Plasma lipid profile and lipid peroxidation in beta-thalassemic children

Journal of Clinical Lipidology (2008) 2, 405– 409

Original Articles

Plasma lipid profile and lipid peroxidation in beta-thalassemic children Mona Ramadan Nasr, MD,* Awatef M. Abdelmaksoud, PhD, Kholooud Salah Eldin Abd El-Aal, PhD, Naglaa Abdul-Zaher Mabrouk, PhD, Wafaa Muhamed Ismael, MSc Pediatric Department, Ahmed Maher Teaching Hospital, Port Said Street, Cairo, Egypt (Dr. Nasr); Clinical Nutrition Department, National Nutrition Institute, Cairo, Egypt (Dr. Abdelmaksoud and Ms. Ismael); Faculty of Science, Ain Shams University, Cairo, Egypt (Drs. Abd El-Aal and Mabrouk) KEYWORDS: ␤-thalassemia; Children; Lipid peroxidation; Lipoproteins; Malondialdehyde

BACKGROUND: Beta-thalassaemic patients who usually have a combination of; chronic hemolytic anemia, iron storage disease, myocarditis, and premature death especially due to heart failure may also have increased oxidation of lipids and abnormal lipoprotein concentrations. OBJECTIVE: To determine plasma lipids, triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and lipid peroxidation product malondialdehyde (MDA) in children with ␤-thalassemia and unaffected control children. Relationships with age, gender, hemoglobin, serum iron, and serum ferritin were examined. Children with ␤-thalassemia (28 males and 15 females, aged 4 to 18 years) and 31 age- and gender-matched healthy controls were studied. RESULTS: In children with ␤-thalassemia, there was an increase in TG, MDA, and the MDA/ LDL-C ratio (P ⫽ .000) and a decrease in TC, LDL-C, HDL-C, and the LDL-C/ TG ratio (P ⬍ .001) compared to unaffected controls. The LDL-C/HDL-C ratio was not different. The MDA/LDL-C ratio was correlated negatively with blood hemoglobin and TC (P ⬍ .05 for each), whereas the LDL-C/ HDL-C ratio was correlated with age, weight, body mass index, and TC (P ⬍ .05, ⬍ .05, ⬍ .05, and ⬍ .01 respectively). CONCLUSION: Despite the derangement in plasma lipid profile in children with ␤-thalassemia accompanied by excess lipid peroxidation, the lipoprotein concentrations do not suggest increased risk. The MDA/LDL-C ratio may prove to be a valuable marker for lipid peroxidation. © 2008 Published by Elsevier Inc. on behalf of National Lipid Association.

Beta-thalassemic red blood cells with unpaired ␣-hemoglobin chains exhibit increased amounts of hemoglobin and iron. This excess hemoglobin/iron promotes per-oxidative damage to cell and organelle membranes through production of oxygen free radicals.1 Oxygen free radicals are

* Corresponding author. E-mail address: [email protected] Submitted October 10, 2008. Accepted for publication October 19, 2008.

capable of reversibly or irreversibly damaging compounds of all biochemical classes, including nucleic acids, proteins, lipoproteins, and lipids.2 Lipid abnormalities have been repeatedly reported in all phenotypes of patients with ␤-thalassemia. In homozygous ␤-thalassemia major, plasma total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C), and high density lipoprotein-cholesterol (HDL-C) were found to be low compared to their controls, whereas plasma triglycerides (TG) did not differ between subjects.3 In ␤-thalassemia intermedia, TC, LDL-C, and HDL-C were

1933-2874/$ -see front matter © 2008 Published by Elsevier Inc. on behalf of National Lipid Association. doi:10.1016/j.jacl.2008.10.008

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even lower and were not influenced by age, gender, hemoglobin, ferritin, or liver injury.4 In ␤-thalassemia minor TC and LDL-C were significantly lower compared to controls, whereas plasma TG and HDL-C did not differ between the two groups.5 LDL-C oxidation in vivo depends on plasma lipid composition. The ratios of LDL-C/TG and LDL-C/ HDL-C can be predictive parameters of LDL oxidation in vivo.6 Livrea et al7 suggested that the level of malondialdehyde (MDA) in ␤-thalassemia patients may represent a sensitive index of the oxidative status of LDL-C in vivo and of its potential atherogenicity. The aim of the present study is to determine TG, TC, HDL-C, LDL-C, the lipid peroxidation product, malondialdehyde (MDA), and lipid ratios of children with ␤-thalassemia. Of particular interest are the potential relationships between these parameters and the blood concentrations of hemoglobin, serum iron, and serum ferritin. We also examined the relationships among duration and frequency of transfusion, as well as age, sex, height, weight, and body mass index (BMI).

