Characterization of molecular forms of N-terminal B-type natriuretic peptide in vitro.

BACKGROUND: The heterogeneity of circulating peptides may influence the interpretation of results from N-terminal profragment of BNP (NT-proBNP) assays. Our objective was to characterize the heterogeneity for better usability of the assays. METHODS: Endogenous proBNP was purified from patient samples and treated with trifluoromethanesulfonic acid (chemical deglycosylation). The human proBNP gene was introduced into rat hearts by adenoviral transfer. Cell lysates and plasma samples containing proBNP-derived peptides were analyzed by chromatography. The fate of exogenous recombinant NT-proBNP added to fresh whole blood samples was followed by immunoassays and chromatography. The main NT-proBNP components were isolated and identified by mass spectrometry. RESULTS: Immunoreactive NT-proBNP in human plasma comprised several molecular forms, as did circulating immunoreactive human NT-proBNP after adenoviral transfer of human proBNP cDNA into rat ventricular myocardium. Incubation of recombinant NT-proBNP(1-76) in human plasma or serum resulted in multiple components with the 2 major components identified as NT-proBNP(1-36) and NT-proBNP(1-62/64). Profiling by different antisera and chromatography indicated masking of the non-mid-region epitopes likely due to formation of oligomers. More than 75% of the original immunoreactivity in the mid-region epitope was retained after 3-week storage of plasma samples at room temperature. CONCLUSIONS: There is marked heterogeneity in immunoreactive NT-proBNP in plasma not related to glycosylation. The mid-region epitope of NT-proBNP is stable even in harsh storage conditions. Careful choice of antibody epitopes can yield extraordinarily robust assays.

Increased fetal cardiac natriuretic peptide secretion in type-1 diabetic pregnancies.

BACKGROUND: We hypothesised that human fetal cardiac natriuretic peptide secretion is increased in type-1 diabetic pregnancies with normal placental hemodynamics, and this increase is related to the first trimester maternal glycemic control. METHODS: Thirty-two neonates of type-1 diabetic mothers and 60 neonates born after uncomplicated pregnancy and labour (control group) were included in this study. Diabetic pregnancies were divided into two subgroups based on the first trimester HbA1c value. Group 1 pregnancies (n =22) had a good glycemic control (HbA1c <7.5%) and Group 2 pregnancies (n =10) had a poor glycemic control (HbA1c > or =7.5%). At delivery, an umbilical artery (UA) blood sample was obtained for assessment of N-terminal peptide of proatrial (NT-proANP) and proB-type (NT-proBNP) natriuretic peptide levels. In diabetic pregnancies, each fetus had normal UA Doppler velocimetry for gestational age. RESULTS: The newborn UA NT-proANP and NT-proBNP concentrations were significantly higher in type-1 diabetic pregnancies than in the control group. Group 2 fetuses with poor glycemic control showed significantly higher UA NT-proANP levels than Group 1 fetuses. UA NT-proANP levels correlated significantly with maternal HbA1c values in the first, second and third trimesters, while UA NT-proBNP levels did not correlate with maternal HbA1c values. CONCLUSIONS: In type-1 diabetic pregnancies, fetal cardiac natriuretic peptide secretion is increased, even in the presence of good glycemic control and normal placental hemodynamics. In addition, fetal NT-proANP levels are already related to maternal glycemic control in the first trimester of pregnancy.

N-terminal probrain natriuretic peptide level inversely correlates with cardiac index after arterial switch operation in neonates.

