Diagnostic Evaluation of Enzyme Activity Related to Steroid Metabolism by Mass Spectrometry-Based Steroid Profiling

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  • ABSTRACT

    Gas chromatography-mass spectrometry (GC-MS) methods have been used extensively in clinical steroid analyses. Evaluating the metabolic ratios of precursors to products by accurate quantification of individual steroid levels in biological samples can reveal the activities of enzymes associated with steroid metabolism. This review article discusses the impact of GC-MS-based steroid profiling on our understanding of the biochemical role of steroids and their metabolic enzymes in hormone-dependent diseases, such as congenital adrenal hyperplasia (CAH), cortisol-mediated hypertension, apparent mineralocorticoid excess (AME), male-pattern baldness, and breast and thyroid cancers. Steroid profiling is a comprehensive analytical technique that can be applied whenever the highest specificity is required and may be a reasonable initial diagnostic approach.


  • KEYWORD

    steroid , metabolite profiling , biomarker , clinical diagnosis , steroidogenesis

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  • [Figure 1.] Steroid hormone metabolic pathways in humans. The arrows mark the conversions of substrates to products by the enzymes shown in red. Data are from reference #15.
    Steroid hormone metabolic pathways in humans. The arrows mark the conversions of substrates to products by the enzymes shown in red. Data are from reference #15.
  • [Figure 2.] Cholesterol biosynthetic pathways. The mevalonate pathway produces lanosterol, which can be converted to cholesterol by either the Bloch pathway, via desmosterol, or the Kandutsch-Russell pathway, via 7-dehydrocholesterol. Two other branches also diverge from the mevalonate pathway. The intermediates and enzymes in this shunt pathway have not yet been fully elucidated; however, they are presumed to follow the Kandutsch-Russell pathway. Abbreviations: MK, mevalonate kinase; PMK, phosphomevalonate kinase; MVD, diphosphomevalonate decarboxylase; FPPS, farnesyl pyrophosphate synthase; SQS, squalene synthase; LDM, lanosterol 14α-demethylase; SC5D, sterol C5-desaturase. Data are from reference #16.
    Cholesterol biosynthetic pathways. The mevalonate pathway produces lanosterol, which can be converted to cholesterol by either the Bloch pathway, via desmosterol, or the Kandutsch-Russell pathway, via 7-dehydrocholesterol. Two other branches also diverge from the mevalonate pathway. The intermediates and enzymes in this shunt pathway have not yet been fully elucidated; however, they are presumed to follow the Kandutsch-Russell pathway. Abbreviations: MK, mevalonate kinase; PMK, phosphomevalonate kinase; MVD, diphosphomevalonate decarboxylase; FPPS, farnesyl pyrophosphate synthase; SQS, squalene synthase; LDM, lanosterol 14α-demethylase; SC5D, sterol C5-desaturase. Data are from reference #16.
  • [Figure 3.] An overview of cortisol metabolism catalyzed by various metabolic enzymes in congenital adrenal hyperplasia (CAH). The enzymes that mediate each conversion are indicated by different colored arrows. Defects in the four main enzymes required for the synthesis of cortisol from cholesterol in the adrenal cortex can lead to CAH, including 3β-hydroxysteroid dehydrogenase (3β-HSD) and three hydroxylases, CYP17A1 (17α-hydroxylase), CYP21A2 (21-hydroxylase), and CYP11B1 (11β-hydroxylase).
    An overview of cortisol metabolism catalyzed by various metabolic enzymes in congenital adrenal hyperplasia (CAH). The enzymes that mediate each conversion are indicated by different colored arrows. Defects in the four main enzymes required for the synthesis of cortisol from cholesterol in the adrenal cortex can lead to CAH, including 3β-hydroxysteroid dehydrogenase (3β-HSD) and three hydroxylases, CYP17A1 (17α-hydroxylase), CYP21A2 (21-hydroxylase), and CYP11B1 (11β-hydroxylase).
