Natriuretic Peptides and Analytical Barriers.
Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) were described in the 1980s as circulating peptides released from the heart and involved in maintaining cardiorenal homeostasis by diuresis and natriuresis (3). ANP is produced predominantly in the atrium and is stored in intracellular granules where it is readily available for release. BNP is synthesized to a greater extent in the ventricular myocardium and is stored in only modest quantities. Thus, BNP requires more time to be synthesized and released and is a slower reacting peptide. Both peptides induce diuresis and natriuresis, and reduce vascular resistance and systemic blood pressure. C-type natriuretic peptide (CNP) is produced in the central nervous system and vascular endothelium, and acts as a paracrine regulator with little known implications for the cardiovascular system. Dendroaspis natriuretic peptide (DNP) is another member of the natriuretic peptide family and urodilantin is a renally secreted cleavage product of ANP (3).
All natriuretic peptides in humans contain a preserved ring structure of 17 amino acid residues and form an intramolecular disulfide bond, which presumably is responsible for receptor binding. Most BNP assays target this ring structure for 1 of their epitopes. The receptors, termed natriuretic peptide receptors (NPRs) are classified as types A and B. Binding of these guanylyl cyclase-coupled receptors leads to an increase in cGMP, which has downstream effects of diuresis and natriuresis, vasodilation, inhibition of the renin-angiotensin-aldosterone system, enhanced myocardial relaxation, inhibition of fibrosis and hypertrophy, promotion of cell survival, and inhibition of the inflammatory response. NPR type C lacks guanylyl cyclase activity and is thought to be a clearance receptor (3).
Conditions of the heart accompanied by volume and pressure overload, such as HF and myocardial infarction, lead to cardiac wall tension and stretch, volume overload, or ischemia and result in increased production and release of natriuretic peptides (4). The use of these biomarkers for diagnostic and prognostic purposes has been delineated (4) and endorsed by guideline authorities for heart care (5). Moreover, recombinant forms of human ANP (carperitide) and BNP (neseritide) were developed as therapeutic agents for HF (6, 7). However, clinicians have lost enthusiasm for these agents due to doubtful efficacy and safety issues (8).
BNP is more stable in vitro and has superior diagnostic performance to ANP, thus BNP and BNP-related peptides have established a role in clinical practice for the diagnosis of risk stratification and follow-up of patients with HF (1, 9).
Brief Relevant Molecular Biology of Natriuretic Peptides
ANP is synthesized as a 151-amino acid prepropeptide, preproANP, which is stored in atrial intracellular granules (10). PreproANP is secreted and cleaved to the mature peptide, ANP, in response to atrial stretch, angiotensin II, endothelin, or sympathetic-mediated stimulation. PreproANP is cleaved to proANP, which is processed by the convertase corin into 2 circulating peptides, ANP 1-28 and the N-terminal fragment, NT-proANP 1-98 (11). NT-proANP is subsequently cleaved into 3 fragments with biological importance. ProANP 1-30 is the long-acting natriuretic peptide, proANP 31-67 has vasodilation properties, and proANP 79-98 or kaliuretic peptide, is involved in potassium excretion. NT-proANP and its cleavage derivatives are all found in circulation. Another member of the ANP family, urodilantin can be isolated from human urine and increases diuresis (12).
BNP is synthesized as a 134-amino acid preproBNP precursor, which is cleaved to the 108-amino acid proBNP by removal of a 26-amino acid signal peptide (13). A peptide fragment of preproBNP containing amino acid residues 17-26 is present in normal individuals and patients with acute MI. It has been proposed as a biomarker of cardiac ischemia. Another peptide of the signal residue containing amino acid residues 16-25 may be a biomarker of ischemia (14).
BNP is encoded by an early response gene allowing transcription to reach maximal levels rapidly (15). Intracellular BNP storage is minimal and it is synthesized de novo when needed and released by ventricular cardiomyocytes.
The processing of proBNP forms an N-terminal proBNP (NT-proBNP) fragment with amino acid residues 1-76 and a C-terminal region active BNP with amino acid residues 77-108 (16). Because of proteolysis of proBNP, BNP and NT-proBNP are produced in a stoichiometric ratio of 1:1. NT-proBNP has no known biologic activity. Despite the 1:1 stoichiometric ratio, the molar plasma concentration of NT-proBNP is higher than the concentration of BNP likely because NT-proBNP has slower clearance from the circulation (17). ProBNP in nonprocessed form is found in healthy individuals and patients with HF (18). ProBNP is post-translationally modified in patients with HF by
0-glycosylation at several threonine and serine residues within the N-terminal region (amino acid residues
1-76), but not within the BNP-portion of proBNP (amino acid residues 77-108) (19). It appears that NT-proBNP is glycosylated in the central region (amino acid residues 28 -56), while the C-terminus region of the molecule (amino acid residues 61-76) is not post-translationally modified (19). ProBNP, however, is glycosylated both in the central region and in the region located close to the cleavage site, specifically at amino acid residues 63-76. This region can be blocked to site-specific antibodies because of steric impediment due to glycosylation (20). The degree of glycosylation of both proBNP and NT-proBNP is highly individual. It is also known to inhibit the activity of furin on the 76 -78 amino acid cleavage site (21).
