|Year : 2015 | Volume
| Issue : 4 | Page : 978-985
Plasma brain natriuretic peptide concentration in β-thalassemia patients
Waleed M Fathy1, Walaa F Abd El-Aziz2, Seham M Ragab3, Rasha T Helal MBBCh 1
1 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Cardiology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
3 Department of Pediatric, Faculty of Medicine, Menoufia University, Menoufia, Egypt
|Date of Submission||28-Jul-2014|
|Date of Acceptance||03-Nov-2014|
|Date of Web Publication||12-Jan-2016|
Rasha T Helal
Ramad Al-Giza Hospital, 12511 Giza
Source of Support: None, Conflict of Interest: None
The aim of this study was to investigate the diagnostic and predictive value of plasma brain natriuretic peptide (BNP) levels in detecting diastolic dysfunction in β-thalassemic patients in comparison with conventional and new Doppler echocardiography indices.
β-Thalassemia major is a unique disease characterized by early diastolic dysfunction related exclusively to iron myocardial deposition. BNP is a sensitive biomarker for the detection of asymptomatic left ventricular (LV) dysfunction and has important diagnostic and prognostic implications.
Materials and methods
This study enrolled 25 b-thalassemic major patients with normal systolic function and 10 age-matched and sex-matched individuals. All participants were studied thoroughly by tissue Doppler echocardiography and blood samples were taken for determination of BNP, ferritin, total iron, and complete blood count. Patients were divided into two groups according to the E mitral/E mitral annulus ratio (E/E') at cutoff point 8.
BNP levels were higher in thalassemic patients compared with the control group. BNP and serum ferritin levels showed a statistically significant increase in group I (E/E' >8) in comparison with group II (E/E' <8). There was a statistically significant positive correlation between BNP and serum ferritin. Using receiver operating characteristic analysis, BNP at a cut point of 28.5 pg/ml was highly accurate (area under curve = 0.86, P = 0.003) in ruling out diastolic dysfunction (E/E' <8), with a sensitivity 88.9% and a specificity of 81.2%.
BNP level is significantly increased in LV diastolic dysfunction and has a predictive value in detecting latent LV diastolic dysfunction in β-thalassemia major.
Keywords: brain natriuretic peptide, diastolic dysfunction, ferritin, thalassemia
|How to cite this article:|
Fathy WM, Abd El-Aziz WF, Ragab SM, Helal RT. Plasma brain natriuretic peptide concentration in β-thalassemia patients. Menoufia Med J 2015;28:978-85
|How to cite this URL:|
Fathy WM, Abd El-Aziz WF, Ragab SM, Helal RT. Plasma brain natriuretic peptide concentration in β-thalassemia patients. Menoufia Med J [serial online] 2015 [cited 2020 Jun 1];28:978-85. Available from: http://www.mmj.eg.net/text.asp?2015/28/4/978/173693
| Introduction|| |
β-Thalassemias are a group of hereditary human diseases caused by more than 200 mutations of the human globin gene, leading to low or absent production of adult b-globin and an excess of a-globin causing ineffective erythropoiesis and lower or absent production of adult hemoglobin (HbA). The conventional treatment for these patients is based on regular blood transfusion and chelation therapy 
Despite progress in chelation therapy, heart failure is still the main cause of death in patients with β-thalassemia major. Hemochromatosis alone or in combination with immunogenetic factors is the main pathogenetic mechanism of heart failure, usually expressed as either left ventricular (LV) systolic or diastolic dysfunction  .
The mammalian heart synthesizes and secretes a family of related peptide hormones (cardiac natriuretic hormones), which have potent diuretic, natriuretic, and vascular smooth muscle-relaxing effects as well as complex interactions with the hormonal and nervous systems  .
The natriuretic peptide system consists of three known peptides that are distinct gene products and include atrial natriuretic peptide and brain natriuretic peptide (BNP), primarily from cardiomyocytes, and C-type natriuretic peptide, chiefly from endothelial cells. These three peptides, following binding to their respective receptors, induce an increase in intracellular cyclic guanosine monophosphate, leading to activation of cyclic guanosine monophosphate-dependent protein kinase  .
