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Letters to the Editor |
Department of Clinical Biochemistry, Rigshospitalet, University of Copenhagen, Denmark
aAddress correspondence to the author at: Department of Clinical Biochemistry, Rigshospitalet, 9 Blegdamsvej, DK-2100, Copenhagen, Denmark. Fax +4535454640, e-mail soerenjunge{at}gmail.com
To the Editor:
It has been difficult to establish an endocrine role for C-type natriuretic peptide (CNP). Release of CNP from the heart remains to be convincingly demonstrated (1)(2)(3)(4)(5), and data concerning CNP in cardiac disease have been conflicting, possibly because of specificity problems in measurement of CNP. All natriuretic peptides (NPs) display strong homology, and antibodies raised against one NP may consequently bind other NPs. With the relatively high plasma concentrations of A- and B-type NPs (ANP and BNP), a CNP assay with even a small degree of ANP or BNP cross-reactivity may produce falsely high CNP concentrations. This is especially relevant in heart failure with augmented cardiac ANP and BNP expression. We examined the selectivity of a widely used CNP RIA.
To evaluate cross-reactivity in the CNP assay, we measured serial dilutions of human CNP-22 (4.5–582 pmol/L), ANP-28 (5–100 000 pmol/L), and BNP-32 (4–81 000 pmol/L) against the calibrators of the CNP assay. We obtained human cardiac tissue samples from a heart failure patient undergoing cardiac transplantation. The local ethics committee approved the use of human tissue, and written informed consent was obtained from the patient before surgery. The extracts were subjected to measurements of CNP, BNP, NT-proBNP, and NT-proCNP using a commercial CNP RIA (Phoenix), a BNP assay (Shionoria), and in-house RIAs directed against the N-terminus of proBNP and proCNP, respectively.
Results are expressed as mean (SE). We used nonlinear regression analysis to fit 4-parameter logistic models to the data from the buffer experiments. Based on the model, we calculated the cross-reactivity as a function of BNP-32 concentration using a 1-phase exponential decay model. Likewise, we calculated cross-reactivity as a function of the BNP concentration by fitting data from the heart extract experiments to a 1-phase exponential decay model. Cross-reactivity was calculated as measured CNP concentration divided by measured BNP concentration.
Serial dilutions of human CNP-22, BNP-32, and ANP-28 in barbital buffer measured with the CNP RIA revealed cross-reactivity for BNP-32 in physiological concentrations (Fig. 1A
). The dilution curves for CNP-22 and BNP-32 were not parallel; cross-reactivity increased with decreasing BNP-32 concentrations, being 5.1% at a BNP-32 concentration of 100 pmol/L and 1.6% at 1000 pmol/L (Fig. 1B
). As expected, BNP and NT-proBNP concentrations were high in the atria and lower in the ventricles (Fig. 1C
). NT-proCNP and particularly CNP concentrations were very low in both atria and ventricles (Fig. 1D
). With only trace amounts of NT-proCNP in the extracts, most of the measured CNP represents cross-reacting BNP. Calculating cross-reactivity as CNP concentrations divided by BNP concentrations in the extracts, we fitted a curve parallel to that obtained in the buffer experiment (Fig. 1B
). Cross-reactivity was estimated to be 3.2% at a BNP concentration of 100 pmol/L and 1.1% at 1000 pmol/L.
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Nearly all reports on CNP measurement in plasma are based on the same RIA. According to the manufacturer, the assay does not exhibit any cross-reactivity with ANP or BNP. Nevertheless, our data indicate substantial BNP cross-reactivity of the CNP assay in buffer with added BNP-32 and in heart extracts (Fig. 1B
). Ideally, chromatographic evaluation of the extracts could resolve the BNP and CNP forms, as coelution of CNP and BNP immunoreactivity would have provided further proof of BNP cross-reactivity. However, the CNP concentration in the heart extracts did not exceed 15 pmol/L, rendering chromatography very difficult. The difference in the degree of cross-reactivity between the buffer with added BNP-32 and the unmodified heart extracts may be partly because immunoreactive BNP in the extracts comprises both BNP-32 and intact proBNP, which may have another cross-reactivity profile. Alternatively, BNP measured by the Shionoria assay may partly have been cross-reacting ANP, which is highly expressed in the cardiac atria. Because N-terminal proCNP, proANP, and proBNP have virtually no sequence resemblance, cross-reactivity of the NT-proCNP assay with proANP and proBNP is highly unlikely. If intact proCNP(1-103) is present in the heart, the NT-proCNP assay would probably also measure it.
The CNP assay may still be valuable for CNP measurement in samples with low BNP concentrations; however, measurements done in samples with a high BNP concentration—such as heart tissue extracts or plasma samples from patients with heart failure—should be interpreted cautiously.
Acknowledgments
Grant/support Funding: This study was supported by a research fellowship position from the Lundbeck Foundation.
Financial Disclosures: None declared.
Acknowledgment: The expert technical assistance of Jan Kirkeby Simonsen is gratefully appreciated.
References
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