The diversity of circulating proBNP-derived peptides is explained in part by proBNP processing, which occurs immediately before, or at the time of release. The processing is facilitated by prohormone convertases. Furin and corin are convertases currently thought to be proBNP-processing enzymes. The evidence is indirect from in vitro studies, which demonstrate the formation of BNP 1-32 (by furin) and BNP 4-32 (by corin). Since corin produces a short BNP form, BNP 4-32, this enzyme is unlikely to be the only one responsible for the processing of proBNP, suggesting that furin also plays a role (22).
Glycosylation residues in the region of the proBNP molecule close to the cleavage site inhibit the processing of proBNP. In in vitro studies, both furin- and corin-mediated processing of proBNP are suppressed by O-glycans located at Thr71. It appears that only proBNP molecules not glycosylated at Thr71 can be processed into BNP and NT-proBNP, probably due to access of enzyme to the unbound cleavage site. Inhibition of processing via glycosylation might be a pathologic phenomenon found in HF.
Degradation Fragments of BNP in Circulation
Although initially it was thought that there were only 2 circulating fragments of BNP, BNP 1-32 and NT-proBNP 1-76, contemporary data have challenged this concept recently (23). Mass spectrometry analyses showed only minute amounts of circulating BNP 1-32. BNP 1-32 is found in patients with advanced HF in only very low levels (14). In blood samples from HF patients multiple N- and C-truncated fragments of BNP are present such as BNP 1-32, BNP 3-32, BNP 4-32, BNP 5-32, BNP 5-31, BNP 1-26, and BNP 1-25 (24). The variety of such fragments is believed to be the result of proteolysis by dipeptidyl peptidase IV (DPP IV), which forms BNP 3-32 and neprilysin (NEP), which forms BNP 5-32. BNP can also be proteolyzed by insulin-degrading enzyme (IDE), but not by NEP and IDE implying that another enzyme is involved in cleavage (25). BNP 4-32 may be the result of corin activity (21). Another protease, peptidyl arginine aldehyde protease, can also degrade BNP, and meprin was shown to lyse BNP in animal models but not in humans (26). Due to BNP instability in EDTA plasma even at low temperatures, protease inhibitors in high concentrations should always be used (23).
ProBNP and Cross-Reactivity
Intact proBNP is found in healthy individuals and patients with HF. Immunoassays used in clinical practice to detect BNP show extensive cross-reactivity with proBNP. The extent of cross-reactivity is different for various assays and different forms of glycosylated or nonglycosylated proBNP. Notably, only BNP and not the precursor proBNP has been shown to have a natriuretic response in patients with HF (27). ProBNP is resistant to proteolysis and inactivation by human kidney membranes (28). The role of proBNP in healthy volunteers and patients with HF may be a physiologic or a pathologic process. ProBNP is a poor stimulator of guanylyl cyclase. There are differential cGMP activating properties of BNP forms and, notably, proBNP 1-108 and NT-proBNP 1-76 have reduced cGMP activity in vitro (29). Some speculate that unprocessed proBNP is a circulating "reserve" of BNP since in vivo cleavage of proBNP produces BNP. Glycosylation of Thr71 in proBNP has inhibitory effects since it prevents proteolysis by both furin and corin. Only proBNP that is not glycosylated at Thr71 can form active BNP1-32.
ProBNP Circulating Forms in Acute and Chronic HF
The highest percentage of glycosylated proBNP is present in patients with chronic HF compared to patients in the acute decompensated and nonacute decompensated groups (30). Another interesting finding is that furin activity but not its concentration is greater in the acute HF group than in the chronic HF group (30). Perhaps there is a differential mechanism of proBNP processing in disease progression in HF patients with increased BNP production in the acute decompensated group of patients with fluid overload. In the chronic HF group, proBNP degradation will
not occur since there is less acute fluid overload, implying that there might be regulatory mechanisms responsible for rapid increases in plasma BNP by degradation of the processing-susceptible proBNP. These findings demonstrate that there is a different natriuretic peptide spectrum in patients with different forms of HF. Thus, assays that detect glycosylated and nonglycosylated forms of proBNP might provide additional diagnostic information.
There is a multitude of immunodetection platforms for natriuretic peptides that are used in clinical practice. All detect multiple forms and resulting interpretation is challenging. It is important to keep in mind both this fact and also the type of HF syndrome involved. Moreover, it appears that BNP and NT-proBNP have the same renal extraction fraction in humans, but there is marked extraction of NT-proBNP across the skeletal muscle (31). Additionally, there are handling differences associated with various assays. Analytical characteristics of currently available commercial assays for natriuretic peptides are presented in Table 1. The location of epitopes on BNP and NT-proBNP molecules is depicted in Fig. 1.
