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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.

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.

This research was aimed to investigate the prevalence and clinical impact of occult HBV infection in thalassemic patients with chronic HCV infection. In this cross-sectional study we have totally examined 60 patients suffering HBV and HCV infections by PCR and RT-PCR methods, respectively, in Kerman province of Iran. ELISA technique (RADIM, Italy) was used to detect anti-HBc, anti-HBs and HBsAg. The serum level of liver enzymes (SGOT, SGPT, DB, TB and ALK) were analyzed in the HCV infected patients (MAN, IRAN). Statistical analyses performed using t-test and Chi-square. We found that 27 cases (out of 60) were infected by HCV but HBV-DNA was not seen in HCV infected patients. Present findings also showed that none of samples were HBsAg positive but 9 (33%) (out of 27) HCV-RNA positive patients were anti-HBc positive and 11 (40.7%) were positive for anti-HBs. We found that SGOT, SGPT, DB, TB and ALK are above normal in 27 (100%), 19(70.3%), 12(44.5%), 15 (55.5%) and 15 (55.5%) RNA-HCV positive patients, respectively. The prevalence of hepatitis C infection is very high in thalassemic patients and based on other studies our results showed that the prevalence of HCV infection in Kerman is more than other provinces of Iran. In contrast with other studies HBV-DNA in these patients could not be detected, hence, it seems that occult HBV infection isn't frequent in Iranian thalassemic patients who suffering from chronic HCV infection.

The enzyme chitotriosidase (ChT), the human analogue of chitinases from non-vertebrate species, is one of the most abundant and indicative proteins secreted by activated macrophages. Its enzymatic activity is elevated in serum of patients suffering from Gaucher's disease type 1 and in some other inherited lysosomal storage disorders, as well as in diseases in which macrophages are activated. The last decade has witnessed the appearance of a substantial number of studies attempting to unravel its cellular functions, which have yet not been fully defined. A great deal of progress has been made in the study of the physiological roles of ChT. This review is looks at the key areas of investigations addressed to further illuminate whether ChT activation might have different functional meanings in various diseases.

Osteoporosis is an important cause of morbidity in beta-thalassemia patients. Bisphosphonates have been recently used for the treatment of osteoporosis in beta-thalassemia. This study is a prospective quasi-experimental study to assess the efficacy and safety of zoledronic acid in thalassemics with osteoporosis. Eighteen thalassemia patients with osteoporosis were given zoledronic acid 4 mg intravenously every 3 months over a period of 12 months. The efficacy of treatment was assessed by measuring (BMD) at the lumbar spine, femoral neck, and hip at baseline, 6, and 12 months. Z-score was used to measure the BMD. Other medical assessments included markers of bone formation and resorption (bone alkaline phosphatase (BAP), osteocalcin (OC), and urinary deoxypyridinoline), and the assessment of pain score, analgesic score, and performance score. Ten thalassemic osteoporotic patients were followed up only with serial BMDs as controls. Both groups had no significant difference with respect to age, gender, and baseline BMD. Patients taking zoledronic acid had a significant increase in their lumbar spine, femoral neck, trochanter, and total hip BMD measurements over the 12-month period. Patients in the control group did not have any significant change in BMD measurements. There was a significant change in the levels of OC and BAP over the 12-month follow-up period. There was also a significant decrease in the number of painful sites experienced by the patients. Treatment of thalassemic osteoporotic patients with zoledronic acid is very effective in increasing BMD at the lumbar spine and hip and in reducing pain; it is also well-tolerated.

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.

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.

In this paper we report a male infant heterozygous for thalassemia with a mild glucose 6 phosphate dehydrogenase deficiency. The molecular basis of this new Class III G6PD variant is a G-->T mutation at nucleotide 34 in the exon 2, which predicts a Val-->Leu aminoacid substitution at codon 12. We designated this variant as G6PD Sinnai from the place of birth of the propositus.

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.

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.

