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Ischemia modified albumin: An oxidative stress marker in β-thalassemia major
Clinica Chimica Acta 413 (2012) 907–910
Contents lists available at SciVerse ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Ischemia modified albumin: An oxidative stress marker in β-thalassemia major Samir M. Awadallah a,⁎, Manar F. Atoum b, Nisreen A. Nimer b, Suleiman A. Saleh b a b
University of Sharjah, College of Health Sciences, Department of Medical Lab Sciences, Sharjah, United Arab Emirates The Hashemite University, Faculty of Allied Health Sciences, Department of Medical Lab Sciences, Zarqa, Jordan
a r t i c l e
i n f o
Article history: Received 12 December 2011 Received in revised form 30 January 2012 Accepted 31 January 2012 Available online 7 February 2012 Keywords: Ischemia modified albumin Thalassemia Oxidative stress
a b s t r a c t Background: Ischemia modified albumin (IMA) is an altered type of serum albumin that forms under conditions of oxidative stress. This study reports on the levels and clinical significance of IMA in patients with βthalassemia major. Methods: Blood specimens were collected from 166 subjects (101 β-thalassemia major patients and 65 healthy controls). Serum levels of IMA, ferritin, malondialdehyde (MDA), ferroxidase, transaminases, total protein, and albumin were determined using conventional methods. Results: Serum levels of IMA (ABSU) were significantly higher in patients than in controls (0.725 ± 0.155 vs 0.554 ± 0.154, p = 0.000). Similarly, higher levels were also observed for ferritin, MDA, ferroxidase, and transaminases. No significant differences were observed between patients and controls with respect to total protein and albumin. Spearman univariate analysis demonstrated significant correlation between IMA and ferritin, MDA, ferroxidase, and transaminases. Multiple linear regression analysis revealed significant association of IMA with ferritin and ferroxidase after adjusting for the other variables (r = 0.343, p = 0.002; r = 0.228, p = 0.029 respectively). MDA however, correlated significantly with ferritin only (r = 0.654, p = 0.000). Conclusion: Our findings suggest that increased levels of IMA in thalassemic patients are likely to be a result of iron-induced oxidative stress and hence its potential significance as a new marker of oxidative stress in such patients. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Thalassemia is a group of inherited disorders that generally affects people of the Mediterranean, Middle East, Africa, India, and South Asia [1]. It is characterized by chronic hemolytic anemia resulting from defective globin chain synthesis and ineffective erythropoiesis. β-thalassemia, the most common type, arises as a result of decreased synthesis or total absence of the beta chain of hemoglobin. Consequently, excess free alpha chain accumulates and precipitates within erythrocytes leading to shortened life span and ineffective erythropoiesis [2,3]. Furthermore, patients with β-thalassemia major require continuous blood transfusion which leads to iron overload with subsequent organ and tissue damage. The status of iron overload and iron-induced oxidative stress has been repeatedly investigated in patients with thalassemia major [4–8]. Many studies reported increased blood levels of the redox active fractions of non-transferrin bound iron (NTBI) and labile plasma iron (LPI) in patients with βthalassemia [3,5,9]. It has also been demonstrated that such patients experience decreased antioxidant capacity and increased products ⁎ Corresponding author at: University of Sharjah, College of Health Sciences, Department of Medical Lab Technology, Sharjah, United Arab Emirates. Tel.: + 971 50 5469636; fax: + 971 6 5057515. E-mail address: [email protected] (S.M. Awadallah). 0009-8981/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2012.01.037
of peroxidative damage [6–8]. Collectively, these findings demonstrate that patients with β-thalassemia are under significant irondriven oxidative stress. The N-terminus residues of human serum albumin tend to bind with transition metals such as cobalt, copper and nickel [10,11] and alterations in this region of albumin hinder its binding capacity to such elements. Reactive oxygen species (ROS) resulting from conditions such as ischemia, hypoxia, acidosis, free radicals, and free iron can decrease the ability of the N-terminus to bind with transition metals [10–13]. Human serum albumin with a decreased binding capacity as a result of ischemic events is referred to as ischemia modified albumin (IMA) [12]. IMA as measured by the albumin cobalt binding test is currently used as an early marker for myocardial ischemia and acute coronary syndrome [14]. However, recent studies have reported increased levels of IMA in conditions other than ischemic heart diseases including diabetes mellitus, hyperlipidemia, chronic renal disease, obesity, and others [15–21]. Accordingly, it has been suggested that elevated levels of IMA may reflect a generalized rather than organ- or tissue-specific state of oxidative stress [21]. Oxidative stress, redox active forms of iron, and generation of ROS are well documented in patients with β-thalassemia major [3–9]. It is therefore possible that under such conditions, the structure of human serum albumin is modified in such a way that permits for excessive production of IMA. However, the relationship between β-
