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Head-to-head comparison of N-terminal pro-brain natriuretic peptide, brain natriuretic peptide and N-terminal pro-atrial natriuretic peptide in diagnosing left ventricular dysfunction
Clinica Chimica Acta 310 Ž2001. 193–197 www.elsevier.comrlocaterclinchim
Head-to-head comparison of N-terminal pro-brain natriuretic peptide, brain natriuretic peptide and N-terminal pro-atrial natriuretic peptide in diagnosing left ventricular dysfunction Angelika Hammerer-Lercher a,) , Elke Neubauer b, Silvana Muller ¨ b, Otmar Pachinger b, Bernd Puschendorf a , Johannes Mair b a
Department of Medical Chemistry and Biochemistry, DiÕision of Clinical Biochemistry, UniÕersity of Innsbruck, Fritz-Pregl-Str. 3, A-6020 Innsbruck, Austria b Department of Internal Medicine, DiÕision of Cardiology, UniÕersity of Innsbruck, Innsbruck, Austria Received 8 January 2001; accepted 15 June 2001
Abstract Brain natriuretic peptide ŽBNP., NT-proBNP and NT-pro-atrial natriuretic peptide ŽNT-proANP. were measured in blood samples from 57 patients using immunoassays and immunoradiometric assays to evaluate the usefulness as diagnostic markers for the detection of heart failure. For the detection of impaired left ventricular ejection fraction ŽLVEF., receiver operating characteristic curves showed that BNP had the best diagnostic performance with an area under curve ŽAUC. of 0.75 " 0.06. However, NT-proBNP ŽAUC: 0.67 " 0.07. and NT-proANP ŽAUC: 0.69 " 0.08. showed no significant difference to BNP. In a further analysis for the detection of resting LVEF - 40%, BNP again was the best marker with an AUC of 0.83 " 0.06. NT-proBNP showed only a slightly smaller AUC Ž0.79 " 0.07.. The AUC for NT-proANP was significantly smaller Ž0.65 " 0.08. compared to BNP. Additionally, BNP and NT-proBNP correlated negatively with the resting LVEF ŽBNP: y0.472, p - 0.001; NT-proBNP: y0.306, p s 0.026., whereas NT-proANP showed no significant correlation. In summary, BNP was the best marker to detect patients with impaired LVEF compared to NT-proBNP and NT-proANP. However, NT-proBNP showed no significant differences to BNP and it is therefore a new promising alternative marker for the detection of left ventricular dysfunction. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Brain natriuretic peptide; N-terminal pro-brain natriuretic peptide; N-terminal pro-atrial natriuretic peptide; Left ventricular dysfunction; Three-dimensional echocardiography; Radionuclide ventriculography
AbbreÕiations: BNP, Brain natriuretic peptide; NT-proBNP, N-terminal proBNP; NT-proANP, N-terminal pro-atrial natriuretic peptide; LVEF, Left ventricular ejection fraction; ROC, Receiver operating characteristic curve; AUC, Area under curve; CHF, Congestive heart failure; ACE, Angiotensin-converting enzyme; LVD, Left ventricular dysfunction; cGMP, Cyclic guanosine monophosphate; 3D, Three-dimensional; RNV, Radionuclide ventriculography; NYHA, New York Heart Association. ) Corresponding author. Tel.: q43-512-507-2876; fax: q43-512-507-3522. E-mail address: [email protected] ŽA. Hammerer-Lercher.. 0009-8981r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 8 9 8 1 Ž 0 1 . 0 0 5 7 8 - 2
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A. Hammerer-Lercher et al.r Clinica Chimica Acta 310 (2001) 193–197
1. Introduction Congestive heart failure ŽCHF. is one of the main causes of hospitalisation in industrialized countries with a 5-year survival rate of only 50%. Its prevalence increases markedly with age w1x. The early diagnosis of CHF is crucial, since an early treatment with inhibitors of the angiotensin-converting enzyme ŽACE. can prevent or retard disease progression w2,3x. Natriuretic peptides have been proposed for early diagnosis and as prognostic markers in left ventricular dysfunction ŽLVD. w1x. In earlier studies it has been demonstrated that natriuretic peptides and their second messenger cyclic guanosine monophosphate ŽcGMP. increase with the severity of CHF w4–8x. Most comparative studies showed that brain natriuretic peptide ŽBNP. and NT-pro-atrial natriuretic peptide ŽNT-proANP. are superior to other natriuretic peptides to diagnose asymptomatic and symptomatic LVD w7,9–11x. An own previous investigation with very well-defined asymptomatic CHF patients ŽNYHA I. revealed that BNP is superior to ANP, NT-ANP and cGMP in diagnosing symptomless left ventricular dysfunction w9x. Recently, a new marker, NT-proBNP, has been proposed as a diagnostic and prognostic alternative and possibly superior marker in heart failure patients w12,13x. The aim of the present study was to directly compare the diagnostic values of the NT-natriuretic peptide fragments, i.e. NT-proBNP Ž8–29. and NTproANP Ž1–98., with BNP in the same study population to evaluate the usefulness of NT-proBNP in a head-to-head comparison with established natriuretic peptide markers.
