Androstenediol Sulfate Research Articles

Novel GC-MS/MS Technique Reveals a Complex Steroid Fingerprint of Subclinical Hypercortisolism in Adrenal Incidentalomas.

Improvement of imaging methods has led to more incidental adrenal tumor findings, especially adenomas. Routine hormonal evaluation uses only a few steroids to evaluate possible hormonal hypersecretion of these adenomas, but a wide spectrum of serum steroid hormone changes has not been published. To measure the serum levels of 83 steroids from patients with unilateral and bilateral adrenal incidentalomas to uncover full steroid profile changes in patients with subclinical hypercortisolism (SH). Cross-sectional study. The study was conducted at a tertiary inpatient clinic. Fifty-two patients with adrenal incidentalomas (unilateral, n = 29; bilateral, n = 23), including nonfunctioning (n = 11) vs SH (n = 41), and 26 age- and sex-matched controls from the general population were included. Eighty-three serum steroids were measured by gas chromatography-tandem mass spectrometry (GC-MS/MS) before and after 1 mg dexamethasone, ACTH, midnight serum cortisol, and urinary free cortisol/24 hour. Of 83 measured steroids, 10 were significantly decreased in patients with SH, including dehydroepiandrosterone sulfate (DHEAS), androsterone sulfate, epiandrosterone sulfate, androstenediol sulfate, conjugated 5α-androstane-3β,17β-diol, and conjugated 5α-androstane-3α,17β-diol. This finding was observed even when unilateral, bilateral, male, and female subgroups were analyzed separately. When we compared routine clinical methods and GC-MS/MS‒measured steroids, the most discriminatory was DHEAS followed by midnight serum cortisol, epiandrosterone sulfate, androsterone sulfate, ACTH, and 16α-hydroxypregnenolone. SH was associated with decreased levels of adrenal androgens, their metabolites, and pregnenolone metabolite. GC-MS/MS is a powerful tool for measuring serum levels of these undescribed changes in steroid metabolism, which are characteristic of SH in adrenal incidentalomas.

Transport of steroid 3-sulfates and steroid 17-sulfates by the sodium-dependent organic anion transporter SOAT (SLC10A6)

The sodium-dependent organic anion transporter SOAT/Soat shows highly specific transport activity for sulfated steroids. SOAT substrates identified so far include dehydroepiandrosterone sulfate, 16α-hydroxydehydroepiandrosterone sulfate, estrone-3-sulfate, pregnenolone sulfate, 17β-estradiol-3-sulfate, and androstenediol sulfate. Apart from these compounds, many other sulfated steroids occur in mammals. Therefore, we aimed to expand the substrate spectrum of SOAT and analyzed the SOAT-mediated transport of eight different sulfated steroids by combining in vitro transport experiments in SOAT-transfected HEK293 cells with LC–MS/MS analytics of cell lysates. In addition, we aimed to better understand the structural requirements for SOAT substrates and so selected structural pairs varying only at specific positions: 3α/3β-sulfate, 17α/17β-sulfate, mono-sulfate/di-sulfate, and 17α-hydroxylation. We found significant and sodium-dependent SOAT-mediated transport of 17α-hydroxypregnenolone sulfate, 17β-estradiol-17-sulfate, androsterone sulfate, epiandrosterone sulfate, testosterone sulfate, epitestosterone sulfate, and 5α-dihydrotestosterone sulfate. However, 17β-estradiol-3,17-disulfate was not transported by SOAT. In conclusionSOAT substrates from the group of sulfated steroids are characterized by a planar and lipophilic steroid backbone in trans-trans-trans conformation of the rings and a negatively charged mono-sulfate group at positions 3′ or 17′ with flexibility for α- or β- orientation. Furthermore, 5α-reduction, 16α-hydroxylation, and 17α-hydroxylation are acceptable for SOAT substrate recognition, whereas addition of a second negatively charged sulfate group seems to abolish substrate binding to SOAT, and so 17β-estradiol-3,17-disulfate is not transported by SOAT.

