MSACL 2016 EU Abstract

Keeping the Flow – Equilibrium Analysis of the Renin-Angiotensin-System and Applications in Diagnosis and Treatment of Hypertension

Oliver Domenig (Presenter)
Medical University of Vienna

Authorship: Marko Poglitsch (1), Cornelia Schwager (1), Dunja van Oyen (1), Claudia Aigner (1), Oliver Domenig (2)
(1) Attoquant Diagnostics GmbH, (2) Medical University of Vienna

Short Abstract Biochemical analysis of the Renin-Angiotensin-System is technically challenging, since angiotensins undergo rapid turnover during blood sampling, introducing analytical artefacts. Unique features of the circulating RAS allow for a novel diagnostic approach, overcoming these pre-analytical issues. RAS-equilibrium analysis can be applied to standard heparin plasma or serum samples and is based on the LC-MS/MS based quantification of up to 10 angiotensin metabolites (RAS-Fingerprint) providing a comprehensive qualitative and quantitative picture of soluble RAS components. The AA2-Ratio, a RAS-Fingerprint derived diagnostic value, has shown to possess a huge potential as a robust and drug tolerant screening test for primary aldosteronism.

Long Abstract

The renin-mediated formation of Angiotensin I from angiotensinogen is a well-characterized enzymatic reaction providing the molecular input for the RAS. Renin is rate limiting and its product Angiotensin I is immediately further metabolized to downstream angiotensin metabolites including Angiotensin II, being a key player in the regulation of fluid balance and blood pressure. All molecular components of the RAS are present in the plasma compartment contribute to a continuous flow of angiotensin metabolites through the peptide cascade in the vascular compartment. The resulting steady-state angiotensin levels are affected by the local molecular environment, which is constituted by angiotensin converting enzymes and receptors present on endothelial surfaces and in the plasma compartment. The high concentration of the pro-hormone angiotensinogen in human plasma results in a long-lasting and stable rate of Angiotensin I formation without significantly affecting plasma angiotensinogen levels during many hours of ex vivo incubation.

This phenomenon can be utilized to overcome pre-analysis issues in the molecular characterization of the human RAS. Ex vivo equilibration of heparin plasma or serum samples result in a stable RAS equilibrium that can be used for molecular characterization of the soluble RAS. The readout is based on the quantification of up to 10 angiotensin metabolites in equilibrium by LC-MS/MS (RAS-Fingerprint) and provides a comprehensive qualitative and quantitative view of the soluble RAS. The quantification of equilibrium angiotensin levels does not require any special sample collection procedure and can be applied to frozen serum and heparin plasma samples. It turned out that ex vivo angiotensin levels represent a measure for the consumption of angiotensin metabolites by the organism, therefore providing a powerful and versatile tool for assessing “physiologically active” Angiotensin II. Investigation of healthy volunteers receiving different RAS blockers revealed, that the ratios between metabolically related peptides directly reflect the activity of the corresponding enzymes. The relative differences between stabilized and equilibrium angiotensin levels were peptide specific, with Angiotensin II showing an up to 20-fold increase from “stabilized“ to “equilibrium”, while Angiotensin I changes were limited to a 5-fold increase, supporting the reported prominent role of Angiotensin II in acting on tissues via its receptors. A strong correlation between equilibrium and stabilized angiotensin levels was observed, suggesting equilibrium Angiotensin II to be a potential surrogate marker for the physiologically active portion of this important hormone.

We further investigated equilibrium angiotensin levels in primary aldosteronism (PA). Importantly, the Aldosterone-to-Ang II-Ratio (AA2-Ratio) has a strong potential to be superior to the ARR in terms of its ability to discriminate between PA and other forms of hypertension, better reflecting the activity of the true direct circulating regulator of aldosterone secretion (i.e. angiotensin II rather than renin), and being less affected by antihypertensive medications including ACE inhibitors. First data obtained in a proof-of-concept study investigating PA positive and negative patients proved the AA2-Ratio to be a powerful and cost-effective diagnostic tool for the diagnosis of PA among hypertensive patients.

The availability of RAS equilibrium analysis as a versatile and clinically applicable diagnostic tool for molecular characterization of the soluble RAS might pave the way for angiotensin peptides to be used in clinical routine testing by solving key pre-analytic issues in terms of analyte stability.

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