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Biomedical subjects

N L Fortier

Publications and source records attributed to N L Fortier.

At least 19 recordsLinked to original sources
PubMed1988

The role of membrane protein sulfhydryl groups in hydrogen peroxide-mediated membrane damage in human erythrocytes.

The formation of spectrin-hemoglobin complex following treatment of red cells with hydrogen peroxide (H2O2) has previously been shown to be associated with alterations in cell shape, decreased membrane deformability and increased recognition of modified cells by anti-IgM immunoglobulin in a phagocytic assay by monocytes. Prior treatment with carbon monoxide completely inhibited the H2O2-associated membrane changes, indicating a role for oxidized hemoglobin in the complex formation. Also, in a cell-free system, blockage of sulfhydryl (SH) groups on purified spectrin by N-ethylmaleimide significantly reduced the complex formation, suggesting a role for SH groups of spectrin in crosslinking process. The present study was undertaken to examine the role of SH blockade by N-ethylmaleimide on intact red cells undergoing oxidative damage. Pretreatment of erythrocytes with N-ethylmaleimide at concentrations ranging from 0.1 to 0.2 mM resulted in decreased lipid peroxidation and spectrin hemoglobin crosslinking. Moreover, pretreatment with N-ethylmaleimide resulted in less marked alterations in cell shape and membrane deformability as well as reduced recognition of peroxidized cells by antiglobulin serum. N-Ethylmaleimide treatment had no effect on methemoglobin formation. Studies with 14C-labeled N-ethylmaleimide showed that over 50% of N-ethylmaleimide was incorporated into spectrin. Pretreatment of cells with higher concentrations of N-ethylmaleimide (over 0.2 mM) was associated with membrane dysfunction independent of H2O2. These results imply that blocking of reactive SH groups leads to reduced interaction of spectrin with oxidized globin. These data, along with our prior observations, indicate that SH groups on spectrin play an important role in hemoglobin oxidation-induced formation of spectrin-hemoglobin complex and the resultant deleterious effects on membrane properties.

Electrophoresis, Polyacrylamide Gel↗
PubMed1987

Antiglobulin serum mediated phagocytosis of normal senescent and oxidized RBC: role of anti-IgM immunoglobulins in phagocytic recognition.

Fresh human monocytes usually do not recognize normal RBCs; however, in our newly developed assay antiglobulin-opsonized normal RBCs were phagocytized. Both anti-IgG and anti-IgM fractions present in the antiglobulin serum were involved but the major opsonin was anti-IgM. The anti-IgM opsonized mainly senescent RBCs and therefore could be used to discriminate young from senescent RBCs. The antiglobulin serum and monospecific anti-IgM increased opsonization of in vitro oxidized and desialylated RBCs, whereas trypsin-treatment of RBCs decreased phagocytosis. The material removed by trypsin from the RBCs surface inhibited the antiglobulin and monospecific anti-IgM phagocytic assay supporting the view that membrane associated elements crossreacted with anti-IgM. These results suggest that both internal cellular events and external removal of sialic acid play a role in the emergence of non-IgG covered epitopes on the surface of senescent and oxidized erythrocytes.

Antibodies, Anti-Idiotypic↗
PubMed1985

Demonstration of haemoglobin associated with isolated, purified spectrin from senescent human red cells.

Employing a direct and sensitive radioimmunoassay (RIA) we have confirmed the presence of haemoglobin associated with isolated, purified spectrin from senescent red cells. Haemoglobin associated with spectrin occurs in the highest amount in cells with an MCHC greater than 36 g/dl and is approximately 3% of the total spectrin extract. Spectrin from the young cells had the least haemoglobin, while an intermediate amount was found in unfractionated, whole red cells. The RIA results were in close approximation with estimation of the haemoglobin-spectrin complex obtained by carefully integrating the Coomassie blue stain profiles from 4% SDS PAGE in densitometric scans from isolated spectrin.

Densitometry↗
PubMed1985

Properties and characterization of vesicles released by young and old human red cells.

We have presently demonstrated morphologic differences between young and senescent red cells following 18 h of metabolic depletion in vitro. Young and old red cells both form echinocytes, whereas only young cells demonstrated myelin forms or microspheres. Furthermore, vesicles were released in greater quantities into the cell-free supernatant from young cells. Isolated vesicles from both young and old red cells contained lipids, intrinsic membrane proteins (especially band 3), and haemoglobin, and they were also enriched in acetylcholinesterase (AChE). Young cells produced more vesicles than old cells but the composition of the low density vesicles was similar except that haemoglobin-spectrin complex was found exclusively in vesicles from young cells. Oxidation of young red cells prior to metabolic depletion prevented both myelin formation and vesicle release.

Acetylcholinesterase↗
PubMed1985

Effect of hydrogen peroxide exposure on normal human erythrocyte deformability, morphology, surface characteristics, and spectrin-hemoglobin cross-linking.

