Co-inheritance of α0 -thalassemia elevates Hb A2 level in homozygous Hb E: Diagnostic implications.

INTRODUCTION: Differentiation of homozygous hemoglobin (Hb) E with and without α0 -thalassemia is subtle on routine hematological ground. We examined in a large cohort of homozygous Hb E if the level of Hb A2 is helpful. METHODS: A total of 592 subjects with homozygous Hb E were recruited from ongoing thalassemia screening program. Additionally, five couples at risk of having fetuses with Hb Bart's hydrops fetalis who were homozygous Hb E were also investigated. Hb analysis was performed using capillary electrophoresis system. Globin genotypes were defined by DNA analysis. RESULTS: Subjects were classified into four groups including pure homozygous Hb E (n=532), homozygous Hb E/α0 -thalassemia (n=48), Hb Constant Spring EE Bart's disease (n=8), and Hb EE Bart's disease (n=4). The levels of Hb A2 were found, respectively, to be 4.97±0.69, 6.64±1.02, 4.86±0.87, and 7.60±1.04%. Among five couples at risk, α0 -thalassemia was identified in three subjects with Hb A2 >6.0%. CONCLUSIONS: Increased Hb A2 level is a useful marker for differentiation of homozygous Hb E with and without α0 -thalassemia. This should lead to a significant reduction in number of referral cases of homozygous Hb E for molecular testing of α0 -thalassemia in routine practice.

Diagnosis of common hemoglobinopathies among South East Asian population using capillary isoelectric focusing system.

INTRODUCTION: We have evaluated an automated capillary isoelectric focusing (cIEF)-based Hb analyzer in diagnosis of hemoglobinopathies commonly found among South East Asian population. METHODS: Study was performed on a cohort of 665 adult Thai subjects and 13 fetal blood specimens obtained at routine thalassemia diagnostic laboratory. Hb analysis was performed using the cIEF system. Thalassemia genotypes were defined by DNA analysis. RESULTS: The system revealed satisfactorily within-run and between-run precision for quantitation of Hb A2 and Hb E (CV: 0.02-0.09%). The reference ranges of Hb A2 and Hb E were 2.6-4.0% and 25.7-33.1%, respectively. The system identified the cases of ß-thalassemia and Hb E disorders correctly. Several thalassemia genotypes and Hb variants were identifiable. However, Hb Constant Spring was separated closely to Hb A2 and Hbs Bart's and H were relatively difficult to be reported due to interfering peaks separating at the same regions. Prenatal diagnosis by fetal blood analysis was found to be accurate for Hb Bart's hydrops fetalis and Hb E-ß0 -thalassemia disease. CONCLUSIONS: The cIEF system could accurately diagnose ß-thalassemia and Hb E carriers and demonstrate many Hb variants found in the region. The system cannot report Hb A2 in the presence of Hb E whereas Hbs Lepore and F are comigrated. Diagnosis of α-thalassemia disease based on Hb H and Hb Bart's might be difficult.

ICSH recommendations for assessing automated high-performance liquid chromatography and capillary electrophoresis equipment for the quantitation of HbA2.

Automated high performance liquid chromatography and Capillary electrophoresis are used to quantitate the proportion of Hemoglobin A2 (HbA2 ) in blood samples order to enable screening and diagnosis of carriers of ß-thalassemia. Since there is only a very small difference in HbA2 levels between people who are carriers and people who are not carriers such analyses need to be both precise and accurate. This paper examines the different parameters of such equipment and discusses how they should be assessed.

Tandem measurements of iron and creatinine by cross injection analysis with application to urine from thalassemic patients.

This work presents development of a method for the dual determination of Fe(III) and creatinine using cross injection analysis (CIA). Two CIA platforms connected in series accommodated sample and reagents plugs aspirated via y-direction channels while water was pumped through the x-direction channel toward a flow-through cell of a diode array UV-vis. detector. Iron was detected from the colorimetric reaction between Fe(II) and 2-(5-bromo-2-pyridylazo)-5-(N-propyl-N-(3-sulfopropyl)amino) aniline (5-Br-PSAA), with prior reduction of Fe(III) to Fe(II) by ascorbic acid. The Jaffe's reaction was employed for the detection of creatinine. Under the optimal conditions, good linearity ranges were achieved for iron in the range 0.5 to 7 mg L(-1) and creatinine in the range 50 to 800 mg L(-1). The CIA system was applied to spot urine samples from thalassemic patients undergoing iron chelation therapy, and was successfully validated with ICP-OES and batchwise Jaffe's method. Normalization of urinary iron excretion with creatinine is useful for correcting the iron concentration between urine samples due to variation of the collected urine volume.

