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Association of haptoglobin phenotypes with ceruloplasmin ferroxidase activity in β-thalassemia major
Clinica Chimica Acta 412 (2011) 975–979
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Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
Association of haptoglobin phenotypes with ceruloplasmin ferroxidase activity in β-thalassemia major Samir M. Awadallah a,⁎, Nisreen A. Nimer b, Manar F. Atoum b, Suleiman A. Saleh b a b
University of Sharjah, College of Health Sciences, Department of Medical Lab Technology, 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 14 December 2010 Received in revised form 15 January 2011 Accepted 1 February 2011 Available online 17 February 2011 Keywords: Thalassemia Ceruloplasmin Haptoglobin Ferroxidase Iron overload
a b s t r a c t Background: Haptoglobin (Hp) and ceruloplasmin (CP) are 2 plasma antioxidants playing a role in preventing iron-induced oxidative damage. This study presents data related to Hp phenotypes and ceruloplasmin ferroxidase activity in relation to iron store markers in patients with β-thalassemia major. Methods: Blood specimens were collected from 196 subjects (124 β-thalassemia major patients and 72 healthy controls). Serum levels of iron, total iron binding capacity (TIBC), ferritin, high sensitivity C-reactive protein (hs-CRP), ceruloplasmin, and ferroxidase activity were determined using conventional methods. Haptoglobin phenotypes were determined by polyacrylamide gel electrophoresis. Results: As expected, the mean levels of iron store markers, except TIBC, were significantly higher in patients than in controls. Ceruloplasmin concentrations (mg/dl) and its ferroxidase activity (U/l) were significantly higher in patients than in controls (57.9 ± 18.8 vs 46.9 ± 14.2 and 159.9 ± 47.8 vs 95.3 ± 20.9; p b 0.001, for CP and Hp, respectively). As for Hp phenotypes, no significant differences were observed between iron store markers and ferroxidase activity among the control group. In the patients group however, significantly higher concentrations of ceruloplasmin and its ferroxidase activity were observed among patients with Hp2-2 phenotype as compared to patients with the other phenotypes. Additionally, correlations according to Hp phenotypes revealed strong association between ceruloplasmin ferroxidase activity and serum ferritin in patients with Hp 2-2 phenotype and not in the others (r = 0.331, p b 0.05). Conclusion: Thalassemia patients with Hp 2-2 phenotype are under greater iron-driven oxidative stress than patients with other phenotypes. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Life-long blood transfusion is a standard protocol used for the treatment and care of patients with β-thalassemia major. Although lifesaving, chronic blood transfusion however, results in iron overload with subsequent organ and tissue damages. Under normal conditions, about 35% of transferrin, the main transport protein, is saturated with iron. Accordingly, as iron loading progresses, transferrin iron binding capacity will be exceeded, and excess iron deposits in various organs and tissues. Furthermore, redox active fractions such as non-transferrin-bound iron (NTBI) and labile plasma iron (LPI) increase in circulation [1,2]. These fractions can catalyze the production of highly reactive oxygen species (ROS) and are associated with significant injury to the liver, heart, and endocrine organs [3,4]. The LPI fraction is the most redox active species that readily enters the cells, and represents the main target for iron chelation [5]. In β-thalassemia major, the status of iron overload and iron-induced oxidative stress had been intensively investigated [6–10].
⁎ Corresponding author. Tel.: +971 50 5469636; fax: +971 6 5057515. E-mail address: [email protected] (S.M. Awadallah). 0009-8981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2011.02.003
Increased levels of serum iron, serum ferritin, and transferrin saturation, and decreased total iron binding capacity (TIBC) are well documented in thalassemia patients. In addition, increased levels of the redox active fractions of NTBI and LPI had been demonstrated by many studies [2,6,7]. Other investigators demonstrated decreased antioxidant capacity and increased products of peroxidative damage in plasma of β-thalassemia patients [8–10]. Ceruloplasmin (CP) and haptoglobin (Hp) are other plasma proteins that are involved in iron homeostasis. CP is a copper-containing α2-globulin that is synthesized in liver and secreted in plasma. CP participate in several biological functions including copper transport, coagulation, angiogenesis, defense against oxidative stress as antioxidant, and iron homeostasis [11]. The antioxidant property of CP is attributed to its ability to prevent lipid oxidation, scavenging superoxide, and sequestering free copper ions [11–13]. Concerning its role in iron metabolism, many studies have demonstrated that CP possesses ferroxidase activity that plays a major role in the homeostasis of iron [14–16]. CP catalyzes the oxidation of the ferrous (Fe 2+) form of iron to the ferric (Fe3+) form, thus reducing the oxidant capacity of toxic ferrous iron by inhibition of the Fenton reaction. Furthermore, the ferroxidase activity of CP plays a major role in promoting the loading of
