Extraction of human alpha atrial natriuretic peptide and its physiological validation

Abstract

A very sensitive and specific radioimmunoassay for human alpha atrial natriuretic peptide (hANP) and a novel extraction method for hANP, have been developed. Antiserum to hANP showed no cross-reactivity with related analogues (e.g., brain natriuretic peptide). The radioimmunoassay can detect 1.2 fmol ANP/assay tube. Using a commercially available tracer, the antiserum binds 0.7 fmol of radioligand at a final dilution of 1:96 000. Production of ANP tracer, using 125I, Iodogen and reversed-phase HPLC separation, produces two products. The first has identical properties to the commercial reagent resulting in an identical antibody titre. The second, however, is more reactive with the antiserum which can be employed at a final dilution of 1:192K. These products represent oxidised and reduced peptides, respectively, inferring that the commercial tracer is oxidised. The recovery of synthetic hANP from plasma over the range of 0–1000 ng/l through Sep-Pak C 18 cartridges, using an extraction method of acetic acid–acetonitrile (4:96) was 89%. Inter- and intra-assay coefficients of variation were 9.5% and 8.2%, respectively. The radioimmunoassay was validated in man by measuring plasma ANP (ng/l) following change of posture and exercise in normal man. Plasma ANP rose from 13.2 (1.0; S.D.) to 20.1 (1.6) from supine to sitting position. Plasma ANP increased to 20.1 (1.6) at rest (sitting) to 34 (2.7) ng/l at peak of exercise, but decreased from 31.2 (2.5) to 21.4 (0.1) ng/l at 3 and 6 min after exercise, respectively. These results confirm that the assay is capable of differentiating changes of concentrations within the physiological range.