Discovery of the endocrine function of the heart, which exerts a fundamental role in regulating cardiovascular homeostasis through atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), has opened up a new field of research that has resulted in multiple clinical applications

A. Clerico

C. Passino

M. Emdin

EMDIN, MICHELE

PASSINO, Claudio

CLERICO, ALDO

Archivio della ricerca della Scuola Superiore Sant'Anna

mppppTatpyiprJournal of the American College of Cardiology Vol. 57, No. 12, 2011© 2011 by the American College of Cardiology Foundation ISSN 0735-1097/$36.00Published by Elsevier Inc. doi:10.1016/j.jacc.2010.09.075EDITORIAL COMMENTNatriuretic PeptideAssays RevisitedDo We NeedPro–B-Type Natriuretic Peptide?*Michele Emdin, MD, PHD,†Claudio Passino, MD,†‡ Aldo Clerico, MD†‡Pisa, ItalyDiscovery of the endocrine function of the heart (1), whichexerts a fundamental role in regulating cardiovascular ho-meostasis through atrial natriuretic peptide (ANP) andB-type natriuretic peptide (BNP), has opened up a new fieldof research that has resulted in multiple clinical applications(2). The human BNP gene encodes for a pre-proBNPolecule of 134 amino acid residues, including a signaleptide of 26 amino acids. The BNP is cleaved out of arohormone molecule of 108 amino acids, the proBNP1-108(proBNP). Before secretion from cardiomyocytes intoblood, proBNP is split by some proteolytic enzymes (prob-ably corin and/or furin) into 2 peptides: the biologicallySee page 1386inactive NH2-terminal peptide fragment proBNP1–76 (NT-roBNP), and the COOH-terminal peptide fragmentroBNP77–108 (BNP), which is the active hormone, bindingspecific receptors (NPR-A, -B, -C) (2,3). In addition tobioactive BNP, a huge number of circulating proBNP-derived fragments can be identified by chromatographicprocedures in human plasma (4), including the intactprecursor proBNP1–108, found in a significant amount instage C and stage D heart failure (HF) patients (3–5). Lianget al. (6) demonstrated that proBNP, constituting a sub-stantial portion of immunoreactive BNP in plasma of HFpatients, possesses significantly lower biological activity thanthe 32-amino acid hormone.The widespread diffusion of automated assays of BNPand NT-proBNP has furnished a novel tool for diagnosis,prognostic evaluation, and guide to treatment of overt HF,*Editorials published in the Journal of the American College of Cardiology reflect theviews of the authors and do not necessarily represent the views of JACC or theAmerican College of Cardiology.From the †Fondazione G. Monasterio CNR-Regione Toscana, Pisa, Italy; and‡Scuola Superiore Sant’Anna, Pisa, Italy. All authors have reported that they have norelationships to disclose.as well for the screening of asymptomatic left ventriculardysfunction in the general population (2,7), with a potentialuse in primary care (8). Recent evidence has challenged,however, the specificity of all commercial assays for BNPand NT-proBNP, indicating a significant cross-reactivitywith the precursor proBNP1–108, variable for each assay (9).his means that, beyond their proven diagnostic accuracynd prognostic value in HF, those assays are not specific forheir declared target, with evident implications for theathophysiological and clinical interpretation of data, notet completely understood.In this issue of the Journal, Macheret et al. (10) measuredn the general community the circulating intact precursorroBNP1–108 by means of a highly specific proBNP1–108sandwich immunoassay (11). They evaluated its ability todetect left ventricular systolic dysfunction, in comparisonwith commercial BNP and NT-proBNP assays, in a cohortof 1,939 subjects (672 healthy subjects and 1,267 stages A to CHF patients, 45 years of age), undergoing clinical andechocardiographic characterization. The method used isbased on a specific monoclonal antibody recognizing thecleavage site of proBNP1–108, without any significant cross-reactivity with either recombinant NT-proBNP1–76 or syn-thetic BNP1–32, combined with a polyclonal antibody di-ected against BNP1–32 (11).Macheret et al. (10) demonstrated that proBNP1–108 wasdetectable in all subjects studied, and its level was dependenton sex, age, heart rate, and