A theoretically sound and practicable equilibrium dialysis method for measuring percentage of free testosterone

Free testosterone measured in serum equilibrated in vitro is considered a good index of biologically available testosterone even though a large part of free testosterone in vivo is derived locally from rapid dissociation of testosterone bound to albumin. The most accurate method for measuring free testosterone, however, is unsettled. The classical method--equilibrium dialysis--has been questioned because of the dilution of serum that it entails and the previous inability to achieve identical results with diluted and undiluted serum. Essentially identical measurements of free testosterone were achieved in diluted and undiluted charcoal-stripped serum by using the dialysis method and calculation reported here. The measured free testosterone in undiluted whole serum from women was only 4-6% lower than the estimated physiological values. These results were obtained using a validated calculation, controlling pH, using physiological bicarbonate buffer at 37 degrees C, maintaining a constant free ligand concentration for dilutions, measuring the water gain by the dialysis bag, and using highly purified labeled testosterone. The mean free testosterone for normal women was 0.17 ng/dl (0.11-0.23) and for hirsute women was 0.49 ng/dl (0.27-0.71). The testosterone not bound to testosterone-estradiol binding globulin, calculated from free testosterone and albumin concentrations, was close to the production rate/min of testosterone. The method should be adaptable to other ligands.     

Non-polar extracts of serum from males contain covert radioimmunoassayable testosterone

Non-polar extracts of sera from human males contain immunoreactive testosterone in a form that is released by mild alkaline hydrolysis. The non-polar derivative shows no immunoreactivity with testosterone antibody prior to hydrolysis. Hydrolyzed non-polar serum extracts from ten adult male volunteers contained 2.0 +/- 0.8 (SD) ng/mL of testosterone. Neither non-polar serum extracts of normal females nor a water blank substituted for non-polar extract of serum yields any immunoreactive testosterone after alkaline hydrolysis. Testosterone palmitate hydrolyzed alone or after addition to non-polar extract of serum yields the expected quantities of radioimmunoassayable testosterone. Previously described conjugates of testosterone are polar and are neither extractable by petroleum ether nor hydrolyzable by alkali. These observations suggest that fatty acid esters of testosterone may be present in serum of human males.     

Effect of testosterone on serum immunoactive inhibin concentrations in intact and hypophysectomized male rats

Intact and hypophysectomized rats were treated with graded doses of testosterone via subcutaneous Silastic implants over a 13-week period. Serum inhibin concentrations fell 50% (P less than 0.001) after 2 weeks of hypophysectomy, remaining suppressed at this level for 13 weeks. The administration of testosterone to hypophysectomized rats (serum testosterone values 2-12 ng/ml; control values 5.5 ng/ml) was without effect on serum inhibin values. In contrast, administration of testosterone to intact animals for 7 weeks resulted in an initial fall (P less than 0.05) in inhibin levels to 50-70% of controls then increasing to reach control levels at higher doses. Serum FSH concentrations were similarly biphasic with increasing dose of testosterone and values for these two hormones were significantly correlated (r = 0.44, P less than 0.01). Segments of seminiferous tubules in culture from rats after various times of hypophysectomy showed a partly suppressed secretion of inhibin. The administration of testosterone did not modify inhibin production although inhibin production was sensitive to FSH. It is concluded that (1) serum inhibin concentrations are partly suppressed after hypophysectomy and testosterone has no effect on serum inhibin values; and (2) the suppression of serum inhibin in intact rats treated with increasing doses of testosterone is attributable to the concomitant fall in serum FSH concentrations.     

Correlation between serum zinc and testosterone: A systematic review

Zinc is a vital trace element for normal function of the living system. In male, zinc is involved in various biological processes, an important function of which is as a balancer of hormones such as testosterone. For this purpose, studies related to the influence of zinc on serum testosterone were selected and summarized, including the effect of dietary zinc deficiency and zinc supplementation on testosterone concentrations. After preliminary searching of papers on databases, 38 papers including 8 clinical and 30 animal studies were included in this review. We concluded that zinc deficiency reduces testosterone levels and zinc supplementation improves testosterone levels. Furthermore, the effect degree of zinc on serum testosterone may vary depending on basal zinc and testosterone levels, zinc dosage form, elementary zinc dose, and duration. In conclusion, serum zinc was positively correlated with total testosterone, and moderate supplementation plays an important role in improving androgen.     

