Intact Human Erythrocytes Research Articles

Effect of pigment packaging on diffuse reflectance spectroscopy of samples containing red blood cells

We present the results of diffuse reflectance measurements made on the surface of a tissue-simulating phantom containing intact human erythrocytes. These measurements indicate that the absorption spectrum of hemoglobin in its natural environment is significantly different from that measured in homogeneous fluid solution, especially in the spectral regions of highest absorption. We show that this difference can be explained by the pigment packaging theory developed by Duysens [Biochim. Biophys. Acta 19, 1 (1956)] and that the adoption of basis spectra that take this effect into account improves the accuracy of fitting diffuse reflectance spectra.

Carbogen breathing significantly enhances the penetration of red light in murine tumours in vivo

We report results of experiments that evaluated the influence of oxygenation on the penetration of red light in tissue, with particular emphasis on 630 and 650 nm laser wavelengths commonly used in photodynamic therapy (PDT) of solid tumours. Direct measurements in tissue-simulating phantoms comprised of intact human erythrocytes suspended in a scattering emulsion demonstrated significant enhancements in fluence rate at depths of 0.5–2 cm from the irradiated surface when the cells were fully oxygenated versus fully deoxygenated. The 630 and 650 nm fluence rates at depth in the homogeneous phantoms continued to increase when examined over a continuous range of oxygen partial pressures from 0 to 160 Torr. When considered as a function of haemoglobin oxygen saturation, the largest increases in fluence rate were observed as the saturation increased beyond 70%. Dramatic increases in optical fluence rate were measured at the base of 1-cm-thick subcutaneous EMT6 mammary carcinomas in vivo when the tumour-bearing mouse was subjected to carbogen through a nose cone. These results indicate that improved tumour oxygenation is important in PDT not only for the maintenance of the oxygen-dependent photochemistry but, through the effects reported here, may also enable more efficient treatment of thicker lesions.

Verapamil prevents impairment in filterability of human erythrocytes exposed to oxidative stress.

Effects of oxidative stress on intact human erythrocytes were investigated using tert-butyl hydroperoxide (tBHP). Exposure of erythrocytes to tBHP caused a marked decrease in filterability in a time-dependent manner. Erythrocytes exposed to tBHP also show an increase in mean corpuscular volume and a remarkable formation of methemoglobin (met-Hb) without any appearance of hemichromes that form Heinz bodies. High performance liquid chromatography demonstrated that the tBHP-treated erythrocytes exhibited an apparent decrease in the membrane phospholipid, phosphatidylethanolamine (PE). The decrease in PE was inhibited by pretreatment with ascorbate, but not with verapamil. SDS-polyacrylamide gel electrophoresis of the tBHP-treated erythrocyte membrane showed a degradation of spectrin, band 3, band 4.2, and band 4.5, accompanied by the appearance of low-molecular-weight products. The degradation of the membrane proteins was not prevented by pretreatment with verapamil or ascorbate. However, the pretreatment with verapamil but not with ascorbate revealed significant inhibition of the tBHP-induced impairment in filterability in the presence of extracellular Ca2+. Thus, the present study shows that verapamil, a potent drug in reperfusion therapy, plays an important role in protection against oxidative injury, based on a close linkage among decreased filterability, met-Hb formation, and impaired membrane integrity.

Different types of glutathionylation of hemoglobin can exist in intact erythrocytes

Glutathionylation of hemoglobin (Hb) was studied by incubation of intact human erythrocytes with 1 mM tert-butylhydroperoxide ( tBHP). Electrophoresis of the membranes showed a time dependent increase of membrane-bound Hb α chain until 10 min, and immunoblotting study showed that membrane-bound Hb α chain reacted with anti-glutathione antibody only after 10 min. Concomitant with the Hb α chain, membrane associated actin, spectrin, and glyceraldehyde 3-phosphate dehydrogenase reacted with the antibody. Cytosolic Hb of the control erythrocytes reacted with anti-glutathione antibody. Together with our previous paper, the present study indicates that at least three different types of glutathionylation of Hb can exist in erythrocytes. The first type is a mixed disulfide bond between reduced glutathione (GSH) and normal Hb. The second type is a disulfide bond between the cysteine 93 of metHb β chain and oxidized glutathione (GSSG), and the third type is a disulfide bond between the other cysteine residues of metHb α chain and/or metHb β chain and GSSG.