We have previously shown that physiologic concentrations of hemin cause marked inhibition of several red blood cell (RBC) enzymes. Because endogenous heme content is elevated in sickle RBCs, we have examined the activity of hemin-sensitive enzymes in these RBCs. One of the hemin-sensitive enzymes, pyrimidine nucleoside monophosphate kinase (PNMK), was shown to have decreased activity in sickle RBCs relative to RBCs of equivalent cell age. The other hemin-sensitive enzymes, including adenylate kinase (AK), pyrimidine 5'-nucleotidase (P5N), 6-phosphogluconate dehydrogenase (6PGD), and aldolase, had activities that were appropriate for cell age. We have also examined the affinity of the hemin-sensitive enzymes to hemin. Using two different methods, PNMK was shown to have the highest binding affinity to hemin. The exquisite sensitivity of PNMK to inhibition by hemin, coupled with the enzyme's high affinity to hemin, may account for the decrease in PNMK activity and the lack of significant decrease in the other hemin-sensitive enzymes in sickle RBCs. These results suggest that the increased endogenous heme content in sickle RBCs may be responsible for the decrease in PNMK activity. Whether the increased endogenous heme content of sickle RBCs can cause hemolysis indirectly by inhibiting RBC enzymes remains to be determined.

FAD-dependent methaemoglobin reductases (MHR) were studied in red cells in heterozygous beta-thalassaemia to investigate how they related to low FAD-dependent glutathione reductase (GR). In contrast to GR, MHR activities were usually normal or increased. In particular, whether expressed in relation to haemoglobin or number of red cells, NADPH-MHR activity was markedly increased in most subjects, probably being a response to increased oxidative stress. Oral riboflavin had no effect on MHR activities, indicating saturation with FAD even though GR was deficient. A strong correlation between percent stimulation of GR by FAD and NADPH-MHR activity indicates that FAD is utilized by MHR at the expense of GR. This could be an important influence on GR in heterozygous beta-thalassaemia. Thus, the low activity resulting from an inherited deficiency of FAD is decreased further.

Glucose-6-phosphate dehydrogenase (G6PD) has been analyzed by gel electrophoresis and by quantitative assay in an unselected sample of 1524 schoolboys from the province of Matera (Lucania) in southern Italy. We have identified 43 subjects with a G6PD variant. Of these, 31 had severe G6PD deficiency, nine had mild to moderate deficiency, and three had a non-deficient electrophoretic variant. The overall rate of G6PD deficiency was 2.6%. The frequency of G6PD deficiency, ranging from 7.2% on the Ionian Coast to zero on the eastern side of the Lucanian Apennines, appears to be inversely related to the distance of each town examined from the Ionian Coast, suggesting that this geographic distribution may reflect, at least in part, gene flow from Greek settlers. Biochemical characterization has shown that most of the G6PD deficiency in this population is accounted for by G6PD Mediterranean. In addition, we have found several examples of two other known polymorphic variants (G6PD Cagliari and G6PD A-); three new polymorphic variants, G6PD Metaponto (class III), G6PD Montalbano (class III), and G6PD Pisticci (class IV); and two sporadic variants, G6PD Tursi (class III) and G6PD Ferrandina (class II). These data provide further evidence for the marked genetic heterogeneity of G6PD deficiency within a relatively narrow geographic area and they prove the presence in the Italian peninsula of a gene (GdA-) regarded as characteristically African.

We have examined aspects of methemoglobin (metHb) reduction in sickle and in thalassemic red blood cells (RBCs). NADH metHb reductase activity in sickle and thalassemic RBCs was significantly increased compared with normal RBCs. Because in vitro enzyme activity does not necessarily represent in vivo activity, we measured the rate of metHb reduction in intact RBCs. Intact thalassemic RBCs demonstrated a significantly increased rate of metHb reduction compared with normal RBCs. In contrast, intact sickle RBCs had a rate of metHb reduction that was similar to normal RBCs and significantly decreased relative to high reticulocyte RBCs of equivalent cell age. To determine the mechanism for the relative impairment of metHb reduction in sickle RBCs, we measured intraerythrocytic NADH, a cofactor in the metHb reduction reaction. Thalassemic RBCs had a significantly increased NADH content relative to normal RBCs. In contrast, sickle RBCs did not have an increase in NADH content. Furthermore, incubating normal RBCs under conditions that increase the NADH content resulted in an increased rate of metHb reduction. In contrast, conditions that decrease the NADH content in normal RBC resulted in a decreased rate of metHb reduction. These data and other results suggest that metHb reduction in intact RBCs is dependent on NADH content, and that the impaired metHb reduction rate in sickle RBCs may be a result of a lack of increase in NADH content. The dependence of metHb reduction on RBC NADH content and the ability to manipulate NADH content in vitro suggest a new strategy for decreasing oxidant damage to sickle RBCs in vivo.