Simultaneous quantification of cholesterol sulfate, androgen sulfates, and progestagen sulfates in human serum by LC-MS/MS

Steroids are primarily present in human fluids in their sulfated forms. Profiling of these compounds is important from both diagnostic and physiological points of view. Here, we present a novel method for the quantification of 11 intact steroid sulfates in human serum by LC-MS/MS. The compounds analyzed in our method, some of which are quantified for the first time in blood, include cholesterol sulfate, pregnenolone sulfate, 17-hydroxy-pregnenolone sulfate, 16-α-hydroxy-dehydroepiandrosterone sulfate, dehydroepiandrosterone sulfate, androstenediol sulfate, androsterone sulfate, epiandrosterone sulfate, testosterone sulfate, epitestosterone sulfate, and dihydrotestosterone sulfate. The assay was conceived to quantify sulfated steroids in a broad range of concentrations, requiring only 300 μl of serum. The method has been validated and its performance was studied at three quality controls, selected for each compound according to its physiological concentration. The assay showed good linearity (R(2) > 0.99) and recovery for all the compounds, with limits of quantification ranging between 1 and 80 ng/ml. Averaged intra-day and between-day precisions (coefficient of variation) and accuracies (relative errors) were below 10%. The method has been successfully applied to study the sulfated steroidome in diseases such as steroid sulfatase deficiency, proving its diagnostic value. This is, to our best knowledge, the most comprehensive method available for the quantification of sulfated steroids in human blood.

Predictors of body composition and body energy changes in response to chronic overfeeding

ObjectiveWe have previously shown that 24 young lean men (12 pairs of identical twins) subjected to a standardized 353 MJ (84 000 kcal) overfeeding protocol over 100 days exhibited individual differences in body weight and composition gains. The mean (+SD) gains in fat mass (FM) and fat-free mass (FFM) were 5.4+1.9 kg and 2.7+1.5 kg for a total body energy (BE) gain of 221+75 MJ representing 63% of the energy surplus consumed. We report here on the most important baseline correlates of these overfeeding-induced changes with the aim of identifying biomarkers of the response.ResultsBaseline maximal oxygen uptake per kilogram body mass was negatively correlated with gains in weight, FM, and BE (all p<0.05). Enzyme activities indicative of skeletal muscle oxidative potential correlated with gains in FM and BE (all p<0.05). Baseline TSH levels in response to a TRH stimulation correlated positively with changes in FM-to-FFM ratio (p<0.05). Plasma concentrations of androstenediol-sulfate, dehydroepiandrosterone, and 17-hydroxy pregnenolone were negatively correlated with gains in FM and BE (0.01<p<0.05), while level of estrone was negatively and androsterone-glucoronide was positively correlated with FFM gains (p<0.05). Baseline leptin and abdominal fat cell size correlated positively with gains in weight, FM, and BE (p<0.05). When compared to the six highest BE gainers, the six lowest gainers exhibited higher thermic effect of a meal (TEM) and plasma levels of total testosterone, cortisol, estradiol, androstenedione, and androstenediol-sulfate (all p<0.05). High baseline levels of total TEM, testosterone, and androstenediol-sulfate were associated with lower FM gains whereas high baseline levels of FT4 and estrone were found in low-FFM gainers.ConclusionAlthough none of the variables exerted individually an overwhelmingly strong influence on overfeeding-induced changes, baseline FFM, maximal oxygen uptake, muscle oxidative capacity, androgens, and leptin levels were the most consistent significant biomarkers of the responsiveness to chronic overfeeding.

Enzymic conversion of 3β-hydroxy-5-ene-steroids and their sulfates to 3-oxo-4-ene-steroids for increasing sensitivity in LC–APCI-MS

A method of increasing the sensitivity of 3β-hydroxy-5-ene (Δ 5)-steroids in liquid chromatography–atmospheric pressure chemical ionization-mass spectrometry (LC–APCI-MS) based on the structural conversion by cholesterol oxidase (ChO) was demonstrated. The Δ 5-steroids were rapidly converted to their 3-oxo-4-ene (Δ 4)-forms by the treatment with ChO and the obtained Δ 4-forms provided 3–14-fold higher sensitivity compared to intact steroids in the positive-APCI-MS. This enzymic conversion method was also applied to the sulfated conjugates of Δ 5-steroids after solvolysis. The method enabled the detection of trace levels of dehydroepiandrosterone and androstenediol 3-sulfate in human serum, which could not be detected by the usual LC–MS.

