Brain Natriuretic Peptide: (NT-pro)BNP

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Structure, properties and matrices

The biosynthesis of proBNP and its derivatives.[1]

Brain natriuretic peptide or B-type natriuretic peptide (BNP) is an endogenous polypeptide that is secreted by the heart ventricles in reponse to excessive strain on heart muscle cells. The name stems from the original discovery of the molecule in procine brain extracts. In humans, BNP is mainly produced in cardiac ventricles. The release of BNP is modulated by calcium ions.[2]

The molecular pathway starts with the 134-amino acid precursor preproBNP that is subsequently processed into the 108-amino acid propeptide, proBNP(1-108). Through several proprotein convertases, such as corin and furin, proBNP is processed into two peptides. These peptides are the N-terminal 76-amino acid peptide proBNP(1-76), usually called NT-proBNP, and the biologically active C-terminal 32-amino acid peptide proBNP(77-108), usually called BNP.[2][3][4]

BNP and proBNP contain a ring structure formed by a disulfide bond between two cysteine residues. The ring structure is essential for the biological activity of the molecules. BNP and, to a lesser extent proBNP, are active ligands for the natriuretic peptide receptors A and B (NPRA and NPRB). These receptors are guanylyl cyclases. BNP and proBNP have a much stronger effect on NPRA than on NPRB. BNP is closely related to atrial natriuretic peptide ANP, having similarities in function and structure.[2][5][6][4]

ProBNP can be O-glycosylated on several sites, mainly the first 76 amino acids, within the golgi apparatus. The glycolysation on the Thr71 site on proBNP prevents cleavage by corin and furin. Other O-glycolysations do not prevent cleavage by corin and furin.[4][7]

ProBNP produced in cardiomyocytes can either be processed into NT-proBNP and BNP within the cell, or can be secreted as an intact proBNP peptide. Especially when the Thr71 is glycosylated the latter seems to occur. In patients with heart failure (HF) the greatly increased production of proBNP can even lead to the secretion of non-glycosylated proBNP. The natriuretic peptide clearance receptor (NPRC) clears the natriuretic peptides by sequestering and/or internalizing the peptide. NT-proBNP is not cleared by NPRCs but instead is thought to be cleared mainly by the kidneys.[4][8]

ProBNP, BNP and NT-proBNP can all be found in the plasma of healthy individuals. ProBNP can also be cleaved after secretion whilst in circulation.[4] BNP is rapidly degraded in vivo by peptidases such as dipeptidyl peptidase IV and neutral endopeptidase. There is no confirmation whether enzymes such as meprin can degrade this peptide hormone in humans. It is also suggested that aldehyde protease can cleave BNP at arginine sites.

The eventual effect of the pathway involving BNP and proBNP is that it increases natriuresis and the dilation of vascular smooth muscle, reducing the effective circulating blood volume as well as the blood pressure.[2][9]

proBNP[7]

AA sequence: HPLGSPGSAS DLETSGLQEQ RNHLQGHKLSE LQVEQTSLEP LQESPRTGV WKSREVATEG IRGHRKMVLY TLRAPR SPKM VQGSGCFGRK MDRISSSSGL GCKVLRRH.

MW 11905.5 Da. pI 10.12

NT-proBNP[7]

AA sequence: HPLGSPGSAS DLETSGLQEQ RNHLQGHKLSE LQVEQTSLEP LQESPRTGV WKSREVATEG IRGHRKMVLY TLRAPR.

MW 8457.4 Da. pI 8.45

BNP[7]

AA sequence: SPKM VQGSGCFGRK MDRISSSSGL GCKVLRRH.

MW 3466 Da. pI 10.95.

Clinical significance

BNP and NT-proBNP are widely used as biomarkers for heart failure (HF) due to their increased production when the heart is under stress. For example, low values of BNP can be used to exclude acute heart failure, and regularly measured levels of BNP and/or NT-proBNP can be used to monitor the progression of chronic heart disease.[1]

Surveys among general practitioners show great interest in (NT-pro)BNP. Out of more than 1600 general practitioners, about 59% indicated that they would be interested to use a (NT-pro)BNP point-of-care test. [10][11]

The table below shows the reference values for BNP and NT-proBNP.

