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Clinical Chemistry 49: 1337-1346, 2003; 10.1373/49.8.1337
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(Clinical Chemistry. 2003;49:1337-1346.)
© 2003 American Association for Clinical Chemistry, Inc.

Proteomics and Protein Markers

Utility of the Amino-Terminal Fragment of Pro-Brain Natriuretic Peptide in Plasma for the Evaluation of Cardiac Dysfunction in Elderly Patients in Primary Health Care

Urban Alehagen1,a, Göran Lindstedt2, Henry Eriksson3 and Ulf Dahlström1

1 Department of Cardiology, Linköping University Hospital, SE-581 85 Linköping, Sweden.

2 Sahlgren Academy at Gothenburg University, SE-413 45 Gothenburg, Sweden.

3 Department of Medicine, Sahlgrenska University Hospital-Östra, SE-416 85 Gothenburg, Sweden.

aAuthor for correspondence. Fax 46-13-222224; e-mail urban.alehagen{at}ihs.liu.se.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Background: The aims of this study were to measure the N-terminal fragment of pro-brain natriuretic peptide (proBNP) in plasma in medical conditions commonly found in primary care and to evaluate the utility of these measurements in identifying impaired cardiac function in elderly patients with symptoms associated with heart failure.

Methods: We studied 415 patients (221 men and 194 women; mean age, 72 years) who had contacted a primary healthcare center for dyspnea, fatigue, and/or peripheral edema. One cardiologist evaluated the patients in terms of history, physical examination, functional capacity, electrocardiography, and suspicion of heart failure. Plasma N-terminal proBNP was measured by an in-house RIA. An ejection fraction <=40% by Doppler echocardiography was regarded as reduced cardiac function. Abnormal diastolic function was defined as an abnormal mitral inflow defined as reduced ratio of peak early diastolic filling velocity to peak filling velocity at atrial contraction (E/A ratio), or as abnormal pulmonary venous flow pattern.

Results: Patients with impaired functional capacity, impaired systolic function, and/or impaired renal function had significantly increased N-terminal proBNP concentrations. By multiple regression analysis, N-terminal proBNP concentrations were also influenced by ischemic heart disease, cardiac enlargement, and certain medications but not by increased creatinine. No gender differences were observed. Patients with isolated diastolic dysfunction attributable to relaxation abnormali-ties had lower concentrations than those with normal cardiac function, whereas those with pseudonormal E/A ratios or restrictive filling patterns had higher concentrations.

Conclusions: Plasma N-terminal proBNP concentrations increase as a result of impaired systolic function, age, impaired renal function, cardiac ischemia and enlargement, and certain medications. Values are high in diastolic dysfunction with pseudonormal patterns, but not in patients with relaxation abnormalities. An increase in plasma N-terminal proBNP might be an earlier sign of abnormal cardiac function than abnormalities identified by currently used echocardiographic measurements.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
Heart failure is a common syndrome in the elderly (1). Manifest heart failure carries a serious prognosis comparable to many malignancies. Early diagnosis and treatment therefore are important. However, the early symptoms of heart failure are nonspecific, often being found in several other conditions. By tradition, the diagnostic strategy includes the examination of heart structure and function by Doppler echocardiography and electrocardiography (ECG).1 Drawbacks with Doppler echocardiography, however, are mainly twofold: (a) low availability in relation to demand, and (b) poor precision leading to misclassification errors attributable to overlap between patients with normal and patients with abnormal function (2).

An alternative approach to the first-line evaluation is to measure endocrine alterations, e.g., natriuretic peptides (3)(4)(5)(6)(7), associated with increased heart load and cardiac dysfunction. Initially, interest was focused on atrial natriuretic peptide (ANP) and the N-terminal fragment of its precursor, proANP. Brain natriuretic peptide (BNP), which was discovered later and is released mainly from the cardiac ventricles in severe heart failure, has been shown to be superior to ANP and N-terminal proANP for the diagnosis of systolic dysfunction. There is increased awareness that BNP, in addition to having antagonistic effects on plasma volume expansion, vasoconstriction, and hypertension, also has important effects on myocyte and cardiac fibroblast metabolism.

