3α-Androstanediol Kinetics in Man

Kinetics of 5alpha-androstane-3alpha, 17beta-diol (3alpha-diol) were studied in man. Clearance rates were determined by both the constant infusion and single injection techniques. Production rates were calculated as the product of clearance rate data and plasma values in the a.m. obtained by a radioimmunoassay specific for 3alpha-diol. Mean metabolic clearance rates were 1,776+/-492 (SD) liters/day in males and 1,297+/-219 (SD) liters/day in females. Metabolic clearance rates by single injection were similar. Calculated production rates are 208+/-26 (SD) mug/day in males and 35+/-11 mug/day in females, which are significantly different. Hepatic extraction of 3alpha-diol determined by hepatic vein catheterization during constant infusion was 76% which was greater than expected from information on in vitro binding in plasma. The kinetic data is of interest since 3alpha-diol has a calculated inner pool (V(1)) volume of 12-14 liters, similar to 17beta-hydroxyandrost-4-en-3-one (testosterone) and 5alpha-androstan-17beta-ol-3-one (dihydrotestosterone), but the calculated outer pool (V(2)) of 33.5 liters is very large as are the metabolic rate and transfer constant. In contrast to testosterone and dihydrotestosterone, 3alpha-diol, although bound to sex hormone binding globulin, has a high metabolic clearance of which a large fraction represents extrahepatic (splanchnic) metabolism. A production rate of 3alpha-diol similar to dihydrotestosterone together with rather unique kinetic characteristics encourages further investigation of the biological role of this potent androgen.     

Metabolic clearance rate and blood production rate of testosterone and dihydrotestosterone in normal subjects, during pregnancy, and in hyperthyroidism

The metabolic clearance rate (MCR) and blood production rate (BP) of testosterone (T) and dihydrotestosterone (DHT), the conversion of plasma testosterone to plasma dihydrotestosterone, and the renal clearance of androstenedione, testosterone, and dihydrotestosterone have been studied in man. In eight normal men, the MCR(T) (516+/-108 [SD] liters/m(2)/day) was significantly greater than the MCR(DHT) (391+/-71 [SD] liters/m(2)/day). In seven females, the MCR(T) (304+/-53 [SD] liters/m(2)/day) was also greater than the MCR(DHT) (209+/-45 [SD] liters/m(2)/day) and both values were less than their respective values in men (P < 0.001). In men the conversion of testosterone into dihydrotestosterone at 2.8+/-0.3% (SD) was greater than that found in females, 1.56+/-0.5% (SD) (P < 0.001). In five pregnant females the MCR(T) (192+/-36 [SD] liters/m(2)/day), the MCR(DHT) (89+/-30 [SD] liters/m(2)/day) and the conversion of testosterone into dihydrotestosterone (0.72+/-0.15%) (SD) were significantly less than the values found in nonpregnant women. In five females with hyperthyroidism, the MCR for testosterone and dihydrotestosterone were similar to those observed in pregnant females, but the conversion of testosterone into dihydrotestosterone (2.78+/-1.7%) (SD) was greater, and similar to that found in men. In men the production of dihydrotestosterone was 0.39+/-0.1 (SD) mg/day, 50% being derived from the transformation of plasma testosterone. In women the production of DHT was 0.05+/-0.028 (SD) mg/day, only 10% coming from testosterone. During pregnancy, the production of testosterone and dihydrotestosterone are similar to that in normal women. In three patients with testicular feminization syndrome (an adult with hyperthyroidism and two children) these two MCRs were greatly reduced compared to the normal females, but the conversion of testosterone into dihydrotestosterone was in the limits of normal male rangeIn the normal subjects the renal clearance of androstenedione was greater than that of testosterone and dihydrotestosterone. Less than 20% of the dihydrotestosterone and less than 10% of the androstenedione in the urine is derived from the plasma dihydrotestosterone and androstenedione.     