A new differential pH technique for glucose-6-phosphate dehydrogenase quantitative determination in whole blood has been evaluated. It is a rapid (90 s/analysis), reproducible (C.V. within-run 3.7%; between-run: 2.8%) and accurate method (in comparison with WHO method: r = 0.970). Reference intervals in non-deficient males were evaluated in 167 non-thalassaemics and in 60 beta-thal heterozygotes. The G6PD activity in beta-thalassaemia carriers is higher than in normals; this is particularly true if the activity is expressed in terms of U/g Hb. The phenotypic distribution measured in females is in agreement with that calculated by the Hardy-Weinberg law based on the incidence of the Gd(-) gene in males.

The activity of 18 red blood cell (RBC) enzymes and reduced glutathione (GSH) content were measured in 70 normal subjects, in 50 heterozygous beta-thalassaemia carriers and in 50 non-thalassaemic patients with haemolytic anaemia and high reticulocyte counts. In addition, pyrimidine 5'nucleotidase (P5N) activity was also determined in 34 patients with hypochromic, microcytic, iron deficiency anaemia. beta-Thalassaemia trait was associated with an increase in almost all of the enzyme activities, except for 2,3-bisphosphoglycerate synthetase (BPGS) and glutathione reductase (GR) which were normal and for acetylcholinesterase (AChE) and P5N which were slightly and markedly decreased respectively. The increases in enzyme activities were similar to those observed in patients with non-thalassaemic reticulocytosis except for glyceraldehydephosphate dehydrogenase (GAPD), phosphoglyceratekinase (PGK), pyruvate kinase (PK), glutathione peroxidase (GPX) and adenylate kinase (AK) which were higher than in non-thalassaemic group of patients with increased number of reticulocytes. No correlation was found between the severity of P5N deficiency and the intensity of basophilic stippling which was present in 46 of 50 thalassaemic carriers here studied. In addition, GSH content and UV absorption spectra of deproteinized thalassaemic RBC extracts were also found to be normal. The present findings provide further information on the metabolic status of RBC in beta-thalassaemia trait and suggest a possible molecular explanation for the frequently observed basophilic stippling in this disease.

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.

We present a new abnormality of the red cell membrane observed in the course of a study on beta-thalassemia. In a child suffering from a beta 0-thalassemia major, the neutral membrane bound phosphatase, instead of having Michaelis-Menten kinetics, displayed a biphasic kinetics consistent with inhibition by substrate excess. This abnormality is discussed along with other erythrocyte membrane alterations that are known to occur in beta-thalassemia.

The European standardized method for measuring porphobilinogen synthase (PBGS, EC 4.2.1.24) activity in peripheral erythrocytes is widely used in epidemiological studies of low-level Pb absorption. We have modified it to include activation with dithiothreitol (DTT). In children with concentrations of Pb in whole blood ranging from 0.29 to 1.93 mumol/L, we found the following statistically significant correlations (r): blood Pb vs nonactivated PBGS activity = -0.773 (p less than 0.005); blood Pb vs DTT-activated PBGS activity = -0.265 (p less than 0.005); blood Pb vs the ratio of DTT-activated/nonactivated PBGS activity = +0.818 (p less than 0.005). At a DTT-activated/nonactivated PBGS activity ratio of 1, which indicates no Pb-dependent inhibition of PBGS, a corresponding blood Pb concentration of 0.24 mumol/L (50 micrograms/L) would be predicted. In individuals with hematological disorders, the DTT-activated/nonactivated ratio better predicted blood concentration of Pb than did the nonactivated PBGS activity, and largely compensated for the high variability in PBGS activity. We also describe techniques to minimize contamination by Zn and Pb and to stabilize enzyme activity during collection, transport, storage, and analysis of samples.