Serum concentrations of androstenediol and androstenediol sulfate, and their relation to cytokine production during and after normal pregnancy

Since it is known that androstenediol (ADIOL) has potent immunoregulatory effects, changes in ADIOL levels during and after pregnancy might affect the maternal immune system. We examined serum concentrations of ADIOL and androstenediol 3-sulfate (ADIOLS) together with IFN-γ and IL-4 production levels during pregnancy and after delivery up to 10–11 months postpartum. The subjects were 73 normal pregnant, 76 normal postpartum, and 28 normal non-pregnant women. ADIOL and ADIOLS were measured using EIA and GC/MS, respectively. The cytokine levels in the supernatant of whole-blood cultures stimulated with phorbol 12-myristate 13-acetate and ionomycin were measured using ELISA. ADIOL levels significantly decreased compared to non-pregnant levels in the first trimester ( P < 0.05) and were reversed in the third trimester ( P < 0.05). After pregnancy, ADIOL levels gradually declined, and a significant decrease was observed at 10–11 months postpartum ( P < 0.05). ADIOLS levels were significantly lower in the third trimester ( P < 0.05) and significantly higher at the first month postpartum ( P < 0.001) compared to non-pregnant women. IFN-γ and IL-4 levels decreased during pregnancy and subsequently increased postpartum. On the other hand, we found significant negative correlations between ADIOL concentrations and production levels of IFN-γ ( P < 0.05) or IL-4 ( P < 0.05). These findings suggest that ADIOL may be involved in modifying the maternal immune response during and after pregnancy.

Simultaneous determination of androstenediol 3-sulfate and dehydroepiandrosterone sulfate in human serum using isotope diluted liquid chromatography–electrospray ionization-mass spectrometry

A simple method for simultaneous determination of androstenediol 3-sulfate (Adiol-3S) and dehydroepiandrosterone sulfate (DHEA-S) in human serum using isotope diluted liquid chromatography–electrospray ionization-ion trap-mass spectrometry (LC–ESI-ion trap-MS) was developed. After addition of deuterated internal standards ([ 2 H 5 ]Adiol-3S and [ 2 H 4 ]DHEA-S), human serum (100 μl) was deproteinized with acetonitrile and then applied to a solid-phase extraction cartridge, Oasis HLB. The obtained steroid sulfates fraction was washed with hexane and then analyzed by LC–ESI-MS operated in the negative ion mode. The quantification ranges of Adiol-3S and DHEA-S were 10–400 ng/ml and 0.05–8 μg/ml, respectively. The method does not require the chemical or enzymatic hydrolysis of the conjugates and purification with high-performance liquid chromatography, and shows satisfactory reproducibility and accuracy. The concentrations of these sulfates in the sera of healthy male volunteers ( n=14) were 19.2–245.3 mg/ml (Adiol-3S) and 0.175–5.16 μg/ml (DHEA-S), and those of patients with prostate cancer ( n=19) were 15.3–182.7 ng/ml (Adiol-3S; four samples, not detectable) and 0.110–2.421 μg/ml (DHEA-S).

Induction of peroxisomal β-oxidation by structural analogues of dehydroepiandrosterone in cultured rat hepatocytes: Structure-activity relationships