Oxidation of hemoglobin to methemoglobin in intact erythrocyte by a hydroperoxide induces formation of glutathionyl hemoglobin and binding of α-hemoglobin to membrane

Biochemical consequences of oxidation of hemoglobin (Hb) in intact human erythrocytes were studied. The incubation of washed erythrocyte with 1 mM tert-butylhydroperoxide induced an increase in glutathionyl-Hb (G-Hb). The formation of G-Hb occurred linearly until 10 min in parallel with the formation of methemoglobin (metHb) after exhaustion of reduced glutathione. The results show that metHb, but not normal Hb, reacts with oxidized glutathione to form G-Hb. G-Hb was confirmed by immunoblotting with anti-glutathione antibody and the formation of G-Hb was accompanied by parallel decrease in β-globin detected with a reversed phase HPLC. Electrophoretic studies showed time-dependent increase in membrane-associated α-Hb until 10 min, indicating that a part of unpaired α-Hb bound to the membrane. Pre-β-globin increased despite the decrease in β-globin and a part of the increase was independent of the decrease in β-globin. Pre-β-globin reacted with anti-glutathione antibody, but it differs from G-Hb in many features.

A novel blood transfusant candidate: intact human erythrocytes containing hemoglobin exclusively nitrosylated in the alpha-subunits.

The transport of oxygen from lungs to peripheral tissues is carried out by hemoglobin (Hb), the main protein component inside red blood cells. However, the modulation of its physiological performance is regulated by the intracellular content of metabolites, mainly 2,3-diphosphoglycerate (DPG), which lower the oxygen affinity of Hb to optimal levels, so that an efficient oxygen delivery is accomplished within a narrow gradient in partial pressures of oxygen between lungs and tissues. However, intracellular levels of DPG in stored blood decay with time, which causes a gradual increase in oxygen affinity, thus imposing a limit to the lifetime of blood for practical use.

Protective effect of melatonin against cytotoxic actions of malondialdehyde: an in vitro study on human erythrocytes

Malondialdehyde (MDA), a by-product of the oxidation of polyunsaturated fatty acids, is strongly cytotoxic. Here we report the in vitro ability of melatonin to protect intact human erythrocytes against the damage induced by the exposure to MDA. MDA at 20 microM caused marked variations in the red blood cell (RBC) membrane. High molecular weight fluorescent adducts were formed within minutes with membrane proteins. A 6-hr incubation led to the oxidation of membrane lipids, as reflected by the formation of conjugated diene (CD) lipid hydroperoxides and oxidation of vitamin E, and to an increase of the high molecular weight fluorescent adducts, which were an indication of MDA finally generated in the cells. Functional damage to the membrane was evident as a leakage of K+ ions into the incubation medium, and an increased resistance to osmotic lysis. A time-dependent hemolysis was observed by exposure of RBCs to 20 microM MDA for 6-12 hr. Melatonin was not a substrate for MDA, therefore it was not able to prevent the early formation of the adducts from the reaction of the MDA in the medium with membrane proteins. Melatonin, however, concentration-dependent prevented the formation of CD lipid hydroperoxides. As a consequence of counteracting the membrane lipid oxidation, the indoleamine prevented the loss of vitamin E and the increase of the fluorescent proteinaceous adducts observed after a 6-hr exposure to MDA. Melatonin also inhibited the K+ loss and returned to normal the osmotic resistance of the erythrocyte in the osmotic fragility test. By protecting membrane lipids and proteins, melatonin effectively prevented the MDA-induced time-dependent hemolysis. In the light of the known radical scavenging properties of melatonin, mechanisms of the cytoprotective effects of melatonin in our system are discussed.

Effects of ascorbate on membrane phospholipids and tocopherols of intact erythrocytes during peroxidation by t-butylhydroperoxide: comparison with effects of dithiothreitol.

