Steroid Sulfates Research Articles

Evaluation of sulfate metabolites as markers of intramuscular testosterone administration in Caucasian and Asian populations

The introduction of alternative markers to the steroid profile can be an effective approach to improving the screening capabilities for the detection of testosterone (T) misuse. In this work, endogenous steroid sulfates were evaluated as potential markers to detect intramuscular (IM) T administration. Fourteen sulfate metabolites were quantified using mixed-mode solid-phase extraction and analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Urine samples after a single IM injection (100mg) of T cypionate to six Caucasian and six Asian healthy male volunteers were analyzed. Principal component analysis (PCA) was used to characterize the sample cohort and to obtain the most useful markers for discrimination between pre- and post-administration samples. For Caucasian volunteers, a separation between pre- and post-administration samples was observed in PCA, whereas for Asian volunteers no separation was obtained. Seventeen ratios between sulfate metabolites were selected and further considered. Detection times (DTs) of each marker were evaluated using individual thresholds for each volunteer. The best results were obtained using ratios involving T and epitestosterone (E) sulfates in the denominator. The best marker was the ratio androsterone sulfate/testosterone sulfate (A-S/T-S) which prolonged the DT 1.2-2.1 times in respect to those obtained using T/E ratio in all Caucasian volunteers and 1.3-1.5 times in two Asian volunteers. Other ratios between A-S or etiocholanolone sulfate and E-S, and sulfates of etiocholanolone, dehydroandrosterone or epiandrosterone, and T-S were also found adequate. These ratios improve the DT after IM T administration and their incorporation to complement the current steroid profile is recommended.

Antisulfatase, Osteogenic, and Anticancer Activities of Steroid Sulfatase Inhibitor EO-33 in Mice.

Steroid sulfatase (STS) is a key enzyme involved in the biosynthesis of estrogens from inactive sulfated steroids. After we reported EO-33 as a potent in vitro STS inhibitor without undesirable estrogenic activity and with osteogenic properties, we are now interested in validating EO-33's in vivo potential to inhibit STS, to prevent bone deterioration, and to reduce estrogen-dependent tumor growth. A scale-up synthesis was first elaborated to prepare the multigram quantity of EO-33 needed to perform in vivo studies. EO-33 blocked the uterine weight stimulated by estrone sulfate in ovariectomized mice by 69% and the STS activity in the liver by 81%. It also produced a selective estrogen receptor modulator effect as assessed by measuring the tibia weight and calcium content. Using a human breast cancer (MCF-7 xenograft) model in nude mice, EO-33 blocked 90% of tumor growth induced by estradiol sulfate, and no toxic effect was observed by assessing the body and liver weights.

Melting Down Protein Stability: PAPS Synthase 2 in Patients and in a Cellular Environment.

Within the crowded and complex environment of the cell, a protein experiences stabilizing excluded-volume effects and destabilizing quinary interactions with other proteins. Which of these prevail, needs to be determined on a case-by-case basis. PAPS synthases are dimeric and bifunctional enzymes, providing activated sulfate in the form of 3′-phosphoadenosine-5′-phosphosulfate (PAPS) for sulfation reactions. The human PAPS synthases PAPSS1 and PAPSS2 differ significantly in their protein stability as PAPSS2 is a naturally fragile protein. PAPS synthases bind a series of nucleotide ligands and some of them markedly stabilize these proteins. PAPS synthases are of biomedical relevance as destabilizing point mutations give rise to several pathologies. Genetic defects in PAPSS2 have been linked to bone and cartilage malformations as well as a steroid sulfation defect. All this makes PAPS synthases ideal to study protein unfolding, ligand binding, and the stabilizing and destabilizing factors in their cellular environment. This review provides an overview on current concepts of protein folding and stability and links this with our current understanding of the different disease mechanisms of PAPSS2-related pathologies with perspectives for future research and application.

