Glucose-6-phosphate dehydrogenase deficiency in Italian blood donors: prevalence and molecular defect characterization.

BACKGROUND: In the countries with high G6PD deficiency prevalence, blood donors are not routinely screened for this genetic defect. G6PD deficiency is often asymptomatic, blood donors may be carriers of the deficiency without being aware of it. The aim of the study was to evaluate the prevalence of G6PD deficiency among the Italian blood donors. DESIGN AND METHODS: From October 2009 to April 2011, 3004 blood donors from a large hospital transfusion centre were screened for G6PD deficiency using differential pH-metry and the characterization of G6PD mutations was performed on G6PD-deficient subjects. The haematological features of G6PD-deficient and normal donors were also compared. RESULTS: Thirty-three subjects (25 men and 8 women) with low G6PD activity were identified, corresponding to 1·1% of the examined blood donor population. The frequencies of class II severe alleles (Mediterranean, Valladolid, Chatham and Cassano) and class III mild alleles (Seattle, A- and Neapolis) were 48% and 43%, respectively. The haematological parameters of G6PD- donors were within normal range; however, the comparison between normal and G6PD- class II donors showed significant differences. CONCLUSION: In Italy, the presence of blood donors with G6PD deficiency is not a rare event and the class II severe variants are frequent. The identification of G6PD-deficient donors and the characterization of the molecular variants would prevent the use of G6PD-deficient RBC units when the haemolytic complications could be relevant especially for high risk patients as premature infants and neonates and patients with sickle cell disease submitted to multiple transfusions.

The effects of insulin and insulin-like growth factors on tumor vascularization: new insights of insulin-like growth factor family in cancer.

It is currently believed that the development of a clinically relevant tumor needs new vessel formation provided by both angiogenesis (primary involving endothelial cells) and postnatal vasculogenesis (primary involving bone marrow-derived cells). Clearly, it is important to identify factors that help to enhance the growth and "health" of tumors, as well as their further vascularization. The Insulin and Insulin-like Growth Factors (IGFs) systems play a key role in cellular metabolism, differentiation, proliferation, transformation and apoptosis, during normal and malignant growth. Moreover, these molecules seem essential in promoting tumor vascularization. Due to the complexity of these systems, the review has been focused on the role of insulin and IGFs signaling in the regulation of tumor angiogenesis and postnatal vasculogenesis. Since targeting on IGF for cancer therapy is rapidly becoming a clinical reality, a better understanding of IGFs-mediated pathways has a relevant impact, providing new insights to help the design of newly developed drugs.

6-Phosphogluconate dehydrogenase deficiency in an Italian family.

A rare case of hereditary erythrocyte enzymopathy, namely 6-phosphogluconate dehydrogenase (6PGD) deficiency, was found in an Italian family. The activity of the enzyme was reduced to 35% in the propositus and her mother, but was normal in the other three members of the family. The 6PGD deficiency was associated with a variable reticulocyte count and recurrent increased unconjugated bilirubinemia without anemia in the propositus, while no clinical or hematological symptoms were evident in her mother. Increased levels of erythrocyte pyruvate kinase (PK) activity and reduced glutathione (GSH) were observed, indicating a slight decrease in mean red blood cell (RBC) age and an activation of reducing systems. The episodic hemolytic events with jaundice observed in the propositus may be the result of a defective RBC ability to counteract conditions of marked oxidative stress. In this report the importance of 6PGD estimation for a proper analysis of glucose-6-phosphate dehydrogenase (G6PD) deficiency is also highlighted. In fact in the present study, the presence of 6PGD deficiency could be mistaken for a partial G6PD deficiency if the assay of G6PD activity was performed without correcting for 6PGD activity.

Acute and long-term effects of low-density lipoprotein (LDL)-apheresis on oxidative damage to LDL and reducing capacity of erythrocytes in patients with severe familial hypercholesterolaemia.

