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European Journal of Heart Failure 2005 7(1):81-86; doi:10.1016/j.ejheart.2004.03.014
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© 2004 European Society of Cardiology

Influence of age on natriuretic peptides in patients with chronic heart failure: a comparison between ANP/NT-ANP and BNP/NT-proBNP

Jochem Hogenhuisa, Adriaan A. Voorsa, Tiny Jaarsmaa, Hans L. Hillegeb, Frans Boomsmac and Dirk J. van Veldhuisen*,a

a Thoraxcenter, Department of Cardiology, University Hospital Groningen P.O. Box 30.001, Groningen 9700 RB, The Netherlands
b Trial Coordination Center, University Hospital Groningen The Netherlands
c Department of Internal Medicine, Erasmus MC Rotterdam, The Netherlands

* Corresponding author. Tel.: +31-50-3612355; fax: +31-50-3614391. E-mail address: d.j.van.veldhuisen{at}thorax.azg.nl


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Background: Natriuretic peptides are currently used in the diagnosis and follow-up of patients with Chronic Heart Failure (CHF). However, it is unknown whether there are different influences of age on atrial natriuretic peptide (ANP)/N-terminal-ANP (NT-ANP) or B-type natriuretic peptide (BNP)/N-terminal-proBNP (NT-proBNP).

Aims: To compare the influence of age and gender on plasma levels of ANP/NT-ANP and BNP/NT-proBNP in CHF patients.

Methods and results: Natriuretic peptides were measured in 311 CHF patients (68±8 years, 76% males, left ventricular ejection fraction (LVEF) 0.23±0.08). All natriuretic peptides were significantly related to age (p<0.05) on multivariate regression analysis, with partial correlation coefficients of 0.18, 0.29, 0.28 and 0.25 for ANP, NT-ANP, BNP and NT-proBNP, respectively. The relative increase of both BNP/NT-proBNP were more pronounced than of ANP/NT-ANP (p<0.01). Furthermore, the relative increase of BNP with age was markedly larger than of NT-proBNP (p<0.01). Levels of all natriuretic peptides were also significantly related to cardiothoracic ratio, renal function and LVEF.

Conclusion: In patients with CHF, BNP/NT-proBNP were more related to age than ANP/NT-ANP, and BNP was more related to age than NT-proBNP. However, in these CHF patients the influence of age on the levels of all natriuretic peptides was modest, and comparable to several other factors.

Key Words: ANP • BNP • Age • Heart failure

Received November 6, 2003; Revised February 11, 2004; Accepted March 26, 2004


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Plasma natriuretic peptides have been shown to be of additional value in the diagnosis [1–4] and prognosis [5] of Chronic Heart Failure (CHF) patients. Research initially focussed on the Atrial Natriuretic Peptides (ANP) and N-terminal ANP (NT-ANP), which are primarily secreted in the atria. In recent years, peptides secreted in the ventricles (B-type Natriuretic Peptide [BNP], and N-terminal proBNP [NT-proBNP]) have been increasingly studied. In a comparative study, the diagnostic value of BNP appeared to be superior to ANP, especially with regard to the positive predictive value (BNP 70%, ANP 55%) [6]. Furthermore BNP and NT-proBNP are better predictors of prognosis after myocardial infarction than ANP and NT-ANP [7]. However, plasma levels of natriuretic peptides are influenced by age and gender in healthy subjects [8–10]. Although natriuretic peptides are largely used in CHF patients, only limited data about the influence of age [11,12] and gender [13] on natriuretic peptides in CHF patients are available. Despite the prognostic and diagnostic superiority of BNP, direct comparative studies on the influence of age on BNP/NT-proBNP and ANP/NT-ANP in CHF patients are lacking. We therefore compared the effects of age and gender on ANP/NT-ANP and BNP/NT-proBNP in a large group of CHF patients.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Study sample
The present analyses used the baseline data of patients included in the PRIME-II study in the Netherlands. PRIME-II was designed to examine the effect of oral ibopamine versus placebo, on all cause mortality in patients with moderate to severe CHF [14]. Systolic dysfunction was an inclusion criterion of the PRIME-II study (left ventricular ejection fraction<35%). The design, and full inclusion and exclusion criteria of the PRIME-II study have been described in detail by Hampton et al. [14].