BACKGROUND: Natriuretic peptide levels are associated with cardiac output and ventricular function. We hypothesized that concomitant measurement of the peptide fragments and the hemodynamic parameters could elucidate the associations of these parameters after pediatric cardiac surgery. METHODS: After approval of the institutional review board and parents' informed consent, we investigated the clinical data of eight neonates undergoing correction of transposition of the great arteries. We measured the level of N-terminal fragments of prohormones of atrial and brain natriuretic peptides (NT-proANP, NT-proBNP) preoperatively, postoperatively and 12, 24, 48, and 72 h after arrival in the intensive care unit. The hemodynamic status was assessed by transpulmonary thermodilution at the same time points. Creatinine and other laboratory values were analyzed in the first 48 h postoperatively. RESULTS: NT-proBNP levels were inversely correlated with cardiac index (CI, r = -0.47, P = 0.030), stroke volume index (r = -0.65, P = 0.005), and global end-diastolic volume index (GEDI; r = -0.63, P = 0.011). There was strong inverse correlation between the change of NT-proBNP levels and the change of CI between two consecutive measurements during the postoperative period (r = -0.79, P = 0.001). The NT-proBNP level 12 h after surgery was strongly correlated with the creatinine level of the postoperative 24th hour (r = 0.81, P = 0.014). CONCLUSIONS: NT-proBNP correlated with the hemodynamic parameters and with the severity of renal dysfunction. Therefore, NT-proBNP is a reliable indicator of the circulatory state and the severity of a low output syndrome after arterial switch operation in neonates.

Plasma N-terminal fragments of natriuretic propeptides predict the risk of cardiovascular events and mortality in middle-aged men.

AIMS: The prognostic significance of N-terminal pro-A-type (NT-proANP) and pro-B-type natriuretic peptides (NT-proBNP) is not well documented in population-based prospective studies. We, therefore, studied if both NT-proANP and NT-proBNP are predictive for overall death, cardiovascular events, and atrial fibrillation (AF) among middle-aged men without heart failure or AF at baseline. METHODS AND RESULTS: Plasma NT-proANP and NT-proBNP were measured in a representative population-based sample of 905 men (age 46-65 years) from eastern Finland. There were 110 deaths [58 cardiovascular and 40 coronary heart disease (CHD)] and 59 cases of AF during a follow-up of 10 years. The multivariable adjusted risk for overall was 1.35-fold (95% CI 1.15-1.57) and 1.52-fold (95% CI 1.21-1.91) for CHD death for each SD (160.8 pmol/L) increment in NT-proANP. The respective risks were 1.26-fold (95% CI 1.12-1.42) and 1.44-fold (95% CI 1.22-1.60) for each SD (58.9 pmol/L) increment in NT-proBNP. The adjusted risks for future AF were 1.46 (P<0.001) and 1.72-fold (P<0.001) for each SD increment in NT-proANP and NT-proBNP, respectively. CONCLUSION: The main finding of the present study is that NT-proANP and NT-proBNP are strong predictors of death from cardiovascular and other causes including AF. These natriuretic peptides add to the prognostic value of conventional risk factors and provide a non-invasive measure for identifying men with high risk of death and its co-morbidities.

BNP as a biomarker in heart disease.

Heart ventricles produce B-type natriuretic peptide (BNP) in response to increased mechanical load and wall stretch. BNP protects the heart from adverse consequences of overload by increasing natriuresis and diuresis, relaxing vascular smooth muscle, inhibiting the renin-angiotensin-aldosterone system, and by counteracting cardiac hypertrophy and fibrosis. BNP is synthesized by human cardiac myocytes as a 108-amino acid prohormone (proBNP), which is cleaved to the 32-residue BNP and the 76-residue N-terminal fragment of proBNP (NT-proBNP). Both can be used as sensitive biomarkers of cardiac dysfunction and well-characterized commercial assays have recently become available. In acute coronary syndromes increased concentrations are strong predictors of recurring myocardial infarction, heart failure, and death. In acute dyspnea, high BNP and NT-proBNP point to a cardiac rather than a pulmonary origin of the symptoms. BNP and NT-proBNP help in the assessment of the severity of ventricular dysfunction and heart failure and as a prognostic predictor, regardless of the primary cause of the condition. They can be used to guide the therapy of heart failure and left ventricular dysfunction. BNP and NT-proBNP work better when they are used for specific clinical purposes, rather than for screening in the general population. Their main strength is the excellent negative predictive value with regard to left ventricular dysfunction and heart failure. BNP and NT-proBNP are nonspecific biomarkers of cardiac dysfunction. Specific diagnostic tools, such as echocardiography, are required to define the actual abnormality.

