Nobuhiro Uyesaka

Augmentation by erythropoietin of the fetal-hemoglobin response to hydroxyurea in sickle cell disease.

BACKGROUND Hydroxyurea increases the production of fetal hemoglobin in patients with sickle cell anemia, inhibiting the polymerization of hemoglobin S and potentially improving vaso-occlusive manifestations and hemolysis. Recombinant erythropoietin increases the number of reticulocytes containing fetal hemoglobin in laboratory animals and in humans. We studied whether hydroxyurea and erythropoietin might have a potentiating effect on the production of fetal hemoglobin in patients with sickle cell disease. METHODS We treated four patients who were receiving hydroxyurea for sickle cell disease (three who were homozygous for sickle cell anemia and one with sickle beta zero-thalassemia) with escalating doses of intravenous erythropoietin for seven weeks, along with oral iron sulfate. Doses of hydroxyurea on four consecutive days were alternated with doses of erythropoietin on three consecutive days. RESULTS There was a 28 percent increase in the number of reticulocytes containing fetal hemoglobin and a 48 percent increase in the percentage of fetal hemoglobin, as compared with the maximal values obtained with hydroxyurea alone. The percentage of erythrocytes containing fetal hemoglobin (F cells) increased from 64 to 78 percent. As compared with hydroxyurea alone, treatment with hydroxyurea and erythropoietin decreased the mean (+/- SD) serum indirect bilirubin level from 0.8 +/- 0.2 to 0.5 +/- 0.1 mg per deciliter (13.3 +/- 2.9 to 8.9 +/- 2.2 mumol per liter) (P = 0.02), suggesting a further decrease in hemolysis. Red-cell filterability improved. CONCLUSIONS Intravenous recombinant erythropoietin with iron supplementation alternating with hydroxyurea elevates fetal-hemoglobin and F-cell levels more than hydroxyurea alone. Such increases decrease intracellular polymerization of hemoglobin S and improve the overall rheologic characteristics of erythrocytes. A reduced dosage of hydroxyurea alternating with erythropoietin may prove less myelotoxic than hydroxyurea given daily or in pulsed-dose regimens. It may also increase levels of fetal hemoglobin in patients with sickle cell disease who have not been helped by hydroxyurea alone.

Effects of superoxide anions on red cell deformability and membrane proteins

The effect of superoxide anions (O2-) on red blood cells (RBC) deformability and membrane proteins was investigated using hypoxanthine-xanthine oxidase system. Exposure of RBC to O2- caused a marked decrease in RBC deformability with a concomitant increase in cell volume and shape changes. The RBC exposed to O2- also displayed pronounced degradation of membrane proteins such as band 3 protein and spectrin; new bands of low molecular weight products appeared as the original membrane proteins tended to diminish, without the appearance of high molecular weight products. Since the membrane proteins are involved in processes regulating membrane properties such as permeability and viscoelasticity, the decreased deformability induced by O2- may be attributable to changes in membrane proteins. Interestingly, resealed ghosts exposed to O2- did not show any significant change in membrane proteins, which suggests the existence of further generation of O2- and subsequent production of other active oxygen species mediated by O2(-)-initiated autoxidation of hemoglobin in intact RBC. Furthermore, electrophoretic analysis suggested that active oxygens increased the endogenous proteolytic susceptibility of RBC. In conclusion, a close linkage was suggested between RBC deformability and the membrane proteins.

Further investigations of red cell deformability with nickel mesh.

Although the filtration method has been widely employed in red cell deformability studies, the structural irregularity of the pores of a Nuclepore polycarbonate membrane has always been a major problem. Anegawa, T. et al. (Clin. Hemorheol., 7, 1987) obtained a higher reproducibility with the filtration method using a newly designed thin metal film with pores engraved by the photofabrication technique. We further studied the pressure - flow rate relationship of red cell suspension employing this nickel mesh. The filtration of red cell suspensions through the nickel mesh was not influenced by leukocytes contamination or added leukocytes up to a leukocyte count of 250 cells/mm3 within an experimental limitation. On the other hand, the flow was greatly influenced by leukocytes contamination when the polycarbonate membrane was used. The nickel mesh was found to be useful in detecting major determinants of red cell deformability, such as cell geometry and internal cellular viscosity, and in detecting abnormalities of red cell deformability in a patient with microangiopathic hemolytic anemia. In conclusion, the present study clearly shows that the nickel mesh is preferable for investigating red cell deformability to the polycarbonate membrane from a quantitative point of view. This material should contribute to the physiologic and clinical investigation of red cell deformability.

