The pyruvate kinase activator mitapivat reduces hemolysis and improves anemia in a ß-thalassemia mouse model.

Anemia in ß-thalassemia is related to ineffective erythropoiesis and reduced red cell survival. Excess free heme and accumulation of unpaired α-globin chains impose substantial oxidative stress on ß-thalassemic erythroblasts and erythrocytes, impacting cell metabolism. We hypothesized that increased pyruvate kinase activity induced by mitapivat (AG-348) in the Hbbth3/+ mouse model for ß-thalassemia would reduce chronic hemolysis and ineffective erythropoiesis through stimulation of red cell glycolytic metabolism. Oral mitapivat administration ameliorated ineffective erythropoiesis and anemia in Hbbth3/+ mice. Increased ATP, reduced reactive oxygen species production, and reduced markers of mitochondrial dysfunction associated with improved mitochondrial clearance suggested enhanced metabolism following mitapivat administration in ß-thalassemia. The amelioration of responsiveness to erythropoietin resulted in reduced soluble erythroferrone, increased liver Hamp expression, and diminished liver iron overload. Mitapivat reduced duodenal Dmt1 expression potentially by activating the pyruvate kinase M2-HIF2α axis, representing a mechanism additional to Hamp in controlling iron absorption and preventing ß-thalassemia-related liver iron overload. In ex vivo studies on erythroid precursors from patients with ß-thalassemia, mitapivat enhanced erythropoiesis, promoted erythroid maturation, and decreased apoptosis. Overall, pyruvate kinase activation as a treatment modality for ß-thalassemia in preclinical model systems had multiple beneficial effects in the erythropoietic compartment and beyond, providing a strong scientific basis for further clinical trials.

The autophagy-activating kinase ULK1 mediates clearance of free α-globin in ß-thalassemia.

In ß-thalassemia, accumulated free α-globin forms intracellular precipitates that impair erythroid cell maturation and viability. Protein quality control systems mitigate ß-thalassemia pathophysiology by degrading toxic free α-globin, although the associated mechanisms are poorly understood. We show that loss of the autophagy-activating Unc-51-like kinase 1 (Ulk1) gene in ß-thalassemic mice reduces autophagic clearance of α-globin in red blood cell precursors and exacerbates disease phenotypes, whereas inactivation of the canonical autophagy-related 5 (Atg5) gene has relatively minor effects. Systemic treatment with the mTORC1 inhibitor rapamycin reduces α-globin precipitates and lessens pathologies in ß-thalassemic mice via an ULK1-dependent pathway. Similarly, rapamycin reduces free α-globin accumulation in erythroblasts derived from CD34+ cells of ß-thalassemic individuals. Our findings define a drug-regulatable pathway for ameliorating ß-thalassemia.

Hereditary spherocytosis is associated with decreased pyruvate kinase activity due to impaired structural integrity of the red blood cell membrane.

Hereditary spherocytosis (HS) is characterised by increased osmotic fragility and enhanced membrane loss of red blood cells (RBC) due to defective membrane protein complexes. In our diagnostic laboratory, we observed that pyruvate kinase (PK) activity in HS was merely slightly elevated with respect to the amount of reticulocytosis. In order to evaluate whether impaired PK activity is a feature of HS, we retrospectively analysed laboratory data sets from 172 unrelated patients with HS, hereditary elliptocytosis (HE), glucose-6-phosphate dehydrogenase (G6PD) or PK deficiency, sickle cell or haemoglobin C disease, or ß-thalassaemia minor. Results from linear regression analysis provided proof that PK activity decreases with rising reticulocyte counts in HS (R2  = 0·15; slope = 9·09) and, less significantly, in HE (R2  = 0·021; slope = 8·92) when compared with other haemolytic disorders (R2  ≥ 0·65; slopes ≥ 78·6). Reticulocyte-adjusted erythrocyte PK activity levels were significantly lower in HS and even declined with increasing reticulocytes (R2  = 0·48; slope = -9·74). In this report, we describe a novel association between HS and decreased PK activity that is apparently caused by loss of membrane-bound PK due to impaired structural integrity of the RBC membrane and may aggravate severity of haemolysis in HS.

Efficacy Of Interrupted And Modified Deferasirox Dose Among Paediatric Patients With Β- Thalassemia Major And High Alanine Aminotransferase Level.