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iron from tissue stores and plasma onto transferrin [17,18]. The importance of CP in iron metabolism was clearly demonstrated by studies conducted on humans as well as animals that were deficient of ceruloplasmin [18–23]. These studies demonstrated massive iron deposition in a number of organs including brain and liver [18–20]. Further studies have demonstrated that CP enhances the efflux of iron out of hepatocytes [17,23], and that CP administered to CP knock-out mice can mobilize iron out of liver restoring normal iron homeostasis [22]. Collectively, these studies suggest that CP is required for efficient iron release from cells and tissues. Haptoglobin (Hp) is a polymorphic α2-glycoprotein that exists in three main phenotypes (Hp 1-1, Hp 2-1, and Hp 2-2) possessing different structural and functional properties [24]. In circulation, the main function of Hp is to bind with free hemoglobin (Hb) preventing loss of iron and kidney damage. It is thus considered a powerful antioxidant as a result of its ability to prevent hemoglobin-driven oxidative damage [24]. Many studies however, had demonstrated that the antioxidative property of Hp and the rate of clearance of free Hb from circulation are phenotype-dependent [25]. It has been demonstrated that Hp 2-2 phenotype has lower hemoglobin binding capacity as compared to the other phenotypes [26,27]. Furthermore, it has been observed that clearance of Hp 1-1-Hb complex is faster than that of Hp 2-2-Hb complex [25]. Therefore, it was postulated that subjects with Hp 2-2 phenotype are under greater oxidative stress as a result of increased redox active iron derived from hemoglobin [28,29]. Once Hp–Hb complex is formed, it is quickly removed from circulation and scavenged by the CD163 receptors of hepatocytes and tissue monocytes and macrophages [30]. Studies have demonstrated that internalization of the Hp–Hb complex by the CD163 receptors is more potent for the Hb–Hp2-2 complex than for Hb bound to Hp 1-1 or 2-1 [28,30]. Accordingly, it was suggested that individuals with Hp 2-2 phenotype are under greater oxidative stress as a result of higher iron accumulation within monocytes or macrophages [25,28]. Taken together, both Cp ferroxidase activity and the inheritance pattern of Hp appear to play a significant role in the homeostasis of iron. However, data related to the role of these two plasma proteins in relation to the status of iron store in patients with beta-thalassemia is very scarce, and not well documented. Therefore, this study was conducted to evaluate the possible interplay between iron overload on one hand and ceruloplasmin ferroxidase activity and haptoglobin phenotypes on the other, in a group of beta-thalassemia major patients. 2. Materials and methods The study involved 124 β-thalassemia major patients (69 males and 55 females) and 72 age- and sex-matched healthy controls (39 males and 33 females). Subjects under age of 8 years were not included in the study. Patients aged 8 to 30 years (mean 15.8 ± 5.2 years), of whom only 12 (9.6%) were above 25 years, were recruited from the thalassemia outpatient clinic at Al Rahma Hospital (Irbid, 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 all were on iron chelation therapy using either deferasiox (Exjade) or deferoxamine. Serum ferritin levels were assessed at least twice a year in thalassemia patients, and the mean results for the last 5 years were used for evaluation. Thirty-nine patients were hepatitis C virus (HCV) positive, and 29 had splenoctomy. Clinical data relevant to cardiac complications, endocrine disorders, or growth retardation were not documented and not involved in the study. Healthy controls aged 8 to 30 years (mean 16.2 ± 3.6 years) were selected from various outpatient clinics at the same hospital. None of the control subjects were thalassemia minors and none had any history of blood transfusion, anemia, liver disease, or active inflammatory conditions. All participants provided informed written consent, which was previously approved by the local research ethics committee.
Blood samples were collected in plain tubes. Thalassemia patients’ blood was collected just before blood transfusion. After clotting, serum was separated by centrifugation and divided into several aliquots and kept at −20 °C until they are analyzed. Serum iron, total iron binding capacity (TIBC), alanine transaminase (ALT), high sensitivity CRP (hs-CRP), and ceruloplasmin 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 [31]. Haptoglobin phenotypes were identified using polyacrylamide gel electrophoresis followed by peroxidase staining (Fig. 1) [32]. The Hp phenotype frequencies were directly calculated from the number of individuals expressing the specific Hp phenotype in both patients and controls. Percent of transferrin saturation was calculated by dividing serum iron by TIBC. Data analysis was carried out using the statistical software package of SPSS ver. 17.0 (SPSS Inc, Chicago, IL). Statistical comparison between groups was assessed by the Kruskal–Wallis test, and results were presented as mean ± SD. Spearman's correlation coefficients were calculated to study the association between relevant biochemical parameters in all investigated groups. Statistical significance was set at p b 0.05. 3. Results Table 1 demonstrates the general characteristics of subjects investigated. As expected, iron store parameters, except TIBC, were significantly higher in patients than in controls. Additionally, the levels of CP, ferroxidase, CRP and ALT were also significantly higher in patients than in controls. No significant differences related to age, gender, and the distribution haptoglobin phenotypes were observed. Furthermore, splenoctomy and/or HCV did not influence the frequencies of Hp phenotypes among the patients group. Table 2 shows the laboratory findings according to Hp phenotypes. Within the control group, no significant differences were observed in all parameters investigated. Among the patients’ group however, the levels of CP and ferroxidase activity were significantly higher in patients with Hp2-2 phenotypes as compared to those with the other phenotypes (pb 0.05 and b0.01, respectively). No significant difference was observed between the other parameter in relation to Hp phenotype. Spearman's correlation analysis was conducted to analyze the correlation between ferritin, ferroxidase, CP, and CRP among the thalassemia group. In all patients combined, no significant correlation was observed between ferroxidase and ferritin, however, significant correlation was observed between ferroxidase activity with CP and CRP (r = 0.330, p b 0.01 and r = 0.179, p b 0.05, respectively), and ferritin with CRP (r= 0.342, p b 0.01). Correlations according to Hp phenotype however, revealed strong correlation of
Fig. 1. Polyacrylamide gel electrophoresis of Hp phenotypes. The gel shows the electrophoretic patterns of the three Hp phenotypes (Hp 1-1, 2-1, and 2-2).