BNP is a hormone with natriuretic and vasodilator properties. It is synthesized by cardiomyocytes in response to elevated ventricular wall stress or ischemia  . There are two main types of BNP that are available for testing. The first is BNP, which is a hormone secreted in the ventricular myocardium during periods of increased ventricular stretch and wall tension  . Once secreted, the BNP precursor is split into the biologically active peptide and the second type of BNP, the more stable, amino-terminal prohormone fragment (N-BNP or NT proBNP)  .
It is already known that BNP levels provide sensitive and reasonably specific tests for the diagnosis of heart failure and LV dysfunction  .
Hence, this study aimed to evaluate the diagnostic and the predictive value of BNP in the detection of LV diastolic dysfunction in β-thalassemia patients.
| Materials and methods|| |
This study was carried out at the Department of Clinical Pathology (Faculty of Medicine, Menoufia University) from January 2012 to September 2013. The patients were selected from among the hematology inpatients of the Department of Pediatric, Faculty of Medicine Menoufia University, and echocardiography was performed at the Department of Cardiology, Faculty of Medicine, Menoufia University.
The study was approved by the ethical committee of the hospital and the patients provided informed consent.
This study included 25 multitransfused b-thalassemic patients. There were 14 males and 11 females ranging in age from 3 to 23 years. In addition, 10 healthy age-matched and sex-matched individuals were included. Patients were classified according to the Doppler echocardiographic parameter E/E' (mitral inflow E wave/E' mitral annulus velocity ratio).
This group included nine b-thalassemic major patients. Patients in this group fulfilled E/E' >8. These patients were suspected to have diastolic dysfunction.
This group included 11 β-thalassemic major patients. Patients in this group fulfilled E/E' <8. These patients did not have diastolic dysfunction.
This group included 10 individuals. Participants in this group had no history of cardiac disease or any chronic disease, and normal echocardiographic findings.
All the participants in the study were subjected to conventional echocardiography, tissue Doppler indexes, serum ferritin, serum iron, complete blood count, and BNP.
Echocardiography in the form of a complete two-dimensional echocardiographic examination was performed using an ultrasound machine (vivid 9; General Electric Medical Systems, Horton, Norway) equipped with a 5 MHz variable-frequency harmonic-phased array transducer, with simultaneous ECG monitoring performed in the left lateral decubitus. Images were recorded in the standard parasternal long axis, apical four-chamber, and two-chamber views.
Routine M-mode, two-dimensional, pulsed, and continuous wave Doppler recordings were obtained for each participant. The left atrial diameter, LV internal cavity dimensions, LV ejection fraction, and LV fractional shortening (FS) were measured from the two-dimensional targeted M-mode echocardiographic tracings in the parasternal long-axis view.
Pulsed wave DTI
The pulsed wave doppler tissue imaging (DTI) was performed using the same machine. To display tissue velocities, from the apical four-chamber and two-chamber views, the Doppler sample volume was placed at four different sites of the mitral annulus: anterior, lateral, septal, and inferior sites to record major velocity time intervals: isovolumetric contraction time (ICT), isovolumetric relaxation time (IRT), and ejection time.
Five milliliters of venous blood was drawn from the cubital vein of every fasting investigated participant into a sterile vacutainer. They were divided as follows:
Two milliliters of blood in an EDTA tube for complete blood count.
One milliliter of blood was allowed to clot for 30 min and centrifuged for 15 min at 3000 rpm and the serum obtained was used for the determination of serum iron.
Two milliliters of blood was allowed to clot for 30 min and centrifuged for 15 min at 3000 rpm and the serum obtained was stored at -80°C until the time of assay of BNP and ferritin level.
