VOLUME II No.1. Jan-June 2005 And Oxidative Stress In 11 β-thalassemia Patients In Jakarta Research Article Blood Lipids And Oxidative Stress In β-thalassemia Patients In Jakarta

Freisleben HJ. 3, Hidayat J. 3 , Handayani S. 3 , Udyaningsih-Freisleben S.K.3 , Kurniati V.3 , Adhiyanto C. 3 , Laksmitawati D.R. 3 , Kusnandar S. 2 , Dillon H.S.D.4 , Munthe B.G. 1 , Wirawan R.2 , Soegianto R.R.3 , Ramelan W3.

Thalassemia Center1 and Clinical Pathology2 , Cipto Mangunkusumo General Hospital, Study Program Biomedical Science3, and SEAMO- TROPMED Nutrion4, Faculty of Medicine, University of Indonesia, Salemba Raya No.4, Jakarta, Indonesia

ABSTRACT A study on thalassemia patients in Jakarta was initiated to obtain a comprehensive picture of metabolic dysregulation, iron overload, oxidative stress, and damage. This paper focuses on blood lipids from a group of 12 transfusion-dependent patients in an age range of 11-25 years (T) an another group of 9 frequently transfused (for at least 15 years) patients aged 17-30 years (L). A third group comprises 6 pateients (aged 4 to 14 years) who had not yet obtained transfusion (N). The 20 controls (C) were voluntary students without diagnosis or clinical signs of thalassemia up to 30 years of age. The hematological result can be summarized that non-transfused thalassemia intermedia patients exert slight signs of oxidative stress, and increased hemoglobin degradation but no significant indication of tissue or cell damage. This picture differs considerably from transfusion-dependent thalassemia major patients and is even worse in long-term transfused patients : highly significant decrease in antioxidants and thiols and tremendous iron overload and cell damage. Total , HDL, and LDL are decreased, whereas and ratios of total cholesterol or LDL to HDL are higher than in controls. Malondialdehyde, a byproduct of peroxidation increases from controls < N < T < L. The relation between plasma lipids and red cell membranes is discussed. Keywords : thalassemia, blood lipids, oxidative stress. ZUSAMMENFASSUNG Eine Studie mit Thalassämiepatienten in Jakarta wurde intiiert, um ein umfassendes Bild von metabolischer Dysregulation, Eisenüberladung, oxidativen Stress und Zellzerstörung zu erhalten. Diese Arbeit beschäftigt sich mit den Blutlipiden einer Fruppe von 12 transfusionsabhängigen Patienten im Alter von 11-25 Jahren (T), und einer weiteren Gruppe von 9 häufig (mindestens 15 Jahre) transfundierten Patienten im Alter 17-30 Jahren (L). Eine dritte Gruppe umfasst 6 Patienten im Alter von 4-14 Jahren, die noch keine Transfusionen erhalten hatten (N). Als Kontrolle (C) dienten 20 freiwillige Studenten bis zu einem Alter von 30 Jahren ohne Diagnose oder klinische Symptome der Thalassämie. Die hämatologischen Ergebnisse können dahingehend zusammengefasst warden, dass nicht transfundierte thalassaemia intermedia Patienten leichte Zeichen von oxidativem Stress und erhöten Hämoglobinabbau haben, jedoch keine signifikanten Anzeichen von Gewebe-oder Zellzerstörung. Dieses Bild unterscheidest sich erheblich von dem der transfusions- abhängigen thalassaemia major Patienten und ist noch schlechter bei den langzeittransfundierten Patienten: ein hochsignifikanter Verlust an Antioxidantien und Thiolen im Serum sowie enorme Eisenüberladung und Zellzerstörung. Totales Cholesterin, HDL und LDL sind erniedrigt, während und das Verhältnis von totalem Cholesterin bzw. LDL zu HDL höher sind als in der Kontrollgruppe. Malondialdehyde, das bei der Lipdperoxidation entsteht, steigt von der kontrolle < N < T < L. Die Beiziehung zwischen Plasmalipiden und Erythrozytenmembranen wird diskutiert.

