- 1.50 ng/g, respectively. As expected, when Flutamide was administered to the rat and the guinea pig, the levels of prostatic steroids remained undetectable while, in men, the DHT content in the prostate was further reduced to undetectable values. In summary, the plasma levels of DHEAS, DHEA, delta 4-dione are markedly different between men and both animal models used and furthermore, measurements of prostatic levels of androgens suggest that the high plasma levels of these steroids are likely responsible for the presence of important amounts of DHT in human prostate after castration.

Endogenous levels of testosterone, 5 alpha-dihydrotestosterone (5 alpha-DHT), androstenedione and oestradiol as well as levels of androgen (AR) and oestrogen (ER) receptors were measured in human primary breast tumour samples. The purification procedure developed allowed simultaneous quantitation of the four steroids, by radioimmunoassay, in small samples with adequate precision, sensitivity and accuracy. The majority of the tumours analysed contained detectable levels of the four steroids in the homogenate or cytosol fractions. There was no significant correlation between steroid content of the tissue and the age of the patient for any of the four steroids. A positive correlation (r = 0.71) was found between the levels of 5 alpha-DHT and testosterone in tumours. In general, tissue steroid concentrations decreased with an increase in dedifferentiation. Fifty-two per cent of the tumours analysed for receptor content were found to be ER positive, and a similar proportion were AR positive. No relationship was observed between AR status and age although receptor concentration was significantly (P = 0.004) higher in post-menopausal women when only receptor positive tumours were evaluated. The mean values for AR and ER were higher in tumours containing both receptors than in tumours showing either receptor alone; there was, however, no significant relationship between concentrations of the two receptors. No correlation was observed between tumour AR or ER status and any of the four steroids measured in either fraction. In addition, the ratio between the combined levels of 5 alpha-DHT and testosterone compared to oestradiol in the same tumour, only showed a maximum value of 40. Thus, in vivo these two androgens are unlikely to influence oestrogen action in human primary breast tumours by interfering with the association of oestradiol with its receptor.

Two peaks of hormone concentration in blood and skeletal muscles (SM) were found, immediately after intensive physical exercises (PE) and the late period of rest. Immediately after PE testosterone (T) and estradiol (E2) concentration in SM increased on 36 and 430%, returning to the initial level in 2 h. E2 and androstenedione content in SM increased from 33 +/- 7 to 89 +/- 3 and from 563 +/- 58 to 767 +/- 38 pg/g tissue accordingly in 48 h, returning to the initial level in 72 h. T content increased in blood 3.7-fold in SM 2.2-fold in 72 h after PE. Androgen receptor (AR) contents in SM cytosol increased (on 28%) in 2 h, then returned to the control level and increased again in 72 h (from 0.6 +/- 0.4 to 1.2 +/- 0.2 fmol/mg protein) after PE. Kd did not change significantly and were 0.33 +/- 0.03 nM. The experiments on female rats showed the same tendency, but T increasing was more marked compared to male rats. The obtained results showed that the hormonal regulation of metabolic processes in SM has a cyclic character and is connected with the changing of hormonal content and hormonal SM receptors.

The outer (glomerulosa and fasciculata) and inner (reticularis) zones of the adrenal cortex of the guinea pig were separated and their steroid content determined. It was found that the concentration of 21-hydroxypregnenolone, deoxycorticosterone, corticosterone, aldosterone, 11-deoxycortisol, and cortisol was significantly higher in the outer cortical region, while the concentration of pregnenolone, 17-hydroxypregnenolone, and dehydroepiandrosterone was significantly higher in the inner zone. The concentration of progesterone, 17-hydroxyprogesterone, and androstenedione was not different in the two zones. Examination of specific steroid ratios suggested the following: (1) 3 beta-ol dehydrogenase/isomerase and 21-hydroxylase activities are reduced in the inner zone, (2) 17-hydroxylase and C17 20 lyase activities appear to be equally active in the two zones (3) 11 beta-hydroxylase activity appears to be more active in the inner zone (4) 21- hydroxypregnenolone , deoxycorticosterone, corticosterone, 11-deoxycortisol, and cortisol along with aldosterone are produced principally in the outer zone.