New updating into hemoglobinopathies.

Thalassemia and abnormal hemoglobin are the most common genetic disorders and are considered health problems in many developing countries. In the last few years, there has been much progress in laboratory diagnosis, treatment and control of thalassemia. The variation in the clinical severity in both α- and ß-thalassemia reflects a genotype-phenotype interaction. This is important for future therapeutic intervention and should be well characterized in each population. The quality of life of the patients is much improved with regular blood transfusion and novel iron chelators. The cure for thalassemia is possible by stem cell transplantation and future gene therapy. It is expected that under multinational collaboration the prevention of thalassemia will happen worldwide.

ICSH recommendations for the measurement of haemoglobin F.

Measurement of the Haemoglobin F in red cell haemolysates is important in the diagnosis of 뫧 thalassaemia, hereditary persistence of fetal haemoglobin (HPFH) and in the diagnosis and management of sickle cell disease. The distribution of Hb F in red cells is useful in the diagnosis of HPFH and in the assessment of feto-maternal haemorrhage. The methods of quantifying Hb F are described together with pitfalls in undertaking these laboratory tests with particular emphasis on automated high-performance liquid chromatography and capillary electrophoresis.

ICSH recommendations for the measurement of haemoglobin A2.

Although DNA analysis is needed for characterization of the mutations that cause ß-thalassaemia, measurement of the Hb A(2) is essential for the routine identification of people who are carriers of ß-thalassaemia. The methods of quantitating Hb A(2) are described together with pitfalls in undertaking these laboratory tests with particular emphasis on automated high-performance liquid chromatography and capillary electrophoresis.

Increase in non-transferrin bound iron and the oxidative stress status in epilepsy patients treated using valproic acid monotherapy.

OBJECTIVE: This study aims to investigate the alteration of iron homeostasis and oxidative stress status in epilepsy patients treated with valproic acid (VPA) monotherapy. MATERIALS: 24 epilepsy patients receiving VPA monotherapy (12 men, 12 women, age 27.5 ± 7.2 y) and 24 sex- and age-matched healthy volunteers were included in the study. METHODS: The level of iron status parameters; serum iron, ferritin, transferrin saturation, non-transferrin bound iron (NTBI), serum level of trace elements (copper, zinc and selenium), concentration of antioxidant parameters, activities of antioxidant enzymes and level of lipid peroxidation product were determined. RESULTS: NTBI was found in the patients although their other iron status parameters were normal. Levels of antioxidant parameters were decreased while activities of antioxidant enzymes were increased. Levels of serum MDA were significantly increased in patients with epilepsy. The daily dose of valproic acid associated was statistically significant: serum concentration of NTBI (r = 0.579; p = 0.003) and MDA (r = 0.465; p = 0.022). A positive correlation existed between NTBI and zinc (r = 0.522; p = 0.009). CONCLUSION: According to our results, VPA treatment in patients with epilepsy contributes to the metabolism of iron, leading to the formation of NTBI and an increase in oxidative stress.

Presumptive diagnosis of common haemoglobinopathies in Southeast Asia using a capillary electrophoresis system.

INTRODUCTION: This study was conducted to examine ability of the Capillarys 2 haemoglobin (Hb) testing system to assist in presumptive diagnosis of common Hb variants found in Southeast Asia including five α-chain and nine ß-chain variants. METHODS: Blood samples with unknown Hb variants sent from other hospitals to our centre for identification were re-analysed using the Capillarys 2 Hb analyser (SEBIA). DNA analyses by allele specific PCR assays were used to establish the final diagnoses. RESULTS: Five α-globin chain variants including Hbs Q-Thailand, Queens, Siam, Constant Spring and Paksé were detected. In heterozygous form, the machine demonstrated clearly two abnormal derivatives of Hbs A and A(2) for the former three variants. Small peaks of Hb Constant Spring and Hb Paksé were observed but could not be differentiated. In contrast, only one abnormal peak of Hb A was observed for ß-globin chain variants including those with more negative charge (Hb J-Bangkok, Hb Hope and Hb Pyrgos) and less negative charge (Hb D-Punjab, Hb S, Hb Korle-Bu and Hb C). Hb Tak, an elongated ß-chain variant was co-separated with Hb F whereas the Hb Malay co-migrated with Hb A in a subject with high Hb A(2) ß- thalassaemia trait. CONCLUSION: The capillary electrophoresis system could clearly demonstrate the presence of abnormal Hbs and provide useful information for presumptive diagnoses in most cases. The Hb analysis results could help in selection of appropriate DNA testing for final diagnoses of these variants.