Introduction inheritance of different kinds of thalassemias and related Thalassemia are inborn disordes of the globin chain hemoglobin anomalies. The influence of the globin haplotype synthesis and the most frequent single gene derived inherited on the clinical expression of thalassemias has been discussed, 3 diseases in the world. More than 3 % of the worlds population elsewhere. carry thalassemia genes, with highest incidence up to 40% in The clinical picture of thalassemias is dominated by South East Asia (αo-thalassemia 30 mill., β-thalassemia 60 mill., chronic anemia caused by ineffective erythropoesis, intra- and HbE/β-thalassemia 84 mill.).1 Various mutations and deletions extramedullary hemolysis, and by iron overload following i) on – and the - globin gene clusters (more than 100 in the β- increased intestinal iron absorption non-transfused thalassemic and about 200 in the β-gene are known) diminish globin chain patients and ii) blood supply to transfusion-dependent patients. synthesis to different extent and result in thalassemia trait or Imbalance excessive globin chains tend to precipitate near the clinical pictures of thalassemia. In the Indonesian population a red blood cell membrane causing either intramedullary cell frequency of –globin genes of about 6-10% is supposed.2 The β- death or increased clearance of the erythrocyte from the thalassemic phenotype shows wide variations between peripheral circulation. Hemochromes, heme (protoporphyrin), transfusion-dependent thalassemia major, different severities and released iron contribute to the damage conferred by the 4,5 of thalassemia intermedia and thalassemia minor or trait and is precipitated globin chains (inclusion bodies). determined by the combination of two different alleles and co- 12 Freisleben HJ DIGM Medical of Journal

Transition metals such as iron, and some iron-containing low measured in the SEAMO-TROPMED Nutrition laboratory molecular weight compounds play an important role in the according to Livrea et al. (1996).10 and thiols were generation of reactive oxygen species (ROS) and free radicals, determined as described elsewhere.11 All other hematological which are supposed to damage cellular and subcellular parameters were performed from citrate blood (10 mL) structures and cause metabolic dysfunction.6-8 Inter alia, the according to standard methods in the Laboratory of Clinical disturbed iron generates oxygen-derived free Pathology.9,11 radicals in thalassemias. This condition, together with impaired natural factors and mechanisms involved in detoxification of Isolation and preparation of red cell membranes, SDS- ROS and free radicals, results in extensive oxidative stress.9,10 PAGE, membrane labeling, and EPR-meassurements were In the frame of this study on thalassemia intermedia accomplished as described.12 and major patients in Jakarta to obtain a comprehensive picture of metabolic dysfunction and cell damage, we now Statistical Evaluation report on blood lipids and lipid peroxidation. Mean values, standars deviations and correlations were calculated using MIcroscoft Excell Statistics and for probability calculation ANOVA and STATS ’98, internet version Materials and Methods 1.1, Decision Analyst, Inc. were used ; p < 0.05 was considered 13 Subjects statistically significant and p < 0.01 higly significant. Twelve transfusion-dependent and not regularly chelated β-thalassemia major patients from Cipto Result Mangunkusumo General Hospital, in an age range of 11 to 25 Blood and hemoglobin analyses, serum , years were investigated (group T). Nine additional patients especially , and thiol status, metabolites (aged 17-30 years) had been regularly transfused for at least 15 and urate, iron status; total (SI), total iron binding years (long-term transfused, group L). generally, deferoxamine capacity (TIBC), unsaturated iron-binding capacity (UIBC), (desferal) chelation is applied