SUN-360 Multi-Steroid Panels to Replace Dynamic Testing for the Diagnosis of Nonclassic 21-Hydroxylase Deficiency

Background: The diagnosis of nonclassic 21-hydroxylase deficiency (N21OHD) is considered in children with precocious pubarche, in the differential diagnosis of polycystic ovary syndrome, and in first-degree relatives of patients with 21OHD. Currently, the diagnosis of N21OHD requires cosyntropin (ACTH)-stimulated serum 17α-hydroxyprogesterone (17OHP4) measurements; in addition, 17OHP4 also derives from the ovaries, and basal 17OHP4 is often inconclusive. Objectives: To determine if a panel of steroid biomarkers can diagnose N21OHD in a single blood draw, circumventing the need for dynamic testing. Methods: Patients undergoing evaluation for N21OHD with ACTH stimulation at two tertiary referral centers were included in this study. The diagnosis of N21OHD was based on stimulated serum 17OHP4 concentrations of >1,000 ng/dl (30 nmol/L). Baseline serum samples were used for the quantitation of 22 steroids (18 unconjugated and 4 sulfated steroids) by liquid chromatography-tandem mass spectrometry. Mann Whitney U test was used for two-group steroids comparison. Logistic regression modeling with lasso penalty, combined with clinical knowledge (relevant due to high correlation between biomarkers) were implemented for the selection of a small set of steroids that best discriminate patients with N21OHD from controls. Results: A total of 72 patients (63 females, 9 males) who underwent ACTH stimulation testing, median age 28 (range 6-70 years) were included in this study. Of these, 24 patients (4 males) were diagnosed with N21OHD; the other 48 patients (5 males) in whom N21OHD was excluded served as controls. Age and sex distribution were similar between the two groups (p = 0.9 and 0.5, respectively). Steroids displaying the largest differences between the two groups included: 11-ketotestosterone, 11-ketoandrostenedione (11KA4), 21-deoxycortisol (21dF), 17OHP4 and 16α-hydroxyprogesterone (all higher in N21OHD), plus 11-deoxycorticosterone (DOC) and corticosterone (lower in N21OHD), p <0.0001 for all. Notably, testosterone, androstenedione and all four sulfated steroids were similar between N21OHD patients and controls, including when data analysis was restricted to females. Logistic regression modeling showed that a three-steroid panel incorporating 21dF, 11KA4 and DOC was highly accurate in diagnosing N21OHD in baseline serum samples (AUC, 0.98) and superior to basal 17OHP4. Other combinations of adrenal-derived steroids also provided better discrimination than basal 17OHP4 (AUC >0.97 vs. 0.93). Conclusion: A limited number of adrenal-specific steroids measured in a single baseline blood draw can diagnose N21OHD with high accuracy. Such steroid panels might circumvent the need of dynamic testing in most patients to diagnose or exclude N21OHD.

SAT-010 Adrenal Androgen Synthesis in Aging Men

Background: The testes are the major source of androgens in men. The adrenal glands are important sources of sex-steroids and precursors during fetal life and adrenarche. The major adrenal C19 steroids, dehydroepiandrosterone and its sulfate (DHEA and DHEAS), peak in the 20s and then decline progressively with aging in both men and women. Testosterone (T) and androstenedione (A4), which are produced by both the adrenal glands and the gonads, also decline with aging. In contrast, we have recently shown that the adrenal-derived 11-oxygenated C19 (11oxyC19) steroids do not decline in aging women. While ovarian estrogen synthesis ceases abruptly by the 6th decade of life in most women, T production from the male testis declines gradually with aging. The adrenal contribution to the pool of circulating androgens and the trajectory of 11oxyC19 steroid production in aging men is not well understood. Objective: To assess the production of adrenal androgens in adult men, across a wide range of ages. Patients and methods: Morning serum was obtained from 107 men, age 20-92 years, during routine outpatient evaluations. Patients with severe comorbidities and those taking hormonal therapy were excluded. We quantified 17 steroids by liquid chromatography-tandem mass spectrometry, including A4, T, 4 steroid sulfates, and the 11oxyC19 steroids; 11β-hydroxyandrostenedione, 11β-hydroxytestosterone, 11-ketoandrostenedione and 11-ketotestosterone. The nonparametric Mann-Whitney U test was used for comparison of steroids between young and old men. Pearson’s correlation test was used to assess the relationship between steroids and age. Results: DHEAS, androstenediol-3-sulfate (AdiolS), pregnenolone sulfate (PregS) and 17-hydroxypregnenlone sulfate (17OHPregS), A4, and T were significantly lower in men older (N=55) vs. younger (N=52) than 60 years (p=0.04 for T, and p<0.001 for all others). Conversely, all four 11oxyC19 steroids were similar between men younger and older than 60 years. DHEAS, AdiolS, PregS and 17OHPregS showed an inverse correlation with age (r=-0.7, -0.6, -0.4 and -0.5, respectively, p<0.0001 for all), and A4 and T also declined modestly with age (r=-0.4 and -0.2, respectively, p<0.01). In contrast, none of the 11oxyC19 steroids were associated with age (p>0.05). Conclusion: Unlike the traditional adrenal androgen precursor DHEAS, the 11oxyC19 steroids do not decline with aging in men. Consequently, the relative contribution of the adrenal gland to the pool of circulating androgens increases in aging men as gonadal function declines. The potential for adrenal-derived 11oxyC19 androgens to mitigate the physiologic consequences of the age-related T decline in men deserves study.