method of Fossti and Prencipe.10 Plasma LDL-cholesterol was calculated using Friedewald et al’s equation (plasma LDL-C ⫽ TC ⫺ [HDL-C ⫹ TG/5]).11 Iron concentration was measured in fresh serum using kits provided from BioSystems (Spain) according to Artiss et al12: serum ferritin was measured by microplate immunoenzymatic assay according to the method of Tielz.13 MDA was determined according to the method of Ohkawa et al.14 4. MDA/LDL-C, LDL-C/TG, and LDL-C/HDL-C ratios were calculated.

Methods Patients The present study included 43 children with ␤-thalassemia (as determined by history, physical examination, and laboratory investigations, including hemoglobin electrophoresis). All patients attended the thalassemia unit of the pediatric department of Ahmed Maher Teaching Hospital, Cairo, Egypt, between December 2004 and December 2005. Patients were 28 males and 15 females, between the ages of 4 and 18 years. All were receiving regular blood transfusion and desferrioxamine chelation therapy. Inclusion criteria were age from ⬎3 to ⱕ18 years and BMI ⬍90th percentile for age and gender. Exclusion criteria included diabeties mellitus, hepatitis B or C, and unstable liver and kidney function test results. Age- and gender-matched healthy children (n ⫽ 31) were enrolled as a control group. After obtaining verbal consent from the patients and controls or their guardians, all underwent the following procedures. 1. Recording of medical history, including the estimated number and frequency of blood transfusion, chelation therapy, and splenectomy, or drug intake that may have affected plasma lipids. 2. Physical examination with anthropometric measurements. 3. Laboratory analysis of venous blood samples after 12 hours of fasting and before blood transfusion. The hemoglobin concentration was measured in fresh heparinized blood and plasma aliquots were frozen at ⫺70°C until further determination of lipid levels, including TC according to Allan et al.8 HDL-cholesterol was measured by the method described by Burstein et al9 and TG by the

Statistical analysis The results were statistically analyzed using SPSS software (version 11; SPSS, Inc., Chicago, IL).The results were expressed as mean ⫾ standard deviation and the independent Student’s t-test was used to compare means. Bivirate correlation between variables using Pearson’s correlation coefficient r value was used. A calculated P value of ⬍ .05 was considered significant.

Results The mean ⫾ standard deviation age of the thalassemic group was 10.73 ⫾ 4.58 years; the male/female ratio was 28/15 compared to the control group, which consisted of 17/14, with a mean age 11.65 ⫾ 3.3 years without a statistically significant difference. There was also a nonsignificant difference between their weights and their BMI. The clinical data from the two groups studied are presented in Table 1. As expected, the serum concentrations of iron, ferritin, MDA, the MDA/LDL-C ratio, and TG were all significantly higher in the thalassemic group (P ⬍ .001). The concentrations of hemoglobin, TC, LDL-C, HDL-C, and the LDL-C/ TG ratio were significantly lower (Table 2) in the thalassemic group compared to the control group (P ⬍ .001). The LDL-C/HDL-C ratio did not differ significantly between the two groups (P ⫽ .432). The correlations of blood lipids, lipid ratios, and MDA with the other clinical parameters are shown in Table 3. TG was correlated inversely with LDL-C (P ⬍ .05). MDA was correlated positively with female gender (P ⬍ .05). The MDA/LDL-C ratio was correlated negatively with blood hemoglobin and TC (P ⬍ .05 for each), whereas the LDLC/HDL-C ratio was correlated with age, weight, BMI, and TC (P ⬍ .05, ⬍ .05, ⬍ .05, and ⬍ .01, respectively).

Discussion The results of our study revealed significantly lower mean values for the plasma concentrations of TC, LDL-C, and HDL-C in the thalassemic group compared to their

Nasr et al Table 1

407 Clinical data from beta-thalassemic children and controls

Clinical data

Groups studied

Age (y), range, mean ⫾ SD Male/female ratio Years requiring transfusions (mean ⫾ SD) Years of chelation Therapy (mean ⫾ SD) Frequency of blood Transfusion (mean ⫾ SD) Type and dose of chelation Therapy Height (cm), mean ⫾ SD Weight (kg), mean ⫾ SD BMI (mean ⫾ SD) No. with splenectomy Length of spleen below left costal margin (cm) (n ⫽ 30) Length of liver below right costal margin (cm)

Thalassemic group (n ⫽ 43)