Simultaneous determination of progesterone, androst-4-enedione, pregnenolone, dehydroepiandrosterone (DHEA) and 17-hydroxyprogesterone has been developed for human cerebral tissue. Before immunoassay, steroids were separated on a Celite column with propylene glycol as stationary phase with hexane containing increasing proportions of dichloromethane as mobile phase. This system allowed separation of steroids of similar polarity, especially of pregnenolone and progesterone. The brain regions studied cortex (prefrontal, parietal and temporal), cerebellum and corpus callosum, were obtained after autopsy from 9 women and 1 man between 76 and 93 years of age. Steroids were found in all regions. The overall concentrations expressed in nmol/kg of tissue were: 10.1, 7.6, 120.7, 19.6 and 10.4 respectively, for progesterone, androst-4-enedione, pregnenolone, dehydroepiandrosterone and 17-hydroxyprogesterone, corresponding to 7.3, 4.9, 74, 6.5 and 9.2 times the plasma levels. These very high concentrations, not previously described in human brain tissue, pose the question of the existence of local biosynthetic pathways independent of the peripheral endocrine gland system as well as that of progressive accumulation of steroids over a lifetime. Concentrations of each steroid in each subject varied little among the various brain regions studied, but there was much variation among the subjects with respect to the concentrations of a given steroid.

The concentrations of progesterone, androstenedione, testosterone, 5 alpha-dihydrotestosterone and androsterone were determined in tissue samples from the human hypothalamus, anterior pituitary, pineal, amygdala and parietal cortex, taken at autopsy from male (n = 4) and female cadavers (n = 4) of various ages. The measurements were performed using radioimmunoassays for the individual steroids after the chromatographic purification of solvent extracts of tissue samples on Lipidex-5000TM. Preliminary qualitative analyses of the chromatographic profiles of various steroids by radioimmunoassay demonstrated the presence of these steroids in various regions of the brain, but an immunoreactive peak corresponding to 17-hydroxyprogesterone was not found. The concentrations (ng/g tissue wet wt.) of all steroids measured were either very low or below the limit of detection in brain tissues taken from male and female infants. In the adult brain, there was no difference in the distribution of steroids between the various regions studied. There was no sex difference in the brain tissue steroid concentrations, with the exception of testosterone which was clearly much higher in brain tissues from men as compared to women. Although testosterone was undetectable in most samples taken from adult women. 5 alpha-dihydrotestosterone could be measured in almost all samples, which suggests that this is the most important androgen in the human brain. When brain tissue steroid levels are compared with serum concentrations, it can be postulated that a state of equilibrium exists between the fraction of serum steroids which are not bound to high-affinity binding proteins and the amount of steroids in brain tissues.

Sex steroids and 5-en-3 beta-hydroxysteroids were determined by radioimmunoassay in specific regions of the human brain, in the anterior and posterior pituitary, in one sensory organ, the retina and in the cranial nerves. Progesterone, androstenedione, testosterone and estrone were found in all areas of the brain and in all the cranial nerves but not in all cases. There was no sex difference except in the case of androstenedione where values were higher in women in some brain areas. Estrone values were always higher than those of estradiol in both men and women. No 5 alpha-dihydrotestosterone was detected in any of the samples studied. The values for pregnenolone, dehydroepiandrosterone and their sulfates were much higher than those of the sex steroids in all areas of the brain and in all the cranial nerves. Values for pregnenolone were greater than those of its sulfate while those of dehydroepiandrosterone were in general equal to or higher than those of its sulfate. The values for pregnenolone were greater than those of dehydroepiandrosterone. There were no obvious regional differences in the concentrations of the 5-en-3 beta-hydroxysteroids either in specific areas of the brain or in the cranial nerves. But there was a definite trend for the free dehydroepiandrosterone values to be higher in women. The possible significance of these observations is discussed.