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thalassemia major and IMA as a marker of oxidative stress has yet to be investigated. 2. Materials and methods The study population consisted of 101 β-thalassemia major patients along with 65 age- and sex-matched healthy controls. In the patients group, the disease was detected at an early age of life (1–2 y) using hemoglobin electrophoresis at alkaline pH (Helena Labs, Beaumont, TX). None of the patients were classified as thalassemia intermedia and none of the controls were thalassemia minor. Subjects under 5 years of age were not included in the study. All patients were recruited from the Blood Bank and Transfusion Center of Zarqa, Jordan. Clinical history and other relevant data were collected from patients' files with prior permission of attending physician. All patients received approximately 350 to 500 ml of packed red blood cells at each transfusion (3 to 4-week intervals) and were all on iron chelation therapy using either deferasiox (Exjade) or deferoxamine. Mean serum ferritin level measured over the last two years was used for evaluation. Clinical data relevant to cardiac complications, endocrine disorders, or growth retardation were not documented and not included in the study. The control population was selected from various outpatient clinics in the same area. None had any history of blood transfusion, anemia, liver disease, or active inflammatory conditions. All participants were informed of the purpose of the study and were asked to sign a written consent, which was previously approved by the local research ethics committee. Blood was collected in plain and EDTA tubes; samples were collected from patients just prior to blood transfusion. Serum was separated by centrifugation and divided into several aliquots and kept at −80 °C until they were analyzed. Serum total protein, albumin, alanine transaminase (ALT), and aspartate transaminase (AST) were determined by automated chemistry analyzer (Hitachi 912, Roche, Germany). Serum ferritin levels were determined by immunoassay analyzer (Elecsys, Roche, Germany). Ferroxidase activity was determined by spectrophotometry using O-dianisidine dihydrochloride method [22]. Lipid peroxidation was measured by determining the malondialdehyde (MDA) production using thiobarbituric acid (TBA) by the method of Buege and Aust [23]. Serum levels of IMA were determined by the albumin cobalt binding test as previously described by Bar-Or et al. [12] and the results were reported as absorbance units (ABSU). Results were expressed as mean ± SD or frequency as per the parameter in question. Data analysis was carried out using the statistical software package of SPSS ver. 17.0 (SPSS Inc, Chicago, IL). Statistical differences between groups were assessed by the Mann–Whitney U test for nonparametric variables. Spearman univariate correlation analysis was conducted between all parameters. Multiple linear regression analysis was performed to determine the level of association (p ≤ 0.05) between IMA vs. the independent variables ferritin, MDA, ferroxidase, ALT, and AST and that of MDA vs. the independent variables age, ferritin, IMA, ferroxidase, ALT, and AST. 3. Results Table 1 shows the general characteristics and lab findings for both patients and healthy controls. The levels of ALT and AST were significantly higher and the levels of Hb were significantly lower in patients as compared with controls. No significant differences were observed between patients and healthy counterpart with regard to serum total protein and albumin. Data was also analyzed separately to test the bearing of HCV and splenectomy as potential sources of variation with respect to all measured parameters. No significant differences were observed between patients with or without HCV or splenectomy.
Table 1 General characteristics of thalassemic patients and healthy controls. Parametera
Thalassemia (101)
Controls (65)
p Value
Age, years Male/Female Hepatitis C, n (%) Splenectomy, n (%) Hb, g/dl TP, g/dl Alb, g/dl ALT, U/l AST, U/l
12.5 ± 7.0 48/53 15 (14.9) 38 (37.6) 7.9 ± 1.3 7.6 ± 0.76 4.6 ± 0.64 47.9 ± 39.5 56.8 ± 47.4
15.8 ± 5.8 32/33 None None 12.4 ± 2.1 7.4 ± 0.68 4.8 ± 0.8 14.5 ± 8.2 18.5 ± 7.4
NSb NS
0.000 NS NS 0.000 0.000
a Hb (hemoglobin), TP (total protein); Alb (albumin); ALT (alanine aminotransferase); AST (aspartate aminotransferase). b NS (not significant).
Consistent with previous reports [7,24] and as shown in Table 2, thalassemic patients are under significant oxidative stress as indicated by the high levels of ferritin, MDA, ferroxidase and IMA. The mean serum levels of all four parameters were significantly higher in patients than in controls (Table 2). Univariate Spearman correlation analysis was conducted to evaluate the association between IMA versus age, ferritin, MDA, ferroxidase, ALT and AST. As shown in Table 3, significant correlation was observed between IMA and all parameters except for age. On the other hand, MDA correlated significantly with all other parameters including age. Interestingly, ferritin levels correlated significantly with all other parameters except for ferroxidase. The only two parameters with which ferroxidase correlated were MDA and IMA. Consistent with the previously noted observation that no significant difference was found between patients and controls in terms of total protein and albumin (Table 1), neither parameter correlated with IMA (data not shown). In the control group, no significant correlations were observed between IMA and all other variables (data not shown). Multiple linear regression analysis was performed to explore the predictors of MDA and IMA levels. Table 4 and Fig. 1 show significant association with both ferritin and ferroxidase when IMA was treated as the dependent variable (r = 0.343, p = 0.002; r = 0.228, p = 0.029 respectively). On the other hand, only ferritin was the predictor when MDA was treated as the dependent variable (r = 0.654, p = 0.000) (Table 5, Fig. 2). 4. Discussion Results of this study point to several significant findings. (i) The levels of IMA are significantly higher in thalassemic patients as compared with healthy controls. (ii) High levels of IMA in thalassemic patients significantly correlate with ferritin, MDA, and ferroxidase. (iii) While ferritin was found to be the only predictor of MDA status in thalassemic patients, both ferritin and ferroxidase were found to be the predictors of the IMA status in such patients. Previous studies have demonstrated that overproduction of ROS resulting from conditions related to ischemia, hypoxia, acidosis, free radicals, and free iron plays a major role in the formation of IMA [10–14]. Several recent studies however have demonstrated that IMA elevates in conditions other than ischemic heart disease [15–21]. In that, increased levels of IMA were reported in conditions
Table 2 Oxidative stress parameters in thalassemia patients and controls. Parametera
Thalassemia (101)
Controls (65)
p Value
Ferritin (μg/l) MDA (μmol/l) Ferroxidase (U/l) IMA (ABSU)
2529 ± 2218 1.90 ± 0.91 136.2 ± 35.8 0.725 ± 0.155
38 ± 26 1.10 ± 0.32 96.5 ± 19.8 0.554 ± 0.154
0.000 0.000 0.000 0.000
a
MDA (malondialdehyde); IMA (ischemia modified albumin).