Complete blood count
Using the Coulter counter model Beckman 750 Int.; (Brea, CA 92821, USA), an automated homogram was performed for all cases; this included hemoglobin estimation (Hb), red blood cell (RBCs) count, total leukocytic count, and platelet count.
Serum ferritin was supplied by Vidaf Int., using kits supplied by GenWay Biotech Inc. (6777 Nancy Ridge Dr, San Diego, CA 92121, USA). The assay system utilizes one rabbit antiferritin antibody for solid-phase (microtiter wells) immobilization and a mouse monoclonal antiferritin antibody in the antibody-enzyme (horseradish peroxidase) conjugate solution, resulting in the ferritin molecules being sandwiched between the solid phase and the enzyme-linked antibodies. After a 45-min incubation at room temperature, the wells were washed with water to remove unbound-labeled antibodies. A solution of TMB reagent was added and incubated at room temperature for 20 min, resulting in the development of a blue color. The color development was stopped with the addition of stop solution, and the color changed to yellow and was measured spectrophotometrically at 450 nm. The concentration of ferritin is directly proportional to the color intensity of the test sample.
Serum iron was assessed by Synchron Cx 5 autoanalyser using kits supplied by Beckman Instrument Inc. (Fullerton, California, USA). Ferric ions are released from transferring by guanidine hydrochloride and reduced to a ferrous state by hydroxylamine. Ferrous ions react with ferrozine, forming a colored complex. To prevent copper interference, cupric ions are bound to thiourea. The color intensity is directly proportional to the iron concentration and is determined by monitoring the increase in absorbance at 546 nm.
Serum brain natriuretic peptide
Serum BNP was provided by RayBiotech Inc. (3607 Parkway Ln #100, Norcross, GA 30092, USA). The samples and standards, which are spiked with biotinylated peptide, are pipetted into the assay plate; the target protein and the labeled peptide interact competitively with the capture antibody. After washing, the bound biotin-peptide then interacts with streptavidin-HRP, producing a color development reaction. The intensity of colorimetric signal is directly proportional to the amount of biotin-peptide and inversely proportional to the amount of target protein in the samples.
Statistical evaluation was carried out using the Statistical Package for the Social Sciences (SPSS) version 14 (SPSS Inc., Chicago, Illinois, USA) for Windows statistics program using the Mann-Whitney U-test and the Pearson correlation test (r value as the coefficient). Data were expressed as mean ± SD. A P value less than 0.05 was considered statistically significant.
| Results|| |
There was no significance between the patient and the control groups according to age and sex (P > 0.05) ([Table 1]).
|Table 1 Comparison between patients and controls in terms of age and sex |
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There was a highly significant reduction in the Hb levels and RBCs count in the patient group compared with the control group (P < 0.001). There was a significant increase in the white blood cells count and platelets in the patient group compared with the control group (P < 0.05). There was a highly significant increase in the serum iron and ferritin level in the patient group compared with the control group (P < 0.001). There was a highly significant increase in the BNP level in the patient group compared with the control group (P < 0.001) ([Table 2]).
|Table 2 Comparison between patients and controls in terms of laboratory data |
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In terms of the echocardiographic parameters, there was a significant increase in aortic diameter and left atrium in the patient group compared with the control group (P < 0.05). There was a highly significant increase in left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), and E in the patient group compared with the control group (P < 0.001). There was no significance in ejection fraction, FS, A, and E/A ratio between the patient group and the control group (P > 0.005). In terms of E/E' (mitral inflow early diastolic to mitral annulus velocity ratio), there was no significant difference between the thalassemic patients and the controls (P > 0.05). There was a highly significant increase in the mean ICT (P < 0.001) in thalassemic patients in comparison with the control group. There was a significant increase in the mean contraction time (CT) and the mean IRT (P < 0.05) in thalassemic patients in comparison with the control group ([Table 3]).