Peroxidation of intact human erythrocytes by t-butylhydroperoxide (tBHP)) was studied. By incubation of the erythrocytes with 1 mM tBHP, reduced glutathione (GSH) was exhausted within 1 min, and tocopherols (Toc) and phospholipids (PL) decreased to nearly their lowest levels (in this study) within 5 min. The rate of decrease of alpha-Toc was faster than that of gamma-Toc, but alpha-Toc was never exhausted. The rates of decrease of Toc were faster than that of PL. Malondialdehyde increased slowly to reach a maximal value at 30 min. Methemoglobin (metHB) reached a maximum at 15 min. The maximal levels of these substances were maintained until 90 min incubation, which indicated that the peroxidation by tBHP had stopped spontaneously until at least 90 min. By the incubation with tBHP for 30 min, phosphatidylethanolamine (PE) and alpha-Toc decreased to about 70 and 30% of control levels, respectively, and gamma-Toc and GSH were almost exhausted. Ascorbate (0.1 mM) afforded protection of 92% to PE, 50% to alpha-Toc, and 65% to gamma-Toc against peroxidation, but ascorbate had no preventive effect at all on the formation of metHB and the decrease of GSH. These results may indicate that ascorbate-mediated protection of the membrane PL against the peroxidation depends primarily on Toc. On the other hand, dithiothreitol (DTT) (5 mM) almost completely prevented the formation of metHB, and DTT completely protected the PL and Toc against peroxidation, indicating the importance of sulfhydryl groups in erythrocytes.

Conversion of a synthetic fructosamine into its 3-phospho derivative in human erythrocytes

Intact human erythrocytes catalyse the conversion of fructose into fructose 3-phosphate with an apparent K(m) of 30 mM [Petersen, Kappler, Szwergold and Brown (1992) Biochem. J. 284, 363-366]. The physiological significance of this process is still unknown. In the present study we report that the formation of fructose 3-phosphate from 50 mM fructose in intact erythrocytes is inhibited by 1-deoxy-1-morpholinofructose (DMF), a synthetic fructosamine, with an apparent K(i) of 100 microM. (31)P NMR analysis of cell extracts incubated with DMF indicated the presence of an additional phosphorylated compound, which was partially purified and shown to be DMF 3-phosphate by tandem MS. Radiolabelled DMF was phosphorylated by intact erythrocytes with an apparent K(m) ( approximately 100 microM) approx. 300-fold lower than the value reported for fructose phosphorylation on its third carbon. These results indicate that the physiological function of the enzyme that is able to convert fructose into fructose 3-phosphate in intact erythrocytes is probably to phosphorylate fructosamines. This suggests that fructosamines, which are produced non-enzymically from glucose and amino compounds, may be metabolized in human erythrocytes.

Sequential phosphorylation of protein band 3 by Syk and Lyn tyrosine kinases in intact human erythrocytes: identification of primary and secondary phosphorylation sites

AbstractTreatment of intact human erythrocytes with pervanadate induces Tyr (Y)-phosphorylation of the transmembrane protein band 3; in parallel, the activity of the immunoprecipitated tyrosine kinases Syk and Lyn is increased. When erythrocytes are incubated with pervanadate together with PP1, a specific inhibitor of Src kinases, including Lyn, the Y-phosphorylation of band 3 is only partially reduced. Indeed, the PP1-resistant phosphorylation of band 3 precedes and is a prerequisite for its coimmunoprecipitation with Lyn, which interacts with the phosphoprotein via the SH2 domain of the enzyme, as proven by binding competition experiments. Upon recruitment to primarily phosphorylated band 3, Lyn catalyzes the secondary phosphorylation of the transmembrane protein. These data are consistent with the view that band 3 is phosphorylated in intact erythrocytes by both PP1-resistant (most likely Syk) and PP1-inhibited (most likely Lyn) tyrosine kinases according to a sequential phosphorylation process. Similar radiolabeled peptide maps are obtained by tryptic digestion of32P-band 3 isolated from either pervanadate-treated erythrocytes or red cell membranes incubated with exogenous Syk and Lyn. It has also been demonstrated by means of mass spectrometry that the primary phosphorylation of band 3 occurs at Y8 and Y21, while the secondary phosphorylation affects Y359 and Y904.