S.M. Awadallah et al. / Clinica Chimica Acta 412 (2011) 975–979 Table 1 General characteristics of patients and controls.
Age, year Male/female Hepatitis C, n (%) Splenoctomy, n(%) Serum iron, μg/dl TIBC, μg/dl Transferrin saturation, % Serum ferritin, μg/l CP, mg/dl Ferroxidase, U/l CRP, mg/dl ALT, U/l Hp phenotypes Hp 1-1, n (%) Hp 2-1, n (%) Hp 2-2, n (%)
Thalassemia (124)
Controls (72)
p value
15.8 ± 5.2 69/55 39 (31.5) 29 (23.4) 240.9 ± 63.0 266.4 ± 63.9 90.8 ± 8.9 2970 ± 1925 57.9 ± 18.8 159.9 ± 47.8 2.5 ± 1.8 41.9 ± 36.7
16.2 ± 3.6 39/33 None None 79.7 ± 29.8 325 ± 44.5 26.5 ± 12.2 35.0 ± 23.7 46.9 ± 14.2 95.3 ± 20.9 1.57 ± 1.5 10.9 ± 7.4
NS NS
0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
12 (9.7) 53 (42.7) 59 (47.6)
7 (9.7) 31 (43.1) 34 (47.2)
NS NS NS
Results are mean ± SD or as indicated in table. NS: No significant.
ferroxidase and CP with ferritin, only in patients with Hp 2-2 (r= 0.331, p b 0.05 and r = 0.344, p b 0.05, respectively) (Fig. 2). Additionally, significant correlation between ferritin and CRP was observed in all three Hp phenotypes, of which Hp 2-2 was the strongest (r= 0.519, p b 0.01). 4. Discussion Results from this study demonstrate for the first time a close association of haptoglobin phenotypes and Cp ferroxidase activity with the iron store status in patients with β-thalassemia major. The results revealed significantly higher serum levels of Cp and its ferroxidase activity in thalassemia patients as compared to healthy controls. Additionally, the levels of Cp and ferroxidase were higher among thalassemic patients with Hp 2-2 phenotype as compared to patients with the other Hp phenotypes. Furthermore, a significant correlation was observed between the levels of Cp and ferroxidase with the levels of serum ferritin among thalassemic patients with Hp 2-2 phenotype only. Many previous studies have demonstrated that most beta-thalassemia patients are under greater oxidative stress as evidenced by decreased plasma antioxidant capacity, increased markers of oxidative damage, and most of all, increased serum levels of redox active forms of NTBI and LPI [6–10]. This status of oxidative stress is attributed to the toxicity of iron overload as a consequence of life-long blood transfusions, increased intestinal absorption of iron, and to the greater intravascular hemolysis [33]. Based on the findings of high levels of serum iron, ferritin, transferrin saturation, and decreased TIBC, it is very
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likely that our patients were under oxidative stress too. Under such conditions of disturbed iron metabolism as well as of increased iron-derived oxidative stress, it is thus possible that the functional activity of Cp, which is a major plasma antioxidant, is altered in a way to oppose and counteract the consequences of iron overload. Firstly however, it should be mentioned here that the increased levels of CP observed in our study were not attributed to acute inflammatory condition. This is due to the fact that the levels of CRP were not high enough to indicate an active or acute inflammatory condition. Accordingly, the high serum levels of Cp and ferroxidase activity observed in our patients might be attributed to several iron overloadrelated mechanisms. First, it is possible that the levels were increased to prevent the formation of reactive oxygen species (ROS) through the Fenton reaction. This is achieved by the antioxidative capacity of Cp through the oxidation of Fe2+ to Fe3+, thus preventing oxidative damage of lipids, proteins and DNA [11]. Second, oxidation of the ferrous form of iron to the ferric form enhances its binding with transferrin and thus its transport to storage and/or utilization sites. Since our results indicated high transferrin saturation percent (N85%), it is also possible that Cp is increased to enhance the binding of iron to substances other than transferrin such as albumin and citrate, thus contributing to the formation of more redox active forms of iron [34]. Third, it is possible that Cp is increased in response to accumulated iron in tissues, thus enhancing efflux of iron into transferrin in plasma. Several studies have provided evidence supporting the function of Cp in mobilization of iron between tissues and plasma [15,17,18,23]. Studies on patients with aceruloplasminemia and hemochromatosis [18–20], demonstrated marked accumulation of iron in various tissues and organs such as liver and brain. The accumulated iron was attributed to the lack ferroxidase activity of Cp that is required for the efflux of iron from tissues to plasma. In addition, studies conducted on animals lacking Cp, demonstrated that administration of Cp restored iron balance in these animals [22]. Fourth, it is possible that Cp levels were increased in response to hypoxia and ineffective erythropoiesis. Results from studies conducted on patients with chronic pulmonary obstructive disease demonstrated significantly increased Cp levels, suggesting an important role of Cp in erythropoiesis under conditions of hypoxia [35]. Such conditions of tissue hypoxia and ineffective erythropoiesis are common features among thalassemic patients. Our results also demonstrated significant correlations between ferroxidase activity with each of CP and CRP, and serum ferritin with CRP in the patient group only. The correlation between ferroxidase and Cp is an expected one, however, the correlations between ferroxidase and CRP (r = 0.179, p b 0.05) and ferritin with CRP (r = 0.342, p b 0.001), might be attributed, in part, to a mild acute inflammatory condition in our patients. Interestingly, findings from this study also demonstrated an existing interplay between Cp and its ferroxidase activity and the
