Oxidant damage of the lipids and proteins of the erythrocyte membranes in unstable hemoglobin disease. Evidence for the role of lipid peroxidation.

Since unstable hemoglobins have been considered a source of reactive oxygen radicals, and oxidative membrane damage a prehemolytic event, we examined the erythrocyte membranes of six patients (three splenectomized) with hemoglobin Köln disease. In the hydrogen peroxide stress test, the patients' erythrocytes generated more than twice the malonyldialdehyde (a lipid peroxidative product) than control erythrocytes. Fluorescence spectra of lipid extracts of the patients' erythrocytes showed an excitation maximum at 400 nm and an emission maximum of 460 nm, characteristic of malonyldialdehyde lipid adducts. Two types of membrane polypeptide aggregates were found in the erythrocytes of the splenectomized patients. The first, which were dissociable by treatment with mercaptoethanol, contained disulfide-linked spectrin, band 3 and globin. The second, not dissociable by mercaptoethanol, had an amino acid composition similar to that of erythrocyte membranes and spectrin (unlike globin) and like that of aggregates produced by the action of malonyldialdehyde on normal erythrocyte membranes. Atomic absorption spectroscopy of hemoglobin Köln erythrocytes showed no increase in calcium content implying that these cross-links were not due to calcium-stimulated transglutaminase. Using a micropipette technique, we demonstrated that erythrocytes containing membrane aggregates from splenectomized patients were less deformable while aggregate-free erythrocytes from non-splenectomized patients had normal deformability. We conclude that the erythrocyte membranes in hemoglobin Köln disease show evidence of lipid peroxidation with production of malonyldialdehyde, and that the nondissociable membrane aggregates formed in this disease are likely cross-linked by malonyldialdehyde. Because the erythrocytes containing membrane aggregates from splenectomized patients with unstable hemoglobin disease show decreased membrane deformability, we hypothesize that this abnormality results in premature erythrocyte destruction in vivo.

Decreased survival in vivo of diamide-incubated dog erythrocytes. A model of oxidant-induced hemolysis.

Erythrocytes from patients with chronic hemolytic variants of glucose-6-phosphate dehydrogenase (G-6-PD) deficiency have structural membrane protein abnormalities accompanied by decreased cell membrane deformability which we postulate represent the consequences of oxidant-induced membrane injury. To evaluate the pathophysiologic significance of oxidant-induced membrane injury, we studied the in vitro and in vivo effects of the thiol-oxidizing agent, diamide, on dog erythrocytes. In vitro incubation of dog erythrocytes with 0.4 mM diamide in Tris-buffered saline for 90 min at 37 degrees C resulted in depletion of GSH, formation of membrane polypeptide aggregates (440,000 and > 50,000,000 daltons) and decreased cell micropipette deformability, abnormalities similar to those observed in the erythrocytes of patients with chronic hemolytic variants of G-6-PD deficiency. In addition, diamide-incubated cells had increased viscosity and increased membrane specific gravity, but no change in ATP. Reinjection of 51Cr-labeled, diamide-incubated cells was followed by markedly shortened in vivo survival and splenic sequestration. Further incubation of diamide-incubated cells in 4 mM dithiothreitol reversed the membrane polypeptide aggregates, normalized micropipette deformability, decreased cell viscosity, prolonged in vivi survival, and decreased splenic sequestration. These studied demonstrate that diamide induces a partially reversible erythrocyte lesion which is a useful model of oxidant-induced membrane injury. They suggest that oxidant-induced erythrocyte membrane injury plays an important role in the pathophysiology of chronic hemolysis which accompanies some G-6-PD variants.

Magnetoencephalography for pediatric epilepsy: how we do it.

Magnetoencephalography (MEG) is increasingly being used in the preoperative evaluation of pediatric patients with epilepsy. The ability to noninvasively localize ictal onset zones (IOZ) and their relationships to eloquent functional cortex allows the pediatric epilepsy team to more accurately assess the likelihood of postoperative seizure freedom, while more precisely prognosticating the potential functional deficits that may be expected from resective surgery. Confirmation of clinically suggested multifocality may result in a recommendation against resective surgery because the probability of seizure freedom will be low. Current paradigms for motor and somatosensory testing are robust. Paradigms allowing localization of those regions necessary for competent language function, though promising, are under continuous optimization. MR imaging white matter trajectory data, created from diffusion tensor imaging obtained in the same setting as the localization brain MR imaging, provide ancillary information regarding connectivity of the IOZ to sites of rapid secondary spread and the spatial relationship of the IOZ to functionally important white matter bundles, such as the corticospinal tracts. A collaborative effort between neuroradiology, neurology, neurosurgery, neuropsychology, technology, and physics ensures successful implementation of MEG within a pediatric epilepsy program.