The Source of Plasma Dihydrotestosterone in Man

The source of plasma dihydrotestosterone (DHT) (17beta-hydroxy-5alpha-androstan-3-one) in humans has been investigated by infusing two potential peripheral precursors, testosterone (T) and androstenedione (A). Metabolic clearance rates (MCR), conversion ratios (CR), transfer constants (rho), and blood production rates (P(B)) were calculated. Plasma testosterone and dihydrotestosterone were measured by competitive binding techniques. The MCR(DHT) was 652 +/-35 (SD) liters/day in five males and 314 +/-63 (SD) liters/day in four adult females. In each individual, the MCR(DHT) was significantly lower than MCR(T) as predicted by testosterone-binding protein affinity studies. The P(B) (DHT) was 302 +/-65 (SD) mug/day in males and 56 +/-26 mug/day in females. Testosterone and androstenedione are precursors (prehormones) for plasma dihydrotestosterone. The conversion ratio CR(BB) (T-DHT), calculated as the ratio of counts per minute per liter of plasma of product to precursor after infusion of labeled precursor, was 5.6 +/-0.6 (SD)% (six subjects) in the male and 3.5 +/-0.4 (SD)% (four subjects) in the female. CR(BB) (A-DHT) after androstenedione infusion to three female subjects averaged 9.2%. No dihydrotestosterone back conversion was detected (< 0.2%). The transfer constants were [rho]BB(T-DHT), 3.9 +/-1.0% (male) and 1.7 +/-0.6% (female), and [rho]BB(A-DHT) average was 13.3% in three female subjects. Using either plasma testosterone and dihydrotestosterone values from our subjects and mean androstenedione values as reported in the literature, approximate contributions can be calculated. Testosterone conversion accounts for at least 70% of plasma DHT in the male, but less than 20% in the normal female. Androstenedione appears to be a major prehormone of plasma dihydrotestosterone accounting for at least two-thirds plasma dihydrotestosterone by peripheral conversion in adult females. In three normal women undergoing tubal ligation, there was an unimpressive gradient between ovarian vein and peripheral plasma dihydrotestosterone. It is suggested that dihydrotestosterone in the blood does not arise from direct secretion but may reflect events occurring in peripheral androgen target tissues.     

Testosterone and Androstenedione Blood Production Rates in Normal Women and Women with Idiopathic Hirsutism or Polycystic Ovaries

The average plasma testosterone concentration of women with either hirsutism or polycystic ovaries and hirsutism was higher (p < 0.01) than that of normal women although the ranges overlapped. Testosterone blood production rates averaged 830 +/- 120 SE and 1,180 +/- 310 SE mug per day in the two groups of hirsute women and 230 +/- 33 SE mug per day in normal women. The ranges did not overlap.The testosterone metabolic clearance rates of hirsute women (1,090 +/- 140 SE L per day) and of men (1,240 +/- 136 SE L per day) were significantly higher than those of normal women (590 +/- 44 SE L per day). These differences persisted when the metabolic clearance rates were corrected for surface area. We suggest that testosterone metabolic clearance rates vary directly with some function of testosterone production.The mean plasma androstenedione levels (2.8 +/- 0.35 SE and 2.8 +/- 0.30 SE mug per L) and production rates (6,060 +/- 450 SE and 7,360 +/- 345 SE mug per day) of the women with hirsutism or polycystic ovaries, respectively, were significantly higher than those of normal women (1.5 +/- 0.22 SE mug per L; 3,300 +/- 830 SE mug per day). The androstenedione metabolic clearance rates were the same in each group. Plasma androstenedione was the precursor of 49% of plasma testosterone in normal women and of 26% of plasma testosterone in hirsute women. Thus, 74% of the plasma testosterone in these subjects must have been either secreted or derived from a precursor that did not enter the plasma androstenedione pool.     

Conversion of blood androgens to estrogens in normal adult men and women

Continuous infusions of Delta(4)-androstenedione-7-(3)H and testosterone-7-(3)H have been used to demonstrate that these androgens are converted to estrone and 17beta-estradiol, and contribute to the circulating blood levels of these estrogens in normal males and females. The conversion ratio (ratio of concentrations of radioactivity of free product steroid [chi(-PRO)] and free precursor steroid [chi(-PRE)], both corrected for recoveries, after an infusion of radioactive precursor steroid) for androstenedione (precursor) to estrone (product) is 0.013 in males and 0.007 in females, and the conversion ratio for testosterone (precursor) to estradiol (product) is 0.0018 in males and 0.005 in females. The transfer constant, [rho](BB) (AE1), for androstenedione conversion to estrone ([rho](BB) (AE1) = per cent of infused androstenedione, precursor, converted to estrone, product, when infusion and measurement are both in blood) is 1.35% in males and 0.74% in females, and the transfer constant, [rho](BB) (TE2), for testosterone conversion to estradiol is 0.39% in males and 0.15% in females.Whether measured as conversion ratio or transfer constant, the peripheral aromatization of androstenedione takes place to a greater degree than that of testosterone, and, for the respective androgens, both the conversion ratio and [rho](BB) value are greater in males than females.For the androgen interconversions, [rho](BB) (AT) is 4.5% in males and 2.2% in females; [rho](BB) (TA) is 8.2% in males and 12.0% in females.Studies on the distribution coefficients (effective concentration in red cells/plasma) for precursor radioactivity were also made. In both males and females the distribution coefficient for androstenedione is 0.16-0.17 while that of testosterone is 0.01-0.03.     