To further define the conditions for forming spectrin-hemoglobin cross-linking in human erythrocyte membranes and to examine its possible effects on membrane function, we incubated normal human erythrocytes for up to 3 h in concentrations of H2O2, varying from 45 to 180 microM, in an azide phosphate buffer, pH 7.4. The chemical changes observed indicated that methemoglobin formation occurred early and at a low concentration (45 microM). Morphologic changes characterized by increased echinocyte formation occurred in a dose-dependent fashion. In addition, decreased cell deformability commensurate with increased membrane rigidity was found. Finally, an increase in cell recognition as determined by monocyte phagocytosis and adherence in vitro, as well as decreased phosphatidylcholine accessibility to bee venom phospholipase A2, was found in H2O2-treated erythrocytes compared with controls. Both of these latter changes were closely correlated with the extent of spectrin-hemoglobin cross-linking. In addition to these protein-mediated interactions, lipid peroxidation also occurred after H2O2 exposure, as shown by generation of fluorescent amino propene derivatives. The addition of the antioxidant, butylated hydroxytoluene, decreased the fluorescent derivatives, but did not prevent the effects on membrane function. This suggests that lipid peroxidation, though present, was not necessary for the membrane changes found. In contrast, spectrin-hemoglobin aggregation and the alterations in membrane function were completely prevented by prior exposure of the erythrocytes to carbon monoxide.

Carbon Monoxide↗
PubMed1983

Spectrin-haemoglobin crosslinkages associated with in vitro oxidant hypersensitivity in pathologic and artificially dehydrated red cells.

Protein crosslinkages are apparent at 215 000-250 000 daltons in electrophoregrams of membranes from hydrogen peroxide treated erythrocytes (British Journal of Haematology, 48, 435, 1981). Hydrogen peroxide is also capable of inducing crosslinkages of identical molecular weights in stage I and II (red) ghosts and in a mixture of purified spectrin and haemoglobin, but not in white ghosts or in either spectrin or haemoglobin alone. Autoradiographic studies using 14C-methaemoglobin and 32P-spectrin confirm the involvement of spectrin and haemoglobin in this reaction. The alpha chains of both proteins are more reactive than the corresponding beta chains: 3 times more reactive in the case of spectrin and 10 times more reactive in haemoglobin. The reaction is almost totally inhibited by NaCN and partially inhibited by N-ethylmaleimide. Direct addition of malondialdehyde to a spectrin-haemoglobin mixture does not result in protein crosslinkage. Metabolic depletion (40 h), in vivo ageing and sucrose dehydration of fresh, normal cells enhance the reaction considerably, whereas in vitro rehydration of xerocytes normalizes their H2O2 sensitivity.

Erythrocyte Membrane↗
PubMed1982

Characteristics of peripheral blood monocytes in hereditary xerocytosis and spherocytosis.

Peripheral blood monocytes isolated from patients with congenital hemolytic anemia, hereditary xerocytosis and spherocytosis, demonstrated in vivo engulfment of red cell and platelet fragments. In addition, morphometric studies performed on these monocytes showed an increase in cytoplasmic/nuclear ratio as well as lysosome and phagosome volumes. The production of carbon dioxide from glucose-1-14C in abnormal monocytes was increased (15-80%) but the intracellular values of beta-glucuronidase and esterase activity were similar to control monocytes. Monocyte locomotion assessed in the presence of chemotactic stimuli was found significantly increased (73 +/- 12 monocytes/oil immersion fields vs. 46 +/- 5 for control monocytes). We concluded that the monocytes in hemolytic anemias associated with increased in vitro red cell fragmentation have some features resembling the 'stimulated' monocytes and that this alteration may be due to red blood cell fragment ingestion.

Anemia, Hemolytic, Congenital↗
PubMed1981

Altered red blood cell surface area in hereditary xerocytosis.

Hereditary xerocytes appear larger than normal red cells in scanning electron micrographs and exhibit a higher ghost packing volume. The major chemical components--protein, phosphorus, cholesterol and sialic acid--are increased uniformly, as are all polypeptides visible on gel electrophoresis patterns of xerocyte membranes. These data are consistent with a xerocyte surface area 15 to 25% above normal. Certain clinical anomalies common to this disorder, including unexpectedly low reticulocyte count and 2,3-diphosphoglycerate level, are discussed in the light of the present findings.

Cholesterol↗
PubMed1981

Red cell membrane response to hydrogen peroxide-sensitivity in hereditary xerocytosis and in other abnormal red cells.

Osmotically resistant red cells associated with some haemolytic anaemias, including hereditary xerocytosis, sickle-cell disease and beta thalassaemia minor, are more sensitive than normal red cells to exogenous in vitro hydrogen peroxide (H2O2). This sensitivity is manifested by a rapid loss of intracellular potassium, shape change, protein aggregation, and methaemoglobin formation at lower concentrations of H2O2 (225 microM) than are required to induce similar effects in normal red cells (450 microM). Malonyldialdehyde (MDA) formation occurs later than the other effects and can be inhibited by the antioxidant, butylated hydroxytoluene (BHT), without affecting protein aggregation or potassium leak. Incubation of normal red cells directly with MDA induces protein aggregation, but only after 1 h of incubation. Although nystatin-sucrose treated normal cells which are dehydrated with altered cation content, and therefore osmotically resistant, do not display abnormal H2O2 hypersensitivity as manifested by excessive potassium permeability, they do show an increase in methaemoglobin formation and protein aggregation similar to xerocytes. These data indicate that membrane protein cross-linking occurring immediately following H2O2 exposure seems independent of either the sulfhydryl or MDA mechanisms, and that the membrane permeability of the abnormal red cells predisposes them to oxidative damage.

