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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 (R2 > 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.

Three new sulfated steroid monoglycosides, leptaochotensosides A–C (1–3), and a new sulfated polyhydroxylated steroid (4) were isolated from the alcoholic extract of the Far Eastern starfish Leptasterias ochotensis. The structures of compounds 1–4 were established by extensive nuclear magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESIMS) analyses and chemical transformations. Although the isolated compounds did not show any apparent cytotoxicity against melanoma RPMI-7951 and breast cancer T-47D cell lines, leptaochotensoside A (1) demonstrated inhibition of T-47D cell colony formation in a soft agar clonogenic assay at nontoxic doses. In addition, this compound decreased the epidermal growth factor (EGF)-induced colony formation of mouse epidermal JB6 Cl41 cells. The cancer preventive action of 1 is realized through regulation of mitogen-activated protein kinase (MAPK) signaling pathway.

Steroid sulfatase (STS) deficiency is the underlying cause of the skin condition known as recessive X-linked ichthyosis (RXLI). RXLI patients show scales on their skin caused by high concentrations of cholesterol sulfate (CS), as they are not capable of releasing the sulfate group from its structure to obtain free cholesterol. CS has been reported, so far, as the sole sulfated steroid with increased concentrations in the blood of RXLI patients. A non-targeted LC-MS approach in negative mode detection (LC-MS precursor ion scan mode) was applied to serum samples of 12 RXLI patients and 19 healthy males. We found that CS was not the only sulfated compound consistently elevated in RXLI patients, because a group of compounds with a m/z of 481 was found in high concentrations too. Further LC-MS/MS demonstrated that the main contributor to the m/z 481 signal in RXLI serum is 27-hydroxycholesterol-3-sulfate (27OHC3S). Accordingly, a new method for 27OHC3S quantification in the context of RXLI has been developed and validated. Other hydroxycholesterol sulfate compounds were elevated as well in RXLI patients.

Disulfated and trisulfated steroids have been synthesized from cholesterol and their acetylcholinesterase inhibitory activity has been evaluated. In our studies we have found that the activity was not only dependent on the location of the sulfate groups but on their configurations. 2β,3α,6β-trihydroxy-5α-cholestan-6-one trisulfate (18) was the most active steroid with an IC50 value of 15.48 μM comparable to that of 2β,3α-dihydroxy-5α-cholestan-6-one disulfate (1). Both compounds were found to be less active than the reference compound eserine. The butyrylcholinesterase activity of 1 and 18 was one magnitude lower than that against acetylcholinesterase revealing a selective inhibitor profile. Fil: Richmond, Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos Aplicados A la Química Orgánica (i); Argentina. Universidad de Buenos Aires; Argentina Fil: Murray, Ana Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Bahía Blanca. Instituto de Química del Sur; Argentina. Universidad Nacional del Sur; Argentina Fil: Maier, Marta Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos Aplicados A la Química Orgánica (i); Argentina. Universidad de Buenos Aires; Argentina

Four new 6E-hydroximinosteroids (1, 2a, 3 and 4) have been synthesized from the correspondingketones, 2b,3b-dihydroxy-5 a -cholestan-6-one (5), 2 a ,3 a -dihydroxy-5 a -cholestan-6-one (6), 2b,3 a -dihydroxy-5 a -cholestan-6-one (7) and 2b,3 -dihydroxy-5 -cholestan-6-one-disulfate (8). The cytotoxicactivity of the steroidal oximes was evaluated against two prostate carcinoma cell lines (PC-3 and LNCaP)and compared with that of five polyhydroxylated sulfated analogs (8–12). Oxime 3 and trisulfated analog11 were the most active compounds with IC 50 values of 10.8 l M (PC-3) and 7.9 l M (LNCaP), respectively. 2014 Elsevier Inc. All rights reserved. 1. IntroductionMarine organisms have become an important source of steroidswith unusual and interesting structures. Among these secondarymetabolites, sulfated polyhydroxysterols have beendescribed fromPorifera and Ophiuroidea (Echinodermata) [1–3], and some raresteroidal oximes have been isolated from Cinachyrella sponges[4,5]. These compounds have attracted considerable interest dueto their broad spectrum of biological activities, such as ligands ofhuman nuclear receptors [6], inhibition of protein tyrosine kinases[7,8], antiviral [9], cytotoxic [10,11], and antifouling activities [12].In recent years, a variety of marine steroidal analogs have beensynthesized in order to develop new bioactive compounds. In pre-vious works [13,14], we have synthesized new di- and trisulfatedsteroids with acetylcholinesterase inhibitory activity and estab-lished that the activity was dependent on the location and the con-figurations of the sulfate groups on rings A and B. In 2009, Cui et al.[15] reported the synthesis of 3b,6b-dihydroxysterol disulfates anddetermined the importance of the cholesterol-type side chain forthe cytotoxic activity.Recently, several 6-hydroximinosteroids have been synthesizedand evaluated for their cytotoxic activity against human tumor celllines in vitro [10,16–20]. These studies have revealed the impor-tance of oxygenated positions in ring A and a cholesterol-type sidechain for an increase in cytotoxic activity.These results prompted us to synthesize three new6E-hydroximinosteroids with hydroxy groups at C-2 and C-3 (