BNP [32 amino acids in length; biological half-life, 23 min (8)] is released from its precursor, proBNP (108 amino acids), in the course of the secretion process in the heart and/or in the circulation, together with its inactive N-terminal fragment, N-terminal proBNP [proBNP 1–76; biological half-life in the circulation, 1–2 h (9)]. Hypothetically, BNP and N-terminal proBNP might equally well reflect the secretory status of proBNP and its cleavage products. However, circulating concentrations also depend on the elimination rates from the plasma compartment. The biologically active peptide is eliminated from plasma through different enzyme-catalyzed pathways and by glomerular filtration (10)(11). In contrast, the biologically inactive fragment, N-terminal proBNP, possibly is largely eliminated via glomerular filtration only, as has been shown to be the case for N-terminal proANP. Factors affecting the various elimination processes may therefore affect the circulating peptide concentrations and their diagnostic properties. In addition, stability in blood specimens differs, and the diagnostic utilities of the two peptides might therefore be different.

The measurement of N-terminal proBNP, first described by Hunt et al. (12), may offer new diagnostic possibilities for elderly patients with suspected heart failure in primary healthcare. The aims of our study were to evaluate such measurements by a newly developed in-house competitive RIA (13). We studied medical conditions common in the elderly in primary care and evaluated the utility of this peptide in identifying impaired cardiac ventricular function in elderly patients with symptoms commonly associated with those of heart failure. We also considered different pathophysiologic factors that have been thought to affect natriuretic peptide concentrations (10)(14)(15)(16)(17)(18)(19).


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
We studied 415 patients from a cohort of 1168 patients who attended primary care because of symptoms and/or signs that might be attributed to heart failure. The design of the study has been described in detail previously (20). In summary, we examined all records (n = 1168; patient age, 65–82 years) of patients with certain symptoms and/or signs who during 1995–1996 had contacted the primary healthcare center in Kinda municipality, which has a mainly rural population of 10 300 inhabitants, located in southeastern Sweden. Symptoms and/or signs were those that might be associated with heart failure (shortness of breath, and/or peripheral edema, and/or fatigue). Of these individuals, all of those for whom heart failure could not be ruled out by careful scrutiny of the patient records were invited to participate in our study (n = 548 individuals). The patient history included questions concerning whether the patients were present or former smokers, and the standard examination procedure included measurement of peak expiratory flow.

Thirty-eight patients decided not to participate because of long transportation distance, severe illness, mental insufficiency, or incapacity. Thus, 510 patients accepted (participation rate, 93%). The study took place in 1996. For 457 patients, we were able to obtain blood samples and perform echocardiographic examinations. Of this latter group, we were also able to assess and interpret both systolic and diastolic cardiac function in 429 patients. Fourteen patients with significant valvular disease that could influence signs and symptoms as well as the concentration of N-terminal proBNP were excluded from the analysis. The final study population thus included 415 patients (Table 1 ).


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  		Table 1. Characteristics of the study population.

All patients visited one cardiologist (U.A.), who recorded patient history, including drug treatment, performed a clinical examination, and assessed New York Heart Association (NYHA) functional class as well as the Boston score as modified according to Remes et al. (21). The modified Boston score system estimates the probability of heart failure based on history, clinical signs, and chest x-ray. Dyspnea was defined from the patient history, whereas presence of peripheral edema was defined on patient history and/or clinical examination. In addition, 12-lead ECG was performed.

The same cardiologist categorized the patients into three groups according to degree of clinical suspicion of heart failure (strong, moderate, and weak-no clinical suspicion), but was blinded at that moment from the results of Doppler echocardiography, chest x-rays, and biochemical measurements.