Characterisation of high affinity binding sites of androgens in primary hepatocellular carcinoma

The reported presence of androgen receptors (AR) in hepatocellular carcinoma (HCC) and foetal liver, but not in normal adult human liver, has been followed by further study of AR employing a new microassay. Tissues examined were: 5 samples of HCC with surrounding normal liver in 3 cases; 5 samples of cirrhotic liver and a single specimen of HCC in a child. High affinity binding of 5 alpha-dihydrotestosterone (DHT) was detected in cytosol (11.5-21 fmol/mg, Kd 1.5 X 10(-10)-3.1 X 10(-11) mol/l) and in nucleosol (8.7-11.4 fmol/mg, 6.7-1.4 X 10(-11) mol/l) of the 5 HCC samples. All other liver samples exhibited non-specific binding only. Competition studies indicated that DHT, testosterone, androstenedione, 5 alpha-androstan-3 beta, 17 beta-diol, androst-5-ene-3 beta,17 beta-diol and cyproterone acetate were acting at the same receptor binding site, relative displacement of 3H-DHT being 100, 85.7, 77.4, 67.8, 34.5 and 60.2 per cent respectively. Presence of 3.5S cytosolic and both 2.8S and 4S nucleosolic receptor patterns were demonstrated in both prostatic and HCC tissue. These studies confirm the presence of a cytosolic and nucleosolic androgen receptor in HCC which possesses similar characteristics to the AR of human prostate.     

Conversion of plasma progesterone to deoxycorticosterone in men, nonpregnant and pregnant women, and adrenalectomized subjects.

During the third trimester of human pregnancy the concentrations of deoxycorticosterone (DOC) in maternal plasma are 4-50 times those in nonpregnant women and men. It has been suggested that the increased amount of DOC in maternal plasma originates in the fetal compartment. We considered an alternate explanation for the high levels of DOC in plasma or near-term pregnant women, viz., that DOC may be derived in part from 21-hydroxylation of maternal plama progesterone. To test this hyposthesis we measured the fractional conversion of plasma progesterone to DOC from the relationship between the 3H:14C ratio of the infused tracers, [3H]progesterone and [14C]-DOC, and the 3H:14C ratio or urinary 3 alpha,21-dihydroxy-5 beta-pregnan-20-one (tetrahydro-DOC). The fractional conversion of plasma progesterone to DOC ([rho](BU)P-DOC), measured in this manner, was 0.007 +/- 0.001 (mean +/- SEM, n = 26) in the subjects of this study. The values for [rho](BU)P-DOC varied widely among subjects (0.002-0.022) but the range of values for [rho](BU)P-DOC was similar among women pregnant with an anencephalic or dead fetus, nonpregnant and adrenalectomized women, and men. The transfer constant of conversion of progesterone to DOC in plasma, [rho](BB)P-DOC, remained constant in a nonpregnant woman during the infusion of nonradiolabeled progesterone at rates of 0-14 mg/h. Based on the results of these studied, we conclude that DOC is formed by extra-adrenal 21-hydroxylation of plasma progesterone and that the rate of formation of DOC by this pathway is proportional to the concentration of progesterone in plasma.     

Familial gynecomastia with increased extraglandular aromatization of plasma carbon19-steroids.