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.

A total of 1,112 randomly selected Saudi Arabs, of both sexes, living in Jeddah and the surrounding areas were screened for the phenotypic distribution of red cell glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD). They were also investigated for haemoglobin and for thalassaemia. Phenotyping of the haemoglobins and the red cell enzymes was carried out by starch gel electrophoresis and the dye-decolouration screening test, while the investigation for thalassaemia was carried out by globin-chain biosynthesis, followed by column chromatography. The red cell Gd- alleles were significantly associated with the sickle-cell gene in both the males (chi 2(1): AS-28.80; SS-4.89) and females (chi 2(1): AS-10.99; SS-13.16). A similar association was also observed between G6PD deficiency and thalassaemias in males (chi 2(1): alpha-thalassaemia - 3.13; beta-thalassaemia - 11.06) and females (chi 2(1): alpha-thalassaemia - 6.63). However, no such association was detected between red cell 6PGD types and haemoglobin genes. The results suggest that the red cell G6PD deficiency, sickle-cell and thalassaemia genes might have evolved as a result of the same ecological factor, probably malaria.

Per cent stimulation of GR activity by FAD in vitro and PNP oxidase activity were measured in G6PD deficiency, heterozygous beta-thalassaemia and controls. It is confirmed that, in contrast to the high stimulation of GR by FAD commonly found in in thalassaemia indicating red-cell deficiency of FAD, and shown here to be greater in the Italian subjects, GR is usually saturated with FAD in G6PD deficiency, leading to high in vitro activity. Unexpectedly, on the other hand, low FMN-dependent PNP oxidase activity due to red-cell deficiency of FMN, confirmed by response to oral riboflavin, was found in the majority of subjects with G6PD deficiency, similar to that found in heterozygous beta-thalassaemia. Whereas this is explained in thalassaemia by an inherited slow red-cell metabolism of riboflavin to FMN, it is suggested that in G6PD deficiency an increased rate of red-cell metabolism of FMN to FAD leads to the low FMN and high FAD. When G6PD deficiency occurs with heterozygous beta-thalassaemia, GR is usually saturated with FAD as in G6PD deficiency alone, unless there is an inherited, very slow red-cell metabolism of riboflavin to FMN. The part played by GR in haemolytic crises in G6PD deficiency is discussed.

5-Aminolaevulinic acid (ALA) synthase activity was measured in highly purified preparations of age-matched human erythroblasts. Enzyme activity in immature normoblasts was four-fold higher than that found in late orthochromatic normoblasts. ALA synthase activity in the immature erythroblasts in primary acquired sideroblastic anaemia (PASA) was reduced and remained unchanged during further erythroid differentiation. The pattern of erythroblast ALA synthase activity in two patients with congenital dyserythropoietic anaemia (CDA) and in one patient with beta-thalassaemia intermedia was similar to that found in PASA. This study has clearly demonstrated reduced erythroblast ALA synthase activity in PASA but has also found reduced enzyme activity in conditions in which ring sideroblasts are not prominent. This would suggest that haem synthesis is abnormal in PASA but that reduced erythroblast ALA synthase activity does not inevitably lead to ring sideroblast formation.

A physiological role for glycosidases in cell membranes has been suggested. Therefore the activities of four glycosidases--beta-galactosidase, beta-glucoronidase, N-acetyl-beta-glucosaminidase and acid phosphatase--were examined in normoblasts and membranes of red blood cells (RBC). The enzymatic assays were based on the hydrolysis of fluorimetric 4-methylumbelliferone from the enzyme substrate. In order to avoid contamination by lysosomal activities derived from RBC, the mature RBC and normoblasts obtained from normal controls and thalassemic patients were separated from other blood elements by cellulose chromatography. The cells were disrupted and lysed by freezing and thawing hypotonic solution. Higher enzymatic activities were found in preparations from thalassemic patients than from normal subjects. With a sucrose density gradient, further separation of normoblasts from RBC membranes was obtained, indicating that the normoblast fraction contributed most of the high specific activity found in the thalassemic preparation. It was concluded that relatively high glycosidase activities are present in normoblasts of thalassemic patients. Lower but significant activities were detected in RBC membranes of normal control subjects and thalassemic patients.