Diabetes mellitus was defined as a fasting blood glucose concentration >=7.0 mmol/L or ongoing treatment for diabetes (diet, oral therapy, or insulin). Hypertension was defined as a blood pressure >160/95 mmHg measured in the right arm with the patient in supine position after at least a 30-min rest (the choice of blood pressure cutoffs took into account that we studied elderly patients and that we measured the blood pressure only on a single occasion). Patients were also defined as hypertensive if they had previously received the diagnosis of hypertension and were receiving antihypertensive medication. Ischemic heart disease was defined as a history of angina pectoris, treatment for angina pectoris, and/or a previous myocardial infarction.

Significant valvular heart disease was defined as moderate or severe mitral or aortic insufficiency according to the integrated Doppler echocardiographic evaluation, including signal intensity and penetration, and signs of left ventricular dilatation and volume overload. A possibly significant aortic stenosis was assumed in the cases with maximal transvalvular velocity of at least 3.5 m/s. Impaired renal function was defined as a serum creatinine concentration exceeding 130 µmol/L.

As part of the patient history, the examining cardiologist recorded all medications. For angiotensin-converting enzyme inhibitor (ACEI) and ß-receptor blockers, this was calculated as a fraction of the optimal daily dose. The optimal daily dose of ACEI was set as follows: captopril, 150 mg; enalapril, 20 mg; lisinopril, 20 mg; and ramipril, 10 mg. For ß-receptor blockers it was set as follows: metoprolol, 200 mg; atenolol, 100 mg; and sotalol, 160 mg.

In vivo investigations included ECG, chest x-ray, and Doppler echocardiography. Doppler echocardiographic examinations (Accuson XP-128c) were performed with the patient in the supine left position. Both M-mode and two-dimensional methodologies were used. Values for systolic function (SFN), expressed as ejection fraction (EF) (22)(23), were categorized into four classes with interclass limits of 30%, 40%, and 50%. A semiquantitative method of assessment was used. Normal SFN was defined as an EF >=50%. Severely impaired SFN was defined as an EF <=30%. For assessment of diastolic function (DFN), we analyzed the mitral ratio of peak early diastolic filling velocity to peak filling velocity at atrial contraction (E/A ratio) and pulmonary venous flow pattern and compared them with age-adjusted decision limits (20).

Blood samples were drawn from fasting patients in the sitting position after a 30-min rest. The samples were collected in prechilled plastic tubes containing EDTA (Terumo EDTA K-3), placed on ice, and centrifuged at 3000g for 10 min at 4 °C. The samples were then immediately stored at -70 °C until analyzed. The N-terminal proBNP measurement was performed at the Research Institute of Internal Medicine, Rikshospitalet, University of Oslo, Norway. This nonextraction in-house assay (13) is a competitive RIA using radiolabeled proBNP 1-21-Tyr-0 (chloramine labeling; Med Probe AS) and an in-house antiserum (raised in rabbits) against proBNP 1-21 conjugated to bovine albumin. As a calibrator we used proBNP 1-21 (Peninsula). Incubations, in a phosphate buffer-bovine albumin matrix, were done for 2 days at 4 °C after the simultaneous addition of sample (calibrator, plasma sample, or control), radioligand, and antiserum. Serial dilution curves of plasma samples from three patients with congestive heart failure were parallel to the calibration curve. No cross-reactivity was demonstrated against ANP, N-terminal proANP, or BNP. As suggested from gel chromatography in nondissociating medium, the assay detects mainly a high-molecular-mass plasma protein fraction (13–14 kDa) and, to a lesser extent, N-terminal proBNP (i.e., proBNP 1–76; molecular mass, 8–9 kDa). The detection limit was 9.7 pmol/L (0 calibrator + 2 SD). The total interassay CV was 15% (mean of 26 pmol/L; n = 10 assay runs during 2 months). The intraassay CVs, as determined from replicate assays in one assay run, were 15% at a mean of 26 pmol/L (n = 7), 7.3% at a mean of 431 pmol/L (n = 10), and 9.7% at a mean of 1400 pmol/L (n = 10).