S.M. Awadallah et al. / Clinica Chimica Acta 413 (2012) 907–910 Table 3 Spearman's univariate correlations in thalassemic patients.
A
Age
Ferritin
Ferroxidase
MDA
IMA
ALT
Ferritin
r = .426 p = .000 r = .105 p = .295 r = .410 p = .000 r = −.031 p = .759 r = .384 p = .001 r = .255 p = .010
r = .175 p = 0.081 r = .764 p = .000 r = .414 p = .000 r = .525 p = .000 r = .425 p = .000
r = .258 p = .009 r = .278 p = .005 r = .131 p = .193 r = .112 p = .260
r = .313 p = .001 r = .445 p = .000 r = .359 p = .000
r = .252 p = .011 r = .239 p = .016
r = .752 p = .000
MDA IMA ALT AST
r = 0.343 p = 0.002
IMA (ABSU)
Parametersa
Ferroxidase
909
Ferritin ( g/L)
a
MDA (malondialdehyde); IMA (ischemia modified albumin); ALT (alanine transaminase); AST (aspartate transaminase).
B
related to oxidative stress including type 2 diabetes [15], hyperlipidemia [16,17], chronic kidney disease [18], obesity [19], and multiple sclerosis [21]. Furthermore, recent in vitro studies have demonstrated that generation of hydroxyl radicals (•OH) by the Fenton reaction was associated with a rapid rise of IMA concentration [13]. Consistent with these observations and taking into account the fact that oxidative stress, production of ROS, impaired antioxidant defense mechanisms, hypoxia, and anemia clearly manifest in thalassemic patients, it is only logical to predict elevated levels of IMA in such patients. In this context, iron-driven oxidative stresses in association with chronic anemia and hypoxia, are the most likely causes that lead to the modification of the N-terminus of serum albumin and thus the formation of increased levels of IMA in thalassemic patients. Albeit, the exact mechanism still need to be elucidated. High serum ferritin is a predictable consequence of continuous blood transfusion in thalassemic patients [25]. Though it may also increase during acute inflammation, it remains the most common indicator of iron overload in thalassemic patients [25]. In agreement with these observations, high levels of serum ferritin noted in our patients are strongly suggestive of a state of iron overload. As a result of iron overload, redox active fractions such as NTBI and LPI are expected to increase in circulation [4,9]. These fractions can catalyze the production of ROS contributing to significant tissue injury. Under such conditions, it is very possible that such redox active forms of iron could contribute to the formation of IMA in thalassemic patient; a finding that has been noted by this study. In further support of this finding, significant correlation was observed between IMA and serum ferritin as evidenced by data from univariate (p = 0.000) (Table 3) and multivariate regression analysis (p = 0.002) (Table 4 and Fig. 1). Ceruloplasmin, a copper-containing plasma protein, exhibits antioxidative ferroxidase activity through different mechanisms. Ceruloplasmin ferroxidase activity converts iron from the ferrous to the ferric form [26] thus preventing the formation of ROS through the Fenton reaction and hence minimizing oxidative damage of lipids, proteins, and DNA [26]. Conversion of ferrous iron to its ferric form is known to enhance iron uptake by transferrin thus facilitating its Table 4 Multiple linear regression analysis of IMA (dependent variable) versus independent variables (ferritin, MDA, ferroxidase, ALT, and AST). Independent variables
Beta (b)
SEb
p Value
Ferritin MDA Ferroxidase ALT AST
0.566 − 0.090 0.202 0.186 − 0.138
0.000 0.025 0.000 0.001 0.001
0.002 NS 0.029 NS NS
b: regression coefficient; SEb: standard error of b.