A new standard fluorescence microsphere for quantitative flow cytometry

A standard fluorescence microsphere has been prepared for quantitative flow cytometry. A uniformly sized (6.53 +/- 0.34 micron in diameter) and perfectly spherical plastic microsphere, Micropearl-FT, was covalently conjugated with fluorescein isothiocyanate (FITC). The characteristic of this microsphere to lyse completely in strong alkaline solutions made it possible to determine the amount of bound FITC easily and reproducibly. The amount of bound FITC depended on the concentration of FITC and the incubation time at pH 9.5 and 37 degrees C. In flow cytometry, the fluorescence intensity of the FITC-conjugated microsphere (FCMS) showed linear dependence on the amount of bound FITC. The fluorescence intensity of FCMS was stable for at least 3 months even in aqueous solutions. Using FCMS, an Epics V was calibrated and the minimum measurable fluorescence intensity was examined. From analysis using this system, the number of FITC-conjugated OKT3 molecules bound to human T lymphocytes was shown to average 4.4 X 10(4) per cell at saturation.

PRODUCTION OF PROSTAGLANDINS E1 AND E2 BY ADULT HUMAN RED BLOOD CELLS

We showed that human adult red blood cells (RBCs) produce prostaglandin E1 (PGE1) and E2 (PGE2). RBCs that were mechanically stressed in the presence of extracellular Ca2+ by being injected rapidly through a fine needle produced PGE1 and PGE2 within 30 min after this mechanical stress. The amounts of PGE1 and PGE2 produced by 1 x 10(9) mechanically stressed RBCs were approximately 50 pg and 100 pg, respectively, which were determined in the cytosolic fraction from sonicated RBCs using a competitive enzyme immunoassay method. A Western blot analysis using anti-cyclooxygenase-2 antibody revealed a band at the 70-kDa position in the samples from RBCs producing PGE1 and PGE2. Treatment with 10 micrograms/mL indomethacin completely inhibited the productions of PGE1 and PGE2. The present results may indicate a new role of RBCs in microcirculation.

Impaired deformability of Heinz body-forming red cells.

Although a decrease in the deformability of red blood cells (RBCs) has been suspected in Heinz body-forming hemolytic anemia, it remains uncertain whether the formation of Heinz bodies themselves impairs RBC deformability or not. To elucidate this question, we used RBCs treated with phenylhydrazine and RBCs from patients with unstable hemoglobin (Hb) disease (Hb Yokohama) to investigate the effect of Heinz body formation on deformability in terms of RBC filterability through nickel mesh and viscosity of the RBC suspension. The phenylhydrazine-treated RBCs exhibited a marked decrease in deformability in a dose-dependent manner. The Heinz body-forming RBCs from the patients also showed a marked decrease in deformability. Thus we confirmed that Heinz body formation impairs RBC deformability. Further, both phenylhydrazine-treated RBCs and RBCs from the patient showed a degradation of spectrin without any cross-linking of membrane proteins, thereby suggesting that the impaired deformability is associated with the oxidative degradation of the cytoskeletal framework. In summary, this study supports the conclusion that RBC deformability is impaired by the presence of Heinz bodies as well as the related oxidative damage involved in their formation.

Application of the transition state theory to water transport across cell membranes

We have applied the transition state theory of Eyring et al. (The Theory of Rate Processes, McGraw-Hill, 1941) to water transport across cell membranes. We have then evaluated free energy (Delta F(not equal)), enthalpy (Delta H(not equal)) and entropy (Delta S(not equal)) of activation for water permeation across membranes, such as Arbacia eggs, Xenopus oocytes with or without aquaporin water channels, mammalian erythrocytes, aquaporin proteoliposomes, liposomes and collodion membrane. Delta H(not equal) was found to be correlated with Delta S(not equal). This is so-called Delta H(not equal) and Delta S(not equal) compensation over the ranges of Delta H(not equal) and Delta S(not equal) from 2 to 22 kcal/mol and from -26 to 45 e.u., respectively, indicating that low Delta H(not equal) values correspond to negative Delta S(not equal). Large positive Delta S(not equal) and high Delta H(not equal) values might be accompanied by reversible breakage of secondary bonds in the membrane, presumably in membrane lipid bilayer. Largely negative Delta S(not equal) and low Delta H(not equal) values for aquaporin water channels, aquaporin proteoliposomes and porous collodion membrane could be explained by the immobilization of permeating water molecules in the membrane, i.e., the partial loss of rotational and/or translational freedoms of water molecules in water channels.

