Human steroid signatures evaluated by gas chromatography-mass spectrometry-based quantitative analytical protocols
저자
발행사항
Seoul : Graduate School, Yonsei University, 2012
학위논문사항
학위논문(박사)-- Graduate School, Yonsei University : Dept. of Chemistry 2012.8
발행연도
2012
작성언어
영어
주제어
발행국(도시)
서울
기타서명
가스 크로마토그래피-질량분석기 기반 최적화된 정량 분석 방법에 의한 스테로이드 인식 기술 연구
형태사항
xv, 200 p. : 삽화(일부천연색) ; 26 cm
일반주기명
지도교수: Myeong Hee Moon
소장기관
Steroid hormones are components of the endocrine system that are biologically active compounds that regulate diverse human physiologies. Abnormalities in steroid metabolism lead to hormonal imbalances, which can be responsible for the development of endocrine diseases. Therefore the comprehensive quantification of steroid hormones in biological specimens is needed to elucidate altered expression of steroids. I have chosen to focus on gas chromatography?mass spectrometry (GC?MS) based techniques because it allows highly sensitive detection of steroids from human urine, which facilitates a non-invasive screening technique for the clinic setting.To optimize gas chromatography?mass spectrometry (GC?MS)-based analytical protocols, 1) solid-phase extraction (SPE), 2) enzymatic hydrolysis, and 3) chemical derivatization methods have been developed to establish analytical protocols in quantitative steroid profiling. The quantitative data obtained were analyzed by hierarchical-clustering analysis (HCA) to generate quantitative steroid signatures, which were visualized in heat maps to show the relative concentrations of steroids within clinical samples from various disease states.For the SPE approach to purification of steriods, β-cyclodextrin (β-CD) polymer was used for sample purification of biological steroids. By taking advantage of the two different ligand binding mechanisms of β-cyclodextrin, molecular inclusion and chemical interaction, a novel SPE sorbent with β-CD resulted in improved sensitivity and selectivity along with effective elimination of urinary interferences compared to the conventional method which uses hydrophobicity-based SPE methods. All steroids tested with the β-CD polymer showed that overall recoveries ranged from 82% to 112% in urine. The hydroxylated estrogens were notable due to their excellent binding capacities (96 ? 106% recovery). The high recoveries are likely due to hydrogen bonding between their phenoilc hydroxyl and the exterior hydroxyl groups of β-CD.For enzymatic hydrolysis, the unexpected transformation of urinary steroids was investigated in the presence of two difference enzyme solutions, β-glucuronidase (E. Coli) and a mixture of β-glucuronidase/arylsulfatase (Helix pomatia). The systematic error showed the conversion of 3β-hydroxysteroids to 3-oxo-steroids in 18 steroids when urinary steroids were hydrolyzed with Helix pomatia, resulting in a misreading of clinical implications. The optimized hydrolysis procedures were suitable for study purposes to prevent false evaluation.After optimizing the sample preparation techniques for both SPE and hydrolysis, the GC?MS-based urinary steroid profile, containing 22 androgens, 18 estrogens, 15 corticoids, 13 progestins, and 2 sterols, was validated and HCA was used to evaluate the observed metabolic changes. This GC?MS method provided a good linearity (r2 < 0.994) with the exception of cholesterol (r2 < 0.983), and precisions (% CV) and accuracies (% bias) ranged from 0.9% to 11.2% and from 92% to 119%, respectively. The comprehensive GC?MS-based steroid signature was applied to urine samples obtained from 59 patients with benign prostatic hyperplasia (BPH) and compared to urine from 41 healthy male subjects. Samples from BPH patients showed altered concentrations of urinary steroids where HCA of the analytes pointed to the activity of the oxidoreductases 5α-reductase, 3α-HSD, 3β-HSD, and 17β-HSD, but not 20α-HSD. The data for these experiments drew on large experimental datasets across multiple subjects for many analytes, which allowed patterns to be rapidly identified in a single visualization.In addition to the steroid signatures, steroid assays were developed for sensitive estrogen profiling and multiplexed cytochrome P450 (CYP) evaluation using trimethylsilyl (TMS) derivatization, extractive ethoxycarbonylation (EOC) with ethylchloroformate and subsequent pentafluoropropionyl (PFP) derivatization which demonstrated pg/mL urinary estrogen quantification. The 19 EOC?PFP estrogens were separated through a high-temperature MXT-1 column which facillitated short analytical run times as compared with the commonly used fused-silica GC column. The limit of quantification was from 0.02 to 0.1 ng/mL for all estrogens analyzed, except for 2-hydroxyestiol (0.5 ng/mL).The utility of a sensitive estrogens profiling technique was demonstrated with urine samples obtained from 100 postmenopausal female patients with osteoporosis. This patient population typically has very low-levels of estrogens and their detection could enable a better understanding of the osteoporosis disease process. Amongst the 19 estrogens monitored, 8 estrogens were found with greater than 90% detection incidence and 6 hydroxylated and methoxyestrogens were detected within approximately 50% of the total samples. The rapid, sensitive, selective and reproducible quantification at pg/mL levels of urinary estrogens could make it possible to examine the impact of estrogen metabolism in postmenopausal osteoporosis.A multiplexed cytochrome P450 (CYP) assay was carried out to detect/quantify regioselective hydroxylated steroids and their precursors, including 26 androgens, 9 estrogens, 5 progestins, and 7 corticoids. The activities of the CYP enzymes from twelve healthy subjects orally administered 600 mg/day rifampicin, for seven days were evaluated. Rifampicin is an agent used to induce the expression of several steroid metabolism enzymes including CYP3A4. All 47 steroids were well separated with the good peak shape using GC?MS. This assay showed a good linearity (r2 > 0.994) in a dynamic range, and the precision (% CV) and accuracy (% bias) of the inter-assay were 3.0 ? 15.6% and 98 ? 109%. Urine samples from subjects administered rifampicin showed distinct differences in analyte quantities reflective of CYP3A4 as well as CYP11B1, CYP19A1, HSD11B, and HSD17B activities. The steroid signatures were generated by HCA and visualized in a heat-map. This multiplexed CYP signatures allowed for simultaneous assessment of CYP1A, CYP1B, CYP2C, CYP3A, CYP11B, CYP17A, CYP19A, and CYP21A in urine samples. This technique represents an improvement in the sensitivity and reduces the time of measurement of key steroid analytes to aid in the diagnosis and study of metabolic diseases.Therefore, GC?MS-based analytical protocols in steroid profiling and the use of hierarchically clustered “steroid signatures” could be useful as a non-invasive screening and/or confirmation technique in clinical diagnosis as well as a better understanding of the pathogenesis of endocrine-related disorders where highly sensitive quantification is required. In addition, the techniques introduced here can be used for the assessment of both drug efficacy and in pharmacogenomics where CYP biomarkers discriminate amongst patient populations.
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