Genotype-steroidogenic phenotype relationships in patients with aldosteronomas and in experimental cell model systems

• Applicant: Professor Jacques W.M. Lenders, Ph.D.
• Subject Area: Endocrinology, Diabetology, Metabolism
• Term: from 2011 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 189897882

• Final Report Year: 2019

Final Report Abstract

The focus of this project was to determine the value of adrenal venous plasma steroids to differentiate between unilateral and bilateral aldosterone excess in patients with PA and to identify whether distinct adrenal venous steroid profiles are associated with mutations in aldosteronomas in patients with PA. We explored the latter associations also by manipulations of gene expression and steroid profiling in cell line models. To achieve these aims we first developed and validated a new laboratory assay (LC-MS/MS) for accurate measurements of adrenal steroids in venous plasma. This method appeared superior to existing immunoassays in several ways, in particular by offering improved analytical specificity and an efficient tool to measure simultaneously a panel of over 15 steroids in a single blood sample. For correct interpretation of steroid results we established LC-MS/MS based reference levels for 16 steroids, including major steroid-specific impacts of gender and age, with minor impact of body mass index and negligible associations with blood pressure status. Differentiation between unilateral and bilateral aldosterone excess in PA patients depends on adrenal venous sampling. We demonstrated that LC-MS/MS based measurements of several steroids, other than cortisol, showed higher selectivity indices than cortisol for assessing correct catheter positioning in adrenal veins, thus providing higher success rates for adrenal venous sampling and outdating cortisol for this purpose. Our studies also showed that LC-MS/MS-based measurements, including use of alternative steroids to cortisol, achieved higher lateralization ratios for differentiating unilateral from bilateral aldosterone excess compared to conventional immunoassay-based measurements. A combination of steroids measured in a venous plasma sample could also classify correctly 80% of all patients into unilateral adenoma or bilateral hyperplasia. More importantly, LC-MS/MS-based peripheral venous steroid profiling (in particular 18- oxocortisol) can be used to identify patients with specific somatic mutations (KCNJ5) in adenomas, thereby offering potential for selecting patients for AVS. If these data can be reproduced and confirmed in a larger prospective study, adrenal venous sampling might also be obviated in a considerable number of patients, thereby considerably reducing the costs of the diagnostic work-up of primary aldosteronism. The results of this KFO project has set the stage for initiating a large multicenter prospective study with a standardized follow-up, the PROSALDO study. Finally, we have developed a continuous cell culture system to investigate the effects of steroidogenic inhibitors. We could show that such a model has advantages over static steroid measurements. Culturing cells under perfusion with flux analyses of steroid metabolites establishes a more realistic model for investigating drug effects. This enables simple and rapid calculations of intracellular fluxes and offers a robust method for drug screening or in vitro investigations of metabolic mechanisms. The method can be used to evaluate specific aldosterone synthase inhibitors at each of the three catalytic steps of the enzyme. Application of this novel approach may accelerate the development of new specific drugs, ultimately opening new therapeutic avenues for tailored drug treatment of PA.

Publications

• An LC-MS/MS Method for Steroid Profiling during Adrenal Venous Sampling for Investigation of Primary Aldosteronism. J Steroid Biochem Mol Biol 2015;145:75-84
  Peitzsch M, Tanja Dekkers T, Haase M, Sweep FC, Siegert G, Lenders JW, Deinum J, Willenberg HS, Eisenhofer G
  
• Pharmacological treatment of aldosterone excess. Pharmacol Ther. 2015;154:120-33
  Deinum J, Riksen NP, Lenders JW
  
• Steroid Hormone Production in Patients with Aldosterone Producing Adenomas. Horm Metab Res. 2015;47:967-72
  Moors M, Williams TA, Deinum J, Eisenhofer G, Reincke M, Lenders JW
  
