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.

Relationship of high-density lipoprotein cholesterol and red blood cell filterability: cross-sectional study of healthy subjects.

The deformability of red blood cells (RBCs) is an important rheologic factor in the maintenance of normal blood flow in the microcirculation. Contrary to the well-known relationship between hyperlipidemia and atherosclerosis, the relationship between RBC rheology and the serum lipid profile has remained controversial and obscure. Moreover, the correlation of high-density lipoprotein (HDL)-cholesterol and RBC deformability has not been fully understood. In the present cross-sectional study of 139 apparently healthy subjects, we investigated whole-cell deformability (filterability) of RBCs in relation to the lipid profile, using a nickel mesh filter with 3.2-microm pores. RBC filterability was independent of gender, age and serum levels of low-density lipoprotein (LDL)-cholesterol. The filterability was significantly proportional to the HDL-cholesterol values (r = 0.382, p < 0.01), whereas it was inversely proportional to the triglyceride levels (r = -0.259, p < 0.01). These findings may provide new insight into the role of HDL-cholesterol not only in preventing atherosclerotic progression but also in improving RBC filterability.

Endothelin-1 improves the impaired filterability of red blood cells through the activation of protein kinase C.

We previously showed that the deformability of human red blood cells (RBCs) is affected by intracellular signaling pathways by examining the effects of Ca2+ influx and the intracellular cAMP level on mechanically-impaired RBC filterability. In the present study, we investigated whether protein kinase C (PKC) participates in the regulation of RBC deformability by affecting membrane properties. The filterability of mechanically-stressed RBCs showed a V-shaped curve depending on the extracellular Ca2+ concentration; the maximum decrease was achieved at 20-40 microM. The PKC activity, as measured in the membrane-rich fraction by an ELISA method using an antibody for the phosphorylated PKC substrate, maximally increased at the extracellular Ca2+ concentration where the filterability showed a marked improvement following the bottom of the V-shaped curve of the impaired filterability. At this Ca2+ concentration, the PKC activator endothelin-1 increased the PKC activity, and a PKC inhibitor (calphostin C) decreased it. Endothelin-1 improved and calphostin C worsened the impaired filterability. A specific type-B endothelin receptor agonist (IRL 1620) also improved the impaired filterability. A Western blot analysis revealed the presence of endothelin receptors in the RBC membrane. These results indicate that PKC improves the impaired filterability and that RBCs are the target of endothelin-1.

Kinetic analysis of the mechanism of action of the multidrug transporter.

To clarify the mechanistic role of PGP (P-glycoprotein) in multidrug transport, we constructed a kinetic model composed of four compartments: (1) the extracellular space; (2) the space in the membrane; (3) the intracellular space; and (4) the pore-like space in the PGP molecule. The kinetics of the concentration of ADM (adriamycin) in each compartment were formulated based on the assumptions that (a) the movement of ADM between two compartments by diffusion is dependent on a dynamic distribution coefficient introduced here, (b) the uptake of ADM into the pore-like structure by the pump mechanism activated by ATP is described by enzyme kinetics, (c) the movement of ADM out of the pore-like structure to the extracellular medium through a valve-like mechanism is also expressed by enzyme kinetics. The mathematical analysis of the exact solution can explain the distinct effects of verapamil and vanadate on the accumulation and release of ADM, where verapamil inhibits the efflux by the valve-like mechanism and vanadate blocks the influx by the pump mechanism. We also performed a numerical calculation with this model for a quantitative explanation and found the valid parameter values to fit the experimental data. These results support the modified hydrophobic vacuum cleaner model.

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.

Sickling of nucleated erythroid precursors from patients with sickle cell anemia.

The pathophysiology of sickle cell anemia is primarily explained in terms of the oxygen-dependent polymerization of sickle hemoglobin (HbS) followed by sickling of erythrocytes. Since the rate and extent of HbS polymerization depend on its intracellular concentration, it has been generally assumed that sickling occurs primarily in mature erythrocytes with their high intracellular hemoglobin concentration. In the present study, we investigated the propensity of nucleated erythroid precursors to undergo sickling; both cultured and fresh marrow-derived erythroid precursors from patients with homozygous sickle cell anemia were studied. The results revealed that upon deoxygenation cultured erythroblasts underwent characteristic morphological deformation in the form of fine, fragile, elongated spicules. Ultrastructural analysis demonstrated highly organized and tightly aligned hemoglobin fibers in the protruded regions. Bone marrow cells examined under partial or complete deoxygenated conditions displayed similar morphological changes. When cultured SS erythroid precursors were exposed to hydroxyurea or butyrate, drugs that may increase fetal hemoglobin (HbF) and inhibit intracellular polymerization, a significant decrease was observed in the propensity of these precursors to undergo sickling, accompanied by a three- to fivefold increase in HbF. These results suggest that, in addition to mature erythrocytes, nucleated erythroid precursors in the bone marrow have the capacity to undergo characteristic sickling as a result of HbS polymerization and may be involved in several aspects of the pathophysiology of sickle cell anemia. Treatment with HbF-stimulating drugs may benefit patients with this disease by inhibiting polymerization-induced sickling of erythroid precursors in the marrow as well as mature erythrocytes in the peripheral blood.