once together with each transferring saturation, and as well as antioxidants, transfusion. Only few patients, who can afford desferal vitamin A, C, E, and β-carotene were measured and have been 9,11 privately, receive chelation theraphy more often. Twenty published previously. Here, the values are just listed in Table controls (group C) were voluntary students without diagnosis or I and Table II, and only mentioned in the text if relevant to clical signs of thalassemia in the same age range (19-30 years). understanding. Since it was difficult to find thalassemia intermedia patients who had not yet been transfused in the same age range as in Serum lipids the two other groups, the age range of group N (six non- Relative to controls, triglycerides are between 1.5-and transfused patients) was from 4 to 14 years. The groups had 2-fold in thalassemic patients (C = 116, N = 156, T = 184, L = 180 random sex distribution. All participants were non-smokers, did [mg/dL]), whereas total cholesterol, LDL, and HDL are lower in not suffer obviously from other metabolic diseases or acute patients (total cholesterol, C = 184, N = 164, T = 105, L = 132 infection, and indicated not to receive specific vitamin [mg/dL]; LDL, C = 104, N = 78, T = 50, L = 79 [mg/dL]; HDL, C = supplementation or medication other than the above 56, N = 41, T = 22, L = 20 [mg/dL]). The LDL/HDL ratio and the transfusion and chelation. ratio of total cholesterol to HDL increase from controls (1.8 and The study was approved by the Ethical Clearance 3.3, respectively) via N (1.9; 4.0) and T (2.3; 4.8) to long-term Board of the Faculty of Medicine, University of Indonesia and transfused patients (3.9; 6.6). Strongly correlated with the all blood samples from controls and patients were obtained on years of transfusions are total cholesterol with the years of fully informed consent. Citrate blood (20 mL) and transfusions are total cholesterol (r=0.89) and LDL-cholesterol ethylenediaminetetraacetate (EDTA) blood (5 mL) were (r=0.91). collected from each subject. From transfusion-dependent Peroxidation products of (poly)-unsaturated fatty acids patients blood samples were collected just before they in serum and membrane lipids such as thiobarbiturate-reactive received a new transfusion. Further information about the substance (TBARs) (e.g. malondialdehyde, MDA) are < 2.0 patients, transfusion and chelation regimens, how long the nmol/mL in controls, 5.0 nmol/mL in non-transfused, 8.7 patients had already been transfused and clinical status etc. nmol/mL in transfused and 9.6 nmol/mL in long-term were taken from the patients’ ward records at Cipto transfused patients. Mangunkusumo General Hospital in Jakarta.

Blood Analysis Hemoglobin (Hb)-screening was performed in the laboratory of the Thalassemia Ward with a VARIANT Hemoglobin Testing System. Lipid-soluble antioxidants were

Volume II No. 1 Jan-June 2005 Blood Lipids And Oxidative Stress In 13 β-thalassemic Patients In Jakarta

Table I : General hematological parameters Group C ± SD N ± SD T ± SD L ± SD Clin. Status Control Th.interm. Th.major Th.major Transfusion None Not yet Regular >15 years Age (years) 19-30 7-14 11-25 17-30 Number N = 20 N = 6 N = 12 N = 9 Blood & hemoglobin analyses Parameter C ± SD N± SD T ± SD L ± SD RBC [106/µL] 5.1 ± 0.58 2.8 ± 1.13 3.0 ± 0.54 3.0 ± 0.59 Significance ** ** ** HCT [ % ] 43.5 ± 5.9 20.7 ± 8.0 21.3 ± 3.9 23.3 ± 4.5 Significance ** ** ** Hb [g/dL] 14.9 ± 1.68 6.6 ± 2.83 7.3 ± 1.23 8.0 ± 1.49 Significance ** ** ** WBC [109/L] 7.8 ± 2.1 8.5 ± 3.7 9.8 ± 3.18 28.5 ± 19.3 Significance n.s. * ; oo ** ; # Lympho [%] 32 ± 9.29 45 ± 15.34 46 ± 16.5 62 ± 13.4 Significance * ** ; o ** Mono [%] 6.2 ± 3.27 10 ± 7.0 5.9 ± 1.98 6.2 ± 2.3 Significance n.s. n.s. n.s. Neutro [%] 61.4 ± 10.57 45.3 ± 14.8 48.3 ± 15.3 31.6 ± 14.3 Significance ** ** ** Platelet [109/L] 261 ± 31 482 ± 231 326 ± 205.5 388 ± 150.8 Significance ** ** ** Protein [g/dL] 7.8 ± 0.41 6.9 ± 1.18 7.5 ± 1.27 7.9 ± 0.79 Significance ** n.s. n.s. Albumin [g/dL] 4.6 ± 0.32 4.3 ± 1.45 4.2 ± 0.54 4.3 ± 0.35 Significance n.s. ** * Globulin [g/dL] 3.2 ± 0.46 2.7 ± 0.55 3.4 ± 0.61 3.6 ± 0.6 Significance + n.s. * ; # SGOT [IU/L] 39.6 ± 6.83 59.4 ± 24.95 88.9 ± 25.2 111.6 ± 44 Significance ** ; + ** ** ; # SGPT [IU/L] 32.4 ± 12.18 46.7 ± 13.9 81.2 ± 34.8 99.3 ± 28.4 Significance * ; + ** ** ; # # C-GT [IU/L] 20.3 ± 5.94 19.5 ± 11.1 42.3 ± 25.0 60.0 ± 25.1 Significance + ** ** ; # # Prot-SH [nmol/mg] 2.0 ± 0.3 1.3 ± 0.87 0.9 ± 0.53 1.1 ± 0.86 Signif. * ** ** Tot.bili.[mg/dL] 0.6 ± 0.21 1.2 ± 0.64 1.9 ± 0.7 1.8 ± 0.37 Significance **;++ ** **; # Con.bili.[mg/dL] 0.18 ± 0.07 0.5 ± 0.3 0.6 ± 0.29 0.6 ± 0.2 Significance ** ** ** Urate [mg/dL] 262 ± 104.34 260 ± 83.94 250 ± 88.9 297 ± 50.4 Significance n.s. n.s. n.s. Prot-SH [nmol/mg] 2.0 ± 0.3 1.3 ± 0.87 0.9 ± 0.53 1.1 ± 0.86 Significance * ** ** Ser. Iron [µg/dL] 84.7 ± 29.85 76.0 ± 31.39 143.9 ± 41.3 179.9 ± 31.1 Sigmificance + + ** ; o ** ; # # TIBC [µg/dL] 295 270 260 250 UIBC [µG/dL] 210 194 116 71 Transf.sat. [%] 37 28 65 76 Ferritin [ng/mL] 95 ± 49.93 191 ± 77.62 3907 ± 3537 5969 ± 5009 Significance ** ; + ** ** ; # Carotene [µmol/L] 0.4 ± 0.15 0.4 0.3 ± 0.29 0.2 ± 0.22 Significance Only 1 value n.s. ** Vit.A [µmol/L] 1.9 ± 0.45 1.3 ± 0.98 1.2 ± 0.44 1.1 ± 0.4 Significance n.s. ** ** Vit E [µmol/L] 21.8 ± 6.48 13.7 ± 9.69 8.9 ± 6.8 6.2 ± 4.5 Significance * ** ** Vit.C [µmol/L] 153.6 ± 48.04 80.0 ± 60.3 62.3 ± 42.1 58.9 ± 44.3 significance ** ** ** Blood & hemoglobin analyses Group C ± SD N ± SD T ± SD L ± SD Legend to table I and Table II : C, control; Clin. Status Control Th.interm Th.major Th.major N,not yet transfused ; T, transfusion- Transfusion None Not yet Regular >15 years Age (years) 19-30 7-14 11-25 17-30 dependent; L, regularly transfused for at Number N = 20 N = 6 N = 12 N = 9 least 15 years (= long-term transfused); ± Triglycer. [mg/dL] 156 ± 55.5 184 ± 47.4 180 ± 42.5 116 ± 51.57 Significance ** + * ** SD, standard deviation; clin. Status, clinical Tot.chol [mg/dL] 164 ± 91.4 105 ± 30.6 132 ± 21.5 184 ± 30.42 Significance + ** ; o ** status; Th.interm., thalassemia intermedia; LDL [mg/dL] 78 ± 31.5 50 ± 35.2 79 ± 33.98 104 ± 27.02 significance, p < 0.05, significant; p < 0.01, Significance n.s. ** * HDL [mg/dL] 41 ± 15.5 22 ± 8.8 20 ± 2.9 highly significant; * , significant vs. C, **, 56 ± 17.0 Significance + + ** ** ; # # highly significant vs.C; +, significant N vs T, LDL/HDL 1.8 1.9 2.3 3.9 Tot.chol/HDL 3.3 4.0 4.8 6.6 + + highly significant; o, significant L vs T; #, MDA [mol/L] 5.0 ± 4.18 8.7 ± 6.0 9.6 ± 8.1 1.96 ± 1.46 Significance ** ** ** significant L vs N, # #, highly significant L vs. N; for further explanations, see text. 14 Freisleben HJ DIGM Medical of Journal

Discussion We investigated four groups, three of them (controls, patterns had also been reported by others in thalassemia transfusion-dependent and long-term transfused patients) in patients,14-15 e.g., hypertriglyceridemia in an 11-month-old the age range of 11 to 30 years; the group of not yet transfused female infant in association with homozygous beta-thalassemia patients was between 7 and 14 years of age. It is very difficult and attention was drawn to a possible alteration of lipid to find not yet transfused thalassemia intermedia patients in metabolism in association with thalassemia major by Ameri et Indonesia older than 20 years. al. (1997).16 In a greek study, no significant abnormalities were Plasma lipids are imbalance in our patients: found in the majority of β-thalassemia major patients triglycerides are higher and total cholesterol, HDL, and LDL investigated, except for low HDL-cholesterol.17 lower than in controls. Abnormal serum lipid and A very detailed study was presented by Maioli et al ., (compared with noncarriers) have an increase of blood (1997)18 ; patients with homozygous