The predefined neurohormonal sub-study consisted of 372 patients, with 311 patients having complete datasets. The study was approved by each local Ethical Committee, and prior to the announcement of the investigation all patients provided informed written consent. The investigation conforms with the principles outlined in the declaration of Helsinki.

2.2. Natriuretic peptide measurement
At baseline in the PRIME-II study, before treatment with the study drug ibopamine was started, blood was collected between 9:00 and 10:00 AM after patients had rested in supine position for >30 min. An intravenous canula was used to transfer blood into chilled 10 ml tubes containing EDTA (19 mg) and aprotinin (1000 kIU). The tubes were centrifuged within 30 min (4 °C, 10 min, 2000xg) and plasma was separated and stored in polyethylene tubes at –70 °C. The plasma natriuretic peptide samples were transported (on dry ice) to the Core Laboratory at the University Hospital Dijkzigt, Rotterdam, the Netherlands, where all measurements were performed. Measurement of ANP (normal value: 15–35 pmol l–1) was performed after SepPak extraction, with commercially available radioimmunoassay kits from the Nichols Institute, Wijchen, The Netherlands, as previously described [15–17]. Plasma NT-ANP (normal value: 150–500 pmol l–1) was determined using a commercially available radioimmunoassay kit (Biotop, Oulu, Finland) [18]. NT-proBNP was measured using a radioimmunoassay kit with reagents including antibody, standards, and radiolabel. The assay uses 50 µl of unextracted plasma and has a standard range of 60–1000 pmol l–1. All samples giving results of >900 pmol l–1 were re-analysed in appropriate dilutions with physiological salt. In 12 consecutive assays, variability was 14%, 11%, 4% and 4% at concentrations of 131, 199, 293 and 901 pmol l–1, respectively. BNP was determined by a commercially available immunoradiometric assay (Shionoria, Osaka, Japan).

2.3. Statistical analyses
To investigate the relation between natriuretic peptides and other patient characteristics, Pearson and Spearman correlation coefficients were calculated when appropriate. For all natriuretic peptides, the following predictor variables were used in univariate analyses: age, gender, NYHA class, the existence of coronary artery disease, dilated cardiomyopathy or hypertension, systolic and diastolic blood pressure, heart rate, left ventricular ejection fraction, cardio-thoracic ratio, sinus rhythm or atrial fibrillation, renal function and the medication that was taken by the patient (β-blockers, ACE inhibitors, diuretics, digoxin and anti-arrhythmics). A multivariate regression analyses was performed to study the relation between natriuretic peptides on the one hand and age and gender on the other. Because the plasma natriuretic peptides had a skewed distribution the natural logarithm was used to get an optimal residual analysis. A p-value <0.15 was required to enter a variable into the multivariate model, and a p-value <0.05 was needed to remove a variable from the model. Partial correlation coefficients of the plasma level of ANP/NT-ANP and BNP/NT-proBNP were calculated with correction for the predictors that were entered in the multivariate regression model.

Age was divided into quartiles, using the first quartile as the reference group. In all quartiles the relative differences between lnANP and lnBNP, lnNT-ANP and lnNT-proBNP, lnANP and lnNT-ANP and lnBNP and lnNT-proBNP were investigated with paired samples t-tests.

Outcomes were considered significant when p<0.05. Results were presented as means±standard deviations except when stated otherwise.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
Demographic and clinical characteristics of the 311 patients are presented in Table 1. On average patients were 68±8 years of age, the majority (76%) of patients of this study was male. A significant age difference between males and females was found (p-value <0.05). The median and ranges of ANP, NT-ANP, BNP and NT-proBNP in the patient population were 103 (12–815) pmol l–1, 1078 (129–4280) pmol l–1, 60 (0.6–502) pmol l–1 and 610 (2–5295) pmol l–1, respectively (Table 2).


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  		Table 1 Characteristics of study population divided in age quartiles

 


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  		Table 2 Natriuretic peptide plasma levels in age quartiles (median [minimum–maximum])

 
Plasma levels of ANP/NT-ANP and BNP/NT-proBNP showed significant positive correlations with age on univariate regression analyses (p-value<0.05). Furthermore, ANP and NT-ANP plasma levels were significantly higher in patients with atrial fibrillation compared to patients with sinus rhythm (p-value<0.05). Although a similar difference was indeed present, it was not statistically significant for BNP and NT-proBNP.

