Status of oxidant, antioxidantand serum enzymes in thalassaemic children receiving multiple blood transfusions.

OBJECTIVE: To determine the levels of oxidant, antioxidant and serum enzymes in thalassaemic children receiving multiple blood transfusions. METHODS: The case-control study was done from February to August 2012, and comprised thalassaemic children receiving multiple blood transfusions at Allied Hospital, Ali Zeb Foundation, and the Thalassaemia Centre in Hilal-e-Ahmar Hospital, Faisalabad, Pakistan. Healthy subjects were also screened for any related disease condition that could prejudice the results. Blood samples were analysed for the values of total oxidant status, total antioxidant capacity, serum malondialdehyde, catalase, paraoxonase, arylesterase, glutathione peroxidase and ceruloplasmin. RESULTS: There were 180 children in the study; 90(50%) cases and 90(50%) controls. Of the cases, 48(53.3%) were under-weight while the weight of 42(46.7%) was in the normal range. The values of total oxidant status and total antioxidant capacity were significantly (p<0.01) higher in thalassaemic children compared to normal values. Serum malondialdehyde and catalase levels were also considerably elevated (p<0.05), suggesting the increased activity of these enzymes. However, the concentrations of serum paraoxonase, arylesterase, glutathione peroxidase were significantly (p<0.01) lower in cases than the controls, displaying diminished activities during multiple blood transfusions in these patients. CONCLUSIONS: Multiple blood transfusions disconcert the levels of oxidants, antioxidants and serum enzymes of thalassaemic children. Oxidative damage is seen because of the increased iron overload in these patients. Hence, regular evaluation of oxidant and antioxidant status should be monitored in thalassaemic patients during initial few years of life.

Cholelithiasis in thalassemia major.

OBJECTIVES: Aim of this study was to evaluate prevalence and characteristics of cholelithiasis in a large population of patients with thalassemia major (TM). METHODS: Data from 858 consecutive patients with transfusion-dependent thalassemia at five major Italian centers were analyzed. In these centers, a complete abdomen ultrasonography is performed yearly after the beginning of the transfusion regimen. The role of co-inheriting Gilbert's syndrome genotype was investigated studying the promoter region of the UGT1-A1 gene by automated sequencing. RESULTS: Thirty percent of TM patients had gallstones. The Gilbert's genotype [homozygosity for (TA)(7) motif at UGT1A promoter gene], influenced both the prevalence of cholelithiasis and the age at which it developed. CONCLUSIONS: Cholelithiasis has a remarkable frequency and precocity in patients with TM and especially in those with (TA)(7)/(TA)(7) UGT1-A1 genotype. An early biliary ultrasonography is recommended from childhood and a closer follow-up in patients with thalassemia and associated Gilbert's syndrome may be indicated.

Preliminary study of the effect of vitamin E supplementation on the antioxidant status of hemoglobin-E carriers.

The antioxidant status of hemoglobin-E carriers was studied pre- and post-treatment with vitamin E for 3 months. Fourteen hemoglobin-E carriers (age = 21.36 +/- 1.08 years, BMI = 18.32 +/- 1.22 kg/m2) were treated with vitamin E 200 I.U. daily for 3 months. Fasting blood samples were collected and analyzed for erythrocyte superoxide dismutase activity, total antioxidant activity, hemoglobin concentration, hematocrit, MCV, Heinz body formation and osmotic fragility test. The blood parameters before and after vitamin E treatment were compared. The results showed that superoxide dismutase activity in the erythrocytes was significantly decreased, while total antioxidant activity in plasma, and the osmotic fragility of the erythrocytes, was significantly increased after vitamin E supplementation. However, hematocrit, MCV, and Heinz body formation did not change significantly. This demonstrated that vitamin E 200 IU could be used as a lipophilic antioxidant in red blood cells and could help increase the level of antioxidant in hemoglobin-E carriers.

Unique profile for erythrocyte membrane acetylcholinesterase in hereditary spherocytosis.

Acetylcholinesterase of human erythrocytes from healthy donors and from patients with hematological disorders was analysed in a search for differential membrane parameters. Two substrates were used to estimate the exposure of acetylcholinesterase active site in the membrane: phenylacetate, a hydrophobic substrate, to determine total enzyme activity, and acetylcholine, an ionic substrate, to measure the externally reactive enzyme. The sensitivity of acetylcholinesterase to added stearic acid was also analysed. Three categories of the disorders studied were discerned: (a) The erythrocyte acetylcholinesterase profile was indistinguishable from normal control in beta-thalassemia minor and groups of patients with autoimmune hemolytic anemia or congenital dyserythropoietic anemia type II. (b) A marked decline in acetylcholinesterase with both substrates and reduced sensitivity to stearic acid were exhibited by the erythrocytes of paroxysmal nocturnal hemoglobinuria, beta-thalassemia major and other autoimmune hemolytic anemia and congenital dyserythropoietic anemia type II patients. Normal erythrocytes, either aged or pretreated to 50 degrees C, also showed similar characteristics. (c) Hereditary spherocytosis was singly differentiated by an elevated acetylcholinesterase activity with acetylthiocholine and by a vastly diminished sensitivity to stearic acid, while activity with phenylacetate was equal to control. This distinct profile may reflect the unique organization of the erythrocyte membrane in hereditary spherocytosis.