Control group (n ⫽ 31)

4–18 10.73 ⫾ 4.58 28/15 8.07 ⫾ 3.67 6.26 ⫾ 3.72 8.49 ⫾ 2.8/y Desferroxamine 40–50 mg/kg, 5 ⫻/wk 128.02 ⫾ 21.1 31.13 ⫾ 14.93 17.95 ⫾ 3.246 13 patients (30%) 5.95 ⫾ 0.89

5–18 11.65 ⫾ 3.33 17/14 None None None None

4.71 ⫾ 0.9

Difference (P value) .34 NS ⬍ .001 ⬍ .001

137.3 ⫾ 16.35 33.1 ⫾ 11.64 17.05 ⫾ 2.22 None 0

⬍ .001

0

⬍ .001

0.043 0.544 0.173

BMI, body mass index; SD, standard deviation.

controls (P ⱕ .001). Hypocholesterolemia has been described in various hematologic disorders, including thalassemia major15,16 and thalassemia intermediate.17 Several mechanisms for hypocholesterolemia in those patients have been proposed: plasma dilution due to anemia, increased cholesterol requirement associated with erythroid hyperplasia, macrophage system activation with release of cytokines,

Table 2

increased cholesterol uptake by reticuloendothelial system and liver injury secondary to iron overload.16,17 In thalassemia minor, lower TC and LDL-C has been reported and attributed to increased uptake of LDL-C by macrophages and histiocytes of the reticuloendothelial system.5 Hartman et al17 and Goldfarb3 have described similar findings. Unlike our study, these previous publications did not report

Laboratory data of the thalassemic group compared to their controls

Lab value*

Groups studied

Whole blood hemoglobin (gm/dl) S. iron (␮g/dl)† S. ferritin (ng/ml)† TG (mg/dL) * TC (mg/dL) HDL-C (mg/dL) LDL-C (mg/dL) MDA (nmol/mL) MDA/LDL-C Ratio LDL-C/TG Ratio LDL-C/HDL-C Ratio

Thalassemic group (n ⫽ 43)

Control group (n ⫽ 31)

Difference P value

5–9.1 7.07 ⫾ 0.92 50–288 125.7 ⫾ 36.1 190–1080 842 ⫾ 479.6 87–178 156 ⫾ 18.7 84–137 100.8⫾13.7 14.6–39.7 27 ⫾ 6 13.1–79.1 37.2 ⫾ 16.1 4.7–35 12.28 ⫾ 6.89 0.1–1.4 0.383 ⫾ 0.307 0.07–0.54 0.205 ⫾ 0.102 0.33–3.71 0.693

11.3–14.6 12.4 ⫾ 0.73 15–82 40.3 ⫾ 15.6 3–37 21.9 ⫾ 12.23 52–99 89 ⫾ 25.5 121–199 155.2 ⫾ 18.6 29–80 49.15 ⫾ 10.5 47–99 73.4 ⫾ 17.3 0.5–3.8 2.04 ⫾ 0.62 0.01–0.23 0.037 ⫾ 0.04 0.28–1 0.502 ⫾ 0.177 0.6–2.75 1.59 ⫾ 0.572

⬍ .001 ⬍ .001 ⬍ .001 ⬍ .001 ⬍ .001 ⬍ .001 ⬍ .001 ⬍ .001 ⬍ .001 ⬍ .001 .432

HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; MDA, malondialdehyde; S., serum; TC, total cholesterol; TG, triglycerides. *Range of values in group and group mean value ⫾ SD. †Measured in serum. Other values measured in heparinized plasma.

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Table 3 Pearson correlation coefficients assessed between parameters measured in cohort of children with ␤-thalassemia (n ⫽ 43)

Female gender Age Years of transfusion Frequency of transfusion Years of chelation chelation Height Weight BMI HB S. iron S. ferritin MDA TG TC HDL-C LDL-C MDA/ LDL-C LDL-C/ TG LDL-C/ HDL-C

Vs TG

Vs TC

Vs HDL-C

Vs LDL-C

0.125 ⫺0.036 ⫺0.027 0.202

0.023 0.138 0.119 ⫺0.125

0.016 ⫺0.281 ⫺0.234 0.043

0.047 0.210 0.182 ⫺0.210

⫺0.079

0.089

⫺0.246

0.183

⫺0.122 ⫺0.089 ⫺0.077 0.109 0.109 0.257 0.07 1 ⫺0.099 ⫺0.015 ⫺0.304* 0.287 ⫺0.523† ⫺0.216