We studied the red cell membrane neutral phosphatase, which is part of the Na+K+ ATPase, in several types of oxidative hemolytic anemias. We used an artificial substrate, the p-nitrophenylphosphate. In controls and in patients heterozygous for various unstable hemoglobins (Hb Hope, Hb Köln, or Hb Hammersmith), the kinetics were of the Michaelis-Menten type. On the contrary, in nearly all patients with alpha- or beta-thalassemia, the kinetics displayed an abnormally biphasic character. The apparent Michaelis constant (KMapp) was significantly decreased. The biphasic character correlated with the imbalance of globin chain synthesis. The beta-mercaptoethanol markedly increased Vmax in controls, but had little effect on the biphasic kinetics. Omission of K+ abolished the biphasic kinetics. The abnormal kinetics failed to appear with another artificial substrate, the 4-methylumbelliferylphosphate, nor did it appear with ATP, the natural substrate. In vitro, H2O2 treatment of normal and thalassemic red cells was unable to induce or exaggerate, respectively, the biphasic kinetics, but generated alterations of a different nature. We suggest that the various kinetic alterations of the phosphatase in thalassemic syndromes originate from the imbalance of globin chain synthesis. However, the involvement of an oxidative process remains to be demonstrated.

Thalassemia is characterized by unequal rates of synthesis of the alpha and beta globin chains that are part of the hemoglobin tetramer. In the type of thalassemia due to a defect in beta-chain synthesis (beta-thalassemia), this imbalance results in a relative exoess of alpha-chains. We have studied the susceptibility of excess free alpha-chains to proteolysis. Incubation of isotopically labeled peripheral blood lysates from individuals with beta-thalassemia trait in the presence of bone marrow or normoblast lysates from thalassemic or hematologically normal individuals resulted in a decrease in the alpha/beta ratio and a loss of free alpha-chain radioactivity. Neither contamination with leukocytes nor higher ATP contents in young erythrocytes appeared to be responsible for this activity in normoblasts and bone marrow. We propose that erythroid precursor cells possess proteolytic activity that is markedly diminished in mature cells. This activity serves an important control function in the regulation of hemoglobin synthesis. It accounts at least in part for the more balanced synthesis of alpha- and beta-chains observed in bone marrow than in peripheral blood in heterozygous beta-thalassemia. It also plays a fine-tuning role in maintaining balanced synthesis in non-thalassemic erythrocytes.

Adenosine triphosphate (ATP) and adenosine diphosphate levels are decreased in erythrocytes from individuals with beta-thalassemia minor. Because 5-phosphoribosyl-1-pyrophosphate (PRPP) is an essential precurosr of adenine nucleotides, we tested the hypothesis that impaired PRPP synthesis is a mechanism for the decreased adenine nucleotide content. Erythrocyte PRPP synthetase activity was significantly decreased, and the Michaelis-Menten constant (Km) for ribose-5-phosphate (R5P) was significantly increased in individuals with alpha-thalassemia minor and those with beta-thalassemia minor. Intact erythrocytes from individuals with alpha-thalassemia and those with beta-thalassemia minor also had an impaired rate of PRPP formation. Both the decrease in PRPP synthetase activity and the impaired PRPP formation were also found in erythrocytes with microcytosis resulting from iron deficiency, indicating that these phenomena may not be specific to thalassemia minor. In all individuals examined, the rate of PRPP formation correlated with ATP content, suggesting that either (1) PRPP synthetase activity is a determinant of ATP content or (2) ATP content is a determinant of PRPP synthetase activity. The depletion of ATP from normal erythrocytes did not affect PRPP synthetase activity, suggesting that ATP content is not a determinant of PRPP synthetase activity. However, a decrease in PRPP synthetase activity did cause an impairment in the rate of adenine nucleotide synthesis, suggesting that PRPP synthetase activity is a determinant of ATP content. Taken together, our results suggest that the decrease in PRPP synthetase activity and the resulting impairment in the rate of PRPP formation are mechanisms for the decreased adenine nucleotide content in thalassemic erythrocytes.