Biomarker Normal range[7] Heart failure cut-off value[8]
BNP 13.9 - 63.7 pg/mL 100 pg/mL
NT-proBNP 68 - 243 pg/mL 125 pg/mL if below 75 years, 450 pg/mL if above 75 years

Concentrations of BNP and NT-proBNP are influenced by age, sex, renal function and obesity.[4][8]

The half-life of proBNP and NT-proBNP in blood is longer than 60 minutes, whereas the half-life of BNP is 15 to 20 minutes and less than 10 minutes for ANP.[4][12] This makes proBNP and NT-proBNP longer-term indicators for HF than BNP.

Many immunoassays for NT-proBNP and BNP suffer from cross reactivity with proBNP. In BNP assays, glycosylated proBNP has been found to cross-react stronger than non-glycosylated proBNP. In NT-proBNP assays, glycosylated proBNP, but not non-glycosylated, cross-reacts heavily (29-249%). All NT-proBNP assays use antibodies supplied by Roche Diagnostics. This causes smaller assay-to-assay differences in NT-proBNP assays as compared to BNP assays. BNP assay-to-assay variabilities can be up to two-fold.[2][4]

Lab protocols

The concentration of BNP in blood is unstable due to protease activity. NT-proBNP is also affected, but to a lesser extent.[7][8]

BNP requires plastic collection tubes and NT-proBNP can be collected in either glass or plastic. For BNP the only reasonable choice is EDTA stabilized whole blood or plasma. For NT-proBNP either serum, heparin plasma or EDTA plasma should suffice, although EDTA gives 10% lower results on tests.[8]

State of the art sensing methods

SensUs focuses on measurement systems that are small and that can be used at the side of patients and at home.

There are presently three systems on the market for (NT-pro)BNP testing that are intended for handheld use:

Company Product Biomarker Sample type Sample volume (µL) Time to result (min) Reportable range (pg/mL)
Roche cobas h232[13][14] NT-proBNP Heparinized venous whole blood 150 8-12 60-9000
Samsung LabGeo IB10[15][16] NT-proBNP EDTA and lithium heparinized whole blood and plasma 500 20 30-5000
Abbott i-STAT[17] BNP EDTA whole blood or plasma 17 12 15-5000

The abovementioned systems are all based on antibody sandwich assays, but they use different readout mechanisms. More information on these assays follows in the next Section.

The precision of concentration determination (CV=coefficient of variation) of these systems is typically about 10%.[14][15][17]

(NT-pro)BNP measurement methods: Past, present, and future

BNP was first described in 1988 by Sudoh et al. who found a natriuretic peptide in a porcine brain similar but not equal to the then known atrial natriuretic peptide ANP.[18] In 1995 it was hypothesized that since proBNP is cleaved resulting in a C-terminal active part called BNP-32 or simply BNP, there should also be an N-terminal part. Hunt et al. tested this hypothesis and indeed found what is now known as NT-proBNP in human blood plasma.[19] In 2008 Seferian et al showed that the central region (28-56 aar) of NT-proBNP is glycosylated, whereas the C-terminal portion of the molecule (61-76 aar) is mostly free of O-glycans. [20] Intact nonprocessed proBNP was also found to be present in the circulation. [21]

First (NT-pro)BNP quantification methods

To measure BNP and NT-proBNP, a combination of size exclusion chromatography, reverse phase high pressure liquid chromatography (RP-HPLC) and a radioimmunoassay (RIA) was used. For the RIA synthesized BNP molecules were iodinated to obtain a standard curve, which could then be compared to plasma samples that went through the same process of SEC, RP-HPLC and RIA.[19]

Already in 1999 the first sandwich immunoassay was developed using antibodies and streptavidin-biotin interaction.[22] Measurement by immunoassay is now the golden standard and the advantages compared to the SEC/RP-HPLC/RIA process are that immunoassays are faster and easier to perform.

Sandwich immunoassays

Principle of sandwich immunoassay.[23]

The principle of a sandwich immunoassay is that a biomarker is captured between two antibodies that are both specific for the biomarker. One of these antibodies is attached to a solid base (capture antibody). The biomarker attaches to this antibody and the area is washed so only the bound (relevant) molecules are left. Thereafter a solution containing another antibody specific for the biomarker is added. This antibody (detection antibody) has a label attached to it, for instance an enzyme or a fluorophore. After a second washing step only the antibody/biomarker/antibody sandwiches are left. In case a fluorescent label is used, light with a specific wavelength excites the fluorophore and the intensity of the emitted light is measured. By comparing these values to a standard curve, the intensity can be linked to the concentration of biomarker in the sample.

