Abstract 702: Accurate Quantification Of Peptides In The Renin Angiotensin Aldosterone System By Mass Spectrometry

Abstract

Aim/Background: The Renin Angiotensin Aldosterone System (RAAS) is responsible for the regulation of vascular hemodynamics. When dysregulated, the RAAS contributes to cardiovascular pathologies, such as hypertension, which affects ~40-50% of the US population. Angiotensin (Ang) peptides are the primary effector molecules of the classical RAAS and alternative RAAS pathways. 1 The accurate quantification of these vasoactive peptides using a high throughput method may provide a better understanding of the mechanisms underlying hypertension that standard blood pressure cuff measurements cannot capture. Methods: A liquid chromatography-mass spectrometry-based method was developed to simultaneously measure four (4) angiotensin peptides (Ang (1-7), Ang (1-9), Ang I, and Ang II) in human serum. The peptides are isolated prior to measurement using a two-step precipitation followed by solid phase extraction. Peptides were quantified using stable isotope labeled peptides as internal standards. Results: Calibration curves spanning a concentration range of 1 - 1000 fmol/mL were established for all peptides. Linear regression analysis established R 2 values ≥ 0.98. Estimated method limits of detection (LOD) are at or below 5 fmol/mL for all 4 peptides. For initial assessments, RAAS peptides were quantified in 25 commercial serum samples. Median values (concentration ranges) were as follows: Ang (1-7) 3.7 fmol/mL (LOD - 37.9 fmol/mL), Ang (1-9) 3.8 fmol/mL (LOD - 16.4 fmol/mL), Ang II 82.0 fmol/mL (LOD - 549.6 fmol/mL), Ang I 27.2 fmol/mL (LOD - 324.0 fmol/mL). Ang II always occurred in the highest concentration, except for one sample where Ang I was the most abundant peptide. In most samples, Ang I was about 4 times lower, and Ang (1-9) and Ang (1-7) were about 25 times lower than Ang II. In one sample, Ang I was about 30 time higher, and Ang (1-7) was about 3 times higher than Ang II. Conclusion: This mass spectrometry method enables accurate RAAS peptide quantification in serum samples. Application of this method will lead to the generation of new data that may provide unique, mechanistic insights into hypertension, as well as renal diseases, arteriosclerosis, and other cardiovascular pathologies associated with the RAAS pathway.