Depletion of proBNP1-108 in patients with heart failure prevents cross-reactivity with natriuretic peptides.

BACKGROUND: After synthesis by cardiomyocytes, precursor proBNP1-108 is cleaved into NT-proBNP and BNP. Recently, cross-reactivity between these assays was discussed. The aim of this study was to characterize the cross-reactivities, through a new biochemical innovative approach consisting in the total depletion of the circulating proBNP1-108 in patients with heart failure (HF). METHODS: This prospective study included 180 patients with chronic HF. BNP and NT-proBNP were dosed with commercial kits. ProBNP1-108 was determined using an ELISA research assay specific to the precursor. ProBNP1-108 depletion was performed by immunocapture with a specific antibody targeting exclusively the ProBNP1-108 hinge region. ProBNP1-108, BNP and NT-proBNP levels were determined before and after depletion using this process in HF patients. RESULTS: Mean age was 74.34 +/-12.5 y, and 69% of patients were males. NYHA classes II and III were the most frequent (32% and 45% respectively). Before depletion, ProBNP1-108, NT-proBNP and BNP levels were 316.8+/-265.9 pg/ml; 6,054.0+/-11,539 pg/ml and 684.3+/-82.1 pg/ml respectively, and were closely correlated with NHYA classes. After immuno-depletion, proBNP1-108 was decreased in mean by 96% (p<0.0001), BNP by 53% (p<0.0001) and NT-proBNP by 5%. The relationship between BNP or NT-proBNP and NHYA classes remained unchanged. CONCLUSION: Current BNP and NT-proBNP assays measured as well proBNP molecule. This cross reactivity percentage has been controversial. Thanks to the removal of circulating proBNP1-108 with our immunodepletion process, we are now able to assess the remaining "true" BNP and NT-proBNP molecules and further evaluate their clinical relevance.

Processing-independent analysis for pro-C-type natriuretic peptide.

C-type natriuretic peptide (CNP) is expressed in several human tissues. We designed a specific processing-independent assay for proCNP-derived products and quantitated the concentrations in human seminal plasma from normal and vasectomized men. Antibodies were raised against the N-terminus of human proCNP 11-27. Samples were incubated with trypsin prior to immunoassay, which allows for the measurement of "total" proCNP irrespective of the degree of post-translational processing. Seminal plasma from normal young men and vasectomized men were collected and quantitated; the molecular heterogeneity was evaluated by gel chromatography. The antiserum displayed high binding affinity. Preanalytical trypsin treatment fully exposed the proCNP 11-27 epitope detected by the antiserum. Seminal plasma from healthy men (n=120) contained ~8-fold higher proCNP concentrations compared to blood plasma (range 104-933 pmol/L, age 18-25 years); gel chromatography suggested the presence of several molecular forms. Parameters associated to male fertility, proCNP concentrations in blood plasma and time of abstinence did not correlate to the seminal proCNP concentrations. Measurement in vasectomized men disclosed seminal proCNP concentrations similar to non-vasectomized men (range 107-705 pmol/L, age 34-44 years). Taken together, our new proCNP assay shows that proCNP is abundantly present in human seminal plasma and that seminal proCNP is secreted from the prostate gland and/or the seminal vesicles.

Identification of amino-terminal pro-C-type natriuretic peptide in human plasma.

We report the first identification of a circulating peptide from the amino-terminal end of proCNP. A specific radioimmunoassay was established based on antisera to the synthetic peptide proCNP(1-15). Extracts of plasma, drawn from patients with congestive heart failure or from sheep with experimental heart failure, were subjected to size exclusion and reverse-phase high-pressure liquid chromatography (HPLC) coupled to radioimmunoassay (RIA). These studies revealed the presence of an immunoreactive peptide with a molecular weight (M(r) approximately 5 kDa) similar to that expected for NT-proCNP(1-50), a potential fragment released during processing of pro(CNP). The same material was isolated from extracts of homogenized ovine pituitary, a tissue known to be a relatively enriched source of CNP. Plasma NT-proCNP levels in 22 patients with congestive heart failure (9.7 +/- 0.5 pmol/L, mean +/- SEM, range 5.4-13.7 pmol/L) were raised (P = 0.003) compared to those in 16 healthy volunteers (7.4 +/- 0.3 pmol/L, range 5.7-10.7 pmol/L) and were higher than levels reported for CNP in similar subjects. This first identification of circulating NT-proCNP opens the possibility of studying the factors regulating CNP production and metabolism in vivo.