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. Author manuscript; available in PMC: 2011 Jul 1.
Published in final edited form as: Br J Haematol. 2010 Apr 16;150(1):102–107. doi: 10.1111/j.1365-2141.2010.08195.x

NT-PROBNP AS A MARKER OF CARDIOPULMONARY STATUS IN SICKLE CELL ANAEMIA IN AFRICA

Zakari Y Aliyu 1,3, Aisha Suleiman 2, Ester Attah 2, Aisha I Mamman 2, Aliyu Babadoko 2, Mehdi Nouraie 3, Laurel Mendelsohn 4, Gregory J Kato 4, Victor R Gordeuk 3, Mark T Gladwin 5
PMCID: PMC2907438  NIHMSID: NIHMS216458  PMID: 20408845

Summary

N-terminal (NT) pro-brain natriuretic peptide (proBNP) ≥160 ng/l has a 78% positive predictive value for pulmonary hypertension and is associated with increased mortality in US sickle cell disease patients, but the importance in sickle cell disease patients in Africa is not known. In a cross-sectional study at Ahmadu Bello University Teaching Hospital, Shika-Zaria, Nigeria, we studied 133 hydroxycarbamide-naïve Nigerian sickle cell anaemia patients aged 18-52 years at steady-state and 65 healthy controls. Twenty-six percent of patients versus 5% of controls had NT-proBNP ≥160 ng/l (P=0.0006). By logistic regression among the patients, human immunodeficiency virus seropositivity, higher serum ferritin and lower haemoglobin or higher lactate dehydrogenase independently predicted elevated NT-proBNP. After adjustment for haemoglobin concentration, elevated NT-proBNP concentration was associated with an estimated 7.8-fold increase in the odds of severe functional impairment, defined as an inability to walk more than 300 m in six min (95% confidence interval 1.5-32.6; P=0.005). Similarly, elevated tricuspid regurgitation velocity was associated with an estimated 5.6-fold increase in the odds of functional impairment (95% confidence interval 1.5-21.0; P=0.011). In conclusion, NT-proBNP elevation is common and is associated with markers of anaemia, inflammation and iron status and with severe functional impairment among sickle cell anaemia patients in Nigeria.

Keywords: sickle cell disease, N-terminal pro-brain natriuretic peptide, six-minute walk, haemolysis, Africa

Introduction

The N-terminal (NT) pro-brain natriuretic peptide (proBNP) hormone mediates natriuresis and vasodilation and down-regulates the rennin-angiotensin-aldosterone axis (Wang, et al 1996). Elevated NT pro-BNP levels predict diminished exercise tolerance and poor prognosis in patients with left ventricular failure (Tsutamoto, et al 1997). NT-proBNP levels are also increased in patients with primary and severe secondary pulmonary hypertension including haemolysis-related pulmonary hypertension (Kragelund, et al 2005, Leuchte, et al 2004, Voskaridou, et al 2007). The degree of NT-proBNP elevation reflects the clinical and haemodynamic status of patients and response to specific therapy (Leuchte, et al 2004). We recently reported that NT-proBNP levels ≥160 ng/l were associated with pulmonary hypertension in adults with sickle cell disease in the United States, with a positive predictive value of 78% (Machado, et al 2006). More recent findings indicate that elevated levels are highly predictive of mortality in the Cooperative Study of Sickle Cell Disease cohort (unpublished observations).

The global burden of sickle cell disease is highest in sub-Saharan Africa, especially in Nigeria where more than 150,000 children are born with the disease annually and about four million people are afflicted with the condition (Aliyu, et al 2008a, Makani, et al 2007). More than 98% of these patients are not treated with hydroxycarbamide (Aliyu et al 2007). We recently reported a 25% prevalence of pulmonary arterial hypertension among sickle cell disease patients in Nigeria based on an elevated echocardiogram-determine tricuspid regurgitation velocity (Aliyu, et al 2008b). Pulmonary hypertension was associated with a decreased functional status in the Nigerian cohort, as assessed by a six-min walk distance of less than 300 m. The vast majority of sickle cell disease patients in Africa have limited access to adequate health care, including echocardiography for the diagnosis and stratification of pulmonary hypertension. As an example, there are less than twelve functional echocardiograms with color and spectral Doppler capabilities in northern Nigeria for a total population of more than 50 million people (Aliyu 2009). Additionally, there is a lack of skilled personnel to complete accurate echocardiography studies. It would be therefore be helpful to have a screening bio-marker that would identify high-risk adult patients in Africa who should be considered for interventions to reduce mortality.