β-thalassemia showed an reticulocytes and plasma levels of interleukin-6 would support abnormal lipoprotein profile with significantly lower plasma these hypotheses.19 levels of total-cholesterol, LDL-cholesterol, HDL-cholesterol, In our study, LDL to HDL as well as total cholesterol to apo A-I, apo B and higher triglyceride concentration than β- HDL ratios increase from controls < non-transfused < thalassemia trait carriers or controls. All lipoprotein subclasses transfused < long-term transfused patients. High total were triglyceride-enriched, while LDLs were also protein- cholesterol to HDL ratio was also reported from Greek enriched and HDLs protein-depleted. 18 thalassemia major patients study mainly due to low HDL- Thalassemia trait carriers disclosed a small but cholesterol.17 significant reduction in apo A-I and apo B plasma levels but only Summarizing literature and our own results it can be minor lipoprotein abnormalities with respect to the controls. stated that cholesterol plasma levels in b-thalassemic patients Lipoprotein composition of bone marrow-transplated are lower than in healthy controls and triglycerides are higher. homozygous The altered lipid pattern can be found in all subclasses of β-thalassemic patients was intermediate between plasma lipids, most prominent in HDL leading to increased LDL homozygous beta-thalassemia and normal subjects. No Lp(a) to HDL or total cholesterol to HDL ratios. This picture increased plasma level modification could be detected in thalassemia trait from trait via heterocygotes (intermedia) and bone marrow carriers and apo(a) plasma levels did not differ among the transpalanted homocygotes (major) to homocygotes (major) groups. However, a higher prevalence of ‘small’ apo(a) isoforms patients, in which latter group it furthermore increases with was present in homozygous β-thalassemic patients.18 Maioli et number of transfusions (group T < group L). Several al., (1997) suggested that an altered hepatic apo(a) synthesis or mechanisms have been discussed in literature for the lower LDL catabolism due to cirrhosis and to diminished or total cholesterol levels and bone marrow and liver appear to glycation may be involved.18 be involved in altered thalassemic lipid synthesis on the In β-thalassemia tarit carriers a partially improved grounds of pathoanatomy and pathobiochemistry of these cardiovascular risk profile was apparent (low hematrocrit, low organs. Moreover, increased LDL catabolism was discussed, LDL-cholesterol and apo B), thus justifying the claim for a low especially by means of RES components. prevalence of ischemic heart disease. Also in asymptomatic What are the influences of theses facts on the heterozygotes the lipid pattern was less markedly affected and patients? In mild forms of thalassemia, especially trait, low related to a diminished cardiovascular risk.18 cholesterol seems to decrease the cardiovascular risk. This Deiana et al (2000)19 investigated whether the LDL- effect is most obvious in patients with certain forms of familial lowering effect of the βo-thalassemia trait was also present in .19 subjects with familial hypercholesterolemia. In Sardinian However, in studies of symptomatic thalassemic patients with the clinical diagnosis of familial patients, the risk of atherosclerosis20 and gallstone formation 21 hypercholesterolemia and different mutations of the LDL are increased although cholesterol levels are low. This may be receptor gene, the plasma LDL cholesterol level was due to significantly decreased HDL levels and increased LDL to significantly lower in subjects with βo-thalassemia trait than in HDL ratios. In addition to metabolic lipid disorder there is an subjects without this trait. The authors suggested that the LDL- important second aspect in thalassemia: oxidative stress and lowering effect of βo-thalassemia may be related to (i) the mild lipid peroxidation. erythroid