Regulation of red blood cell filterability by Ca2+ influx and cAMP-mediated signaling pathways.

To investigate the mechanism of the regulation of human red blood cell deformability, we examined the deformability under mechanical stress. Washed human red blood cells were rapidly injected through a fine needle, and their filterability was measured using a nickel mesh filter. The decrease in filterability showed a V-shaped curve depending on the extracellular Ca2+ concentration; the maximum decrease was achieved at approximately 50 muM. The decreased filterability was accompanied by no change in cell morphology and cell volume, indicating that the decrease in filterability can be ascribed to alterations of the membrane properties. Ca2+ entry blockers (nifedipine and felodipine) inhibited the impairment of filterability under mechanical stress. Prostaglandins E1 and E2, epinephrine, and pentoxifylline, which are thought to modulate the intracellular adenosine 3',5'-cyclic monophosphate (cAMP) level of red blood cells, improved or worsened the impaired filterability according to their expected actions on the cAMP level of the cells. These results strongly suggest that the membrane properties regulating red blood cell deformability are affected by the signal transduction system, including Ca(2+)-dependent and cAMP-mediated signaling pathways.

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.

Filterability of mixtures of sickle and normal erythrocytes.

We investigated the deformability of sickle (SS) cells from 25 patients and mixtures of these SS cells with blood type-compatible normal (AA) cells, using a nickel mesh filtration system, with the aim of determining optimal goals for exchange therapy. We found that for air-equilibrated SS/AA cell mixtures the fraction of dense cells (MCHC > 37 g/dl) is the determinant factor in filterability and that the dense cells contribute in a linear fashion to the loss of filtration up to 15% dense cells (y = -4.41x + 98.23, r = 0.945, P < 0.0001). The slope of this effect is approximately 25 times steeper than that of the relationship between filtration and percent nondense (MCHC < 37/g/dl) SS cells (y = -0.17x + 106.53, r = 0.772, P < 0.0001). A comparison of the proportion of high fluorescence reticulocytes to total reticulocytes (HFR ratio), indicating an elevation of immature reticulocytes, between six nontransfused patients and six exchange-transfused patients showed significant higher values in the nontransfused individuals (0.154 +/- 0.051 versus 0.070 +/- 0.054, P < 0.003). These results may have implications regarding targets for exchange transfusion therapy. Further studies of the effect on transfusion, both simple and exchange, on the numbers of dense cells and the proportions and populations of reticulocytes and the rheological characteristics of the erythrocyte subpopulations seems warranted.

Contributions of sickle hemoglobin polymer and sickle cell membranes to impaired filterability.

Sickle cell anemia is a disease of abnormal rheology caused by acute and reversible, as well as chronic and irreversible, changes in the properties and deformability of sickle erythrocytes. Deformability is determined by several factors, including intracellular sickle hemoglobin polymerization, the abnormal membrane properties of sickle cells, and the abnormal rheological properties of the soluble concentrated hemoglobin solution within dense sickle red blood cells. In this study, we used a 5-microns pore nickel mesh filter to evaluate quantitatively the effects of these factors on the filterability of erythrocytes containing sickle hemoglobin. We used sickle trait and sickle/beta(+)-thalassemia cells, because they have minimal membrane abnormalities or density heterogeneity, to investigate the effects of polymer formation on rheological properties. We found that filterability of these cells is sensitive to small amounts of intracellular polymer and that impaired filtration is linearly related to oxygen-dependent polymer formation, up to a polymer fraction of 0.3. By increasing the proportion of dense cells in populations of normal cells or cells from individuals with sickle syndromes and equilibrating these cells with gas ligands, we estimate that polymerization, even at 95% saturation, contributes twice as much to impaired filterability of sickle erythrocytes as the abnormal membranes in homozygous sickle cell disease. At lower saturation values, the effects of polymer are even greater. The viscosity of the concentrated hemoglobin in dense cells had the smallest effect, over physiologically relevant saturation values. These results emphasize the importance of sickle hemoglobin polymerization in the pathogenesis of sickle cell disease and should help define its pathophysiology and responses to therapy in quantitative terms.

Sickle cell rheology is determined by polymer fraction--not cell morphology.

Sickle cell disease pathophysiology is mediated by acute and chronic impairment of cell flexibility due to the formation of intracellular sickle hemoglobin (Hb S) polymer as cells are partially deoxygenated in the microcirculation. We have recently developed a method to measure the relationship between the formation of intracellular polymerized Hb S and cell filtration. In this study, we have used this method to examine whether sickle cell morphology, independent of Hb S polymer fraction, had an effect on cell rheology. We primarily use sickle trait (AS) and Hb S-beta(+)-thalassemia (S-beta(+)-thal) erythrocytes with low hemoglobin F levels, which have normal membranes and few or no dense cells, to remove these confounding effects. We find that the relationship between filtration and the percentages of each "type" of morphological deformation of AS erythrocytes was different from that of the S-beta(+)-thal erythrocytes. In addition, we find that while the filtration of AS erythrocytes as a function of oxygen saturation was similar, whether measured during deoxygenation or reoxygenation, the relationship between the percentages of each type of deformed erythrocyte and oxygen saturation demonstrated hysteresis during oxygenation-deoxygenation experiments. Transmission electron microscopy, for both elongated and irregularly shaped cells, showed that similarly distorted cells could have very different amounts and alignment of polymer. These results suggests that cell morphology per se is not strongly related to filtration, whereas calculated intracellular Hb S polymer fraction predicts loss of filtration of AS and S-beta(+)-thal erythrocytes well. Measured or calculated polymer fraction values would appear to be a better parameter for the study of sickle cell disease pathophysiology and response to treatment than cell morphology studies.