We evaluated a family in which gynecomastia occurred in five males in two generations. In each affected subject, gynecomastia and male sexual maturation began at an early age. The ratio of the concentration of plasma estradiol-17 beta to that of plasma testosterone was elevated in each affected subject. In the three siblings with gynecomastia, the transfer constant of conversion of androstenedione to estrone (i.e., the fraction of plasma androstenedione that was converted to estrone as measured in the urine) was 10 times that of normal persons. The transfer constant of conversion of testosterone to estradiol-17 beta in the one subject studied also was 8-10 times that of normal men, whereas the transfer constants of conversion of estrone to estradiol-17 beta and of estradiol-17 beta to estrone were normal. Despite the elevation in extraglandular aromatase activity, there was a normal response of the hypothalamic-pituitary axis to provocative stimuli. This is the second documentation of gynecomastia that is associated with increased extraglandular aromatase activity, and the first time that the defect was found to be familial with a probable X-linked (or autosomal dominant, sex limited) mode of inheritance.     

3 alpha, 17 beta-androstanediol glucuronide in plasma. A marker of androgen action in idiopathic hirsutism.

Biologically active androgens and peripheral androgen metabolites in plasma were measured in 25 women with idiopathic hirsutism (IH). Plasma testosterone was not significantly elevated. Free testosterone however was increased although the elevation was not impressive (10.9 +/- 6.6 SD vs. 3.3 +/- 1.5 ng/dl) and one-fourth of the cases had normal unbound testosterone. Dihydrotestosterone (DHT) values were elevated (23.5 +/- 14 vs. 12.5 +/- 3.59) but again over half of the values were within the normal range. In our series of mild to moderate cases, 3 alpha-diol was not at all discriminatory. However, plasma 3 alpha-diol glucuronide was markedly increased (604 +/- 376 vs. 40 +/- 10 ng/dl), and elevated in all but one mild case. Previous studies document that DHT is the important androgen in skin and formation of DHT and 3 alpha-diol is markedly increased in vitro in IH. Since 3 alpha-diol glucuronide is derived largely from extrasplanchnic events, beta-glucuronidase is present in skin, and androgen stimulates formation of the enzyme in extrasplanchnic tissue, we conclude that 3 alpha-diol glucuronide is a marker of peripheral androgen action and markedly elevated in IH.     

Low ratio of androstenedione to testosterone in plasma and saliva of hirsute women.

To gain insight into the passage of androstenedione (A4) through the salivary gland, we measured concentrations of plasma total (TA4), free (FA4), and salivary (SA4) androstenedione during administration of dexamethasone and synthetic corticotropin to control subjects and hirsute women and compared these data with plasma total (TT), free (FT), and salivary testosterone (ST) concentrations. TA4 and FA4 were significantly lower in control subjects throughout (0, 15, 45, and 75 min) (P less than 0.05). SA4 in control subjects was significantly lower only in the follicular phase at 0 and 15 min. There was no significant difference between the increments in SA4 in response to corticotropin. The concentration of SA4 in the control women at 0 min was not significantly different from the concentration of FA4. At 45 and 75 min after corticotropin administration, however, SA4 was slightly higher than FA4 (P less than 0.01). In hirsute women, however, the concentration of SA4 was significantly lower than FA4 at all times (P less than 0.05). TT, FT, and ST concentrations were about twofold higher in the hirsute women than in control subjects throughout. In both groups, ST concentrations were three times as high as FT concentrations (P less than 0.001). The SA4:ST ratio was significantly lower than the FA4:FT ratio in both groups (P less than 0.001) because of higher ST than FT concentrations and similar or even lower SA4 concentrations in both groups. Both FA4:FT and SA4:ST ratios were lower in hirsute women, except for the FA4:FT ratio in control subjects in the luteal phase. Our data are compatible with 17-hydroxysteroid oxidoreductase activity in the salivary gland. If the difference in the ratios of A4 to T between hirsute women and control subjects is attributed to this hypothetical enzymatic activity, it would suggest a more rapid conversion of A4 to T in the hirsute group.     

Endocrine effects of combined treatment with an LHRH agonist in association with flutamide in metastatic prostatic carcinoma

