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Biomedical Research

Role of Tumor necrosis factor alpha, Malondialdehyde and serum Iron in Anemic Tuberculosis Patients

Author(s): Rashmi kulkarni, Ajit Deshpande, Kiran Saxena, Akhouri RS Sinha, Meena Verma

Vol. 22, No. 1 (2011-01 - 2011-03)

Rashmi kulkarni1, Ajit Deshpande2, Kiran Saxena3, Akhouri RS Sinha4, Meena Verma5, Ravi Saxena6

(1) Department of Biochemistry Shri Aurobindo Institute of Medical Sciences, India
(2) Department of Community Medicine Shri Aurobindo Institute of Medical Sciences, India
(3) Department of Biochemistry Chirayu Medical College Hospital, Bhopal, India
(4) Department of Biochemistry College of Medical sciences, Amargadh. India
(5) Department of Biochemistry MGM Medical College, Indore, India
(6) Department of Physiology. College of medical sciences, Amargadh, India

Abstract

The role of Tumor Necrosis Factor alpha, Malondialdehyde and serum Iron in inducing anemia in Tuberculosis patients was studied. The present study was conducted in the Department of Biochemistry, Shri Aurobindo Institute of Medical Sciences, Indore. The study comprised of sixty-eight anemic pulmonary tuberculosis patients. Levels of Hemoglobin, Iron, TIBC, Ferritin, MDA and TNF α were analyzed in all of them. The inflammatory parameters TNF α and Ferritin along with oxidative marker malondialdehyde were found to be increased in anemic tuberculosis patient and levels of Hb, iron and TIBC was decreased. Overall correlation shows that the levels of Hb, Iron and TIBC were negatively correlated to the levels of Ferritin, MDA and TNF α. Ferritin was negatively correlated with Iron (r = -0.261, p<0.05) and showed a positive correlation with MDA. (r = 0.304, p < 0.05). For Ferritin and TNF α correlation was positive, it was not significant (r = 0.114, NS).There was a positive significant correlation between Iron and TIBC. (r = 0.666, p<0.01) and a negative significant correlation between the TNF α levels and the hemoglobin concentration (r = -.273, p<0.05). Hypoferremia was induced as a consequence of inflammatory response and action of free radicals in anemic tuberculosis patients

Key word: Anemia, Hypoferremia, Ferritin, TNF α , Malondialdehyde
Accepted September 19 2010

Introduction

Active tuberculosis infection is generally associated with major abnormalities in heamopoiesis [1]. Anemia is a common complication of pulmonary tuberculosis, the reported prevalence ranging from 16 to 76% in different studies [2]. The precise mechanism of anemia in pulmonary tuberculosis patients (PTB) is not clearly known however, anemias due to inflammation and iron deficiency have been implicated [3,4]. The pathogenesis of anemia of chronic inflammation may evolve due to cytokine-mediated defense against microbial pathogens, effectively withholding iron from microbes, which incidentally also deprive erythroid precursors of their iron supply [5,6,7]. The development of hypoferremia thus results in the development of iron-restricted erythropoiesis and anemia. Cytokine also induced alterations in cellular iron homeo-stasis are mediated in part by the increased production of nitric oxide (NO) and reactive oxygen species (ROS) [8].

The present study was taken to find out the role of Tumor Necrosis Factor alpha, Malondialdehyde and serum Iron in inducing Anemia in Tuberculosis Patients.

Material and Method

Present study was conducted in the Department of Biochemistry, Shri Aurobindo Institute of Medical sciences, Indore. Sixty-eight patients of anemic pulmonary tuberculosis were selected. The patients of pulmonary tuberculosis were taken from Shri Aurobindo Institute of Medical Sciences and Manorama Raje Tuberculosis Hospital, Indore. Pulmonary tuberculosis patient falling in DOTS category CAT I and aged between 18-55 yrs and hemoglobin (Hb) less than <13 g/dL for Men and HB <12 g/dL for non-pregnant Women was included. Tuberculosis patient with other disease or disorder and patients with incomplete data records and moderate to severe injury or surgery during the previous month were excluded. Those patients who have taken antibiotics for cough like Amoxicillin, Septran, Ciprofloxacilln etc were included but those patients with history of prior anti-TB treatment were not included. Patient with extra pulmonary involvement, blood donors, subjects with history of smoking and who had taken iron supplementation were excluded. The study was approved by the Institutional and Human Ethical Committee of the Sri Aurobindo Institute of Medical sciences, Indore. Informed consent was taken from each subject and then they were enrolled in the study.

5ml of fasting venous blood samples were drawn from subjects, each sample was divided into two parts: the first part was collected in EDTA while the second sample was collected in a plain tube and left to clot. Serum was obtained after centrifuge. The serum samples were aliquoted and stored according to the instructions in the kits, until the various assays described below were performed.

