Measurement of the total proANP product in mammals by processing independent analysis

The cellular processing of natriuretic propeptides is attenuated in heart disease, resulting in release of a mixture of unprocessed precursor, partially processed fragments, and the bioactive hormone. Here, we report a species-independent method for quantification of pro-atrial natriuretic peptide (proANP) and its products irrespective of variable post-translational processing. The processing-independent assay (PIA) was developed raising mono-specific antibodies against the C-terminus of sequence 1–16 in proANP. The assay procedure included plasma extraction followed by tryptic cleavage, which releases the assay epitope from the N-terminal region. The PIA was tested in elderly patients with symptoms of heart failure (n = 450), in pigs with acute myocardial infarction (n = 21), and in normal dogs and dogs with heart failure (n = 77). The epitope specificity permitted reliable measurement in man, dog, cat and pig. In human plasma, the PIA correlated well with an established proANP analysis (r = 0.86, P < 0.0001) but with a 5.5-fold difference in plasma level (P < 0.0001). In pigs, the PIA measured 9.2-fold higher concentrations compared to a human assay (804 versus 87 pmol/L, P < 0.0001). The basal proANP concentration was 396 pmol/L in dogs: a dramatic increase was seen in canine heart failure. Our new processing- and species-independent proANP assay allows for the measurement of the total proANP product, irrespective of changes in post-translational maturation. We suggest that this tool should be used for comparative studies between human patients and porcine and canine models of human cardiac disease.

► We report a method for quantification of proANP and its products irrespective of variable post-translational processing.
► Our new proANP assay allows for measurement irrespective of changes in post-translational maturation.
► The epitope specificity permits reliable measurement in man, dog, cat and pig.
► This tool should be used for comparative studies between human, porcine and canine cardiac disease.