BACKGROUND: Abnormal liver function tests lead to interruptions of Deferasirox therapy. The aim of this study is to assess the efficacy of deferasirox dose 30 mg /kg /day in maintaining cardiac protective level of serum ferritin of <2500 ng/ml among patients who received interrupted and modified doses. METHODS: A retrospective cohort study was conducted in Ibn Al Atheer paediatric hospital in Mosul city, Iraq, utilizing the monthly reading of serum ferritin level during the period started in February 2013 to march 2014 using documented patients' records. Group A, patients included thirty-five patients with ß- thalassemia major whose Deferasirox dose of 30 mg/kg/day was interrupted and modified due to ≥ 5-fold raise in alanine aminotransferase during any month of the study period. Compared group B patients included 40 children who received constant median deferasirox dose 30 mg/kg/day throughout one year of study period. Serum ferritin and alanine aminotransferase levels were routinely analysed every month among those patients. RESULTS: Interrupted and modified Deferasirox dose of 30 mg/kg/day significantly (p=0.000) increase the frequency of having mean serum ferritin >2500 ng/ml, and was associated with 55 times relative risk of having mean serum ferritin >2500 ng/ml compared to group B with steady median deferasirox dose. CONCLUSIONS: Interrupted and modified deferasirox dose of 30 mg/kg/day has a significant adverse effect on cardiac protective level of serum ferritin.

Association of thalassemia major and gingival inflammation: A pilot study.

OBJECTIVES: This cross-sectional study aimed to investigate the relationship between thalassemia major (TM) and gingival inflammation through the salivary, serum, and gingival crevicular fluid (GCF) levels of matrix metalloproteinase (MMP)-8, MMP-9 and tissue inhibitor of MMP (TIMP)-1. METHODS: Biofluid samples and full-mouth clinical periodontal recordings were obtained from 29 otherwise healthy patients with TM and 25 systemically healthy (SH) individuals. Biofluid samples were evaluated by immunofluorometric assay (IFMA) and enzyme-linked immunoassays (ELISAs). Data were tested statistically by Kolmogorov Simirnov, Mann-Whitney U tests, Spearman correlation analysis. RESULTS: Age, smoking status, bleeding on probing, plaque index were similar in the study groups, but probing depth, gender data exhibited significant differences (p=0.037 for both). Salivary MMP-8, MMP-9, TIMP-1 concentrations were significantly higher in the TM than SH group (p=0.014; p<0.001; p=0.042, respectively). Serum TIMP-1 concentrations were significantly higher; MMP-8/TIMP-1, MMP-9/TIMP-1 molar ratios were significantly lower in the TM than SH group (p<0.001; p=0.005; p=0.022, respectively). Very few GCF samples revealed biochemical data above the detection limits. Numerous correlations were found between clinical periodontal parameters and biochemical data. CONCLUSIONS: It may be suggested that TM may exacerbate the local inflammatory response as manifested in salivary MMP-8, MMP-9, TIMP-1 levels.

Molecular characterization and phenotypical study of ß-thalassemia in Tucumán, Argentina.

The main hereditary hemoglobin (Hb) disorder in Argentina is ß-thalassemia (ß-thal). Molecular studies performed in the center of the country exhibited a marked prevalence of the codon 39 (C > T) and IVS-I-110 (G > A) mutations. The northwest region of Argentina has a different demographic history characterized by an important Spanish influx. Seventy-one ß-thal carriers attending the Instituto de Bioquímica Aplicada, Tucumán, Argentina, were investigated for ß-globin gene mutations by real-time polymerase chain reaction (RT-PCR). To examine the genotype-phenotype relationship, mean corpuscular volume (MCV), mean corpuscular Hb (MCH) and Hb A2 were measured. In order to recognize ß-thal, Mentzer Index, Shine & Lal and Red Cell Distribution Width Index (RDWI), were calculated. The ethnic background of subjects revealed that 82.0% of the population was of Italian, Spanish and Arab origin. Seven mutations were detected: codon 39 (45.0%), IVS-I-1 (G > A) (22.5%), IVS-I-110 (16.3%), IVS-II-1 (G > A) (4.1%), IVS-I-1 (G > T) (2.0%), IVS-I-6 (T > C) (2.0%) and IVS-II-745 (G > C) (2.0%). In three families (6.1%), ß-thal mutations were not determined. These results differed from other Argentinian studies because at present codon 39 and IVS-I-1 are the most prevalent; MCV, MCH and Hb A2 did not correlate with the type of mutation (ß(0)/ß(+)). Values of MCV (67.0 fL) and Hb A2 (4.85%) were unable to discriminate between them. Significant differences (p < 0.05) in MCV, MCH and Shine & Lal were observed between the undetermined group and the three most common mutations. These data show different patterns of ß-thal mutations in the center and northwest regions of Argentina. Differences might represent the influence of Spanish immigration.

