Specific and non-specific measurements of tissue angiotensin II cascade members

Although angiotensin II (ANG II) has been the focal regulatory peptide of the renin-angiotensin system, its proteolytic fragments have recently been demonstrated to have biological effects. Conventional measurement of angiotensins involves radioimmunoassay (RIA), which is a sensitive binding technique capable of measuring low physiological concentrations. However, ANG II antibody cross-reacts with ANG II and its fragments (ANG II cascade), rendering RIA measurement alone to be a non-specific measure of immunoreactive ANG II (ir-ANG II). On the other hand, high-performance liquid chromatography (HPLC) is capable of separating immunoreactive ANG II cascade members, but may not be sensitive enough to detect these low peptide concentrations often present in biological samples. Consequently, a reverse-phase HPLC method, with triethylammoniun formate as an ion-pair reagent, was developed to separate ANG II and its fragments, ANG III, ANG IV and ANG V. This HPLC separation was applied to extracts from normal canine hearts and ANG II cascade immunoreactive fractions were collected. Collected fractions were quantified by RIA, with the use of separate standard curves. The isocratic HPLC separation of ANG II, ANG III, ANG IV and ANG V was achieved in less than 5 min with adjacent peaks having baseline resolution. Measured cardiac left ventricle ANG III, ANG IV and ANG V concentrations (mean+/-SD) were 5.3+/-2.2,4.0+/-1.0 and 3.1+/-1.0 fmol/g (n=9), respectively. There was a significant difference (P=0.003, n=9) between left ventricular immunoreactive ANG II and 'true' ANG II, corrected for recovery rates of 86.2+/-22.5 and 53.5+/-16.2 fmol/g, respectively. We conclude that the combination of HPLC with RIA ensures the specific measurement of the ANG II cascade family members while non-chromatographic processing of tissue renders ANG II measurement non-specific. In addition, the use of triethylammonium formate as mobile phase additive is superior in the HPLC separation of the angiotensins.