Steroids in Reproduction: LC-MS-MS and GC (-MS) Based Steroidomics
Final Report Abstract
Steroid determination with highest reliability was a prerequisite for the realization of this research group. In contrast to immunoassays, analytical methods based on mass spectrometry (MS) currently present the most specific qualitative and quantitative methods for steroid determination. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) presents the analytical first line approach for the analysis of the intact conjugated steroid. Thus, during the first funding period, we laid a foundation by successfully developing a new LC-MS/MS based multi-targeted method for the simultaneous determination of six intact steroid sulfates (estrone sulfate, estradiol sulfate, 5-androstenediol sulfate, pregnenolone sulfate, dehydroepiandrosterone sulfate, 16-hydroxydehydroepiandrosterone sulfate) from various biological matrices. During the second funding period, we were able to further expand the capacity of the first method by further seven steroid sulfates (cholesterol sulfate, 17-hydroxy-pregnenolone sulfate, androsterone sulfate, epiandrosterone sulfate, testosterone sulfate, epitestosterone sulfate, dihydrotestosterone sulfate) thus achieving the hitherto most comprehensive method for characterizing the “sulfated steroidome” in biological samples. Regarding unconjugated steroids, multi-targeted methods for the simultaneous determination of estriol, estrone, estradiol, 17-hydroxy-pregnenolone, dehydroepiandrosterone, 16-hydroxydehydroepiandrosterone, 4-androstenedione, testosterone, progesterone, 17-hydroxyprogesterone, 11-deoxycortisol, cortisol, androsterone, pregnenolone, androstanediol and dihydrotestosterone were developed for both LC-MS/MS and gas chromatography-mass spectrometry (GC-MS). For these metabolites, due to its high specificity, GC-MS/MS proved advantageous as a first line research tool in characterizing steroid metabolomes in biological specimens. In a translational approach, we have successfully applied our method to study the sulfated steroidome in patients with steroid sulfatase deficiency (recessive X-linked ichthyosis). These patients are a good model to understand the biosynthesis of sulfated steroids and the physiological role of steroid sulfatase. Their steroid disease signature confirmed the coexistence of two steroidogenic pathways: one for unconjugated steroids and another one for sulfated steroids. Our method allowed for complete discrimination of patients from controls and currently presents the only tool of metabolic diagnosis of this condition. In a subsequent study, we could further demonstrate for the first time that patients with steroid sulfatase deficiency have high levels of oxysterol sulfates. Particularly 27-hydroxycholesterol-3-sulfate proved to be the main elevated compound presenting a novel diagnostic marker of this disease. Our group has acted with great success as central analytical platform for steroid metabolomics and has provided analytical service for all collaborators within the research group and even beyond. The gas chromatography-tandem mass spectrometry unit (GC-MS/MS) enabled further expansion of our methodological portfolio. This unique analytical instrumentation with the complementary capacities of LC-MS and GC-MS techniques proved indispensable for characterizing the steroid metabolome in wild-type and Slc10a6-(Soat)-knockout mice, as well as in the in vitro and in vivo metabolic experiments. The extremely positive reception of our results in the scientific community and the growing request for national and international collaborations let us gratefully conclude that the analytical achievements within this DFG research group have propelled our laboratory to the forefront of steroid analytics and steroid research worldwide.
Publications
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The Journal of Steroid Biochemistry and Molecular Biology (2016): LC-MS based analytics and applications in steroid research. J Steroid Biochem Mol Biol 162: 1-134; special issue
Wudy SA and Choi MH (editors)
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(2013) Introduction to gas chromatography-mass spectrometry. Methods in Molecular Biology 1065:27-44
Sánchez-Guijo A, Hartmann MF and Wudy SA
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(2013) Profiling intact steroid sulfates and unconjugated steroids in biological fluids by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Analyst 138:3792-3801
Galuska CE, Hartmann MF, Sanchez-Guijo A, Bakhaus K, Geyer J, Schuler G, Zimmer KP and Wudy SA
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(2014) Determination of free cortisol and free cortisone in human urine by on-line turbulent flow chromatography coupled to fused-core chromatography-tandem mass spectrometry (TFC-HPLC-MS/MS). Analytical and Bioanalytical Chemistry 406:793-801
Sánchez-Guijo A, Hartmann MF, Shi L, Remer T and Wudy SA
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(2015) High levels of oxysterol sulfates in serum of patients with steroid sulfatase deficiency. J Lipid Res 56:403-412
Sánchez-Guijo A, Oji V, Hartmann MF, Schuppe HC, Traupe H and Wudy SA
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(2015) Simultaneous quantification of cholesterol sulfate, androgen sulfates, and progestagen sulfates in human serum by LC-MS/MS. J Lipid Res 56:1843-1851
Sánchez-Guijo A, Oji V, Hartmann MF, Traupe H and Wudy SA
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(2016) Changes in the Metabolome in Response to Low-Dose Exposure to Environmental Chemicals Used in Personal Care Products during Different Windows of Susceptibility. PLoS One 11:e0159919
Houten SM, Chen J, Belpoggi F, Manservisi F, Sánchez-Guijo A, Wudy SA and Teitelbaum SL
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(2016) Profiling of bile acids in bovine follicular fluid by fused-core-LC-MS/MS. J Steroid Biochem Mol Biol 162:117-125
Sánchez-Guijo A, Blaschka C, Hartmann MF, Wrenzycki C and Wudy SA
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(2016) Role of steroid sulfatase in steroid homeostasis and characterization of the sulfated steroid pathway: Evidence from steroid sulfatase deficiency. Mol Cell Endocrinol 437:142-153
Sánchez-Guijo A, Neunzig J, Gerber A, Oji V, Hartmann MF, Schuppe HC, Traupe H, Bernhardt R and Wudy SA
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(2016) Steroid LC-MS has come of age (Editorial). J Steroid Biochem Mol Biol 162:1-3
Wudy SA and Choi MH
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(2017) The Art of Measuring Steroids - Principles and Practice of Current Hormonal Steroid Analysis. J Steroid Biochem Mol Biol
Wudy SA, Schuler G, Sánchez-Guijo A, Hartmann MF