The plasma levels of pituitary hormones (LH, FSH and prolactin) as well as testosterone were determined in 62 patients treated with combined therapy using the LHRH agonist [D-Trp6, des-Gly-NH2(10)]LHRH ethylamide and the antiandrogen Flutamide. Plasma radioimmunoassayable LH and FSH levels increased to 534% (p less than 0.01) and 150% (p less than 0.01) of control, respectively, during the first 5 days of treatment, while, afterwards, a marked inhibition was observed which remained constant at approximately 30-50% of control values during the whole period of treatment. All patients showed a decrease of plasma testosterone concentration to approximately 10% of control levels. Detailed determinations of plasma testicular and adrenal steroid levels were then performed in 15 patients. Our data indicate that, except for the blockade of testicular 17-hydroxyprogesterone secretion, the combined therapy has no effect on plasma C-21 steroid levels. However, adrenal C-19 steroids, namely dehydroepiandrosterone and its sulfate, androst-5-ene-3 beta, 17 beta-diol and androstenedione were decreased to approximately 50% of control values (p less than or equal to 0.01). The main testicular steroids, testosterone and dihydrotestosterone, which were increased during the first 10 days of combined administration, rapidly decreased and reached approximately 10% of control values at later time intervals. The present study extends our previous observations indicating that the combined antihormonal treatment affects both testicular and adrenal steroidogenesis. Moreover, we have demonstrated that, up to at least 2 years, this treatment, in addition to decreasing the serum levels of testicular androgens, causes an inhibition of the plasma levels of C-19 steroids from adrenal origin.     

Formation of bile acids in man: conversion of cholesterol into 5β-cholestane-3α,7α,12α-triol in liver homogenates

The mechanisms of the conversion of cholesterol into bile acids in man were studied by examining the metabolism of cholesterol-1,2-(3)H, cholest-5-ene-3beta,7alpha-diol-7beta-(3)H, tritiumlabeled 7alpha-hydroxycholest-4-en-3-one, 7alpha,12alpha-dihydroxycholest-4-en-3-one, and cholest-5-ene-3beta,7alpha,12alpha-triol in fractions of liver homogenates. The 20,000 g supernatant fluid catalyzed the conversion of cholesterol into cholest-5-ene-3beta,7alpha-diol, 7alpha-hydroxycholest-4-en-3-one, 7alpha-12alpha-dihydroxycholest-4-en-3-one, and 5beta-cholestane-3alpha,7alpha,12alpha-triol. In the presence of microsomal fraction fortified with NAD(+), cholest-5-ene-3beta,7alpha-diol was converted into 7alpha-hydroxycholest-4-en-3-one, and when this fraction was fortified with NADPH small amounts of cholest-5-ene-3beta-7alpha,12alpha-triol were formed. 7alpha-Hydroxycholest-4-en-3-one was metabolized into 7alpha-12alpha-dihydroxycholest-4-en-3-one in the presence of microsomal fraction fortified with NADPH and into 5beta-cholestane-3alpha,7alpha-diol in the presence of 100,000 g supernatant fluid. Cholest-5-ene-3beta,7alpha,12alpha-triol was converted into 7alpha,12alpha-dihydroxycholest-4-en-3-one in the presence of microsomal fraction fortified with NAD(+). The 100,000 g supernatant fluid catalyzed the conversion of 7alpha,12alpha-dihydroxycholest-4-en-3-one into 5beta-cholestane-3alpha,7alpha,12alpha-triol. The sequence of reactions in the conversion of cholesterol into 5beta-cholestane-3alpha,7alpha-diol and 5beta-cholestane-3alpha,7alpha,12alpha-triol, the subcellular localization of the enzymes, and the cofactor requirements were found to be the same as those described for rat liver.     

Dihydrotestosterone in prostatic hypertrophy: I. The formation and content of dihydrotestosterone in the hypertrophic prostate of man

To explore the relation between androgens and prostatic hypertrophy in man, the concentrations of testosterone, dihydrotestosterone, and androstenedione and the rate of conversion of testosterone to dihydrotestosterone have been measured in normal and hypertrophic prostate tissue. First, a double isotope derivative technique was adapted for the measurement of tissue androgen content in 15 normal and 10 hypertrophic prostates. Although there was no significant difference in the content of androstenedione and testosterone between the two types of tissue, the content of dihydrotestosterone was significantly greater in the hypertrophic tissue (0.60 +/-0.10 mug/100 g) than in the normal glands (0.13 +/-0.05 mug/100 g). Second, a regional study was performed in three normal prostates and four glands with early hypertrophy, and it was demonstrated that the dihydrotestosterone content was two and three fold greater in the periurethral area where prostatic hypertrophy usually commences than in the outer regions of the gland. Finally, the rate of conversion of testosterone to dihydrotestosterone has been measured under standardized conditions in tissue slices from 4 normal and 20 hypertrophic prostates. There was no significant difference in the rate of dihydrotestosterone formation between the two types of gland (6.0 +/-0.8 and 7.8 +/-0.5 mumumoles/15 mg of tissue per hr). While the mechanism by which dihydrotestosterone accumulation occurs remains unexplained, it is possible that the local accumulation of dihydrotestosterone may be involved in the pathogenesis of prostatic hypertrophy in man.     

