Epiandrosterone Sulfate Research Articles

Abstract LB-193: Prediagnostic breast cancer metabolites in Mexican Americans: a nested case control study

Abstract Breast cancer (BrCa) is the most common invasive cancer in Hispanic women. Although at decreased risk, Hispanic women are diagnosed at younger ages and with more aggressive disease than non-Hispanic Whites. Early detection is key in BrCa survival; however, there are no pre-diagnostic circulating biomarkers for early detection. This pilot nested case-control study based on our Mexican-American Cohort (MAC) study evaluates the role of global metabolic expression in predicting BrCa risk. MAC includes ∼23,000 participants in the Houston area with baseline blood specimens and epidemiological data. Using mass spectrometry-based global protein expression, 435 plasma metabolites were analyzed by Metabolon in 30 invasive BrCa cases (14 pre- (preM) and 16 post-menopausal (postM)) diagnosed 1-5 years after enrollment and 40 healthy controls (matched to cases on age, menopausal status) and length of follow-up). Mean age of preM women was 37.6 years (range 32-43) compared to 63.4 for postM (range 50-81). Using principal component analysis, we evaluated differences by disease and menopausal status. We found 76 metabolites that differed significantly (P<0.05) between cases and controls among postM women compared to only 13 metabolites in preM women. Using a pathway-based approach, we found that the majority of case-control differences were in metabolites related to hormone, lipid and energy metabolism. The specific metabolites differed by menopausal status. In postM cases, levels of steroid hormones epiandrosterone sulfate, androsterone sulfate, and 5-alpha-androstan-3beta,17beta-diol disulfate were higher than in controls; among preM cases, steroid hormones 5-alpha-pregnan-3beta,20alpha-diol disulfate and 5-alpha-pregnan-3alpha,20beta-diol disulfate 1 levels were higher. PostM cases had significantly (P<0.05) lower levels in fatty acids valerylcarnitine and butyrylcarnitine and higher levels of monoacylglycerols and glycerol 3-phosphate (G3P) than controls; there were no significant differences in these metabolites in preM cases and controls. The statistically significant (P<0.05) decreases in 3-phosphoglycerate, pyruvate, sarcosine (N-methylglycine), citrate, and malate which are all associated with glycolysis and the tricarboxylic acid cycle found in cases compared to controls irrespective of menopausal status, suggest energy metabolism may be associated with BrCa risk. In summary, our findings suggest: (1) case-control differences in plasma levels of specific metabolites associated with key functional pathways; (2) the specific metabolites and number of relevant metabolites differs by menopausal status; and (3) these differences are detectable years prior to diagnosis. Future studies exploring these associations with tumor hormone receptor status, dietary intake and anthropometric measurements will aid in further identifying key pre-diagnostic plasma markers for BrCa. Citation Format: Sara S. Strom, Hua Zhao, Abenaa Brewster, Yuko Yamamura. Prediagnostic breast cancer metabolites in Mexican Americans: a nested case control study. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-193. doi:10.1158/1538-7445.AM2015-LB-193

An omics investigation into chronic widespread musculoskeletal pain reveals epiandrosterone sulfate as a potential biomarker.

Chronic widespread musculoskeletal pain (CWP) is common, having a population prevalence of 10%. This study aimed to define the biological basis of the CWP/body mass association by using a systems biology approach. Adult female twins (n = 2444) from the TwinsUK registry who had extensive clinical, anthropometric, and "omic" data were included. Nontargeted metabolomics screening including 324 metabolites was carried out for CWP and body composition using dual-energy X-ray absorptiometry. The biological basis of these associations was explored through a genome-wide association study and replicated in an independent population sample (Cooperative Health Research in the Region of Augsburg [KORA] study, n = 2483). A causal role for the genetic variants identified was sought in CWP using a Mendelian randomisation study design. Fat mass/height2 was the body composition variable most strongly associated with CWP (TwinsUK: P = 2.4 × 10(-15) and KORA: P = 1.59 × 10(-10)). Of 324 metabolites examined, epiandrosterone sulfate (EAS) was highly associated with both CWP (P = 1.05 × 10(-09) in TwinsUK and P = 3.70 × 10(-06) in KORA) and fat mass/height2. Genome-wide association study of EAS identified imputed single nucleotide polymorphism rs1581492 at 7q22.1 to be strikingly associated with EAS levels (P ≤ 2.49 × 10(-78)), and this result was replicated in KORA (P = 2.12 × 10(-9)). Mendelian randomization by rs1581492 genotype showed that EAS is unlikely to be causally related to CWP. Using an agnostic omics approach to focus on the association of CWP with body mass index, we have confirmed a steroid hormone association and identified a genetic variant upstream of the CYP genes, which likely controls this response. This study suggests that steroid hormone abnormalities result from pain rather than causing it, and EAS may provide a biomarker that identifies subgroups at risk of CWP.