body mass index. Furthermore,they found that the degree of sensitivity and specificity ofthe proBNP assay for the detection of left ventriculardysfunction was comparable to 2 commercial assays forBNP and NT-proBNP (10).This study confirms and extends previous evidence (5)that the intact precursor of biologically active BNP (i.e., theproBNP1–108) circulates in plasma of both healthy subjectsand HF patients, and underscores the diagnostic usefulnessof its assay (5), which has been previously shown to holdprognostic value in patients with systolic HF, too (12).Moreover, circulating levels of proBNP1–108 are age- andsex-dependent and progressively increase with disease se-verity in HF patients, as previously reported for BNP andNT-proBNP measured by commercial immunoassays (2,3,13).Two potential drawbacks are evidenced by the Macheretstudy (10): the automated proBNP immunoassay is notcommercially available, and may not be considered, at thismoment, for current clinical use, especially in emergencydepartments. Moreover, this study was not able to demon-strate a better diagnostic accuracy of proBNP for leftventricular dysfunction, as compared with a currently usedNT-proBNP assay.However, the high specificity of this proBNP assay fortargeted molecules and its overall good diagnostic accuracyas shown in this study pave the way for a future use of thisbiomarker, as an alternative to other BNP assays currentlyused for screening of left ventricular dysfunction in asymp-ecamtaHNmbpistdeufecotnsrrrcipmC(vetbpanfiFlcworpm(craacwsvifspBo1397JACC Vol. 57, No. 12, 2011 Emdin et al.March 22, 2011:1396–8 ProBNP Assay in the General Populationtomatic subjects at intermediate to high risk for HF, or fordifferential diagnosis in patients with dyspnea. Evidence isgrowing, supporting an out-of-hospital use of natriureticpeptide assays: their implementation in screening programsand diagnostic algorithms in primary care might contributeto decrease the apparent disparity between the generalpractitioner and the specialist approach to disease manage-ment (8).Indeed, the findings by Macheret et al. (10) indicate thatthe greater part of B-type natriuretic peptides assayed in HFpatients is devoid of biological activity. As a whole, theseresults (10) support the hypothesis that the post-translational maturation processing of ventricular proBNPis not efficient in HF (2,15,16).The BNP gene is expressed in both atrial and ventricularmyocytes of normal and diseased hearts (1,2,14). Ventricu-lar myocytes do not usually display any evident secretorygranules at electron microscopy in normal heart, whereassome granules, similar to the atrial ones, can be found insamples of ventricular myocardium collected during surgeryor in endocardial biopsies (2,3). That suggests that normalventricular myocardium may produce only a limited amountof biologically active BNP1–32 in response to an acute,fficacious stimulation, probably through a constitutive se-retory pathway, while the amount of hormone producednd secreted after chronic stimulation in patients with HFight be greatly increased through an up-regulated secre-ory pathway (10,14). According to this hypothesis, a hugemount of immunoreactive BNP circulating in plasma ofF patients and measurable with conventional BNP andT-proBNP immunoassays is not biologically active. Thatay add to the understanding of the resistance to theiological effects of natriuretic hormones (the “natriureticaradox”), which plays an important role in the pathophys-ology of sodium retention and systemic and renal vasocon-triction observed in severe HF, substantially contributingo disease progression (2,3,15).At present, the exact reason for proBNP1–108 processingeficiency in HF is unknown, but an enzymatic deficiency,ither due to saturation or constitutive dysfunction, mightnderlie the progression from asymptomatic cardiac dys-unction to overt HF (2). The hypothesis of a “cardiacndocrine dysfunction” may support the high variability oflinical HF phenotypes, given a similar etiology and extentf cardiac damage/dysfunction. However, it is well knownhat infusion of the same ANP or BNP dose has a reducedatriuretic effect in HF patients, as compared to