The applicability to red blood cells of two widely used methods for spectrophotometric assay of superoxide dismutase activity has been tested, in view of the demand for routine screening of the level of this enzyme in various circumstances of hematological interest. The nitro blue tetrazolium reduction method was found to be inapplicable, even after removal of hemoglobin by chloroform-ethanol extraction. On the other hand, the epinephrine oxidation method gave reliable values after chloroform-ethanol extraction. The enzyme content in normal adults was found to be 6.2 +/- 1.4 X 10(15) gr. per red blood cell. No significant change in the enzyme content was found in 19 patients with beta-thalassemia major and 5 patients with beta-thalassemia intermedia, indicating no influence on the level of this enzyme by the increased flux of oxygen radicals which is to be expected in thalassemic red blood cells.

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.

Pyrimidine 5'-nucleotidase (P5'N) partial deficiency has been described in several hematological disorders and also in the beta-thalassemic trait. To check if the P5'N deficiency in thalassemia was acquired we used thalassemic red cells (from either homo- or heterozygous subjects), whose P5'N activity was lower than in control cells. After separation on a density gradient, activity in lighter cells was similar to controls and less than 50% in denser cells. The most probable mechanism for this faster inactivation involves enzyme -SH groups modification by oxidation and reaction with monofunctional aldehydes produced by membrane lipid peroxidation. In vitro challenge of thiol enzymes as pyruvate kinase (PK), adenylate kinase (AK) and P5'N with increasing concentrations of GSSG, hexanal (HEX) and 4-hydroxynonenal (HNE), showed that HNE is the most powerful among the enzyme inhibitors tested and that P5'N activity is a more sensitive index of -SH groups damage, when compared to PK and AK.

A child with triose phosphate isomerase deficiency has congenital nonspherocytic hemolytic anemia, mental subnormality, motor impairment, growth failure, and cardiac failure. The deficiency state is characterized by moderately reduced red cell triose phosphate isomerase activity and marked instability of the abnormal enzyme to heat. The stability characteristics of triose phosphate isomerase in cultured fibroblasts define the homozygous and heterozygous states with sufficient precision to allow prenatal diagnosis of the disorder. Successful prenatal identification of a heterozygote and an unaffected fetus in utero is described.

The 'antioxidant' enzymes superoxide dismutase (SD), catalase and glutathione peroxidase (GSH-Px) were found greatly elevated in red blood cells of subjects with beta-thalassaemia minor and similar to normal values in red blood cells of subjects with beta-thalassaemia major. These findings allows us to speculate that red cells in beta-thalassaemia minor react to the increased oxidant threat with augmented antioxidant enzyme activities. The normal levels of antioxidant enzymes in beta-thalassaemia major seem to be due to the presence of normal red cells owing to multiple transfusions.

We have recently demonstrated that phosphoribosylpyrophosphate (PRPP) synthetase activity is decreased in RBC from individuals with thalassemia minor. Because NAD biosynthesis requires PRPP, the product of the PRPP synthetase reaction, we have investigated NAD synthesis in thalassemic RBC. NAD synthesis was measured in intact RBC both by following the accumulation of unlabeled NAD and by following the incorporation of 14C-nicotinic acid into NAD. Using both assay systems, we demonstrate that NAD synthesis is decreased significantly in thalassemic RBC compared to either normal or high reticulocyte red cells. Although this suggested that NAD content should be decreased in thalassemic RBC, no significant difference in NAD content was found among thalassemic, normal, or high reticulocyte red cells. Mechanisms for the lack of a significant decrease in NAD content in thalassemic RBC are discussed. These results indicate that NAD synthesis is impaired in thalassemic RBC possibly as a result of their decrease in PRPP synthetase activity. Our data provide evidence that thalassemic RBC have secondary metabolic abnormalities in addition to their primary defect in hemoglobin synthesis.