Abbott’s i-STAT BNP test cartridge uses a sandwich ELISA (enzyme-linked immunosorbent assay) with an electrochemical sensor. This sensor is coated with antibodies to which the BNP binds when the sample is added. The second antibody is conjugated to an alkaline phosphatase enzyme and this conjugate complex dissolves into the sample once it is loaded. The wash fluid that is used to remove the remainder of the sample and unbound antibody/enzyme conjugate contains a substrate for the alkaline phosphatase enzyme. Enzymes that are part of the antibody/BNP/antibody sandwich are not washed away, so they can cleave the substrate present in the wash fluid and as a result release an electrochemically detectable product. The electrochemical (amperometric) sensor measures this enzyme product, and the signal is proportional to the concentration of BNP in the sample.[24]

The Roche cobas h232 point of care system uses a sandwich immunoassay with gold nanoparticles as labels. A test strip (CARDIAC® NT-proBNP assay) contains monoclonal and polyclonal antibodies against epitopes of the NT-proBNP molecule of which one is gold-labelled and the other biotinylated. The optical system of the instrument detects and measures the intensity of the signal line by means of a digital camera. [25][26]

The Samsung LABGEO IB10 is an NT-proBNP measurement system that has recently come onto the market. We have not yet been able to find which assay principles it uses.

Possible future sensing methods

In the future the point-of-care testing devices will become smaller, faster, easier to use (e.g. finger-prick based), and more economical, so that testing can be done at any place and even at home. The use of the devices and the test result must become very simple and intuitive. In the far future, devices may also become available for in-vivo sensing, i.e. sensors worn on or in the body, much like present-day subcutaneous sensors for continuous glucose monitoring.