Several studies have suggested that the oxidative modification of low-density lipoprotein (LDL) could play a key role in the early stages of atherosclerosis. The susceptibility of LDL to oxidation has been found to be greater in patients with coronary heart disease. Familial hypercholesterolaemia (FH) is a powerful clinical model in which to study the predictive role of LDL in atherogenesis. LDL-apheresis is a treatment that is able to decrease lipid levels in plasma. This study was aimed at investigating the reducing capacity of erythrocytes and the in vitro susceptibility to oxidation of LDL isolated from patients with homozygous, heterozygous and double-heterozygous FH, who were treated fortnightly with LDL-apheresis or left untreated. In 14 FH patients, at baseline and after a cycle of treatment, the susceptibility of LDL to oxidative modification was analysed by studying the kinetics of conjugate diene formation. Plasma hydroperoxides, polyunsaturated fatty acid content, LDL electrophoretic mobility on agarose, the titre of auto-antibodies against oxidized LDL and serum paraoxonase activity were also measured. Furthermore, in order to evaluate a potential relationship between LDL oxidation and redox status, erythrocyte GSH and ATP levels were determined in FH patients treated regularly or never treated previously by LDL-apheresis. Unlike in the control group, the oxidative status of LDL in all FH patients was modified by LDL-apheresis, as revealed by the higher negative charge and the increase in levels of hydroperoxides and antibodies against oxidized LDL in the plasma. Our findings suggest both an acute effect and a long-term effect of LDL-apheresis in FH patients treated with dextran sulphate cellulose apheresis. The acute effect of LDL-apheresis on the susceptibility to oxidation of plasma and LDL was demonstrated by significant decreases in plasma hydroperoxide content, total LDL concentration and polyunsaturated fatty acid content. The increased resistance of LDL to oxidation was shown by prolongation of the lag time (P<0.05) in samples after a single cycle of treatment. The long-term effect of LDL-apheresis was demonstrated by the comparable values for lag phases (obtained from the kinetics of conjugate diene formation) in patients under active treatment and controls. Compared with healthy controls and untreated patients, the erythrocyte GSH content was significantly higher (P

Aging and red blood cell membrane: a study of centenarians.

Successful aging, characterized by little or no loss in physiological functions, should be the usual aging process in centenarians. It is known that well-preserved physiological functions depend on the proper functioning of cell systems. In this article we focus on cell membrane integrity and study the red blood cell membrane to evaluate the effect of physiological aging in centenarians. Fifteen healthy, self-sufficient centenarians, mean age 103 years, were examined by assessing hemocytometric values and some relevant characteristics of the erythrocyte membrane, i.e., the cholesterol/phospholipid molar ratio, the distribution of phospholipid classes and their fatty acid composition, the integral and skeletal protein profiles. The centenarians showed a significant decrease in the red blood cell count (p < 0.0002), hemoglobin (p < 0.0002), and hematocrit (p < 0.0005). The red blood cell membrane showed a significantly increased cholesterol/phospholipid molar ratio (p < 0.01), with a concomitant increase in polyunsaturated fatty acids in phosphatidylcholine (p < 0.001) and, to a lesser extent, in phosphatidylethanolamine. The electrophoretic pattern of membrane proteins was qualitatively normal compared to controls but the densitometric analysis showed a significant increase in the integral protein band 4.2 (p < 0.05) and in the skeletal protein actin (p < 0.001). Extreme longevity seems to be associated with a substantial integrity of the erythrocyte membrane. Moreover, the evident increase in polyunsaturated fatty acids and in actin are likely to improve the membrane fluidity and to strengthen the membrane structure.

[Glucose-6-phosphate dehydrogenase deficiency and hereditary hemolytic anemia]. / Deficit di glucosio-6-fosfato deidrogenasi ed anemia emolitica ereditaria.

G6PD deficiency is the most common enzymopathy in the world. The highest frequency values are found in tropical Africa, in the Middle East, in some areas of the Mediterranean, in tropical and sub-tropical Asia and in Oceania. This genetic defect shows sex linked inheritance and a marked heterogeneity. At least 400 abnormal variants with different biochemical characteristics and about 100 diverse mutations have been identified. In most cases the phenotypic expression is a marked decrease in erythrocyte G6PD activity. The most common clinical consequences are neonatal jaundice and sporadic haemolytic crises caused by a number of drugs, by infections or by ingestion of fava beans. A few cases of chronic non-spherocytic haemolytic anaemia associated with rare molecular variants have been reported. Early diagnosis, education and epidemiologic surveillance have been proved to be cornerstones in the prevention of the haemolytic disease. Therefore they should be taken into account in the national health programmes, especially in the countries with high prevalence rates.