Newborn female and male C57BL6 mice were decapitated at birth or at different times during the first 24 h after birth and testosterone was determined by radioimmunoassay in plasma and testes. In newborn females, plasma testosterone is low and does not significantly change over the first 24 h after birth. In contrast, in newborn males, plasma testosterone more than doubles during the first 2 h after birth and then falls rapidly to remain relatively low for the remainder of the 24 h period after birth. The increase in plasma testosterone is of almost certain testicular origin since it follows a decrease in testicular testosterone content. It seems likely that the increase in plasma testosterone in male mice which reaches its peak at 2 h after birth is involved in an essential way in the development of well-documented sex differences in gonadotropin secretion and behavior.

A radioimmunoassay system for serum 16 alpha-hydroxyandrost-4-ene-3,17-dione was developed with the use of rabbit antiserum against 16 alpha-hydroxyandrost-4-ene-3,17-dione-3-(O-carboxymethyl)oxime which was conjugated with bovine serum albumin. The antiserum was highly specific for 16 alpha-hydroxyandrost-4-ene-3,17-dione, with cross reactions to other steroids being less than 0.8% except for androst-4-ene-3,17-dione(3.4% cross reaction). Use of LH-20 column chromatography, however, clearly separated these two steroids. Pregnancy sera were measured with this assay system after an addition of labelled internal standard, extraction and separation by column chromatography. The lower limit of detection for 16 alpha-hydroxyandrost-4-ene-3,17-dione was 2 pg/tube. The mean recovery rate of the added standard was 98.3 +/- 8.8% (mean +/- SE). Intra- and inter-assay coefficients of variation were 8.6% (n = 6) and 12.1% (n = 7), respectively.

As an extension of our studies on the influence of age on testicular function and with the aim of detecting whether the decline in testosterone production by aged testes is accompanied by a block in the biosynthetic chain leading from cholesterol to testosterone, we determined in the testis of young and elderly men, who died suddenly either from a cardiac incident or from accident, intratesticular steroids: pregnenolone, 17 hydroxypregnenolone (3 beta, 17 alpha-dihydroxy-5-pregnen-20-one), dehydroepiandrosterone, androstenediol, (5-androsten-3 beta, 17 beta-diol), progesterone, 17 hydroxyprogesterone, androstenedione, 17 beta-estradiol as well as testosterone, dihydrotestosterone (5 alpha-androstan-17 beta-ol-3-one) and androstanediol (5 alpha androstane-3 alpha, 17 beta-diol). The intratesticular steroid pattern in elderly men was essentially characterized by a decrease of the 5-ene steroid concentration, whereas we did not observe a decrease in the 4-ene steroids, progesterone concentration being even significantly higher in the aged testes. There was no evidence for a decrease in either lyase or 17-hydroxylase activity. It is suggested that the steroid pattern as observed in the aged testes is the consequence of a decreased oxygen supply, due to a decreased testicular perfusion.

Rats were made bilaterally cryptorchid at 21 days of age; sham-operated rats were used as controls. At 35 days, the animals were injected i.m. with saline or with 10 IU hCG. Progesterone, 17-hydroxyprogesterone, androstenedione and testosterone were measured in both testes and plasma under basal conditions and 2, 4, 8, 12, 24 and 72 h respectively after injection. The plasma levels and intratesticular contents of the steroids were generally lower in cryptorchid rats. The patterns of the steroid response to hCG were similar in both groups: in the testes and in the plasma, they increased acutely following hCG injection (except testicular androstenedione), then, after 72 h, returned to normal values in the plasma but remained higher than the basal values in the testes. These results suggest that there are no gross abnormalities in the testicular steroidogenic pathways and that the mechanism of action of hCG on the Leydig cells is unaltered in bilaterally cryptorchid immature rats.