男性排精后血清睾酮水平的周期性变化

为了解男性排精后可能存在的性激素变化过程,在排精后连续禁欲的情况下,对28人血清睾酮（T）水平进行了两个时段的每天定时检测,检测发现,在排精后连续禁欲的第2-5天,睾酮水平变化不明显;在禁欲的第7天,睾酮水平出现一个明显的高峰,峰值平均提高至底线的145.7%,第7天后如果再继续禁欲,没有再发现明显的变化,排精是这一周期性现象的前提和开始,没有排精就没有这种独特的周期性变化,这些结果说明男性排精会引起睾酮水平的周期性变化,这种变化的形式以禁欲第7天出现一个高峰为标志,这些结果证实了血清睾酮水平这种周期性变化现象的存在以及排精与这一现象之间的联系。     

Direct radioimmunoassay (RIA) of salivary testosterone: correlation with free and total serum testosterone

Simple and sensitive direct RIA for determination of salivary testosterone was developed by using RSL NOSOLVEX TM (125 1) kit produced by Radioassay System Laboratories (Carson, California). In addition, a relationship between salivary and serum free and total testosterone concentrations was studied in randomly selected 45 healthy subjects, 5 females on oral contraceptive pills and 28 hypertensive patients on various treatment regimens. The lowest weight of testosterone detectable by our modified method was equivalent to 1 pg/ml of saliva, taking into account analytical variability. Intra- and interassay coefficients of variation were 5.09 +/- 2.7% and 8.2 +/- 5.9% respectively. Statistically significant correlations were found between salivary and serum free testosterone (r = 0.97) and salivary and serum total testosterone concentrations (r = 0.70-0.87). The exception to this was a group of hypertensive females in which no correlation (r = 0.14) between salivary and total serum testosterone was found. It is also of interest that, while salivary testosterone was significantly increased in subjects taking oral contraceptives and most of the hypertensive patients the total serum testosterone concentration was in normal range. Our findings suggest that determination of salivary testosterone is a reliable method to detect changes in the concentration of available biologically active hormone in the circulation.     

Gender differences in the validity of testosterone measured in saliva by immunoassay

We rigorously evaluated gender differences in the measurement validity of salivary testosterone. Matched serum, saliva, and finger stick blood spot specimens were collected from 40 (20 males) young adults (aged 18-27 years). Saliva was assayed for testosterone by two independent (isotopic and non-isotopic) immunoassay methods. Serum was assayed by commercially available immunoassay kits for free and total testosterone. An immunoassay was developed for the measurement of testosterone in dried blood spots and is presented in detail so as to be reproducible from this report. Regardless of assay method, salivary testosterone levels are modestly correlated with serum levels for males but not necessarily for females. Blood spot assay results were highly correlated with serum total and free testosterone for both males and females. Substitution of saliva assay results for serum values substantially underestimates known testosterone-behavior associations, and this effect is much more pronounced for females than for males. The findings have important implications for the use and potential misuse of noninvasive measures of testosterone, and with respect to statistical power, the probability of observing significant testosterone-behavior relationships.     

Effect of serum and serum lipoproteins on testosterone production by adult rat Leydig cells in vitro.

The effect of serum factors other than luteinizing hormone on Leydig cell testosterone secretion was examined using an in vitro bioassay system based on the stimulation of purified adult rat Leydig cells during a 20 h incubation in the presence of a maximal dose of human chorionic gonadotrophin (hCG). Charcoal-extracted serum and testicular interstitial fluid (IF) from normal adult male rats were separated into lipoprotein and lipoprotein-deficient fractions by density ultracentrifugation. Stimulatory bioactivity was found in the lipoprotein fraction of both serum and IF, although the levels of lipoprotein and corresponding bioactivity recovered from IF were significantly lower (25%) than those of serum. There was no difference between the effects of serum lipoproteins on Leydig cell testosterone production stimulated by either hCG or dibutyryl cAMP. In time-course studies, the serum lipoprotein fraction had no effect on hCG-stimulated testosterone production in vitro at 3.0 or 6.0 h, but partially prevented the normal decline in hCG-stimulated testosterone production after 6.0 h. In contrast, unfractionated serum was stimulatory at all time-points. In the absence of hCG, the lipoprotein fraction was stimulatory at both 6.0 and 20 h, although not at 3.0 h. The lipoprotein-deficient protein fraction of serum had no effect on hCG-stimulated testosterone production alone, but significantly enhanced the bioactivity of the lipoprotein fraction, and caused a dose-dependent stimulation of testosterone production in the presence of a constant concentration of serum lipoproteins. Both a stimulatory peak of activity (apparent MW 40-80 kDa), and a large MW (> 100 kDa) inhibitor of testosterone production were identified in serum after fractionation by gel filtration (Sephadex G-100). The data indicate that (i) the stimulatory effect of serum on short-term hCG-stimulated Leydig cell testosterone production in vitro is predominantly due to the serum lipoprotein fraction, possibly by providing additional precursors for testosterone synthesis, (ii) the biological activity of the lipoproteins is influenced by both stimulatory and inhibitory serum proteins in addition to luteinizing hormone, and (iii) that serum lipoproteins may be involved in supporting Leydig cell steroidogenesis in vivo.     