|Table 3 Comparison between patients and controls in terms of echocardiographic parameters |
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In terms of the BNP level, there was a highly significant difference between the two patient groups (P < 0.001). In terms of the ferritin level, there was a significant difference between the two patient groups (P < 0.005). There was no significant difference in the Hb level or serum iron between the two patient groups (P > 0.005) ([Table 4]).
|Table 4 Comparison between patients groups in terms of laboratory parameters |
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For the lab and echo profile of the population study, there was a statistically positive correlation between BNP and ferritin (r = 0.63 and P < 0.001), E (r = 0.62 and P < 0.001), mean ICT (r = 0.48 and P = 0.003), mean CT (r = 0.68 and P < 0.001), and mean IRT (r = 0.45 and P = 0.007) ([Table 5]).
|Table 5 Correlation between brain natriuretic peptide and serum ferritin and some echocardiographic parameters |
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However, there was no significant correlation between BNP and E/E' or E/A (P > 0.05).
| Discussion|| |
β-Thalassemia major is a unique disease characterized by very early diastolic dysfunction, which appears early in the patient's life  and is related mainly to myocardial iron deposition. Myocardial iron deposition does not solely account for altered LV relaxation, although it appears to directly lead to LV myocardial restriction with elevated pulmonary artery pressure and right ventricular dilatation  .
The blood indices (Hb concentration and RBCs count) were lower in thalassemic patients than the controls. However, white blood cells and platelets counts were higher in thalassemic patients than the controls, which is in agreement with Weatherall  . This may be attributed to marrow hyperplasia in TM patients.
In terms of the ferritin level, there was a highly significant increase in the patient group compared with the control group (P < 0.001), which is in agreement with Lekawanvijit and Chattipakorn  . This can be attributed to chronic hemolysis and repeated blood transfusion.
M-mode echocardiography of the patients studied showed a statistically highly significant increase in LV diastolic dimensions (LVEDD and LVESD) in the patients studied compared with the control group (P < 0.001). This is in agreement with the result reported by Garadah and colleagues, who studied cardiac dimensions in 38 thalassemic patients and 38 normal controls. They found that the LV dimensions at the end of systole (LVESD) and diastole (LVEDD) were significantly larger in TM compared with the control group  . The cardiac dilatation may be because of chronic anemia and hyperdynamic circulation.
LV diastolic function was defined by the pattern of transmitral inflow on spectral Doppler interrogation, consisting of the E/A ratio, E wave deceleration time, and IRT  . Diastolic flow from the left atrium to the LV across the mitral valve has two components - the E wave, which reflects early diastolic filling, and the A wave, in late diastole, which reflects atrial contraction  .
There was a significantly higher E wave in the patient group compared with the control group (P < 0.05), which is in agreement with Garadah et al.  , attributable to an abnormal LV relaxation pattern.
There was no significant difference in A or the E/A ratio in the thalassemic group compared with the control group. This is in agreement with Noori and Mehralizadeh  , who reported no significant difference in the E/A ratio between TM group and the control group. This is also in agreement with Gharzuddine et al.  , who reported that the E/A ratio did not differ in 50 thalassemic patients and 29 healthy control participants, Similar results were published by Iarussi et al.  . However, the result differs from that of Garadah and colleagues, who reported a significantly higher E/A ratio in the TM patient group compared with the control group, indicating a restrictive pattern of cardiomyopathy. This may be explained by the low mean age range of patients and the lack of patients with heart failure symptoms.
None of our patients showed impairment in LV systolic function, manifested by normal LVEF, which was greater than 55% and FS greater than 25%. This is in agreement with Spirito et al.  , who showed that diastolic filling is altered in an early stage of this disease when iron overload has not yet caused systolic dysfunction and LV dilatation. The normal LVEF indicates that TM patients have minimal deleterious effect of iron overload on myocardial systolic function  . However, several studies have reported a significantly lower LVEF in thalassemic patients in comparison with healthy age-matched and sex-matched individuals  .
TDI of the studied patients showed a statistically significant increase in IRT, one of the diastolic function indices, in the patients studied compared with the control group, which reflects a reduction in ventricular relaxation because of iron overload. This is in agreement with Gharzuddine and colleagues , .
