Comparative determinations of non SHBG-bound serum testosterone, using ammonium sulfate precipitation, Concanavalin A binding or calculation in men

BackgroundTestosterone (T) circulates tightly bound to sex hormone-binding globulin (SHBG) and weakly to albumin. Non-SHBG-bound T is considered as the bioavailable T (BT) and was recommended for the evaluation of androgen disorders. Two methods, BT calculating from T, SHBG and albumin or BT measurement using ammonium sulfate precipitation of [SHBG-T] complex have been widely used. Using SHBG separation with Concanavalin-A (ConA) was recently proposed as a more specific method. The aim of this work was to compare these three methods in male patients.MethodsSerum samples of 131 consecutive untreated men (15–81 years) referred for suspicion of hypogonadism were collected. Total T was measured by GC–MS, SHBG by immunoradiometric assay. Level of BT was assayed using ammonium sulfate precipitation, ConA separation and calculated using the Issam web calculator.ResultsOnly few differences were found between ammonium sulfate or ConA BT measurements. However, we found much higher serum calculated BT than assayed BT with an increasing bias when BT levels increased.ConclusionsMeasurement of BT using ConA separation could be recommended. The results are equivalent to those obtained using ammonium sulfate precipitation. This method eliminates the possible non specific albumin precipitation that can occur with ammonium sulfate when the assay conditions are not rigorously controlled.     

A new specific and sensitive time resolved-fluoroimmunoassay of 11-deoxycortisol in serum

A biotinylated 11-deoxycortisol tracer was synthesized from 11-deoxycortisol-3-carboxymethyloxime and the conjugate obtained by acylation of biotinylaminopropylammonium trifluoroacetate. This biotinylated tracer was used to develop an 11-deoxycortisol time-resolved-fluoroimmunoassay (TR-FIA). The tracer was quantified after adding streptavidine-Europium. A TR-FIA sensitive standard curve, with displacement of 20, 50, and 80% of tracer was obtained with 12.4, 70.7, and 512.8 pg of 11-deoxycortisol, respectively. After extraction followed by Celite chromatography, purified serum samples were simultaneously assayed by RIA and TR-FIA. The results obtained by the two methods were practically identical, however, this new specific, non-isotopic 11-deoxycortisol assay has the advantage of being more sensitive than RIA, thus well-suited to accurate measurement in endocrinological studies, particularly when serum 11-deoxycortisol levels in patients are just above the highest normal values. Moreover, this non-isotopic assay is cheaper than RIA.     

Specific radioimmunoassay of estrone sulfate. Application to measurement in male plasma

We described a new, specific and easy to use radioimmunoassay (RIA) of estrone sulfate (E1S) in males. After synthesis of an E1S-6-(O-carboxymethyl) oxime hapten then coupling to BSA, we obtained a specific anti-E1S antiserum. Although the cross-reactivity of DHEAS with our anti-E1S antiserum was low (CR=0.002%), we confirmed the absolute necessity of separating plasma DHEAS from plasma E1S, before E1S RIA, because in plasma, DHEAS is present at levels 3-6000-fold higher than E1S, which generally is ignored. Thus, we elicited an easy separation of DHEAS from E1S, by a fast chromatography on in-house minicolumns. This new RIA, was applied to the determination of E1S plasma normal values in males. In 27 young men (<35 years), mean+/-S.D. were 1.97nmol/l+/-1.07nmol/l and in 63 untreated healthy aged men (>55 years), 1.80nmol/l+/-1.21nmol/l. No significant difference was seen between young and older subjects. The ranges of E1S plasma levels in these subjects were rather large and the ratios between the highest and the lowest E1S plasma levels were seven in the young group and 23.4 in the older group. No decrease of E1S plasma levels was observed with ages. Contrary to large interindividual E1S plasma level variations, the intraindividual variations have been found to be no significant. Correlations between E1S and unconjugated estrogens, E2 and E1 were 0.22 (P=0.016) and 0.51 (0.001), respectively.     

Systemic Compensatory Response to Neonatal Estradiol Exposure Does Not Prevent Depletion of the Oocyte Pool in the Rat

The formation of ovarian follicles is a finely tuned process that takes place within a narrow time-window in rodents. Multiple factors and pathways have been proposed to contribute to the mechanisms triggering this process but the role of endocrine factors, especially estrogens, remains elusive. It is currently hypothesized that removal from the maternal hormonal environment permits follicle formation at birth. However, experimentally-induced maintenance of high 17β-estradiol (E2) levels leads to subtle, distinct, immediate effects on follicle formation and oocyte survival depending on the species and dose. In this study, we examined the immediate effects of neonatal E2 exposure from post-natal day (PND) 0 to PND2 on the whole organism and on ovarian follicle formation in rats. Measurements of plasma E2, estrone and their sulfate conjugates after E2 exposure showed that neonatal female rats rapidly acquire the capability to metabolize and clear excessive E2 levels. Concomitant modifications to the mRNA content of genes encoding selected E2 metabolism enzymes in the liver and the ovary in response to E2 exposure indicate that E2 may modify the neonatal maturation of these organs. In the liver, E2 treatment was associated with lower acquisition of the capability to metabolize E2. In the ovary, E2 depleted the oocyte pool in a dose dependent manner by PND3. In 10 µg/day E2-treated ovaries, apoptotic oocytes were observed in newly formed follicles in addition to areas of ovarian cord remodeling. At PND6, follicles without any visible oocyte were present and multi-oocyte follicles were not observed. Our study reveals a major species-difference. Indeed, neonatal exposure to E2 depletes the oocyte pool in the rat ovary, whereas in the mouse it is well known to increase oocyte survival.     