Sequential phosphorylation of protein band 3 by Syk and Lyn tyrosine kinases in intact human erythrocytes: identification of primary and secondary phosphorylation sites

Treatment of intact human erythrocytes with pervanadate induces Tyr (Y)-phosphorylation of the transmembrane protein band 3; in parallel, the activity of the immunoprecipitated tyrosine kinases Syk and Lyn is increased. When erythrocytes are incubated with pervanadate together with PP1, a specific inhibitor of Src kinases, including Lyn, the Y-phosphorylation of band 3 is only partially reduced. Indeed, the PP1-resistant phosphorylation of band 3 precedes and is a prerequisite for its coimmunoprecipitation with Lyn, which interacts with the phosphoprotein via the SH2 domain of the enzyme, as proven by binding competition experiments. Upon recruitment to primarily phosphorylated band 3, Lyn catalyzes the secondary phosphorylation of the transmembrane protein. These data are consistent with the view that band 3 is phosphorylated in intact erythrocytes by both PP1-resistant (most likely Syk) and PP1-inhibited (most likely Lyn) tyrosine kinases according to a sequential phosphorylation process. Similar radiolabeled peptide maps are obtained by tryptic digestion of32P-band 3 isolated from either pervanadate-treated erythrocytes or red cell membranes incubated with exogenous Syk and Lyn. It has also been demonstrated by means of mass spectrometry that the primary phosphorylation of band 3 occurs at Y8 and Y21, while the secondary phosphorylation affects Y359 and Y904.

A GBP 130 derived peptide from Plasmodium falciparum binds to human erythrocytes and inhibits merozoite invasion in vitro.

The malarial GBP 130 protein binds weakly to intact human erythrocytes; the binding sites seem to be located in the repeat region and this region's antibodies block the merozoite invasion. A peptide from this region (residues from 701 to 720) which binds to human erythrocytes was identified. This peptide named 2220 did not bind to sialic acid; the binding site on human erythrocyte was affected by treatment with trypsin but not by chymotrypsin. The peptide was able to inhibit Plasmodium falciparum merozoite invasion of erythrocytes. The residues F701, K703, L705, T706, E713 (FYKILTNTDPNDEVERDNAD) were found to be critical for peptide binding to erythrocytes.

Evaluation of Hypotonic Preswelling Method for Encapsulation of Enalaprilat in Intact Human Erythrocytes

The hypotonic preswelling method for encapsulation of drugs in intact human erythrocytes was evaluated using enalaprilat as a model peptidelike drug. Several process variables, including volume, concentration, pH, and method of addition of drug solution, type of erythrocyte-suspending medium, temperature, initial packed density of erythrocytes, and individual process steps, were exploited with respect to their effects on the loading parameters (i.e., loaded amount, efficiency of entrapment, and cell recovery). In addition, the probable mechanism by which the erythrocytes were loaded by enalaprilat at the point of lysis was shown to be a simple concentration gradient–based diffusion through membrane openings occurring on hemolysis. Finally, the adopted method was validated, and the results showed a considerable degree of reproducibility and recovery for the entire loading procedure.

Membrane microextension: a possible mechanism for establishing molecular contact in electrofusion

True cell membrane contact is an essential condition for electro-pulsed cell fusion, but initial morphological perturbation leading to true contact is still not clear. Dielectrophoresis mediated compression and fusogenic pulse induced compaction of cells led to rapid merger of tight membranes, and deprived direct microscopic view of surface membrane perturbation. Freely suspending cells with large and different cell–cell gaps may proceed to electrofusion with perturbed membrane and initiates fusion events at different time. These pulsed exposed cells can be used for capturing changes in the membrane surface and early electrofusion events. Early stage of fusion of freely suspended intact human erythrocytes exposed to single exponential decay pulse was studied by scanning electron microscopy (SEM). Field pulse induces small membrane bumps. Interaction of bumps on adjacent membranes lead to true membrane contact and form bridges between the membranes as microextension, combining both membranes into a topologically single structure. Some fusion products showed expanded fusion zones, which suggest indication of open lumen at contact area.