Alteration in the metabolism of dihydrotestosterone in elderly men with prostate hyperplasia.

In vivo androgen kinetics were determined in six young (21--49 yr) and elderly men (62-77 yr) with prostatge hyperplasia (BPH). Steady-state infusions of [14C]testosterone and [3H]androstanediol (3 alpha diol) were given, which allowed determination of the conversions testosterone leads to dihydrotesterone (DHT) in equilibrium or formed from 3 alpha diol. These infusions also yield metabolic clearance data which, together with meaurement of nonisotopic steroid levels, yield estimations of blood production rates. The production rate for testosterone was 6.04 +/- 1.66 vs. 3.69 +/- 0.62 mg/d, whereas the production rate for 3 alpha diol was 319 +/- 57 and 193 +/- 34 micrograms/d (P < 0.05 both groups). The irreversible conversion rate of testosterone to DHT was 3.1 +/- 0.4 and 3.5 +/- 0.9% (NS). The back conversion of 3 alpha diol to dHT was high (68 +/- 25 vs. 81 +/- 17, NS) indicating that 3 alpha diol might cause BPH as a result of conversion to DHT in vivo. The conversion of DHT to 3 alpha diol is reduced in the elderly group (15.8 +/- 2.6 and 6.3 +/- 1.4, P < 0.001). Since DHT formation in the prostate is a key event in the development of BPH and blood DHT appears to be a measure of extrasplanchnic sexual target tissue activity, our in vivo studies suggest that the tissue increase in DHT may result from reduced metabolism and the activity of 3 alpha-oxidoreduction favors the oxidative pathway in elderly men.     

Sex hormone binding globulin: binding capacity and studies on the binding of cyproterone acetate and other steroids

Sex hormone binding globulin in blood is quantitated by ammonium sulphate precipitation technique with two radioactive ligands, [3H]testosterone and [3H5dihydrotestosterone. Plasma levels in normal men, normal women, women taking oral contraceptives containing oestrogens, and pregnant women are presented, measured with both ligands. Binding experiments with these ligands and several competing steroids show marked differences in displacement ability. For cyproterone acetate 100-500 ng per tube is required to obtain even a slight displacement of the radioactive ligands.     

Elevated serum concentrations of androgens in women with pregnancy-induced hypertension

Alterations of steroid hormone profiles have been suggested to be involved in the pathophysiology of pregnancy-induced hypertension (PIH). The aim of our study was first to investigate serum concentrations of testosterone, dihydrotestosterone, androstenedione and dehydroepiandrostenedione sulfate in women with PIH and normotensive pregnant women and secondly to evaluate an association between elevated serum concentrations of androgens and the development of severe disease. Serum concentrations of androgens were measured in 40 patients with PIH and 40 normotensive pregnant women, matched for gestational age, determined by enzyme linked immunosorbent assay. Multivariate logistic regression models were used to analyze the influence of elevated serum concentrations of androgens on the occurrence of PIH and the development of severe disease. The median serum concentrations of androstenedione and testosterone were significantly elevated in women with PIH compared to controls (6.3 and 5.0 ng/ml, 1.8 and 1.1 ng/ml, p = 0.005 and p = 0.04, respectively). The difference between the median serum concentrations of dihydrotestosterone and dehydroepiandrostenedione sulfate in women with PIH and controls was not significant. Elevated serum concentrations of androstenedione revealed a significant influence on the odds of presenting with PIH (p = 0.043) and were significantly associated with the development of severe disease (p = 0.014). Women with PIH have elevated serum concentrations of androstenedione and testosterone. Moreover, elevated serum concentrations of androstenedione are associated with development of severe disease.     

Combined radioimmunoassay of four steroids in one ml of plasma: II. Androgens.