The structural requirements of dehydroepiandrosterone (DHEA) for the induction of peroxisomal β-oxidation were studied in cultured rat hepatocytes. The hepatocytes were incubated for 5 days with various steroids, including 3- and 17-substituted analogues and 5-hydrogenated analogues of DHEA, and the activities of peroxisomal β-oxidation and carnitine acetyltransferase were measured. Among the steroids examined, DHEA, DHEA sulfate (DHEAS), dehydroandrosterone sulfate, androstenediol 3-sulfate, epiandrosterone sulfate and androsterone sulfate significantly induced the enzymes; 4.6- to 14.2-fold for β-oxidation and 5.1- to 10.9-fold for carnitine acetyltransferase at 50 μM. All of the sulfated steroids were more effective than the corresponding unsulfated forms. DHEAS was the most potent inducer. The 3-sulfuric group was required for the marked induction of peroxisomal β-oxidation, and the 17-carbonyl group was also important. Furthermore, the relatively planar conformation of the steroidal hydrophobic backbone was crucial for inducing the enzyme. The configuration of the 3-sulfuric group (β-configuration) and the presence of a double bond at position C5 were not primary determinants for the action of DHEAS. On the other hand, the introduction of bulky substituents to position C17 or aromatization of ring A led to a loss of inducing activity. Thus, there are strict structural requirements for the DHEA induction of peroxisomal β-oxidation, suggesting the presence of a certain specific recognition site in the cell for DHEAS, which mediates the peroxisome proliferator action of DHEA.

INDUCTION OF PEROXISOMAL β-OXIDATION ENZYMES BY DEHYDROEPIANDROSTERONE SULFATE

Induction of peroxisomal β-oxidation enzymes by dehydroepiandrosterone (DHEA) was studied in primary cultures of rat hepatocytes. Treatment of cultured hepatocytes with DHEA and its sulfate (DHEAS) for up to 5 days resulted in a progress increase in peroxisomal β-oxidation and camitine acetyltransferase activity, the magnitude of which was dose-related. Western blot analyses confirmed the induction of acyl-CoA oxidase, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme and carnitine acetyltransferase in the treated cells. Induction of fatty acid ω-hydroxylase proteins (P4504As) was also revealed. DHEAS was a good inducer of these enzymes, and its effects coorelated well with those of DHEA; moreover, DHEAS was more potent than DHEA. Studies with structural analogues of DHEA revealed strict structural requirements for DHEA induction of peroxisomal β-oxidation. Of the steroids examined, dehydroandrosterone sulfate, androstenediol 3-sulfate, epiandrosterone sulfate and androsterone sulfate, besides DHEA and DHEAS, significantly induced the enzyme. All of the sulfated steroids were more potent than the corresponding unsulfated forms. DHEAS was the most potent inducer. The 3-sulfuric group was required for the marked induction of peroxisomal β-oxidation, and the 17-carbonyl group was also important. The presence of a double bond at position C5 was not a primary determinant for the action of DHEAS, but the relatively planar conformation of the steroidal hydrophobic backbone was crucial for inducing the enzyme. Certain metabolites, such as 16a-hydroxyDHEA, androstenedione and DHEA glucuronide, also caused enzyme induction, however the magnitude of which was lower than that seen with DHEA and DHEAS. When hepatocytes were co-treated with acetaminophen, a potent sulfate scavenger, or chlorate, an inhibitor of PAPS formation, the DHEA induction of peroxisomal β-oxidatiown as suppressed, whereas induction by DHEAS and clofibric acid was not affected. These findings support the idea that DHEA, after administration, undergoes sulfate-conjugation in the cell to gain peroxisome proliferator activity.

Direct radioimmunoassay of androstenediol-3-sulfate in the serum of normal men

A commercially available antidihydrotestosterone antiserum was used for the direct radioimmunoassay of androstenediol-3-sulfate (ADS) in human serum. Aliquots of 1 or 2 μl male serum (mean age of 40 subjects, 38.2 ± 5.0 years) were diluted and extracted with ethanol for assay. The tracer, [7- 3H]ADS, was prepared by sodium borohydride reduction of [7- 3H]dehydroepiandrosterone sulfate (DS). Significantly cross-reacting steroids were testosterone, DS, androsterone sulfate, and epiandrosterone sulfate, which combined to produce a mean overestimation of ADS of 4.3 μg/dl in male serum. Mean serum ADS was 23.6 ± 10.0 μg/dl (SD) in 20 fresh-frozen sera versus 28.4 ± 9.7 μg/dl (SD) in 20 long-term (24.4 ± 1.2 years) frozen specimens, showing stability on long-term frozen storage. Androstenediol-3-sulfate also showed a strong correlation with serum DS (r = 0.75). The possible physiologic significance of ADS is discussed, particularly in terms of the known estrogenicity ofunconjugated androstenediol. (Steroids 56:320–324, 1991)

Effect of burn trauma on adrenal and testicular steroid hormone production.