All NT-proBNP assays use the same antibodies and calibrators. These are distributed by Roche, thus there is an advantage of unifying these detection platforms. There are only minor differences between the commercial NT-proBNP assays with variation across methods that is <10%. Despite this standardization, however, assay harmonization remains incomplete (32). The International Collaborative of NT-proBNP Study involving 1256 patients proposed a cutoff value for NT-proBNP assays below or above 125 ng/L. A value >300 ng/L was optimal with the existing assays for the exclusion of acute HF (33, 34). This study included a blend of chronic and acute HF patients. Reference limits also vary by age and sex of patients analyzed (35).
While the first generation of NT-proBNP immunoassays uses polyclonal antibodies to amino acids 1-21 and 39-50, the second generation uses monoclonal antibodies (MAb) recognizing the central region of NT-proBNP: amino acids 22-28 and 42-46 (27-31). Many studies have demonstrated the negative effect of glycosylation for antibody recognition to the middle area of the NT-proBNP molecule. The NT-proBNP immunoassays currently available show important cross-reactivity with nonglycosylated proBNP and do not detect glycosylated NT-proBNP and proBNP peptides owing to the presence of O-glycans in the epitopes recognized by the antibodies in the nonglycosylated form. Clinicians should be aware that these detection platforms underestimate the concentration of circulating biomarker considerably. Some studies suggest that the underestimation might be up to 10-fold (19, 35). Some recommend that future NT-proBNP platforms use deglycosylation of blood samples before assay. Perhaps more robust deglycosylation explains why some patients such as those with renal failure have discordantly high values for NT-proBNP. If alternative assays for NT-proBNP are developed, they will need to assess carefully the extent to which the antibodies are influenced by glycosylation.
All BNP assays use different antibodies and materials for peptide detection; thus there is a paucity of standardization and reports show differences of up to 50% across various BNP detection platforms, even when the same antibodies are used on equipment (2).
The most frequent method used clinically is the "sandwich" assay that uses 2 antibodies specific for 2 distantly located epitopes of the BNP peptide. One of these antibodies is always specific for the intact cysteine ring, which is thought to be the active form, while the other antibody recognizes either the C-terminus of the peptide (Abbott AxSYM and Architect, Shionogi IRMA) or for the N-terminus (Alere Triage and Beckman Access). Assays using antibodies specific to the C-terminus will not detect BNP molecules that are degraded in this region, whereas assays using antibodies specific to the N-terminus of BNP molecule will not measure peptides processed in the N-terminus region. The "Single Epitope Sandwich" Immunoassay (SES-BNP[TM]) designed by HyTest and implemented by ET healthcare is different. The assay uses 1 MAb (24C5) specific to the relatively stable ring fragment of the BNP molecule (epitope 11- 17), which is located within the biologically active cysteine ring, and the second MAb (Ab-BNP2), which recognizes the immune complex of 24C5 with BNP only. There is no space between epitopes, and cleavage between the epitopes does not affect detection since only 1 epitope is needed. This approach stabilizes the immune complex increasing the affinity for its antigen and leads to a very highly sensitive assay, which recognizes both intact BNP and recombinant glycosylated and nonglycosylated forms of proBNP.
The cutoff value for BNP to exclude acute HF with high negative predictive value is 100 ng/L, with the caveat that each assay should, however, have its own cutoff value due to the diversity and lack of standardization of BNP immunoassays. There are systematic differences amongst various BNP assays which partly can be explained by the multitude of proBNP peptides. There is also a lack of reference materials for assay calibration. A common calibrator would improve the harmonization of these assays. Indeed, there is extensive variation depending upon the nature of the natriuretic peptide calibrators used and their glycosylation (Table 2). Cross-reactivity of BNP with proBNP was also demonstrated; this is particularly important in HF patients since proBNP is more prevalent in these patients (36). A summary of cross-reactivity of BNP and NT-proBNP assays currently in clinical use is presented in Fig. 2. Additionally, it was revealed that all BNP immunoassays share substantial cross-reactivity with proBNP, as proBNP shares the same structure (part of BNP) as BNP. Considering that proBNP is the major BNP-immunoreactive form in the circulation of HF patients, this cross-reactivity is clinically relevant. Moreover, some BNP assays will not measure proBNP when it is glycosylated due to spatial obstruction to antibody recognition. Thus, most of the BNP measured is either nonprocessed proBNP or degradation product of BNP.
The PARADIGM-HF trial [Prospective Comparison of ARNI (angiotensin receptor neprisylin inhibitor) with ACEI (angiotensin-converting enzyme inhibitor) to Determine Impact on Global Mortality and Morbidity in HF] showed better outcomes with the neprilysin inhibitor and angiotensin receptor blocker, LCZ696, compared to the angiotensin-converting enzyme inhibitor, enalapril alone in patients with HF with New York Heart Association functional class II (37).