hyperplasia, which would increase the LDL removal Livrea et al., (1998)20 conducted a by the bone marrow, and (ii) the chronic activation of the differentiated study on the oxidative modification of low monocyte-macrophage system, causing an increased secretion density and the atherosclerotic risk in thalassemia of some cytokines (interleukin-1, interleukin-6, and tumor intermedia patients. Conjugated diene lipid hydroperoxide (CD) necrosis factor-alpha) known to affect the hepatic secretion levels were three-fold higher and lysil residues of apo B-100, and the receptor-mediated removal of apolipoprotein B- vitamin E, and β-carotene were decreased in LDL from patients containing lipoproteins. The observation that the as compared with age-matched healthy controls. In patients, hypercholesterolemic subjects with βo-thalassemia trait LDL-CD showed a strong inverse correlation with LDL vitamin E Volume II No. 1. Jan-June 2005 Blood Lipids And Oxidative Stress In 15 β-thalassemia Paients In Jakarta and a negative trend with LDL-carotene. In the plasma of interface becomes more fluid. There are phase separation thalassemia patients, malondialdehyde (MDA) was increased by tendencies between protein-rich and lipid domains in these about twofold indicating increased LPO, while plasma vitamin E RBC membranes with loss in structural integrity and was decreased by 52% versus controls LDL-CD were inversely homogeneity26,27 with reorientation of integral proteins to the correlated with plasma vitamin E and correlated positively periphery and increase in polarity in the inner hydrophobic correlated with MDA. Also plasma ferritin was positively region of thalassemic RBC membranes. This means that besides correlated with LDL-CD , whereas no correlation was found conformational changes in membrane proteins there are between the age of the patients and plasma MDA or LDL-CD.20 changes in lipid structure possibly with formation of hydrophilic Clinical evidence of vascular complications in patients matched pores. Band-3 degradation may strongly be involved12,28 and with elevated LDL-CD over mean levels in patients. From their certainly lipid peroxidation with generation of hydrophilic results, the authors suggest that the level of plasma MDA in β- derivatives of unsaturated fatty acids also contributes to the thalassemia patients may represent a sensitive index of the increase in membrane polarity. Concomitantly, a loss in fluidity oxidative status of LDL in vivo and of its potential occurs because of loss in unsaturated lipid residues and atherogenicity.20 increase in the ratio of saturated vs. unsaturated In our study, plasma lipid soluble antioxidants, vitamin phospholipids27 and re-distribution of phospholipids27 between E (α-tocopherol), vitamin A (retinol), β-carotene and the water- the inner and outer leaftlets. This mean, not only the other soluble are lower in patients than in controls. For gradient but also the gradient of polarity across the membrane example, plasma vitamin E decreases by 42% in non-transfused, gets lost in RBC membranes of transfusion-dependent by 56% in transfused, and by 73% in long-term transfused thalassemia patients including also the loss of asymmetry, patients. Similar tendency of decreased plasma antioxidants in fluidity-viscosity gradients and the energetic barrier.27,29 thalassemia patients has been reported by others.10,22,23 In Plasma vitamin E, MDA and LDL/HDL ratio or the ratio parallel, reactive thiol groups in the plasma and in proteins of total cholesterol to HDL, respectively, are suitable indices to decrease.10 Vice versa, parameters of lipid peroxidation the modification of blood lipids in thalassemic patients and the increase, measured as TBARs, e,g., MDA, a byproduct of lipid increased atherosclerotic and cardiovascular risk and are also peroxidation and a parameter of membrane damage. The indices to the loss of structural integrity