Radio-immunoassay of atrial natriuretic peptide (ANP) and characterization of ANP immunoreactivity in human plasma and atrial tissue

Abstract

A sensitive radio-immunoassay (RIA) was developed to determine the occurrence of atrial natriuretic peptide (ANP) in plasma and atrial extracts from patients undergoing open heart surgery. The immunoreactive ANP (irANP) was characterized by high-pressure liquid chromatography coupled with RIA. The plasma irANP response to releasing stimuli during the operation was determined in simultaneously sampled venous and arterial blood, in order to evaluate any differences.

The antiserum recognized the intact ring-structure of α-humanANP (α-hANP) and its propeptide γ-hANP, as well as β-hANP, an anti-parallel dimer of α-hANP. Less bioactive N-or C-terminal fragments of α-hANP, or an N-terminal fragment of the propeptide, γ-hANP 1–67, did not cross-react with the antiserum. Sep Pak C₁₈-extraction of plasma resulted in an 80% recovery of synthetic α-hANP. The assay had a sensitivity of 1.9 pmol 1⁻¹, well below the venous plasma concentrations of irANP found in healthy volunteers (7.4 ± 1.3 pmol 1⁻¹, mean ± SEM, n = 19), and the local standard was identical to an international standard of α-hANP. In atrial extracts three major peaks of irANP were identified as α-, β-and γ-hANP, with γ-hANP as the most abundant form. In plasma α-hANP dominated, but in two cases high plasma levels of β-hANP were seen, reflecting the high atrial content in these patients. In peripheral arterial blood, irANP was on an average 56% ± 20% (p < 0.001, n = 18) higher than in venous blood; this was associated with more distinct arterial irANP responses to releasing stimuli during the operation.

Key Words:

• cardiopulmonary bypass
• chromatography
• high-pressure liquid
• heart atrium
• heart failure
• congestive
• peptides
• protein precursors