Reversal of cardiac iron loading and dysfunction in thalassemic mice by curcuminoids.

Non-transferrin bound iron (NTBI) is found in plasma of ß-thalassemia patients and causes oxidative tissue damage. Cardiac siderosis and complications are the secondary cause of death in ß-thalassemia major patients. Desferrioxamine (DFO), deferiprone (DFP) and deferasirox (DFX) are promising chelators used to get negative iron balance and improve life quality. DFP has been shown to remove myocardial iron effectively. Curcuminoids (CUR) can chelate plasma NTBI, inhibit lipid peroxidation and alleviate cardiac autonomic imbalance. Effects of CUR on cardiac iron deposition and function were investigated in iron-loaded mice. Wild type ((mu)ß(+/+) WT) and heterozygous ß-knockout ((mu)ß(th-3/+) BKO) mice (C57BL/6) were fed with ferrocene-supplemented diet (Fe diet) and coincidently intervened with CUR and DFP for 2 months. Concentrations of plasma NTBI and malondialdehyde (MDA) were measured using HPLC techniques. Heart iron concentration was determined based on atomic absorption spectrophotometry and Perl's staining methods. Short-term electrocardiogram (ECG) was recorded with AD Instruments Power Lab, and heart rate variability (HRV) was evaluated using MATLAB 7.0 program. Fe diet increased levels of NTBI and MDA in plasma, nonheme iron and iron deposit in heart tissue significantly, and depressed the HRV, which the levels were higher in the BKO mice than the WT mice. CUR and DFP treatments lowered plasma NTBI as well as MDA concentrations (p <0.05), heart iron accumulation effectively, and also improved the HRV in the treated mice. The results imply that CUR would be effective in decreasing plasma NTBI and myocardial iron, alleviating lipid peroxidation and improving cardiac function in iron-loaded thalassemic mice.

Effects of green tea on iron accumulation and oxidative stress in livers of iron-challenged thalassemic mice.

Liver is affected by secondary iron overload in transfusions dependent b-thalassemia patients. The redox iron can generate reactive oxidants that damage biomolecules, leading to liver fibrosis and cirrhosis. Iron chelators are used to treat thalassemias to achieve negative iron balance and relieve oxidant-induced organ dysfunctions. Green tea (GT) (Camellia sinensis) catechins exhibit anti-oxidation, the inhibition of carcinogenesis, the detoxification of CYP2E1-catalyzed HepG2 cells and iron chelation. The purpose of this study was to investigate the effectiveness of GT in iron-challenged thalassemic mice. Heterozygous BKO type-thalassemia (BKO) mice (C57BL/6) experienced induced iron overload by being fed a ferrocene-supplemented diet (Fe diet) for 8 weeks, and by orally being given GT extract (300 mg/kg) and deferiprone (DFP) (50 mg/kg) for a further 8 weeks. Liver iron content (LIC) was analyzed by TPTZ colorimetric and Perl's staining techniques. Concentrations of liver reduced glutathione (GSH), collagen and malondialdehyde (MDA) were also measured. Dosages of the GT extract and DFP lowered LIC in the Fe diet-fed BKO mice effectively. The extract did not change any concentrations of liver glutathione, collagen and MDA in the BKO mice. Histochemical examination showed leukocyte infiltration in the near by hepatic portal vein and high iron accumulation in the livers of the iron-loaded BKO mice, however GT treatment lowered the elevated iron deposition. In conclusion, green tea inhibits or delays the deposition of hepatic iron in regularly iron-loaded thalassemic mice effectively. This will prevent the iron-induced generation of free radicals via Haber-Weiss and Fenton reactions, and consequently liver damage and fibrosis. Combined chelation with green tea would be investigated in beta-thalassemia patients with iron overload.

Efficacy of curcuminoids in alleviation of iron overload and lipid peroxidation in thalassemic mice.