3.1. Multivariate predictors of natriuretic peptide plasma levels
Left ventricular ejection fraction, cardio-thoracic ratio and renal function added significant value to the multivariate regression model (p-value<0.05) for all tested natriuretic peptides. Gender was significantly related to ANP and NT-ANP in the multivariate model, while this was not the case for BNP and NT-proBNP (Table 3). Use of diuretics was only related to ANP, and systolic blood pressure was only related to NT-ANP and NT-proBNP. The existence of coronary artery disease was only related to NT-proBNP. The multivariate partial correlation coefficients in relation to age were 0.18, 0.29, 0.28 and 0.25 for ANP, NT-ANP, BNP and NT-proBNP, respectively (Table 3). The interaction term between age and gender did not add significant value to the multivariate regression model of all tested natriuretic peptides.


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  		Table 3 Partial correlation coefficients for natriuretic peptides in relation with age and gender (corrected for CT-ratio, LVEF, plasma Creatinine, use of diuretics, SBP, CAD)

 
3.1.1. Direct comparisons
To directly compare the age dependency between ANP/NT-ANP and BNP/NT-proBNP, we divided our population in quartiles. The relative increase in lnBNP was significantly larger than the relative increase in lnANP, and the relative increase in lnNT-proBNP was significantly larger than the relative increase in lnNT-ANP (Fig. 1). In addition, we directly compared lnANP to lnNT-ANP and lnBNP to lnNT-proBNP. The relative increase of lnANP and lnNT-ANP with age was similar (Fig. 1). However, the relative increase of lnBNP with age was significantly larger than the relative increase of lnNT-proBNP with age (Fig. 1).


Figure 1
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  		Fig. 1 Comparison of lnANP vs. lnBNP (A), lnNT-ANP vs. lnNT-proBNP (B), lnBNP vs. lnNT-proBNP (C) and lnANP vs. lnNT-ANP (D) in their relative increase in age quartiles related to reference group (38–62 years). Legend: *p<0.01, {dagger}p<0.001.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Plasma natriuretic peptides are of added value both in the diagnosis [1–4] and prognosis [5] of CHF. Direct comparative studies indicated both diagnostic [6] and prognostic [7] superiority of BNP/NT-proBNP over ANP/NT-ANP. Although age-dependency of plasma levels of both ANP/NT-ANP and BNP/NT-proBNP has been demonstrated, direct comparative studies on age-dependency between ANP/NT-ANP and BNP/NT-proBNP in CHF patients were not available. In a large group of CHF patients, we demonstrated that BNP/NT-proBNP were influenced by age to a larger extent than ANP/NT-ANP. We also demonstrated that the relative increase of BNP with age was significantly larger than NT-proBNP.

4.1. Relation between ANP/NT-ANP, BNP/NT-proBNP and age in healthy adults
The relation between age and levels of natriuretic peptides is well described in healthy subjects [9,19]. In a large group of healthy adults (n=911), Wang et al. calculated multivariate correlations between ANP/NT-ANP, BNP and age. After multivariate adjustment, a 10-year increase in age was associated with a 1.4 fold increase in BNP levels and a 1.2 fold increase in NT-ANP [10]. Another large study in healthy subjects (n=216) showed a weak but significant relationship between age and both ANP and BNP [8]. Raymond et al. [20] analysed a healthy sub group (n=130) of a large sample of the general population, and found a strong positive relationship between NT-proBNP and age.

4.2. Relation between ANP/NT-ANP, BNP/NT-proBNP and age in CHF patients
In contrast to healthy subjects, the relation between age and natriuretic peptides is less well described in CHF patients. This seems contradictory, since natriuretic peptides are generally used in CHF patients. Interestingly, Dutka et al. [11] demonstrated that although ANP levels increased with age in healthy subjects, in CHF patients, ANP levels appeared to decrease with age. In contrast, an increase of NT-ANP with age was demonstrated in a large sample of CHF patients [12]. Although the multivariate correlation was only modest, NT-ANP increased approximately threefold from the age of 40 to 80 years [12]. Differences between these studies might be related to differences between ANP and NT-ANP, although in the current study, both ANP and NT-ANP increased with age, and we did not find a difference between the relative increase of either ANP or NT-ANP with age. To our knowledge, only one small study has demonstrated an age-dependency of BNP and NT-proBNP in CHF patients (n=92) [21]. In this study of Masson et al. [21], the increase in plasma levels of BNP and NT-proBNP with age was similar. We confirmed this age dependency of both BNP and NT-proBNP in a large group of patients classified in NYHA functional class III/IV. However, we demonstrated that the relative increase of BNP with age was significantly larger than the relative increase of NT-proBNP with age. The difference between our study and the results by Masson et al. might be related to the severity of CHF (NYHA III–IV and mainly NYHA II, respectively), and the difference in age (68 vs. 57 years, respectively), although these explanations remain highly speculative.

