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B-type natriuretic peptide (BNP) and the amino-terminal fragment (NTproBNP) correlate with clinical variables, but have not been simultaneously studied in a large number of pediatric patients with pulmonary arterial hypertension (PAH). The purpose of our investigation was to compare BNP and NTproBNP with clinical indicators of disease in a pediatric PAH population for which biomarkers are much needed.
We retrospectively compared BNP and NTproBNP levels with exercise capacity, echocardiographic data, and hemodynamics in PAH patients under 21 years-old. Two hundred sixty three blood samples from 88 pediatric PAH patients were obtained, with BNP and NTproBNP drawn at the same time.
There was a correlation between BNP and NTproBNP with mean pulmonary arterial pressure/mean arterial pressure (mPAP/mSAP) ratio (r=0.40 p<0.01, r=0.45 p<0.01, respectively), mean right atrial pressure (mRAP) (r=0.48 p<0.01, r=0.48 p<0.01), and tricuspid regurgitant (TR) velocity (r=0.36 p<0.01, r=0.41 p<0.01). BNP and NTproBNP are associated with 6 minute walking distance, mPAP, mPAP/mSAP ratio, mRAP, pulmonary vascular resistance index (PVRI), and TR velocity when investigated longitudinally. On the average, a 1 unit increase in log BNP or NTproBNP was associated with 4.5 unitsxm2 or 3.4 unitsxm2 increase in PVRI, respectively. There was a strong correlation between log BNP and log NTproBNP measurements (r= 0.87, p<0.01).
In pediatric PAH, BNP and NTProBNP are strongly correlated and predict changes in clinical variables and hemodynamics. In a cross-sectional analysis, NTproBNP correlated with echocardiographic and exercise data better than BNP; NTproBNP showed less within patient variability over time, therefore NTproBNP can add additional information towards predicting these clinical measurements.
Three major natriuretic peptides (NPs) have been discovered including atrial natriuretic peptide, B-type natriuretic peptide (BNP), and C-type natriuretic peptide. These NPs are activated to protect from volume or pressure overload in cardiovascular disease. The overload of the right or left ventricle leads to synthesis of the intracellular prohormone in the myocardium. The pro-BNP is cleaved into the biologically active hormone, BNP, and an inactive amino-terminal fragment (NTproBNP).1,2 Despite BNP and NTproBNP being equally secreted, their plasma concentrations are not 1:1. BNP has a half-life of approximately 20 min; in contrast, NTproBNP, has a longer half-life of approximately 1 to 2 h. The difference causes NTproBNP to have a higher circulating level and slower fluctuations compared with BNP.3 BNP has a potential role by counteracting increased ventricular volume due to vasoconstriction, sodium retention, and activation of the renin-angiotensin-aldosterone system.4 The measurement of BNP and NTproBNP are utilized in the decision making process in various clinical settings. The diagnostic value of both peptides has been well established in symptomatic and/or asymptomatic patients with chronic heart failure.5–10 BNP has important clinical implications to identify high-risk pulmonary arterial hypertension (PAH) adults and to guide treatment.11 In addition, elevated plasma BNP concentrations are associated with increased mortality in patients with PAH.12–14 Likewise, NTproBNP can also be of prognostic value in predicting poor long-term outcome in patients with severe PAH.15–17 In addition, NTproBNP is cleared by the kidney and may be falsely evaluated with significant renal disease. Few studies have directly compared BNP and NTproBNP, and these comparisons have not been made in PAH. The purpose of our investigation was to compare BNP and NTproBNP with clinical indicators of disease in a pediatric PH population for which biomarkers are much needed.
This single-center observational study was performed between November, 2007 and October, 2010 at The Children’s Hospital, Denver. All patients were enrolled in an IRB approved protocol entitled Prospective Evaluation of Adolescents and Children with Pulmonary Arterial Hypertension (PEACH). In total, there were 88 PAH patients with a median age of 10 years-old (IQR 5–15 years-old) with 46 (52%) of patients being female. Only observations with both BNP and NTproBNP measured at the same time were included. Observations from patients older than 21 years of age were excluded. All patients had a mean pulmonary artery pressure (mPAP) over 25 mmHg, pulmonary capillary wedge pressure under 15 mmHg at right heart catheterization, normal systemic blood pressure, and renal function. PAH due to ischemic heart diseases, cardiomyopathy, and pulmonary fibrosis were excluded by further diagnostic tests including echocardiography, high-resolution computed tomography scan, and pulmonary function tests.
