Analysis of circulating forms of proBNP and NT-proBNP in patients with severe heart failure.

BACKGROUND: The specific forms of pro-B-type natriuretic peptide (proBNP) that occur in human blood are not yet clear. We demonstrated the presence of several proBNP forms in human plasma with a new affinity chromatography method that can be used in combination with nano-liquid chromatography electrospray ionization tandem mass spectrometry (nano-LC-ESI-MS/MS). METHODS: For affinity chromatography, we coupled Fab' fragments of polyclonal sheep antibodies specific for N-terminal proBNP (NT-proBNP) epitope 1-21 to silica beads. We connected a column (10 mm x 0.8 mm inner diameter) packed with these beads to a trypsin reactor and used a preconcentrator in combination with a fritless nanospray column to perform MS analyses of proBNP forms in preextracted and non-preextracted samples of plasma from patients with severe heart failure (HF). We used Western blotting in deglycosylation experiments to confirm the shifts in proBNP and NT-proBNP masses. RESULTS: Tandem MS experiments demonstrated the presence of both NT-proBNP and circulating proBNP in preextracted samples of plasma from patients with severe HF, and Western blotting analyses revealed 2 bands of approximately 23 kDa and 13 kDa that shifted after deglycosylation to positions that corresponded to the locations of recombinant proBNP and synthetic NT-proBNP. CONCLUSIONS: We obtained clear evidence for circulating proBNP in patients with severe HF and provided the first demonstration of O-glycosylation of NT-proBNP. The higher molecular masses for NT-proBNP and proBNP observed in the Western blotting analyses than those expected from calculations can be explained by O-glycosylation of these peptides in vivo.

NT-proBNP concentrations indicate cardiac disease in pediatric patients.

OBJECTIVE: To test the performance of N-terminal pro B-type natriuretic peptide to distinguish from cardiac and non-cardiac disease in the pediatric patient population. METHOD: NT-proBNP concentrations were retrospectively analysed in 102 pediatric patients (median age: 5.96 years; 0-18 years) with cardiac diseases comprising left-to-right-shunt lesions (n=42), left heart lesions (n=47) and right heart lesions (n=13) and in 65 pediatric patients (median age: 3.37 years; 0.03-18 years) with acute infection, minor trauma or neurological disorder. RESULTS: NT-proBNP levels between patients without heart disease and patients with heart disease differed significantly with a median NT-proBNP value of 224.9 ng/l, 108.7 ng/l-945.6 ng/l (25th-75th percentile) versus 76.7 ng/l, 35.0 ng/l-122.4 ng/l, p<0.0001. The diagnostic performance of NT-proBNP to differentiate between patients with and without cardiac diseases was high with an area under curve of 0.81 (95% confidence intervals 0.75-0.87). At a cut-off value of 134 ng/l the specificity was 83% (95% CI: 74-92%). The presence of heart failure (p<0.0001) had a significant impact on NT-proBNP concentrations. CONCLUSIONS: NT-proBNP measurement is a helpful addition to identify pediatric patients with heart disease.

Utility of N-terminal pro-B-type natriuretic peptide to differentiate cardiac diseases from noncardiac diseases in young pediatric patients.

BACKGROUND: Previous studies comparing children with cardiac disease with children with lung disease or healthy children indicated that natriuretic peptides are promising markers in pediatric patients. The aim of this study was to further clarify the diagnostic usefulness of N-terminal pro-B-type natriuretic peptide (NT-proBNP) measurements in a less preselected population of children younger than 3 years, a population in which clinical symptoms are frequently unspecific. METHODS: NT-proBNP concentrations (Roche Diagnostics) were measured in sera of 142 pediatric patients (age range, 33-1070 days) presenting at the Gynaecologic and Pediatric Hospital (Linz, Austria) between January 2003 and January 2004. ROC curve analysis for the diagnostic performance of NT-proBNP, the Mann-Whitney U-test for group comparison, and linear regression analysis for influencing factors were performed. RESULTS: NT-proBNP concentrations were significantly increased in infants with cardiac diseases [median (25th-75th percentile), 3681 (1045-13557) ng/L; n = 23] compared with infants with other diseases [241 (116-542) ng/L; n = 119], and ROC analysis revealed good performance for NT-proBNP in differentiating between infants with and without cardiac diseases [mean area under the curve (AUC) with 95% confidence interval (CI), 0.87 (0.76-0.94)]. A subgroup analysis of exactly age- and sex-matched infants was performed, which revealed results comparable to those for the whole study population [mean (95% CI) AUC, 0.84 (0.68-0.93)]. CONCLUSION: In a heterogeneous group of pediatric patients < 3 years of age, NT-proBNP showed good diagnostic performance to distinguish between cardiac diseases and various noncardiac diseases.