The effects of burn trauma in men on the production of adrenal and testicular steroids was investigated. Whereas there were significant increases in serum cortisol levels and urinary 17-hydroxycorticosteroid excretion soon after thermal injury, there were significant decreases in serum dehydroepiandrosterone sulfate, dehydroepiandrosterone, androstenedione, and testosterone concentrations during the first 4 weeks following burn trauma. Serum androstenediol and androstenediol sulfate levels also were reduced, though insignificantly, 10-23 days postburn. Serum LH levels were unchanged during the postburn interval. Since urinary 17-ketosteroid excretion was normal or below normal rather than increased, the decline in serum C19-steroid levels probably resulted from decreased glandular secretion rather than increased rates of metabolism and excretion. Low dehydroepiandrosterone sulfate and/or testosterone levels were found in some men several months after recovery from their burns. These data suggest that thermal injury leads to acute inhibition of adrenal and testicular C19-steroid secretion, but stimulation of adrenal glucocorticosteroid production, and that endocrine function in many instances is not normalized after complete healing of the burned surfaces. The mechanisms and physiological consequences of such changes in the steroid milieu of men after burn trauma are unknown.

699 INCREASED CHOLESTEROL SULFATE IN PLASMA AND RED BLOOD CELL MEMBRANES OF STEROID SULFATASE (STS) DEFICIENT PATIENTS

STS deficiency is an inborn error of metabolism due to a relatively common mutation on the short arm of the X chromosome. The phenotype of affected individuals includes decreased estriol production during fetal life and ichthyosis postnatally. Although increased levels of several steroid sulfates have been observed in amniotic fluid, maternal urine, and cord blood from such pregnancies, no consistent substrate abnormalities have been found beyond the perinatal period. Specifically, normal radioimmunoassayable plasma pregnenolone sulfate (PS), dehydroepiandrosterone sulfate (DS), and androstenediol sulfate (AS) have been reported. Utilizing gas chromatography (GC), we have found cholesterol sulfate (CS) to be strikingly elevated in plasma and red blood cell membranes of patients with STS deficiency. 80% methanol extracts were purified by solvent partition and TLC, subjected to solvolysis and quantitated by GC. Recoveries were monitored with 3H-CS. High levels of CS with normal levels of PS, DHEAS, and AS may indicate that desulfation is quantitatively more important for CS than for the other steroid sulfates. CS is known to affect membrane stability and the present observations may help to explain the pathogenesis of STS deficiency and X-linked ichthyosis.

Estrone sulphate in rat brain: uptake from blood and metabolism in vivo.

It Has been well established that the estrogens, estrone and estradiol, enter the brain from the circulatory system. These compounds are concentrated and retained in the hypothalamus and preoptic regions, particularly in the nuclei of the cells in these regions (McEwen, ZIGMOKD, AZMITIA and Weiss, 1970; Zigmond and Mcewen, 1970). In a previous communication from this laboratory we indicated that a small portion of blood bound dehydroepiandrosterone sulfate enters the brain intact and then is converted to free dehydroepiandrosterone and to androstenediol sulfate (Kishimoto and Hoshi, 1972). Estrone sulfate is the most abundant estrogen in plasma at a concentration of 0.349 pg/ml of plasma, a level which is as much as 10‐fold higher than that of free estrone and estradiol in both male and female plasma (Loriaux, Ruder and Lipsett, 1971). Therefore, we deemed it important to establish whether estrone sulfate also enters the brain and if it acts as the source of other brain estrogens. In this communication, we wish to report the results obtained by intracardiac and intracerebral injection of [3H] estrone [35S] sulfate into the developing rat.