Comparative intermolecular cross-relaxation studies of human hemoglobin in red blood cells and bovine serum albumin in solution

Intermolecular cross‐relaxation rate (CR) spectra [1/TIS(HDO) or 1/TIS(H2O) vs f2(ppm) profiles] for bovine serum albumin [BSA; molecular weight (MW), 66 kDa] solution, partially hydrolyzed BSA gel (BSA*gel) and packed human red blood cells (RBCs) with normal or unstable hemoglobin (Hb; MW, 65 kDa) were studied using f2 irradiation ranging from – 100 to 100 ppm at γH2/2π of 250 Hz. The CR spectra for BSA*gel (pD 4.01, 0.10 M NaCl, 4.83 and 14.39%) exhibited different features in the off‐resonance region (below – 2.00 and above 12.0 ppm) relative to that for BSA solution (pD 7.14, 0.10 M NaCl, 14.39%), indicating the association of BSA* molecules in the gel state. The CR spectrum for packed RBCs was compared with those for BSA*gel and BSA solution (14.39%) by correcting for differences in protein concentration. The corrected CR spectrum for packed normal RBCs in the off‐resonance region was similar to that for BSA solution, indicating that the physical characteristics of Hb in normal RBCs may be in a solution‐like state. Our results on normal RBCs were approximately consistent with the previously reported thermodynamic and hydrodynamic findings that Hb in RBCs and/or in concentrated solution seems to be in a suspension of hard scaled particles. Copyright

Electron spin resonance studies of erythrocyte membrane in spinocerebellar degeneration

Erythrocyte membrane fluidity was examined by electron spin resonance spectra using nitroxide fatty acid spin labels in spinocerebellar degeneration (SCD). Subjects with SCD, motor neuron disease (MND) and controls did not differ in fluidity of the deep site (hydrophobic region) of the erythrocyte membrane. However, the fluidity of the shallow site (hydrophilic region) in the erythrocyte membrane was significantly less fluid in SCD than in controls and MND (outer hyperfine splitting of 5-nitroxide stearic acid: SCD 54.70 +/- 0.43 G, controls 53.57 +/- 0.41 G, MND 53.54 +/- 0.35 G, P less than 0.001). Serum HDL-cholesterol and membrane fluidity correlated significantly in controls, but not in SCD. A significant negative correlation between age and membrane fluidity was found in SCD, but not in controls. These data suggest that membrane abnormality exists in SCD and may be concerned with aging.

Quantitative characterization of P-glycoprotein-mediated transport in mdr1-gene-transfected lymphoma cells.

We have established a quantitative flow cytometry system to elucidate the causal role of P-glycoprotein in the phenomenon of multidrug resistance. We have used this method to analyze the accumulation and release of adriamycin (ADM) in intact L5178Y and L5178Y/VMDR/C.06 (L5178Y/R) cells, by determining the effect of sodium orthovanadate (Na3VO4), verapamil, bovine serum albumin (BSA) and physiologically operative materials on the cells. Based on the experiments, we prepared a standard solution that contained NaCl, D-glucose, L-cysteine, HCO3- and BSA, which was sufficient to perform transport experiments. In particular, BSA caused a decrease in ADM accumulation and a facilitation of the rate of ADM release in both L5178Y and L5178Y/R cells, probably due to its relatively high affinity for ADM as compared to the cell membrane. In multidrug-resistant L5178Y/R cells, sodium orthovanadate, a strong ATP-binding inhibitor, caused a marked increase in the accumulation of ADM, whereas vanadate-treated drug-sensitive L5178Y cells showed little increase in ADM accumulation. In a release (0-trans exit) experiment, vanadate-treated L5178Y/R cells exhibited an apparent decrease in ADM release (increase in ADM retention), to a level which was almost the same as L5178Y cells. We thus confirmed that the P-glycoprotein-mediated efflux system is coupled with P-glycoprotein-associated ATP-hydrolysis. Further, verapamil, a potent inhibitor of P-glycoprotein-mediated transport, facilitated the ADM accumulation in L5178Y/R cells up to the level of L5178Y and vanadate-treated L5178Y/R cells. A more important finding is that, in the release experiment, verapamil-treated L5178Y/R cells exhibited a much greater ADM retention than drug-sensitive L5178Y and vanadate-treated L5178Y/R cells. These findings, in particular the potent effect of verapamil on drug-resistant cells, may afford new insight into the pathophysiology of the phenomenon of multidrug resistance and the mechanism of action of the multidrug transporter.