Haemogram was estimated by automated cell counter. (Sysmex KX) Serum iron and total iron-binding capacity (TIBC) were estimated on an autoanalyser from Rosh diagnostics using commercial test kits from Randox Laboratories, UK. Ferritin was estimated by direct immunoenzymatic determination by using test kits from Diametra, Italy. Serum TNF α was estimated by using a commercially available immunoassay Kit from DIA-CLONE,France. Serum Malondialdehyde was estimated spectrophotometrically by thiobarbituric acid method of Wilbur K M et al (1949) [9].

Correlations were calculated by Pearson’s correlation coefficients (two-tailed) using SPSS Version 10. P value ≤0.05 was used as a threshold of significance.

Result

The levels of Hb, iron and TIBC was decrease than normal ranges and ferritin, MDA and TNF values found to be increased than normal ranges in anemic tuberculosis patients (Table 1)

Pearson correlations (Table no 2) were carried out on the different variables, which showed that the decreasing levels Hb, Iron and TIBC was negatively correlated to the increasing levels Ferritin, MDA and TNF α.

The statistically significant correlation was found between Hb and TNF α (r = -0.273, p<0.05), Iron and TIBC ( r = 0.666, p<0.01) , Iron and ferritin (r = -0.261, p< 0.05) and Ferritin and MDA (r = 0.304, p &tl; 0.05).

Table 1. Mean ± SD values for hemoglobin, iron related parameters and inflammation and oxidative marker in anemic tuberculosis patient group

Parameters Normal ranges Anemic TB Patients
N=68
Mean ± SD
Hb (g/dl) Males > 13 Females > 12 9.08 ± 1.34
Iron (μmol/l) Males = 10.6 8.3 Females = 6.6- 26.0 7.38 ± 1.79
TIBC (μg/dl) 250- 450 178.01 ± 44.0
Ferritin (ng/mL) Males = 6 350 Females = 20 400 321.25 ± 77.8
MDA (nmol/ml) >3 5.82 ± 0.92
TNF (pg/ml) > 8 57.55 ± 14.7

Table 2. Pearson’s Correlations between the Variables

  IRON
r value
TIBC
r value
Ferritin
r value
MDA
r value
TNF
r value
Hb (g/dl) 179 172 033 120 273 ٭
IRON (μmol/l) - 666 ٭ ٭ 261 ٭ 040 128
TIBC (μg/dl) - - 114 139 147
Ferritin (ng/mL) - - - 304 ٭ 114
MDA (pg/ml) - - - - 131

r = Correlation coefficient. r ٭٭ = Correlation significant at p<0.01 and r ٭ = Correlation significant at p<0.05

Discussion

Anemia is one of the abnormalities found in TB Patients due to poor nutritional causes and increased in cytokines level 1. Therefore, both anemia of inflammation as well as iron deficiency anemia may coexist in pulmonary tuberculosis. Our findings were consistent with the previous reports that there was decrease in Hb, Iron and TIBC levels and increased levels of ferrtin, TNF α and MDA in tubercu-losis patients [10, 11, 12, 13, 14 ,15].

Hypoferremia in disease associated anemia appears to be the result of a complex network of cytokines, immune cells, and acute-phase proteins [16]. TIBC values are usually high in iron-deficiency anemia and low in anemia of inflammation [17,18]. The low TIBC concentrations seen in our present patients might have been the result of an acute-phase response. Ferritin concentration in blood is considered a specific indicator of body iron stores [19], however, the concentrations can rise following an inflammatory response, irrespective of iron status [20,21,23,].

Endogenous TNF α response during inflammation is a sufficient proximal stimulus to mediate an associated anemia with decreased RBC survival and impaired erythropoiesis [24].

Recombinant TNF α and recombinant interleukin 1 (IL-1) cause a significant decrease in serum iron levels accom-panied by an increase in circulating ferritin concentration and a decrease in soluble transferrin receptor levels [25,26]. Increased levels of TNF α , ferritin and decreased iron levels in present study and their correlation with each other was in accordance with the finding that tumour necrosis factor alpha induce hypoferraemia and ferritin production [16]. Similar findings was stated by Bayes et al [11] for C-reactive protein (CRP), iron and ferritin.

The increased levels of TNF α and MDA and decreased levels of Hb, Iron, TIBC reveals that the MDA along with TNF α was involve in redistribution of iron. These findings are in concordance with the literature [26]. Cytokines affect iron homeostasis in monocytes by inducing the formation of short-lived radicals such as nitric oxide (NO) and reactive oxygen species (ROS) which affect the post-transcriptional regulation of iron homeostasis via iron regulatory proteins (IRPs) [27,28].

In anemic tuberculosis patients disturbances in iron ho-meostasis may be one of the cause to the shift of iron from a transferrin-bound available state to a ferritin-incorporated storage state. These changes are the components of a systemic inflammatory response and action of free radical, which may be one of the major factors in the pathogenesis of anemia.

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Correspondence to:
Rashmi A Kulkarni

Department of Biochemistry,
Shri Aurobindo Institute of Medical Sciences
Indore, Madhya Pradesh, India

Biomedical Research 2011; 22 (1): 69-72

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