Serum YKL-40 levels and chitotriosidase activity in patients with beta-thalassemia major.

BACKGROUND: YKL-40 association with human disease has been the object of many years of investigation. ß-thalassemia patients are affected by hepatic siderosis, which determines a fibrotic process and tissue remodelling. Chitotriosidase has been found to be increased in thalassemic patients returning to normal in patients submitted to bone marrow transplantation. YKL-40 is associated with macrophage activation in liver and in other tissues. The aim of the study was to analyse the level of serum YKL-40 and plasma chitotriosidase activity of patients with beta-thalassemia to assess whether their expression correlates with liver disease and degree of liver siderosis. METHODS: Expression of YKL-40 and chitotriosidase as a marker of inflammation in 69 thalassemic patients were evaluated. We sought to investigate whether these two chitinases could be considered as a significant biomarker to evaluate therapy effectiveness. RESULTS: Surprisingly we found normal value of YKL-40. We, also, analysed chitotriosidase activity in the same patients that was slightly increased as a consequence of macrophage activation. CONCLUSIONS: These data would suggest a good treatment for these patients.

Gγ-Xmn I polymorphism: a significant determinant of ß-thalassemia treatment without blood transfusion.

ß-thalassemia is characterized by impaired ß-chain synthesis leading to ineffective erythropoiesis, severe anemia, and a need for blood transfusion. Presence of Xmn I polymorphism (-158 C-T nucleotide change) in γ-globin gene is associated with a higher fetal hemoglobin and a lesser clinical severity. This prospective study attempted to find out the effect of hydroxyurea (HU) on ß-thalassemia patients in the presence or absence of Xmn I polymorphism. A total of 143 consecutive ß-thalassemia patients received HU (16 mg/kg/d). Sixty-four (44.7%) had Xmn I polymorphism (either homozygous or heterozygous). Patients were evaluated at a median duration of 3 years (range, 6 mo to 9 y). Responders became transfusion independent after 6 months, partial responders had a least 50% reduction in transfusion requirement and nonresponders had no significant reduction. Of the 64 patients with Xmn I polymorphism, 44 (69%) showed response (P<0.01), whereas in those who lacked Xmn I polymorphism (n=79), only 17 (21%) were responders. This study showed that the presence of Xmn I polymorphism in ß-thalassemia is a predictor of response to HU and highlights the possibility of managing this subset of patients without blood transfusion.

Clinical severity of ß-thalassaemia/Hb E disease is associated with differential activities of the calpain-calpastatin proteolytic system.

Earlier observations in the literature suggest that proteolytic degradation of excess unmatched α-globin chains reduces their accumulation and precipitation in ß-thalassaemia erythroid precursor cells and have linked this proteolytic degradation to the activity of calpain protease. The aim of this study was to correlate the activity of calpain and its inhibitor, calpastatin, with different degrees of disease severity in ß-thalassaemia. CD34(+) cells were enriched from peripheral blood of healthy individuals (control group) and patients with mild and severe clinical presentations of ß(0)-thalassaemia/Hb E disease. By ex vivo cultivation promoting erythroid cell differentiation for 7 days, proerythroblasts, were employed for the functional characterization of the calpain-calpastatin proteolytic system. In comparison to the control group, enzymatic activity and protein amounts of µ-calpain were found to be more than 3-fold increased in proerythroblasts from patients with mild clinical symptoms, whereas no significant difference was observed in patients with severe clinical symptoms. Furthermore, a 1.6-fold decrease of calpastatin activity and 3.2-fold accumulation of a 34 kDa calpain-mediated degradation product of calpastatin were observed in patients with mild clinical symptoms. The increased activity of calpain may be involved in the removal of excess α-globin chains contributing to a lower degree of disease severity in patients with mild clinical symptoms.

Decreased blood catalase activity is not related to specific beta-thalassemia mutations in Hungary.

