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

Abstract

The steroid disulfates ( 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,20-diol, pregn-5-ene-3β,21-diol (STSD, neonatal PORD) and 5α-pregnane-3β,20-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,20-diol and 5ξ-pregn-7-ene-3ξ,17,20-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.

Human steroid biosynthesis, metabolism and excretion are differentially reflected by serum and urine steroid metabolomes: A comprehensive review

“…Notably, bis-conjugation with the same (e.g. bis-sulfation) [[158], [159], [160], [161], [162]] or two different groups (e.g. sulfate and glucuronic acid) are possible [163].…”

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

“…These advances are exemplified by the CIL scan method for the direct and untargeted detection of urinary steroid bis(sulfate) metabolites [26]. The CIL scan method has been employed to study the endogenous steroid bis(sulfate) profile, including during pregnancy, to identify markers associated with sports doping [26], and for the analysis of maternal urine to provide discriminating prenatal diagnosis of inborn errors of steroid biosynthesis associated with SLOS, STSD, and PORD [27]. Integral to the development of the CIL scan method was the interplay between chemical synthesis and MS method development.…”

“…This method revealed a wide range of endogenous bis(sulfates) including examples from the estrane, androstane, and pregnane steroid families. The CIL scan method was applied to identify metabolites associated with sports doping and has also been employed in the analysis of maternal urine for the prenatal diagnosis of inborn errors of steroid biosynthesis associated with Smith-Lemli-Opitz Syndrome (SLOS), Steroid Sulfatase Deficiency (STSD), and cytochrome P450 Oxido-Reductase Deficiency (PORD) [27].…”

Sulfation pathways from red to green

“…In addition to investigations at the systems level, newly developed technologies will certainly provide the cutting edge in sulfation pathway research. On the one hand, these are analytical nature-advanced methods to detect, characterize, and quantitate various sulfo-conjugates such as peptides (99), steroids (202), or plant secondary metabolites (170). On the other hand, these also represent novel chemo-synthetic approaches to obtain good purity sulfated nucleotides (203), steroid reference material (204), or sugars (205).…”