Tissue steroid levels in response to reduced testicular estrogen synthesis in the male pig, Sus scrofa.

Production of steroid hormones is complex and dependent upon steroidogenic enzymes, cofactors, receptors, and transporters expressed within a tissue. Collectively, these factors create an environment for tissue-specific steroid hormone profiles and potentially tissue-specific responses to drug administration. Our objective was to assess steroid production, including sulfated steroid metabolites in the boar testis, prostate, and liver following inhibition of aromatase, the enzyme that converts androgen precursors to estrogens. Boars were treated with the aromatase inhibitor, letrozole from 11 to 16 weeks of age and littermate boars received the canola oil vehicle. Steroid profiles were evaluated in testes, prostate, and livers of 16, 20, and 40 week old boars using liquid chromatography/mass spectrometry. Testis, prostate, and liver had unique steroid profiles in vehicle-treated animals. Only C18 steroid hormones were altered by treatment with the aromatase inhibitor, letrozole; no significant differences were detected in any of the C19 or C21 steroids evaluated. Testis was the only tissue with significantly decreased free estrogens following treatment with the aromatase inhibitor; estrone and estradiol concentrations were lower (p < 0.05) in testes from 16, 20, and 40 week letrozole-treated boars. However, concentrations of the sulfated conjugates, estrone-sulfate and estradiol-sulfate, were significantly decreased (p<0.05) in 16 and 20 week boar testes, prostates, and livers from letrozole-treated boars. Hence, the distribution of estrogens between the free and conjugated forms was altered in a tissue-specific manner following inhibition of aromatase. The results suggest sulfated testicular estrogens are important estrogen precursors for the prostate, potentially enabling peripheral target tissues to synthesize free estrogens in the male pig.

Measurement of serum steroid profiles by HPLC-tandem mass spectrometry

Accurate investigations of adrenal hormone levels play a vital role in pediatric endocrinology for the detection of steroid-related disorders. In this study, we developed and validated a simultaneous assay for eight serum steroids, i.e., DHEA, androstenedione, testosterone, progesterone, 17‑hydroxyprogesterone, DHEA‑sulfate, pregnenolone‑sulfate and cholesterol-sulfate, using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Serum samples were prepared by liquid-liquid extraction with methyl t‑butyl ether. Quantitation by LC-MS/MS was performed in multiple reaction monitoring mode with an electrospray ionization source. The run time was 10 min. Analytical performance was evaluated, including imprecision, linearity, ion suppression, carry over and detection capabilities. Serum specimens from 59 children and 120 adults were analyzed to compare the distribution of steroid levels between the groups. All hormones were effectively extracted and separated using our method. The method was essentially free from potential interference and ion suppression. Within-run and between-run imprecision values were <20%. The lower limits of quantification varied from 0.025 to 10 ng/mL. The results were generally good and correlated with those obtained using immunoassay techniques. We developed the HPLC-MS/MS method for the simultaneous measurement of free and sulfated steroid hormones. The performance of the developed method was generally acceptable. Thus, this method may provide a novel approach to steroid profiling in children of age before adrenarche.

Sulfate-based lipids: Analysis of healthy human fluids and cell extracts.