Abstract 160: Modulation of the serum metabolomic profiles of breast cancer patients after receiving limonene intervention.

Abstract Background. Limonene is a bioactive food component found in citrus peel oil that has demonstrated chemopreventive and chemotherapeutic activities in preclinical studies. To better characterize its mechanism of action in the clinical setting, we measured the serum metabolomic profiles of breast cancer patients before and after receiving a short-term limonene intervention. Methods. Forty women with stage 0 - II breast cancer received 2 g QD limonene daily for 2 - 6 weeks (21.5 ± 8.8 days) before surgical resection of their tumor. Pre/post intervention blood draws were taken to assess serum metabolomic profiles. Metabolomic analysis was performed on the gas chromatography mass spectrometry and liquid chromatography mass spectrometry platforms established at Metabolon, Inc. Results. Of the 397 identified biochemicals, 71 changed significantly (p<0.05) from baseline to post limonene intervention. Pathway analysis revealed several key metabolic changes. Significant increases were noted in eight bile acid conjugates following limonene intervention, including a 3-fold elevation in taurocholate, taurodeoxycholate, and glycoursodeoxycholate. There were also significant decreases in a number of steroid hormones including dehydroisoandrosterone sulfate (DHEA-S), 4-androsten-3beta, 17beta-diol disulfate, epiandrosterone sulfate, androsterone sulfate, and pregnen-diol disulfate. In addition, markers of collagen remodeling or degradation were elevated following limonene intervention. Increased circulating vitamin C, improved insulin sensitivity and glycemic control, and reduced inflammatory potential were also noted following limonene treatment. An increase in plasma levels of ten different glycerophosphocholines and four carnitines were all significantly correlated to a decrease in cyclinD1 (all p's<0.01 and Rˆ2>0.4) in an exploratory analysis. Conclusions. This global metabolomic profiling study revealed several novel mechanistic insights into the potential clinical activities of limonene. Efforts are ongoing to correlate these metabolic changes to carcinogenesis, clinical characteristics, and risk markers measured in breast tissue and serum. Citation Format: Jessica A. Miller, Kirk Pappan, Chengcheng Hu, Elizabeth J. Want, Hector Keun, Julie E. Lang, H-H Sherry Chow. Modulation of the serum metabolomic profiles of breast cancer patients after receiving limonene intervention. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 160. doi:10.1158/1538-7445.AM2013-160

Determination of salivary 17-ketosteroid sulfates using liquid chromatography–electrospray ionization-mass spectrometry

A simple and sensitive liquid chromatography–electrospray ionization-mass spectrometric (LC–ESI-MS) method for the simultaneous quantification of representative 17-ketosteroid sulfates, dehydroepiandrosterone sulfate (DHEAS), androsterone sulfate and epiandrosterone sulfate, in human saliva has been developed and validated. The saliva was deproteinized with acetonitrile, purified using a solid-phase extraction cartridge and subjected to LC–MS. Deuterium-labeled DHEAS was used as the internal standard and quantification was based on the selected ion monitoring (SIM) mode of each deprotonated molecule. This method allowed the reproducible and accurate quantification of the salivary sulfates using a 100-μl sample; the intra- and inter-assay coefficients of variation were below 6.8 and 6.3%, respectively, and the % accuracy values were quantitative for all the sulfates. The limits of quantitation for all the sulfates were 100 pg/ml. No significant matrix effect or change in the measured value by freeze/thaw repetitions was observed. The developed method was applied to clinical studies, and produced satisfactory results.