Junctional sites of erythrocyte skeletal proteins are specific targets of tert-butylhydroperoxide oxidative damage.

The oxidative denaturation of the erythrocyte membrane, which is considered a major cause of the haemolytic process, was evaluated upon 'in vitro' oxidative stress with tertbutylhydroperoxide. Biochemical and ultrastructural analyses were performed to point out the effect of this substance on the skeletal network, which is mainly responsible for red cell shape and viability. Moreover, cell morphology was observed by scanning electron microscopy and membrane rigidity assessed by EPR measurements. The most relevant features of the membrane denaturation were, (i) lipid peroxidation, as assessed by malonidialdehyde production, (ii) spectrin and ankyrin degradation with simultaneous globin binding to the membrane, as evidenced by electrophoretic pattern of red cell ghosts. These phenomena were related to the drug concentration in the incubation medium, and accompanied by depletion of intracellular reduced glutathione. The denaturation of protein components hindered the release of spectrin in a hypotonic extraction medium and could be only partially reversed by dithiothreitol. The extensive membrane protein and lipid degradation, at high drug concentration, was coherent with a marked increase of membrane order (membrane 'rigidity'). No clustering of intramembrane proteins was shown by the transmission electron microscopy images. At the same time scanning electron microscopy demonstrated shrinking and disco-stomatocytic deformation of erythrocytes. Ultrastructural analysis of the membrane skeleton by fluorescence-labelling of spectrin and actin, allowed to point out that exposure to t-BHP caused the marginalization of spectrin and the rearrangement of actin molecules with formation of micro aggregates, so that a detachment of actin from the spectrin network was suggested. In addition to the generalized damage of red cell membrane, tertbutylhydroperoxide was found to induce a specific alteration of the skeletal network at the horizontal junction sites involving spectrin, actin, and protein 4.1 and thus to modify the cytoskeletal assembly. This effect on the membrane skeletal components was consistent with the hypothesis that oxidative stress plays a key role in the haemolytic process.

A pilot programme of external quality assessment for general haematology in Italy.

In the years 1984-1989 the Istituto Superiore di Sanità organized an EQAS for haematology (SVEQE) in Italy. A series of trials for haemocytometry, abnormal haemoglobins, HbA2, HbF, red cell G6PD and peripheral blood films, were carried out with the participation of 126 hospital laboratories, in different regions. SVEQE was an educative programme, aiming at promotion of quality assurance (QA) in laboratory haematology. At the same time an attempt was made to survey the analytical methods and instruments and to estimate the "state of the art" by the dispersion of all results. Participant laboratories were not scored for their performances. The operative protocol was harmonized to the guidelines established by WHO and ICSH; the trial specimens were prepared from normal or pathologic blood samples provided by blood banks or hospital departments. The trials for haemocytometry demonstrated a wide use of completely automated analyzers and in a steady state of performance during about five years. CVs, mainly for WBV and PLT, were somewhat higher than in other countries, where national QA systems have been established for a long time. Such discrepancies were not surprising in a pilot programme and were likely to be caused by inadequate internal quality control. The exercises for abnormal haemoglobins, HbA2, HbF and G6PD pointed out the need of using standardized methods according to the recommendations of ICSH. A large number of participating laboratories took part in the trial for blood cell morphology, being convinced of the educative function of this exercise; it is important to continue with systematic surveys, even including rare haematological disorders amongst the selected cases.

A monoclonal antibody monitoring band 3 modifications in human red blood cells.

Morphologic and metabolic erythrocyte modifications are thought to be the basis of cell removal from circulating blood. A significant role has been ascribed to the immunological network which may remove aged or misshapen erythrocytes through the binding of specific autoantibodies. Along this line recent observations indicate that a senescence antigen appears in consequence of postsynthetic modifications of band 3, one of the most important erythrocyte membrane proteins, which accounts for many functional activities of the red cells. On this basis, we raised a mouse hybridoma anti-band 3 monoclonal antibody (B6 MoAb) of the IgG2a class which monitors band 3 differences among normal red blood cells separated by Percoll density gradient. These differences are outlined by the decrease of B6 MoAb binding to band 3 monomer, the appearance of an 80-90 kDa new band, lighter than band 3, and the increase of low molecular weight fragments in the 4.5 region. The B6 MoAb appears to be very useful in detecting modifications of band 3 since it bind to a 19 kDa Chy-Try fragment estimated to be sensitive to aging.