Neprilysin is a ubiquitously expressed membrane-bound protease, found mostly in the kidney, which cleaves the amino bonds of hydrophobic residues. It is implicated in the cleavage of glucagon, enkephalins, substance P, neurotensin, oxytocin, bradykinin, and amyloid [beta]. Both human ANP and human CNP are substrates for neprilysin, while human BNP is not. Specific neprilysin inhibitors fail to block BNP degradation by human kidney membranes, suggesting that neprilysin does not regulate BNP. Thus, the effect of LCZ696 should be more prominent on ANP than BNP. Since proBNP is the major active form resulting from BNP synthesis, one should question whether proBNP rather than BNP is the substrate of LCZ696 activity; however, recent data suggest that proBNP is relatively resistant to neprilysin in vitro. of interest, in that study, glycosylation had no impact. In addition, assays specific to the amino terminus (amino acids 11-17) are less susceptible to BNP cleavage by neprilysin than epitopes specific for amino acids 14-21. Currently it is not clear how BNP and NT-proBNP concentrations are affected by treatment with LCZ696 in different conditions. The PARADIGM-HF trial showed that after treatment with valsartan-sacubitril BNP levels increased initially while NT-proBNP sustainably decreased. Why this occurred is unclear since human BNP appears not to interact with neprilysin. By 8 months, the increases in BNP had abated. It is claimed that because of this NT-proBNP should be used exclusively with valsartan-sacubitril (37). Such a suggestion is premature. We do not know how representative the changes in NT-proBNP with this novel agent are compared to other agents. In addition, it may be after additional studies that metrics about how to use BNP assays will be developed (38,39). Increases in circulating BNP might inhibit proBNP production, thus decreases in the NT-proBNP concentration might not be due to improvements in cardiac function. In addition, increases in BNP have been reported to reduce neprilysin activity acutely, which may provide a governor on its effects. Furthermore, measurement of BNP or NT-proBNP individually may not be sufficient for diagnostic and prognostic purposes with this new agent.
HyTest designed an assay based on a capture MAb specific for the region 26-32 of the BNP peptide and a detection antibody specific for the fragment 13-20 of proBNP peptide (18). This highly sensitive immunoassay is not affected by the glycosylation of proBNP molecules, since the epitopes are not glycosylation sites.
A specific MAb recognizes the hinge region of the proBNP molecule 75-80. This antibody along with a polyclonal antibody recognizing the BNP part of the BNP peptide constitutes a sandwich immunoassay for the measurement of proBNP. This method was performed on the BioPlex 2200 Analyzer Multiplex System (Bio-Rad). However, O-glycan residues at Thr71 may impede the hinge-specific antibody interaction. Thus, measured proBNP may underestimate the amount of proBNP present. In acutely decompensated HF patients, proBNP assays are similar to NT-proBNP and BNP assays without incremental value.
Another reason why various assays may differ from one another is related to preanalytical issues. Proteolytic degradation of the BNP molecule appears immediately upon blood collection. The stability of the sample is method dependent with different stabilities of the epitopes targeted by the specific assay. BNP is unstable when collected in glass tubes because of activation of kallikreins and this degradation may be dependent on the specificities of antibodies used in the measurement method (40). It is recommended that BNP blood samples be collected in plastic tubes only. For BNP assays, EDTA plasma is the only recommended specimen. For the Elecsys NT-proBNP assay, serum is the recommended specimen. There are substantial differences between serum and plasma natriuretic peptide concentrations measured with various detection platforms. The type of anticoagulant used is also important. BNP may be stored at room temperature for 24 h or at 30 [degrees]C for 12 h. In EDTA, overall plasma BNP values are stable at 20 [degrees]C for 1 month but the forms may vary over time. The addition of the protease inhibitor aprotinin, increases the storage time for BNP samples. The NT-proBNP assay is relatively stable during storage in serum, heparinized plasma, or EDTA plasma when stored at room temperature or 4 [degrees]C for at least 72 h or for up to 1 year when stored at -80 [degrees]C. Additionally, for both BNP and NT-proBNP assays, validation for the effect of freeze-thaw cycles upon stability needs to be performed (40).
Natriuretic peptides are established biomarkers for the diagnosis and prognosis of patients with HF. Owing to a high diversity of natriuretic peptides circulating in patients with various forms of HF, along with a complex molecular biology and detection-related issues with the currently widely used clinical detection platforms, challenges arise when clinicians are confronted with result interpretation of these immunoassays. The introduction of novel therapeutic agents in HF which target natriuretic peptides requires a thorough understanding of issues related to biologic activity, detection platforms and pathophysiology of the HF spectrum. Moreover, there are various HF types associated with distinct and differential expression of specific forms of natriuretic peptides. There are large differences between assays that result from the lack of common reference material and antibody diversity. Given the lack of harmonization across assays, providers need to be cautious when interpreting results measured with different assays and ideally should use the same detection platform for meaningful follow-up and comparison. Furthermore, preanalytic issues should also be taken into account. Newer generation assays should try to improve these impediments.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition Of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:
Employment or Leadership: None declared.
Consultant or Advisory Role: A.S. Jaffe, Beckman-Coulter, Abbott, Alere, Roche, Diadexus, Siemens, Lpath, Dart Neurosciences, Neu roGenomeX, Inc., and theheart.org.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: None declared.
Expert Testimony: None declared.
Patents: None declared.
(1.) Yancy CW, Jessup M, Bozkurt B, Butler J, Casey DE Jr, Drazner MH, et al. 2013 ACCF/AHA guideline for the management of heart failure: a report of the American College of Cardiology Foundation/American Heart Association Task Force on practice guidelines. Circulation 2013;128:e240 -327.