Some relationships between membrane phospholipid domains, conformational order, and cell shape in intact human erythrocytes

A novel method developed in this laboratory [D.J. Moore et al., Biochemistry 35 (1996) 229–235; D.J. Moore et al., Biochemistry 36 (1997) 660–664] to study the conformational order and the propensity for domain formation of specific phospholipids in intact human erythrocytes is extended to two additional species. Acyl chain perdeuterated 1,2-dilauroylphosphatidylethanolamine (diC 12PE-d 46) was incorporated preferentially (in separate experiments) into the inner leaflet of stomatocytic erythrocytes and into the outer leaflet of echinocytic erythrocytes, while acyl chain perdeuterated 1,2-dipentadecanoylphosphatidylcholine (diC 15PC-d 58) was incorporated into the outer leaflet of echinocytic erythrocytes. The conformational order and phase behavior of the incorporated molecules were monitored through FT-IR studies of the temperature dependence of the CD 2 stretching vibrations. For both diC 12PE-d 46 and diC 15PC-d 58, the gel→liquid crystal phase transition persisted when these lipids were located in the outer leaflet of echinocytic cells, a result indicative of the persistence of phospholipid domains. In each case, the transition widths were broadened compared to the pure lipids, suggestive of either small domains or the presence of additional molecular components within the domains. The conformational order of diC 12PE-d 46 differed markedly depending on its location and the morphology of the cells. When located predominantly in the inner membrane of stomatocytes, the phase transition of this species was abolished and the conformational order compared with pure lipid vesicles at the same temperature was much lower. The current results along with our previous studies provide a sufficient experimental basis to deduce some general principles of phospholipid conformational order and organization in both normal and shape-altered erythrocytes.

Inhibition of Human Erythrocyte (Na +-K +)ATPase by Organic Hydroperoxides and Protection by Ascorbic Acid and Butylated Hydroxytoluene

1. The in vitro effects of cumene hydroperoxide and t-butyl hydroperoxide on intact human erythrocyte membrane (Na +-K +)ATPase activities have been studied. 2. (Na +-K +)ATPase activities on erythrocyte membranes decreased in agreement with the results of chemiluminescence experiments. 3. Our results demonstrated that the organic hydroperoxides inhibit the activity of (Na +-K +) ATPase enzyme and that the antioxidants used prevent this inhibition.

Protection and Recycling of α-Tocopherol in Human Erythrocytes by Intracellular Ascorbic Acid

Ascorbic acid can recycle α-tocopherol from the tocopheroxyl free radical in lipid bilayers and in micelles, but such recycling has not been demonstrated to occur across cell membranes. In this work the ability of intracellular ascorbate to protect and to recycle α-tocopherol in intact human erythrocytes and erythrocyte ghosts was investigated. In erythrocytes that were 80% depleted of intracellular ascorbate by treatment with the nitroxide Tempol, both 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) and ferricyanide oxidized α-tocopherol to a greater extent than in cells not depleted of ascorbate. In contrast, in erythrocytes in which the intracellular ascorbate concentration had been increased by loading with dehydroascorbate, loss of α-tocopherol was less with both oxidants than in control cells. Protection against AAPH-induced oxidation of α-tocopherol was not prevented by extracellular ascorbate oxidase, indicating that the protection was due to intracellular and not to extracellular ascorbate. Incubation of erythrocytes with lecithin liposomes also generated an oxidant stress, which caused lipid peroxidation in the liposomes and depleted erythrocyte α-tocopherol, leading to hemolysis. Ascorbate loading of the erythrocytes delayed liposome oxidation and decreased loss of α-tocopherol from both cells and from α-tocopherol-loaded liposomes. When erythrocyte ghosts were resealed to contain ascorbate and challenged with free radicals generated by AAPH outside the ghosts, intravesicular ascorbate was totally depleted over 1 h of incubation, whereas α-tocopherol decreased only after ascorbate was substantially oxidized. These results suggest that ascorbate within the erythrocyte protects α-tocopherol in the cell membrane by a direct recycling mechanism.