4.3. Direct comparisons between ANP/NT-ANP and BNP/NT-proBNP
Clerico et al. described the age dependency of ANP and BNP of healthy adults in the same paper. Although they found significant correlations with age for both peptides (ANP: r=0.350; BNP: r=0.254), no direct comparisons were performed [8]. Another study reported on the age dependency of both BNP and NT-ANP. Again, significant increases with age were described in healthy subjects (BNP: 1.4 fold and NT-ANP: 1.2 fold increase per age decade), but no statistical comparisons between the natriuretic peptides were shown [10].

So, although age-dependency of both ANP/NT-ANP and BNP/NT-proBNP has been well described, to our knowledge this is the first direct comparative study. Since age dependency may be a disadvantage, natriuretic peptides without age dependency will be favoured over the ones with age dependency. We found significant differences between natriuretic peptides in favour of ANP/NT-ANP over BNP/NT-proBNP and also of NT-proBNP over BNP, although differences appeared to be generally small.

4.4. Relation between ANP/NT-ANP, BNP/NT-proBNP and gender
In healthy adults, gender differences in plasma levels of natriuretic peptides are found, with females having higher plasma levels [9,10,13]. However, we only found a gender difference for ANP/NT-ANP and not for BNP/NT-proBNP. This might reflect the age difference between men and women; because BNP/NT-proBNP were shown to be more affected by ageing compared to ANP/NT-ANP, this age difference could be more powerful in BNP/NT-proBNP.

4.5. Interpretation of the findings
Age dependency of natriuretic peptides has been clearly shown in a healthy population [8–10,19,20]. We demonstrated that the influence of age on natriuretic peptides in CHF patients was modest, and comparable to several other factors, such as cardio-thoracic ratio, renal function and left ventricular ejection fraction. Therefore, indexing natriuretic peptides for age seems reasonable in the diagnosis of CHF. However, when natriuretic peptides are used for prognosis or to guide medical treatment in patients already diagnosed with CHF, we do not recommend routinely indexing natriuretic peptides for age.

4.6. Limitations
First, the current study was relatively small. The majority of the patient population was male (76%), and in some age quartiles less than 20 females were present. Therefore, power was too small to draw definite conclusions regarding gender differences between ANP/NT-ANP and BNP/NT-proBNP.

Second, several other factors appeared to be related to the levels of some natriuretic peptides, but not to others. This is in contrast to the age-dependency, which was demonstrated with all natriuretic peptides. Since we cannot clearly explain these findings, we think that this might have been a chance finding.

Third, measurements of plasma levels of NT-proBNP were performed using a non-commercially available assay developed by Prof. O. Vuolteenaho (Oulu, Finland). The disadvantage of using this assay is that no solid validation information is available. Because different assays have different outcomes, current results are only valid for the natriuretic peptide plasma levels determined with the same assays.

4.7. Conclusions
The present analysis confirmed the positive relation between (NT-) ANP, BNP/NT-proBNP and age in a large group of CHF patients. In addition, the influence of age appeared to be more pronounced on levels of BNP/NT-proBNP than on ANP/NT-ANP. Also, the relative increase of BNP with age was significantly larger than of NT-proBNP. Nevertheless, many other factors were also related to plasma levels of both ANP/NT-ANP and BNP/NT-proBNP, and partial correlation coefficients were relatively low.


   Acknowledgements
 
Prof. Dr. D.J. van Veldhuisen is a Clinical Established Investigator of the Netherlands Heart Foundation (Grant D97.017). The authors are indebted to the Trial Coordination Centre for the statistical support.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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