2. Materials and methods 2.1. Subjects After giving informed consent, 57 consecutive patients Ž45–80 years. with stable chronic heart failure who underwent three-dimensional Ž3D. echocardiography and radionuclide ventriculography ŽRNV. at rest to assess left ventricular ejection fraction ŽLVEF. were enrolled in this study. Sixteen patients were in the New York Heart Association ŽNYHA. class I, 31 were in NYHA class II and 10 were in
NYHA class III, respectively. Most of the patients Ž n s 54. suffered from coronary artery disease and 12 patients had hypertension. Creatinine concentrations were within normal range in all but two patients. Drug treatment comprised ACE inhibitors Ž n s 25 patients., b-blockers Ž n s 21., nitrates Ž n s 19., diuretics Ž n s 10. and calcium channel blockers Ž n s 5.. The patient population was divided into two subgroups: first, in patients with normal ŽLVEF ) 48% according to 3D-echocardiograpy and LVEF ) 55% according to RNV. and impaired resting LVEF ŽLVEF - 48% according to 3D-echocardiograpy and LVEF - 55% according to RNV., and second, in patients with resting LVEF below and above 40% Žaccording to RNV.. EDTA blood samples were drawn after a standardized period of rest in a supine body position. After blood withdrawal, samples were stored at 4 8C Žup to 1 h. until centrifugation at 4 8C and 2000 = g for 10 min. The plasma was subsequently stored at y20 8C Žfor up to 1 month. before assay of BNP, NT-proBNP and NT-proANP. 2.2. Assays All peptides were measured using commercially available immunoassays without plasma extraction procedures. BNP was measured by an immunoradiometric assay ŽCat. No. IC-1049, Shionogi, Japan. w14x. In brief, samples were thawed and aliquots were incubated with 125 I-BNP marked antibody together with anti-human BNP antibody coated beads for approximately 20 h. After aspirating the tubes, the remaining radioactivity bound to the beads was measured with a gamma scintillation counter. NTproBNP w15x and NT-proANP w16x were measured by enzyme immunoassays ŽCat. No. BI-20852 and BI20892 Biomedica, Vienna, Austria.. In short, for NT-proBNP measurement, a limiting amount of NT-proBNP specific antibody was incubated with an anti sheep-specific antibody adsorbed to the wells for 5 h. After washing the wells, aliquots of diluted samples were incubated together with the biotinylated peptide Žtracer. overnight at 4 8C. After a washing step, the peroxidase-conjugated streptavidin that binds to the biotin of the bound tracer was added and incubated for 1 h. After removal of unbound conjugate through washing, substrate was added to
A. Hammerer-Lercher et al.r Clinica Chimica Acta 310 (2001) 193–197
the wells and incubated for 20 min in the dark. Bound biotinylated NT-proBNP Ž8–29. was quantified by an enzyme catalysed colour change, and the absorption was determined photometrically. For measurement of NT-proANP, aliquots of diluted samples and biotinylated NT-proANP were incubated simultaneously for 150 min at 37 8C. After washing, a streptavidin–peroxidase conjugate was added and incubated for 1 h at 37 8C. Another washing step was followed by an incubation of tetramethylbenzidine for 20 min at room temperature in the dark. The absorbance was read at 450 with 690 nm as reference.