IMA (ABSU)
r = 0.228 p = 0.029
Ferroxidase (U/L) Fig. 1. Partial regression plot between IMA (dependent variable) and 5 independent variables: ferritin, ferroxidase, MDA, ALT, and AST. IMA correlated significantly with ferritin (A) and ferroxidase (B). The X and Y axes are expressed as residual values after adjusting for variation in the independent variables.
transport, storage, and/or utilization. Furthermore, ferroxidase activity promotes the efflux of iron from tissues into plasma as means of minimizing iron-induced tissue damage [27–29]. It has been suggested that under conditions of iron overload and as a result of transferrin saturation, iron could bind with albumin thus contributing to the formation of more redox active forms of iron like NTBI [30–32]. Based on these findings, increased ferroxidase activity as observed in thalassemic patients [24] (Table 2) could be explained in the context of indirectly enhancing oxidative stress and hence the significant correlation with IMA (Table 4, Fig. 1). It is worth emphasizing that this is the first study reporting on the association between ferroxidase activity and IMA in thalassemic patients. MDA, a product of lipid peroxidation, significantly elevates in thalassemic patients through different mechanisms including excess amount of iron binding to erythrocytes [8] and free α-globin chain precipitation and the consequent generation of intracellular ROS [6,33,34]. It has also been suggested that increased liver lipid peroxidation as a result of ferritin accumulation could raise the rate of leakage of MDA into the circulation [7]. Hence, the association between Table 5 Multiple linear regression analysis of MDA (dependent variable) vs. independent variables (age, ferritin, IMA, ferroxidase, ALT, and AST). Independent variables
Beta (b)
SEb
p Value
Age Ferritin IMA Ferroxidase ALT AST
0.111 0.754 − 0.045 0.093 0.151 − 0.187
0.009 0.000 0.430 0.002 0.003 0.002
NS 0.000 NS NS NS NS
b: regression coefficient; SEb: standard error of b.
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r = 0.654 p = 0.000
Fig. 2. Partial regression plot of MDA (dependent variable) showing significant correlation with ferritin after adjusting for age, IMA, ferroxidase, ALT, and AST. The X and Y axes are expressed as residual values after adjusting for variation in the independent variables.
ferritin and MDA as previously reported [7] and as observed in this study (Table 5 and Fig. 2) can be readily explained. In conclusion, findings reported by this study suggest that IMA is a general rather than a tissue-specific oxidative stress marker. The significant association of IMA with ferritin and ferroxidase indicates the importance of including it as a new marker of oxidative stress in thalassemia. Acknowledgments This work was supported by research grants from the University of Sharjah (No. 090506) and the Hashemite University in Jordan (No. AM/16/13/10). The authors wish to thank Mrs. Kefah Abu Ashaikh of Zarqa Blood Bank for her technical expertise. References [1] Weatherall DJ. Hemoglobinopathies worldwide: present and future. Curr Mol Med 2008;8:592–9. [2] Olivieri NF. The beta-thalassemias. N Engl J Med 1999;341:99–109. [3] Nathan DG, Gunn RB. Thalassemia: the consequences of unbalanced hemoglobin synthesis. Am J Med 1966;41:815–30. [4] Koren A, Fink D, Admoni O, Tennenbaum-Rakover Y, Levin C. Non-transferrin bound labile plasma iron and iron overload in sickle cell disease: a comparative study between sickle cell disease and β-thalassemic patients. Eur J Haematol 2010;84:72–8. [5] Chakraborty D, Bhattacharyya M. Antioxidant defense status of red blood cells of patients with b-thalassemia and E/beta-thalassemia. Clin Chim Acta 2001;305: 123–9. [6] Kassab-Chekir A, Laradi S, Ferchichi S, et al. Oxidant, antioxidant status and metabolic data in patients with beta thalassemia. Clin Chim Acta 2003;338:79–86. [7] Walter PB, Fung EB, Killilea DW, et al. Oxidative stress and inflammation in iron overloaded patients with β-thalassaemia or sickle cell disease. Br J Haematol 2006;135:254–63. [8] Livrea MA, Tesoriere L, Pintaudi AM, et al. Oxidative stress and antioxidant status in beta-thalassemia major: iron overload and depletion of lipid-soluble antioxidants. Blood 1996;88:3608–14.