The different etiologies of PAH were as follows: idiopathic PAH (n=35), PAH associated with congenital heart disease (n=39), and PAH associated with connective tissue disease (n=1). Two-dimensional echocardiographic data included tricuspid regurgitant (TR) jet velocities for estimating pulmonary artery systolic pressure, right ventricular diastolic dimension, and left ventricular fractional shortening (LVFS) by the modified Simpson’s method. Right heart catheterization was performed with a balloon tipped, flow directed Swan-Ganz catheter. We measured mean right atrial pressure (mRAP), mPAP, mean systemic arterial pressure (mSAP), and pulmonary capillary wedge pressure. The pulmonary vascular resistance index (PVRI) was calculated. Cardiac output was obtained using thermo-dilution and cardiac index (CI) was calculated. If the patient had a significant intra-cardiac defect, cardiac output was obtained by the Fick method with oxygen consumption assessed by the LaFarge method. Pulmonary function tests were performed under room air at rest. We measured peak oxygen consumption (peak VO2) during cardiopulmonary test and calculated a ratio of minute ventilation (VE) to carbon dioxide output (VCO2) as ventilator efficiency (VE/VCO2).
The plasma BNP and serum NTproBNP level were obtained within 5 days of right heart catheterization. Echocardiographic evaluation, six-minute walk test with the maximum distance (6MWD), and cardiopulmonary exercise test within 90 days around the blood draw were included. In general, for those patients undergoing cardiac catheterization, the blood test was performed during the procedure. Echocardiography, pulmonary function tests, and 6MWD were most often performed the day before the catheterization. Blood for measurement of plasma BNP concentrations was collected in ethylenediaminetetraacetic acid tubes. In contrast, NTproBNP was collected in a serum sample tube. Plasma BNP was assayed on i-STAT® system using the two-site enzyme-linked immunosorbant assay (Abbott Laboratories, IL, USA) and serum NTproBNP level was measured on an electrochemiluminescence immunoassay (Mayo medical laboratories; ProBNP II, Roche Diagnostics, Indianapolis, IN, USA). Temporal changes were expressed as percentage of the baseline values. The minimum measurable level of BNP is 15 pg/ml.
The marker measurements were log (base 10) transformed and anchored at 1. Descriptive statistics were calculated using mean ± standard deviations or medians and inter-quartile range (IQR) for continuous variables and percentages for categorical variables. Cross-sectional correlations were determined using Spearman’s rank correlation coefficients. Associations were quantified using slope estimates from mixed effect regression models with a random subject effect to address the serial measurements contained in the full dataset. To estimate and compare the gender specific changes with age in the BNP and NTproBNP marker variables simultaneously, a bivariate mixed model with piece-wise linear regression and subject specific coefficients was used.18 All analyses were performed using SAS version 9.2 software (SAS Institute Inc., Cary, NC, 2008). A p-value of < 0.01 was considered to be statistically significant.
Clinical characteristics of the 88 pediatric patients using data from their first observation are shown in Table 1. Fifty-two % of patients were female between the ages of 1 month and 21 years. During the median follow-up period of 13.3 months (range 0.2 to 34 months), only 3 patients (3%) were initiated on epoprostenol infusion therapy. All patients survived owing to various vasodilator therapies; in addition, none of the patients received lung transplantation. The median mPAP was 38 mmHg (interquartile range (IQR), 27 to 54 mm Hg), the median mRAP value of 6.0 mm Hg (IQR, 4 to 8 mm Hg), the median PVRI was 7.5 units xm2 (IQR; 4.2–12.3 units xm2), and the median CI was 4.1 l/min/m2 (IQR; 3.3–5.3 l/min/m2). In our series, we obtained 263 blood samples from 88 patients. The median plasma BNP concentration was 38 pg/ml (IQR; 20–75 pg/ml) and median serum NTproBNP concentration was 179 pg/ml (IQR; 72–393 pg/ml).
The gender specific change in marker concentrations versus age was investigated by visual inspection of the plotted data with a scatterplot smoother to describe the general trend (Figure 1; 263 samples in 88 patients). Until around age 12 there is a decreasing trend in both NPs markers with increasing age without large gender differences. After the age of 12, the NPs concentrations for males begin to increase whereas the concentrations in the females decreased more slowly. A bivariate mixed effects model was used to estimate and compare these trends across gender and marker (BNP versus NTproBNP). A piece-wise linear regression with a knot at 12 years old was used to describe the trends versus age. NTproBNP had significantly higher concentrations compared to BNP across the full age range. The slope of the BNP and NTproBNP levels decreased up to age 12, but was not significantly different between BNP and NTproBNP (p=0.15). After age 12, both NP concentrations begin to increase in males. This increase was faster than the increase in females, although these gender differences were not statistically significant (BNP p = 0.56, NTproBNP p = 0.80).