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2.5. Statistics Receiver-operating characteristic ŽROC. plot analysis was carried out to compare the diagnostic utility
2.3. Echocardiography Left ventricular volume indices, as end-systolic and end-diastolic volumes and stroke volume, were measured using native harmonic 2D imaging with biplane Simpson’s rule with apical four-chamber view and two-chamber view and 3D echocardiography. A commercial ultrasound system Sequoia C 256 ŽAcuson Mountainview, USA. equipped with a 3.5r1.75 MHz receivertransmit transducer was used. For 3D freehand echocardiography, a magnetic field system ŽAscension Technology, Burlington, VT. was used to track the position and orientation of the image planes w17x. The LVEF calculated by 3D echocardiography, which is more accurate, was used for comparison with natriuretic peptide concentrations. 2.4. Radionuclide Õentriculography In short, as previously described w9x, rest LVEF was determined by ECG-gated equilibrium RNV in supine body posture. Apex SR camera and an Elscint Apex 1 computer system ŽElscint Medical Technology, Austria. were used for data acquisition. Red blood cells were labelled by an in vitro labelling method with 700–900 MBq technetium-99 m as described before w9x. Time–activity curves were obtained in phase mode using 16 frames per cycle. LVEF was determined Žafter background correction. independently by two experienced technicians using a semiautomatic operator-interactive computer program.
Fig. 1. Receiver-operating-characteristic ŽROC. curves for BNP, NT-proBNP and NT-proANP to ŽA.: correctly identify patients with impaired LVEF ŽLVEF - 48% according to 3D-echocardiograpy and LVEF - 55% according to RNV. and to ŽB.: correctly identify patients with resting LVEF - 40% ŽRNV..
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of the natriuretic peptides, and Spearman rank correlation coefficients were calculated. A p value of - 0.05 was considered to indicate statistical significance.
3. Results ROC analysis showed that among the evaluated natriuretic peptides, BNP had the best diagnostic performance to detect patients with either impaired LVEF or resting LVEF - 40% ŽFig. 1.. The area under curve ŽAUC. was greater than those of the NT-natriuretic peptides. In the subanalysis of normal versus impaired LVEF, AUC Ž"S.E.. were 0.75 Ž"0.06. for BNP, 0.67 Ž"0.07. for NT-proBNP, and 0.69 Ž"0.08. for NT-proANP, respectively. In the subanalysis of patients with LVEF - 40% versus ) 40%, BNP showed an AUC of 0.83 Ž"0.06., NT-proBNP of 0.79 Ž"0.07. and NT-proANP of 0.65 Ž"0.08.. There was a significant difference between BNP and NT-proANP for the detection of resting LVEF - 40% ŽRNV.. However, NT-proBNP did not differ significantly from BNP in both subanalysis. Sensitivities, specificities, positive and negative predictive values and efficiencies for the detection of LVEF below 40% ŽRNV. are listed in Table 1. Optimal discriminator values according to ROC analysis were 41 ŽBNP., 488 ŽNT-proBNP. and 2150 pmolrl ŽNT-proANP.. Values given in Table 1 are based on these optimal discriminator values. Additionally, BNP Ž r s y0.472; p - 0.001. as well as NT-proBNP Ž r s y0.306; p s 0.026., but not NTproANP, showed a significant inverse correlation with LVEF. Table 1 Diagnostic performance of BNP, NT-proBNP and NT-proANP at optimal discriminator values Ž41, 488 and 2150 pmolrl, respectively. for detection of LVEF - 40% versus ) 40%
Sensitivity Specificity Positive predictive value Negative predictive value Efficiency
BNP
NT-proBNP
NT-proANP
0.73 0.77 0.70
0.70 0.73 0.61
0.59 0.61 0.48
0.79
0.80
0.71
0.75
0.72
0.60