The present analysis aimed to determine the prevalence, risk factors and cardiopulmonary consequences of elevated serum NT-proBNP concentration among adult patients with sickle cell anaemia in Africa.

Methods

Patients and controls

We previously reported echocardiographic findings in 208 consecutive sickle cell disease patients aged 10 to 52 years at steady state and 94 healthy non-matched controls who were studied in Nigeria in a cross-sectional manner (Aliyu, et al 2008b). The study was approved by the National Heart Lung Blood Institute and Howard University Institutional Review Boards and the Ethical and Scientific Committee of Ahmadu Bello University, Zaria, Nigeria, and was designed to comply with the Fair Benefit Framework for international research (Participants in the 2001 Conference on Ethical Aspects of Research in Developing Countries, 2002). The research subjects were Nigerians 10 years of age or older of both genders who volunteered and provided written informed consent. Patients were recruited from the community but regional referrals were also accepted. Patients with sickle cell disease were required to have electrophoretic and or high pressure liquid chromatography documentation of haemoglobin SS, SC, Sβ0-thalassaemia, Sβ+-thalassaemia or other major sickling phenotypes. The control group was required to have electrophoretic documentation of haemoglobin A or AS phenotype. Criteria for exclusion from this study included subjects unable to understand the investigational nature of the study or to give informed consent. The present report includes a subgroup of 133 hydroxycarbamide-naïve Nigerian patients with sickle anaemia (haemoglobin SS) and 65 healthy controls who were ≥18 years of age, to determine the associations of NT-proBNP and cardiopulmonary findings in an older cohort. Routine evaluation, echocardiography and six-min walk test were performed and circulating NT-proBNP concentrations were determined.

Echocardiography

Cardiac measurements were performed with 2D transthoracic echocardiogram according to the guidelines of the American Society of Echocardiography (Lang, et al 2005). Tricuspid regurgitation was assessed in the parasternal right ventricular inflow, parasternal short-axis, and apical four-chamber views to determine the highest velocity, which reflects systolic pulmonary artery pressure (Quinones, et al 2002). Peak velocities of the E wave and A wave and the ratio of the E wave to the A wave were measured in a standard manner (Lang, et al 2005, Quinones, et al 2002) to estimate any coexisting left ventricular diastolic dysfunction, a potential cause of pulmonary hypertension and an independent risk factor for death in patients with sickle cell disease (Sachdev, et al 2007). Elevated systolic pulmonary artery pressure was defined prospectively as tricuspid regurgitation velocity ≥2.5 m/sec (Gladwin, et al 2004).

Laboratory measurements

Haemoglobin levels, leucocyte and platelet counts, reticulocyte counts, lactate dehydrogenase (LDH), aspartate aminotransferase, direct bilirubin, serum creatinine, and haemoglobin F were assessed using standard methodologies. Serum ferritin levels were measured by a microparticle enzyme immunoassay (IMX System, Abbot Diagnostics, Abbott Park, Illinois, USA; normal range 20–200 μg/l). Serum NT-proBNP levels were evaluated using an electrochemiluminescence immunoassay (Roche Diagnostics GmbH, Mannheim, Germany) in samples that were collected in the morning of the Doppler echocardiograpic examination, and were then centrifuged and stored within 4–6 h of venipuncture.

Statistical analysis

Sickle cell disease patients were categorized into two groups, based on NT-proBNP levels, and then were compared using the Kruskal-Wallis test for continuous variables and the chi square test or Fisher exact test for categorical variables. For multivariate statistical analyses, continuous variables that followed a skewed distribution were transformed in the best manner to approximate a normal distribution. In sickle cell patients, associations of demographic, physical examination, echocardiography parameters including tricuspid regurgitation velocity and NT-proBNP as continuous variables were assessed using Spearman correlation. Stepwise logistic regression models were used to assess the associations of sets of variable with NT-proBNP dichotomized a priori as ≥160 ng/l. An arbitrary level of 5% statistical significance was assumed to be significant. The logistic regression then was confirmed using robust standard error estimation. Statistical analysis was done using Stata 10.1 (StataCorp, College station, TX).

Role of the funding source

The research was supported by NIH intramural and extramural funds as follows: the NHLBI, NIH Division of Intramural Research; a bench to bedside grant from the Office of Rare Diseases and the Clinical Center, NIH; grant nos. 2 R25 HL003679-08 and 1 R01 HL079912-03 from NHLBI, NIH, Bethesda, MD.