Effect ofGinkgo bilobaExtract on Plasma Steroid Concentrations in Healthy Volunteers: A Pilot Study

To determine if a standardized ginkgo supplement significantly alters concentrations of circulating androgenic steroids in humans. Open-label, fixed-treatment order, crossover study. University general clinical research center. Eleven healthy volunteers (six men, five women). Volunteers received ginkgo biloba 240 mg/day for 14 days. Plasma concentrations of cortisol, 11-deoxycortisol, 17alpha-hydroxyprogesterone, testosterone, dihydrotestosterone, dehydroepiandrosterone sulfate, sex hormone-binding globulin, androstenedione, and free testosterone, as well as free androgen index and combined concentrations of androsterone sulfate and epiandrosterone sulfate, were analyzed in all subjects before and after their 14-day course of ginkgo biloba. Ginkgo biloba did not significantly alter endogenous steroid levels compared with baseline values (p < 0.05). A 14-day oral administration of a widely used, standardized ginkgo extract at a generally advocated dosage of 240 mg/day did not significantly alter concentrations of major circulating steroids in men and women.

Circulating levels of isoflavones and markers of 5α-reductase activity are higher in Japanese compared with New Zealand males: What is the role of circulating steroids in prostate disease?

Epidemiological evidence implicates dietary isoflavone intake as protective against prostate disease. A putative mechanism is attenuated circulating androgen levels in male populations consuming an isoflavone rich diet. We investigated this hypothesis by collecting plasma from 60 Japanese and 60 New Zealand males aged between 21 and 31 years each consuming their traditional diets. We measured plasma testosterone, dihydrotestosterone (DHT), androstenedione, dehydroepiandrosterone sulfate (DHEAS), the combined levels of androsterone sulfate and epiandrosterone sulfate (AoS/epiAoS), sex hormone-binding globulin, and cortisol and corticosteroid-binding globulin as well as the isoflavones genistein and equol. Plasma genistein and equol levels were several times higher in Japanese males as would be expected from an isoflavone rich diet. However, androstenedione, DHEAS, calculated free testosterone and paradoxically markers of 5α-reductase, DHT and AoS/epiAoS were all also significantly higher in Japanese rather than the New Zealand male counterparts. All other comparisons were not significant. Plasma DHT and DHEAS correlated positively with plasma equol and plasma AoS/epiAoS correlated positively with genistein levels. Taken together the results suggest that, rather than reduced levels of steroidogenesis, Japanese males may have increased 5α-reductase activity and possibly altered 17β OH steroid dehydrogenase activity. Significantly the positive association between isoflavones levels and 5α-steroids is counter-intuitive to isoflavone intake offering prostate protection, unless this is postulated to occur through other mechanisms.

Effects of St John's wort (Hypericum perforatum L.) extract on plasma androgen concentrations in healthy men and women: a pilot study

St John's wort extract (SJW; Hypericum perforatum L.) is taken extensively as a putative herbal antidepressant. It has been shown to induce the activity of cytochrome P-450 3A4 (CYP3A4) and to increase the clearance of numerous drugs and steroids such as cortisol and ethinyl estradiol. This study was conducted to determine if SJW exposure also alters the concentrations of circulating androgenic steroid hormones. The study was conducted using healthy volunteers (6M, 6F) studied before and after a 14-day treatment period with a SJW preparation previously demonstrated to induce the activity of CYP3A4. Plasma concentrations of testosterone, dihydrotestosterone (DHT), dehydroepiandrosterone sulfate (DHEAS), sex hormone-binding globulin (SHBG) and the combined concentrations of androsterone sulfate (AoS) and epiandrosterone sulfate (epiAoS) were measured by immunoassay methods. The results of analysis demonstrated that SJW did not significantly alter the majority of the androgens studied (p > 0.05) although the combined concentrations of the 5alpha-reduced steroids, AoS and epiAoS, significantly declined following treatment in all subjects (p = 0.02), and in males (p = 0.04). Furthermore, the testosterone to DHT ratio was increased in both men and women. Although the latter increase did not reach statistical significance, it is also consistent with the possible inhibition of 5alpha-reductase by SJW. It is concluded that despite significant induction of CYP3A4, short term administration of SJW does not significantly alter the concentrations of most circulating androgens in men and women but may produce a dimunition in some of the circulating 5alpha-reduced androgens.