A pilot External Quality Assessment Scheme for haemocytometry in Italy.

BACKGROUND: An Italian EQA scheme for haemocytometry, organized by the Istituto Superiore di Sanità, has been active for about five years (1984-1989). The aims of this programme were to evaluate the state of the art and to introduce in Italy a scheme recommended by ICSH. N. 126 public laboratories from different provinces joined voluntarily the programme and trials for haemoglobinometry (A01, A02), full blood count (B01-B08) and platelet count (D01-D03) were performed. METHODS: Materials for testing consisted of blood lysate, preserved blood preparation containing native red cells and pseudoleukocytes, suspension of fixed platelets. The performances of laboratories was evaluated by consensus values (median, mean and standard deviation) and individual deviation index. RESULTS: The instrument survey demonstrated that fully automated systems had the highest frequency. Non Gaussian distributions of results were often obtained and this was particularly true for WBC, PLT and MCV. The overall variability was lower than 5.5% for Hb, RBC and MCH and lower than 9.3% for other erythrocyte parameters; WBC and PLT counts displayed a higher dispersion (CV* = 9.8% and 25.4%); the spreading of results was strongly reduced in the homogeneous group of Coulter counters. In the course of the programme CV*s didn't show any relevant modification, a steady state performance being apparent. As regards the nature of variability, the random component was prevalent for all parameters, with the exception of MCV. CONCLUSIONS: This pilot programme allowed to demonstrate the practicability of a national EQAS for haemocytometry according to the ICSH guidelines. Materials for testing showed acceptable stability, homogeneity and commutability. As regards analytical equipment as well as analytical variability, hospital centers participating in these EQA trials were comparable with laboratories taking part in similar EQAS of other European countries.

Oxidative erythrocyte membrane damage in hereditary spherocytosis.

The occurrence, in Hereditary Spherocytosis, of an oxidative damage to red blood cell membranes was studied by "in vitro" treatment of the erythrocytes with tert-butylhydroperoxide, methylene blue, or phenylhydrazine. Spherocytes were found to be more sensitive than normal erythrocytes to the action of these drugs. Tert-butylhydroperoxide caused a more intense lipid peroxidation as well as more extensive membrane protein alterations, namely spectrin degradation, formation of high molecular weight aggregates, and globin binding to the membrane. Marked spectrin degradation was also induced by methylene blue and by phenylhydrazine, which differed from each other for their effects on the generation of membrane-bound globin and of intermediate proteolysis products. Spectrin appeared therefore to be, in Hereditary Spherocytosis, a highly sensitive target to oxidative stress, a phenomenon which may, also "in vivo", increase the rate of spectrin loss thus enhancing erythrocyte fragility.

Membrane alterations in G6PD- and PK-deficient erythrocytes exposed to oxidizing agents.

After in vitro treatment of normal, glucose-6-phosphate dehydrogenase-deficient or pyruvate kinase-deficient human erythrocytes with three different oxidizing agents, the extent of lipid peroxidative degradation and the alterations of membrane proteins were evaluated. Exposure to tert-butylhydroperoxide induced, most markedly in G6PD- and PK-deficient erythrocytes, a reduction of protein bands 1, 2, 2.1, 3, 4.1, 4.2, and 5, with the appearance of high-molecular-weight aggregates and of "new" polypeptide components in the 29- to 23-kDa region and with a marked increase of membrane-bound globin. Malonyldialdehyde production was highest in G6PD-deficient cells and relatively low in PK-deficient ones. Methylene blue, which had similar but less relevant effects on lipid peroxidation, in G6PD-deficient erythrocytes caused a conspicuous appearance of high-molecular-weight aggregates and a simultaneous relevant decrease of bands 1 and 2 and of membrane-bound globin; it brought about an almost opposite effect in PK-deficient red cells. Acetylphenylhydrazine, which under our conditions appeared the mildest agent, failed, in normal and PK-deficient erythrocytes, to increase malonyldialdehyde production or to alter membrane proteins, whereas it caused, in G6PD-deficient cells, a slight decrease of bands 1 and 2, a more pronounced decrease of band 3, and a marked increase of bands 4.5 and 4.9.