The descriptive data are presented as a percentage or as the mean (SD). In the case of continuous variables, analyses were made using the Student unpaired two-sided t-test, whereas the {chi}2 test was used for discrete variables. A P value <0.05 was considered statistically significant. All statistical tests were performed on log10-transformed peptide concentration values because the values did not follow a gaussian distribution. After ROC analysis, the area under curve (AUC) was calculated according to Hanley (24). Multiple regression analysis was undertaken to test the independent prediction of increased natriuretic peptide concentration. All data were analyzed using generally available statistical analysis software packages [Statistica, Ver. 6.0 (Statsoft Inc), or Analyze-it, Ver. 1.63 (Analyze-it Software Ltd)]. The Ethics Committee of the University Hospital of Linköping approved the study protocol.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
The mean concentration of N-terminal proBNP was 88 pmol/L in the final study group of 415 patients. The distribution was markedly skewed to the right, 1 SD being 98 pmol/L (Table 1Up ). The mean values and their 95% confidence intervals for the different patient groups are given in Tables 2 , 4 , and 5 and are further analyzed below.


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  		Table 2. Plasma N-terminal proBNP concentration in relation to cardiac function by Doppler echocardiography, and fractions of patients with different echocardiographic findings in the different quartiles of N-terminal proBNP concentration.


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  		Table 4. Plasma N-terminal proBNP concentration in different clinical groups, and fractions of patients in the different quartiles of N-terminal proBNP concentration.


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  		Table 5. Plasma N-terminal proBNP concentration in different patient groups and fractions of patients in the different quartiles of N-terminal proBNP concentration.1

The distributions of values for plasma N-terminal proBNP in relation to echocardiographic findings are given in Table 2Up . We found a statistically highly significant difference in mean plasma concentration between patients with normal cardiac function and patients with EF <30% (t = 7.64; P <0.0001) as well as patients with EF <40% (t = 8.00; P <0.0001). The group of patients with 40% < EF < 50% was not significantly different from the group with normal cardiac function in terms of N-terminal proBNP concentration (P = 0.06).

In a ROC curve analysis of increased plasma concentration of N-terminal proBNP as an indicator of impaired systolic ventricular function by Doppler echocardiography, the AUC for patients with EF <30% was 0.92, whereas the AUC for patients with EF <40% was 0.77 (Fig. 1 ).



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  		Figure 1. ROC curves for the different clinical groups.

(A), patients with EF <30% (9 of 415 patients; AUC = 0.92). (B), patients with EF <40% (54 of 415 patients; AUC = 0.77). (C), NYHA class III patients (43 patients; AUC = 0.78). (D), patients with Boston scores of 7–9 (33 patients; AUC = 0.77). The chosen cutoff values for N-terminal proBNP were 250 pmol/L (A), 100 pmol/L (B), and 40 pmol/L (C).

Among the 92 patients with isolated diastolic dysfunction, relaxation abnormalities were present in 79 and a pseudonormal E/A mitral flow pattern was observed in 13. Patients with relaxation abnormalities had significantly lower mean N-terminal proBNP concentrations than those with normal cardiac function (t = 3.67; P = 0.0003). There was a significantly higher mean plasma concentration of N-terminal proBNP in patients with a pseudonormal E/A mitral flow pattern compared with patients with normal cardiac function (t = 3.65; P = 0.0003) and those with relaxation abnormalities (t = 5.29; P <0.001). Multiple regression analysis revealed that findings of EF <30% and EF 30–39% were strong predictors of increased plasma concentrations of N-terminal proBNP (t = 7.19; P <0.001 and t = 6.10; P <0.001, respectively), whereas EF 40–49% or a pseudonormal mitral E/A ratio were not. Because it was possible to explain only 23% of the variations in N-terminal proBNP concentration with the results from echocardiography according to the multiple regression analysis, it is obvious that other factors influence the concentration of N-terminal proBNP in a substantial way.