Studies on neurosteroids XIV. Levels of dehydroepiandrosterone sulfate in rat brain and serum determined with newly developed enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) of dehydroepiandrosterone sulfate (DHEAS), one of the neurosteroids, has been developed for measuring its brain and serum levels in rats without deconjugation. 11 alpha-Hemiglutaryloxy-DHEAS was newly synthesized, conjugated with bovine serum albumin (BSA), and immunized to rabbits for the production of anti-DHEAS antibodies. A bridge-heterologous ELISA system employing the sequential saturation method exhibited a high sensitivity with a midpoint of 100 pg. Although the antibody significantly cross-reacted with epiandrosterone sulfate, it easily discriminated the unconjugated steroids and pregnenolone sulfate, which is reported to exist in the brain at a much higher level when compared with DHEAS. The brain homogenate or serum was treated with hexane to remove the lipophilic compounds and purified with an OASIS HLB cartridge. The DHEAS levels were then determined by ELISA. The overall recovery rate through the pretreatment was a satisfactory and constant (81.8 +/- 3.4% for brain, 89.3 +/- 3.0% for serum, mean +/- standard deviation). This ELISA afforded a satisfactory serial dilution study and recovery test. The intra- and inter-assay coefficients of variation were lower than 13%, which showed the precision of the proposed method. The applied method showed that DHEAS was not detected in some brain samples and its levels were much lower than those previously reported and than its serum levels.

Production and characterization of monoclonal antibodies to dehydroepiandrosterone sulfate: Application to direct enzyme-linked immunosorbent assays of dehydroepiandrosterone sulfate and androsterone/epiandrosterone sulfates in plasma

Mice were immunized with 5-androstene-3β-ol-7,17-dione-7-CMO:bovine serum albumin (DHEA-7-O-CMO-BSA) or 5-androstene-3β-ol-17-one hemisuccinate-bovine serum albumin (DHEA-3HS-BSA) conjugates and monoclonal antibodies were produced, characterized, and selected for maximum DHEAS binding. Of these hybridomas, four clones from DHEA-3HS-BSA-immunized mice had acceptable criteria for the development of a competitive enzyme-linked immunosorbent assay (ELISA) for DHEAS in plasma. One hybridoma supernatant from DHEA-7-O-CMO-BSA-immunized mice showed 360% cross-reactivity to both androsterone sulfate and epiandrosterone sulfate. This allows the possibility of the direct determination of androsterone sulfate and epiandrosterone sulfate in plasma after correction for the DHEAS contribution. Both ELISAs employ a DHEA-3HS-thyroglobulin conjugate adsorbed to the wells of a standard 96-well microtiter plate. DHEAS in the standards or diluted plasma sample competes with immobilized DHEA-3HS-thyroglobulin for antibody-binding sites. Antibody is detected with anti-mouse-Ig peroxidase by further washing, adding o-phenylenediamine substrate, and reading the absorbance at 492 nm. The ELISAs are simple, reproducible, and reliable and, to our knowledge, they are the first tests employing monoclonal antibodies to DHEAS.