Table 2 Laboratory findings according to Haptoglobin phenotypes (mean ± SD). Controls (72)a
Thalassemia (124)
Age, year Serum iron, μg/dl TIBC, μg/dl Trans saturation,% Serum ferritin, μg/l CP, mg/dl Ferroxidase, U/l CRP, mg/dl ALT, U/l a b c
Hp 1-1 (12)
HP 2-1 (53)
Hp 2-2 (59)
Hp 1-1 (7)
HP 2-1 (31)
Hp 2-2 (34)
16.9 ± 5.3 255.2 ± 44.5 271.4 ± 45.4 94.3 ± 3.4 3682 ± 1459 50.8 ± 18.7 121.9 ± 25.9 2.9 ± 2.2 44.4 ± 41.6
16.0 ± 5.9 232 ± 61 253 ± 59 92 ± 9.8 2924 ± 2381 53.8 ± 17.2 133.8 ± 27.0 2.2 ± 1.6 40.8 ± 33.4
15.3 ± 4.6 246 ± 68 277 ± 69 89.2 ± 8.9 2868 ± 1438 62.9 ± 19.3b 191.0 + 46.7c 2.6 ± 1.8 42.6 ± 39.6
18.1 ± 2.5 75.3 ± 30.0 327 ± 38.5 24.7 ± 11.4 54.4 ± 33.6 42.3 ± 9.6 97.1 ± 12.7 2.7 ± 1.8 22.8 ± 18.6
16.8 ± 3.6 80.2 ± 31.1 318 ± 49.7 26.5 ± 13.4 31.6 ± 17.8 43.8 ± 11.6 94.7 ± 15.8 1.5 ± 1.2 9.9 ± 3.6
15.2 ± 3.5 80.1 ± 29.3 331 ± 40.9 25.0 ± 13.4 34.2 + 24.9 52.1 ± 15.4 104.3 ± 18.8 1.4 ± 1.5 9.4 ± 2.8
Except for age, all parameters of controls are significantly different from their counterparts of thalassemia. p b 0.05 for Hp 2-2 versus the other Hp phenotypes. p b 0.01 for Hp 2-2 versus the other Hp phenotypes.
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specific markers of iron overload such as NTBI, LPI, and intracellular ferritin, may need to be thoroughly investigated. Acknowledgments Hp 1-1: Hp 2-1: Hp 2-2:
Work of this study was supported by research grant # 4/2010 from the College of Graduate Studies and Research at the Hashemite University, Jordan. The authors wish to thank Sultan Medical Laboratories in Amman, Jordan, for their assistance in using their autoanalyzers. References
Fig. 2. Correlation of serum ferritin levels with Cp ferroxidase activity in beta thalassemia patients in relation to Hp phenotype. Correlation coefficient and p values: Hp 1-1: r = −0.441, p = 0.151; Hp 2-1: r = 0.061, p = 0.663; Hp 2-2: r = 0.331, p = 0.010.
inheritance pattern of haptoglobin in thalassemic patients. On one hand, the levels of Cp and ferroxidase were significantly higher in patients with Hp 2-2 phenotype than their counterparts, and on the other, the levels correlated significantly with serum ferritin in the same group of patients (Table 2 and Fig. 2). Such correlation however, was not observed when patients were combined (irrespective of Hp phenotype). Many studies have reported that Hp phenotypes are directly or indirectly involved in disease processes influenced by iron metabolism such as hemochromatosis, atherosclerosis and cardiovascular disease [25]. None however, had investigated the role of Hp phenotypes in relation to iron metabolism in beta-thalassemia major. The observed high levels of Cp and ferroxidase activity and their significant correlation with serum ferritin among Hp 2-2 thalassemic patients might be related to the poor antioxidative property of Hp 2-2 phenotype [24,26,27], which is mainly attributed to its low serum concentration and to its decreased affinity to free Hb in circulation [26,27]. In fact, serum levels of haptoglobin in chronic hemolytic anemias including beta-thalassemia are known to be severely reduced. Thus, it would be expected that free Hb and its associated heme-driven iron would be greatly increased in the circulation among individuals with Hp 2-2 phenotype. Accordingly, the increased serum levels of Cp and its ferroxidase activity observed in our Hp 2-2 patients might be the result of excess free Hb and heme iron in circulation. Furthermore, studies on the monocyte–macrophage CD163 receptor, which functions as Hb scavengers, demonstrated that these receptors have greater affinity to the Hb-Hp2-2 complex than Hb complexes formed with other phenotypes [25,30]. In this regard, studies on Hp 22 healthy individuals demonstrated potent internalization of heme iron, resulting in greater intracellular accumulation of ferritin as compared to the other Hp phenotypes [25,28]. Furthermore, studies on patients with hereditary hemochromatosis showed increased serum iron and ferritin among Hp 2-2 male patients [36]. Such situation is probably exaggerated in Hp 2-2 thalassemic patients, where excessive amounts of intracellular ferritin and probably labile iron do exist. Accordingly, Cp ferroxidase activity in Hp 2-2 patients may increase in attempt to enhance efflux and mobilization of iron from cells into plasma. In conclusion, results from this study suggest that iron-derived oxidative stress and Cp ferroxidase activity are affected by the Hp 2-2 phenotype inβ-thalassemia major. However, since this is the first time such findings were reported and no previous work to compare with, more confirmatory studies are needed to consolidate the findings. Therefore, assessment of such interplay, especially in relation to more
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Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
Association of haptoglobin phenotypes with ceruloplasmin ferroxidase activity in β-thalassemia major Samir M. Awadallah a,⁎, Nisreen A. Nimer b, Manar F. Atoum b, Suleiman A. Saleh b a b
University of Sharjah, College of Health Sciences, Department of Medical Lab Technology, 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
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Article history: Received 14 December 2010 Received in revised form 15 January 2011 Accepted 1 February 2011 Available online 17 February 2011 Keywords: Thalassemia Ceruloplasmin Haptoglobin Ferroxidase Iron overload