• Computational analysis of liquid chromatography-tandem mass spectrometric steroid profiling in NCI H295R cells following angiotensin II, forskolin and abiraterone treatment. J Steroid Biochem Mol Biol. 2016;155(Pt A):67-75
  Mangelis A, Dieterich P, Peitzsch M, Richter S, Jühlen R, Hübner A, Willenberg HS, Deussen A, Lenders JW, Eisenhofer G
  
• Genotype-Specific Steroid Profiles Associated With Aldosterone-Producing Adenomas. Hypertension. 2016;67:139-145
  Williams TA, Peitzsch M, Dietz AS, Dekkers T, Bidlingmaier M, Riester A,Treitl M, Rhayem Y, Beuschlein F, Lenders JW, Deinum J, Eisenhofer G, Reincke M
  
• Mass Spectrometry-Based Adrenal and Peripheral Venous Steroid Profiling for Subtyping Primary Aldosteronism. Clin Chem. 2016;62:514-24
  Eisenhofer G, Dekkers T, Peitzsch M, Dietz AS, Bidlingmaier M, Treitl M,Williams TA, Bornstein SR, Haase M, Rump LC, Willenberg HS, Beuschlein F, Deinum J, Lenders JW, Reincke M
  
• Primary Aldosteronism Surgery Outcome (PASO) investigators. Outcomes after adrenalectomy for unilateral primary aldosteronism: an international consensus on outcome measures and analysis of remission rates in an international cohort. Lancet Diabetes Endocrinol. 2017;5:689-99
  Williams TA, Lenders JWM, Mulatero P, Burrello J, Rottenkolber M, Adolf C, Satoh F, Amar L, Quinkler M, Deinum J, Beuschlein F, Kitamoto KK, Pham U, Morimoto R, Umakoshi H, Prejbisz A, Kocjan T, Naruse M, Stowasser M, Nishikawa T, Young WF Jr, Gomez-Sanchez CE, Funder JW, Reincke M
  
• Reference intervals for plasma concentrations of adrenal steroids measured by LC-MS/MS: Impact of gender, age, oral contraceptives, body mass index and blood pressure status. Clin Chim Acta. 2017;470:115-24
  Eisenhofer G, Peitzsch M, Kaden D, Langton K, Pamporaki C, Masjkur J,Tsatsaronis G, Mangelis A, Williams TA, Reincke M, Lenders JWM, Bornstein SR
  
• Immunohistopathology and Steroid Profiles Associated With Biochemical Outcomes After Adrenalectomy for Unilateral Primary Aldosteronism. Hypertension. 2018; 72:650-57
  Meyer LS, Wang X, Sušnik E, Burrello J, Burrello A, Castellano I, Eisenhofer G, Fallo F, Kline GA, Knösel T, Kocjan T, Lenders JWM, Mulatero P, Naruse M, Nishikawa T, Peitzsch M, Rump LC, Beuschlein F, Hahner S, Gomez-Sanchez CE, Reincke M, Williams TA
  
• A steady state system for in vitro evaluation of steroidogenic pathway dynamics: Application for CYP11B1, CYP11B2 and CYP17 inhibitors. J Steroid Biochem Mol Biol. 2019;188:38-47
  Mangelis A, Jühlen R, Dieterich P, Peitzsch M, Lenders JW, Hahner S, Schirbel A, Eisenhofer G
  
• Classification of microadenomas in patients with primary aldosteronism by steroid profiling. J Steroid Biochem Mol Biol. 2019 Jan 14
  Yang Y, Burrello J, Burrello A, Eisenhofer G, Peitzsch M, Tetti M, Knösel T, Beuschlein F, Lenders JW, Mulatero P, Reincke M, Williams TA
  
• The Primary Aldosteronism Surgical Outcome Score for the Prediction of Clinical Outcomes After Adrenalectomy for Unilateral Primary Aldosteronism. Ann Surg. 2019 Jan 18
  Burrello J, Burrello A, Stowasser M, Nishikawa T, Quinkler M, Prejbisz A, Lenders JWM, Satoh F, Mulatero P, Reincke M, Williams TA