MSACL 2024 Abstract

High Throughput DESI Tissue Analysis of Angiotensin II Metabolism in Mice Kenneth L. Virgin (1,2), Nicolás M. Morato (2), R. Graham Cooks (2) (1) Indiana University School of Medicine, Indianapolis, IN (2) Purdue University, Lafayette, IN Kenneth Virgin, BS Biochemistry (Presenter) Purdue University, Indiana University School of Medicine >> POSTER (PDF)

Presenter Bio: Medical student at Indiana University School of Medicine doing research on intraoperative mass spectrometry and high throughput DESI MS with Aston Labs at Purdue University.

INTRODUCTION: Angiotensin metabolism is essential for blood pressure regulation. Many antihypertensive drugs target the production and action of angiotensin II. However, angiotensin II is only part of an intricate cascade that is known to induce adverse effects such as dry cough or angioedema when disrupted. A technique is needed to rapidly analyze multiple metabolites simultaneously for new drug design and potentially patient-specific treatment. High throughput desorption electrospray ionization (HT-DESI) can analyze complex solutions of small metabolites at a rate of less than 1 second per sample with no additional sample preparation necessary. We hypothesized that HT-DESI could quantitatively monitor the production of angiotensin metabolites in mouse tissue.

METHODS: We prepared mouse tissue homogenates of liver, brain, jejunum, and kidney. We added angiotensin II to each sample to saturate tissue enzymes and quenched aliquots of the reaction over the space of one hour. A 50 nL aliquot of each solution was transferred to a PTFE-coated glass slide and analyzed under ambient conditions using a Waters DESI source and QTOF Synapt G2 Si mass spectrometer. No internal standard was used.

We then calibrated angiotensin metabolites as well as bradykinin and bradykinin fragment in the range of 10 to 100 uM using DADLE as an internal standard. Calibration samples were then analyzed with HT-DESI as stated previously.

RESULTS: Angiotensin II was almost completely converted into angiotensin III, IV, and (1-7) within an hour of incubation in tissue homogenate. The relative intensity of angiotensin II metabolites was markedly different between different tissue types suggesting tissue-specific metabolism consistent with current literature. The data showed very little noise between samples, but we did not include an internal standard to quantify the concentration changes.

Subsequent calibration of angiotensin metabolites with DADLE as an internal standard showed linearity in the range of 10 to 100 uM for bradykinin R^2 = 0.994, bradykinin fragment R^2 = 0.992, angiotensin II R^2 = 0.993, angiotensin III R^2 = 0.996, angiotensin IV R^2 = 0.991, angiotensin (1-7) R^2 = 0.998. Angiotensin converting enzyme II has a Km of 10-15 uM which is in the range of our calibration.

Tissue homogenates will be reanalyzed with DADLE as an internal standard to quantify tissue enzyme kinetics with and without ACE inhibitors enalapril and lisinopril and the ARB losartan.

DISCUSSION: The present data demonstrate the ability of HT-DESI to rapidly monitor angiotensin metabolism without additional sample preparation. Additionally, HT-DESI can quantify metabolite concentration with the use of an internal standard. By analyzing the substrate metabolism in vivo, one could rapidly assess the metabolic impact of ACE inhibitors or other drugs on multiple metabolic pathways.

This technology could accelerate the design of new antihypertensive compounds as well as open the path to design patient-specific drugs based on individual metabolic variations.