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Warfarin is one of the most commonly used oral anticoagulants. Many medications have been shown to influence the prothrombin time in patients treated with warfarin by various mechanisms. We observed a major bleeding episode that resulted from a probable interaction between levamisole (Ergamisol), 5-fluorouracil, and warfarin. It is conceivable that many patients who are predisposed to thromboembolism because of cancer and surgery will be taking this combination of medications.

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The rotational dynamics of TEMPAMINE can be used to study directly the intracellular environment. The extracellular signal from TEMPAMINE is broadened away by the use of potassium ferricyanide which does not enter the cell. The EPR signal which results when 1 mM TEMPAMINE, 120 mM ferricyanide, and erythrocytes are mixed together arises from TEMPAMINE only in the intracellular aqueous space. The relative viscosity measured by the motion of TEMPAMINE in various control environments is: water at 37 degrees C = 1; human plasma at 37 degrees C = 1.1; internal aqueous environment of washed erythrocytes or whole blood at 37 degrees C = 4.92 +/- 0.32. Erythrocytes can be fractionated by density. In sickle-cell anemia (SS), the percentage of cells we find with density greater than 1.128 g/ml is 15-40%, in normals (AA) and sickle trait (AS) 1%. By direct spin-label measurements with TEMPAMINE we show, for the first time, that the relative internal viscosity (eta mu) of these dense erythrocytes is markedly elevated and density-dependent. Our results show that (1) eta mu increases with increasing cell density; (2) eta mu obtained from sickle cells is higher than eta mu obtained from normal cells at a given density, and this effect is greater at 37 degrees C than at 20 degrees C; (3) eta mu is proportional to MCHC, but eta mu in erythrocytes is higher than eta mu obtained from in vitro preparations of hemoglobin S at equivalent concentrations. We conclude that the relative internal viscosity of erythrocytes is affected by three factors: the state of cell hydration, the amount of hemoglobin polymer present, and the potential interactions of the cell membrane with intracellular hemoglobin.

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An unusual form of red cells (called sequestrocytes) that circulate in the peripheral blood of patients with sickle cell anemia has been identified. In wetmount light microscopy preparations, sequestrocytes appear as massively vacuolated erythrocytes. We have shown by transmission electron microscopy and scanning electron microscopy that the morphology of sequestrocytes can be accounted for on the basis of transcellular cross-bonding of the cell membrane. Initially, endocytotic vesicles span the cytoplasmic space, forming putative cytoskeletal fusion zones. Points of fusion of the membrane skeleton bridging the cytoplasmic compartment expand laterally to form linear fusion zones that entrap lakes of hemoglobin within pseudovacuoles. Sequestrocytes can be isolated in the densest layer (1.159 g ml-1 or greater) of an arabinogalactan density gradient. These cells can be generated in increased numbers in sickle cell blood by incubating samples in 1.5 mM-acetylphenylhydrazine (APH) solution for two hours at 37 degrees C. Their formation is partially blocked by incubation with the reducing agent, dithiothreitol (DTT). Our results suggest that these cells represent an expression of oxidative membrane injury in sickle cell anemia.

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The amount and activity of superoxide dismutase (SOD) (EC 1.15.1.1) were measured in red cells collected from 50 white controls, 101 black controls, 50 patients with sickle hemoglobin (SS Hb), 12 with sickle trait, and 11 with other sickling hemoglobinopathies. Red cells from normal black subjects had more SOD amount and activity than normal whites (1.77 U/mg Hb and 2.96 micrograms/mg Hb vs. 1.47 U/mg Hb and 2.64 micrograms/mg Hb, respectively) or blacks with SS Hb or other sickling hemoglobinopathies. Patients with more severe manifestations of SS Hb had lower levels of SOD activity than those with milder symptoms but had the same amount of enzyme protein. Individuals with sickle trait had amounts and activities of SOD comparable to black controls. An alteration in defense to free radical oxygen may play a role in the severity of symptoms experienced by patients with homozygous sickle cell disease.