A modified screening procedure to detect pyruvate kinase deficiency.

Beutler's screening procedure was used to detect pyruvate kinase deficiency in the local population. In this test, hemolysate and the reagent mixture are incubated and then placed at a spot on filter paper to be examined for fluorescence. Complete nonfluorescence marks the reaction endpoint, and fluorescence beyond 30 minutes indicates pyruvate kinase deficiency. It was difficult to determine this endpoint due to uneven sedimentation of unhemolyzed red cells on the spot. In this modified technique, the leukocyte-depleted red cell suspension was frozen and thawed for complete red cell lysis before being used for the test. Using both techniques, 493 health individuals and 126 anemic patients were screened for pyruvate kinase deficiency. By the conventional technique, 3.7% remained fluorescent after 30 minutes, whereas by the modified technique, none were fluorescent after 30 minutes. Quantitative assay indicated that all samples had pyruvate kinase activity levels greater than the lower limit of the reference range. We also demonstrated that blood samples from individuals with thalassemia trait were primarily responsible for the aberrant results from the conventional screening procedure.

Serum immunoreactive trypsin in beta-thalassaemia major.

To assess the exocrine pancreatic function in beta-thalassemia major with iron overload, serum immunoreactive trypsin (IRT) was measured in 38 patients with this condition. In 23 (60%) patients' IRT was abnormal: it was subnormal in 16 patients and supranormal in seven. Whereas subnormal IRT concentrations were more frequent in patients of more than 12 years old, supranormal IRT concentrations were more frequent in younger patients. These data provide the first antemortem evidence of exocrine pancreatic damage in this condition. They also suggest that this acinar cell damage is initially associated with a rise in IRT which is followed by a fall in the serum concentration of this enzyme.

Comparison of erythrocyte antioxidative enzyme activities between two types of haemoglobin H disease.

The activities of erythrocyte antioxidative enzymes were measured in two groups of patients with different genotypes of haemoglobin (Hb) H disease: 21 with alpha-thalassaemia 1 or alpha-thalassaemia 2 (alpha-thalassaemia 1/2) and 21 with alpha-thalassaemia 1/Hb Constant Spring (HbCS). They were compared with 21 normal subjects. Both genotypes of Hb H disease had increased activities of erythrocyte superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase when compared with those of controls. Comparison of the two genotypes showed that subjects with alpha-thalassaemia 1/Hb CS, the more severe disease, had higher SOD and GSH-Px activities but lower catalase activity than those with alpha-thalassaemia 1/2. This indicates that there are compensatory mechanisms in Hb H erythrocytes to cope with increased generation of oxygen free radicals as a result of increased excess beta chain.

Iron state in alpha and beta thalassaemia trait.

The iron state was examined in two groups of pregnant women who were carriers of alpha and beta thalassaemia genes. In both groups the haematological expression of the disease--namely, haemoglobin and mean cell haemoglobin concentrations--was nearly identical. In patients with alpha thalassaemia the ferritin concentrations and percentage of ferritin deficiency was the same as in normal pregnant patients, whereas in those with beta thalassaemia the ferritin concentrations were usually much higher and iron deficiency four times less common. This variance appears to be explained by different degrees of extravascular or intravascular haemolysis between the two thalassaemias as assessed by the activities of serum alpha hydroxybutyrate dehydrogenase.

Increased erythrocyte superoxide dismutase activities in beta 0-thalassaemia/haemoglobin E and in haemoglobin H diseases.

Erythrocyte superoxide dismutase activities were measured in 45 subjects, 15 each of beta 0-thalassaemia/haemoglobin (Hb) E disease, Hb H disease, and normal. The erythrocyte superoxide dismutase activities were significantly higher in the patients with beta 0-thalassaemia/Hb E and Hb H diseases than in the normal subjects. The increase of erythrocyte superoxide dismutase activities is most likely due to abnormalities specific to thalassaemic red cells rather than an increased number of younger red cells for reticulocytes and nucleated red blood cells did not affect the enzyme activity. Patients with beta 0-thalassaemia/Hb E disease with lower haemoglobin concentration had significantly higher superoxide dismutase activities. In all 45 subjects haemoglobin concentrations and superoxide dismutase activities were inversely correlated (r = -0.60 (p less than 0.001)). This indicates that the amounts of superoxide generated in the red cells may, at least partly, determine severity of red cell damage and thus severity of disease; the increased superoxide dismutase activity in thalassaemia is a response to superoxide generated in greater amounts because of accumulation of excessive globin chains and iron in the red cells. The superoxide dismutase activities in Hb H disease, an alpha-thalassaemic disease, were found to be strikingly increased, higher than in beta 0-thalassaemic disease or other conditions.