of thalassemic red cell influence of alternations in cholesterol and triglyceride membranes. In the group of long-term transfused patients, metabolism and of lipid modifications by peroxidation ferritin levels and signs of membrane disintegration are processes on erythrocyte membrance were investigated by EPR highest. Although a clear correlation cannot be drawn between spectroscopy and SDS-PAGE of isolated red cell membranes.12 these two observations (because ‘membrane disintegration’ is It should be mentioned that hemoglobin analysis of composed from more than one parameter), red cell membrane thalassemia major patients who have been regularly transfused disintegration increased in parallel with plasma ferritin levels, for years is often not consistent with the clinical picture which in turn increase with in long-term transfusion. because their blood contains mixtures of endogenous and Finally, another aspect of altered lipid metabolism and exogenous (=transfused) erythrocytes and Hemoglobin. generally decreased plasma cholesterol levels must be However, blood samples of transfused patients were always considered, i.e., the effect on biosynthesis of steroids in these drawn immediately before tansfusions, which means that patients. This may explain, why thalassemia patients often exogenous erythrocytes and hemoglobin were already widely exhibit deficits in cholesterol-derived hormonal and neuronal degraded. In β-thalassemic RBC, excessive α-globin chains transmitters.30 precipitate associated with the cytoskeleton and the membrane. They are unstable, oxydizable, and iron ions into Acknowledgement the environment of the erythrocyte membrane.24,25 This study was supported by the URGE Project of the Interpreting our own data it can be stated that the Indonesian Government. The authors are furthermore thalassemic red cell membrane is more rigid than that of gratefully obliged to Prof. Dr. Iskandar Wahidayat, normal erythrocyte. However, in transfused patients the Departement of Pediatric Hematology, Cipto Mangunkusumo interpretation is much more complicated: rigidity further General Hospital, Faculty of Medice, Salemba, Jakarta for his increase in the inner, hydrophobic moiety, whereas the polar support in this study.

16 Freisleben HJ DIGM Medical of Journal

References patients with beta-thalassemia major; an epidemiological study in young adults from Greece. Lipids Health Disease.2004;3:3-10. 1. WHO, Bull World Health Org. 1983;61:63-80. 18. Maioli M, Vigna GB, Tonolo G, Brizzi P, Ciccarese M, Donega P, et al. 2. Sofro ASM. Molecular pathology of β-thalassemia in Indonesia. Plasma lipoprotein composition, apolippprotein(a) concentration and Southeast Asian J Trop med Public Health. 1995;26 Suppl 1:221-4. isoforms in beta-thalassemia. . 1997 May;131(1):127- 3. Lie-Injo LE, Cai SP, Wahidayat I, Moeslichan S, Lim ML, Evangelista L, 33. et.al. Beta-thalassemia mutations in Indonesia and their linkage to 19. Deiana L, Garuti R, Pes GM, Carru C, Errigo A, Rolleri M, Pisciotta L, different haplotypes. Am J Hum Genet, 1989;45:971-5. Masturzo P, Cantafora A, Calandra S, Bertolini S. Influence of beta(0)- 4. Shinar E, Rachmilewitz EA. Oxidative denaturation of red blood cells in thalassemia on the phenotypic expression of heterozygous familial thalassemia. Semin Hematol. 1990;27:70-82. hypercholesterolemia: a study of patients with familial 5. Williamson D. The unstable hemoglobins. Blood Rev.1993;7:146-63. hypercholesterolemia from Sardinia. Arterioscler Thromb Vasc 6. Hershko C, Graham G, Bates GW, Rachmilewitz EA. Non-specific serum Biol.2000 Jan;20(1):236-43. iron in thalassemia: an abnormal serum iron fraction of potential 20. Livrea MA, Tesoriere L, Maggio A, D’Arpa D, Pintaudi AM, Pedone E. toxicity. Br J Haematol.1978;40:255-63. Oxidative Modification of Low-Density Lipoproteins and Atherogenetic 7. Britton RS. Metal-induced hepatotoxity. Semin liver Dis. 1996;16:3-12. Risk in β-Thalassemia. Blood.1998 Nov 15:92(10):3936-42. 