a b s t r a c t Background: Haptoglobin (Hp) and ceruloplasmin (CP) are 2 plasma antioxidants playing a role in preventing iron-induced oxidative damage. This study presents data related to Hp phenotypes and ceruloplasmin ferroxidase activity in relation to iron store markers in patients with β-thalassemia major. Methods: Blood specimens were collected from 196 subjects (124 β-thalassemia major patients and 72 healthy controls). Serum levels of iron, total iron binding capacity (TIBC), ferritin, high sensitivity C-reactive protein (hs-CRP), ceruloplasmin, and ferroxidase activity were determined using conventional methods. Haptoglobin phenotypes were determined by polyacrylamide gel electrophoresis. Results: As expected, the mean levels of iron store markers, except TIBC, were significantly higher in patients than in controls. Ceruloplasmin concentrations (mg/dl) and its ferroxidase activity (U/l) were significantly higher in patients than in controls (57.9 ± 18.8 vs 46.9 ± 14.2 and 159.9 ± 47.8 vs 95.3 ± 20.9; p b 0.001, for CP and Hp, respectively). As for Hp phenotypes, no significant differences were observed between iron store markers and ferroxidase activity among the control group. In the patients group however, significantly higher concentrations of ceruloplasmin and its ferroxidase activity were observed among patients with Hp2-2 phenotype as compared to patients with the other phenotypes. Additionally, correlations according to Hp phenotypes revealed strong association between ceruloplasmin ferroxidase activity and serum ferritin in patients with Hp 2-2 phenotype and not in the others (r = 0.331, p b 0.05). Conclusion: Thalassemia patients with Hp 2-2 phenotype are under greater iron-driven oxidative stress than patients with other phenotypes. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Life-long blood transfusion is a standard protocol used for the treatment and care of patients with β-thalassemia major. Although lifesaving, chronic blood transfusion however, results in iron overload with subsequent organ and tissue damages. Under normal conditions, about 35% of transferrin, the main transport protein, is saturated with iron. Accordingly, as iron loading progresses, transferrin iron binding capacity will be exceeded, and excess iron deposits in various organs and tissues. Furthermore, redox active fractions such as non-transferrin-bound iron (NTBI) and labile plasma iron (LPI) increase in circulation [1,2]. These fractions can catalyze the production of highly reactive oxygen species (ROS) and are associated with significant injury to the liver, heart, and endocrine organs [3,4]. The LPI fraction is the most redox active species that readily enters the cells, and represents the main target for iron chelation [5]. In β-thalassemia major, the status of iron overload and iron-induced oxidative stress had been intensively investigated [6–10].
⁎ Corresponding author. Tel.: +971 50 5469636; fax: +971 6 5057515. E-mail address: [email protected] (S.M. Awadallah). 0009-8981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2011.02.003
Increased levels of serum iron, serum ferritin, and transferrin saturation, and decreased total iron binding capacity (TIBC) are well documented in thalassemia patients. In addition, increased levels of the redox active fractions of NTBI and LPI had been demonstrated by many studies [2,6,7]. Other investigators demonstrated decreased antioxidant capacity and increased products of peroxidative damage in plasma of β-thalassemia patients [8–10]. Ceruloplasmin (CP) and haptoglobin (Hp) are other plasma proteins that are involved in iron homeostasis. CP is a copper-containing α2-globulin that is synthesized in liver and secreted in plasma. CP participate in several biological functions including copper transport, coagulation, angiogenesis, defense against oxidative stress as antioxidant, and iron homeostasis [11]. The antioxidant property of CP is attributed to its ability to prevent lipid oxidation, scavenging superoxide, and sequestering free copper ions [11–13]. Concerning its role in iron metabolism, many studies have demonstrated that CP possesses ferroxidase activity that plays a major role in the homeostasis of iron [14–16]. CP catalyzes the oxidation of the ferrous (Fe 2+) form of iron to the ferric (Fe3+) form, thus reducing the oxidant capacity of toxic ferrous iron by inhibition of the Fenton reaction. Furthermore, the ferroxidase activity of CP plays a major role in promoting the loading of
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iron from tissue stores and plasma onto transferrin [17,18]. The importance of CP in iron metabolism was clearly demonstrated by studies conducted on humans as well as animals that were deficient of ceruloplasmin [18–23]. These studies demonstrated massive iron deposition in a number of organs including brain and liver [18–20]. Further studies have demonstrated that CP enhances the efflux of iron out of hepatocytes [17,23], and that CP administered to CP knock-out mice can mobilize iron out of liver restoring normal iron homeostasis [22]. Collectively, these studies suggest that CP is required for efficient iron release from cells and tissues. Haptoglobin (Hp) is a polymorphic α2-glycoprotein that exists in three main phenotypes (Hp 1-1, Hp 2-1, and Hp 2-2) possessing different structural and functional properties [24]. In circulation, the main function of Hp is to bind with free hemoglobin (Hb) preventing loss of iron and kidney damage. It is thus considered a powerful antioxidant as a result of its ability to prevent hemoglobin-driven oxidative damage [24]. Many studies however, had demonstrated that the antioxidative property of Hp and the rate of clearance of free Hb from circulation are phenotype-dependent [25]. It has been demonstrated that Hp 2-2 phenotype