A new monoclonal antibody to an age sensitive band 3 transmembrane segment.

The appearance of band 3 structural modifications related to aging could be evidenced by means of monoclonal antibodies against senescence antigen. Hence in the attempt to provide an immunological marker of erythrocyte aging, we raised a monoclonal antibody against native band 3 (B6 MoAb), which seems to detect differences in the band 3 molecule from erythrocytes of different ages separated by density gradient. Densitometric evaluation of immunoblotting patterns indicates that the in vivo aging is associated with band 3 monomer degradation. The Percoll separated fractions show a significant increase of those proteolytic fragments that bind the B6 antibody. Finally, protease digestions of unsealed membrane ghosts have been performed to test the binding site of the B6 antibody on the band 3 molecule. The data show that the B6 antibody binds a 19 KDa chymotryptic-tryptic fragment which corresponds to a segment of the looped membrane domain whose steric structure appears to be sensitive to age.

Characterization of differences in insulin receptors from young and old red blood cells.

It has been demonstrated that young RBCs (reticulocytes and early mature erythrocytes) possess more insulin receptors than old RBCs (late mature erythrocytes) but it is not yet known whether insulin receptors on young and old RBCs are regulated similarly. In the present investigation insulin receptors on young and old RBCs have, therefore, been studied in five normal male subjects before and after 2 days dexamethasone ingestion (0.5 mg tablet every 6 h) and, in the same subjects, before and 5 h after ingestion of 75 g glucose. The results obtained clearly demonstrate that dexamethasone increases insulin receptor concentration while glucose ingestion increases both insulin receptor affinity and concentration on young RBCs. By contrast, neither stimuli modify insulin receptors on old RBCs. Studies on RBCs are usually performed on the whole RBC population not taking into account this differential responsiveness of receptors on young versus old RBCs; consequently, this phenomenon might be responsible of the fact that some data reported on RBCs are not in agreement with those reported on monocytes or adipocytes and it should be taken into consideration when using RBCs to evaluate insulin receptor regulation.

Homozygous NADH-methemoglobin reductase and aspartylglucosaminidase deficiencies in a moderately retarded Sicilian child.

We report on a 10-year-old boy with generalized deficiency of both NADH-methemoglobin reductase and aspartylglucosaminidase. Although the two enzymatic defects, both autosomal recessive traits, are associated with severe mental retardation, the patient was less retarded than his sister who had only aspartylglucosaminuria.

Red blood cell age, pyruvate kinase activity, and insulin receptors. Evidence that monocytes and RBCs may behave differently.

Data emerging from insulin receptor studies performed on red blood cells (RBCs) and monocytes from the same subject are not always in agreement; dichotomy might occur since variations in mean RBC age are not taken into account or because insulin receptors on the two cell types behave differently. In the present investigation RBCs from normal male subjects were separated into five populations of different mean age by means of centrifugation of RBCs on a discontinuous gradient of buffered Percoll for 10 min at 1000 X g. Insulin binding varied significantly depending upon the RBC population tested and was closely correlated to the activity of pyruvate kinase (r2 = 0.86), a well-known marker of RBC age. These data suggested that pyruvate kinase assay might be helpful in studies of RBCs. To confirm this hypothesis, RBCs from 10 normal male subjects and 13 male patients with hemolytic anemia were studied; insulin binding was correlated to pyruvate kinase activity. By adjusting insulin binding to 2 X 10(9) RBCs/ml the range of data was abnormally high, but it became acceptable after adjusting insulin binding to pyruvate kinase activity (0.75 U/2 X 10(9) RBCs). The overall data indicated that insulin binding was highly correlated to pyruvate kinase activity (r2 = 0.82) but only slightly to reticulocyte number (r2 = 0.56) since not only reticulocytes but also erythrocytes lose receptors during maturation. Pyruvate kinase activity was measured in RBCs from normal men and from normally menstruating women at the seventh and twenty-fourth days of the cycle; results demonstrated that adjustment of data, according to mean RBC age, broadens dichotomy of monocyte and RBC data.(ABSTRACT TRUNCATED AT 250 WORDS)