Of our patients with normal cardiac function according to echocardiography, only 51% were categorized as NYHA class I and 6% were categorized as NYHA class III. We found a highly significant difference in mean N-terminal proBNP plasma concentrations between these functional classes despite their normal cardiac function according to Doppler echocardiography [NYHA I vs NYHA III, t = 4.87 (P <0.0001); NYHA II vs NYHA III, t = 5.1 (P <0.0001)]. The median (SD) values were as follows: for NYHA class I, 55.6 (50.9) pmol/L; for NYHA class II, 53.0 (46.2) pmol/L; and for NYHA class III, 135.0 (103.1) pmol/L. In a multiple regression analysis in which other possible factors that might influence the concentration of N-terminal proBNP were included in addition to the echocardiographic results, the following factors showed predictive power: age >75 years, ischemic heart disease, enlargement of heart on chest x-ray, NYHA functional class III, and treatment with diuretics (furosemide >=40 mg/day) or ß-receptor blockers (>50% of optimal daily dose; Table 3 ).


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  		Table 3. Multiple regression analysis using log10 N-terminal proBNP as dependent variable.1

There was a significant difference in mean plasma concentrations of N-terminal proBNP between the different age groups (age <70 vs age >79 years, t = 6.32; P <0.001; Table 4Up ). The correlation between mean plasma concentration of N-terminal proBNP and age when all patients with known influencing concomitant diseases had been excluded was significant (r = 0.53; P <0.05). We found no gender differences in our patients.

A history of ischemic heart disease was recorded in 144 patients (35%). As shown in Table 4Up , patients with ischemic heart disease had a higher mean plasma N-terminal proBNP concentration (t = 4.38; P <0.0001) compared with nonischemic patients. Ischemic heart disease also strongly correlated with N-terminal proBNP in a multiple regression analysis (P <0.001). Hypertension or diabetes mellitus had no predictive power on plasma concentration of N-terminal proBNP (Table 3Up ).

Serum creatinine was increased (>130 µmol/L) in 31 patients (8%), who had significantly higher mean N-terminal proBNP concentration (t = 5.01; P <0.0001) than those with serum creatinine <130 µmol/L (Table 4Up ). Univariate regression analysis revealed a significant correlation between serum creatinine and N-terminal proBNP concentrations (r = 0.21), but there was no correlation between these variables in multiple regression analysis (Table 3Up ).

We found peripheral edema in 154 patients (37%) at examination. These patients had significantly higher mean plasma concentrations of N-terminal proBNP compared with patients without peripheral edema (t = 2.69; P = 0.008). The situation was the same for dyspnea [161 patients (39%); t = 2.06; P = 0.04] and for rales [43 patients (10%); t = 2.72; P = 0.007]. In a multiple regression analysis, however, jugular distension, dyspnea, rales, and peripheral edema were not significant predictors of increased N-terminal proBNP. Two separate multiple regression analyses were done: one including age, sex, etiology, chest-x-ray, symptoms and/or signs, and treatment, and one that also included echocardiographic results. No major differences in results were noted.

Functional capacity was evaluated by NYHA functional class and the modified Boston scoring system. A majority of patients (n = 193; 47%) were categorized as NYHA functional class I, whereas 181 patients (44%) were categorized as class II and 41 (10%) as class III (Table 5Up ). Compared with patients in NYHA class I, those in NYHA class II had significantly higher N-terminal proBNP concentrations (t = 3.42; P = 0.0007), and those in NYHA class III had even higher N-terminal proBNP concentrations (t = 5.53; P <0.0001). Furthermore, NYHA functional class III was an independent predictor of increased plasma N-terminal proBNP concentrations in a multiple regression analysis (P = 0.004).

Concerning the modified Boston score, 383 patients (92%) had scores of 1–6, whereas 32 patients (8%) had scores of 7–9 (t = 6.06; P <0.0001).

To determine the usefulness of peptide measurements in patients with severe functional impairment (NYHA class III or modified Boston score 7–9), we performed a ROC analysis. The AUC was 0.78 for patients classified as NYHA class III and 0.77 for patients with a modified Boston score of 7–9 (Fig. 1Up ).