4. Discussion The current results concur with our previous findings w9x, demonstrating that BNP was a superior marker compared with ANP, NT-ANP, and cGMP for the identification of patients with asymptomatic LVD. This also is in accordance with the results of Motwani et al. w18x, Yamamoto et al. w7x, and Choy et al. w19x who all showed that the inverse relation of LVEF with BNP was stronger than with ANP or NT-proANP. Our findings are not consistent with those of Lerman et al. w20x who found that NT-proANP was a powerful marker of symptomless LVD. Recently, Arnlov et al. w21x reported increased levels of NT-proANP in patients with LVD. However, in these two above mentioned studies BNP was not measured, and thus the predictive power of NT-proANP and BNP cannot be compared. Furthermore, we found a slightly better AUC for BNP compared with NT-proBNP, but there was no significant difference between the two markers. Richards et al. w12x showed that the relation of NT-proBNP with LVEF was comparable with that of BNP and was stronger than for the other measured hormones. Moreover, in his study, the sensitivity for prediction of death and LVD of NT-proBNP Ž91%. slightly exceeded that of BNP Ž86%., whereas specificity and positive and negative predictive values were equal. Hunt et al. w22x also demonstrated that NT-proBNP and BNP were significantly correlated with each other and inversely with LVEF. The levels of NT-proBNP were approximately fourfold higher than BNP levels in patients with moderate LVD. Richards et al. w12x and Hunt et al. w22x used radioimmunoassays, which need extraction of plasma samples for the determination of natriuretic peptides. We used a highly specific, sensitive and precise immunoradiometric assay to determine concentrations of BNP w14x and recently developed immunoassays for NTproBNP Ž8–29. w15x and NT-proANP Ž1–98. w16x, which do not need an extraction step before peptide measurement. Consequently, time for measuring either BNP, NT-proBNP or NT-proANP is shorter, and there is no imprecision arising from different recovery amounts of natriuretic peptides due to extraction of plasma samples. In the present study, BNP and NT-proBNP were comparable useful markers of LVD with a better diagnostic performance of
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BNP compared with NT-proANP. Yet, NT-proBNP showed a marginally smaller AUC than BNP. In conclusion, BNP was tendentiously the best marker to detect patients with impaired LVEF. The differences between BNP and NT-proBNP were not statistically significant and, therefore, NT-proBNP is a promising alternative marker for diagnosing heart failure, and it is worthy of further evaluation.
Acknowledgements This study was supported by a research grant from Abbott Laboratories ŽNorth Chicago, IL, USA..
References w1x Mair J, Friedl W, Thomas S, Puschendorf B. Natriuretic peptides in assessment of left-ventricular dysfunction. Scand J Clin Lab Invest, Suppl 1999;230:132–42 wReviewx. w2x SOLVD Investigators. Effect of enalapril on mortality and the development of heart failure in asymptomatic patients with reduced left ventricular ejection fraction. N Engl J Med 1992;327:685–91. w3x Packer M, Cohn JN. Consensus recommendations for the management of chronic heart failure. Am J Cardiol 1999; 83:1A–32A. w4x Burnett Jr. JC, Kao PC, Hu DC, et al. Atrial natriuretic peptide elevation in congestive heart failure in human. Science 1986;231:1145–7. w5x Haug C, Metzele A, Koch M, Hombach V, Gruner A. ¨ Plasma brain natriuretic peptide and atrial natriuretic peptide concentrations correlate with left ventricular end-diastolic pressure. Clin Cardiol 1993;16:553–7. w6x Jakob G, Mair J, Vorderwinkler KP, et al. Clinical significance of urinary cyclic guanosine monophosphate in diagnosis of heart failure. Clin Chem 1994;40:96–100. w7x Yamamoto K, Burnett JC, Jougasaki M, et al. Superiority of brain natriuretic peptide as a hormonal marker of ventricular systolic and diastolic dysfunction and ventricular hypertrophy. Hypertension 1996;28:988–94. w8x Tsutamoto T, Wada A, Maeda K, et al. Attenuation of compensation of endogenous cardiac natriuretic peptide system in chronic heart failure—prognostic role of plasma brain natriuretic peptide concentration in patients with chronic symptomatic left ventricular dysfunction. Circulation 1997; 96:509–16.