This analysis utilizes the most recent sample pair from each patient, allowing each patient to equally contribute to the correlation estimate. There was a strong correlation between log BNP and log NTproBNP measurements with a cross-sectional correlation coefficient equal to 0.87 (p<0.01). Although the measurements are highly correlated, the NTproBNP values are detected in higher quantities compared to BNP, as expected (Figure 2; 88 samples in 88 patients).
Table 2 shows the correlations between both peptide levels and clinical variables using cross-sectional analysis. The NTproBNP levels had a higher correlation with 6MWD compared to BNP levels (r = −0.49, p<0.01, r = −0.32, p=0.04, respectively). Both NTproBNP and BNP were significantly associated with mPAP/mSAP (r = 0.45, p<0.01, r = 0.40, p=0.01, respectively), mRAP (r=0.48, p<0.01, r=0.48, p<0.01, respectively) and TR velocity (r=0.41, p<0.01, r=0.36, p<0.01, respectively). Interestingly, hemodynamic parameters, including mPAP and PVRI, were not significantly associated with NPs in PAH patients. With sensitivity analysis, the correlations were calculated using only measurements taken on the same day as blood collection. The correlations between 6MWD and the markers became more comparable (BNP r = −0.41 and NTproBNP r = −0.42), the difference between correlations with LVFS doubled (BNP r = 0.54 and NTproBNP r = 0.26) while the correlations with other echo markers were not as dramatically affected. For all the hemodynamic variables, except CI, BNP continued to have higher correlations compared to NTproBNP (mPAP; r = 0.37, p=0.02, r = 0.26, p=0.11, PVRI; r = 0.23, p=0.16, r = 0.13, p=0.45, respectively). There were not adequate samples sizes to perform the sensitivity analysis for the exercise variables.
Using all patient data available, assessment of conditional associations was performed to determine the association between the clinical variables with a 1 unit change in log BNP or NTproBNP (Figure 3). The t-statistic corresponding to the slope estimate was used to compare the magnitude of the associations between BNP and NTproBNP. The sign of the t-values correspond to the direction of the association. For example, the negative 6MWD t-value indicates that as log BNP increases the 6MWD decreases. NTproBNP had larger t-values for 6MWD, TR velocity, peak VO2 and VE/VCO2 compared to BNP. Whereas BNP had only slightly larger t-values for mPAP and CI compared to NTproBNP.
Table 3 shows the associations between BNP markers and clinical variables using all patient data accounting for repeated measures. Both peptides, BNP and NTproBNP were associated with 6MWD (BNP; slope = −49.0 m, 99% confidence limits (CL); −88.1 – −9.9, NTproBNP; −49.7 m, CL −84.0 – −15.4), mPAP (BNP; 12.9 mmHg, CL 3.1−22.6, NTproBNP; 9.0 mmHg, CL 1.23−16.9), mPAP/mSAP (BNP; 0.24, CL 0.08−0.40, NTproBNP; 0.20, CL 0.07−0.32), mRAP (BNP; 3.0 mmHg, CL 1.3−4.7, NTproBNP; 2.5 mmHg, CL 1.2−3.8), PVRI (BNP; 4.5 unitsxm2, CL 0.4−8.7, NTproBNP; 3.4 unitsxm2, CL 0.1−6.6), and TR velocity (BNP; 0.34m/s, CL 0.07–0.60, NTproBNP; 0.40m/s CL 0.17–0.62) (Figure 4; 214 samples in 69 patients). In this study, there was less variability in the association estimates of NTproBNP compared to BNP, which can be seen by comparing the standard error of estimates. Intraclass correlation coefficients (ICC) were calculated for each marker, BNP and NTproBNP had ICC values of 0.52 and 0.61, respectively. The ICC corresponds to the proportion of total variation that is between subjects, with a higher value indicating the concentrations within a patient are more similar. The associations were re-calculated after adjusting for the potential confounding effects of age and gender due to the results presented earlier related to the significant changes in the markers with the variables. The magnitude and significance of all associations were robust after adjusting for age and gender.