Results

Clinical characteristics of patients and controls

We have previously reported the clinical characteristics of patients and controls from which the present sub-group was selected (Aliyu, et al 2008b). All 133 of the patients with sickle cell disease in the present report were haemoglobin S homozygotes. Ninety percent of the patients reported less than five units of whole blood transfused during their lifetime and none were receiving hydroxycarbamide. The patients with sickle cell disease were younger and more commonly of the female gender than the controls.

Median (interquartile range) NT-proBNP concentrations were significantly higher in 133 patients with sickle cell anaemia than in the 65 control subjects: 93 ng/l (53 to 166 ng/l) versus 33 ng/l (14 to 57 ng/l; P <0.0001) (Figure 1) and 26% of patients versus 5% of controls had elevated concentrations (≥160 ng/ml) (P = 0.0006).

Figure 1.

Figure 1

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Distribution of circulating NT-proBNP concentrations in patients with sickle cell disease and control participants. The lines indicate the median concentrations.

Clinical associations of elevated NT-proBNP concentrations in patients with sickle cell disease

In bivariate analyses, elevated NT-proBNP was associated with lower haemoglobin, albumin and transferrin concentrations, higher serum ferritin concentration and higher proportion of human immunodeficiency virus type 1 (HIV-1) seropositvity but not higher LDH concentration (Table 1). Only six of the sickle cell anaemia patients were HIV-1 seropositive, but, nevertheless, NT-proBNP concentrations were higher in HIV-positive than HIV-negative patients (medians of 229 ng/l versus 92 ng/l, P = 0.011) and similarly high NT-proBNP concentrations were more frequent in HIV-positive patients than HIV-negative patients (67% vs. 25%, P = 0.022). Multivariate logistic regression confirmed that HIV-1 seropositivity had a significant, independent association with elevated NT-proBNP (Table 2).

Table 1. Distribution of demographic and clinical variables in sickle cell anaemia patients according to NT-proBNP concentration*.

N NT-proBNP
<160 (ng/l)		 N NT-proBNP
≥160 (ng/l)		 P**
Age (years) 98 24 (20-27) 35 23 (21-29) 0.9
Male gender, no, (%) 98 47 (48%) 35 14 (40%) 0.4
Systolic blood pressure (mm Hg) 58 118 (110-125) 21 113 (104-120) 0.17
HIV positive, no (%) 97 2 (2%) 35 4 (11%) 0.023
Haemoglobin (g/l) 97 86 (78-94) 35 82 (69-86) 0.006
Lactate dehydrogenase (u/l) 98 388 (321-517) 35 382 (324-477) 0.6
Alanine aminotransferase (u/l) 98 17 (13-22) 35 17 (14-22) 0.9
Albumin (g/l) 98 44 (41-46) 35 42 (39-44) 0.006
Creatinine (μmol/l) 98 53 (44-62) 35 53 (44-62) 0.6
Ferritin (μg/l) 95 180 (77-411) 35 570 (175-658) 0.001
Transferrin (mg/l) 98 203 (174-236) 35 168 (151-197) 0.0001
Tricuspid regurgitation velocity (m/s) 82 2.2 (1.8-2.4) 30 2.3 (1.9-2.5) 0.5
Mitral valve E/A ratio 71 1.6 (1.4-1.9) 24 1.5 (1.4-2.0) 0.6
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*

Results are in median (interquartile range) unless otherwise indicated

**

P values <0.005 are statistically significant after Bonferonni correction for multiple comparisons.

Table 2. Independent predictors of elevated NT-proBNP concentration (≥160 ng/l) among sickle cell anaemia patients (N = 129)*.

Odds
ratio		 95% confidence
interval		 P
Age (increase of 1 year) 0.4 0.2-1.01 0.053
HIV-1 seropositive 6.9 1.0-46.3 0.046
Ferritin (log) 2.0 1.2-3.2 0.004
Haemoglobin (decrease of 10 g/l) 6.7 1.4-32.3 0.016
Age and haemoglobin** interaction 1.5 1.0-2.4 0.050
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*

One outlier was removed from analysis.

**

Increase of 10 g/l

Area under receiver operating characteristic curve = 0.79; P value for goodness of fit = 0.6.