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w9x Friedl W, Mair J, Thomas S, Pichler M, Puschendorf B. X X Natriuretic peptides and cyclic guanosine 3 ,5 -monophosphate in asymptomatic and symptomatic left ventricular dysfunction. Heart 1996;76:129–36. w10x Cowie MR, Struthers AD, Wood DA, Coats AJS, Thompson SG, Poole-Wilson PA. Value of natriuretic peptides in assessment of patients with possible new heart failure in primary care. Lancet 1997;350:1349–53. w11x McDonagh TA, Robb SD, Murdoch DR, et al. Biochemical detection of left-ventricular dysfunction. Lancet 1998;351:9– 13. w12x Richards AM, Nicholls MG, Yandle TG, et al. Plasma N-terminal pro-brain natriuretic peptide and adrenomedullin. New neurohormonal predictors of left ventricular function and prognosis after myocardial infarction. Circulation 1998; 97:1921–9. w13x Talwar S, Squire B, Davies JE, Barnett DB, Ng LL. Plasma N-Terminal pro-brain natriuretic peptide and the ECG in the assessment of left-ventricular systolic dysfunction in a high risk population. Eur Heart J 1999;20:1736–44. w14x Kono M, Yamauchi A, Tsuji T, et al. An immunoradiometric assay for brain natriuretic peptide in human plasma. Jpn J Nucl Med Technol 1993;13:2–7. w15x Mißbichler A, Hawa G, Woloszczuk W, Schmal N, Hartter E. Enzyme immunoassays for proBNP fragments Ž8–29. and Ž32–57.. J Lab Med 1999;23Ž4.:241–4. w16x Hartter E, Khalafpour S, Mißbichler A, Hawa G, Woloszczuk W. Enzyme immunoassays for fragments Žepitopes. of human proatrial natriuretic peptides. Clin Chem Lab Med 2000; 38Ž1.:27–32. w17x Leotta DF, Munt B, Bolson MS, et al. Quantitative three-dimensional echocardiography by rapid imaging from multiple transthoracic windows: in vitro validation and initial in vivo studies. J Am Soc Echocardiogr 1997;10:830–9. w18x Motwani JG, McAlpine H, Kennedy N, Struthers AD. Plasma brain natriuretic peptide as an indicator for angiotensin-converting-enzyme inhibition after myocardial infarction. Lancet 1993;341:1109–13. w19x Choy AMJ, Darbar D, Lang CC, et al. Detection of left ventricular dysfunction after acute myocardial infarction: comparison of clinical, echocardiographic, and neurohormonal methods. Br Heart J 1994;72:16–22. w20x Lerman A, Gibbons RJ, Rodeheffer FJ, et al. Circulating N-terminal atrial natriuretic peptide as a marker for symptomless left-ventricular dysfunction. Lancet 1993;341:1105– 9. w21x Arnlov J, Lind L, Stridsberg M, Andren B, Lithell H. N-terminal atrial natriuretic peptide and left ventricular geometry and function in a population sample of elderly males. J Intern Med 2000;247Ž6.:699–708. w22x Hunt PJ, Richards AM, Nicholls MG, Yandle TG, Doughty RN, Espiner EA. Immunoreactive amino-terminal pro-brain natriuretic peptide ŽNT-proBNP.: a new marker of cardiac impairment. Clin Endocrinol 1997;47:287–96.
Head-to-head comparison of N-terminal pro-brain natriuretic peptide, brain natriuretic peptide and N-terminal pro-atrial natriuretic peptide in diagnosing left ventricular dysfunction Angelika Hammerer-Lercher a,) , Elke Neubauer b, Silvana Muller ¨ b, Otmar Pachinger b, Bernd Puschendorf a , Johannes Mair b a
Department of Medical Chemistry and Biochemistry, DiÕision of Clinical Biochemistry, UniÕersity of Innsbruck, Fritz-Pregl-Str. 3, A-6020 Innsbruck, Austria b Department of Internal Medicine, DiÕision of Cardiology, UniÕersity of Innsbruck, Innsbruck, Austria Received 8 January 2001; accepted 15 June 2001