The cardiologist who performed clinical examinations of the patients also scored the patients in three different classes according to the degree of suspicion of heart failure based on history, clinical examination, and ECG (Fig. 2 ). There was no clinical suspicion of heart failure in 62% of the patients, whereas there was a strong suspicion in 10% of the patients. We observed a statistically highly significant difference in mean plasma concentrations of N-terminal proBNP between these different patient groups (Table 5Up ).



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  		Figure 2. Relationship between log plasma N-terminal proBNP concentration and examining clinician’s suspicion of heart failure.

Shown is the complete study group without exclusions (n = 415). As stated in the text, the evaluation was based on history, clinical examination, and ECG results. {blacksquare}, mean values; error bars represent the 5th (top bar) and 95th (bottom bar) centiles. Differences between weak or no suspicion of heart failure vs moderate suspicion (t = 3.29; P = 0.001) and between moderate suspicion of heart failure vs strong suspicion (t = 6.91; P <0.001) were significant.

All patients had chest x-ray. In a majority of the patients (59%), no signs of enlargement of the heart were apparent. The mean plasma concentration of N-terminal proBNP was significantly higher in those with enlarged hearts compared with those with no enlargement of the heart on chest x-ray (t = 7.74; P <0.0001). Enlargement of the heart on chest x-ray was a significant predictor of increased concentration of N-terminal proBNP in a multiple regression analysis (P <0.001).

In the group with severe systolic impairment (EF <30%), 78% had enlarged hearts on chest x-ray. In those with normal cardiac function by Doppler echocardiography, 32% had enlarged hearts. In the quartile of patients with the most pronounced increase in left ventricular end-diastolic dimension, only 52% had been classified as having an enlarged heart on chest x-ray.

Treatment with ß-receptor blockers, ACEI, diuretics, and/or digoxin was associated with higher concentrations of N-terminal proBNP compared with absence of treatment, but in a multiple regression analysis only diuretics and ß-receptor blockers (with >50% of optimal daily dose) were significant predictors of increased plasma concentrations of N-terminal proBNP (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
This study deals with approximately one-third of a representative population of primary-care patients, i.e., those presenting with symptoms and/or signs compatible with heart failure. In our view, the patient records gave complete and reliable information concerning contacts with healthcare. Of our patients, 48% had systolic and/or diastolic dysfunction and 22% had isolated diastolic dysfunction according to echocardiographic examination. The group of patients with severely impaired cardiac function (EF <30%) was rather small (2.2%).

Several immunoassays for N-terminal proBNP (proBNP 1–76) have been described during recent years. There is poor agreement in values for reference individuals between these reports, the difference between reported mean or median values being up to ~100-fold. Therefore, it is not possible to use the same decision limits for N-terminal proBNP concentrations in the diagnosis of heart failure with the different immunoassays. The mean (SD) plasma concentration of N-terminal proBNP was 59 (51) pmol/L in the group of patients with normal SFN and DFN by Doppler echocardiography and without certain disorders, abnormalities, or drug treatment (n = 39). The mean value was 73 (56) pmol/L for this group before exclusions (n = 214; Table 2Up ), i.e., similar to that obtained with a newly introduced immunoenzymometric assay (25).

As shown by ROC curves, the diagnostic sensitivity and specificity of N-terminal proBNP were high in identifying patients with severe impairment of the left ventricular SFN (EF <30%) and lower in the group with moderate impairment (EF <40%). Similar findings have been reported for BNP. The mean plasma concentration of N-terminal proBNP in patients with EF <40% was highly significantly different from that in patients with normal cardiac function and from the mean for patients with isolated diastolic dysfunction.