Haemoglobin concentration tended to increase with increasing age, suggesting that the patients who survived to the older age range may have had milder disease (data not shown). By logistic regression analysis, there was an interaction between haemoglobin and age in determining NT-proBNP category (P = 0.050). After adjusting for this interaction and the effects of age, HIV seropositivity and ferritin, each 10 g/l decline in haemoglobin concentration was associated with an estimated 6.7-fold increase in the odds of elevated NT-proBNP (95% confidence interval [CI], 1.4-32.3; P = 0.016) (Table 2). Serum ferritin concentration tends to increase and serum transferrin concentration tends to decrease with increased iron stores or inflammation. In keeping with this expectation, there was a strong inverse correlation between serum concentrations of ferritin and transferrin in the research participants (R = −0.57; P <0.001), and transferrin could substitute for ferritin in the logistic regression model of increased NT-proBNP (data not shown).

The haemoglobin concentration serves as a reflection of both the degree of anaemia and the degree of haemolysis in sickle cell disease. Serum LDH concentration is marker of haemolysis in sickle cell disease (Kato, et al 2006). In keeping with this, the LDH concentration correlated inversely with haemoglobin concentration among the patients with sickle cell anaemia in this study (r = −0.36, P <0.001). To determine if the independent relationship of lower haemoglobin concentration with elevated NT-proBNP reflected an association of increased haemolysis with elevated NT-proBNP, log LDH was substituted for haemoglobin in the model shown in Table 2. In keeping with the observations with haemoglobin concentration, LDH concentration tended to decrease with increasing age (data not shown) and there was an interaction between LDH and age in determining NT-proBNP category (P = 0.011). After adjusting for this interaction and the effects of age, HIV seropositivity and ferritin, higher LDH concentration was associated with increased estimated odds of having elevated NT-proBNP (P = 0.016).

Association of elevated NT-proBNP with cardiopulmonary findings

The median (interquartile range) tricuspid regurgitation velocity was 2.3 m/s (1.9 to 2.5 m/s) in 30 patients with elevated NT-proBNP concentration compared to 2.2 m/s (1.8 to 2.4 m/s) in 82 patients with normal NT-proBNP concentration (P = 0.5) (Table 1). Tricuspid regurgitation velocity ≥2.9 m/s was present in three (10%) of the individuals with elevated NT-proBNP versus 2 (2%) of those with normal NT-proBNP concentration (P = 0.086). The median (interquartile range) mitral valve E/A ratio was 1.5 (1.4 to 2.0) in 24 patients with elevated NT-proBNP concentration compared to 1.6 (1.4 to 1.9) in 71 patients with normal NT-proBNP concentration (P = 0.6) (Table 1). Mitral valve E/A ratio <1.0, a cut-off which has been used to reflect left ventricular dysfunction in adults with sickle cell disease (Sachdev, et al 2007), was present in none of 24 of the individuals with elevated NT-proBNP versus 2 of 71 (3%) with normal NT-proBNP concentration (P = 0.4).

The median (interquartile range) six-min walk distance was 382 m (307 to 432 m) in 31 patients with elevated NT-proBNP concentration compared to 387 m (342 to 428 m) in 85 patients with normal NT-proBNP concentration (P = 0.6). Severe functional impairment, defined as an inability to walk greater than 300 m in six min, was present in 23% of individuals with elevated NT-proBNP versus 7% of those with normal NT-proBNP (P = 0.019) (Figure 2). A six-min walk distance of less than 300 m is associated with increased risk of hospitalization and one-year morality in the setting of non-sickle cell disease-related left ventricular failure (Bittner, et al 1993), but has not been validated as a cut-off for increased complications in the setting of sickle cell disease. After adjustment for haemoglobin concentration, elevated NT-proBNP concentration was associated with an estimated 7.8-fold increase in the odds of severe functional impairment, defined as an inability to walk more than 300 m in six min (95% CI, 1.3 - 32.6; P = 0.005). In parallel, elevated tricuspid regurgitation velocity of ≥2.5 m/s was associated with an estimated 5.6-fold increase in the odds of inability to walk more than 300 m in six min (95% CI, 1.5 - 21.0; P = 0.011). Inability to walk more than 300 m in patients was observed in none of the two patients with E/A ratio <1.0 versus 11 of 85 patients (13%) with mitral valve E/A ratio of 1.0 or higher (P = 0.6).

Figure 2.

Figure 2

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Proportions of sickle cell disease participants with severe functional limitation, defined as inability to walk >300 m in six min.

Discussion

In the present study, NT-proBNP concentrations were three-fold higher in patients with sickle cell disease than control subjects in Africa, more than 25% of sickle cell disease patients had elevated NT-proBNP concentrations (≥160 ng/l) compared to 5% of controls, and elevated NT-proBNP concentration was associated with increased severe functional impairment, defined as an inability to walk more than 300 m in six min. In parallel, elevated tricuspid regurgitation velocity was also associated with increased severe functional impairment. However, in contrast to studies in the US (Machado, et al 2006), NT-proBNP concentrations did not correlate significantly with tricuspid regurgitation velocity in this study.