Abstract Brain natriuretic peptide ŽBNP., NT-proBNP and NT-pro-atrial natriuretic peptide ŽNT-proANP. were measured in blood samples from 57 patients using immunoassays and immunoradiometric assays to evaluate the usefulness as diagnostic markers for the detection of heart failure. For the detection of impaired left ventricular ejection fraction ŽLVEF., receiver operating characteristic curves showed that BNP had the best diagnostic performance with an area under curve ŽAUC. of 0.75 " 0.06. However, NT-proBNP ŽAUC: 0.67 " 0.07. and NT-proANP ŽAUC: 0.69 " 0.08. showed no significant difference to BNP. In a further analysis for the detection of resting LVEF - 40%, BNP again was the best marker with an AUC of 0.83 " 0.06. NT-proBNP showed only a slightly smaller AUC Ž0.79 " 0.07.. The AUC for NT-proANP was significantly smaller Ž0.65 " 0.08. compared to BNP. Additionally, BNP and NT-proBNP correlated negatively with the resting LVEF ŽBNP: y0.472, p - 0.001; NT-proBNP: y0.306, p s 0.026., whereas NT-proANP showed no significant correlation. In summary, BNP was the best marker to detect patients with impaired LVEF compared to NT-proBNP and NT-proANP. However, NT-proBNP showed no significant differences to BNP and it is therefore a new promising alternative marker for the detection of left ventricular dysfunction. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Brain natriuretic peptide; N-terminal pro-brain natriuretic peptide; N-terminal pro-atrial natriuretic peptide; Left ventricular dysfunction; Three-dimensional echocardiography; Radionuclide ventriculography
AbbreÕiations: BNP, Brain natriuretic peptide; NT-proBNP, N-terminal proBNP; NT-proANP, N-terminal pro-atrial natriuretic peptide; LVEF, Left ventricular ejection fraction; ROC, Receiver operating characteristic curve; AUC, Area under curve; CHF, Congestive heart failure; ACE, Angiotensin-converting enzyme; LVD, Left ventricular dysfunction; cGMP, Cyclic guanosine monophosphate; 3D, Three-dimensional; RNV, Radionuclide ventriculography; NYHA, New York Heart Association. ) Corresponding author. Tel.: q43-512-507-2876; fax: q43-512-507-3522. E-mail address: [email protected] ŽA. Hammerer-Lercher.. 0009-8981r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 8 9 8 1 Ž 0 1 . 0 0 5 7 8 - 2
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A. Hammerer-Lercher et al.r Clinica Chimica Acta 310 (2001) 193–197
1. Introduction Congestive heart failure ŽCHF. is one of the main causes of hospitalisation in industrialized countries with a 5-year survival rate of only 50%. Its prevalence increases markedly with age w1x. The early diagnosis of CHF is crucial, since an early treatment with inhibitors of the angiotensin-converting enzyme ŽACE. can prevent or retard disease progression w2,3x. Natriuretic peptides have been proposed for early diagnosis and as prognostic markers in left ventricular dysfunction ŽLVD. w1x. In earlier studies it has been demonstrated that natriuretic peptides and their second messenger cyclic guanosine monophosphate ŽcGMP. increase with the severity of CHF w4–8x. Most comparative studies showed that brain natriuretic peptide ŽBNP. and NT-pro-atrial natriuretic peptide ŽNT-proANP. are superior to other natriuretic peptides to diagnose asymptomatic and symptomatic LVD w7,9–11x. An own previous investigation with very well-defined asymptomatic CHF patients ŽNYHA I. revealed that BNP is superior to ANP, NT-ANP and cGMP in diagnosing symptomless left ventricular dysfunction w9x. Recently, a new marker, NT-proBNP, has been proposed as a diagnostic and prognostic alternative and possibly superior marker in heart failure patients w12,13x. The aim of the present study was to directly compare the diagnostic values of the NT-natriuretic peptide fragments, i.e. NT-proBNP Ž8–29. and NTproANP Ž1–98., with BNP in the same study population to evaluate the usefulness of NT-proBNP in a head-to-head comparison with established natriuretic peptide markers.