[9] Cabantchik ZI, Breuer W, Zanninelli G, Cianciulli P. LPI-labile plasma iron in iron overload. Best Pract Res Clin Haematol 2005;18:277–87. [10] Chan B, Dodsworth N, Woodrow J, Tucker A, Harris R. Site-specific N-terminal auto-degradation of human serum albumin. Eur J Biochem 1995;227:524–8. [11] Bar-Or D, Curtis G, Rao N, Bampos N, Lau E. Characterization of the Co(2 +) and Ni(2 +) binding amino-acid residues of the N-terminus of human albumin. Eur J Biochem 2001;268:42–7. [12] Bar-Or D, Lau E, Winkler JV. Novel assay for cobalt–albumin binding and its potential as a marker for myocardial ischemia — a preliminary report. J Emerg Med 2000;19:311–5. [13] Roy D, Quiles J, Gaze DC, Collinson P, Kaski JC, Baxter GF. Role of reactive oxygen species on the formation of the novel diagnostic marker ischaemia modified albumin. Heart 2006;92:113–4. [14] Sbarouni E, Georgiadou P, Voudris V. Ischemia modified albumin changes — review and clinical implications. Clin Chem Lab Med 2011;49:177–84. [15] Kaefer M, Piva SJ, De Carvalho JA, et al. Association between ischemia modified albumin, inflammation and hyperglycemia in type 2 diabetes mellitus. Clin Biochem 2010;43:450–4. [16] Duarte MM, Rocha JB, Moresco RN, et al. Association between ischemia-modified albumin, lipids and inflammation biomarkers in patients with hypercholesterolemia. Clin Biochem 2009;42:666–71. [17] Valle Gottlieb MG, da Cruz IB, Duarte MM, et al. Associations among metabolic syndrome, ischemia, inflammatory, oxidatives, and lipids biomarkers. J Clin Endocrinol Metab 2010;95:586–91. [18] Cichota LC, Moresco RN, Duarte MM, da Silva JE. Evaluation of ischemia-modified albumin in anemia associated to chronic kidney disease. J Clin Lab Anal 2008;22: 1–5. [19] Piva SJ, Duarte MM, Da Cruz IB, et al. Ischemia-modified albumin as an oxidative stress biomarker in obesity. Clin Biochem 2011;44:345–7. [20] Bhagavan NV, Ha JS, Park JH, et al. Utility of serum fatty acid concentrations as a marker for acute myocardial infarction and their potential role in the formation of ischemia-modified albumin: a pilot study. Clin Chem 2009;55:1588–90. [21] Borderie D, Allanore Y, Meune C, Devaux JY, Ekindjian OG, Kahan A. High ischemia modified albumin concentration reflects oxidative stress but not myocardial involvement in systemic sclerosis. Clin Chem 2004;50:2190–3. [22] Schosinsky H, Lehmann HP, Beeler MF. Measurement of ceruloplasmin from its oxidase activity in serum by use of o-dianisidine dihydrochloride. Clin Chem 1974;20:1556–63. [23] Buege JA, Aust SD. Microsomal lipid peroxidation. Methods Enzymol 1978;52: 302–10. [24] Awadallah SM, Nimer NA, Atoum MF, Saleh SA. Association of haptoglobin phenotypes with ceruloplasmin ferroxidase activity in β-thalassemia major. Clin Chim Acta 2011;412:975–9. [25] Olivieri NF, Brittenham GM. Iron-chelating therapy and the treatment of thalassemia. Blood 1997;89:739–61. [26] Floris G, Medda R, Padiglia A, Musci G. The physiopathological significance of caeruloplasmin. Biochem Pharmacol 2000;60:1735–41. [27] Young SP, Fahmy M, Golding S. Ceruloplasmin, transferrin and apotransferrin facilitate iron release from human liver cells. FEBS Lett 1997;411:93–6. [28] Attieh ZK, Mukhopadhyay CK, Seshadri V, Tripoulas NA, Fox PL. Ceruloplasmin ferroxidase activity stimulates cellular iron uptake by a trivalent cation-specific transport mechanism. J Biol Chem 1999;274:1116–23. [29] Sarkar J, Seshadri V, Tripoulas NA, Ketterer ME, Fox PL. Role of ceruloplasmin in macrophage iron efflux during hypoxia. J Biol Chem 2003;278:44018–24. [30] Silva Andre MN, Hider Robert C. Influence of non-enzymatic post-translation modifications on the ability of human serum albumin to bind iron. Implications for non-transferrin-bound iron speciation. Biochim Biophys Acta 2009;1794: 1449–58. [31] Esposito BP, Breuer W, Sirankapracha P, Pootrakul P, Hershko C, Cabantchik ZI. Labile plasma iron in iron overload: redox activity and susceptibility to chelation. Blood 2003;102:2670–7. [32] Gutteridge JM, Rowley DA, Griffiths E, Halliwel B. Low-molecular-weight iron complexes and oxygen radical reactions in idiopathic hemochromatosis. Clin Sci 1985;68:463–7. [33] Naithani R, Chandra J, Bhattacharjee J, Verma P, Narayan S. Peroxidative stress and antioxidant in children with beta thalassemia major. Pediatr Blood Cancer 2006;46(7):780–5. [34] Scott MD, Van den Berg JJM, Repka T, et al. Effect of excess β-hemoglobin chains on cellular and membrane oxidation in model β-thalassemic erythrocytes. J Clin Invest 1993;91:1706–12.
