Simultaneous separation of angiotensin and endothelin peptide families by high-performance liquid chromatography: application to the specific radioimmunoassay measurement of angiotensin II or endothelin-1 from tissue.

Currently available measurements of endogenous angiotensin II (ANG II) and endothelin-1 (ET-1) concentrations by radioimmunoassay (RIA) lack specificity to ANG II or ET-1. ANG II and ET-1 antibodies cross-react with immune-reactive angiotensin and endothelin family members, respectively. We have therefore developed an ion-pair reversed-phase high-performance liquid chromatography (HPLC) for simultaneously separating angiotensin and endothelin peptides and enhancing RIA specificity in the measurement of ANG II and ET-1. The developed HPLC separation was applied to canine myocardium extracts; ANG II or ET-1 fractions were collected and quantified by RIA. Elution times for both peptide families, ANG I, ANG LI, ANG III, ANG IV, ANG II(4-8), bET-1, ET-1, ET-2 and ET-3 were within 25 min. In normal canine myocardium from the right atrium, right ventricle, left atrium and left ventricle, ANG II concentrations were 39+/-11, 28+/-21, 31+/-11 and 21+/-8 fmol/g and ET-1 concentrations were 43+/-16, 42+/-19, 55+/-21 and 57+/-34 fmol/g (mean+/-SD, N=7), respectively. The combination of HPLC with RIA renders the measurement of ANG II or ET-I specific and convenient, and saves time. This HPLC separation may be applied to the specific measurement of other immune-reactive angiotensin and endothelin peptides. (C) 2000 Elsevier Science B.V. All rights reserved.