0.163 0.228 0.226 0.071 ⫺0.033 0.119 0.236 ⫺0.099 1 0.259 0.877† ⫺0.402* 0.822† 0.633†

⫺0.195 ⫺0.141 ⫺0.103 0.029 0.031 0.048 0.033 ⫺0.015 0.259 1 ⫺0.100 0.061 ⫺0.044 ⫺0.518†

0.216 0.251 0.259 ⫺0.053 ⫺0.085 0.041 0.207 ⫺0.304* 0.877† ⫺0.100 1 ⫺0.460† 0.955† 0.865†

Vs MDA

Vs MDA/LDL-C

Vs LDL-TC/TG

Vs LDL-C/HDLC

0.229 0.049 ⫺0.010 0.014

0.027 0.167 0.149 ⫺0.235

0.018 0.307* 0.258 ⫺0.112

⫺0.097

0.061

0.107

0.250

0.154 0.154 ⫺0.004 ⫺0.123 0.286 0.117 1 0.07 0.236 0.033 0.207 0.224 0.170 0.138

⫺0.014 ⫺0.101 ⫺0.215 ⫺0.374* 0.104 ⫺0.052 0.224 0.287 ⫺0.402† 0.061 ⫺0.460† 1 ⫺0.469† ⫺0.425†

0.200 0.251 0.287 ⫺0.035 ⫺0.089 ⫺0.017 0.170 ⫺0.523† 0.822† ⫺0.044 0.955** ⫺0.469† 1 0.807†

0.289 0.311* 0.312* 0.049 ⫺0.068 0.058 0.138 ⫺0.216 0.633† ⫺0.518† 0.865† ⫺0.425† 0.807† 1

0.343* 0.287 0.154 0.173

HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; MDA, malondialdehyde; S., serum; TC, total cholesterol; TG, triglycerides. Data are presented as r values. *Significant at P ⬍ .05. †Significant P ⬍ .01.

correlations of plasma TC, LDL-C, and HDL-C with age, gender, blood hemoglobin, duration of transfusion, frequency of transfusion, or serum ferritin. These low levels of TC and LDL-C may protect patients with ␤-thalassemia against coronary heart disease.18 However, HDL-C, which has antioxidant action and may play a protective role against LDL-C oxidation,6 was found to be significantly lower in our patients compared to their controls (P ⬍ .001). Shalev et al19 reported similarly low HDL-C in ␤-thalassemia. This reduced concentration of HDL-C may contribute to the oxidative capacity of blood and along with excess serum iron and ferritin may underly the highly significant increase of MDA in our patients with ␤-thalassemia. This process may also worsen the anemia of ␤-thalassemia by altering the ␣-hemoglobin chains that are more prone to denaturation and oxidation.20 In our study, there was a nonsignificant correlation between MDA, MDA/ LDL-C ratio, and HDL-C, but the study of Kondo et al21 found a negative correlation between HDL-C and the MDA/ LDL-C ratio in patients with high TG concentrations. TG in our patients were significantly higher than the controls (P ⬍ .001). This elevation has been postulated to be caused by a compromise in lipolytic activity due the hepatic disease.22 The plasma TG was negatively correlated with LDL-C (P ⬍ .05) but did not correlate with HDL-C. The MDA/ LDL-C ratio was correlated negatively with blood hemoglobin (P ⬍

.05); the cause may be the decrease of LDL-C in cases of chronic anemia as suggested by Shalev et al.19 The LDL-C/ TG ratio has been suggested to be an important predictor of LDL oxidation.6 This ratio in our patients was significantly decreased compared to our controls (P ⫽ .000) and negatively correlated with the MDA/LDL-C ratio (P ⬍ .01). This result may support the hypothesis of Brizzi et al23; that serum iron and TG are involved in the pathogenesis of LDL-C oxidation. A high LDL-C/HDL-C ratio with high plasma TG is associated with very high risk of CAD in adult patients.24 In our children with ␤-thalassemia, TG was increased, but the LDL-C/HDL-C ratio did not differ significantly from children with normal hemoglobin (P ⫽ 0.432), suggesting that the coronary artery diseases risk related to lipoprotein measures may not be elevated in our thalassemic children. This LDL-C/HDL-C ratio was correlated significantly with age, weight, and BMI (P ⬍ .05 in each group) and with LDLC/TG and TC (P ⬍ .01 in each).

Acknowledgment The work of this study is a part of PhD thesis of the biochemist, Wafaa M. Ismael, a member of the National

Nasr et al Nutrition Institute (NNI) [GOTHI] under supervision of the rest of the authors. We acknowledge the support of the Pediatric Department, Ahmed Maher Teaching Hospital, NNI.