Contents lists available at SciVerse ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Ischemia modified albumin: An oxidative stress marker in β-thalassemia major Samir M. Awadallah a,⁎, Manar F. Atoum b, Nisreen A. Nimer b, Suleiman A. Saleh b a b
University of Sharjah, College of Health Sciences, Department of Medical Lab Sciences, Sharjah, United Arab Emirates The Hashemite University, Faculty of Allied Health Sciences, Department of Medical Lab Sciences, Zarqa, Jordan
a r t i c l e
i n f o
Article history: Received 12 December 2011 Received in revised form 30 January 2012 Accepted 31 January 2012 Available online 7 February 2012 Keywords: Ischemia modified albumin Thalassemia Oxidative stress
a b s t r a c t Background: Ischemia modified albumin (IMA) is an altered type of serum albumin that forms under conditions of oxidative stress. This study reports on the levels and clinical significance of IMA in patients with βthalassemia major. Methods: Blood specimens were collected from 166 subjects (101 β-thalassemia major patients and 65 healthy controls). Serum levels of IMA, ferritin, malondialdehyde (MDA), ferroxidase, transaminases, total protein, and albumin were determined using conventional methods. Results: Serum levels of IMA (ABSU) were significantly higher in patients than in controls (0.725 ± 0.155 vs 0.554 ± 0.154, p = 0.000). Similarly, higher levels were also observed for ferritin, MDA, ferroxidase, and transaminases. No significant differences were observed between patients and controls with respect to total protein and albumin. Spearman univariate analysis demonstrated significant correlation between IMA and ferritin, MDA, ferroxidase, and transaminases. Multiple linear regression analysis revealed significant association of IMA with ferritin and ferroxidase after adjusting for the other variables (r = 0.343, p = 0.002; r = 0.228, p = 0.029 respectively). MDA however, correlated significantly with ferritin only (r = 0.654, p = 0.000). Conclusion: Our findings suggest that increased levels of IMA in thalassemic patients are likely to be a result of iron-induced oxidative stress and hence its potential significance as a new marker of oxidative stress in such patients. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Thalassemia is a group of inherited disorders that generally affects people of the Mediterranean, Middle East, Africa, India, and South Asia [1]. It is characterized by chronic hemolytic anemia resulting from defective globin chain synthesis and ineffective erythropoiesis. β-thalassemia, the most common type, arises as a result of decreased synthesis or total absence of the beta chain of hemoglobin. Consequently, excess free alpha chain accumulates and precipitates within erythrocytes leading to shortened life span and ineffective erythropoiesis [2,3]. Furthermore, patients with β-thalassemia major require continuous blood transfusion which leads to iron overload with subsequent organ and tissue damage. The status of iron overload and iron-induced oxidative stress has been repeatedly investigated in patients with thalassemia major [4–8]. Many studies reported increased blood levels of the redox active fractions of non-transferrin bound iron (NTBI) and labile plasma iron (LPI) in patients with βthalassemia [3,5,9]. It has also been demonstrated that such patients experience decreased antioxidant capacity and increased products ⁎ Corresponding author at: University of Sharjah, College of Health Sciences, Department of Medical Lab Technology, Sharjah, United Arab Emirates. Tel.: + 971 50 5469636; fax: + 971 6 5057515. E-mail address: [email protected] (S.M. Awadallah). 0009-8981/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2012.01.037
of peroxidative damage [6–8]. Collectively, these findings demonstrate that patients with β-thalassemia are under significant irondriven oxidative stress. The N-terminus residues of human serum albumin tend to bind with transition metals such as cobalt, copper and nickel [10,11] and alterations in this region of albumin hinder its binding capacity to such elements. Reactive oxygen species (ROS) resulting from conditions such as ischemia, hypoxia, acidosis, free radicals, and free iron can decrease the ability of the N-terminus to bind with transition metals [10–13]. Human serum albumin with a decreased binding capacity as a result of ischemic events is referred to as ischemia modified albumin (IMA) [12]. IMA as measured by the albumin cobalt binding test is currently used as an early marker for myocardial ischemia and acute coronary syndrome [14]. However, recent studies have reported increased levels of IMA in conditions other than ischemic heart diseases including diabetes mellitus, hyperlipidemia, chronic renal disease, obesity, and others [15–21]. Accordingly, it has been suggested that elevated levels of IMA may reflect a generalized rather than organ- or tissue-specific state of oxidative stress [21]. Oxidative stress, redox active forms of iron, and generation of ROS are well documented in patients with β-thalassemia major [3–9]. It is therefore possible that under such conditions, the structure of human serum albumin is modified in such a way that permits for excessive production of IMA. However, the relationship between β-
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thalassemia major and IMA as a marker of oxidative stress has yet to be investigated. 2. Materials and methods The study population consisted of 101 β-thalassemia major patients along with 65 age- and sex-matched healthy controls. In the patients group, the disease was detected at an early age of life (1–2 y) using hemoglobin electrophoresis at alkaline pH (Helena Labs, Beaumont, TX). None of the patients were classified as thalassemia intermedia and none of the controls were thalassemia minor. Subjects under 5 years of age were not included in the study. All patients were recruited from the Blood Bank and Transfusion Center of Zarqa, Jordan. Clinical history and other relevant data were collected from patients' files with prior permission of attending physician. All patients received approximately 350 to 500 ml of packed red blood cells at each transfusion (3 to 4-week intervals) and were all on iron chelation therapy using either deferasiox (Exjade) or deferoxamine. Mean serum ferritin level measured over the last two years was used for evaluation. Clinical data relevant to cardiac complications, endocrine disorders, or growth retardation were not documented and not included in the study. The control population was selected from various outpatient clinics in the same area. None had any history of blood transfusion, anemia, liver disease, or active inflammatory conditions. All participants were informed of the purpose of the study and were asked to sign a written consent, which was previously approved by the local research ethics committee. Blood was collected in plain and EDTA tubes; samples were collected from patients just prior to blood transfusion. Serum was separated by centrifugation and divided into several aliquots and kept at −80 °C until they were analyzed. Serum total protein, albumin, alanine transaminase (ALT), and aspartate transaminase (AST) were determined by automated chemistry analyzer (Hitachi 912, Roche, Germany). Serum ferritin levels were determined by immunoassay analyzer (Elecsys, Roche, Germany). Ferroxidase activity was determined by spectrophotometry using O-dianisidine dihydrochloride method [22]. Lipid peroxidation was measured by determining the malondialdehyde (MDA) production using thiobarbituric acid (TBA) by the method of Buege and Aust [23]. Serum levels of IMA were determined by the albumin cobalt binding test as previously described by Bar-Or et al. [12] and the results were reported as absorbance units (ABSU). Results were expressed as mean ± SD or frequency as per the parameter in question. Data analysis was carried out using the statistical software package of SPSS ver. 17.0 (SPSS Inc, Chicago, IL). Statistical differences between groups were assessed by the Mann–Whitney U test for nonparametric variables. Spearman univariate correlation analysis was conducted between all parameters. Multiple linear regression analysis was performed to determine the level of association (p ≤ 0.05) between IMA vs. the independent variables ferritin, MDA, ferroxidase, ALT, and AST and that of MDA vs. the independent variables age, ferritin, IMA, ferroxidase, ALT, and AST. 3. Results Table 1 shows the general characteristics and lab findings for both patients and healthy controls. The levels of ALT and AST were significantly higher and the levels of Hb were significantly lower in patients as compared with controls. No significant differences were observed between patients and healthy counterpart with regard to serum total protein and albumin. Data was also analyzed separately to test the bearing of HCV and splenectomy as potential sources of variation with respect to all measured parameters. No significant differences were observed between patients with or without HCV or splenectomy.