G6PD/PK ratio: a reliable parameter to identify glucose-6-phosphate dehydrogenase deficiency associated with microcytic anemia in heterozygous subjects.

OBJECTIVES: To determine if measuring the ratio of glucose-6-phosphate dehydrogenase (G6PD) to pyruvate kinase (PK) is more reliable than only measuring G6PD activity to identify heterozygous G6PD- individuals with associated microcytic anemia in the Calabrian population, which shows high frequencies of both the thalassaemia (thal) trait and G6PD deficiency. DESIGN AND METHODS: Measurement of G6PD and PK activities was carried out on 205 samples of whole blood from Calabrian subjects of both sexes (age range 10-50 years) using a double starter differential pH-metry technique. RESULTS: The G6PD/PK ratio is able to differentiate G6PD- heterozygous individuals from the normal population. G6PD/PK values also allowed us to easily identify the G6PD- heterozygous subjects with microcytic anaemia. Student's t test shows that G6PD/PK ratio is more reliable in both sample groups, relative to G6PD activity in normal subjects. CONCLUSIONS: G6PD/PK ratio is a reliable diagnostic parameter for mass screening for G6PD deficiency.

"Lysosomal" enzyme activities in red blood cells of normal individuals and patients with homozygous beta-thalassaemia.

Four hydrolases, beta-galactosidase, beta-glucuronidase, beta-N-acetylglucosaminidase and acid phosphatase were examined in red blood cells (RBC) of normal donors and patients with homozygous beta-thalassaemia. Highly sensitive fluorimetric substrates were used to determine the specific activities of these enzymes. In order to avoid contamination by lysosomal activities derived from white blood cells (WBC), the mature RBV were separated from other blood elements by cellulose chromatography. The hydrolase activities in normal RBC were detected only in their plasma membranes and were found to be considerably lower than in WBC or platelets. In thalassaemic RBC, hydrolase activities were present in both plasma membranes and in the soluble fraction. The normoblast fraction contributed most of the hydrolase activity found in these preparations, suggesting the presence of lysosomal particles in thalassaemic RBC. No differences in the enzymatic activities were found when purified membranes of mature RBC from thalassemic and normal preparations were compared. The origin and roles of these hydrolytic enzymes in normal and thalassaemic RBC membranes are not known.

Prevalences of thalassemia/hemoglobinopathies and G-6-PD deficiency in malaria patients.

It seems that thalassemia and/or hemoglobinopathies and glucose-6-phosphate dehydrogenase deficiency (G-6-PD) have some protective effects against malaria infection. To verify this, hemoglobin typing and methehoglobin reduction test were performed on 115 malaria patients and compared with controls. It was found that the number of thalassemia/hemoglobinopathies in the malaria group and in the control group were not significantly different and also occurrence of G-6-PD deficiency in the malaria group was not different from that of the controls. Clinical manifestations of malaria in any group are quite similar. It is concluded that there is no protective effect against malaria in thalassemia/hemoglobinopathies or G-6-PD deficiency.

[The glucose-6-phosphate dehydrogenase/6-phosphogluconate dehydrogenase ratio in the identification of glucose-6-phosphate dehydrogenase heterozygosity]. / Il rapporto G-6PD/6-PGD nella identificazione dell' eterozigosi per la G-6PD.

Deficiency in human G-6PD is a widespread X-linked disorder, which is mainly characterized by susceptibility to hemolytic anaemia after the ingestion of certain drugs or toxic substances (e.g. pyrimidine derivates contained in fava beans). G-6PD deficiency in hemizygous males in easily detectable since enzymatic activity is almost absent. In heterozygous subjects the determination of enzymatic activity on red cell lysate cannot detect a partial G-6PD deficiency. Cytochemical methods as methemoglobin reduction test or tetrazolium reduction test are more sensitive than spectrophotometric quantitative test, but are not suitable for screening purposes. We measured both G-6PD activity and 6-PGD activity in G-6PD heterozygous females and we evaluated the G-6PD/6PGD ratio. We tested this ratio also in thalassemic traits and in G-6PD heterozygotes with thalassemic trait in order to detect the interference of thalassemic pathology with the phenotypic expression of the gene for G-6PD. We found that the mean G-6PD values were statistically reduced in G-6PD heterozygous females; on the contrary the measurement of true G-6PD activity alone is not a good tool for discriminating heterozygous subjects with and without thalassemic trait. Actually 100% and 79% of values observed were in the normal range +/- 2 DS respectively. The mean G-6PD/6-PGD ratio in heterozygotes for G-6PD deficiency with and without thalassemic trait was lower than normal and the individual values of G-6PD/6-PGD ratio were in the normal range +/- 2 DS only in a few subjects (8.3% and 10.7% respectively).(ABSTRACT TRUNCATED AT 250 WORDS)