Measurement of Immunoreactive Angiotensin-(1–7) Heptapeptide in Human Blood

Background: The renal enzyme renin cleaves from the hepatic α2-globulin angiotensinogen angiotensin-(1–10) decapeptide [Ang-(1–10)], which is further metabolized to smaller peptides that help maintain cardiovascular homeostasis. The Ang-(1–7) heptapeptide has been reported to have several physiological effects, including natriuresis, diuresis, vasodilation, and release of vasopressin and prostaglandins. Methods: To investigate Ang-(1–7) in clinical settings, we developed a method to measure immunoreactive (ir-) Ang-(1–7) in 2 mL of human blood and to estimate plasma concentrations by correcting for the hematocrit. A sensitive and specific antiserum against Ang-(1–7) was raised in a rabbit. Human blood was collected in the presence of an inhibitor mixture including a renin inhibitor to prevent peptide generation in vitro. Ang-(1–7) was extracted into ethanol and purified on phenylsilylsilica. The peptide was quantified by radioimmunoassay. Increasing doses of Ang-(1–7) were infused into volunteers, and plasma concentrations of the peptide were measured. Results: The detection limit for plasma ir-Ang-(1–7) was 1 pmol/L. CVs for high and low blood concentrations were 4% and 20%, respectively, and between-assay CVs were 8% and 13%, respectively. Reference values for human plasma concentrations of ir-Ang-(1–7) were 1.0–9.5 pmol/L (median, 4.7 pmol/L) and increased linearly during infusion of increasing doses of Ang-(1–7). Conclusions: Reliable measurement of plasma ir-Ang-(1–7) is achieved with efficient inhibition of enzymes that generate or metabolize Ang-(1–7) after blood sampling, extraction in ethanol, and purification on phenylsilylsilica, and by use of a specific antiserum.