Using partially specific antisera combined with a 1 step celite microcolumn chromatography, androstenedione (A), 5alpha-dihydrotestosterone (DHT), testosterone (T), and androst-5-ene-3beta, 17beta-diol (delta 5-diol) could be measured in the same 1 ml aliquot of plasma. The chromatographic step removed known interfering steroids and conferred specificity to the assay. After correction for recovery the sensitivities, expressed as ng/ml of plasma, were respectively: 0.025 for A, 0.05 for DHT, 0.025 for T, and 0.1 for delta5-diol. Recovery experiments, using steroid-free plasma to which various amounts of each steroid were added and then measured in the assay in 12 replicates, confirmed adequate accuracy and precision. The ability to measure multiple androgens in small volumes of plasma should permit comprehensive evaluation of the role of these steroids in health and disease.     

Concentrations of salivary testosterone and plasma total, non-sex-hormone-binding globulin-bound, and free testosterone in normal and hirsute women during administration of dexamethasone/synthetic corticotropin.

Preliminary data indicate that the concentration of circulating cortisol may affect the binding of testosterone to plasma proteins. This interdependency was evaluated by the assessment of plasma cortisol, salivary (ST) and plasma total testosterone (TT), testosterone not bound to sex-hormone-binding globulin (n-SHBGT), and free testosterone (FT) in normal and in hyperandrogenemic women during combined dexamethasone/synthetic corticotropin administration. The concentrations of TT, FT, and ST significantly increased (P less than 0.001) during this dynamic test both in the controls and in hirsute women. Remarkably, however, n-SHBGT remained virtually unchanged in normal women in the follicular phase, decreasing in the luteal phase, and decreasing even more markedly in the hirsute women. Both the normal and the hirsute women showed a statistically significant negative correlation between the percentage of n-SHBGT and plasma total cortisol (P less than 0.001). The apparent decrease of n-SHBGT was the result of displacement of T from corticosteroid-binding globulin (CBG) by the increase in cortisol after the infusion of synthetic corticotropin: the CBG-bound T was measured as n-SHBGT because CBG was not precipitated with 50% saturated ammonium sulfate. Our results indicate that ST or FT better represents the clinical status of androgenicity than does n-SHBGT, as assessed by ammonium sulfate precipitation, because n-SHBGT also depends on the concentration of cortisol.     

Androstenedione and Its Conversion to Plasma Testosterone in Congenital Adrenal Hyperplasia

The plasma concentration, production rate, and conversion ratio of androstenedione and testosterone were studied in seven children with congenital adrenal hyperplasia (CAH) of the 21-hydroxylase type. Plasma androstenedione and testosterone measured by double isotope derivative assay and estimated blood production rates were manyfold increased in the untreated state, markedly suppressed with glucocorticoid, and increased after the administration of ACTH.The metabolic clearance rate when corrected for body size and the conversion ratio of androstenedione to testosterone were similar to previously determined values in normal adults. Consideration of the androgen concentrations and conversion ratios indicates that in children with CAH, 76% of the plasma testosterone in prepubertal females and 36% in males are derived from peripheral conversion of blood androstenedione. The calculated amount of testosterone unaccounted for by peripheral conversion is similar to normal prepubertal values. This approach indicates that virilization in these children results from increased levels of testosterone but that the major source in CAH of this potent androgen is androstenedione secreted by the adrenal cortex.     

Development and validation of a LC-MS/MS method for the establishment of reference intervals and biological variation for five plasma steroid hormones

BackgroundWith liquid chromatography–tandem mass spectrometry (LC-MS/MS) increasingly being used for the quantification of steroid hormones, there is a need for studies that re-establish reference intervals and biological variation in well-defined cohorts.MethodsA plasma steroid hormone profiling method using LC-MS/MS for quantification of progesterone, 17-hydroxyprogesterone, androstenedione, testosterone and dihydrotestosterone was developed and validated. For reference interval assessment, 280 well-characterized healthy subjects from the LifeLines cohort were selected, including 40 women using oral contraceptive pills (OCP). The biological variation was examined in 30 healthy individuals. Samples were collected over a period of 4 months with 4 week intervals.ResultsThe developed method proved to be robust and sensitive. The reference interval levels in men are higher, whereas in women the levels tend to decrease with increasing age. In addition, women using OCP had lower levels of 17-OH-progesterone and androstenedione. The biological variation is generally higher in women compared to men, especially with regard to the inter-individual variation.ConclusionsThe gender-specific determination of the reference intervals, together with the observation that the biological variation demonstrated a high degree of variation, allows interpretation of data on individual and group level for improved biochemical characterization of patients in clinical practice.