Presence in Human Erythrocyte Membranes of a Novel Form of Sialidase Acting Optimally at Neutral pH

AbstractThe feature of intact human erythrocytes and erythrocyte white ghosts is a unique sialidase activity with acidic optimal pH (acidic sialidase). The treatment of white ghosts with mildly alkaline isotonic solutions at 37°C, like that used to produce resealed ghosts, is accompanied by the expression, together with the acidic sialidase, of a novel sialidase with a pH optimum of 7.2 (neutral sialidase) that remained masked in the inside-out vesicles prepared from white ghosts. Exhaustive treatment of resealed ghosts with Bacillus Thuringiensis phosphatidylinositol-specific phospholipase C causes an almost complete release of the acidic sialidase, with the neutral enzyme remaining totally unaffected. The treatment of resealed ghosts with 1.2% Triton X-100 resulted in the solubilization of only the neutral sialidase, whereas 3.6% octylglucoside also solubilized the acidic sialidase. The neutral enzyme affected not only the artificial substrate but also any sialoderivatives of a ganglioside, glycoprotein, and oligosaccharide nature; the acidic enzyme did not affect sialoglycoproteins. Erythrocyte endogenous gangliosides were hydrolyzed by both sialidases, whereas the endogenous sialoglycoproteins responded to only the neutral enzyme. It was definitely proved that the acidic sialidase is located on the outer erythrocyte membrane surface, so presumably the neutral enzyme has the same location. It could be that the newly discovered neutral sialidase has a physiologic role in the releasing of sialic acid from erythrocytes during the erythrocyte aging process, leading to eventual phagocytosis by macrophages.

A1 H spin echo and 51V NMR study of the interaction of vanadate with intact erythrocytes

The action of vanadate on intact human erythrocytes was studied by 1H spin echo and 51V NMR spectroscopy as a model for the behaviour of vanadium(V) complexes in experimental diabetes. Vanadate is reduced by the intact erythrocyte at the expense of intracellular glutathione which rapidly depletes from the intracellular volume. Using the blocking agent 4,4′-diisothio-cyanatostilbene-2,2′-disulfonic acid (DIDS), which specifically blocks the anion transporter, vanadate reduction could be inhibited and glutathione depletion arrested. Thus, for the reaction with the intact cell to occur, vanadium(V) must cross the cell wall, possibly via the anion transporter. Nitrofurantoin was used to inhibit glutathione reductase in the erythrocyte suspensions. Under these conditions, treatment of the cells with vanadate induced glutathione oxidation prior to depletion. A study of the reaction of vanadate with haemolysate indicates that, without the influence of the membrane, rapid oxidation of glutathione to glutathione disulfide by the vanadyl cation occurs with no glutathione depletion, and that under these conditions vanadate reduction is incomplete. This study generates a model for the behaviour of vanadium complexes in vivo, providing a basis for the rational design and synthesis of new vanadium-based agents as insulin mimics. In essence, vanadium is transported across the membrane as vanadate(V), is reduced in situ by glutathione, and becomes complexed to a wide range of intracellular binding sites. Exchange reactions between glutathione and sulfhydryl groups present on haemoglobin and membrane lead to the depletion of glutathione from the cytosol.

Design of an adenosine phosphorylase (AP) by active site modification of purine nucleoside phosphorylase (PNP)

The C(8) and C(5') positions of base and nucleoside substrates of human erythrocytic purine nucleoside phosphorylase (PNP) are promising sites for the development of an inhibitor of this enzyme. The substrate analog, 8-aminoguanine (8-AG), has the lowest dissociation constant (Ki = 0.2–1.2 μM) of any compound reported to date and Vmax = 16 per cent (relative to guanine). Other C(8) substituents decreased the affinity for PNP and, with the exception of the methyl and sulfhydryl groups, abolished substrate activity. Halogens or a thiomethyl group at C(5') of inosine resulted in unchanged or improved affinities (Ki = 10–30 μM) and greatly decreased substrate activity (Vmax < 1 per cent relative to inosine). The Ki of formycin B was reduced from 100 μM to 10 μM or less by substitution of a halogen at C(5'). Phosphorolysis of purine nucleosides was inhibited significantly by 8-AG in intact human erythrocytes and murine Sarcoma 180, L1210 and L5178Y cells. Although a Ki value of 17 μM was determined for 8-aminoguanosine, it was equally effective in inhibiting PNP activity in intact cells. The nucleoside was cleaved to 8-AG which was not a substrate for guanase or hypoxanthine-guanine phosphoribosyltransferase. Despite its low substrate activity (Vmax < 0.2%). 5′-deoxy-5′-iodoinosine was cleaved by intact L1210 and L5178Y cells.