2. Materials and methods 2.1. Subjects After giving informed consent, 57 consecutive patients Ž45–80 years. with stable chronic heart failure who underwent three-dimensional Ž3D. echocardiography and radionuclide ventriculography ŽRNV. at rest to assess left ventricular ejection fraction ŽLVEF. were enrolled in this study. Sixteen patients were in the New York Heart Association ŽNYHA. class I, 31 were in NYHA class II and 10 were in
NYHA class III, respectively. Most of the patients Ž n s 54. suffered from coronary artery disease and 12 patients had hypertension. Creatinine concentrations were within normal range in all but two patients. Drug treatment comprised ACE inhibitors Ž n s 25 patients., b-blockers Ž n s 21., nitrates Ž n s 19., diuretics Ž n s 10. and calcium channel blockers Ž n s 5.. The patient population was divided into two subgroups: first, in patients with normal ŽLVEF ) 48% according to 3D-echocardiograpy and LVEF ) 55% according to RNV. and impaired resting LVEF ŽLVEF - 48% according to 3D-echocardiograpy and LVEF - 55% according to RNV., and second, in patients with resting LVEF below and above 40% Žaccording to RNV.. EDTA blood samples were drawn after a standardized period of rest in a supine body position. After blood withdrawal, samples were stored at 4 8C Žup to 1 h. until centrifugation at 4 8C and 2000 = g for 10 min. The plasma was subsequently stored at y20 8C Žfor up to 1 month. before assay of BNP, NT-proBNP and NT-proANP. 2.2. Assays All peptides were measured using commercially available immunoassays without plasma extraction procedures. BNP was measured by an immunoradiometric assay ŽCat. No. IC-1049, Shionogi, Japan. w14x. In brief, samples were thawed and aliquots were incubated with 125 I-BNP marked antibody together with anti-human BNP antibody coated beads for approximately 20 h. After aspirating the tubes, the remaining radioactivity bound to the beads was measured with a gamma scintillation counter. NTproBNP w15x and NT-proANP w16x were measured by enzyme immunoassays ŽCat. No. BI-20852 and BI20892 Biomedica, Vienna, Austria.. In short, for NT-proBNP measurement, a limiting amount of NT-proBNP specific antibody was incubated with an anti sheep-specific antibody adsorbed to the wells for 5 h. After washing the wells, aliquots of diluted samples were incubated together with the biotinylated peptide Žtracer. overnight at 4 8C. After a washing step, the peroxidase-conjugated streptavidin that binds to the biotin of the bound tracer was added and incubated for 1 h. After removal of unbound conjugate through washing, substrate was added to
A. Hammerer-Lercher et al.r Clinica Chimica Acta 310 (2001) 193–197
the wells and incubated for 20 min in the dark. Bound biotinylated NT-proBNP Ž8–29. was quantified by an enzyme catalysed colour change, and the absorption was determined photometrically. For measurement of NT-proANP, aliquots of diluted samples and biotinylated NT-proANP were incubated simultaneously for 150 min at 37 8C. After washing, a streptavidin–peroxidase conjugate was added and incubated for 1 h at 37 8C. Another washing step was followed by an incubation of tetramethylbenzidine for 20 min at room temperature in the dark. The absorbance was read at 450 with 690 nm as reference.
195
2.5. Statistics Receiver-operating characteristic ŽROC. plot analysis was carried out to compare the diagnostic utility
2.3. Echocardiography Left ventricular volume indices, as end-systolic and end-diastolic volumes and stroke volume, were measured using native harmonic 2D imaging with biplane Simpson’s rule with apical four-chamber view and two-chamber view and 3D echocardiography. A commercial ultrasound system Sequoia C 256 ŽAcuson Mountainview, USA. equipped with a 3.5r1.75 MHz receivertransmit transducer was used. For 3D freehand echocardiography, a magnetic field system ŽAscension Technology, Burlington, VT. was used to track the position and orientation of the image planes w17x. The LVEF calculated by 3D echocardiography, which is more accurate, was used for comparison with natriuretic peptide concentrations. 2.4. Radionuclide Õentriculography In short, as previously described w9x, rest LVEF was determined by ECG-gated equilibrium RNV in supine body posture. Apex SR camera and an Elscint Apex 1 computer system ŽElscint Medical Technology, Austria. were used for data acquisition. Red blood cells were labelled by an in vitro labelling method with 700–900 MBq technetium-99 m as described before w9x. Time–activity curves were obtained in phase mode using 16 frames per cycle. LVEF was determined Žafter background correction. independently by two experienced technicians using a semiautomatic operator-interactive computer program.
Fig. 1. Receiver-operating-characteristic ŽROC. curves for BNP, NT-proBNP and NT-proANP to ŽA.: correctly identify patients with impaired LVEF ŽLVEF - 48% according to 3D-echocardiograpy and LVEF - 55% according to RNV. and to ŽB.: correctly identify patients with resting LVEF - 40% ŽRNV..
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196
of the natriuretic peptides, and Spearman rank correlation coefficients were calculated. A p value of - 0.05 was considered to indicate statistical significance.