Table 1 General characteristics of thalassemic patients and healthy controls. Parametera
Thalassemia (101)
Controls (65)
p Value
Age, years Male/Female Hepatitis C, n (%) Splenectomy, n (%) Hb, g/dl TP, g/dl Alb, g/dl ALT, U/l AST, U/l
12.5 ± 7.0 48/53 15 (14.9) 38 (37.6) 7.9 ± 1.3 7.6 ± 0.76 4.6 ± 0.64 47.9 ± 39.5 56.8 ± 47.4
15.8 ± 5.8 32/33 None None 12.4 ± 2.1 7.4 ± 0.68 4.8 ± 0.8 14.5 ± 8.2 18.5 ± 7.4
NSb NS
0.000 NS NS 0.000 0.000
a Hb (hemoglobin), TP (total protein); Alb (albumin); ALT (alanine aminotransferase); AST (aspartate aminotransferase). b NS (not significant).
Consistent with previous reports [7,24] and as shown in Table 2, thalassemic patients are under significant oxidative stress as indicated by the high levels of ferritin, MDA, ferroxidase and IMA. The mean serum levels of all four parameters were significantly higher in patients than in controls (Table 2). Univariate Spearman correlation analysis was conducted to evaluate the association between IMA versus age, ferritin, MDA, ferroxidase, ALT and AST. As shown in Table 3, significant correlation was observed between IMA and all parameters except for age. On the other hand, MDA correlated significantly with all other parameters including age. Interestingly, ferritin levels correlated significantly with all other parameters except for ferroxidase. The only two parameters with which ferroxidase correlated were MDA and IMA. Consistent with the previously noted observation that no significant difference was found between patients and controls in terms of total protein and albumin (Table 1), neither parameter correlated with IMA (data not shown). In the control group, no significant correlations were observed between IMA and all other variables (data not shown). Multiple linear regression analysis was performed to explore the predictors of MDA and IMA levels. Table 4 and Fig. 1 show significant association with both ferritin and ferroxidase when IMA was treated as the dependent variable (r = 0.343, p = 0.002; r = 0.228, p = 0.029 respectively). On the other hand, only ferritin was the predictor when MDA was treated as the dependent variable (r = 0.654, p = 0.000) (Table 5, Fig. 2). 4. Discussion Results of this study point to several significant findings. (i) The levels of IMA are significantly higher in thalassemic patients as compared with healthy controls. (ii) High levels of IMA in thalassemic patients significantly correlate with ferritin, MDA, and ferroxidase. (iii) While ferritin was found to be the only predictor of MDA status in thalassemic patients, both ferritin and ferroxidase were found to be the predictors of the IMA status in such patients. Previous studies have demonstrated that overproduction of ROS resulting from conditions related to ischemia, hypoxia, acidosis, free radicals, and free iron plays a major role in the formation of IMA [10–14]. Several recent studies however have demonstrated that IMA elevates in conditions other than ischemic heart disease [15–21]. In that, increased levels of IMA were reported in conditions
Table 2 Oxidative stress parameters in thalassemia patients and controls. Parametera
Thalassemia (101)
Controls (65)
p Value
Ferritin (μg/l) MDA (μmol/l) Ferroxidase (U/l) IMA (ABSU)
2529 ± 2218 1.90 ± 0.91 136.2 ± 35.8 0.725 ± 0.155
38 ± 26 1.10 ± 0.32 96.5 ± 19.8 0.554 ± 0.154
0.000 0.000 0.000 0.000
a
MDA (malondialdehyde); IMA (ischemia modified albumin).
S.M. Awadallah et al. / Clinica Chimica Acta 413 (2012) 907–910 Table 3 Spearman's univariate correlations in thalassemic patients.