Whole blood glutathione peroxidase was found significantly increased relative to controls in a group of 21 black patients with sickle cell anaemia. One control group consisted of 15 normal black subjects. A second control group, consisting of 21 black patients with various abnormal haemoglobins including alpha-thalassaemia, also showed a tendency to enhanced glutathione peroxidase activity, confirming previous reports that elevated glutathione peroxidase levels are secondary to a variety of haemolytic conditions, rather than typical of sickle cell anaemia.

Red cell acid phosphatase polymorphism was studied by starch gel electrophoresis in 70 b-thalassemia patients and in 310 healthy Greeks. Our results gave the following gene frequencies; b-thalassemia patients: pa 0.321, pb 0.643, pc 0.036; healthy Greeks: pa 0.302, p b 0.653, pc 0.045. No statistically significant differences were found between the two groups.

Human reticulocytes obtained from patients suffering from various haemolytic disorders convert exogenous [1-14C]-arachidonic acid to 15-hydroxy-5,8,11,13(Z,Z,Z,E)-eicosatetraenoic acid (15-HETE). Immunological studies (dot blot, Western blot) indicated that human reticulocytes contain a lipoxygenase which cross-reacts with a polyclonal antiserum against the rabbit reticulocyte lipoxygenase. Northern blotting with a cloned lipoxygenase cDNA probe shows that the specific mRNA is also present. Reaction of the lipoxygenase with submitochondrial particles caused inactivation of respiratory enzymes. The occurrence of an erythroid cell specific lipoxygenase of similar type in reticulocytes of various mammals and man suggests the general role of this enzyme in the maturational degradation of mitochondria.

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.

We undertook a four year study of 128 thalassaemic patients who had undergone several transfusions, to determine the incidence of hepatitis B virus markers and the activities of transaminases in their sera each month. The results showed that the possibility of these patients contracting hepatitis B virus infection is still high, although on only one occasion was a transient antigenaemia found, indicating low viral replication. Furthermore, the probability of contact with hepatitis B virus increases with the number of transfusions and, therefore, with age. About 25% of these patients were positive for hepatitis B markers and 80% for other hepatitis markers including the case of cytomegalovirus hepatitis.

The haem pathway enzyme uroporphyrinogen-I-synthase (UPGS) was assayed in erythrocyte samples from twenty normal, twenty beta-thalassaemia heterozygotic and twenty beta-thalassaemia homozygotic subjects, after partial separation of the erythrocytes according to their age. UPGS erythrocyte enzyme concentration activity was significantly higher in the young than in the old erythrocytes of normal (66.5 +/- 11.8 v. 45 +/- 9.5 nmol h-1 1(-1), mean +/- SD, P less than 0.001) and beta-thalassaemia heterozygotic subjects (70.1 +/- 18.7 v. 49.8 +/- 14.5 nmol h-1 1(-1), P less than 0.001), but not in patients with homozygous beta-thalassaemia (46.0 +/- 12.8 v. 44.1 +/- 12.5 nmol h-1 1(-1), P = 0.65). Furthermore, UPGS enzyme concentration of both young and old erythrocytes of homozygous beta-thalassaemia was significantly lower than that of the young (P less than 0.001) but similar to that of the old (P greater than 0.2) erythrocytes of either normal or beta-thalassaemia heterozygotic subjects. Since severe chronic haemolysis due to haemoglobinopathies is associated with increased UPGS enzyme concentration, these results suggest that UPGS activity may be suppressed in homozygous beta-thalassaemia.

GPT polymorphism was studied in 500 voluntary blood donors from the Bologna population. The following phenotype frequencies were obtained: GPT 1 = 29.60%, GPT 1-2 = 49.80% and GPT 2 = 20.60%. The frequencies of the alleles were: GPT1 - 0.545 and GPT2 = 0.455. Analysis of 24 informative families has excluded linkage between GPT and beta-thalassaemia.