Direct Immunochemiluminescent Assay for proBNP and Total BNP in Human Plasma proBNP and Total BNP Levels in Normal and Heart Failure.

[Background]Recent studies have shown that in addition to brain (or B-type) natriuretic peptide (BNP) and the N-terminal proBNP fragment, levels of intact proBNP are also increased in heart failure. Moreover, present BNP immunoassays also measure proBNP, as the anti-BNP antibody cross-reacts with proBNP. It is important to know the exact levels of proBNP in heart failure, because elevation of the low-activity proBNP may be associated with the development of heart failure. [Methodology/Principal Findings]We therefore established a two-step immunochemiluminescent assay for total BNP (BNP+proBNP) and proBNP using monoclonal antibodies and glycosylated proBNP as a standard. The assay enables measurement of plasma total BNP and proBNP within only 7 h, without prior extraction of the plasma. The detection limit was 0.4 pmol/L for a 50-µl plasma sample. Within-run CVs ranged from 5.2%–8.0% in proBNP assay and from 7.0%–8.4% in total BNP assay, and between-run CVs ranged from 5.3–7.4% in proBNP assay and from 2.9%–9.5% in total BNP assay, respectively. The dilution curves for plasma samples showed good linearity (correlation coefficients = 0.998–1.00), and analytical recovery was 90–101%. The mean total BNP and proBNP in plasma from 116 healthy subjects were 1.4±1.2 pM and 1.0±0.7 pM, respectively, and were 80±129 pM and 42±70 pM in 32 heart failure patients. Plasma proBNP levels significantly correlate with age in normal subjects. [Conclusions/Significance]Our immunochemiluminescent assay is sufficiently rapid and precise for routine determination of total BNP and proBNP in human plasma

The Effects of Super-Flux (High Performance) Dialyzer on Plasma Glycosylated Pro-B-Type Natriuretic Peptide (proBNP) and Glycosylated N-Terminal proBNP in End-Stage Renal Disease Patients on Dialysis

Background Plasma BNP levels are predictive of prognosis in hemodialysis patients. However, recent studies showed that the current BNP immunoassay cross-reacts with glycosylated proBNP, and the NT-proBNP assay underestimates glycosylated NT-proBNP. In addition, the recently developed high performance dialyzer removes medium-sized molecular solutes such as β2-microgloburin. We therefore investigated the effects of high performance dialysis on measured levels of glycosylated proBNP, glycosylated NT-proBNP and other BNP-related peptides in end-stage renal disease (ESRD) patients on hemodialysis. Method The relationships between clinical parameters and BNP-related molecule were also investigated. We used our newly developed immunoassay to measure plasma total BNP and proBNP in 105 normal subjects and 36 ESRD patients before and after hemodialysis. Plasma NT-proBNP was measured using Elecsys II after treatment with or without deglycosylating enzymes. We also measured plasma ANP and cGMP using radioimmunoassays. Results All the measured BNP-related peptides were significantly higher in ESRD patients than healthy subjects. Total BNP (?38.9%), proBNP (?29.7%), glycoNT-proBNP (?45.5%), nonglycoNT-proBNP (?53.4%), ANP (?50.4%) and cGMP (?72.1%) were all significantly reduced after hemodialysis, and the magnitude of the reduction appeared molecular weight- dependent. Both the proBNP/total BNP and glycoNT-proBNP/nonglycoNT-proBNP ratios were increased after hemodialysis. The former correlated positively with hemodialysis vintage and negatively with systolic blood pressure, while the latter correlated positively with parathyroid hormone levels. Conclusion These results suggest that hemodialysis using super-flux dialyzer removes BNP-related peptides in a nearly molecular weight-dependent manner. The ProBNP/total BNP and glycoNT-proBNP/nonglycoNT-proBNP ratios appear to be influenced by hemodialysis-related parameters in ESRD patients on hemodialysis.ArticlePLOS ONE. 9(3): e92314(2014)journal articl

Changes in plasma levels of individual parameters during hemodialysis.

<p>(A–G) Individual changes in the levels of total BNP (A), proBNP (B), mature BNP (C), nonglycoNT-proBNP (D), glycoNT-proBNP (E), ANP (F) and cGMP (G) in ESRD patients during hemodialysis. Values are means ± SE. *p<0.001 vs. before hemodialysis. Before, before hemodialysis; After, after hemodialysis. (H) Reduction ratios for total BNP, proBNP, mature BNP, nonglycoNT-proBNP, glycoNT-proBNP, ANP and cGMP in ESRD patients during hemodialysis. Values are means ± SE.</p

Recovery of standard glycosylated proBNP and BNP added to human plasma.

<p>Recovery of standard glycosylated proBNP and BNP added to human plasma.</p

Correlations between cGMP and natriuretic peptide-related molecules in ESRD patients before and after hemodialysis.

<p><i>R</i> is the correlation coefficient. Values are expressed as log₁₀ of each level of the indicated molecules.</p

Cross-reactivity between proBNP and BNP.

<p>Cross-reactivity between proBNP and BNP.</p

Changes in plasma levels of individual parameters during hemodialysis.

<p>(A–G) Individual changes in the levels of total BNP (A), proBNP (B), mature BNP (C), nonglycoNT-proBNP (D), glycoNT-proBNP (E), ANP (F) and cGMP (G) in ESRD patients during hemodialysis. Values are means ± SE. *p<0.001 vs. before hemodialysis. Before, before hemodialysis; After, after hemodialysis. (H) Reduction ratios for total BNP, proBNP, mature BNP, nonglycoNT-proBNP, glycoNT-proBNP, ANP and cGMP in ESRD patients during hemodialysis. Values are means ± SE.</p

Clinical profiles of the healthy subjects and ESRD patients enrolled in this study.

<p>Clinical profiles of the healthy subjects and ESRD patients enrolled in this study.</p

ProBNP/total BNP ratios and their correlation with the indicated clinical parameters.

<p>(A) Individual changes in proBNP/total BNP ratios in ESRD patients during hemodialysis: Before, before hemodialysis; After, after hemodialysis. (B) ProBNP/total BNP ratios in ESRD patients during hemodialysis expressed as means ± SE. (C–F) Correlation between proBNP/total BN ratios and hemodialysis vintage (C), left atrial diameter (LAD) (D), systolic blood pressure before hemodialysis (preBPs) (E) and mean blood pressure before hemodialysis (pre mBP) (F). <i>R</i> is the correlation coefficient between the indicated parameters.</p

Schematic diagram of the total BNP and proBNP assay systems.

<p>BC203(Fab') is a common capture antibody in both systems. KY-BNP-II(Fab') is the detection antibody for the total BNP assay, and 18H5(Fab') is the detection antibody for the proBNP assay. ALP: Alkaline phosphatase; CDP-Star EmeraldII (Chemiluminescent Substrate): Disodium 2-chloro-5-(4-methoxy-spiro{1,2-dioxetane-3,2′-(5′-chloro)-tricyclo [3,3,1,13,7]decan}-4-yl)-1-phenyl phosphate.</p