Non-transferrin bound iron (NTBI) is detectable in plasma of beta-thalassemia patients and participates in free-radical formation and oxidative tissue damage. Desferrioxamine (DFO), deferiprone (DFP) and deferasirox (DFX) are iron chelators used for treatment of iron overload; however they may cause adverse effects. Curcuminoids (CUR) exhibits many pharmacological activities and presents beta-diketone group to bind metal ions. Iron-chelating capacity of CUR was investigated in thalassemic mice. The mice (C57BL/6 stain); wild type ((mu)beta(+/+)) and heterozygous beta-knockout ((mu)beta(th-3/+)) were fed with ferrocene-supplemented diet for 2 months, and coincidently intervened with CUR (200 mg/kg/day) and DFP (50 mg/kg/day). Plasma NTBI was quantified using NTA chelation/HPLC method, and MDA concentration was analyzed by TBARS-based HPLC. Hepatic iron content (HIC) and total glutathione concentration were measured colorimetrically. Tissue iron accumulation was determined by Perl's staining. Ferrocene-supplemented diet induced occurrence of NTBI in plasma of thalassemic mice as well as markedly increased iron deposition in spleen and liver. Treatment with CUR and DFP decreased levels of the NTBI and MDA effectively. Hepatic MDA and nonheme iron content was also decreased in liver of the treated mice whilst total glutathione levels were increased. Importantly, the CUR and DFP reduced liver weight index and iron accumulation. Clearly, CUR is effective in chelation of plasma NTBI in iron-loaded thalassemic mice. Consequently, it can alleviate iron toxicity and harmfulness of free radicals. In prospective, efficacy of curcumin in removal of labile iron pool (LIP) in hepatocytes and cardiomyocytes are essential for investigation.

Bioequivalence study of a film-coated tablet of deferiprone in healthy Thai volunteers.

OBJECTIVE: To evaluate the bioequivalence of a single dose of deferiprone tablet manufactured locally (GPO-L-ONE, GPO, Thailand) with a reference formulation (Ferriprox, ApoPharma, Canada). VOLUNTEERS AND METHODS: A randomized, single dose, two-treatment, two-period, two- sequence crossover study was conducted in 24 healthy volunteers. Each subject received a single dose of 3 tablets of 500 mg deferiprone of both formulations with a two-week washout period. Blood samples were collected at 12 points for 480 min. Serum deferiprone levels were analyzed using high performance liquid chromatography (HPLC) method. Pharmacokinetic parameters were calculated from serum concentration-time curve and applying the non-compartment model. Statistical comparisons of Cmax, AUC(0-t), AUC(0-inf) values were evaluated after logarithmic transformation. Other pharmacokinetic parameters were tested non-parametrically. RESULTS: The C(max) value (mean +/- SD) for reference and test product was 32.4 +/- 13.2 and 27.8 +/- 12.8 microg/ml, respectively. Mean ratio (test/reference) of C(max) was 0.852 with 90% CI of 0.772 - 0.934. Mean ratio (test/reference) of AUC(0-t) was 0.962 with 90% CI of 0.914 to 1.012, and of AUC(0-inf) was 0.966 with 90% CI of 0.918-1.016. Both formulations were well tolerated and no adverse effects were observed. CONCLUSION: The 90% CI of mean ratio of AUC(0-t) and AUC(0-inf) fell within the acceptable range (0.80 - 1.25) for bioequivalent eligibility. Regarding the efficacy of deferiprone, which depends on AUC rather than C(max), 90% CI of mean ratio of C(max) was within the acceptable range of WHO criteria for bioequivalence study (0.75 - 1.33). Therefore the two film-coated formulations of deferiprone tablet were proven bioequivalent in healthy Thai volunteers.

Effect of green tea on iron status and oxidative stress in iron-loaded rats.

Plasma non-transferrin bound iron (NTBI) is potentially toxic and contributes to the generation of reactive oxygen species (ROS), consequently leading to tissue damage and organ dysfunction. Iron chelators and antioxidants are used for treatment of thalassemia patients. Green tea (GT) contains catechins derivatives that have many biological activities. The purpose of this study was to investigate the iron-chelating and free-radical scavenging capacities of green tea extract in vivo. Rats were injected ip with ferric citrate together with orally administered GT extract (GTE) for 4 months. Blood was collected monthly for measurement of iron overload and oxidative stress indicators. Plasma iron (PI) and total iron-binding capacity (TIBC) were quantified using bathophenanthroline method. Plasma NTBI was assayed with NTA chelation/HPLC. Plasma malonyldialdehyde (MDA) was determined by using the TBARS method. Erythrocyte oxidative stress was assessed using flow cytometry. Levels of PI, TIBC, NTBI and MDA, and erythrocyte ROS increased in the iron-loaded rats. Intervention with GT extract markedly decreased the PI and TIBC concentrations. It also lowered the transferrin saturation and effectively inhibited formation of NTBI. It also decreased the levels of erythrocyte ROS in week 4, 12 and 16. Therefore, green tea extract can decrease iron in plasma as well as eliminate lipid peroxidation in plasma, and destroy formation of erythrocyte ROS in the rats challenged with iron. The bifunctional effects could be beneficial in alleviating the iron and oxidative stress toxicity. In prospective, these GTE activities should be further examined in thalassemic animals or humans.