Sulfate-based lipids (SL) have been proposed as players in inflammation, immunity and infection. In spite of the many biochemical processes linked to SL, analysis on this class of lipids has only focused on specific SL sub-classes in individual fluids or cells leaving a range of additional SL in other biological samples unaccounted for. This study describes the mass spectrometry screening of SL in lipid extracts of human fluids (saliva, plasma, urine, seminal fluid) and primary human cells (RBC, neutrophils, fibroblasts and skin epidermal) using targeted precursor ion scanning (PIS) approach. The PIS 97 mass spectra reveal a wide diversity of SL including steroid sulfates, sulfoglycolipids and other unidentified SL, as well as metabolites such as taurines, sulfated polyphenols and hypurate conjugates. Semi-quantification of SL revealed that plasma exhibited the highest content of SL whereas seminal fluid and epithelial cells contained the highest sulphur to phosphorous (S/P) ratio. The complexity of biofluids and cells sulfateome presented in this study highlight the importance of expanding the panel of synthetic sulfate-based lipid standards. Also, the heterogenous distribution of SL provides evidence for the interplay of sulfotransferases/sulfatases, opening new avenues for biomarker discovery in oral health, cardiovascular, fertility and dermatology research areas.

Synthesis of steroid bisglucuronide and sulfate glucuronide reference materials: Unearthing neglected treasures of steroid metabolism

Doubly or bisconjugated steroid metabolites have long been known as minor components of the steroid profile that have traditionally been studied by laborious and indirect fractionation, hydrolysis and gas chromatography-mass spectrometry (GC–MS) analysis. Recently, the synthesis and characterisation of steroid bis(sulfate) (aka disulfate or bis-sulfate) reference materials enabled the liquid chromatography-tandem mass spectrometry (LC–MS/MS) study of this metabolite class and the development of a constant ion loss (CIL) scan method for the direct and untargeted detection of steroid bis(sulfate) metabolites. Methods for the direct LC–MS/MS detection of other bisconjugated steroids, such as steroid bisglucuronide and mixed steroid sulfate glucuronide metabolites, have great potential to reveal a more complete picture of the steroid profile. However, access to steroid bisglucuronide or sulfate glucuronide reference materials necessary for LC–MS/MS method development, metabolite identification or quantification is severely limited. In this work, ten steroid bisglucuronide and ten steroid sulfate glucuronide reference materials were synthesised through an ordered combination of chemical sulfation and/or enzymatic glucuronylation reactions. All compounds were purified and characterised using NMR and MS methods. Chemistry for the preparation of stable isotope labelled steroid {13C6}-glucuronide internal standards has also been developed and applied to the preparation of two selectively mono-labelled steroid bisglucuronide reference materials used to characterise more completely MS fragmentation pathways. The electrospray ionisation and fragmentation of the bisconjugated steroid reference materials has been studied. Preliminary targeted ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis of the reference materials prepared revealed the presence of three steroid sulfate glucuronides as endogenous human urinary metabolites.

Steroid sulfatase promotes invasion through epithelial-mesenchymal transition and predicts the progression of bladder cancer.

Androgen signal has been recently suggested to be associated with the progression of bladder cancer. Steroid sulfatase (STS) is a steroid sulfate activation enzyme, considered to be one of the key enzymes in the androgen signaling pathway. However, the role of STS in bladder cancer has not been elucidated. The purpose of the present study was to determine the clinical and functional significance of STS in bladder cancer. Immunohistochemical analysis of surgical specimens obtained by radical cystectomy (n=114) demonstrated that overexpression of STS was associated with the invasion of bladder cancer, as evidenced by the incidence of STS-positive cancers (11.5 and 37.1% in non-muscle invasive and muscle invasive bladder cancers, respectively; P=0.003). STS-positive cancer demonstrated shorter recurrence-free survival and cancer-specific survival (P=0.0027 and 0.0030, respectively). Furthermore, knockdown of STS significantly reduced cell migration and invasion capacities of bladder cancer cells (P<0.001 and P=0.005, respectively), accompanied by the upregulation of E-cadherin and downregulation of vimentin. In summary, the present study demonstrated that STS promotes the invasion capability of bladder cancer via regulation of the epithelial-mesenchymal transition, and may be a useful marker for predicting the progression of bladder cancers.

Higher steroid sulfation is linked to successful weight loss in obese children.