INTRODUCTION: Thalassemia erythrocytes are exposed to oxidative stress especially to hydrogen peroxide, which is regulated with the enzyme catalase. The aim of this study was to examine blood catalase activity and the relationship of blood catalase and beta-thalassemia gene mutations. METHODS: Blood catalase activity, hemoglobin, HbA(2) , HbF, and beta-globin gene mutations were determined in 43 Hungarian patients with beta-thalassemia trait. RESULTS: Compared to controls, the beta-thalassemia trait patients showed a low mean (P < 0.001) of blood catalase (men: 84 ± 29 MU/L vs. sex-matched controls: 118 ± 18 MU/L and women: 74 ± 18 MU/L vs. 108 ± 114 MU/L) and a low mean of blood catalase-to-blood hemoglobin ratio (men: 0.72 ± 0.22 MU/g vs. 0.85 ± 0.12 MU/g, women: 0.77 ± 0.26 MU/g vs. 0.84 ± 0.11 MU/g). The HbA(2) determination showed high sensitivity and specificity for the detection of beta-thalassemia trait patients. Mutation analyses revealed 13 beta-thalassemia trait mutations, of which six have not been reported before in Hungarian beta-thalassemia trait patients. Each group of mutations revealed decreased (P < 0.01) mean of blood catalase and catalase-to-hemoglobin ratio. Acatalasemia mutations were not found in beta-thalassemia trait patients. CONCLUSION: The decrease in blood catalase activity might be due to the damaging effects of free radicals on the catalase protein. Consequently, these beta-thalassemia trait patients may be relatively susceptible to damage caused by oxidative stress.

Absence of JAK2V617F mutation in patients with beta-thalassemia major and thrombocytosis due to splenectomy.

The report of Janus Kinase 2 (JAK2) mutations in myeloid malignancies with high frequency in myeloproliferative neoplasms has been well known since 2005. By monitoring allele burden, it is found that the expression of JAK2V617F mutation is increasing significantly from essential thrombocytosis to polycythemia vera. Furthermore, JAK2 abnormalities are reported in the majority of unexplained thrombotic episodes. Thalassemic syndromes are characterized by ineffective erythropoiesis and thrombocytosis, mainly due to splenectomy. The high incidence of thromboembolic events has led to the identification of a prothrombotic state in these patients. The contribution of JAK2 mutations to the hypercoagulable state of thalassemic patients is still unknown. Furthermore, the potential role of Janus Kinase mutations in hepcidin expression and consequently in ineffective erythropoiesis is still under investigation. This study was scheduled to determine whether the presence of JAK2V617F mutation in thalassemic patients is associated with thrombocytosis. We studied 20 patients DNA with beta-thalassemia for JAK2V617F mutation by using RG-PCR method. None of the patients were positive for this particular mutation. More studies are needed to prove the role of JAK2 in ineffective erythropoiesis, iron metabolism and thrombocytosis and to determine if using JAK2 inhibitors in thalassemic patients can be a potential therapeutic option.

Chelation therapy with desferrioxamine does not normalize ferritin level but attenuates oxidative damage and improves total antioxidant level in Malaysian Chinese beta-thalassaemia major patients.

Beta-thalassaemia major causes severe anaemia and patients with it may be transfusion-dependent for life. Regular blood transfusions cause iron-overload that leads to oxidative damage which can hasten mortality. The objective of this research was to study the oxidant-antioxidant indices in beta-thalassaemia major patients at the University of Malaya Medical Centre (UMMC) who were on desferrioxamine-chelation or without chelation therapy. Blood was collected from 39 Chinese patients and 20 controls. Plasma and peripheral blood mononuclear cell lysates (PBMC) were extracted and biochemical tests to evaluate oxidative stress were performed. Oxidative stress was evident in these patients as advanced oxidized protein products (AOPP) and lipid hydroperoxides were elevated, whereas glutathione peroxidase activity and the ferric reducing antioxidant power (FRAP) were reduced. The catalase activity in the patients' PBMC was elevated, possibly as a compensatory mechanism for the reduced glutathione peroxidase activity in both red blood cells and PBMC. The lower FRAP and higher AOPP levels in the non-chelated patients compared with the chelated patients were indicative of a lower oxidative stress level in the chelated patients. The ferritin levels in the chelated and non-chelated patients were high and the mean levels of liver enzyme activities in the majority of patients were elevated regardless of chelation therapy. In conclusion, this study indicates that desferrioxamine chelation therapy does not normalize ferritin level but attenuates oxidative damage and improves total antioxidant level in Malaysian Chinese beta-thalassaemia major patients.