References

  1. 1.0 1.1 Cardiac biomarker testing in the clinical laboratory: Where do we stand? General overview of the methodology with special emphasis on natriuretic peptides, Aldo Clerico, Claudio Passino, Maria Franzini, Michele Emdin, June 2014, Clinica Chimica Acta, http://dx.doi.org/10.1016/j.cca.2014.06.003
  2. 2.0 2.1 2.2 2.3 2.4 Brain natriuretic peptide (last modified 9 July 2016), Wikipedia (Encyclopedia), retrieved from https://en.wikipedia.org/wiki/Brain_natriuretic_peptide, consulted on 26 August 2016
  3. N-terminal prohormone of brain natriuretic peptide (last modified 13 June 2016), Wikipedia (Encyclopedia), retrieved from https://en.wikipedia.org/wiki/N-terminal_prohormone_of_brain_natriuretic_peptide, consulted on 1 October 2016
  4. 4.0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 State of the art of immunoassay methods for B-type natriuretic peptides: An update, Aldo Clerica et al., December 2014, Critical Reviews in Clinical Laboratory Sciences
  5. Atrial natriuretic peptide receptor (last modified 29 June 2016), Wikipedia (Encyclopedia), retrieved from https://en.wikipedia.org/wiki/Atrial_natriuretic_peptide_receptor, consulted on 1 October 2016
  6. Atrial natriuretic peptide (last modified 20 August 2016), Wikipedia (Encyclopedia), retrieved from https://en.wikipedia.org/wiki/Atrial_natriuretic_peptide, consulted on 1 October 2016
  7. 7.0 7.1 7.2 7.3 7.4 7.5 TechNotes | Human ProBNP and proBNP-derived peptides (BNP and NT-proBNP), HyTest Ltd, August 2010 updated November 2015
  8. 8.0 8.1 8.2 8.3 8.4 Tietz Fundamentals of clinical chemistry and molecular diagnostics, Carl A. Burtis, David E. Burtis, 2015, Seventh Edition pages 643-646
  9. Medical Physiology: A Cellular and Molecular Approach, updated second edition Walter F.Boron, Emile L. Boulpaep, 2012, Elsevier saunders, pages 68-69
  10. Current and future use of point-of-care tests in primary care: an international survey in Australia, Belgium, The Netherlands, the UK and the USA, : Howick J, Cals JWL, Jones C, et al., BMJ Open 2014, http://bmjopen.bmj.com/content/4/8/e005611
  11. Sneltesten in de huisartspraktijk Huidig gebruik en behoefte aan testen in de toekomst, Cals JWL et al., Ned Tijdschr Geneeskd. 2014;158:A8210
  12. Metabolic clearance rate and plasma half life of alpha-human atrial natriuretic peptide in man. Yandle, T. G., Richards, A. M., Nicholls, M. G., Cuneo, R., Espiner, E. A., & Livesey, J. H. (1986). Life Sciences, 38(20), 1827-1833. doi:10.1016/0024-3205(86)90137-2
  13. cobas h 232 POC system (last modified 1 July 2016), cobas, retrieved from http://www.cobas.com/home/product/point-of-care-testing/cobas-h-232.html, consulted on 27 November 2016
  14. 14.0 14.1 The new cobas h 232 POC system (last modified 2016), cobas, retrieved from http://www.cobas.com/content/dam/cobas_com/pdf/product/cobas-h-232-POC-system/cobas-h-232-specifications.pdf, consulted on 27 November 2016
  15. 15.0 15.1 Samsung LABGEO IB10 (last modified 2015), Samsung, retrieved from http://www.samsung.com/global/business-images/insights/2015/HME_IVD_LABGEO_IB10_EN_Catalog_TFS_v4.5_low_151125-0.pdf, consulted on 27 November 2016
  16. Samsung LABGEO IB10 Compact Immunoassay Analyzer, Medical Equipment Centre, retrieved from http://www.medicalequipmentcentre.com/samsung-medison-analyzer.html, consulted on 27 November 2016
  17. 17.0 17.1 i-STAT (last modified June 2015), Abbott POC, retrieved from https://www.abbottpointofcare.com/shared/static-assets/other/030349_C_CartridgeMenu.pdf, consulted on 27 November 2016
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  20. Immunodetection of glycosylated NT-proBNP circulating in human blood. Seferian, K.R., Tamm, N.N., Semenov, A.G., Tolstaya, A.A., Koshkina, E.V., Krasnoselsky, M.I., Postnikov, A.B., Serebryanaya, D.V., Apple, F.S., Murakami, M.M., et al. (2008). Clin Chem 54:866-873, doi: 10.1373/clinchem.2007.100040
  21. Assay of brain natriuretic peptide (BNP) in human plasma: evidence for high molecular weight BNP as a major plasma component in heart failure. Yandle, T.G., Richards, A.M., Gilbert, A., Fisher, S., Holmes, S., and Espiner, E.A. (1993). J Clin Endocrinol Metab 76, 832-838, doi: 10.1210/jcem.76.4.8473392
  22. Development of a novel, N-terminal-proBNP (NT-proBNP) assay with a low detection limit. Karl, J., Borgya, A., Gallusser, A., Huber, E., Krueger, K., Rollinger, W., & Schenk, J. (1999). Scandinavian Journal of Clinical and Laboratory Investigation. Supplementum, 230(2), 177–81. https://doi.org/10.2144/000113897
  23. Retrieved 3 February 2017 from https://thescientificdigest.wordpress.com/2015/07/06/quantitatively-sensing-proteins-on-your-smartphone/
  24. CARTRIDGE & TEST INFORMATION SHEETS, B-TYPE NATRIURETIC PEPTIDE/ (BNP). Retrieved 23-1-2017 from https://www.pointofcare.abbott/us/en/offerings/support/technical-documentation/cartridge-test-information-sheets
  25. SKUP Scandinavian evaluation of laboratory equipment for primary health care, SKUP, 2013, p. 4,17,8,10,11
  26. Präsenzdiagnostik der Akutparameter – einfach , schnell und präzise cobas h 232 POC System Einfach schnell und präzise. Retrieved 19-1-2017 from http://www.roche-diagnostics.ch/content/dam/corporate/roche-dia_ch/documents/broschueren/professional_diagnostics/praxislabor/kardiale_marker/cobas-h-232-poc-system/05585066001_DE_EA_cobas-h-232-Broschuere.pdf