Single assay for amino-terminal fragments of cardiac A- and B-type natriuretic peptides.

BACKGROUND: High circulating concentrations of N-terminal fragments of A- and B-type natriuretic peptides (NT-proANP and NT-proBNP) identify patients with impaired cardiac function. ProANP-derived peptides are particularly sensitive to increased preload of the heart and proBNP-derived peptides to increased afterload; therefore, combining the information from the ANP and BNP systems into a single analyte could produce an assay with increased diagnostic and prognostic power. METHODS: We prepared a hybrid peptide containing peptide sequences from both NT-proBNP and NT-proANP (referred to as NT-proXNP) by recombinant techniques and used it to develop a RIA combining weighed concentrations of NT-proANP and NT-proBNP into a new virtual analyte, NT-proXNP. We used the novel method to measure the circulating concentrations in healthy persons and in patients with cardiac disorders. We also characterized the assay by HPLC analysis of the immunoreactive molecular forms in human plasma and serum. RESULTS: The results from the novel assay correlated well with independent home-made NT-proANP and NT-proBNP assays (r2 = 0.75-0.85) as well with the arithmetic sum of NT-proANP and NT-proBNP (r2 = 0.92). Patients with valvular heart disease (VHD) and coronary artery disease (CAD) had significantly increased NT-proXNP concentrations. The areas under the curve (AUC) of the NT-proXNP assay in detecting VHD and CAD (0.961 and 0.924, respectively) were significantly larger than the AUC of either NT-proANP (0.947 and 0.872) or NT-proBNP (0.913 and 0.782) assays. HPLC analysis showed that the novel NT-proXNP assay detects two major classes of circulating immunoreactivity corresponding to peptides derived from NT-proANP and NT-proBNP. CONCLUSIONS: Our novel immunoassay mimics the physiologic signaling system working in the body by converging the information obtained from the activation of ANP and BNP into a single virtual analyte, NT-proXNP. It appears to have a diagnostic efficiency equal to or slightly better than that of individual NT-proANP or NT-proBNP assays.

Molecular heterogeneity has a major impact on the measurement of circulating N-terminal fragments of A- and B-type natriuretic peptides.

BACKGROUND: The N-terminal fragments of A- and B-type natriuretic peptides (NT-proANP and NT-proBNP) are powerful markers of cardiac function. The current assays require refinement with regard to standardization with native calibrators and the ability to detect the actual circulating forms. METHODS: The following peptides were prepared with recombinant methods: NT-proANP, NT-proBNP, proBNP1-108, and Tyr0-proBNP77-108. Fifteen peptides of 13-22 amino acids, spanning the sequences of NT-proANP and NT-proBNP, were prepared by solid-phase peptide synthesis. Two immunoassays for NT-proANP and four for NT-proBNP were set up, each with a different epitope specificity. The assays were applied for the measurement of NT-proANP and NT-proBNP in healthy individuals and in patients with acute myocardial infarction. The circulating molecular forms were analyzed by gel-filtration and reversed-phase HPLC. RESULTS: According to the HPLC analyses, circulating NT-proANP consists mainly of the full-length peptide, with some degradation at both ends. In contrast, circulating NT-proBNP is very heterogeneous. Most immunoreactive NT-proBNP is significantly smaller in size than NT-proBNP1-76, with truncation at both termini. The smallest fragments can be detected by assays directed at the central part of NT-proBNP only; assays directed at the ends gave 30-40% lower values. Despite the difference, the various assays correlated reasonably well with each other (r2 = 0.77-0.85). In patients with acute myocardial infarction, NT-proANP and NT-proBNP concentrations were 1.8-2.3 and 4.2-4.5 times higher than in healthy individuals. The development of heart failure further increased the concentrations. CONCLUSIONS: Molecular heterogeneity of the circulating forms causes a serious risk of preanalytical errors in assays for NT-proBNP and, to a lesser extent, NT-proANP. The development of a sandwich assay for NT-proBNP would be especially challenging. The most robust and reliable assays use antibodies directed at the central portions of NT-proANP or NT-proBNP.