In the group with normal SFN and DFN by Doppler echocardiography, we found a highly significant difference in mean plasma concentrations of N-terminal proBNP in patients categorized as NYHA class I compared with those categorized as NYHA class III. From this finding we conclude that some patients with normal cardiac function according to Doppler echocardiography have impaired functional capacity. Furthermore, because the mean concentration of N-terminal proBNP differed significantly between the different NYHA classes, this indicates to us that the difference in NYHA class indeed reflected differences in degree of cardiac impairment and that the peptide measurement shows cardiac impairment earlier than Doppler echocardiography. The latter examination assesses structural changes associated with the development of cardiac dysfunction, whereas natriuretic peptides respond to changes in left ventricular filling pressure and, probably, to subtle biochemical alterations occurring early in the development of cardiac insufficiency. In our view, these considerations indicate that the correlation between these principally different methods of assessing cardiac function would be expected to be poor in a patient sample such as the one studied here. This explains much of the confusion in the literature on natriuretic peptides in primary healthcare patients when Doppler echocardiography was taken as the golden standard for cardiac function assessment.

We observed a significant correlation between age and plasma N-terminal proBNP concentrations in our study, as has previously been demonstrated convincingly for BNP (26). The recent study of N-terminal proBNP, using processing-independent analysis, also found a marked increase in values in individuals above the age of 70 years (27). To exclude the possibility that the difference with age might be attributable to a higher prevalence of disease in older persons, we excluded patients with a history of ischemic heart disease, hypertension, and impaired renal function, those being treated with different drugs that influence the plasma concentration of N-terminal proBNP, and those with signs of cardiac dysfunction according to Doppler echocardiography. The correlation between age and plasma N-terminal proBNP concentration was significant also after these exclusions (r = 0.53; P <0.001), indicating that age itself has an important effect. In a multiple regression analysis, age >75 years predicted an increased plasma concentration of N-terminal proBNP. Future studies will be needed to determine whether age-adjusted reference intervals and decision limits should be implemented. A gender difference in natriuretic peptide concentrations has been shown in some studies, with higher values in women (17). Other studies showed no difference(28). We found no gender difference, in good agreement with the findings of Schulz et al. (13), who used the same method as in our study, and those of Goetze et al. (27).

Ischemic heart disease is one of the main etiologic factors for developing heart failure, and it is therefore not surprising that in a multiple regression analysis ischemic heart disease was found to be a strong predictor of increased N-terminal proBNP concentrations (Table 3Up ). One clinical consequence of this finding is that an "unexplained" increase in natriuretic peptide concentration, i.e., the patient has no demonstrable decrease in cardiac function or other factors that might cause such an increase, might support a decision to clarify whether ischemic heart disease is the cause of nonspecific or diffuse chest symptoms.

We found a highly significant difference in the mean plasma concentration of N-terminal proBNP between those with serum concentrations at or above the limit (130 µmol/L) compared with those with values below the limit, similar to reports of others indicating that renal impairment increases plasma concentrations of natriuretic peptides (10)(29). None of our patients had increased plasma concentration because of cor pulmonale (30). Functional impairment as evaluated by the NYHA classification or the Boston score system reflected increased N-terminal proBNP concentrations.

On the basis of patient history, ECG results, and clinical examinations, the examining clinician categorized the patients according to his suspicion of heart failure. Significantly increased plasma concentrations of N-terminal proBNP were associated with a higher degree of suspicion, demonstrating that although the clinical signs of heart failure are quite nonspecific, the "clinical feeling" of the experienced cardiologist is not to be underestimated compared with sophisticated scoring systems such as the modified Boston scoring system. However, in a multiple regression analysis, none of the major clinical symptoms of heart failure, including dyspnea, were found to be a predictor of increased N-terminal proBNP. One possible explanation of this rather surprising finding is that, in this study population, dyspnea was common not only in heart failure, but also in respiratory disease not related to cardiac impairment and in patients with suboptimal physical fitness. Our results underline the important role of natriuretic peptide testing in the clinical evaluation of dyspnea.

Enlargement of the heart showed strong predictive power of increased plasma concentrations of N-terminal proBNP. The results are interesting in relation to the findings that inappropriately high left ventricular mass is a risk factor for cardiovascular events (31). It should be noted that in the group with impaired SFN (EF <40%) and a dilated left ventricle, 33% did not show any enlargement on the chest x-ray. However, in the group with normal cardiac function and no sign of dilatation of the left ventricle, 32% showed enlargement on the chest x-ray. The evaluation of heart size, and the use of this information to judge the possible presence of heart failure, is obviously fraught with difficulties.

