An alternative strategy for the radioimmunoassay of angiotensin peptides using amino-terminal-directed antisera: measurement of eight angiotensin peptides in human plasma

Abstract

We describe here a method of measuring angiotensin peptides and their carboxy-truncated metabolites in human plasma using N-terminal-directed antisera. Antisera raised against N-acetylated angiotensin (Ang) II and N-acetylated Ang III analogues were used to develop two radioimmunoassay. Extracted plasma samples were acetylated prior to separation of cross-reacting angiotensin peptides by high-performance liquid chromatography (HPLC). Fractions were assayed with both antisera to obtain measurements for eight angiotensin peptides. Angiotensin levels measured in normal males were (fmol/ml plasma means .+-. s.e.m., n = 14): Ang-(1-7) 1.0 .+-. 0.2, Ang II 13.9 .+-. 2.0, Ang-(1-9) < 0.4, Ang I 19.5 .+-. 2.4, Ang-(2-7) < 1.1, Ang III 2.9 .+-. 1.0, Ang-(2-9) < 2.1, Ang-(2-10) 2.4 .+-. 0.8. Hypertensive patients receiving angiotensin converting enzyme (ACE) inhibitor therapy (n = 8) had an increase in Ang I to 187.3 .+-. 107.2 fmol/ml (P = 0.002), and a reduction in Ang II to 4.8 .+-. 1.2 fmol/ml (P < 0.001). Furthermore, these patients showed a ninefold increase in Ang-(1-7) to 9.7 .+-. 4.3 fmol/ml (P < 0.001), indicating a role for prolylendopeptidase in the metabolism of AngI in vivo. The N-temrinal assays have demonstrated that carboxy-trucated metabolites of AngI and AngII make little contribution to plasma angiotensin peptides, except during ACE inhibitor therapy. Furthermore, these antisera allow the measurement of AngI and AngII in the same radioimmunoassay of fractions from HPLC, providing a highly reliable estimate of the AngII:AngI ratio.