Androgen control of male sex behavior in the crested newt (Triturus cristatus carnifex Laur.): Castration and sex steroid administration

Castration significantly lowers serum testosterone in sexually active male Triturus cristatus. Replacement therapy by implants of testosterone in silastic capsules elevates the serum testosterone level to higher values than normal. Sex behavior is depressed by castration and partially reinstated by replacement therapy with testosterone. 5α-dihydrotestosterone was the only testosterone metabolite showing some behavioral effectiveness in castrates; estradiol and 5β-dihydrotestosterone failed to elicit sex behavior.     

Diurnal variations of serum testosterone levels in intact and gonadectomized male and female rhesus monkeys.

A radioimmunoassay for serum testosterone which does not require chromatographic separation was used to measure the diurnal variations in intact and orchidecomized males and intact and ovariectomized females. The intact male rhesus monkey shows a distinctive diurnal variation in serum levels of testosterone characterized by lower values during the day and a marked increase in the early evening (1900-2200 hr). The testosterone levels remain high throughout most of the lights-off period in the intact male. In contrast to the intact male, the markedly lowered serum levels of testosterone in the orchidectomized male were higher during the day and consistently showed a nadir during the early evening (2000-2200 hr). The evening nadir of testosterone levels was 51.0% lower than the 24-hr mean whereas the maximum serum level was 46.4% higher. A similar circadian pattern of testosterone was seen in both the intact and ovariectomized females. The testosterone values were higher during the day and consistently showed a nadir during the early evening. These results suggest that the adrenal secretion of testosterone varies in a diurnal pattern characterized by an early evening nadir. This adrenal pattern is overshadowed by a much larger gonadal rhythm in the intact male.     

Interrelationship of serum testosterone, dominance and ovarian cyclicity status in female African elephants

The African elephant population in North American zoos is not self-sustaining, in part due to the prevalence of ovarian acyclicity. While little is known about the cause of this condition, earlier research has shown that females without cyclic corpus luteum (CL) function rank higher in the dominance hierarchy than females with cyclic CL function. The goal of this study was to measure longitudinal serum testosterone concentrations in captive female African elephants to determine if there is a relationship among serum testosterone concentrations, social dominance rank and ovarian cyclicity status. Weekly blood samples from 49 female African elephants (24 having and 25 not having cyclic CL function at 22 facilities) were collected over a 12-month period and analyzed for serum testosterone using an enzymeimmunoassay. A progesterone radioimmunoassay was used to quantify serum progestagen concentrations and categorize ovarian cyclicity status. The dominance hierarchy of individual elephants within each herd was assessed by a written temperament survey, which identified 19 dominant, 15 middle and 15 subordinate females. No clear patterns of serum testosterone secretion were observed in females with and without cyclic CL function. Furthermore, no significant relationships were found among serum testosterone concentrations, dominance rank, and ovarian cyclicity status. These data suggest that increased circulating testosterone concentrations are not associated with greater rates of ovarian acyclicity or dominance status in captive female African elephants.     

Reduced activity of androgen biosynthesis in the testes of rats with analbuminemia