By Doppler echocardiography, most patients did not have severely impaired cardiac function. However, we observed several individuals with increased N-terminal proBNP that was not attributable to pharmacotherapy or renal dysfunction but with normal results from echocardiographic measurements. We suggest that this apparent discrepancy may be explained as follows.

The main sources of BNP and N-terminal proBNP in cardiac dysfunction are cells in the left ventricular wall, although there is some secretion from the left atrium. Increased wall tension in the left ventricle is considered a major stimulant for the release of natriuretic peptides (32), with Omland et al. (33) having found a good correlation between circulating BNP concentration and filling pressure of the left ventricle. Heart failure probably starts with an increase in left ventricular filling pressure, which in turn increases the left ventricular wall tension. Doppler echocardiography at this stage, however, reveals no evidence of impaired ventricular function.

In contrast to other investigators (34), we found no increase in N-terminal proBNP concentration when the complete group of patients with isolated diastolic dysfunction was compared with patients with normal cardiac function. However, the group was heterogeneous as judged from echocardiographic examinations, with the majority of our patients having relaxation disturbances. This represents mild diastolic dysfunction with only slightly increased filling pressures. The patients with pseudonormal E/A mitral flow or restrictive filling pattern had more severe dysfunction and, as expected, had increased concentrations of N-terminal proBNP (Table 2Up ). Interestingly, as a group, the patients with relaxation disturbances had decreased N-terminal proBNP concentrations, i.e., there was a bimodal distribution of values in diastolic dysfunction. At present we can only speculate the reasons for this unexpected finding. Natriuretic peptides act antagonistically to angiotensin II-induced proliferation of cardiac fibroblasts (35). Patients with relaxation disturbances have increased myocardial fibrosis and increased interstitial matrix formation. There is some similarity between these morphologic changes and those observed in mice with targeted disruption of the BNP gene (36). In the group with the disrupted BNP allele, multifocal fibrotic lesions appeared in the myocardium in spite of an absence of signs of hypertension or ventricular hypertrophy. Hypothetically, BNP may act as an antifibrotic factor in humans similar to what Ogawa et al. (37) proposed for mice. Low BNP secretion in relation to physiologic demands might therefore predispose to myocardial fibrosis, which would indicate a clinical interest in low BNP concentrations in relation to the functional status of the heart. In support of these suggestions, Tsuruda et al. (38) recently showed that BNP is produced in cultured cardiac fibroblasts and decreases collagen biosynthesis. In addition, Chen et al. (39) recently demonstrated, in experimental cardiac failure, that increasing BNP concentrations through combined vasopeptidase inhibition and BNP administration effectively increased cardiac output by decreasing systemic vascular resistance and unloading of the heart.

In conclusion, measurement of N-terminal proBNP offers the clinician a useful instrument in the evaluation of potential heart failure patients, but several variables influencing its plasma concentration must be taken into account. Possibly, both high and low values may be relevant. There is suggestive evidence to indicate that N-terminal proBNP measurement has higher diagnostic sensitivity than Doppler echocardiography in the early stages of heart failure.


   Acknowledgments
 
N-terminal BNP measurements were performed at The Research Institute for Internal Medicine, University of Oslo (Oslo, Norway), for which we thank Dr. Christian Hall. The study was supported by grants from The County Council of Östergötland, The Swedish Heart and Lung Foundation, and The Research Foundation of the University of Linköping. We would like to thank Kerstin Gustavsson, heart failure nurse, for help in the study.


   Footnotes
 
1 Nonstandard abbreviations: ECG, electrocardiography; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; NYHA, New York Heart Association; ACEI, angiotensin-converting enzyme inhibitor; SFN, systolic function; EF, ejection fraction; DFN, diastolic function; E/A, ratio of peak early diastolic filling velocity to peak filling velocity at atrial contraction; and AUC, area under the curve.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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