healthyubjects. This observation suggests that there are also othereceptorial and post-receptorial mechanisms causing theesistance to natriuretic hormone action, including down-egulation of A and B natriuretic receptors, up-regulation oflearance NPR-C receptors, or post-receptor issues, such asncreased activity of cyclic guanosine monophosphate-hosphodiesterase, which metabolizes cyclic guanosineonophosphate (2,16). aConversely, it is theoretically conceivable that theOOH-terminal fragment including the cysteine bondi.e., the active hormone BNP1–32) may be produced even inivo from the circulating intact precursor proBNP by plasmanzyme degradation. Although there is no demonstrationhat circulating proBNP can be processed in vivo in humanlood, a soluble form of corin, a transmembrane serinerotease able to cleave both proANP and proBNP, may belso capable of processing the circulating intact precursor ofatriuretic hormones (17). Dong et al. (18) recently con-rmed that soluble corin is measurable in human blood.urthermore, these investigators reported that plasma corinevels are significantly lower in HF patients than in healthyontrols and that the reduction in plasma enzyme correlatesith the severity of the disease (18). These preliminarybservations, if confirmed, suggest a more complex scenarioegarding the “natriuretic paradox”: the circulating intactrecursor proBNP might be actually considered a prohor-one, processed into the active peptide, when necessary17,18). This hypothesis assumes that the peripheral pro-essing of circulating proBNP would be submitted toegulatory rules, possibly altered with HF progression. Thisbnormality might be considered as a possible future ther-peutic target, for example, by studying drugs inducing theleavage of the prohormone into active BNP.Although the assay of proBNP (a more stable moleculeith higher molecular weight than derived peptides) hasome theoretical advantages, from an analytical point ofiew, once automated assays will be available, 1 importantssue could facilitate its diffusion in the clinical setting. As auture perspective, the simultaneous measurement in theame plasma sample with 2 methods, 1 specific for the intactrecursor proBNP1–108, and the other for active peptideNP1–32, could allow a more accurate estimate of the trueactivity of cardiac natriuretic hormone system as comparedwith the single assay of either peptide (4,9), likely extendingour present understanding of pathophysiological and clinicalchanges due to cardiac endocrine dysfunction on HF evo-lution. Indeed, a recent study of ambulatory patients withchronic systolic HF showed that the combined assessmentof conventional BNP and proBNP immunoassays providesadditional information in determining the risk of adverseclinical outcomes, particularly in patients with low BNPvalues (12). Conversely, the proposal by Macheret et al. (10)of considering proBNP/NT-proBNP ratio, based on anelectrochemiluminescent immunoassay of NT-proBNP, asa useful interpretative tool is questionable. Actually, thisratio does not reflect either the ratio between the prohor-mone and the biologically active hormone or the truerelationship between the prohormone and NT-proBNPpeptide level, because of the cross-reactivity of the NT-proBNP method used with proBNP, as mentioned by theauthors themselves.In summary, a proBNP1–108 assay shows several featuresf the ideal biomarker (in vivo and in vitro stability,dequate analytical-functional sensitivity, reproducibility,inpru11398 Emdin et al. JACC Vol. 57, No. 12, 2011ProBNP Assay in the General Population March 22, 2011:1396–8and accuracy) (11), whereas others are still lacking (com-plete assay automation, demonstration of low biologicalvariation, reference range and cut-off values tested for sex,age, and ethnicity dependence, extensively proven gooddiagnostic and prognostic accuracy, evaluation of cost effec-tiveness). Our interest in this “novel” natriuretic peptideassay is in its specificity for the molecule targeted, whereasat present all conventional BNP and NT-proBNP immu-noassays, showing some cross-reactivity with the intactprecursor proBNP, may be considered, at the same time,accurate markers of HF presence and severity, and