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Human adrenal glands, and those of some primates, secrete high concentrations of conjugated steroids, particularly 5-androsten-3 ␤ -ol-17-one-3-sulfate [dehydroepiandrosterone sulfate (DHEAS)], which reaches different peripheral tissues through blood ( 1 ). Sulfonation increases the solubility of steroids in water-based biological fl uids such as blood or urine. Sulfated steroids, also known in the literature as steroid sulfates or steroid sulfonates, represent the most abundant fraction of steroids in human blood. Because the physiological activity has been traditionally assigned to the unconjugated steroids, the classical dogma assumes that sulfated steroids are inactive forms of steroids which require sulfate cleavage to be active . In this context, human steroid sulfatase (STS) is the enzyme that desulfates the sulfated steroids to obtain free forms. The complementary reaction, sulfonation, is provided by sulfotransferase enzymes ( Fig. 1 ). This cycle allows for the regulation and excretion of steroids.The concept of steroid sulfates as inactive compounds is under revision. A specifi c transporter for sulfated steroids in the human testis was recently described ( 2 ). Moreover, Abstract Steroids are primarily present in human fl uids in their sulfated forms. Profi ling of these compounds is important from both diagnostic and physiological points of view . Here, we present a novel method for the quantifi cation of 11 intact steroid sulfates in human serum by LC-MS/MS. The compounds analyzed in our method, some of which are quantifi ed for the fi rst time in blood, include cholesterol sulfate, pregnenolone sulfate, 17-hydroxy-pregnenolone sulfate, 16-␣ -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 l 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 quantifi cation ranging between 1 and 80 ng/ml. Averaged intra-day and between-day precisions (coeffi cient 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 quantifi cation of sulfated steroids in human blood. -S á nchez-Guijo, A., V. Oji, M.

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Metabolic acidosis induces elevated glucocorticoid (GC) levels. However, the influence of less strong daily acid loads on GCs is largely unexplored. To investigate this, we studied whether higher acid loads in children, fully within the normal range of habitual diets, associate with endogenous GCs. In a specific quasi-experimental design, we examined 200 6- to 10-year-old healthy participants of the Dortmund Nutritional and Anthropometric Longitudinally Designed (DONALD) Study equally divided to either high or low 24-hour renal net acid excretion. Major urinary GC metabolites were analyzed by gas chromatography-mass spectrometry to assess daily adrenal GC secretion and metabolites of tissue cortisol catabolism (6β-hydroxycortisol and 20α-dihydrocortisol). Liquid chromatography-mass spectrometry was used to quantify urinary free cortisol and cortisone. After confounder adjustment, significant positive associations were unmasked for urinary potential renal acid load and net acid excretion with adrenal GC secretion, free cortisone, free cortisone plus cortisol, 6β-hydroxycortisol, and 20α-dihydrocortisol. An inverse association emerged for an enzymatic marker (5β-reductase) of irreversible GC inactivation. Our data suggest that existing moderate elevations in diet-dependent acid loads suffice to raise GCs and affect cortisol metabolism. Thus, potential detrimental effects of high acid loading appear to be mediated, in part, by increased GC activity via increased GC secretion and/or reduced GC inactivation. Higher cortisone levels, directly available for intracrine activation to cortisol may play a special role.

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Within the combined DFG research project "Sulfated Steroids in Reproduction" an analytical method was needed for determining sulfated and unconjugated steroids with highest specificity out of different biological matrices such as aqueous solution, cell lysate and serum. With regard to this analytical challenge, LC-MS-MS presents the technique of choice because it permits (1) analysis of the intact steroid conjugate, (2) allows for simultaneous determination of multiple analytes (profiling, targeted metabolomics approach) and (3) is independent of phenomena such as cross-reactivity. Sample work up consisted of incubation of sample with internal standards (deuterium labeled steroids) followed by solid phase extraction. Only serum samples required a protein precipitation step prior to solid phase extraction. The extract was divided in two parts: six steroid sulfates (E1S, E2S, AS, 16-OH-DHEAS, PREGS, DHEAS) were analyzed by C18aQ-ESI-MS-MS in negative ion mode and eleven unconjugated steroids (E3, 16-OH-DHEA, E1, E2, (4)A, DHEA, T, 17-OH-PREG, Prog, An, PREG) were analyzed by C18-APCI-MS-MS in positive ion mode. For steroid sulfates, we found high sensitivities with LoQ values ranging from 0.08 to 1 ng mL(-1). Unconjugated steroids showed LoQ values between 0.5 and 10 ng mL(-1). Calibration plots showed excellent linearity. Mean intra- and inter-assay CVs were 2.4% for steroid sulfates and 6.4% for unconjugated steroids. Accuracy - determined in a two-level spike experiment - showed mean relative errors of 5.9% for steroid sulfates and 6.1% for unconjugated steroids. In summary, we describe a novel LC-MS-MS procedure capable of profiling six steroid sulfates and eleven unconjugated steroids from various biological matrices.

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