  • [Figure 4.] GC-MS total ion chromatograms of cortisol and cortisone and their metabolites (10 pg each in the standards) in two extracts, that is, human serum and urine samples. Data are from reference #4.
    GC-MS total ion chromatograms of cortisol and cortisone and their metabolites (10 pg each in the standards) in two extracts, that is, human serum and urine samples. Data are from reference #4.
  • [Figure 5.] Reconstructed ion chromatogram (RIC) for the determination of DHT, epitestosterone, and testosterone in hair. Comparison of bald and control subject hair samples by selected-ion monitoring and chromatographed on an Ultra-1 (17 m × 0.2 mm i.d. × 0.11 mm film thickness) capillary column. The selected ion for DHT (tR = 12.78, a) was m/z 586, and the ions for epitestosterone (tR = 11.60, b) and testosterone (tR = 13.22, c) were m/z 584. Data are from reference #41.
    Reconstructed ion chromatogram (RIC) for the determination of DHT, epitestosterone, and testosterone in hair. Comparison of bald and control subject hair samples by selected-ion monitoring and chromatographed on an Ultra-1 (17 m × 0.2 mm i.d. × 0.11 mm film thickness) capillary column. The selected ion for DHT (tR = 12.78, a) was m/z 586, and the ions for epitestosterone (tR = 11.60, b) and testosterone (tR = 13.22, c) were m/z 584. Data are from reference #41.
  • [Figure 6.] Steroid metabolism differences in balding and normal Koreans and Caucasians. The line within the box represents the median, the lower boundary of the box indicates the 25th percentile, and the upper boundary of the box indicates the 75th percentile. The whiskers above and below indicate the maximum and minimum steroid levels, respectively. The dots above and below indicate the plot outliers with the 10th and 90th percentiles, respectively. Abbreviations: 3α-HSD, 3α-hydroxysteroid dehydrogenase; 17α-HSD, 17α- hydroxysteroid dehydrogenase; 17α-HSD, 17α- hydroxysteroid dehydrogenase; NK, normal Korean; BK, balding Korean; NC, normal Caucasian; BC, balding Caucasian. Data are from reference #43.
    Steroid metabolism differences in balding and normal Koreans and Caucasians. The line within the box represents the median, the lower boundary of the box indicates the 25th percentile, and the upper boundary of the box indicates the 75th percentile. The whiskers above and below indicate the maximum and minimum steroid levels, respectively. The dots above and below indicate the plot outliers with the 10th and 90th percentiles, respectively. Abbreviations: 3α-HSD, 3α-hydroxysteroid dehydrogenase; 17α-HSD, 17α- hydroxysteroid dehydrogenase; 17α-HSD, 17α- hydroxysteroid dehydrogenase; NK, normal Korean; BK, balding Korean; NC, normal Caucasian; BC, balding Caucasian. Data are from reference #43.
  • [Figure 7.] Sex-based differences in the 2-hydroxyestrone to 2- hydroxy-17β-estradiol metabolic ratio. This metabolic ratio could help determine 17β-hydroxysteroid dehydrogenase activity in estrogen metabolism. The line within the box represents the median, the lower boundary of the box indicates the 25th percentile, and the upper boundary of the box indicates the 75th percentile. The whiskers above and below indicate the maximum and minimum steroid levels, respectively. The dots above and below indicate the plot outliers with the 10th and 90th percentiles, respectively. Data are from reference #52.
    Sex-based differences in the 2-hydroxyestrone to 2- hydroxy-17β-estradiol metabolic ratio. This metabolic ratio could help determine 17β-hydroxysteroid dehydrogenase activity in estrogen metabolism. The line within the box represents the median, the lower boundary of the box indicates the 25th percentile, and the upper boundary of the box indicates the 75th percentile. The whiskers above and below indicate the maximum and minimum steroid levels, respectively. The dots above and below indicate the plot outliers with the 10th and 90th percentiles, respectively. Data are from reference #52.