has lower hemoglobin binding capacity as compared to the other phenotypes [26,27]. Furthermore, it has been observed that clearance of Hp 1-1-Hb complex is faster than that of Hp 2-2-Hb complex [25]. Therefore, it was postulated that subjects with Hp 2-2 phenotype are under greater oxidative stress as a result of increased redox active iron derived from hemoglobin [28,29]. Once Hp–Hb complex is formed, it is quickly removed from circulation and scavenged by the CD163 receptors of hepatocytes and tissue monocytes and macrophages [30]. Studies have demonstrated that internalization of the Hp–Hb complex by the CD163 receptors is more potent for the Hb–Hp2-2 complex than for Hb bound to Hp 1-1 or 2-1 [28,30]. Accordingly, it was suggested that individuals with Hp 2-2 phenotype are under greater oxidative stress as a result of higher iron accumulation within monocytes or macrophages [25,28]. Taken together, both Cp ferroxidase activity and the inheritance pattern of Hp appear to play a significant role in the homeostasis of iron. However, data related to the role of these two plasma proteins in relation to the status of iron store in patients with beta-thalassemia is very scarce, and not well documented. Therefore, this study was conducted to evaluate the possible interplay between iron overload on one hand and ceruloplasmin ferroxidase activity and haptoglobin phenotypes on the other, in a group of beta-thalassemia major patients. 2. Materials and methods The study involved 124 β-thalassemia major patients (69 males and 55 females) and 72 age- and sex-matched healthy controls (39 males and 33 females). Subjects under age of 8 years were not included in the study. Patients aged 8 to 30 years (mean 15.8 ± 5.2 years), of whom only 12 (9.6%) were above 25 years, were recruited from the thalassemia outpatient clinic at Al Rahma Hospital (Irbid, 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 all were on iron chelation therapy using either deferasiox (Exjade) or deferoxamine. Serum ferritin levels were assessed at least twice a year in thalassemia patients, and the mean results for the last 5 years were used for evaluation. Thirty-nine patients were hepatitis C virus (HCV) positive, and 29 had splenoctomy. Clinical data relevant to cardiac complications, endocrine disorders, or growth retardation were not documented and not involved in the study. Healthy controls aged 8 to 30 years (mean 16.2 ± 3.6 years) were selected from various outpatient clinics at the same hospital. None of the control subjects were thalassemia minors and none had any history of blood transfusion, anemia, liver disease, or active inflammatory conditions. All participants provided informed written consent, which was previously approved by the local research ethics committee.
Blood samples were collected in plain tubes. Thalassemia patients’ blood was collected just before blood transfusion. After clotting, serum was separated by centrifugation and divided into several aliquots and kept at −20 °C until they are analyzed. Serum iron, total iron binding capacity (TIBC), alanine transaminase (ALT), high sensitivity CRP (hs-CRP), and ceruloplasmin 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 [31]. Haptoglobin phenotypes were identified using polyacrylamide gel electrophoresis followed by peroxidase staining (Fig. 1) [32]. The Hp phenotype frequencies were directly calculated from the number of individuals expressing the specific Hp phenotype in both patients and controls. Percent of transferrin saturation was calculated by dividing serum iron by TIBC. Data analysis was carried out using the statistical software package of SPSS ver. 17.0 (SPSS Inc, Chicago, IL). Statistical comparison between groups was assessed by the Kruskal–Wallis test, and results were presented as mean ± SD. Spearman's correlation coefficients were calculated to study the association between relevant biochemical parameters in all investigated groups. Statistical significance was set at p b 0.05. 3. Results Table 1 demonstrates the general characteristics of subjects investigated. As expected, iron store parameters, except TIBC, were significantly higher in patients than in controls. Additionally, the levels of CP, ferroxidase, CRP and ALT were also significantly higher in patients than in controls. No significant differences related to age, gender, and the distribution haptoglobin phenotypes were observed. Furthermore, splenoctomy and/or HCV did not influence the frequencies of Hp phenotypes among the patients group. Table 2 shows the laboratory findings according to Hp phenotypes. Within the control group, no significant differences were observed in all parameters investigated. Among the patients’ group however, the levels of CP and ferroxidase activity were significantly higher in patients with Hp2-2 phenotypes as compared to those with the other phenotypes (pb 0.05 and b0.01, respectively). No significant difference was observed between the other parameter in relation to Hp phenotype. Spearman's correlation analysis was conducted to analyze the correlation between ferritin, ferroxidase, CP, and CRP among the thalassemia group. In all patients combined, no significant correlation was observed between ferroxidase and ferritin, however, significant correlation was observed between ferroxidase activity with CP and CRP (r = 0.330, p b 0.01 and r = 0.179, p b 0.05, respectively), and ferritin with CRP (r= 0.342, p b 0.01). Correlations according to Hp phenotype however, revealed strong correlation of
Fig. 1. Polyacrylamide gel electrophoresis of Hp phenotypes. The gel shows the electrophoretic patterns of the three Hp phenotypes (Hp 1-1, 2-1, and 2-2).
