title: Computational analysis of liquid chromatography-tandem mass spectrometric steroid profiling in NCI H295R cells following angiotensin II, forskolin and abiraterone treatment.
abstract: Adrenal steroid hormones, which regulate a plethora of physiological functions, are produced via tightly controlled pathways. Investigations of these pathways, based on experimental data, can be facilitated by computational modeling for calculations of metabolic rate alterations. We therefore used a model system, based on mass balance and mass reaction equations, to kinetically evaluate adrenal steroidogenesis in human adrenal cortex-derived NCI H295R cells. For this purpose a panel of 10 steroids was measured by liquid chromatographic-tandem mass spectrometry. Time-dependent changes in cell incubate concentrations of steroids - including cortisol, aldosterone, dehydroepiandrosterone and their precursors - were measured after incubation with angiotensin II, forskolin and abiraterone. Model parameters were estimated based on experimental data using weighted least square fitting. Time-dependent angiotensin II- and forskolin-induced changes were observed for incubate concentrations of precursor steroids with peaks that preceded maximal increases in aldosterone and cortisol. Inhibition of 17-alpha-hydroxylase/17,20-lyase with abiraterone resulted in increases in upstream precursor steroids and decreases in downstream products. Derived model parameters, including rate constants of enzymatic processes, appropriately quantified observed and expected changes in metabolic pathways at multiple conversion steps. Our data demonstrate limitations of single time point measurements and the importance of assessing pathway dynamics in studies of adrenal cortical cell line steroidogenesis. Our analysis provides a framework for evaluation of steroidogenesis in adrenal cortical cell culture systems and demonstrates that computational modeling-derived estimates of kinetic parameters are an effective tool for describing perturbations in associated metabolic pathways.
authors.affiliation: Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Carl Gustav Carus, Medical Faculty Carl Gustav Carus, Technische Universität Dresden, Fetscherstraße 74, 01307 Dresden, Germany. Electronic address: Anastasios.Mangelis@uniklinikum-dresden.de.
authors.forename: Anastasios
authors.initials: A
authors.lastname: Mangelis
authors.affiliation: Institute of Physiology, University Hospital Carl Gustav Carus, Medical Faculty Carl Gustav Carus, Technische Universität Dresden, Fetscherstraße 74, 01307 Dresden, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Dieterich
authors.affiliation: Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Carl Gustav Carus, Medical Faculty Carl Gustav Carus, Technische Universität Dresden, Fetscherstraße 74, 01307 Dresden, Germany.
authors.forename: Mirko
authors.initials: M
authors.lastname: Peitzsch
authors.affiliation: Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Carl Gustav Carus, Medical Faculty Carl Gustav Carus, Technische Universität Dresden, Fetscherstraße 74, 01307 Dresden, Germany.
authors.forename: Susan
authors.initials: S
authors.lastname: Richter
authors.affiliation: Department of Pediatrics, University Hospital Carl Gustav Carus, Medical Faculty Carl Gustav Carus, Technische Universität Dresden, Fetscherstraße 74, 01307 Dresden, Germany.
authors.forename: Ramona
authors.initials: R
authors.lastname: Jühlen
authors.affiliation: Department of Pediatrics, University Hospital Carl Gustav Carus, Medical Faculty Carl Gustav Carus, Technische Universität Dresden, Fetscherstraße 74, 01307 Dresden, Germany.
authors.forename: Angela
authors.initials: A
authors.lastname: Hübner
authors.affiliation: Department of Internal Medicine III, University Hospital Carl Gustav Carus, Medical Faculty Carl Gustav Carus, Technische Universität Dresden, Fetscherstraße 74, 01307 Dresden, Germany; Division of Endocrinology and Metabolism, Rostock University Medical Center, Ernst-Heydemann-Str. 6, 18057 Rostock, Germany.
authors.forename: Holger S
authors.initials: HS
authors.lastname: Willenberg
authors.affiliation: Institute of Physiology, University Hospital Carl Gustav Carus, Medical Faculty Carl Gustav Carus, Technische Universität Dresden, Fetscherstraße 74, 01307 Dresden, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Deussen
authors.affiliation: Department of Internal Medicine III, University Hospital Carl Gustav Carus, Medical Faculty Carl Gustav Carus, Technische Universität Dresden, Fetscherstraße 74, 01307 Dresden, Germany; Department of General Internal Medicine, Radboud University Medical Center, Geert Grooteplein 8, 6525 Nijmegen, The Netherlands.
authors.forename: Jacques W M
authors.initials: JW
authors.lastname: Lenders
authors.affiliation: Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Carl Gustav Carus, Medical Faculty Carl Gustav Carus, Technische Universität Dresden, Fetscherstraße 74, 01307 Dresden, Germany; Department of Internal Medicine III, University Hospital Carl Gustav Carus, Medical Faculty Carl Gustav Carus, Technische Universität Dresden, Fetscherstraße 74, 01307 Dresden, Germany.
authors.forename: Graeme
authors.initials: G
authors.lastname: Eisenhofer
mesh_terms: D000302:Adrenal Cortex / Q000166:cytology / Q000378:metabolism; D000736:Androstenes / Q000494:pharmacology*; D000804:Angiotensin II / Q000494:pharmacology*; D001692:Biological Transport; D002460:Cell Line / Q000187:drug effects / Q000378:metabolism; D049109:Cell Proliferation; D002853:Chromatography, Liquid / Q000379:methods; D005576:Colforsin / Q000494:pharmacology*; D006801:Humans; D053858:Metabolic Networks and Pathways; D008954:Models, Biological; D013256:Steroids / Q000032:analysis / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000736:Androstenes; D013256:Steroids; D000804:Angiotensin II; D005576:Colforsin; C089740:abiraterone
keywords: Computational analysis; LC–MS/MS; Steroid profiling; Steroidogenesis