(2.) Clerico A, Zaninotto M, Prontera C, Giovannini S, Ndreu R, Franzini M, et al. State of the art of BNP and NT-proBNP immunoassays: the Cardio Ormo Check study. Clin Chim Acta 2012;414:112-9.
(3.) Pandey KN. Biology of natriuretic peptides and their receptors. Peptides 2005;26:901-32.
(4.) Rubattu S, Sciarretta S, Valenti V, Stanzione R, Volpe M. Natriuretic peptides: an update on bioactivity, potential therapeutic use, and implication in cardiovascular diseases. Am J Hypertens 2008;21:733-41.
(5.) Thygesen K, Mair J, Mueller C, Huber K, Weber M, Plebani M, et al. Recommendations for the use of natriuretic peptides in acute cardiac care: a position statement from the Study Group on Biomarkers in Cardiology of the ESC Working Group on Acute Cardiac Care. Eur Heart J 2012;33:2001-6.
(6.) Colucci WS, Elkayam U, Horton DP, Abraham WT, Bourge RC, Johnson AD, et al. Intravenous nesiritide, a natriuretic peptide, in the treatment of decompensated congestive heart failure. Nesiritide Study Group. N Engl J Med 2000;343:246-53.
(7.) Crozier IG, Nicholls MG, Ikram H, Espiner EA, Gomez HJ, Warner NJ. Haemodynamic effects of atrial peptide infusion in heart failure. Lancet 1986;2:1242-5.
(8.) Pleister AP, Baliga RR, Haas GJ. Acute study of clinical effectiveness of nesiritide in decompensated heart failure: nesiritide redux. Curr Heart Fail Reports 2011;8:226 -32.
(9.) Yancy CW, Jessup M, Bozkurt B, Butler J, Casey DE, Jr., Drazner MH, et al. 2013 ACCF/AHA guideline for the management of heart failure: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. JACC 2013; 62:e147-239.
(10.) Oikawa S, Imai M, Ueno A, Tanaka S, Noguchi T, Nakazato H, et al. Cloning and sequence analysis of cDNA encoding a precursor for human atrial natriuretic polypeptide. Nature 1984;309:724-6.
(11.) Yan W, Wu F, Morser J, Wu Q. Corin, a transmembrane cardiac serine protease, acts as a pro-atrial natriuretic peptide-converting enzyme. Proc Natl Acad Sci U S A 2000;97:8525-9.
(12.) Schulz-Knappe P, Forssmann K, Herbst F, Hock D, Pipkorn R, Forssmann WG. Isolation and structural analysis of "urodilatin", a new peptide of the cardiodilatin-(ANP)-family, extracted from human urine. Klin Wochenschr 1988;66:752-9.
(13.) Sudoh T, Maekawa K, Kojima M, Minamino N, Kangawa K, Matsuo H. Cloning and sequence analysis of cDNA encoding a precursor for human brain natriuretic peptide. Biochem Biophys Res Commun 1989;159:142734.
(14.) Hawkridge AM, Heublein DM, Bergen HR 3rd, Cataliotti A, Burnett JC Jr, Muddiman DC. Quantitative mass spectral evidence for the absence of circulating brain natriuretic peptide (BNP-32) in severe human heart failure. Proc Natl Acad Sci USA 2005;102: 17442-7.
(15.) Nakagawa O, Ogawa Y, Itoh H, Suga S, Komatsu Y, Kishimoto I, et al. Rapid transcriptional activation and early mRNA turnover of brain natriuretic peptide in cardiocyte hypertrophy. Evidence for brain natriuretic peptide as an "emergency" cardiac hormone against ventricular overload. J Clin Invest 1995;96:1280-7.
(16.) Kambayashi Y, Nakao K, Mukoyama M, Saito Y, Ogawa Y, Shiono S, et al. Isolation and sequence determination of human brain natriuretic peptide in human atrium. FEBS Lett 1990;259:341-5.
(17.) Pemberton CJ, Johnson ML, YandleTG, Espiner EA. Deconvolution analysisof cardiac natriuretic peptides during acute volume overload. Hypertension 2000;36: 355-9.
(18.) Seferian KR, Tamm NN, Semenov AG, Mukharyamova KS, Tolstaya AA, Koshkina EV, et al. The brain natriuretic peptide (BNP) precursor is the major immunoreactive form of BNP in patients with heart failure. Clin Chem 2007;53:866-73.
(19.) Seferian KR, Tamm NN, Semenov AG, Tolstaya AA, Koshkina EV, Krasnoselsky MI, et al. Immunodetection of glycosylated NT-proBNP circulating in human blood. Clin Chem 2008;54:866-73.
(20.) Semenov AG, Postnikov AB, Tamm NN, Seferian KR, Karpova NS, Bloshchitsyna MN, et al. Processing of probrain natriuretic peptide is suppressed by O-glycosylation in the region close to the cleavage site. Clin Chem 2009; 55:489-98.
(21.) Semenov AG, Tamm NN, Seferian KR, Postnikov AB, Karpova NS, Serebryanaya DV, et al. Processing of proB-type natriuretic peptide: furin and corin as candidate convertases. Clin Chem 2010;56:1166-76.