Direct comparison of relaxation and cGMP production in human coronary by-pass grafts in response to stimulation with natriuretic peptides and a nitric oxide donor.

In the present study, we investigated the vasodilator properties of A-type, B-type and C-type natriuretic peptides (ANP, BNP and CNP respectively) and the NO (nitric oxide) donor sin-1 (3-morpholino-sydnonimine) in human by-pass grafts. In contrast with previous studies, the same vessel was used to demonstrate a direct link between cGMP production and functional relaxation. Remnants of the IMA (internal mammary artery) and SV (saphenous vein) were obtained from 82 patients undergoing coronary artery by-pass grafting. The responses to cumulative concentrations of ANP, BNP, CNP and sin-1 in vessel rings pre-contracted with a thromboxane A2 agonist (U46619) were measured in an organ bath. Additionally, intracellular cGMP production after single submaximal dose application of these drugs to vessel rings was determined by a RIA. ANP (P=0.001) and sin-1 (P<0.001) caused significant concentration-dependent relaxation of the IMA. In the SV, only sin-1 (P<0.001) induced marked concentration-dependent relaxation. At a single submaximal concentration, significant relaxation as well as intracellular cGMP production were found in response to ANP, BNP and sin-1 in the IMA. In contrast, in the SV, only sin-1 significantly induced cGMP production and relaxation. There was a moderate, but significant, correlation between intracellular cGMP net production and net relaxation in the IMA. In conclusion, ANP, as the most powerful relaxant of all the natriuretic peptides tested on the IMA, may be a possible alternative vasorelaxant to overcome peri-operative vasospasm in this artery. In contrast with sin-1, ANP and BNP were not effective vasorelaxants of the SV. Net relaxation in response to natriuretic peptides correlated with cGMP net concentrations in the IMA.

Thermal preconditioning protects the human internal mammary artery from hypoxia/re-oxygenation-induced damage.

1. Preconditioning has been demonstrated to ameliorate ischaemia/reperfusion injury in several cells and tissues. Therefore, in the present study we investigated whether preconditioning of human bypass grafts, internal mammary artery (IMA) and saphenous vein (SV) induces heat shock protein (Hsp) expression and reduces apoptosis in response to subsequent hypoxia/re-oxygenation damage in both vessels. 2. Internal mammary artery and SV rings, obtained from 30 patients (median age 66.5 years) undergoing coronary artery bypass grafting, were either incubated for 30 min at 42 degrees C (preconditioned) or kept in a standard incubator at 37 degrees C (not preconditioned). Six hours later, graft segments were exposed to 90 min hypoxia followed by a 30 min re-oxygenation period. Western blot, real-time quantative polymerase chain reaction analysis and apoptosis detection by the Terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling method were performed. 3. Heat-preconditioned IMA showed significantly increased protein expression of Hsp72 after hypoxia/re-oxygenation treatment compared with controls (median 9.1 vs 5.0 microg/mg total protein; P = 0.048). Expression of Hsp73 was weak and Hsp60 was not detectable in the IMA. 4. In the SV, neither protein nor mRNA expression of Hsp were significantly different between preconditioned and not preconditioned veins. 5. There were significantly fewer apoptotic cells in the intima of the preconditioned compared with not preconditioned IMA (P = 0.041) after hypoxia/re-oxygenation injury, whereas in the SV apoptosis was not significantly prevented by preconditioning. 6. Mild heat preconditioning before hypoxia/re-oxygenation injury is a stimulus for Hsp72 protein expression and a reduction in apoptosis in the human IMA.

B-type natriuretic peptide as a marker of allograft rejection after heart transplantation.