ObjectiveLittle information is available on the steroid sulfates profile in obese children. Therefore, we examined whether sulfated steroids are linked with weight status and associated comorbidities in obese children.MethodsWe analyzed 66 obese children (mean age 10.5 ± 2.5 years, 57.6% female, 53.9% prepubertal, mean BMI 27.0 ± 4.6 kg/m2, 50% with BMI-SDS reduction >0.5, 50% without BMI-SDS reduction) who participated in an outpatient 1-year intervention program based on exercise, behavior and nutrition therapy. We measured intact sulfated steroids (cholesterol sulfate (CS), pregnenolone sulfate (PregS), 17αOH pregnenolone sulfate (17OH-PregS), 16αOH dehydroepiandrosterone sulfate (16OH-DHEAS), DHEAS, androstenediol-3-sulfate, androsterone sulfate and epiandrosterone sulfate) by LC–MS/MS, and insulin resistance index HOMA, lipids, blood pressure at baseline and 1 year later.ResultsAll sulfated steroids except 17OH-PregS, 16OH-DHEAS, androsterone sulfate and epiandrosterone sulfate were higher in boys compared to girls. Concentrations of CS before intervention were higher in children who lost weight. After 1 year of treatment, both groups showed increased levels of DHEAS, 16OH-DHEAS and androstenediol-3-sulfate, but PregS was only increased in children with weight loss. None of the steroid sulfates was significantly related to cardiovascular risk factors or HOMA except 17OH-PregS, which was associated with systolic blood pressure both in cross-sectional (β-coefficient: 0.09 ± 0.07, P = 0.020) and longitudinal analyses (β-coefficient: 0.06 ± 0.04, P = 0.013) in multiple linear regression analyses.ConclusionsSince higher steroid sulfation capacity was associated with successful weight intervention in children disruption of sulfation may be associated with difficulties to lose weight. Future studies are necessary to prove this hypothesis.

Identification of zebrafish steroid sulfatase and comparative analysis of the enzymatic properties with human steroid sulfatase

Steroid sulfatase (STS) plays an important role in the regulation of steroid hormones. Metabolism of steroid hormones in zebrafish has been investigated, but the action of steroid sulfatase remains unknown. In this study, a zebrafish sts was cloned, expressed, purified, and characterized in comparison with the orthologous human enzyme. Enzymatic assays demonstrated that similar to human STS, zebrafish Sts was most active in catalyzing the hydrolysis of estrone-sulfate and estradiol-sulfate, among five steroid sulfates tested as substrates. Kinetic analyses revealed that the Km values of zebrafish Sts and human STS differed with respective substrates, but the catalytic efficiency as reflected by the Vmax/Km appeared comparable, except for DHEA-sulfate with which zebrafish Sts appeared less efficient. While zebrafish Sts was catalytically active at 28 °C, the enzyme appeared more active at 37 °C and with similar Km values to those determined at 28 °C. Assays performed in the presence of different divalent cations showed that the activities of both zebrafish and human STSs were stimulated by Ca2+, Mg2+, and Mn2+, and inhibited by Zn+2 and Fe2+. EMATE and STX64, two known mammalian steroid sulafatase inhibitors, were shown to be capable of inhibiting the activity of zebrafish Sts. Collectively, the results obtained indicated that zebrafish Sts exhibited enzymatic characteristics comparable to the human STS, suggesting that the physiological function of STS may be conserved between zebrafish and humans.

Enhancing the Steroid Sulfatase Activity of the Arylsulfatase from Pseudomonas aeruginosa

Steroidal sulfate esters play a central role in many physiological processes. They serve as the reservoir for endogenous sex hormones and form a significant fraction of the steroid metabolite pool. The analysis of steroid sulfates is thus essential in fields such as medical science and sports drug testing. Although the direct detection of steroid sulfates can be readily achieved using liquid chromatography–mass spectrometry, many analytical approaches, including gas chromatography–mass spectrometry, are hampered due to the lack of suitable enzymatic or chemical methods for sulfate ester hydrolysis prior to analysis. Enhanced methods of steroid sulfate hydrolysis would expand analytical possibilities for the study of these widely occurring metabolites. The arylsulfatase from Pseudomonas aeruginosa (PaS) is a purified enzyme capable of hydrolyzing steroid sulfates. However, this enzyme requires improvement to hydrolytic activity and substrate scope in order to be useful in analytical applications. These imp...