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We have conducted a series of studies using discontinuous arabinogalactan density gradient ultracentrifugation of erythrocytes from the peripheral blood of: patients with sickle cell anemia (SCA), in and out of pain crisis; hydrated SCA, hemoglobin SC, and normal individuals all of whom were pain-free; and patients with SCA given short courses of oral vasodilator compounds. Our results indicate that in pain crisis patients develop an echinocytic change that is most prominent in the denser layers (specific gravity greater than 1.128 g/ml) of the discontinuous gradient and effects both irreversibly sickled cells (ISC) and non-ISC. This echinocytic change, we have previously shown, correlates with a decrease in erythrocyte reduced glutathione, a major intraerythrocyte anti-oxidant compound. We could find no consistent change in the percentage of dense cells in pain crisis versus out of crisis. However, out of crisis, hydration led to a marked increase in the percentage of dense erythrocytes in sickle cell anemia and in a HbSC patient, compared to the same individual out of crisis and on ad lib fluids. There was no consistent change in echinocytic forms. Because hydration may be expected to have produced an increase in intravascular volume and vasodilation, we determined whether short courses of three different vasodilators would increase the dense fraction in patients with SCA who were pain-free. There was no change in percentage of dense erythrocytes or in the percentage of echinocytes. We concluded that painful crisis occurs in association with an echinocytic change that may be induced by oxidant injury and that in the pain-free state, hydration, but not short courses of vasodilator drugs, increased the percentage of dense erythrocytes but not the degree of echinocytosis they displayed. The differential effect of hydration, with respect to painful crisis, may indicate that these dense cells are bound to vascular endothelium or trapped in blood vessels at the time of crisis but mobilized by hydration in the out-of-crisis state.

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The uptake of 32P from exogenous 32Pi into membrane proteins of sickle erythrocytes has been analyzed. The phosphorylation of spectrin is normal in sickle cells. There is, however, a substantial increase in 32P in the sialoproteins of the membrane, which can be demonstrated after fractionation or selective proteolysis. Normal and sickle erythrocytes were separated on Stractan gradients and average cell age was determined using the remaining pyruvate kinase activity as a marker. The altered phosphorylation of sickle cells was not seen in young normal cells, suggesting that it was not related to cell age. The altered phosphorylation was also not correlated with the level of reticulocytes in these fractions. This result is further evidence for abnormalities in the sialoproteins of sickle erythrocytes and is the first demonstration of altered sialoprotein phosphorylation in the red cell.

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Nuclear magnetic resonance spectroscopy has become a powerful tool for metabolic investigations on living cell suspensions. However, unless mechanical means are used to maintain the cells in dispersion, settling occurs during the NMR experiment. Because high packed-cell volumes are generally used to produce maximum NMR signals, settling may be inapparent to the eye, leading to unrecognized artifactual changes in NMR spectra. Such artifacts include time-dependent loss of signal intensity when the sample volume approximates the sensitive volume of the NMR probe, and time-dependent increase in signal intensity when the sample volume exceeds the sensitive volume. Through the addition of the polysaccharide arabinogalactan, increasing the buoyant density of the suspending medium to approach that of the cells, we have eliminated cell settling and improved the quality of 31P NMR spectra of human erythrocytes.

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An increase in the number of irreversibly sickled cells (ISCs) in pain crisis has been found by some investigators but not others. We have used the technique of discontinuous arabinogalactan density-gradient ultracentrifugation of whole blood to study ISCs from patients with sickle cell anemia (SCA) during pain crisis and again when pain free (5-331 days after crisis). Nine patients have been studied through ten episodes of pain crisis. Five layers with densities from 1.128 g/mL to 1.158 g/mL have been used. Careful classification of the cells using Nomarsky optics demonstrated highly significant changes occurring in the layers of the gradient. The changes involve the appearance of an increased percentage of echinocytic ISCs and echinocytic cells that were not ISCs, especially in the denser gradient layers, during crisis, and their replacement by normal-appearing discocytes in the pain-free state. There was no change in ISCs that were not echinocytic. Data collected previously demonstrated that reduced glutathione activity correlated with increased echinocytosis in our gradient layers. This indicates that the echinocytic change may occur as a result of oxidant stress. Hemoglobin F levels and the percentage of hemoglobin F from reticulocytes showed no consistent change to coincide with the rise in normal-appearing discocytes in the lightest layers after crisis. Our data indicate that pain crisis occurs in association with an echinocytic change, which may be induced by oxidant injury. The rise in normal-appearing cells after crisis may reflect increased hemoglobin F production in some patients but mainly relates to the disappearance of these echinocytic erythrocytes.

