Response to Do Commercially Available Assay Kits for B-Type Natriuretic Peptide Measure Pro-BNP1-108, as Well as BNP1-32?
We thank Nishikimi et al1 for their comments regarding the detection of pro-BNP1-108 by the Shionogi radioimmunoassay and the cGMP-generating activity of pro-BNP1-108 in our recent publication.
We reported that both the Shionogi assay and the Biosite assay clearly detected some but <5% of recombinant glycosylated pro-BNP1-108 when added to normal human plasma.2 In contrast, Nishikimi et al1 report that, in their unpublished studies, the Shionogi assay detected pro-BNP1-108 with only slightly reduced sensitivity as compared with BNP1-32. We do not yet know the reasons for this discrepancy, but sample handling, assay conditions, and glycosylation status of pro–B-type natriuretic peptide (BNP) may account for some of the differences. We speculate that protein folding of the long N-terminus of pro-BNP1-108 could sterically inhibit binding of antibodies directed against the ring and C-terminus of BNP1-32. However, if indeed the Shionogi assay detects a considerable amount of pro-BNP, that would strengthen our argument that current assays are not specific and that better methodologies should be used, such as mass spectrometry.3
Nishikimi et al1 state, and we fully agree, that the 17 amino-acid ring of BNP1-32 is essential for cGMP-activating actions. In our study, BNP1-32 and BNP3-32 strongly activated cGMP in both cultured cardiomyocytes and cardiac fibroblasts. In contrast, neither NT-pro-BNP1-76, which lacks the 17 amino-acid ring, nor pro-BNP1-108 significantly activated cGMP, despite the latter having an intact ring. Of note, Figure 4B of our article shows a slight increase in cGMP with pro-BNP1-108 as compared with no treatment; however, this increase did not reach statistical significance and was only ≈20% of that induced by BNP1-32; therefore, the issue at hand is likely one of statistical power. Our view is that binding of pro-BNP1-108 to the natriuretic peptide A receptor is sterically inhibited by the long N-terminus. Indeed, while Liang et al4 recently reported that pro-BNP1-108 induced cGMP generation in cultured endothelial cells and vascular smooth muscle cells, pro-BNP1-108 was ≈6 times less potent than BNP1-32.
These findings support our major points, that some of the conventional radioimmunoassays are not specific for the known molecular forms of BNP and that this may be relevant, because these forms of BNP differ in their cGMP-generating properties and, by implication, in their biological activity. Clearly, more specific methodologies than the radioimmunoassay are needed to quantitate the circulating forms of BNP, which may provide us with a better measure of secretion, enzymatic activation, biological activity, and degradation of pro-BNP–derived peptides. We agree with Nishikimi et al1 that this information could be of enormous clinical relevance.
Sources of Funding
This work was supported by grants from the National Institutes of Health (RO1 HL36634, HL0711, and PO1 HL76611) and the Mayo Foundation.
Nishikimi T, Minamino N, Horii K, Matsuoka H. Do commercially available assay kits for B-type natriuretic peptide measure pro-BNP1-108, as well as BNP1-32? Hypertension. 2007; 50: e163.
Heublein DM, Huntley BK, Boerrigter G, Cataliotti A, Sandberg SM, Redfield MM, Burnett JC Jr. Immunoreactivity and guanosine 3′5′-cyclic monophosphate activating actions of various molecular forms of human B-type natriuretic peptide. Hypertension. 2007; 49: 111–119.
Hawkridge AM, Heublein DM, Bergen HR 3rd, Cataliotti A, Burnett JC Jr, Muddiman DC. Quantitative mass spectral evidence for the absence of circulating brain natriuretic peptide (BNP-32) in severe human heart failure. Proc Natl Acad Sci. U S A. 2005; 102: 17442–17447.