B-type natriuretic peptide (BNP) concentrations, a cardiac hormone released upon cardiac stress, was monitored in patients after heart transplantation. Rejection was assessed by the International Society for Heart and Lung Transplantation (ISHLT) criteria. BNP was assessed by a cross-sectional approach. We found significantly (p = 0.024) increased concentrations during rejection episodes of ISHLT grade 2 and higher. BNP yielded only a moderate diagnostic accuracy (area under the receiving characteristic curve: mean = 0.76, 95% confidence interval 0.53-0.92) to detect clinically significant episodes of rejection, which was too low to replace endomyocardial biopsies. Acute rejection episodes were associated with marked BNP increases and a significant decrease in case of successful treatment in the individual long-term monitoring in the majority of patients. BNP monitoring seems to be a useful addition in the individual follow-up of heart transplant recipients to rule out significant rejection.

Nonenzymatic glycation of histones in vitro and in vivo.

Purified histones in solution, purified nuclei, or whole endothelial cells in cell culture were used to study the reactivity of histones with various sugars. The sugar incubation of purified histones produced nonenzymatic glycation and formation of histone cross-links showing disappearance of individual histone molecules and appearance of dimers and polymers in SDS-PAGE. In solution, core histones react considerably faster with sugars as compared to H1 histones. In sugar-incubated nuclei where histones are nucleosomally organized, H1 histones, which are located at the periphery of the nucleosome, and H2A-H2B dimers, which are associated with the central H3(2)-H4(2) tetramer, are more reactive as compared to H3 and H4 histones, which are most protected from the glycation reaction. Our in vivo experiments using endothelial cells show that high concentrations of ribose are able to generate protein cross-links paralleled by apoptotic cell death. High concentrations of glucose or fructose do not increase histone glycation or cell death, even after 60 days of incubation of endothelial cells. In long-time glucose- or fructose-treated cells, under nondenaturing and nonreducing SDS-PAGE conditions part of the H3 histones shifted away from their normal location. Because it is known that the mitochondrial production of reactive oxygen species (ROS) increases after hyperglycaemia, we hypothesize that ROS could be responsible for the formation of a disulphide bridge between the side chain of the cysteine residues of H3 molecules.

Natriuretic peptides as markers of mild forms of left ventricular dysfunction: effects of assays on diagnostic performance of markers.

BACKGROUND: We compared the performance of different natriuretic peptides to diagnose mild forms of left ventricular dysfunction (LVD) and investigated the influence of measuring B-type natriuretic peptide (BNP) and N-terminal proBNP (NT-proBNP) with different assays on the diagnostic performance of these markers. METHODS: We measured BNP (Triage BNP), NT-proBNP (Biomedica), and N-terminal pro-A-type natriuretic peptide (NT-proANP; Biomedica) in 130 consecutive patients (age range, 28-83 years) with clinically suspected mild LVD. In patients with sufficient sample volume, we measured BNP and NT-proBNP with additional assays (Shionoria and Roche, respectively). RESULTS: For identifying patients with mild systolic LVD, BNP and NT-proBNP were the best markers, with mean (95% confidence interval) areas under the curves (AUC) of 0.78 (0.63-0.89) and 0.75 (0.58-0.87), respectively. However, the diagnostic performance of NT-proANP [AUC, 0.64 (0.48-0.77)] was significantly worse than that of BNP (P = 0.014). Both BNP assays (Triage and Shionoria) and both NT-proBNP assays (Biomedica and Roche) performed equally well for the diagnosis of systolic LVD despite the poor agreement between NT-proBNP assays. In patients with isolated diastolic LVD, the diagnostic performance of the Triage BNP [AUC, 0.70 (0.56-0.81)] was significantly better (P = 0.006) than that of Biomedica NT-proBNP [0.49 (0.34-0.65)]. Furthermore, the performance of the Biomedica NT-proBNP assay was significantly worse (P = 0.03) than that of the Roche NT-proBNP assay for diagnosis of isolated diastolic LVD. CONCLUSIONS: The performance of BNP for the diagnosis of systolic or diastolic LVD is not affected by the assay used, whereas the performance of NT-proBNP for the diagnosis of isolated diastolic LVD is assay dependent.