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To assess the possibility that the membrane lesion seen in sickle cells involves oxidant damage, we investigated the relationship between irreversibly sickled cells and glutathione levels and Heinz body production. We found that the mean level of red cell glutathione, whether expressed as micromoles per gram of hemoglobin or micromoles per milliliter of cell water, was significantly decreased in 39 patients with sickle cell anemia. A weak but significant negative correlation between glutathione levels and irreversibly sickled cell counts in the patients was observed. Stractan density gradient studies were performed to determine glutathione differences in normal- and abnormal-appearing sickle cells. The percent of abnormal cells in each of the five stractan gradient fractions was negatively correlated with both the micromole per gram of hemoglobin and micromole per milliliter of cell water levels of glutathione. Another indicator of susceptibility to oxidant stress, Heinz body formation, was increased in patients with sickle cell anemia vs controls in response to oxidant stress. Surprisingly, we found that the majority of Heinz bodies were in the non-ISC fraction of cells, rather than in the irreversibly sickled cells.

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A 20-year-old black man with known sickle cell anemia was admitted to the hospital with the signs and symptoms of right middle lobe pneumonia. Because of a hemoglobin value of 3.9 g/dL, he received a transfusion of 3 units of packed erythrocytes. Seven days later, hypertension, headache, and one grand mal seizure occurred. Evaluation of his condition gave normal findings. Similar events, some terminating in cerebral hemorrhage and death, have been reported in patients with hemolytic anemia after they received multiple transfusions. To our knowledge, this patient represents the first adult with sickle cell anemia who had this syndrome. Prompt antihypertensive therapy may have been of benefit.

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We studied the calmodulin-like activity (CaM) level in hemolyzed samples of whole blood after determining the percentage and absolute number of reticulocytes present. Twenty-six samples from 25 people with a range of reticulocyte counts were studied. CaM levels correlated with the percentage (r = 0.63, P less than .01) and absolute number (r = 0.64, P less than .01) of reticulocytes present, indicating that the calmodulin-like activity level in whole blood was inversely related to erythrocyte age.

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Patients with sickle cell anemia may be zinc-deficient. Zinc deficiency has been reported to cause delayed dark adaptation. We have studied dark adaptation in 13 patients with homozygous sickle cell anemia. Six patients had delayed dark adaptation. Their mean plasma and neutrophil zinc levels were 93.83 +/- 8.26 micrograms/100 microliters and 37.33 +/- 11.88 micrograms/10(10) cells, respectively, compared to the corresponding mean values for seven patients with normal dark adaptation of 105.86 +/- 13.31 and 86.71 +/- 19.53. For the six patients with delayed dark adaptation, the lower neutrophil zinc value, but not the plasma value, was significant (p less than 0.01). Three of the six were treated with oral zinc, with improvement in their dark adaptation. Neutrophil zinc levels correlated inversely (r = -0.77, p less than 0.01) with log relative threshold at 30 min. We conclude that patients with sickle cell anemia may show decreased ability to dark-adapt that is related to zinc deficiency.

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An increase in plasma ammonia level in human volunteers on restricted zinc intake, and in zinc-deficient rats, has recently been reported. Inasmuch as zinc deficiency has been observed in sickle cell anemia patients, we measured plasma ammonia levels in such subjects. In this report we document hyperammonemia in sickle cell anemia patients that was corrected with zinc therapy.

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We have documented a rise in pCO2 when erythrocytes are haemolysed by freeze-thaw using solid CO2 and methanol. This is partially corrected by preventing leakage of CO2 into the system. The remaining increment is partially explained by stimulation of the hexose monophosphate shunt when erythrocytes are haemolysed by this technique.

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