The current study shows the presence of diastolic dysfunction among our TM patients with various degrees represented by elevated E wave and IRT (diastolic function indices), with preserved systolic function represented by normal ejection fraction and FS (systolic function indices).
Natriuretic peptides are mainly released in response to increased cardiac volume and pressure overload  . BNP has emerged as a sensitive biomarker for the diagnosis, prognosis, and treatment of heart failure  . It has been found that BNP show strong and near-identical test performance in ruling out severe heart failure and in the estimation of prognosis  . It has been suggested that BNP predicts future atrial fibrillation and future heart failure over and above echocardiographic parameters  .
In the current work, we found a highly significant increase in the BNP levels in the TM patients studied compared with the control group (P < 0.001). This is in agreement with Lubien et al.  .
According to the latest heart failure guidelines of the European Society of Cardiology (ESC), E/E' ratio = 15 is a high filling pressure. The gray zone is E/E' 8-15. This indicates another investigative modality  .
There was no significant difference in E/E' between the TM patients studied and the control group. This is different from what was reported by Kremastinos et al.  , who found that there was a significant increase in E/E' in the TM patients compared with the control group. This may be explained by the low mean age of our studied patients and the fact that one of the groups was already on antifailure treatment.
We divided our patients according to the E/E' ratio into two subgroups, subgroup A (E/E' >8) and subgroup B (E/E' <8), and measured the plasma levels of BNP in both subgroups. We observed a highly significant increase in the plasma BNP level in subgroup A (E/E' >8) compared with subgroup B (E/E' <8) (P < 0.001). This proves the ability of the BNP to diagnose LV diastolic dysfunction. This is in agreement with Kremastinos et al.  , who measured the BNP in 70 TM patients divided into three groups according to E/E' (E/E' <8, E/E' 8-15, E/E' >15) and found that BNP levels increased significantly in both groups E/E' larger than 8 compared with the group E/E' lower than 8.
Using receiver operating characteristic curve analysis, BNP at a cut point of 28.5 was highly accurate (area under the receiver operating characteristic curve = 0.86, P = 0.003) in ruling out diastolic dysfunction (E/E' <8) with a sensitivity of 88.9% and a specificity of 81.2%, with a positive predictive value of 72.7% and a negative predictive value of 92%.
Because of the lack of assay standardization, no generally applicable reference intervals for BNP can be defined at present. Currently, BNP reference and decision limits must be determined separately for each assay  . As a general guideline, in young, healthy adults, 90% will have BNP levels less than 25 pg/ml  . In healthy neonates, BNP concentrations are much higher than those in adults (~10- to 20-fold) and are highest immediately after birth, decrease through maturation, and reach adult levels at about 1 month of age  , which is in agreement with the values of our study.
There was a significant positive correlation between BNP and serum ferritin, LV diastolic function indices (E and IRT), and LV systolic function indices (CT and ICT). This indicates the predictive value of BNP in detecting LV diastolic and systolic dysfunction in TM patients. This is in agreement with Kremastinos et al.  , but there was no significant correlation between BNP and E/E' or E/A.
| Conclusion|| |
BNP level is significantly increased in LV diastolic dysfunction as this was assessed in a disease with isolated diastolic dysfunction such as b-thalassemia. Also, BNP is sensitive for the detection of latent LV diastolic dysfunction even in the early stages of disease progression ([Figure 1]).
|Figure 1 Receiver operating characteristic (ROC) curve of brain natriuretic peptide (BNP) in ruling out diastolic dysfunction. Area under the curve = 0.86, SE = 0.08. P value = 0.003, 95% confidence interval (CI) = 0.71-1.01. At cutoff point 28.5, sensitivity = 88.9%, specificity = 81.2%, positive predictive value = 72.7, negative predictive value = 92.9%.|
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| Acknowledgements|| |
Conflicts of interest
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]