S.M. Awadallah et al. / Clinica Chimica Acta 412 (2011) 975–979 Table 1 General characteristics of patients and controls.
Age, year Male/female Hepatitis C, n (%) Splenoctomy, n(%) Serum iron, μg/dl TIBC, μg/dl Transferrin saturation, % Serum ferritin, μg/l CP, mg/dl Ferroxidase, U/l CRP, mg/dl ALT, U/l Hp phenotypes Hp 1-1, n (%) Hp 2-1, n (%) Hp 2-2, n (%)
Thalassemia (124)
Controls (72)
p value
15.8 ± 5.2 69/55 39 (31.5) 29 (23.4) 240.9 ± 63.0 266.4 ± 63.9 90.8 ± 8.9 2970 ± 1925 57.9 ± 18.8 159.9 ± 47.8 2.5 ± 1.8 41.9 ± 36.7
16.2 ± 3.6 39/33 None None 79.7 ± 29.8 325 ± 44.5 26.5 ± 12.2 35.0 ± 23.7 46.9 ± 14.2 95.3 ± 20.9 1.57 ± 1.5 10.9 ± 7.4
NS NS
0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
12 (9.7) 53 (42.7) 59 (47.6)
7 (9.7) 31 (43.1) 34 (47.2)
NS NS NS
Results are mean ± SD or as indicated in table. NS: No significant.
ferroxidase and CP with ferritin, only in patients with Hp 2-2 (r= 0.331, p b 0.05 and r = 0.344, p b 0.05, respectively) (Fig. 2). Additionally, significant correlation between ferritin and CRP was observed in all three Hp phenotypes, of which Hp 2-2 was the strongest (r= 0.519, p b 0.01). 4. Discussion Results from this study demonstrate for the first time a close association of haptoglobin phenotypes and Cp ferroxidase activity with the iron store status in patients with β-thalassemia major. The results revealed significantly higher serum levels of Cp and its ferroxidase activity in thalassemia patients as compared to healthy controls. Additionally, the levels of Cp and ferroxidase were higher among thalassemic patients with Hp 2-2 phenotype as compared to patients with the other Hp phenotypes. Furthermore, a significant correlation was observed between the levels of Cp and ferroxidase with the levels of serum ferritin among thalassemic patients with Hp 2-2 phenotype only. Many previous studies have demonstrated that most beta-thalassemia patients are under greater oxidative stress as evidenced by decreased plasma antioxidant capacity, increased markers of oxidative damage, and most of all, increased serum levels of redox active forms of NTBI and LPI [6–10]. This status of oxidative stress is attributed to the toxicity of iron overload as a consequence of life-long blood transfusions, increased intestinal absorption of iron, and to the greater intravascular hemolysis [33]. Based on the findings of high levels of serum iron, ferritin, transferrin saturation, and decreased TIBC, it is very
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likely that our patients were under oxidative stress too. Under such conditions of disturbed iron metabolism as well as of increased iron-derived oxidative stress, it is thus possible that the functional activity of Cp, which is a major plasma antioxidant, is altered in a way to oppose and counteract the consequences of iron overload. Firstly however, it should be mentioned here that the increased levels of CP observed in our study were not attributed to acute inflammatory condition. This is due to the fact that the levels of CRP were not high enough to indicate an active or acute inflammatory condition. Accordingly, the high serum levels of Cp and ferroxidase activity observed in our patients might be attributed to several iron overloadrelated mechanisms. First, it is possible that the levels were increased to prevent the formation of reactive oxygen species (ROS) through the Fenton reaction. This is achieved by the antioxidative capacity of Cp through the oxidation of Fe2+ to Fe3+, thus preventing oxidative damage of lipids, proteins and DNA [11]. Second, oxidation of the ferrous form of iron to the ferric form enhances its binding with transferrin and thus its transport to storage and/or utilization sites. Since our results indicated high transferrin saturation percent (N85%), it is also possible that Cp is increased to enhance the binding of iron to substances other than transferrin such as albumin and citrate, thus contributing to the formation of more redox active forms of iron [34]. Third, it is possible that Cp is increased in response to accumulated iron in tissues, thus enhancing efflux of iron into transferrin in plasma. Several studies have provided evidence supporting the function of Cp in mobilization of iron between tissues and plasma [15,17,18,23]. Studies on patients with aceruloplasminemia and hemochromatosis [18–20], demonstrated marked accumulation of iron in various tissues and organs such as liver and brain. The accumulated iron was attributed to the lack ferroxidase activity of Cp that is required for the efflux of iron from tissues to plasma. In addition, studies conducted on animals lacking Cp, demonstrated that administration of Cp restored iron balance in these animals [22]. Fourth, it is possible that Cp levels were increased in response to hypoxia and ineffective erythropoiesis. Results from studies conducted on patients with chronic pulmonary obstructive disease demonstrated significantly increased Cp levels, suggesting an important role of Cp in erythropoiesis under conditions of hypoxia [35]. Such conditions of tissue hypoxia and ineffective erythropoiesis are common features among thalassemic patients. Our results also demonstrated significant correlations between ferroxidase activity with each of CP and CRP, and serum ferritin with CRP in the patient group only. The correlation between ferroxidase and Cp is an expected one, however, the correlations between ferroxidase and CRP (r = 0.179, p b 0.05) and ferritin with CRP (r = 0.342, p b 0.001), might be attributed, in part, to a mild acute inflammatory condition in our patients. Interestingly, findings from this study also demonstrated an existing interplay between Cp and its ferroxidase activity and the