Beta-globin gene cluster polymorphisms are strongly associated with severity of HbE/beta(0)-thalassemia.

We evaluated the contribution of 67 single nucleotide polymorphisms (SNPs) within the beta-globin gene cluster to disease severity in groups of 207 mild- and 305 severe unrelated patients from Thailand with Hemoglobin E (HbE)/beta(0)-thalassemia and normal alpha-globin genes. Our analysis showed that these SNPs comprise two distinct linkage disequilibrium blocks, one containing the beta-globin gene and the other extending from the locus control region (LCR) to the delta gene, which are separated by a recombination hotspot in the narrow region of the beta-globin gene promoter. Forty-five SNPs within the interval including the LCR region and the delta gene showed strong association with disease severity. The strongest association was observed with the XmnI polymorphism located 158-bp upstream to the G gamma gene (p = 4.6E-12). Carriers of the T allele of XmnI were more likely to have a milder disease course and higher level of fetal hemoglobin (HbF) in both the mild (p = 0.005) and severe (p = 8.7E-06) patient groups. Haplotype analysis revealed that the T allele of XmnI was nearly always in cis with the HbE allele. The high frequency of this haplotype may be favored by positive selection against malarial infection. Further studies are needed to validate this hypothesis and determine whether XmnI or another closely linked variant modulates severity and HbF levels in patients with beta(0)-thalassemia/HbE disease.

Curcumin contributes to in vitro removal of non-transferrin bound iron by deferiprone and desferrioxamine in thalassemic plasma.

Non-transferrin-bound iron (NTBI) is detectable in plasma of beta-thalassemia patients with transfusional iron overload. This form of iron may cause oxidative tissue damage and increased iron uptake, into several vital organs. Removal of NTBI species is incomplete and transient using standard intermittent desferrioxamine (DFO) or deferiprone (DFP) monotherapy. Combinations of these or other chelators may improve the protection time from NTBI and increase removal of harmful NTBI species. Curcuminoids from Curcuma longa L. is a naturally occurring phytochemical which shows a wide range of pharmacological properties including anti-oxidative, anti-inflammatory, anti-cancer and iron-chelating activities. In this study, the curcuminoids was investigated for NTBI chelation in thalassemic plasma in vitro and for the potential to improve NTBI removal when used with other chelators. Curcumin bound Fe(3+) to form a Fe(3+)-curcumin complex with a predominant absorption at 500 nm. The chemical binding of curcumin was dose- and time-dependent and more specific for Fe(3+) than Fe(2+). Using a HPLC-based NTBI assay without an aluminium blocking step, curcumin shuttled the iron from Fe(3+)-NTA complex, giving underestimated NTBI values. At equivalent concentrations DFO, DFP and curcumin decreased plasma NTBI with the order of DFP>DFO>curcumin. None of these chelators removed NTBI completely, but curcumin appeared to increase the rate of NTBI removal when added to DFP. It is proposed that the beta-diketo moiety of curcumin participates in the NTBI chelation.

Epigallocatechin-3-gallate and epicatechin-3-gallate from green tea decrease plasma non-transferrin bound iron and erythrocyte oxidative stress.

Beta-thalassemia patients suffer from secondary iron overload caused by increased iron absorption and multiple blood transfusions. Excessive iron catalyzes free-radical formation, causing oxidative tissue damage. Non-transferrin bound iron (NTBI) detected in thalassemic plasma is highly toxic and chelatable. Desferrioxamine and deferiprone are used to treat the iron overload, but many side effects are found. Epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) in green tea (GT) show strong antioxidant properties. We separated the EGCG and ECG from GT extract using an HPLC, and examined their iron-binding and free-radical scavenging activities. They bound Fe(3+) rapidly to form a complex with a predominant absorption at 560 nm. EGCG and ECG bound chemical Fe(3+) and chelated the NTBI in a time- and dose dependent manner. They also decreased oxidative stress in iron-treated erythrocytes. In conclusion, EGCG and ECG could be natural iron chelators that efficiently decrease the levels of NTBI and free radicals in iron overload.