Sex-Dimorphic and Sex Hormone-Dependent Role of Steroid Sulfatase in Adipose Inflammation and Energy Homeostasis.

Steroid sulfatase (STS), a desulfating enzyme that converts steroid sulfates to hormonally active steroids, plays an important role in the homeostasis of sex hormones. STS is expressed in the adipose tissue of both male and female mice, but the role of STS in the development and function of adipose tissue remains largely unknown. In this report, we show that the adipose expression of Sts was induced in the high-fat diet (HFD) and ob/ob models of obesity and type 2 diabetes. Transgenic overexpression of the human STS in the adipose tissue of male mice exacerbated the HFD-induced metabolic phenotypes, including increased body weight gain and fat mass, and worsened insulin sensitivity, glucose tolerance, and energy expenditure, which were accounted for by adipocyte hypertrophy, increased adipose inflammation, and dysregulation of adipogenesis. The metabolic harm of the STS transgene appeared to have resulted from increased androgen activity in the adipose tissue, and castration abolished most of the phenotypes. Interestingly, the transgenic effects were sex specific, because the HFD-fed female STS transgenic mice exhibited improved metabolic functions, which were associated with attenuated adipose inflammation. The metabolic benefit of the STS transgene in female mice was accounted for by increased estrogenic activity in the adipose tissue, whereas such benefit was abolished upon ovariectomy. Our results revealed an essential role of the adipose STS in energy homeostasis in sex- and sex hormone-dependent manner. The adipose STS may represent a therapeutic target for the management of obesity and type 2 diabetes.

Characterizing the steroidal milieu in amniotic fluid of mid-gestation: A LC–MS/MS study

Growth and development of an embryo or fetus during human pregnancy mainly depend on intact hormone biosynthesis and metabolism in maternal amniotic fluid (AF). We investigated the hormonal milieu in AF and developed a liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for the determination of 14 sulfated and 6 unconjugated steroids in AF. 65 A F samples (male: female = 35: 30) of mid-gestation ranging from 16th week of gestation to 25th week of gestation were analyzed. Reference data of 20 steroid levels in AF of healthy women were provided. 13 sulfated and 3 unconjugated steroids were for the first time quantified in AF by LC–MS/MS. Highest concentrations were found for pregnenolone sulfate (PregS: mean ± SD, 8.6 ± 3.7 ng/mL), 17α-hydroxypregnenolone sulfate (17OHPregS: 4.9 ± 2.0 ng/mL), epitestosterone sulfate (eTS: 7.3 ± 3.6 ng/mL), 16α-hydroxydehydroepiandrosterone sulfate (16OH-DHEAS: 21.5 ± 10.7 ng/mL), androsterone sulfate (AnS: 9.2 ± 7.4 ng/mL), estrone sulfate (E1S: 3.0 ± 3.0 ng/mL), estriol 3-sulfate (E3S: 8.1 ± 4.0 ng/mL) and estriol (E3: 1.2 ± 0.4 ng/mL). Only testosterone (T) showed a significant sex difference (p < 0.0001). Correlations between AF steroids mirrored the steroid metabolism of the feto-placental unit, and not only confirmed the classical steroid pathway, but also pointed to a sulfated steroid pathway.

Methods in endogenous steroid profiling – A comparison of gas chromatography mass spectrometry (GC–MS) with supercritical fluid chromatography tandem mass spectrometry (SFC-MS/MS)