Oxidative stress modulates heme synthesis and induces peroxiredoxin-2 as a novel cytoprotective response in ß-thalassemic erythropoiesis.

BACKGROUND: ß-thalassemic syndromes are inherited red cell disorders characterized by severe ineffective erythropoiesis and increased levels of reactive oxygen species whose contribution to ß-thalassemic anemia is only partially understood. DESIGN AND METHODS: We studied erythroid precursors from normal and ß-thalassemic peripheral CD34(+) cells in two-phase liquid culture by proteomic, reverse transcriptase polymerase chain reaction and immunoblot analyses. We measured intracellular reactive oxygen species, heme levels and the activity of δ-aminolevulinate-synthase-2. We exposed normal cells and K562 cells with silenced peroxiredoxin-2 to H(2)O(2) and generated a recombinant peroxiredoxin-2 for kinetic measurements in the presence of H(2)O(2) or hemin. RESULTS: In ß-thalassemia the increased production of reactive oxygen species was associated with down-regulation of heme oxygenase-1 and biliverdin reductase and up-regulation of peroxiredoxin-2. In agreement with these observations in ß-thalassemic cells we found decreased heme levels related to significantly reduced activity of the first enzyme of the heme pathway, δ-aminolevulinate synthase-2 without differences in its expression. We demonstrated that the activity of recombinant δ-aminolevulinate synthase-2 is inhibited by both reactive oxygen species and hemin as a protective mechanism in ß-thalassemic cells. We then addressed the question of the protective role of peroxiredoxin-2 in erythropoiesis by exposing normal cells to oxidative stress and silencing peroxiredoxin-2 in human erythroleukemia K562 cells. We found that peroxiredoxin-2 expression is up-regulated in response to oxidative stress and required for K562 cells to survive oxidative stress. We then showed that peroxiredoxin-2 binds heme in erythroid precursors with high affinity, suggesting a possible multifunctional cytoprotective role of peroxiredoxin-2 in ß-thalassemia. CONCLUSIONS: In ß-thalassemic erythroid cells the reduction of δ-aminolevulinate synthase-2 activity and the increased expression of peroxiredoxin-2 might represent two novel stress-response protective systems.

Association of haptoglobin phenotypes with ceruloplasmin ferroxidase activity in ß-thalassemia major.

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 ß-thalassemia major. METHODS: Blood specimens were collected from 196 subjects (124 ß-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<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<0.05). CONCLUSION: Thalassemia patients with Hp 2-2 phenotype are under greater iron-driven oxidative stress than patients with other phenotypes.

Bilirubin concentrations in thalassemia heterozygotes in university students.

OBJECTIVES: To investigate the difference of bilirubin concentrations between α- and ß-thalassemia carriers and the role of variation status in the UDP-glucuronosyltransferase (UGT) 1A1 gene on such a difference. METHODS: A total of 2713 university freshmen who attended a regular physical examination were enrolled in underwent screenings for thalassemias. Finally, 123 subjects whose mean corpuscular volume was ≤80 fL and who had no iron deficiency anemia were tested by PCR and PCR-restriction fragment length polymorphism (RFLP) for α- and ß-thalassemias, respectively, and tested by PCR-RFLP for the five known variations of the UGT1A1 gene. RESULTS: Among the 123 subjects, 76 and 47 were diagnosed with heterozygous α-thalassemia and with heterozygous ß-thalassemia, respectively. Between the α- and ß-thalassemia heterozygotes, variation status of the UGT1A1 gene was not statistically different (P = 0.898), while hemoglobin and bilirubin concentrations differed significantly (P = 0.005 and 0.001, respectively). Bilirubin concentrations were significantly higher among individuals with compound heterozygous variations/homozygous variation in the UGT1A1 gene than in those possessing the wild type and heterozygous variation (P < 0.001 for both α- and ß-thalassemia heterozygotes). Compound heterozygous variations/homozygous variation in the UGT1A1 gene and anemia were the main causes of hyperbilirubinemia in α- and ß-thalassemia heterozygotes, respectively. CONCLUSIONS: The difference in bilirubin concentrations between α- and ß-thalassemia heterozygotes may be attributable to more bilirubin being produced in ß-thalassemia heterozygotes than in α-thalassemia heterozygotes, while variation status of the UGT1A1 gene affects bilirubin concentrations in both α- and ß-thalassemia heterozygotes.

