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 radioimmunoassays. 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, mean +/- s.e.m., n = 14): Ang-(1-7) 1.0 +/- 0.2, Ang II 13.9 +/- 2.0, Ang-(1-9) less than 0.4, Ang I 19.5 +/- 2.4, Ang-(2-7) less than 1.1, Ang III 2.9 +/- 1.0, Ang-(2-9) less than 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 less than 0.001). Furthermore, these patients showed a ninefold increase in Ang-(1-7) to 9.7 +/- 4.3 fmol/ml (P less than 0.001), indicating a role for prolylendopeptidase in the metabolism of Ang I in vivo. These N-terminal assays have demonstrated that carboxy-truncated metabolites of Ang I and Ang II make little contribution to plasma angiotensin peptides, except during ACE inhibitor therapy. Furthermore, these antisera allow the measurement of Ang I and Ang II in the same radioimmunoassay of fractions from HPLC, providing a highly reliable estimate of the Ang II:Ang I ratio.