On the Mechanism of Interaction of Steroids with Human Glucose 6-Phosphate Dehydrogenase

Previous studies have demonstrated that certain 17- and 20-ketosteroids are potent inhibitors of the NADP-linked oxidation of glucose 6-phosphate by mammalian glucose 6-phosphate dehydrogenases, This inhibition is uncompetitive with respect to both NADP+ and glucose 6-phosphate, In order to elucidate the detailed mechanism of this rare type of inhibition, we examined the effects of steroids on human glucose 6-phosphate dehydrogenase catalyzing the reverse reaction, i,e,, the reduction of the gluconolactone by NADPH. To circumvent problems associated with the known instability of 6-phospho-δ-gluconolactone, the natural product of glucose 6-phosphate oxidation, the more stable 6-phospho-γ-gluconolactone was used. Dehydroepiandrosterone, epiandrosterone, 16α-bromoepiandrosterone, and dehydroepiandrosterone sulfate all inhibited the reverse reaction uncompetitively with respect to both NADPH and the γ-lactone. The Ki values for each of these steroids, determined by varying either coenzyme or substrate in both the forward and the reverse reactions, are very similar, These results demonstrate that steroids inhibit glucose 6-phosphate dehydrogenase by binding to the ternary enzyme-coenzyme-substrate ternary complex(es). This is the first direct demonstration that uncompetitive inhibition of a two-substrate enzyme, by compounds other than its substrates or products, can occur by binding of the inhibitor to a ternary enzyme complex.

Specific binding of dehydroepiandrosterone sulfate to rat liver cytosol: A possible association with peroxisomal enzyme induction

Incubation of [ 3H]dehydroepiandrosterone sulfate (DHEAS) with rat liver cytosol demonstrated its specific binding with a dissociation constant of 72 ± 14 nM and a maximal binding capacity of 312 ± 105 fmol/mg cytosol protein. The binding correlated with the amount of cytosol protein, and depended on time, temperature and pH, with equilibrium being reached after 6 h at 0°C and pH 7.5. Boiling or treatment of the cytosol with proteases or sulfhydryl-blocking reagents affected the binding. The apparent molecular mass of the binding entity was estimated to be 160–230 kDa by HPLC gel filtration. In competitive binding studies, free steroids, including dehydroepiandrosterone (DHEA), sulfatase substrates and ligands of organic anion binders such as ligandin and fatty acid binding protein, had no effect on the [ 3H]DHEAS binding. Peroxisome proliferators also had no effect, except Wy-14,643. Competition with various steroids related to DHEAS revealed strict structural requirements for DHEAS binding, in which epiandrosterone sulfate was almost as effective as unlabeled DHEAS in inhibiting [ 3H]DHEAS binding. These findings indicated the presence of a binding protein highly specific to DHEAS in rat liver cytosol. The DHEAS binding in liver cytosol was 2-fold higher in male than in female rats. The cytosolic DHEAS binding was highest in the liver, followed by the kidney and heart. The possibility of association between the DHEAS binding and DHEA induction of peroxisomal β-oxidation is discussed.

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)

Dehydroepiandrosterone sulfate quantification in serum using high-performance liquid chromatography/ mass spectrometry and a deuterated internal standard: a technique suitable for routine use or as a reference method

A thermospray high-performance liquid chromatography / mass spectrometry method for determination of serum dehydroepiandrosterone sulfate is described. The steroid was measured intact using [7,7- 2H 2]dehydroepiandrosterone sulfate us internal standard. The analysis was carried out in the negative ion mode by determining the peak height ratio of the molecular anions of the analyte and internal standard. The method was used to determine the steroid in serum from 15 male and female normal adults and the following values were obtained: males, 272 ± 45 μg/dl (range, 197 to 331 μg/dl) and females, 215 ± 67 μg/dl (range, 107 to 347 μg/dl). In addition, dehydroepiandrosterone sulfate was measured by high-performance liquid chromatography / mass spectrometry and radioimmunoassay (a commercial kit) on 25 individuals of all age groups. There was strong correlation between the values obtained, but the radioimmunoassay values were generally double those obtained by high-performance liquid chromatography / mass spectrometry. Three other steroid sulfates, androsterone sulfate, epiandrosterone sulfate, and androst-5-ene-3β, 17β-diol sulfate, were also assayed. In males, these had mean values of 112, 44, and 13 μg/dl and, in females, they had mean values of 84, 25, and 6 μgl dl, respectively. Radioimmunoassay cross-reactivity measurement for these steroids (as reference compounds) showed that they were unlikely to contribute greatly to the discrepancy between radioimmunoassay and high-performance liquid chromatography / mass spectrometry values. (Steroids 55:472–478, 1990)