(22.) Semenov AG, Seferian KR. Biochemistry of the human B-type natriuretic peptide precursor and molecular aspects of its processing. Clin ChimActa 2011;412:85060.
(23.) Miller WL, Phelps MA, Wood CM, Schellenberger U, Van Le A, Perichon R, Jaffe AS. Comparison of mass spectrometry and clinical assay measurements of circulating fragments of B-type natriuretic peptide in patients with chronic heartfailure. Circ Heart Fail 2011;4: 355-60.
(24.) Brandt I, Lambeir AM, Ketelslegers JM, Vanderheyden M, Scharpe S, De Meester I. Dipeptidyl-peptidase IV converts intact B-type natriuretic peptide into its des-SerProform. Clin Chem 2006;52:82-7.
(25.) Ralat LA, Guo Q, Ren M, Funke T, Dickey DM, Potter LR, Tang WJ. Insulin-degrading enzyme modulates the natriuretic peptide-mediated signaling response. J Biol Chem 2011;286:4670-9.
(26.) Dickey DM, Potter LR. Human B-type natriuretic peptide is not degraded by meprin A. Biochem Pharmacol 2010;80:1007-11.
(27.) Liang F, O'Rear J, Schellenberger U, Tai L, Lasecki M, Schreiner GF, et al. Evidence for functional heterogeneity of circulating B-type natriuretic peptide. JACC2007; 49:1071-8.
(28.) Dickey DM, Potter LR. ProBNP (1-108) is resistant to degradation and activates guanylyl cyclase-A with reduced potency. Clin Chem 2011;57:1272-8.
(29.) Heublein DM, Huntley BK, Boerrigter G, Cataliotti A, Sandberg SM, Redfield MM, Burnett JC, Jr. Immunoreactivity and guanosine 3\5'-cyclic monophosphate activating actions of various molecular forms of human B-type natriuretic peptide. Hypertension 2007;49: 1114-9.
(30.) Vodovar N, Seronde MF, Laribi S, Gayat E, Lassus J, Boukef R, et al. Post-translational modifications enhance NT-proBNP and BNP production in acute decompensated heartfailure. Eur HeartJ 2014;35:3434-41.
(31.) Schou M, Dalsgaard MK, Clemmesen O, Dawson EA, Yoshiga CC, Nielsen HB, et al. Kidneys extract BNP and NT-proBNP in healthy young men. J Appl Physiol (1985) 2005;99:1676-80.
(32.) Prontera C, Zaninotto M, Giovannini S, Zucchelli GC, Pilo A, Sciacovelli L, et al. Proficiency testing project for brain natriuretic peptide (BNP) and the N-terminal part of the propeptide of BNP (NT-proBNP) immunoassays: the Cardio Ormocheckstudy. Clin Chem Lab Med 2009; 47:762-8.
(33.) Januzzi JL, van Kimmenade R, Lainchbury J, Bayes-Genis A, Ordonez-Llanos J, Santalo-Bel M, et al. NT-proBNP testing for diagnosis and short-term prognosis in acute destabilized heart failure: an international pooled analysisof 1256 patients: the International Collaborative of NT-proBNP Study. Eur Heart J 2006;27: 330-7.
(34.) Jernberg T, Stridsberg M, Venge P, Lindahl B. N-terminal pro brain natriuretic peptide on admission for early risk stratification of patients with chest pain and no ST-segment elevation. JACC 2002;40:437-45.
(35.) Nishikimi T, Ikeda M, Takeda Y, Ishimitsu T, Shibasaki I, Fukuda H, et al. The effect of glycosylation on plasma N-terminal proBNP-76 levels in patients with heart or renal failure. Heart 2012;98:152-61.
(36.) Luckenbill KN, Christenson RH, Jaffe AS, Mair J, Ordonez-Llanos J, Pagani F, et al. Cross-reactivity of BNP, NT-proBNP, and proBNP in commercial BNP and NT-proBNP assays: preliminary observations from the IFCC Committee for Standardization of Markers of Cardiac Damage. Clin Chem 2008;54:619-21.
(37.) McMurray JJ, Packer M, Desai AS, Gong J, Lefkowitz MP, RizkalaAR, et al. Angiotensin-neprilysin inhibition versus enalapril in heart failure. N Engl J Med 2014; 371:993-1004.
(38.) Semenov AG, Katrukha AG. Different susceptibility of B-type natriuretic peptide (BNP) and BNP precursor (proBNP) to cleavage by neprilysin: the N-terminal part does matter. Clin Chem 2016;62(4):617-22.
(39.) Mair J, Lindahl B, Giannitsis E, Huber K, Thygesen K, Plebani M, et al. Will sacubitril-valsartan diminish the clinical utility of B-type natriuretic peptide testing in acute cardiac care? Eur HeartJ Acute Cardiovasc Care [Epub ahead of print 2016 Jan 12].
(40.) Apple FS, Panteghini M, Ravkilde J, Mair J, Wu AH, Tate J, et al. Quality specificationsfor B-type natriuretic peptide assays. Clin Chem 2005;51:486-93.