Anemia, Hemolytic, Congenital↗
PubMed1980

Red blood cell associated IgG in normal and pathologic states.

We studied the anti-IgG-induced agglutination of both normal and abnormal red blood cells (RBC) using a sensitive, automated antiglobulin test. Normal RBC agglutinated strongly with anti-IgG antibody, indicating that IgG was present on the erythrocyte membrane. Young RBC, recovered by centrifugation from a normal RBC population, agglutinated with anti-IgG less than the old cells, suggesting that immunoglobulin G accumulated gradually on the RBC membrane in vivo. The degree of anti-IgG-induced RBC agglutination correlated negatively with the reticulocyte count and positively with the concentration of plasma IgG. RBC from patients with hypogammaglobulinemia appeared to have a low subnormal quantity of membrane-bound IgG, whereas the reverse was the case in hypergammaglobulinemia. During hemolytic episodes, RBC of patients with hereditary spherocytosis agglutinated poorly with anti-IgG, apparently due to predominance of young RBC. RBC of patients with nonspherocytic. Coombs-negative, nonimmune hemolytic anemia usually also agglutinated poorly with anti-IgG. However, in some cases of active hemolytic anemia, decreased agglutination with anti-IgG was not observed, suggesting that these young RBC had increased amounts of membrane-bound IgG.

Agammaglobulinemia↗
PubMed1979

Increased erythrocyte lipid peroxidation in hereditary xerocytosis.

Xerocytosis is a chronic hemolytic anemia with abnormal membrane function manifested by an increase in passive potassium permeability. Xerocytes demonstrate a greater susceptibility to hydrogen peroxide manifested by the production of malondialdehyde (MDA). Xerocyte membrane phospholipid and fatty acid analysis is normal except for a slight increase in phosphatidyl choline, a commensurate decrease in sphingomyelin, as well as a decrease in linoleic acid. Metabolism and glutathione stability are normal as well as plasma vitamin E levels in patients with xerocytosis. The increased susceptibility to oxidant stress is exaggerated in the "older aged" xerocyte population and correlated well with decreased intracellular potassium concentration.

Anemia, Hemolytic, Congenital↗
PubMed1979

Red cell membrane in hemolytic disease. Studies on variables affecting electrophoretic analysis.

Significant alterations in the spectrin: band 3 and band 4.1a: band 4.1b ratios and an occasional decrease in the peak height of band 4.2 with respect to band 4.1 were found in electrophoretic patterns of red cell membranes from patients with hereditary xerocytosis. Electrophoretic comparison of whole cell, cytoplasm and membrane polypeptides implied that atypical partitioning at hemolysis could account for some, but not all, of the alterations seen in membrane patterns of xerocytes. A decrease in band 4.2 peak height as well as a variation in the profile of band 3 were produced in controls by specific manipulations of the electrophoresis protocol. Metabolic depletion of normal cells produced the type of alterations in bands 3 and 4.1 found in xerocyte membranes, whereas Heinz body production, addition of calcium to the hemolysis buffer and incubation of membranes in detergent under conditions designed to promote proteolysis did not. The presence of a higher peak height of band 4.1b with respect to that of band 4.1a in membranes of patients with various other red cell disorders correlated with an increase in the percentage of reticulocytes in peripheral circulation. The appearance of both band 3 and 4.1 abnormalities in the patterns of control cells which had been enriched in young cells by density gradient centrifugation suggested that these alterations in hemolytic disease are related to the predominance of young cells in the population.

Adenosine Triphosphate↗
PubMed1978

Physiologic features of hemolysis axxociated with altered cation and 2,3-diphosphoglycerate content.

A hemolytic disorder characterized by altered RBC cation composition (increases Na, decreases K), reduced monovalent cation content (decreased Na + K/liter RBC), and decreased levels of 2,3-diphosphoglycerate (2,3-DPG) is described. The etiology of these RBC abnormalities was not elucidated following extensive laboratory evaluation, although two important physiologic principles were manifested by this case: (1) Hemolysis was relatively well compensated (41% hematocrit) despite a significantly decreased RBC survival (51 Cr t 1/2 = 10.5 days). This effect presumably was due to reduced 2,3-DPG content (1.9 mumol/ml RBC) and the associated increase in whole blood oxygen affinity (P50 = 19.6 mm hg). (2) RBC size and water content were normal in spite of marked cation depletion. This anomaly was thought to reflect the osmotic effects of reduced polyvalent anion (2,3-DPG) content.

Adolescent↗