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NT-proBNP

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== Structure, properties and matrices ==
[[File:BNP_pathway.png|thumb|400px|The biosynthesis of proBNP and its derivatives.<ref name="Arti1">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</ref>]]
'''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.<ref name="WikiBNP">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</ref>

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'''.<ref name=" WikiBNP "></ref><ref name="WikiNT-proBNP">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</ref><ref name="Arti2">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</ref>

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.<ref name=" WikiBNP "></ref><ref name="WikiNPRs">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</ref><ref name="WikiNPs">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</ref><ref name="Arti2"></ref>

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.<ref name="Arti2"></ref><ref name="Arti3">TechNotes | Human ProBNP and proBNP-derived peptides (BNP and NT-proBNP), HyTest Ltd, August 2010 updated November 2015</ref>

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.<ref name="Arti2"></ref><ref name="Tietz">Tietz Fundamentals of clinical chemistry and molecular diagnostics, Carl A. Burtis, David E. Burtis, 2015, Seventh Edition pages 643-646</ref>

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.<ref name="Arti2"></ref> 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.<ref name=" WikiBNP "></ref><ref name="MedPhys">Medical Physiology: A Cellular and Molecular Approach, updated second edition Walter F.Boron, Emile L. Boulpaep, 2012, Elsevier saunders, pages 68-69</ref>

'''proBNP'''<ref name="Arti3"></ref>

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

MW 11905.5 Da. pI 10.12

'''NT-proBNP'''<ref name="Arti3"></ref>

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

MW 8457.4 Da. pI 8.45

'''BNP'''<ref name="Arti3"></ref>

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.<ref name="Arti1"></ref>

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. <ref name="Survey1">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 </ref><ref name="Survey2">Sneltesten in de huisartspraktijk Huidig gebruik en behoefte aan testen in de toekomst, Cals JWL et al., Ned Tijdschr Geneeskd. 2014;158:A8210 </ref>

The table below shows the reference values for BNP and NT-proBNP.
{| class="wikitable" style="width:75%"
|-
!width=25%| Biomarker
!width=25%| Normal range<ref name="Arti3"></ref>
!width=25%| Heart failure cut-off value<ref name="Tietz"></ref>
|-
| 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.<ref name="Arti2"></ref><ref name="Tietz"></ref>

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.<ref name="Arti2"></ref><ref name="Arti4">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</ref> 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.<ref name=" WikiBNP "></ref><ref name="Arti2"></ref>

== Lab protocols ==

The concentration of BNP in blood is unstable due to protease activity. NT-proBNP is also affected, but to a lesser extent.<ref name="Arti3"></ref><ref name="Tietz"></ref>

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.<ref name="Tietz"></ref>

== 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:
{| class="wikitable" style="width:75%"
|-
!width=14.28%| Company
!width=14.28%| Product
!width=14.28%| Biomarker
!width=14.28%| Sample type
!width=14.28%| Sample volume (µL)
!width=14.28%| Time to result (min)
!width=14.28%| Reportable range (pg/mL)
|-
| Roche
| cobas h232<ref name="cobas website">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</ref><ref name="cobas specifications">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</ref>
| NT-proBNP
| Heparinized venous whole blood
| 150
| 8-12
| 60-9000
|-
| Samsung
| LabGeo IB10<ref name="Samsung LabGeo IB10 catalog">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</ref><ref name="Samsung LabGeo IB10 ">Samsung LABGEO IB10 Compact Immunoassay Analyzer, Medical Equipment Centre, retrieved from http://www.medicalequipmentcentre.com/samsung-medison-analyzer.html, consulted on 27 November 2016</ref>
| NT-proBNP
| EDTA and lithium heparinized whole blood and plasma
| 500
| 20
| 30-5000
|-
| Abbott
| i-STAT<ref name="Abbott i-STAT">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</ref>
| 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%.<ref name="cobas specifications"></ref><ref name="Samsung LabGeo IB10 catalog"></ref><ref name="Abbott i-STAT"></ref>

== (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.<ref name="NewNP">A new natriuretic peptide in porcine brain. Sudoh, T., Kangawa, K., Minamino, N., & Matsuo, H. (1988). Nature. 332, 78 - 81, doi:10.1038/332078a0</ref> 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.<ref name="amino-terminal">The amino-terminal portion of pro-brain natriuretic peptide (Pro-BNP) circulates in human plasma. Hunt, P. J., Yandle, T. G., Nicholls, M. G., Richards, A. M., & Espiner, E. A. (1995). Biochemical and Biophysical Research Communications. 214(3), 1175-1183, http://dx.doi.org/10.1006/bbrc.1995.2410</ref> 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. <ref name="glycoNT">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</ref> Intact nonprocessed proBNP was also found to be present in the circulation. <ref name="BNPassay">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</ref>

'''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.<ref name="amino-terminal"></ref>

Already in 1999 the first sandwich immunoassay was developed using antibodies and streptavidin-biotin interaction.<ref name="novelNT">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</ref> 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'''

[[File:sandwich_immunoassay.png|thumb|400px|Principle of sandwich immunoassay.<ref name="SIA">Retrieved 3 February 2017 from https://thescientificdigest.wordpress.com/2015/07/06/quantitatively-sensing-proteins-on-your-smartphone/</ref>]]
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.<ref name="Abbott">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</ref>

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. <ref name="Scan">SKUP Scandinavian evaluation of laboratory equipment for primary health care, SKUP, 2013, p. 4,17,8,10,11</ref><ref name="cobas">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</ref>

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 ==

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