Efficacy and safety of a new testosterone-in-adhesive matrix patch applied every 2 days for 1 year to hypogonadal men

OBJECTIVES: To study long-term efficacy and safety of a testosterone-in-adhesive matrix patch, delivering 4.8 mg of testosterone daily. METHODS: Randomized, open label, multicenter 1-year study. 224 hypogonadal patients were included. 188 received 2 patches of 60 cm2 every 48 h and 36 patients had IM testosterone enanthate injection every 3 weeks. T, bioavailable T (BT), DHT, E2, LH, FSH and SHBG and clinical symptom scores (AMS and MSF-4) were assessed at 3, 6 and 12 months. RESULTS: In the patch group, T serum levels were above 3 ng/mL in 85% of patients and remained stable over time. BT, DHT and E2 levels were restored within physiological range. BT/T ratio varied from 20 to 70%. In the IM group, the percentages of "normalized" patients appeared to be lower, although the two groups cannot be adequately compared due to the kinetic profile of T following IM administration, resulting in greater variations of serum T levels, blood samplings occurring randomly at time of peak, trough, or in between. A significant correlation was found between T, BT and the MSF-4 changes. BT levels were significantly related to total AMS score. PSA values showed a mean (S.D.) increase of 0.13 (0.38), 0.23 (0.79) and 0.30 (1.47)ng/mL at weeks 14, 27 and 53, respectively. The patch was well tolerated with no negative impact either on lipid profile, or red blood cells. Administration site reactions occurred in 35 patients (18.8%). Adhesiveness was good (>or=75%) in >90% patients over the 1 year application period. CONCLUSION: Two 60 cm2 patches, allowed constant physiological levels of sexual hormones over time. This new patch was well tolerated, easy to use, well accepted by the patients and displayed a very good adhesiveness. Clinical efficacy was more related to BT than to T.     

Effects of insulin-like growth factor I (IGF-I) on enzymatic activity in human adrenocortical cells. Interactions with ACTH

Cells obtained from 6 adult human adrenals or adrenal fragments were cultured in serum-free synthetic medium (McCoy's) in order to study the isolated effects of IGF-I on steroidogenesis and its interactions with ACTH. After addition of peptide, changes in the activities of steroidogenic enzymes were assessed by measuring certain steroids in the spent medium. These included pregnenolone, 17-hydroxypregnenolone (17-OH-Preg), dehydroepiandrosterone (DHA), 17-hydroxyprogesterone (17-OH-P), androstenedione (AD), 11-deoxycortisol and glucocorticoids (chiefly cortisol and its immediate precursors, 11-deoxycortisol and 17-OH-P) and cortisol itself.

The steroid responses obtained with repeated doses of IGF-I (40 ng/ml ≈ 10⁻⁹ M), added at 0, 48 and 72 h, over 4 days' culture were quite different from those obtained with repeated doses of ACTH (0.25 ng/ml ≈ 10⁻¹⁰ M). All the steroids measured increased with time of culture under the influence of ACTH and, apart from pregnenolone which peaked, tended to reach a plateau. With IGF-I, by contrast, DHA, AD, 11-deoxycortisol and glucocorticoid production increased initially, then decreased progressively, whereas pregnenolone, 17-OH-Preg and 17-OH-P production was either absent or negative.

Cumulative steroid production over 4 days reached similar levels in response to a single dose of IGF-I and/or ACTH, with two major exceptions: pregnenolone dropped significantly with IGF-I [46% ± 6 (SEM) as opposed to 93% ± 11 with ACTH, P < 0.005, N = 5], as did 17-OH-P (48% ± 11 vs 113% ± 8 with ACTH, P < 0.001, N = 6). Increased formation of down-stream metabolites (DHA, AD, 11-deoxycortisol and glucocorticoids) would suggest that IGF-I induced stimulation of the 17-, 21- and 11β-hydroxylases.

The responses to ACTH stimulation of cells which 4 days previously had been pre-treated with an initial and single dose of IGF-I and/or ACTH emphasized the impact of IGF-I on the 3-hydroxylation steps in cortisol biosynthesis. Compared with ACTH pre-treatment, the effects of which faded in the long term, pre-treatment with IGF-I resulted in a significantly increased steroidogenic response (P between < 0.05 and < 0.01). With the single exception of pregnenolone (43% ± 4.7), production of all the metabolites was amplified: 17-OH-Preg: 348% ± 88; DHA: 643% ± 127; 17-OH-P: 193% ± 36; AD: 725% ± 200; 11-deoxycortisol: 573% ± 110; cortisol: 1000%.