Chelation therapy with desferrioxamine does not normalize ferritin level but attenuates oxidative damage and improves total antioxidant level in malaysian Chinese β-thalassaemia major patients / La terapia de quelación con deferoxamina no normaliza los niveles de ferritinaa pero atenúa el daño oxidativo y mejora el nivel antioxidante total en los pacientes sinomalayos que padecen de talasemia β

Beta-thalassaemia major causes severe anaemia and patients with it may be transfusion-dependent for life. Regular blood transfusions cause iron-overload that leads to oxidative damage which can hasten mortality. The objective of this research was to study the oxidant-antioxidant indices in β-thalassaemia major patients at the University of Malaya Medical Centre (UMMC) who were on desferrioxaminechelation or without chelation therapy. Blood was collected from 39 Chinese patients and 20 controls. Plasma and peripheral blood mononuclear cell lysates (PBMC) were extracted and biochemical tests to evaluate oxidative stress were performed. Oxidative stress was evident in these patients as advanced oxidized protein products (AOPP) and lipid hydroperoxides were elevated, whereas glutathione peroxidase activity and the ferric reducing antioxidant power (FRAP) were reduced. The catalase activity in the patients' PBMC was elevated, possibly as a compensatory mechanism for the reduced glutathione peroxidase activity in both red blood cells and PBMC. The lower FRAP and higher AOPP levels in the non-chelated patients compared with the chelated patients were indicative of a lower oxidative stress level in the chelated patients. The ferritin levels in the chelated and non-chelated patients were high and the mean levels of liver enzyme activities in the majority of patients were elevated regardless of chelation therapy. In conclusion, this study indicates that desferrioxamine chelation therapy does not normalize ferritin level but attenuates oxidative damage and improves total antioxidant level in Malaysian Chinese β-thalassaemia major patients.

La beta-talasemia mayor causa anemia severa, y los pacientes con este padecimiento pueden hacerse dependientes de las transfusiones de sangre por el resto de sus vidas. Las transfusiones regulares de sangre dan lugar a una sobrecarga de hierro que conduce al dano oxidativo, el cual a su vez puede acelerar la mortalidad. El objetivo de esta investigación fue estudiar las tasas de oxidantesantioxidantes en pacientes de beta-talasemia mayor en el Centro Médico de la Universidad de Malaya, tanto aquellos bajo tratamiento de quelación con deferoxamina, como aquellos sin terapia de quelación alguna. Se recogieron muestras de sangre de 39 pacientes chinos y 20 controles. Se extrajeron plasma y lisados de celulas mononucleares perifericas (CMSP), y se realizaron pruebas bioquimicas para evaluar el estrés oxidativo. El estrés oxidativo era evidente en estos pacientes en forma de productos avanzados de oxidación de proteinas (PAOP), y los hidroperoxidos de lipidos eran elevados, en tanto que la actividad de glutatión peroxidasa y el poder reductor ferrico/antioxidante (FRAP) era reducida. La actividad de la catalasa en los pacientes de CMSP era elevada, posiblemente como un mecanismo compensatorio frente a la actividad de glutatión peroxidasa reducida tanto en los globulos rojos como en las CMSP. Los niveles más bajos de FRAP y los más altos de PAOP en los pacientes no quelados en comparación con los pacientes quelados, indicaban un bajo nivel de estrés oxidativo en los pacientes quelados. Los niveles de ferritina tanto en los pacientes quelados como en los no quelados, eran altos, y los niveles promedio de actividades enzimaticas del higado fueron elevados en la mayoria de los pacientes, independientemente de la terapia de quelación. En conclusión, este estudio indica que la terapia de quelación con deferoxamina no normaliza el nivel de ferritina, pero en cambio atenua el daño oxidativo, y mejora el nivel antioxidante total en los pacientes sinomalayos afectados por la betatalasemia mayor.

Plasma levels of lipoprotein-associated phospholipase A(2) are increased in patients with ß-thalassemia.

Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) is an independent cardiovascular risk factor. We investigated the plasma levels of Lp-PLA(2) activity and mass as a function of plasma lipid levels, LDL subclass profile, and oxidative stress in patients with ß-thalassemia. Thirty-five patients with ß-thalassemia major (ß-TM) and 25 patients with ß-thalassemia intermedia (ß-TI) participated in the study. Lp-PLA(2) activity and mass were measured in total plasma, in apolipoprotein (apo)B-depleted plasma (HDL-Lp-PLA(2)), and in LDL subclasses. Lp-PLA(2) activity produced and secreted from peripheral blood monocytes in culture was also determined. Patients with ß-thalassemia are characterized by a predominance of small-dense LDL particles, increased oxidative stress, and very high plasma levels of Lp-PLA(2) mass and activity, despite low LDL-cholesterol levels. A significant positive correlation between plasma Lp-PLA(2) activity or mass and 8-isoprostane (8-epiPGF2a) and ferritin levels as well as intima-media thickness (IMT) values was observed. An increase in secreted and cell-associated Lp-PLA(2) activity from monocytes in culture was observed in both patient groups. The HDL-Lp-PLA(2) activity and mass as well as the ratio of HDL-Lp-PLA(2)/plasma Lp-PLA(2) were significantly higher in both patient groups compared with the control group. In conclusion, patients with ß-thalassemia exhibit high plasma Lp-PLA(2) levels, attributed to increased enzyme secretion from monocytes/macrophages and to the predominance of sdLDL particles in plasma. Plasma Lp-PLA(2) is correlated with carotid IMT, suggesting that this enzyme may be implicated in premature carotid atherosclerosis observed in ß-thalassemia.

Prolidase activity and oxidative status in patients with thalassemia major.

AIM: Prolidase is a specific imidodipeptidase involved in collagen degradation. The increase in the enzyme activity is believed to be correlated with the increased intensity of collagen degradation. The study aimed to evaluate the relationship between prolidase activity and oxidative status in patients with thalassemia major. METHODS: Comparison was made between 87 patients diagnosed with thalassemia major and 33 healthy children of similar age and gender. Mean age of the subjects was 7.5+/-4.3 years in the group of patients with thalassemia major and 8.9+/-3.1 years in the control group. Serum prolidase activity was measured spectrophotometrically. Oxidative status was determined using total oxidant status (TOS), total antioxidant capacity (TAC), and oxidative stress index (OSI) measurement. RESULTS: Prolidase activity was significantly increased in patients with thalassemia major (53.7+/-8.7 U/l) compared to the control group (49.2+/-7.2 U/l, P<0.001). TOS was significantly increased in the patient group (5.31+/-3.14 mmol H2O2 equiv./l) compared to the control group (3.49+/-2.98 mumol H2O2 equiv./l) and the OSI was also significantly increased in the patient group (3.86+/-3.28 arbitrary unit) compared to the control group (2.53+/-2.70 arbitrary unit) (P<0.0001 and P<0.001, respectively), while there were no significant differences between the patient (1.61+/-0.30 mumol Trolox equiv./l) and control (1.64+/-0.33 mumol Trolox equiv./l) groups with respect to TAC. CONCLUSION: Significant increases in prolidase activity in patients with thalassemia major may constitute a key parameter in demonstrating a disorder of the collagen metabolism.

Paraoxonase and arylesterase activity with oxidative status in children with thalassemia major.

PURPOSE: The aim of this study was to study paraoxonase and arylesterase activities along with oxidative status parameters, and to find out whether there is any increased susceptibility to atherogenesis, which might be reflected with increased oxidative stress and decreased serum paraoxonase/arylesterase activity in beta-thalassemia major (BTM) patients. PATIENTS AND METHODS: Eighty-seven patients with BTM and 33 healthy individuals were enrolled in the study. RESULTS: Paraoxonase and arylesterase activities were significantly lower in BTM patients than controls (for all P<0.0001), whereas total oxidant status, total peroxide concentration levels, and oxidative stress index were significantly higher (P<0.0001, <0.0001, and <0.001, respectively). Correlations were found between serum iron and ferritin and levels of total oxidant status in BTM patients. Significant correlation was found with serum total peroxide concentration levels and paraoxonase and arylesterase activities in patients with BTM. CONCLUSIONS: It was seen that oxidative stress increases, while serum paraoxonase activity is decreased in BTM patients. Decrease in paraoxonase activity seems to be associated with both the degree of oxidative stress and anemia. BTM patients may be more prone to development of atherogenesis because of low serum paraoxonase/arylesterase activity.