Abstract 3385: Cyclic GMP Activating Actions and Assay Detection of Altered Forms of BNP in vitro
Background: We recently reported with mass spectrometry that biologically active BNP-32 that functions via the second messenger cGMP may be absent in plasma of some humans with severe heart failure (HF). This recent observation supports the speculation that different forms of BNP with potentially differential activity may circulate in human HF and be detected by conventional assays. Here we evaluated cGMP production in cultured cardiac fibroblasts (CF) of four BNP forms reported to circulate in human HF with the hypothesis that some would demonstrate reduced cGMP activating properties. We also determined the ability of three widely used conventional assays to detect these BNP forms in vitro.
Methods: We determined the ability of proBNP (1–108), NT-proBNP (1–76) and BNP-30 (79–108) on an equimolar basis to activate cGMP in cultured CFs compared to the biologically active mature BNP-32 (77–108). We also assessed the ability of three commercially available assays (Roche [NT-proBNP]; Biosite [BNP-32]; and Shionogi[BNP-32]) to detect these four forms of BNP which were spiked into normal human plasma
Results: In cultured CFs, BNP-32 (10 – 6M) activated cGMP (0.126±0.013 pmol/ml). BNP-30 demonstrated a similar cGMP activating property (0.137±0.004 pmol/ml, p<0.05). In contrast, the cGMP response to proBNP 1–108 (0.06±0.006 pmol/ml) and NT-proBNP 1–76 (0.05±0.004 pmol/ml) was not different from untreated CFs and was significantly reduced compared to BNP-32 (p<0.05). The Roche NT-proBNP assay detected both NT-proBNP 1–76 and proBNP 1–108 while Biosite BNP-32 and Shionogi BNP-32 assays detected mature BNP-32, BNP-30 and proBNP 1–108.
Conclusion: These findings demonstrate differential cGMP activating properties of BNP forms in vitro and importantly that proBNP 1–108 and NT-proBNP 1–76 have reduced cGMP activity in vitro which may have biological relevance to human HF. We also report that widely used commercial assays for NT-proBNP and BNP-32 cannot differentiate between pro, processed or degraded forms and thus may not thoroughly identify circulating BNP forms in human HF.