The present study also found that, in multivariate analyses, elevated NT-proBNP was independently associated with either lower haemoglobin concentration or higher LDH concentrations among Nigerian patients with sickle cell disease. The observations that elevated NT-proBNP was independently associated with HIV seropositivity and either higher serum ferritin or lower transferrin levels suggest that elevated NT-proBNP may be associated with inflammation and increased iron status as well as degree of anaemia in sickle cell disease patients in Africa. Pulmonary hypertension is a recognized complication of HIV disease that has also been reported in Africa (Ntsekhe and Hakim 2005).

Pulmonary hypertension is common in patients with sickle cell disease and is related mechanistically to haemolysis and NO scavenging (Ataga, et al 2004, Castro, et al 2003, Gladwin, et al 2004). A high prevalence of pulmonary hypertension has also been reported in other haemolytic anaemias (Aliyu, et al 2008a). We previously reported that adult sickle cell disease patients in the US with circulating concentration of NT-proBNP of 160 ng/l or higher had a 78% chance of having pulmonary hypertension. This report was based on the NT-proBNP levels measured in 230 patients in the NIH Sickle Cell Disease-Pulmonary Hypertension Screening Study (2001-2005) and in 121 patients enrolled in the Multicentre Study of Hydroxycarbamide in Sickle Cell Anaemia (MSH) Patients’ Follow-up Study (starting in 1996) (Machado, et al 2006). Although the median echocardiogram-determined tricuspid regurgitation velocity, which reflects systolic pulmonary artery pressure, and the proportion with the highest tricuspid regurgitation velocity measurements were slightly higher in the Nigerian sickle cell disease patients with elevated NT-proBNP in the present study, these findings were not statistically significant. The reason for the difference between the findings in Nigeria and the US is not clear, but may partially be related to the smaller sample size and younger age of the Nigerian patients.

Functional impairment characterized by poor exercise tolerance is a common feature of sickle cell disease. The six-min walk test provides information regarding functional capacity, response to therapy and prognosis across a range of chronic cardiopulmonary conditions. (Rasekaba, Lee 2009 et al.; Miyamoto, Nagaya et al. 2000) A distance less than 300 m is associated with increased mortality in chronic heart failure (Bittner, et al 1993). The six-min walk test is simple, requires inexpensive equipment, is reproducible, and is considered safe because patients are self-limited during exercise. In the present study, elevated NT-proBNP concentration was associated with an increase in the odds of severe functional impairment in sickle cell disease patients, defined as an inability to walk more than 300 m in six min, and increased tricuspid regurgitation velocity was also associated with an increase in the odds of such a severe functional impairment. Our findings thus point to both NT-proBNP and tricuspid regurgiation velocity as potential markers for functional impairment and decreased quality of life in African patients with sickle cell disease.

Circulating NT-proBNP concentration has now emerged as a strong independent risk factor for death in sickle cell disease patients in the US (Machado, et al 2006). This has important clinical and public health implications considering the global burden of sickle cell disease (Aliyu, et al 2008a). There is therefore a need for broad-based screening programmes of sickle cell disease patients, especially in parts of the world with large populations of sickle cell disease patients, for the purpose of early identification and intensification of management of sickle cell disease with hydroxycarbamide, simple or exchange transfusion, anticoagulation therapy and iron chelation therapy among other modalities with a goal of ameliorating the risk of death. There is a need for a simple, reproducible, easy to use and relatively affordable biomarker as a predictor of mortality and other complications among sickle cell disease patients in Africa, and serum NT-proBNP concentration has the potential to serve as such a biomarker.

In conclusion, NT-proBNP elevation is common among adults with sickle cell disease in Nigeria and is associated with markers of anaemia, inflammation and iron status. Further studies are needed to determine if NT-proBNP elevation predicts increased mortality in Africa. The present information supports the use of elevated NT-proBNP values to select potentially high-risk patients for inclusion in intervention trials in Africa.

Acknowledgments

Supported by the NHLBI, NIH Division of Intramural Research, by a bench to bedside grant from the Office of Rare Diseases and the Clinical Center, NIH, and by grant nos. 2 R25 HL003679-08 and 1 R01 HL079912-03 from NHLBI, NIH, Bethesda, MD.

Footnotes

Conflict of Interest: None

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