Perfusion of the Boar Testis with Androstenediol Sulfate in vivo and Studies on the in vitro Metabolism of Androgens by Porcine Blood Cells

An attempt was made to see if androstenediol sulfate, a normal secretory product of the boar testis, is important in testicular steroidogenesis in this species when perfused in vivo through the testis. Approximately 5 μCi of 7α-3H-androstenediol sulfate were administered via the spermatic artery to four, mature, anesthetized boars. Almost all of the radioactivity extracted from the spermatic vein blood and from the testis was associated with the unaltered substrate. Less than 0.2% of the substrate was isolated as 3H-dehydroepiandrosterone sulfate from the testicular extracts of one boar. An unsuccessful effort was made to characterize a similar amount of a metabolite present in extracts from spermatic vein blood of the same animal. The total amount of radioactivity which was associated with the free steroids represented about 0.2% of the administered dose of 3H-androstenediol sulfate given to each boar.17β-Hydroxysteroid dehydrogenase activity was demonstrated in a series of incubations of steroids with blood cells of the boar. Unconjugated Δ5-steroids were converted to a greater extent than were the Δ4-steroids (3.33 and 3.91% for DHA and androstenediol, respectively; 1.23 and 0.92% for androstenedione and testosterone, respectively). Androstenediol sulfate was converted to dehydroepiandrosterone sulfate (0.42%) but the reverse reaction was not observed. These findings were considered in relation to the in vivo studies. It was concluded that androstenediol sulfate in arterial blood entering the boar testis was not metabolized appreciably.

The isolation and identification of androstenediol sulfate from spermatic vein blood and testes of the boar.

A method is described for the extraction and purification of steroid sulfates from testicular tissues and blood of the boar. Androstenediol sulfate (3β, 17β-dihydroxy-5-androstene 3-sulfate) was isolated from extracts of both spermatic vein blood and boar testis. Final identification was achieved by infrared spectroscopy, as the ammonium salt of androstenediol 3-sulfate 17-acetate. Additional evidence of identity was obtained with the 3-monosulfate of androstenediol-7α-3H as an internal standard in several chromatographic systems for steroid sulfates, and for free steroids after hydrolysis. Androstenediol sulfate was not detected in peripheral or spermatic arterial blood, and it was concluded that androstenediol sulfate is a major secretory product of the boar testis.

Comparative roles of dehydroepiandrosterone sulfate and androstenediol sulfate as precursors of testicular androgens.

The relative conversion of dehydroepiandrosterone sulfate and 3H-androstenediol- 3-sulfate testosterone to and androstenedione by microsomal preparations of rat testes was studied. In 2 hr, in the presence of DPN, TPN and TPNH, 44% of androstenediol–3-sulfate was converted to testosterone and 29% to androstenedione compared to 15% and 43%,respectively, of dehydroepiandrosterone sulfate. In addition, small amounts of the following 5α reduced steroids were isolated as products of both substrates: epiandrosterone and dihydrotestosterone; from androstenediol–3-sulfate only: androsterone, 5α-androstane-3β,17β-diol and αandrostane- α,17β-diol. The much greater conversion of androstenediol–3-sulfate to testosterone suggests this to be the preferable precursor. The identification of 5α-androstane-3β,17β-diol as a distant product of androstenediol–3-sulfate metabolism is consistent with the previous suggestion that this potent inhibitor of testicular steroid sulfatase may act by feedback inhibition to control the ...

Reduction of dehydroepiandrosterone sulfate in the liver during ethanol metabolism

Superfusion of guinea pig liver slices with a solution containing dehydro-epiandrosterone sulfate resulted in the formation of 5-androstene-3β,17β-diol sulfate. When ethanol, 2.2–43.5 mM, was added to the superfusion fluid, there was a consistent increase (between 1.3 and 4.5 times) in androstenediol sulfate production. The same magnitude of increase occurred during superfusion with 1,1-dideuteroethanol. In addition, there was a prompt incorporation of deuterium into the androstenediol sulfate to a maximum of a 42 atom percent excess. In the presence of 4-bromopyrazole, an inhibitor of alcohol dehydrogenase (alcohol:NAD oxidoreductase, EC 1.1.1.1), dideuteroethanol did not bring about an increase in deuterated or nondeuterated androstenediol sulfate production. It is suggested that the increased concentration of NADH during ethanol metabolism caused an increased reduction of the 17-ketosteroid to the 17β-hydroxysteroid. The results offer an explanation for the increase of the plasma concentration of 17β-hydroxysteroid sulfates observed after administration of alcohol to humans.