Evaluation of plasma enzyme activities using gas chromatography-mass spectrometry based steroid signatures

Abstract

The simultaneous quantification of 65 plasma steroids, including 22 androgens, 15 estrogens, 15 corticoids and 13 progestins, was developed using gas chromatography-mass spectrometry (GC-MS). The extraction efficiency of the catechol estrogens was improved by the addition Of L-ascorbic acid in several steps. All steroids, as their trimethylsilyl derivatives, were well separated with good peak shapes within a 50 min run. The devised method provided good linearity (correlation coefficient, r(2) > 0.993), while the limit of quantification ranged from 0.2 to 2.0 ng mL(-1). The precision (% CV) and accuracy (% bias) were 2.0-12.4% and 93.5-109.2%, respectively. The metabolic changes were evaluated by applying this method to plasma samples obtained from 26 healthy male subjects grouped according to the pre- and post-administration of dutasteride, which inhibits 5 alpha-reductase isoenzyme types 1 and 2. The levels of three plasma steroids, Such as dihydrotestosterone, 5 alpha-androstanedione and allotetrahydrocortisol, were decreased significantly after drug administration, while the levels of testosterone and 5 beta-androstane-3 beta, 17 alpha-diol were increased. In addition, the ratios of the steroid precursors and their metabolites, which represent the activities of the related enzymes, were z-score transformed for visualization in heat maps generated using Supervised hierarchical clustering analysis. These results validated the data transformation because 5 alpha-reductase is an indicator for the biological actions of dutasteride. GC-MS base quantitative visualization might be found in the integration with the mining biomarkers in drug evaluations and hormone-dependent diseases. (C) 2009 Elsevier B.V. All rights reserved.