inaccu-rate indexes of BNP biological activity (9). Diagnosticmanufacturers should set up specific immunoassay methodsfor biologically active BNP1–32 peptide, to overcome thismportant drawback. Thereafter, designed studies would beecessary to estimate and compare the diagnostic andrognostic accuracy of specific assays for different B-typeelated peptides: BNP, NT-proBNP, and intact proBNP,sed either alone or in combination.Reprint requests and correspondence: Dr. Michele Emdin,Cardiovascular Medicine Division, Fondazione G. MonasterioCNR-Regione Toscana, Via Giuseppe Moruzzi 1, Pisa 56124,Italy. E-mail: emdin@ftgm.it.REFERENCES1. de Bold AJ, Borenstein HB, Veress AT, Sonnenberg H. A rapid andpotent natriuretic response to intravenous injection of atrial myocardialextract in rats. Life Sci 1981;28:89–94.2. Clerico A, Recchia FA, Passino C, Emdin M. Cardiac endocrinefunction is an essential component of the homeostatic regulationnetwork: physiological and clinical implications. Am J Physiol HeartCirc Physiol 2006;290:H17–29.3. Goetze JP. Biosynthesis of cardiac natriuretic peptides. Results ProblCell Differ 2010;50:97–120.4. Seferian KR, Tamm NN, Semenov AG, et al. The brain natriureticpeptide (BNP) precursor is the major immunoreactive form of BNP inpatients with heart failure. Clin Chem 2007;53:866–73.5. Mogelvang R, Goetze JP, Schnohr P, et al. Discriminating betweencardiac and pulmonary dysfunction in the general population withdyspnea by plasma pro-B-type natriuretic peptide. J Am Coll Cardiol2007;23;50:1694–701.6. Liang F, O’Rear J, Schellenberger U, et al. Evidence for functionalheterogeneity of circulating B-type natriuretic peptide. J Am CollCardiol 2007;49:1071–8.7. Hunt SA, Abraham WT, Chin MH, et al. 2009 focused updateincorporated into the ACC/AHA 2005 guidelines for the diagnosisand management of heart failure in adults: a report of the AmericanCollege of Cardiology Foundation/American Heart Association TaskForce on Practice Guidelines. J Am Coll Cardiol 2009;53:e1–90.8. Emdin M, Fontana M, Poletti R, et al. Natriuretic peptide testing inprimary care patients. Clin Chem Lab Med 2008;46:1533–42.9. Luckenbill KN, Christenson RH, Jaffe AS, et al. Cross-reactivity ofBNP, NT-proBNP, and proBNP in commercial BNP and NT-proBNP assays: preliminary observations from the IFCC Committeefor Standardization of Markers of Cardiac Damage. Clin Chem2008;54:619–21.0. Macheret F, Boerrigter G, McKie P, et al. Pro–B-type natriureticpeptide1–108 circulates in the general community: plasma determinantsand detection of left ventricular systolic dysfunction. J Am CollCardiol 2011;57:1386–95.11. Giuliani I, Rieunier F, Larue C, et al. Assay for measurement of intactB-type natriuretic peptide prohormone in blood. Clin Chem 2006;52:1054–61.12. Dries DL, Ky B, Wu AH, Rame JE, Putt ME, Cappola TP.Simultaneous assessment of unprocessed ProBNP1-108 in addition toprocessed BNP32 improves identification of high-risk ambulatorypatients with heart failure. Circ Heart Fail 2010;3:220–7.13. Clerico A, Emdin M. Diagnostic accuracy and prognostic relevance ofthe measurement of the cardiac natriuretic peptides: a review. ClinChem 2004;50:33–50.14. McGrath MF, de Bold AJ. Determinants of natriuretic peptide geneexpression. Peptides 2005;26:933–43.15. Chen HH. Heart failure: a state of brain natriuretic peptide deficiencyor resistance or both! J Am Coll Cardiol 2007;49:1089–91.16. Forfia PR, Lee M, Tunin RS, Mahmud M, Champion HC, Kass DA.Acute phosphodiesterase 5 inhibition mimics hemodynamic effects ofB-type natriuretic peptide and potentiates B-type natriuretic peptideeffects in failing but not normal canine heart. J Am Coll Cardiol2007;49:1079–88.17. Knappe S, Wu F, Masikat MR, et al. Functional analysis of thetransmembrane domain and activation cleavage of human corin: designand characterization of a soluble corin. J Biol Chem 2003;278:52363–70.18. Dong N, Chen S, Yang J, et al. Plasma soluble corin in patients withheart failure. Circ Heart Fail 2010;3:207–11.Key Words: biomarker y BNP y heart failure y NT-proBNP yproBNP.

Natriuretic Peptide assays revisited do we need pro-B-type natriuretic Peptide?

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