Table 2 Laboratory findings according to Haptoglobin phenotypes (mean ± SD). Controls (72)a
Thalassemia (124)
Age, year Serum iron, μg/dl TIBC, μg/dl Trans saturation,% Serum ferritin, μg/l CP, mg/dl Ferroxidase, U/l CRP, mg/dl ALT, U/l a b c
Hp 1-1 (12)
HP 2-1 (53)
Hp 2-2 (59)
Hp 1-1 (7)
HP 2-1 (31)
Hp 2-2 (34)
16.9 ± 5.3 255.2 ± 44.5 271.4 ± 45.4 94.3 ± 3.4 3682 ± 1459 50.8 ± 18.7 121.9 ± 25.9 2.9 ± 2.2 44.4 ± 41.6
16.0 ± 5.9 232 ± 61 253 ± 59 92 ± 9.8 2924 ± 2381 53.8 ± 17.2 133.8 ± 27.0 2.2 ± 1.6 40.8 ± 33.4
15.3 ± 4.6 246 ± 68 277 ± 69 89.2 ± 8.9 2868 ± 1438 62.9 ± 19.3b 191.0 + 46.7c 2.6 ± 1.8 42.6 ± 39.6
18.1 ± 2.5 75.3 ± 30.0 327 ± 38.5 24.7 ± 11.4 54.4 ± 33.6 42.3 ± 9.6 97.1 ± 12.7 2.7 ± 1.8 22.8 ± 18.6
16.8 ± 3.6 80.2 ± 31.1 318 ± 49.7 26.5 ± 13.4 31.6 ± 17.8 43.8 ± 11.6 94.7 ± 15.8 1.5 ± 1.2 9.9 ± 3.6
15.2 ± 3.5 80.1 ± 29.3 331 ± 40.9 25.0 ± 13.4 34.2 + 24.9 52.1 ± 15.4 104.3 ± 18.8 1.4 ± 1.5 9.4 ± 2.8
Except for age, all parameters of controls are significantly different from their counterparts of thalassemia. p b 0.05 for Hp 2-2 versus the other Hp phenotypes. p b 0.01 for Hp 2-2 versus the other Hp phenotypes.
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specific markers of iron overload such as NTBI, LPI, and intracellular ferritin, may need to be thoroughly investigated. Acknowledgments Hp 1-1: Hp 2-1: Hp 2-2:
Work of this study was supported by research grant # 4/2010 from the College of Graduate Studies and Research at the Hashemite University, Jordan. The authors wish to thank Sultan Medical Laboratories in Amman, Jordan, for their assistance in using their autoanalyzers. References
Fig. 2. Correlation of serum ferritin levels with Cp ferroxidase activity in beta thalassemia patients in relation to Hp phenotype. Correlation coefficient and p values: Hp 1-1: r = −0.441, p = 0.151; Hp 2-1: r = 0.061, p = 0.663; Hp 2-2: r = 0.331, p = 0.010.
inheritance pattern of haptoglobin in thalassemic patients. On one hand, the levels of Cp and ferroxidase were significantly higher in patients with Hp 2-2 phenotype than their counterparts, and on the other, the levels correlated significantly with serum ferritin in the same group of patients (Table 2 and Fig. 2). Such correlation however, was not observed when patients were combined (irrespective of Hp phenotype). Many studies have reported that Hp phenotypes are directly or indirectly involved in disease processes influenced by iron metabolism such as hemochromatosis, atherosclerosis and cardiovascular disease [25]. None however, had investigated the role of Hp phenotypes in relation to iron metabolism in beta-thalassemia major. The observed high levels of Cp and ferroxidase activity and their significant correlation with serum ferritin among Hp 2-2 thalassemic patients might be related to the poor antioxidative property of Hp 2-2 phenotype [24,26,27], which is mainly attributed to its low serum concentration and to its decreased affinity to free Hb in circulation [26,27]. In fact, serum levels of haptoglobin in chronic hemolytic anemias including beta-thalassemia are known to be severely reduced. Thus, it would be expected that free Hb and its associated heme-driven iron would be greatly increased in the circulation among individuals with Hp 2-2 phenotype. Accordingly, the increased serum levels of Cp and its ferroxidase activity observed in our Hp 2-2 patients might be the result of excess free Hb and heme iron in circulation. Furthermore, studies on the monocyte–macrophage CD163 receptor, which functions as Hb scavengers, demonstrated that these receptors have greater affinity to the Hb-Hp2-2 complex than Hb complexes formed with other phenotypes [25,30]. In this regard, studies on Hp 22 healthy individuals demonstrated potent internalization of heme iron, resulting in greater intracellular accumulation of ferritin as compared to the other Hp phenotypes [25,28]. Furthermore, studies on patients with hereditary hemochromatosis showed increased serum iron and ferritin among Hp 2-2 male patients [36]. Such situation is probably exaggerated in Hp 2-2 thalassemic patients, where excessive amounts of intracellular ferritin and probably labile iron do exist. Accordingly, Cp ferroxidase activity in Hp 2-2 patients may increase in attempt to enhance efflux and mobilization of iron from cells into plasma. In conclusion, results from this study suggest that iron-derived oxidative stress and Cp ferroxidase activity are affected by the Hp 2-2 phenotype inβ-thalassemia major. However, since this is the first time such findings were reported and no previous work to compare with, more confirmatory studies are needed to consolidate the findings. Therefore, assessment of such interplay, especially in relation to more
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