In various fields of endocrinology, the determination of steroid hormones synthesised by the human body plays an important role. Research on central neurosteroids has been intensified within the last years, as they are discussed as biomarkers for various cognitive disorders. Their concentrations in cerebrospinal fluid (CSF) are considered to be regulated independently from peripheral fluids. For that reason, the challenging matrix CSF becomes a very interesting specimen for analysis. Concentrations are expected to be very low and available amount of CSF is limited. Thus, a comprehensive method for very sensitive quantification of a set of analytes as large as possible in one analytical aliquot is desired.However, high structural similarities of the selected panel of 51 steroids and steroid sulfates, including numerous isomers, challenges achievement of chromatographic selectivity.Since decades the analysis of endogenous steroids in various body fluids is mainly performed by gas chromatography (GC) coupled to (tandem) mass spectrometry (MS(/MS)). Due to the structure of the steroids of interest, derivatisation is performed to meet the analytical requirements for GC–MS(/MS). Most of the laboratories use a two-step derivatisation in multi-analyte assays that was already published in the 1980s. However, for some steroids this elaborate procedure yields multiple isomeric derivatives. Thus, some laboratories utilize (ultra) high performance liquid chromatography ((U)HPLC)–MS/MS as alternative but, even UHPLC is not able to separate some of the isomeric pairs. Supercritical fluid chromatography (SFC) as an orthogonal separation technique to GC and (U)HPLC may help to overcome these issues.Within this project the two most promising methods for endogenous steroid profiling were investigated and compared: the “gold standard” GC–MS and the orthogonal separation technique SFC-MS/MS. Different derivatisation procedures for gas chromatographic detection were explored and the formation of multiple derivatives described and confirmed. Taken together, none of the investigated derivatisation procedures provided acceptable results for further method development to meet the requirements of this project. SFC with its unique selectivity was able to overcome these issues and to distinguish all selected steroids, including (pro‐)gestagens, androgens, corticoids, estrogens, and steroid sulfates with appropriate selectivity. Valued especially in the separation of enantiomeric analytes, SFC has shown its potential as alternative to GC. The successful separation of 51 steroids and steroid sulfates on different columns is presented to demonstrate the potential of SFC in endogenous steroid profiling.

Neurosteroid Transport in the Brain: Role of ABC and SLC Transporters.

Neurosteroids, comprising pregnane, androstane, and sulfated steroids can alter neuronal excitability through interaction with ligand-gated ion channels and other receptors and have therefore a therapeutic potential in several brain disorders. They can be formed in brain cells or are synthesized by an endocrine gland and reach the brain by penetrating the blood–brain barrier (BBB). Especially sulfated steroids such as pregnenolone sulfate (PregS) and dehydroepiandrosterone sulfate (DHEAS) depend on transporter proteins to cross membranes. In this review, we discuss the involvement of ATP-binding cassette (ABC)- and solute carrier (SLC)-type membrane proteins in the transport of these compounds at the BBB and in the choroid plexus (CP), but also in the secretion from neurons and glial cells. Among the ABC transporters, especially BCRP (ABCG2) and several MRP/ABCC subfamily members (MRP1, MRP4, MRP8) are expressed in the brain and known to efflux conjugated steroids. Furthermore, several SLC transporters have been shown to mediate cellular uptake of steroid sulfates. These include members of the OATP/SLCO subfamily, namely OATP1A2 and OATP2B1, as well as OAT3 (SLC22A3), which have been reported to be expressed at the BBB, in the CP and in part in neurons. Furthermore, a role of the organic solute transporter OSTα-OSTβ (SLC51A/B) in brain DHEAS/PregS homeostasis has been proposed. This transporter was reported to be localized especially in steroidogenic cells of the cerebellum and hippocampus. To date, the impact of transporters on neurosteroid homeostasis is still poorly understood. Further insights are desirable also with regard to the therapeutic potential of these compounds.

Correction to: Halistanol sulfates I and J, new SIRT1-3 inhibitory steroid sulfates from a marine sponge of the genus Halichondria.

The original article can be found online at https://doi.org/10.1038/ja.2017.145 .

SULFATION PATHWAYS: Alternate steroid sulfation pathways targeted by LC-MS/MS analysis of disulfates: application to prenatal diagnosis of steroid synthesis disorders.