Vlad C. Vasile 1 and Allan S. Jaffe (1), (2) *
 Division of Cardiovascular Diseases, Department of Medicine, Rochester, MN;  Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, MN.
* Address correspondence to this author at: Cardiovascular Division, Gonda Bldg. 5th Floor, Mayo Clinic, 200 First St. SW, Rochester, MN 55905. Fax 507-266-0228; e-mail email@example.com.
Received June 8, 2016; accepted June 30, 2016.
Previously published online at DOI: 10.1373/clinchem.2016.254714
 Nonstandard abbreviations: HF, heart failure; ANP, Atrial natriuretic peptide; BNP, B-type natriuretic peptide; CNP, C-type natriuretic peptide; DNP, Dendroaspis natriuretic peptide; NPRs, natriuretic peptide receptors.
Caption: Fig. 1. Epitope locations on BNP and NT-proBNP utilized by commercially available assays.
Caption: Fig. 2. Cross-reactivity of NT-proBNP and BNP assays [data from Luckenbill et al. (36)]. M, Mitsubishi; S, Scios; P, Peptide Institute; H, HyTest; R, Roche.
Table 1. Analytical characteristics of commercially available MR-proANP, BNP, and NT-proBNP assays per the manufacturer. Capture antibody Detection antibody Assay BNP Abbott Architect, N[H.sub.2] terminus COOH terminus, murine AxSYM iSTAT and part of the ring MAb, aa 26-32 structure (Scios), murine MAb, aa 5-13 Alere (b) Triage BNP N[H.sub.2] terminus BNP (Biosite), murine and part of the ring omniclonal AB, epitope structure (Scios), not characterized murine MAb, aa 5-13 Beckman Coulter (b) BNP (Biosite), murine N[H.sub.2] terminus Access, Access 2, Omniclonal AB, epitope and part of the ring DxI not characterized structure (Scios), murine MAb, aa 5-13 Siemens (Bayer) ACS COOH terminus (BC-203) Ring structure 180, Advia Centaur, (Shionogi), murine (KY-hBNPII) Advia Centaur CP MAb, aa 27-32 (Shionogi), murine MAb Siemens (Dade Ring structure COOH terminus Behring) Dimension (KY-hBNPII) murine (BC-203), murine MAb, VISTA, Dimension ExL MAb, aa 14-21 aa 27-32 Shionogi COOH terminus Ring structure (BC-203), murine MAb, (KY-hBNPII), murine aa 27-32 MAb TosohSTAIA-PACK BNP COOH terminus Ring structure (BC-203), murine MAb, (KY-hBNPII), murine aa 27-32 MAb Assay 1-76 NT-proBNP Alere Triage Murine MAb, aa 27-31 Sheep MAb, aa 42-46 NT-proBNP bioMerieux N[H.sub.2] terminus Central molecule, NT-proBNPI VIDAS polyclonal sheep AB, polyclonal sheep AB, aa 1-21 aa 39-50 NT-proBNP2 VIDAS Murine MAb, aa 27-31 Sheep MAb, aa 42 -46 Mitsubishi Chemical N[H.sub.2] terminus Central molecule, PATHFAST polyclonal sheep AB, polyclonal sheep AB, aa 1-21 aa 39-50 Nanogen LifeSign Monoclonal (mouse) and Polyclonal sheep AB DXpress Reader polyclonal (goat) Abs Ortho Clinical N[H.sub.2] terminus Central molecule, Diagnostics Vitros polyclonal sheep AB, polyclonal sheep AB, ECi aa 1-21 aa 39-50 Radiometer AQT90 N[H.sub.2] terminus Central molecule, FLEX NT-proBNP polyclonal sheep AB, polyclonal sheep AB, aa 1-21 aa 39-50 Response Biomedical Murine MAb, aa 27-31 Central molecule, RAMP polyclonal sheep AB, aa 39-50 Roche NT-proBNP I N[H.sub.2] terminus Central molecule, Elecsys, E170 polyclonal sheep AB, polyclonal sheep AB, aa 1-21 aa 39-50 NT-proBNP II MAb, aa 27-31 Sheep MAb, aa 42-46 Elecsys, E170 Siemens (Dade N[H.sub.2] terminus Central molecule, Behring) Dimension monoclonal sheep AB, Sheep MAb, aa 42-46 RxL, Stratus CS, aa 22-28 Dimension VISTA, Dimension EXL with LM Siemens (DPC) N[H.sub.2] terminus Central molecule, Immulite 1000,2000 polyclonal sheep AB, polyclonal sheep AB, 2500 aa 1-21 aa 39-50 Assay MR-proANP Thermo Fisher Polyclonal sheep AB, Monoclonal rat AB, aa Scientific KRYPTOR aa 50-72 of NT-proANP 73-90 of NT-proANP Standard material FDA (a) cleared-yes/ Assay BNP no/claim Abbott Architect, Synthetic BNP 32 Assist in diagnosis of AxSYM iSTAT HF; assess severity of disease Alere (b) Triage BNP Recombinant BNP Aid in diagnosis and severity assessment of HF; risk stratification of patients with ACS and HF; FDA cleared Beckman Coulter (b) Recombinant BNP Diagnosis HF; assess Access, Access 2, severity HF; risk ACS; DxI risk HF Siemens (Bayer) ACS Synthetic BNP Aid in diagnosis and 180, Advia Centaur, assessment of severity Advia Centaur CP of HF; predict survival and likelihood of future HF in ACS patients Siemens (Dade Synthetic BNP 32 Aid in diagnosis and Behring) Dimension assessment of severity VISTA, Dimension ExL of HF; predict survival and likelihood of future HF in ACS patients; pending FDA clearance Shionogi Synthetic BNP Not FDA cleared TosohSTAIA-PACK BNP Synthetic BNP Not FDA cleared Assay 1-76 NT-proBNP Alere Triage Synthetic NTproBNP 1-76 Aid in diagnosis of NT-proBNP HF; risk stratification of patients with ACS and HF; assessment of increased risk of cardiovascular events and mortality in patients at risk for HF who have stable CAD; not currently available in the US bioMerieux Synthetic NTproBNP 1-76 Diagnosis HF NT-proBNPI VIDAS NT-proBNP2 VIDAS Synthetic NTproBNP 1-76 Not FDA cleared Mitsubishi Chemical Synthetic NTproBNP 1-76 Aid diagnosis of CHF; PATHFAST assess severity CHF; risk stratification in ACS and stable CAD Nanogen LifeSign Synthetic 1-76 NTproBNP Diagnosis HF DXpress Reader Ortho Clinical Synthetic 1-76 NTproBNP Aid diagnosis of CHF; Diagnostics Vitros risk stratification of ECi ACS and CHF; risk assessment of CV events and mortality in patients at risk for HF with stable CAD; assess severity in HF Radiometer AQT90 Synthetic 1-76 NTproBNP Diagnosis HF; risk FLEX NT-proBNP stratification of patients with ACS and HF; not FDA cleared Response Biomedical Synthetic 1-76 NTproBNP Diagnosis HF; assess RAMP severity HF Roche NT-proBNP I Synthetic 1-76 NTproBNP Diagnosis HF; assess Elecsys, E170 severity HF; risk ACS; risk HF NT-proBNP II Synthetic 1-76 NTproBNP Treatment monitoring Elecsys, E170 in LVD Siemens (Dade Synthetic 1-76 NTproBNP Aid in the diagnosis Behring) Dimension of CHF and assessment RxL, Stratus CS, of severity; risk Dimension VISTA, stratification of Dimension EXL with patients with ACS and LM HF Siemens (DPC) Synthetic 1-76 NTproBNP Not FDA cleared Immulite 1000,2000 2500 Assay MR-proANP Thermo Fisher Synthetic 50-90 Not FDA cleared Scientific KRYPTOR (a) FDA, US Food and Drug Administration; CHF, congestive heart failure; ACS, acute coronary syndrome; CV, cardiovascular; CAD, coronary artery disease; LVD, left ventricular dysfunction; aa, amino acid; AB, antibody; MR-proANP, midregional pro-ANP. (b) Both the Alere and Beckman systems use the same 2 antibodies but due to their different assay formats, designation of the monoclonal and omniclonal antibodies as capture and detection antibody is not absolute.(Adapted with permission from IFCC.International Federation of Clinical Chemistry and Laboratory Medicine, website http://www. ifcc.org.) Table 2. Percentage recoveries and cross-reactivities by BNP and NT-proBNP assays for each peptide [data from Luckenbill et al. (36)]. Assay Sa BNP P BNP S proBNP Architect 151 (142-160) (b) 98 (85-110) 38(37-38) AxSYM 184(164-205) 124(117-130) 34 (28-39) Centaur 194(189-199) 137(133-141) 17(17-18) Access 199(192-205) 130(129-130) 24 (24-25) Triage 135(115-156) 79(78-80) 19(18-20) NT-proBNP Dimension <1 (0-0) <1 (0-0) <1 (0-0) Vitros <1 (0.6-0.8) <1 (0.03-0.4) 2 (1.6-2.2) Elecsys <1 (0.2-0.8) <1 (0-0.04) 1 (0.7-2) Assay HproBNP H NT-proBNP R NT-proBNP Architect 6(2-11) <1 (0-0.8) 4 (4-5) AxSYM 9(3-15) <1 (0-0.5) 4 (3-5) Centaur 14(10-17) <1 (0-0.3) 7 (5-9) Access 13(7-19) <1 (0-0.4) 6 (5-7) Triage 5(0-12) <1 (0-0.2) 3 (2-4) NT-proBNP Dimension 249 (230-267) 243 (235-251) 95 (91-99) Vitros 55 (52-59) 127(124-130) 71 (68-74) Elecsys 29 (28-30) 131 (126-137) 47 (45-49) (a) S, Scios; P, Peptide Institute; H, HyTest; R, Roche. (b) 95% CIs are in parentheses.
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|Author:||Vasile, Vlad C.; Jaffe, Allan S.|
|Date:||Jan 1, 2017|
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