Our findings strongly suggest that IGF-I plays a major rôle in the regulation of steroidogenesis by promoting and maintaining enzymatic activity (17, 21- and 11β-hydroxylases) via which the function of ACTH is achieved, viz., biosynthesis of cortisol.     

Persistent Organochlorine Pollutants with Endocrine Activity and Blood Steroid Hormone Levels in Middle-Aged Men

Background

Studies relating long-term exposure to persistent organochlorine pollutants (POPs) with endocrine activities (endocrine disrupting chemicals) on circulating levels of steroid hormones have been limited to a small number of hormones and reported conflicting results.

Objective

We examined the relationship between serum concentrations of dehydroepiandrosterone, dehydroepiandrosterone sulphate, androstenedione, androstenediol, testosterone, free and bioavailable testosterone, dihydrotestosterone, estrone, estrone sulphate, estradiol, sex-hormone binding globulin, follicle-stimulating hormone, and luteinizing hormone as a function of level of exposure to three POPs known to interfere with hormone-regulated processes in different way: dichlorodiphenyl dichloroethene (DDE), polychlorinated biphenyl (PCB) congener 153, and chlordecone.

Methods

We collected fasting, morning serum samples from 277 healthy, non obese, middle-aged men from the French West Indies. Steroid hormones were determined by gas chromatography-mass spectrometry, except for dehydroepiandrosterone sulphate, which was determined by immunological assay, as were the concentrations of sex-hormone binding globulin, follicle-stimulating hormone and luteinizing hormone. Associations were assessed by multiple linear regression analysis, controlling for confounding factors, in a backward elimination procedure, in multiple bootstrap samples.

Results

DDE exposure was negatively associated to dihydrotestosterone level and positively associated to luteinizing hormone level. PCB 153 was positively associated to androstenedione and estrone levels. No association was found for chlordecone.

Conclusions

These results suggested that the endocrine response pattern, estimated by determining blood levels of steroid hormones, varies depending on the POPs studied, possibly reflecting differences in the modes of action generally attributed to these compounds. It remains to be investigated whether this response pattern is predictive of the subsequent occurrence of disease.     

Upregulation of endothelin 1 and its precursor by IL-1beta, TNF-alpha, and TGF-beta in the PC3 human prostate cancer cell line

Increasing evidence indicates that endothelin 1 (ET-1) is implicated in prostate tumour progression. However, data on ET-1 regulation in human prostate and prostate cancer cell lines are lacking. In this study, regulation of ET-1 and its precursor big ET-1, using PC3 cells, a human bone metastatic prostatic carcinoma cell line, was addressed. ET-1 and big ET-1 assays demonstrated greater secretion of both peptides in the presence of 10% fetal calf serum (FCS) as compared with 0.5% FCS. Incubation of PC3 cells in the absence and presence of various cytokines and growth factors known to be implicated in prostate stroma-epithelium interactions, revealed that IL-6, FGF7/KGF and FGF2/bFGF had no effect on ET-1 and big ET-1 secretion, whereas interleukin 1beta (IL-1beta), tumour necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta) stimulated their secretion in a concentration-dependent manner. Binding experiments indicated the presence of specific ET-1 receptors in PC3 cells: Kdapp = 1.1 x 0.2 x 10(-10)M, Bmax = 2660 +/- 390 sites/cell. Data analysis demonstrated the presence of only the ETA receptor subtype in PC3 cells. In conclusion, our results indicate that the implication of ET-1 in prostate cancer is likely to be mediated via paracrine/autocrine control of cell factors.     

Testicular adrenal rest tumours in salt wasting congenital adrenal hyperplasia (in vivo and in vitro studies)

We describe the case of a 20-year-old patient with salt-wasting congenital adrenal hyperplasia (CAH) related to 21-hydroxylase deficiency. Bilateral craggy testicular tumours were found, requiring histological evaluation. Prior to the surgical procedure, the patient was treated with dexamethasone (he presented cortisol deficiency) and was stimulated with ACTH. High levels of 11beta-OH steroids measured in the gonadal vein, compared with peripheral blood samples suggested the presence of adrenal rests. Incubation of the tumours (which could not be differentiated histologically, from Leydig tissue), with radioactive steroid precursors was carried out. The results revealed the testicular tumours were of adrenal tissue origin, associated with 21-hydroxylase deficiency. The patient's non-compliance to glucocorticoid treatment was the main cause of his hypogonadotropic hypogonadism.     

Development of a plasma 17alpha-hydroxyprogesterone time resolved-fluorescence immunoassay involving a new biotinylated tracer

A biotinylated 17alpha-hydroxyprogesterone probe (3) was prepared from 17alpha-hydroxyprogesterone-3-carboxymethyloxime and conjugate obtained by acylation of biotinylaminopropylammonium trifluroacetate. This new tracer was used in the development of a 17alpha-hydroxyprogesterone time-resolved fluoroimmunoassay using streptavidin-europium. The new method was compared to a long-standing radioimmunoassay method and found to be more sensitive and economical.