In this single center retrospective cohort study, we sought to investigate and compare the characteristics between BNP and NTproBNP in pediatric PH patients with variations in disease severity. We found that BNP and NTproBNP levels had a strong correlation with each other and there were significant age and gender related differences in both peptides. Additionally, both peptides significantly, but moderately correlated with TR velocity and hemodynamics such as mRAP, when considered in a cross sectional analysis. NTproBNP correlated better with variables not collected on the same day, such as 6MWD and echocardiographic data, moreover, the difference in the correlation estimates lessened with the constraint that the measurements be taken on the same day. In contrast, BNP tended to correlate better with the catheterization variables such as mPAP and PVRI as the blood samples were collected during catheterization. Therefore, NTproBNP may have advantages if the time between collection and measurements are greater owing to a longer half-life. However, mPAP and PVRI were significantly associated with both peptides when evaluated longitudinally. The longitudinal associations yielded similar results. None of the associations with the clinical variables were statistically different between BNP and NTproBNP. Our studies suggested both markers may be equally useful for making decisions in a clinical setting. This study is interesting because this is the first report to evaluate both BNP and NTproBNP in a relatively large pediatric PAH population.
Previous studies in PAH have shown that each peptide significantly correlated with hemodynamics. A circulating BNP was significantly correlated with mPAP (r = 0.73), total pulmonary resistance (r = 0.79), and mean RAP (r = 0.79) in 26 patients with idiopathic or thromboembolic pulmonary hypertension,19 but the number of patients in this study was small and the population was more clearly defined compared the population used here. In addition, elevated BNP level was a significant prognostic factor.13,20 Likewise, the elevation of serum NTproBNP level was useful in identifying the poor long-term prognosis in idiopathic PAH.13,16,17
Both peptides can be easily and non-invasively measured, therefore, they are widely used as reliable biomarkers in patients with PAH. However, there are few data comparing BNP and NTproBNP. The elimination of BNP in plasma is shorter compared with NTproBNP.21 For this reason, the plasma BNP level may be more useful for assessing more real-time hemodynamic changes in pediatric patients with PAH. In contrast, NTproBNP is eliminated only via the kidneys, resulting in a significantly longer half-life 22,23 and higher concentrations compared to BNP. A previous report showed that such different clearance mechanisms might contribute to the superiority of NTproBNP as a mortality marker.24 In our present study, we were unable to determine whether BNP and NTproBNP is a more sensitive predictor of outcome. However, our results performed on the concentrations collected serially over time, indicate the increased stability of NTproBNP concentrations compared to BNP and would corroborate this finding.
BNP and NTproBNP concentrations at 0 days of age were higher (20-fold for NTroBNP and 18-fold for BNP) compared to adult levels in a prior study 25 and both peptides immediately decrease after birth.26,27 Koch et al. reported that plasma concentration of BNP had sex related differences in the second decade of life,28 and even in adult studies, both peptides were related with age and gender.29–32 Likewise, we found concentrations in both BNP and NTproBNP levels decreased until age 12, after which they began to increase at slightly different rates between males and females. The associations with the clinical variables were however robust after adjusting for these potential confounding effects. In addition, a recent report showed that the decreasing trend of NTproBNP was different from BNP.33 This result was replicated in the presented work as well. Therefore, interpretation of NPs in the clinical setting should include consideration of age- and gender-related differences.
Several limitations should be mentioned. First, as previously described, of the 88 patients, all patients survived, and did not receive lung transplantation. Therefore, we could not evaluate the predictive value of both peptides due to the low number of events. The present study was an observational cohort study from a single center, therefore, clinical findings regarding the prognostic impact of these NPs remain to be confirmed in a larger multi-site study. Second, there was heterogeneity in the study population because our patients included a variety of associated forms of PAH. Third, the patients were individually treated with various vasodilator therapies according to their conditions. Despite these limitations, we found that both biomarkers can be noninvasively assessed as promising tools for the evaluation of pediatric patients with PAH.
In conclusion, BNP and NTProBNP levels strongly correlated and were associated with clinical variables and hemodynamics in pediatric patients with variations in severity of PAH. Both concentrations were affected by age and gender. BNP tended to correlate better with catheterization variables. In a cross-sectional analysis, NTproBNP correlated with echocardiographic and exercise data better than BNP. NTproBNP showed less within patient variability over time, therefore NTproBNP can add additional information towards predicting these clinical measurements. There was a strong correlation between the BNP and NTproBNP levels and both biomarkers may be equally useful for evaluating disease severity, although due to different half-lives, one may be more appropriate in certain contexts. However, given the relatively low magnitude of the correlations, these biomarkers do not replace the clinical parameters, but may provide additional information. The measurements of both natriuretic peptides may serve as useful markers in the assessment of disease in patients with PAH.
This study was supported by the Jayden DeLuca Foundation, the Leah Bult Foundation, UL1 RR025780 Colorado Clinical Translational Science Institute, National Center for Research Resources, and National Institutes of Health.
Disclosures: The authors have no conflicts of interest to disclose.