Rheologic and pathophysiologic significance of red cell passage through narrow pores.

To elucidate the pathophysiologic significance of red blood cell (RBC) filterability, we measured RBC rheology with our own designed nickel mesh with 3-microns pores, smaller than the previously used 5-microns pores. Vertical and cylindrical pores with no pore coincidence were regularly distributed across the filter, the pore entrances of which showed a round and rather smooth transition to the pore inside. An advantage of the nickel mesh is the repeated use (at least 100 times) of the same filter possible after ultrasonic washing. A very low concentration of RBC, i.e., 3 x 10(4) cells per cubic millimeter (hematocrit value of approximately 0.3%), was sufficient for a typical test to examine RBC filterability. The filtration of the dilute RBC suspension was not influenced by contaminating or added leukocytes up to a leukocyte count of approximately seven cells per cubic millimeter; therefore, measurements can be performed using conventionally washed RBCs. This may be practically relevant to routine use, such as in a clinical laboratory. As compared with filtration through 5-micron pores, filtration through 3-micron pores was found to be very sensitive in detecting major determinants of RBC deformability, particularly, changes in viscoelastic properties of the cell membrane, surface area/volume ratio of the cell, perturbing effects of lysophosphatidylcholine, and osmolality of the medium. The 3-micron filtration method revealed a marked impairment in the filterability of Heinz body-containing RBCs from patients with unstable hemoglobin (Hb) disease (Hb Yokohama). Thus, 3-micron-filtration measurements may contribute to several subfields of hematology.

Effect of low-osmolality contrast media on red cell filterability.

The effects of low-osmolality contrast media (CM) on red blood cell (RBC) filterability were investigated using a recently developed nickel mesh filtration method. The conventional hypertonic CM iothalamate, low-osmolality iohexol, and the recently synthesized iomeprol were studied. Among them, the osmolality of iomeprol was the lowest. The impact of CM osmolality, viscosity, and iodine content on the RBC filterability was analyzed. Under equal iodine content or viscosity condition, the filterability order of RBCs suspended in CM was iomeprol > iohexol >> iothalamate, because of the osmolality of CM. Iomeprol caused small echinocytic changes but these had a negligible influence on RBC filterability. In conclusion, the osmotic effect of CM on RBC filterability is more predominant than the other CM effects, and iomeprol is the preferred CM for RBC filterability.

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.

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.

Possible role of red cell deformability and microvasculature in microcirculation.

The roles of the deformability of red blood cells (RBC) and the microvasculature in the maintenance of blood flow were investigated in terms of the pressure (P)-flow rate (Q) relationships in human RBC suspension perfusions of bullfrog hind limb. Although isotonicity for the bullfrog is approximately 215 mOsm/kgH2O, perfusions in intact hind limbs showed no change in the P-Q relationship at test solution osmolalities ranging from approximately 150 to approximately 300 mOsm/kgH2O. The deformability of RBC was examined in glutaraldehyde-fixed hind limbs. Perfusion of fixed limbs with RBC suspension revealed minimum resistance to flow at osmolalities of approximately 250 to approximately 420 mOsm/kgH2O, whereas the same experiment in intact limbs revealed minimum flow resistance at osmolalities of approximately 200 to approximately 300 mOsm/kgH2O. It was noteworthy that the reduction of RBC deformability was not observed in intact limbs at osmolalities of approximately 250 to approximately 200 mOsm/kgH2O. Heinz body-forming RBC from a patient with unstable hemoglobin (Hb) disease (Hb Yokohama) exhibited a marked reduction in deformability as compared with normal RBC in fixed limbs, while there was no discernible difference between the two types of RBC in intact limbs, thereby suggesting that the microvascular bed can compensate, to an appreciable extent, for the impaired deformability of RBC, probably via its distensibility and/or a wall effect. The present study has considerable implications concerning the link between in vitro experiments and the in vivo situation, including the hemodynamic characteristics of RBC suspensions such as the effective viscosity.

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.

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.

Separation of human neutrophils in self-generated continuous density gradients of Percoll.

Human neutrophils were separated into two fractions using a continuous density gradient of Percoll solution. A marked decrease in O2- production was observed in the low-density neutrophil fraction. There was little erythrocyte contamination in the high-density neutrophil fraction (less than 0.5%), and thus the hypotonic or ammonium chloride lysis of erythrocytes was not necessary.