8. Brili SV, Tzonou Al, Castelanos SS, Anggeli CJ, tentolouris CA, Pitsavos 21. Borgna-Pignatti C, Rigon F, Merlo L, Chakrok R, MiccioloR, Perseu L, et CE, et al. The effect of iron overload in the hearts of patients with al. Thalassemia minor, the Gilbert mutation, and the risk of gallstones. beta-thalassemia. Clin Cardiol.1997;20:541-6. Haematologica.2003;88:1106-9. 9. Handayani S, Adhiyanto C, Fani IR, Hidayat C, Ratih D, Kurniati V, et al. 22. Zannos-Mariolea L, Tzortzatou F, Dendaki-Svolaki K, Katerellos CH, Imbalance antioxidant status in transfusion-dependent thalassemia Kavallari M, Matsaniotis N. Serum vitamin E levels with beta- patients in Jakarta, Indonesia. In: Nesaretnam K, Packer L, editors. thalassemia major: preliminary reprt. Brit J Haematol.1974;26:193-9. Micronutrients and Health-Molecular Biological Mechanism. 23. Rachmilewitz EA Shifter A, Kahane I. Vitamin E deficiency in – Champaign: AOCS Press;2001.p.290-8. thalassemia major: changes in hematological and biochemical 10. Livrea MA, Tesoriere L, Pintaudi AM, Calabrese A, Maggio A, Freisleben parameters after a therapeutic trial with –tocopherol. Am J Clin Nutr. HJ, et al. Oxidative stress and antioxidant status in β-thalassemia 1979;32:1850-8. major: iron overload and deplation of lipid-soluble antioxidants. 24. Chiu DTJ, van den Berg J, Kuypers FA, Hung IJ, Wie JS, Liu TZ. Blood.1996;88:3608-14. Correlation of membrane lipid peroxidation with oxidation of 11. Laksmitawati DR, Handayani S, Udyaningsih-Freisleben SK, Kurniati V, hemoglobin variants: possibly related to the rates of hemin release. Adhiyanto C, Hidayat J, et al. Iron status and oxidative stress in β- Free Rad Biol Med.1996;21:89-95. thalassemia patients in Jakarta. Biofactors.2004;19(1,2):53-62. 25. Repka T, Shalev O, Reddy R, Yuan J, Abrahamov A Rachmilewitz EA, et 12. Udyaningsih-Freisleben SK, Kurniati V, Prasetyo PB, Handayani S, al. Nonrandom association of free iron with the membranes of sickle Adhiyanto C, Soegianto RR,. Et al. Isolated erythrocyte membranes of and beta-thalassemic erythrocytes. Blood. 1993;82;3204-10. transfusion-dependent and non-transfused thalassemia patients in 26. Arduini A, Stern A, Storto S, Belfiglio M, Mancinelli G, Scurti R, et al. Jakarta-investigated by electron paramagnetic resonance Effect of oxidative stress on membrane phospholipids and protein spectroscopy. BioFactors.2004;19(1,2):87-100. organization in human erythrocytes. Arch Biochem 13. Trampisch HJ, Windeler J, editors. Medizinische Statistik. Berlin, Biophys.1989;273:112-20. Heidelberg: Springer Verlag; 1997. 27. Rachmilewitz EA. Pathologic changes of red blood cell membranes in 14. Giardini O, Murgia F, Martino F, Mannarino O, Corrado G, Maggiono G. thalassemia. Birth Defects. 1982;18:219-22. Serum Lipid Pattern in β-thalassaemia. Acta haemat.1978;60:100-7. 28. Mannu F, Arese P, Cappellini MD, Fiorelli G, Cappadoro M, Giribaldi G, 15. Maioli M, Cuccuru GB, Pranzetti P, Pacifico A, Cherchi GM. Plasma et al. Role of hemichrome binding to erythrocyte membrane in the lipids and lipoprotein pattern in beta-thalassemia major. Acta generation of band-3 alterations in –thalassemia intermedia Haematol. 1984;71:106-10. erhythrocytes. Blood. 1995;86:2014-20. 16. Ameri MR, Alebouyeh M, Ziai M, Conn RB. Hypertriglyceridemia in 29. Zimmer G, Freisleben HJ. Membrane fluidity determinations from homozygous beta-thalassemia. Helv Paediatr Acta.1977 Jun;32(1):83- viscosimetry. In: Aloia RC, Curtain CC, Gordon LM, editors. Advances in 86. Membrane Fluidity. Vol. I. Methods for Studying Membrane 17. Chrysohoou C, Panagiotaks DP, Pitsavos C, Kosma K, Barbetseas J, Fluidity.New York: Liss; 1988.p.297-318. Karagiorga M, et al., Distribution of serum lipids and lipoproteins in 30. Namazi MR. Minor thalassemia may be a risk factor for impulsiveness. Med Hypotheses. 2003;60:335-6.