References 1. Naithani R, Chanadra J, Bhattachrjee J, Verma, P, Narayan S. Peroxidative stress and antioxidant enzymes in children with beta-thalassemia major. Pediatr Blood Cancer. 2006;46:780 –785. 2. Cross CE, Halliwell B, Borish ET, et al. Oxygen radicals in human disease. Ann Intern Med. 1987;107:526 –545. 3. Goldfarb AW, Rachmilewitz EA, Eisenberg S. Abnormal low and high density lipoprotein in homozygous beta-thalassaemia. Br J Haematol. 1991;79:481– 486. 4. Amendola G, Danise P, Todisco N, et al. Lipid profile in beta-thalassemia intermedia patients: correlation with erythroid bone marrow activity. Int J Lab Hematol. 2007;29:172–176. 5. Maioli M, Pettinato S, Cherchi GM, et al. Plasma lipid in betathalassemia minor. Atherosclerosis 1989;75:245–248. 6. Brizzi P, Tonolo G, Carusillo F, et al. Plasma lipid composition and LDL oxidation. Clin Chem Lab Med. 2003;41:56 – 60. 7. Livrea MA, Tesoriere A, Maggio D, et al. Oxidative modification of low-density lipoprotein and atherogenetic risk in ␤-thalassemia. Blood 1998;92:3936 –3942. 8. Allan C, Deacon ET, Peter JG, et al. Determination of total cholesterol. Clin Chem. 1979;25:976 –984. 9. Burstein M, Scholink HR, Morfin R. Rapid method for the isolation of lipoproteins from human serum by precipitation with polyanions. Scand J Clin Lab Invest. 1980;40:583–595. 10. Fossati P, Prencipe L. Determination of triglycerides. Clin Chem. 1982;28:2077–2080. 11. Friedewald WT, Levy RI, Fredriek-San DS. Estimation of concentration of low density lipoprotein separated by three methods. Clin Chem. 1972:28:2077.

409 12. Artiss JD, Vinogradov S, Zak B. Spectrophotometeric study of several sensitive reagents for serum iron. Clin Biochem. 1981;14: 311–315. 13. Tielz, N. Textbook of clinical chemistry. 3rd Ed. Philadelphia, PA: WB Saunders, 1999. 14. Ohkawa H, Ohishi W, Yagi K. Determination of lipid peroxidation by MDA. Anal Biochem. 1979;95:351. 15. Wasterman MP. Hypocholesterolemia and anaemia. Br J Haematol. 1975;31:87–94. 16. Papanastasiou DA, Siorokou T, Haliotis FA. Beta-thalassemia and factors affecting the metabolism of lipids and lipoproteins. Haematologia (Budap) 1996;27:143–153. 17. Hartman C, Tamary H, Tamir A, et al. Hypocholesterolemia in children and adolescents with B-thalassemia inermedia. J Pediatr. 2002; 141:543–547. 18. Calandra S, Bertotini S, Pes GM, et al. Beta-thalassemia is a modifying factor of the clinical expression of familial hypercholesterolemia. Semin Vasc Med. 2004;4:271–278. 19. Shalev H, Kapelushnik J, Moser A, Knobler H, Hannah T. Hypocholesterolemia in chronic anemia with increased erythropoitic activity. Am J Hematol. 2007; 82:199 –202. 20. Scott MD, van den Berg JJ, Repka T, et al. Effect of excess alphahemoglobin chains on cellular and membrane oxidation in model beta-thalassemic erythrocytes. J Clin Investig. 1993; 91:1706 –1712. 21. Kondo A, Li J, Manabe M, et al. Relationship between high-density lipoprotein- cholesterol and malondialdehyde-modified low-density lipoprotein concentration. J Atheroscler Thromb. 2003;10:72–78. 22. Cherchi GM, Baggi MA, Coinu R, et al. Post heparin lipase activity in ␤-thalassemia major: Preliminary data. Boll Soc Ital Biol Sper. 1983; 59:1739 –1743. 23. Brizzi P, Isaja T, D’Agata A, et al. Oxidized LDL antibodies (OLAB) in patients with beta-thalassemia major. J Atheroscler Thromb. 2002; 9:139 –144. 24. Lemieux I, Lamarche B, Couillard C, et al. Total cholesterol/HDL cholesterol ratio vs LDL cholesterol / HDL cholesterol ratio as indices of ischemic heart disease risk in men. Arch Intern Med. 2001;161: 2685–2692.