The steroid disulfates (aka bis-sulfates) are a significant but minor fraction of the urinary steroid metabolome that have not been widely studied because major components are not hydrolyzed by the commercial sulfatases commonly used in steroid metabolomics. In early studies, conjugate fractionation followed by hydrolysis using acidified solvent (solvolysis) was used for the indirect detection of this fraction by GC-MS. This paper describes the application of a specific LC-MS/MS method for the direct identification of disulfates in urine, and their use as markers for the prenatal diagnosis of disorders causing reduced estriol production: STSD (steroid sulfatase deficiency), SLOS (Smith-Lemli-Opitz syndrome) and PORD (P450 oxidoreductase deficiency). Disulfates were detected by monitoring a constant ion loss (CIL) from the molecular di-anion. While focused on disulfates, our methodology included an analysis of intact steroid glucuronides and monosulfates because steroidogenic disorder diagnosis usually requires an examination of the complete steroid profile. In the disorders studied, a few individual steroids (as disulfates) were found particularly informative: pregn-5-ene-3β,20S-diol, pregn-5-ene-3β,21-diol (STSD, neonatal PORD) and 5α-pregnane-3β,20S-diol (pregnancy PORD). Authentic steroid disulfates were synthesized for use in this study as aid to characterization. Tentative identification of 5ξ-pregn-7-ene-3ξ,20S-diol and 5ξ-pregn-7-ene-3ξ,17,20S-triol disulfates was also obtained in samples from SLOS affected pregnancies. Seven ratios between the detected metabolites were applied to distinguish the three selected disorders from control samples. Our results show the potential of the direct detection of steroid conjugates in the diagnosis of pathologies related with steroid biosynthesis.

Abstract P6-09-12: A functional single nucleotide polymorphisms in ABCC11, rs17822931, is associated with the risk of breast cancer in Japanese

Abstract Background: ATP binding cassette (ABC) transporters are members of membrane protein of transporters, genetic variations of which may influence their activity. ABCC11, a member of ABC transporters, has been known to export steroid sulfates such as E2-17bG and E3S that are precursor of active estradiol. Therefore, rs17822931, a functional single nucleotide polymorphisms (SNP) in the ABCC11, may play a role in carcinogenesis of breast cancer (BC) via estrogen exposure. Three previous studies about the association of this polymorphism with BC risk reported inconsistent results. In this study, we aimed to evaluate the association between ABCC11 rs17822931 and BC risk in a Japanese population. Subjects and Methods: We conducted a case-control study in 697 BC patients and 1394 age- and menopausal status- matched controls within the framework of the Hospital-based Epidemiological Research Program at Aichi Cancer Center II (HERPACC II). The odds ratio (OR) and 95 % confidence interval (CI) were calculated using the conditional logistic model adjusted for potential confounders to evaluate the association. We evaluated heterogeneity by BC subtype using Cochran's Q test, and that by estrogen exposure by multiplicative assumption in the conditional logistic regression model, which included an interaction term for genotype and estrogen exposure variable. Results: Compared with A-allele, G allele was inversely associated with BC risk (a per allele OR=0.76, 95% CI, 0.61–0.94, P = 0.012). In the stratified analysis, we found that this association was observed only in estrogen receptor (ER) positive BC. A per-allele OR for ER positive BC was 0.65 (95% CI 0.49–0.86) while that for ER negative BC was 1.10 (95% CI 0.77-1.72). We found a statistically significant heterogeneity by ER status (p for interaction = 0.047). We did not observe any heterogeneity in the stratified analysis by other tumor characteristics. When stratifying by factors related with estrogen exposure, we found that this polymorphism had a different impact on BC risk by levels of exposure. Compared to women with low exposure to estrogen, those with high exposure had a stronger impact of this polymorphism. Per allele ORs for women who had ever and never breastfeeding were 0.82 (95% CI, 0.65-1.04) and 0.45 (0.26-0.79), respectively. We found suggestive heterogeneity between rs17822931 and history of breastfeeding on BC risk (p for interaction = 0.093). Interestingly, we found heterogeneous impact of rs17822931 by the levels of soy food consumption which is well known that may exert their effects as estrogen antagonists (p=0.005). We observed that ORs for &amp;gt;50, 30-49 and &amp;lt;30g/day of soy intake were 0.98 (0.65-1.46), 0.82 (0.59-1.14) and 0.45 (0.29-0.69), respectively. Conclusion: Current case-control study showed the significant association between rs17822931 and the risk of BC, especially ER-positive BC, in Japanese women. Compared to women with low estrogen efflux activity with A allele, those with high efflux activity with G allele may have a lower risk of BC, especially in women with high estrogen exposure. To the best of our knowledge, this is the first study reporting the association between rs17822931 on ABCC11 and risk of BC considering levels of estrogen exposure. Citation Format: Ishiguro J, Ito H, Tsukamo M, Iwata H, Nakagawa H, Matsuo K. A functional single nucleotide polymorphisms in ABCC11, rs17822931, is associated with the risk of breast cancer in Japanese [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-09-12.