Steroid metabolism in Nagase Analbuminemia Rats (NAR), a mutant strain established from Sprague-Dawley rats, was studied. NAR are characterized by lack of serum albumin and hyperlipidemia. Total testosterone concentration in the serum of NAR was lower than that of normal rats, while the serum free testosterone, LH and FSH concentrations were similar. The half lives of 14C-labeled testosterone administered intravenously in NAR and normal Sprague-Dawley (SD) rats were 4.4 and 4.1 min, respectively. The plasma clearance rates of testosterone in NAR and normal rats were 34.7 and 39.1 ml/min per kg body weight. On Sephadex G-100 chromatography, a mixture of [3H]testosterone and normal rat serum gave two protein peaks eluted in the void volume and the albumin fraction, and the radioactivity was eluted all in the albumin fraction. In contrast, a mixture of [3H]testosterone and NAR serum gave a single protein peak eluted in the void volume and the radioactivity was mainly eluted with this protein peak. The association constants of testosterone to NAR and normal rat sera were 1.25 and 2.24 X 10(4) M-1. Enzyme activities related to the synthesis of testosterone by the testicular microsomal fractions of NAR and normal rats were examined. The activities of 3 beta-hydroxysteroid dehydrogenase, 5-ene-4-ene isomerase, 17 alpha-hydroxylase, C-17-C-20 lyase and 17 beta-hydroxysteroid dehydrogenase were lower in NAR than in normal rats. The activity for synthesis of testosterone from pregnenolone by the testicular microsomal fraction of NAR was about 40% of that of normal rats. These findings indicate that the low serum concentration of testosterone in NAR is mainly attributable to decreased biosynthesis of testosterone in the testes.     

Evidence for a major role of inhibin in the feedback control of FSH in the male rat

Adult rats (16-18/group) received a single intratesticular injection of 25, 100 or 400 microliters glycerol solution (7:3 in distilled water, v/v). Half of the rats in each group were given implants of testosterone, a testosterone-filled Silastic capsule (1.5 cm length) to provide serum values of testosterone within the normal range. After 1 week all animals were killed by decapitation. Serum concentrations of gonadotrophins, testosterone and immunoactive inhibin as well as testicular concentrations of testosterone and bioactive inhibin were determined. Testicular histology was studied in Paraplast-embedded tissue stained with PAS and haematoxylin-eosin. Glycerol treatment caused a dose-dependent ablation of spermatogenesis in a distinct area around the site of injection. Serum concentrations of FSH increased proportionally with increasing spermatogenic damage while serum LH and testosterone remained unaltered except with the highest glycerol dose. The rise in serum FSH was significantly correlated with serum (r = -0.70, P less than 0.001) and testicular (r = -0.66, P less than 0.001) concentrations of inhibin. A less pronounced correlation was found between LH and serum inhibin (r = 0.48). No correlation was found between the concentrations of LH and testicular inhibin or between serum concentrations of FSH and serum testosterone in the 25 and 100 microliters groups. Maintenance of low to normal serum testosterone concentrations by means of Silastic implants blocked the elevation of FSH in glycerol-treated animals but failed to affect significantly serum FSH in untreated rats. In all testosterone treated rats testicular inhibin concentrations were markedly reduced in the presence of lowered concentrations (7-14%) of testicular testosterone and unaltered serum FSH concentrations.     

Decreased serum testosterone concentration in male heroin and methadone addicts

The serum concentrations of testosterone, estradiol-17β, FSH and LH were measured in 22 male subjects addicted to heroin or methadone. Serum testosterone concentration was decreased in many of these subjects without consistent abnormalities in the other hormones. It is suggested that decreased sexual function in male addicts may be partially due to a decrease in serum testosterone concentration.     

慢性应激状态下大鼠睾酮水平测定

目的通过对于慢性应激模型和运动疲劳模型大鼠的血清睾酮水平的对比研究,探讨慢性应激过程中睾酮水平变化规律及其与发生疲劳的关系。方法建立大鼠慢性不可预测性应激模型及运动性疲劳模型;应用大鼠轨迹追踪系统对不同应激时间大鼠进行活动量观测,应用放射性免疫法对各组大鼠进行血清总睾酮和游离睾酮进行测定。结果与正常组比较,慢性应激大鼠第1周组活动量(运动轨迹长度,下同)增加(P0.01),第2周组活动量下降(P0.01)。各实验组大鼠游离睾酮与总睾酮之间均存在线性相关。慢性应激各组随应激时间延长,其游离睾酮与总睾酮比值呈逐渐下降趋势,并且能够达到运动疲劳组水平。结论慢性应激条件可以明显引起大鼠睾酮水平下降。提示分层检测慢性疲劳综合征人群血清总睾酮及游离睾酮的水平以及进行动态性研究可能为本病症的诊断提供生化指标。以游离睾酮与总睾酮比值为指标检测雄性激素水平,能够更好地反映干预因素对动物生理上的影响。     

Androgenic regulation of luteinizing hormone secretion: relationship to androgen binding in sheep pituitary

