Measurement of immunoreactive angiotensin-(1-7) heptapeptide in human blood

Background: The renal enzyme renin cleaves from the hepatic alpha (2)-globu lin angiotensinogen angiotensin-(1-10) decapeptide [Ang-(1-10)], which is f urther metabolized to smaller peptides that help maintain cardiovascular ho meostasis. The Ang-(1-7) heptapeptide has been reported to have several phy siological effects, including natriuresis, diuresis, vasodilation, and rele ase of vasopressin:and prostaglandins. Methods: To investigate Ang-(1-7) in clinical settings, we developed a meth od to measure immunoreactive (ir-) Ang-(1-7) in;2 mt of human blood and to estimate plasma concentrations by correcting for the hematocrit. A:sensitiv e and specific antiserum against Ang-(1-7) was raised in a rabbit. Human bl ood was collected in the presence Of an inhibitor mixture including a renin inhibitor to prevent peptide generation in vitro. Ang(1-7) was extracted i nto ethanol and purified on phenylsilylsilica. The peptide was quantified b y radioimmunoassay. Increasing doses of Ang-(1-7) were infused into volunte ers, and plasma concentrations of the peptide were measured. Results: The detection limit for plasma ir-Ang-(1-7) was I:pmol/L. CVs for high and low blood concentrations were 4% and: 20%, respectively, and betwe en-assay CVs were 8% and 13%, respectively. Reference values for human plas ma 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 eff icient inhibition of enzymes that generate or metabolize Ang-(1-7) after bl ood sampling, extraction in ethanol, and purification on phenylsilylsilica, and by use of a specific antiserum. (C) 2001 American Association for Clin ical Chemistry.