3. Results ROC analysis showed that among the evaluated natriuretic peptides, BNP had the best diagnostic performance to detect patients with either impaired LVEF or resting LVEF - 40% ŽFig. 1.. The area under curve ŽAUC. was greater than those of the NT-natriuretic peptides. In the subanalysis of normal versus impaired LVEF, AUC Ž"S.E.. were 0.75 Ž"0.06. for BNP, 0.67 Ž"0.07. for NT-proBNP, and 0.69 Ž"0.08. for NT-proANP, respectively. In the subanalysis of patients with LVEF - 40% versus ) 40%, BNP showed an AUC of 0.83 Ž"0.06., NT-proBNP of 0.79 Ž"0.07. and NT-proANP of 0.65 Ž"0.08.. There was a significant difference between BNP and NT-proANP for the detection of resting LVEF - 40% ŽRNV.. However, NT-proBNP did not differ significantly from BNP in both subanalysis. Sensitivities, specificities, positive and negative predictive values and efficiencies for the detection of LVEF below 40% ŽRNV. are listed in Table 1. Optimal discriminator values according to ROC analysis were 41 ŽBNP., 488 ŽNT-proBNP. and 2150 pmolrl ŽNT-proANP.. Values given in Table 1 are based on these optimal discriminator values. Additionally, BNP Ž r s y0.472; p - 0.001. as well as NT-proBNP Ž r s y0.306; p s 0.026., but not NTproANP, showed a significant inverse correlation with LVEF. Table 1 Diagnostic performance of BNP, NT-proBNP and NT-proANP at optimal discriminator values Ž41, 488 and 2150 pmolrl, respectively. for detection of LVEF - 40% versus ) 40%
Sensitivity Specificity Positive predictive value Negative predictive value Efficiency
BNP
NT-proBNP
NT-proANP
0.73 0.77 0.70
0.70 0.73 0.61
0.59 0.61 0.48
0.79
0.80
0.71
0.75
0.72
0.60
4. Discussion The current results concur with our previous findings w9x, demonstrating that BNP was a superior marker compared with ANP, NT-ANP, and cGMP for the identification of patients with asymptomatic LVD. This also is in accordance with the results of Motwani et al. w18x, Yamamoto et al. w7x, and Choy et al. w19x who all showed that the inverse relation of LVEF with BNP was stronger than with ANP or NT-proANP. Our findings are not consistent with those of Lerman et al. w20x who found that NT-proANP was a powerful marker of symptomless LVD. Recently, Arnlov et al. w21x reported increased levels of NT-proANP in patients with LVD. However, in these two above mentioned studies BNP was not measured, and thus the predictive power of NT-proANP and BNP cannot be compared. Furthermore, we found a slightly better AUC for BNP compared with NT-proBNP, but there was no significant difference between the two markers. Richards et al. w12x showed that the relation of NT-proBNP with LVEF was comparable with that of BNP and was stronger than for the other measured hormones. Moreover, in his study, the sensitivity for prediction of death and LVD of NT-proBNP Ž91%. slightly exceeded that of BNP Ž86%., whereas specificity and positive and negative predictive values were equal. Hunt et al. w22x also demonstrated that NT-proBNP and BNP were significantly correlated with each other and inversely with LVEF. The levels of NT-proBNP were approximately fourfold higher than BNP levels in patients with moderate LVD. Richards et al. w12x and Hunt et al. w22x used radioimmunoassays, which need extraction of plasma samples for the determination of natriuretic peptides. We used a highly specific, sensitive and precise immunoradiometric assay to determine concentrations of BNP w14x and recently developed immunoassays for NTproBNP Ž8–29. w15x and NT-proANP Ž1–98. w16x, which do not need an extraction step before peptide measurement. Consequently, time for measuring either BNP, NT-proBNP or NT-proANP is shorter, and there is no imprecision arising from different recovery amounts of natriuretic peptides due to extraction of plasma samples. In the present study, BNP and NT-proBNP were comparable useful markers of LVD with a better diagnostic performance of
A. Hammerer-Lercher et al.r Clinica Chimica Acta 310 (2001) 193–197
BNP compared with NT-proANP. Yet, NT-proBNP showed a marginally smaller AUC than BNP. In conclusion, BNP was tendentiously the best marker to detect patients with impaired LVEF. The differences between BNP and NT-proBNP were not statistically significant and, therefore, NT-proBNP is a promising alternative marker for diagnosing heart failure, and it is worthy of further evaluation.
Acknowledgements This study was supported by a research grant from Abbott Laboratories ŽNorth Chicago, IL, USA..
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