Castrated ram lambs (wethers) were investigated for sensitivity to androgen feedback and to determine whether this feedback inhibition of luteinizing hormone (LH) was associated with changes in pituitary androgen receptors. Administration of Silastic capsules containing either dihydrotestosterone or testosterone was found to produce dose-dependent inhibitory effects on serum LH levels in wethers. Physiological dosages of these androgens (i.e., those that produce serum levels of dihydrotestosterone [0.24 ng/ml] or testosterone [2.1 ng/ml] similar to those of intact rams) resulted in differential inhibition of serum LH and LH content of the anterior pituitary. Whereas the inhibitory effect of dihydrotestosterone on pituitary LH content was much more dramatic than that seen with testosterone, the high dosage of testosterone also produced a substantial decrease in pituitary LH content. Responses of the pituitary to changes in serum androgen were compared to responses of the seminal vesicle, which served as a control androgen target organ. Androgen levels were positively correlated with seminal vesicle weights, but pituitary weights were unaffected by castration and/or androgen replacement. Treatments with dihydrotestosterone were associated with decreased cytosol androgen binding activity (i.e., receptors) in pituitary and seminal vesicle, suggesting that both of these tissues were sites of androgen action. Although testosterone inhibited serum LH levels, pituitary cytosol androgen receptors were not affected by changes in serum testosterone. We conclude from these data that dihydrotestosterone is a physiological regulator of pituitary LH secretion in the ram and that further study is needed to investigate the complex actions of testosterone and its metabolites on pituitary function.     

Serum of patients with prostatic cancer or benign prostatic hypertrophy contains nonpolar testosterone.

We have previously described a nonpolar form of radioimmunoassayable serum testosterone (NPT) not measured by available antitestosterone antibodies. It is detected by mild alkaline hydrolysis of the petroleum ether extract of serum and subsequent radioimmunoassay. The properties of NPT are consistent with that of a fatty acid ester of testosterone or dihydrotestosterone. The serum of young males contains 1 to 3 ng/ml NPT, but it is not detected in female serum. We measured serum testosterone and NPT levels in 36 men between 58 and 87 years of age. Seventeen subjects with advanced prostatic cancer (NPT 1.70 +/- 1.44 ng/ml) were compared with a control group consisting of six patients with benign prostatic hypertrophy (BPH) and 13 patients with no prostatic disease (NPT 0.72 +/- 0.46, P = 0.017). There was no significant difference between BPH patients and patients with no prostatic disease; the results were pooled. The concentration of NPT in prostatic cancer patients but not in controls was inversely correlated with that of testosterone. Immunoassayable testosterone was present in the serum of two orchiectomized patients and, therefore, cannot derive solely from the testes.     

Correlation Between Serum Testosterone Level and Concentrations of Copper and Zinc in Hair Tissue

Testosterone deficiency is associated with late-onset hypogonadism. Micronutrients including copper (Cu) and zinc (Zn) influence testosterone synthesis. The association between micronutrient concentrations in hair tissue and serum testosterone was studied in Korean men. The subjects were 88 men 40-60?years of age who visited the health promotion center and an outpatient clinic of family medicine at a university hospital from March 2006 to February 2008. Population sociological features of the subjects were acquired by self-administered surveys and interview, height and weight were measured, serum total testosterone was determined in the morning, and Cu and Zn were quantified from hair tissue collected in the morning. Subjects with normal testosterone group had a significantly higher Zn level compared to low testosterone group (P?=?0.003). Significant negative correlations were evident between total testosterone and Cu level (r?=?-0.252, P?=?0.022), and the Cu/Zn ratio (r?=?-0.288, P?=?0.008). Normal testosterone is associated with a higher Zn level. Decreased serum testosterone is significantly associated with a high level of Cu and elevated Cu/Zn ratio in hair tissue.     

雄貉睾丸宽度和血清睾酮水平的季节性变化及其与某些繁殖特性的关系

作者测定并分析了43只雄貉睾丸宽度、血清睾酮水平的季节性变化。结果表明:睾丸宽度和血清睾酮水平呈明显的年周期季节性变化(p<0.01)。秋分(9月)时,睾丸宽度开始增大(p<0.05 );血清睾酮水平在10月开始升高(p<0.05)。各月雄貉的平均睾酮水平与平均睾丸宽度是极显著的正相关(r=0.83,p<0.01 n=11)。雄貉繁殖季节初期,血清睾酮水平与其首、末次的交配日期呈显著负相关(r=-0.525和r=-0.476,p<0.05,n=19)。