Erythrocyte membrane protein changes in glucose-6-phosphate dehydrogenase mutants with chronic hemolytic disease: an example of postsynthetic modification of membrane proteins.

1. G6PD mutants with CHD have decreased GSH, despite reticulocytosis, and increased membrane polypeptide aggregates. Aggregates increase logarithmically with decrease in RBC GSH. 2. These aggregates contain spectrin and can be depolymerized by disulfide reducing agents. Disulfide bonds between spectrin molecules and between the cytoskeleton and the cytoplasmic protein rigidify the red cell membrane and decrease RBC survival. 3. Direct oxidative damage of the RBC membrane, not Heinz body formation, explains the hemolytic anemia of G6PD mutants with CHD. This membrane damage may constitute a useful model system of oxidant-induced injury of other cells, and is an example of postsynthetic modification of membrane proteins by a nonmembrane gene.

Sucrose density gradient analysis of erythrocyte membranes in hemolytic anemias.

To investigate the membrane abnormalities that may play a pathophysiologic role in several hemolytic anemias we determined the density distribution on sucrose density gradients of human red blood cell (RBC) membranes from patients with these disorders, from normal controls, and from incubated normal RBC. We analyzed the fractions for membrane-adsorbed hemoglobin (Hb), globin, and nonglobin cytoplasmic proteins. The relationship between the cytoplasmic proteins adsorbed on the membranes and the specific gravity (SG) of the membranes was linear. An increase in SG of the entire membrane population was seen in Hb C disease due to adsorbed Hb. Subpopulations of membranes with increased SG due to adsorption of nonglobin protein were evident in the membranes from two splenectomized patients with hemolytic glucose-6-phosphate dehydrogenase (G6PD) variants. Dense membrane subpopulations found in RBC membranes from three splenectomized patients with Hb Köln were associated with adsorbed globin, while similar subpopulations in RBC membranes from three splenectomized patients with hereditary spherocytosis demonstrated increased SG due to adsorbed Hb. Splenectomized normals had no such abnormality in membrane density. Sucrose density gradients demonstrate that membrane bound cytoplasmic protein is characteristic of the RBC membranes in several hemolytic disorders. Additionally, gradients are useful for the isolation and further analysis of those subpopulations of RBC membranes with abnormal SG and exaggerated membrane protein abnormalities.

Mechanisms of decreased erythrocyte deformability and survival in glucose 6-phosphate dehydrogenase mutants.

We have studied the nature of the oxidative lesion of the erythrocyte membrane in glucose-6-phosphate dehydrogenase (G6PD) mutants with chronic hemolysis, comparing these membranes with those from normal red cells (RBC) subjected to oxidative stress in vitro. Disulfide-linked polypeptide aggregates are found in membranes from fresh RBC of these G6PD mutants and from aerobically incubated normal erythrocytes. As further evidence of oxidative damage, increased disulfide bonds were found in the RBC membranes from both the mutants and incubated normal RBC. The intermolecular bonds which cross-link membrane polypeptides to form the observed aggregates, however, only accounted for a fraction of the membrane disulfide bonds present. Thus, most of the disulfide bonds in the G6PD mutants were intramolecular. These intramolecular disulfide bonds were widely distributed on the membrane polypeptides, but were found to be concentrated on cytoskeletal anchoring proteins, bands 2.1-2.3, using [14C] iodoacetamide labelling of the sulfhydryls involved in disulfide bonds. The intermolecular bonds, on the other hand, were concentrated in spectrin. When G6PD mutant membranes were examined on sucrose density gradients, a subpopulation of dense membranes was observed which resembled the membranes of oxidatively stressed normal RBC both in increased density and in increased binding of nonhemoglobin cytoplasmic protein. To study the relationship between sulfhydryl oxidation, membrane density and RBC viscosity the sulfhydryl oxidant diamide (diazine dicarboxylic acid bis-[dimethylamide]) was used. Diamide treated erythrocytes, like the G6PD mutants, had decreased GSH, increased polypeptide aggregates, increased viscosity, but no change in ATP. We conclude that in G6PD mutants with chronic hemolysis oxidative damage includes aggregate formation due to intermolecular disulfide bonds, and intramolecular disulfide bond formation associated with increased binding of non-hemoglobin cytoplasmic proteins to the membrane. The relative importance of intermolecular and intramolecular disulfide bond formation and the mechanism whereby these changes may produce decreased RBC deformability and survival remain to be determined.