A
Age
Ferritin
Ferroxidase
MDA
IMA
ALT
Ferritin
r = .426 p = .000 r = .105 p = .295 r = .410 p = .000 r = −.031 p = .759 r = .384 p = .001 r = .255 p = .010
r = .175 p = 0.081 r = .764 p = .000 r = .414 p = .000 r = .525 p = .000 r = .425 p = .000
r = .258 p = .009 r = .278 p = .005 r = .131 p = .193 r = .112 p = .260
r = .313 p = .001 r = .445 p = .000 r = .359 p = .000
r = .252 p = .011 r = .239 p = .016
r = .752 p = .000
MDA IMA ALT AST
r = 0.343 p = 0.002
IMA (ABSU)
Parametersa
Ferroxidase
909
Ferritin ( g/L)
a
MDA (malondialdehyde); IMA (ischemia modified albumin); ALT (alanine transaminase); AST (aspartate transaminase).
B
related to oxidative stress including type 2 diabetes [15], hyperlipidemia [16,17], chronic kidney disease [18], obesity [19], and multiple sclerosis [21]. Furthermore, recent in vitro studies have demonstrated that generation of hydroxyl radicals (•OH) by the Fenton reaction was associated with a rapid rise of IMA concentration [13]. Consistent with these observations and taking into account the fact that oxidative stress, production of ROS, impaired antioxidant defense mechanisms, hypoxia, and anemia clearly manifest in thalassemic patients, it is only logical to predict elevated levels of IMA in such patients. In this context, iron-driven oxidative stresses in association with chronic anemia and hypoxia, are the most likely causes that lead to the modification of the N-terminus of serum albumin and thus the formation of increased levels of IMA in thalassemic patients. Albeit, the exact mechanism still need to be elucidated. High serum ferritin is a predictable consequence of continuous blood transfusion in thalassemic patients [25]. Though it may also increase during acute inflammation, it remains the most common indicator of iron overload in thalassemic patients [25]. In agreement with these observations, high levels of serum ferritin noted in our patients are strongly suggestive of a state of iron overload. As a result of iron overload, redox active fractions such as NTBI and LPI are expected to increase in circulation [4,9]. These fractions can catalyze the production of ROS contributing to significant tissue injury. Under such conditions, it is very possible that such redox active forms of iron could contribute to the formation of IMA in thalassemic patient; a finding that has been noted by this study. In further support of this finding, significant correlation was observed between IMA and serum ferritin as evidenced by data from univariate (p = 0.000) (Table 3) and multivariate regression analysis (p = 0.002) (Table 4 and Fig. 1). Ceruloplasmin, a copper-containing plasma protein, exhibits antioxidative ferroxidase activity through different mechanisms. Ceruloplasmin ferroxidase activity converts iron from the ferrous to the ferric form [26] thus preventing the formation of ROS through the Fenton reaction and hence minimizing oxidative damage of lipids, proteins, and DNA [26]. Conversion of ferrous iron to its ferric form is known to enhance iron uptake by transferrin thus facilitating its Table 4 Multiple linear regression analysis of IMA (dependent variable) versus independent variables (ferritin, MDA, ferroxidase, ALT, and AST). Independent variables
Beta (b)
SEb
p Value
Ferritin MDA Ferroxidase ALT AST
0.566 − 0.090 0.202 0.186 − 0.138
0.000 0.025 0.000 0.001 0.001
0.002 NS 0.029 NS NS
b: regression coefficient; SEb: standard error of b.
IMA (ABSU)
r = 0.228 p = 0.029
Ferroxidase (U/L) Fig. 1. Partial regression plot between IMA (dependent variable) and 5 independent variables: ferritin, ferroxidase, MDA, ALT, and AST. IMA correlated significantly with ferritin (A) and ferroxidase (B). The X and Y axes are expressed as residual values after adjusting for variation in the independent variables.
transport, storage, and/or utilization. Furthermore, ferroxidase activity promotes the efflux of iron from tissues into plasma as means of minimizing iron-induced tissue damage [27–29]. It has been suggested that under conditions of iron overload and as a result of transferrin saturation, iron could bind with albumin thus contributing to the formation of more redox active forms of iron like NTBI [30–32]. Based on these findings, increased ferroxidase activity as observed in thalassemic patients [24] (Table 2) could be explained in the context of indirectly enhancing oxidative stress and hence the significant correlation with IMA (Table 4, Fig. 1). It is worth emphasizing that this is the first study reporting on the association between ferroxidase activity and IMA in thalassemic patients. MDA, a product of lipid peroxidation, significantly elevates in thalassemic patients through different mechanisms including excess amount of iron binding to erythrocytes [8] and free α-globin chain precipitation and the consequent generation of intracellular ROS [6,33,34]. It has also been suggested that increased liver lipid peroxidation as a result of ferritin accumulation could raise the rate of leakage of MDA into the circulation [7]. Hence, the association between Table 5 Multiple linear regression analysis of MDA (dependent variable) vs. independent variables (age, ferritin, IMA, ferroxidase, ALT, and AST). Independent variables
Beta (b)
SEb
p Value
Age Ferritin IMA Ferroxidase ALT AST
0.111 0.754 − 0.045 0.093 0.151 − 0.187
0.009 0.000 0.430 0.002 0.003 0.002
NS 0.000 NS NS NS NS
b: regression coefficient; SEb: standard error of b.
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r = 0.654 p = 0.000
Fig. 2. Partial regression plot of MDA (dependent variable) showing significant correlation with ferritin after adjusting for age, IMA, ferroxidase, ALT, and AST. The X and Y axes are expressed as residual values after adjusting for variation in the independent variables.
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