Protective effect of quercetin and rutin on photosensitized lysis of human erythrocytes in the presence of hematoporphyrin

Photosensitized hemolysis of human erythrocytes by hematoporphyrin was suppressed by flavonols such as quercetin and rutin at submillimolar concentrations. The suppression of photohemolysis was accompanied by inhibition of lipid peroxidation by the reagents. Quercetin and rutin were photooxidized in the presence of hematoporphyrin and the photooxidation was partially suppressed by 1 mM NaN3, a quencher of singlet molecular oxygen. Flavonols were also oxidized by radicals formed during degradation of lauroyl peroxide. These results indicate that flavonols can function as antioxidants in biological systems by terminating radical chain reactions and removing singlet molecular oxygen. A pharmacological function of flavonols, decrease of the increased permeability and fragility of capillary, was discussed in relation to their antioxidative functions.     

Aging human erythrocytes. Differential sensitivity of young and old erythrocytes to hemolysis induced by peroxide in the presence of thyroxine.

Human erythrocytes were separated according to cell age using albumin density gradients. In the presence of glucose (100 mg%), young cells were able to effectively protect themselves against thyroxine-peroxide induced hemolysis; old cells exhibited less protection. Hemolysis in heterogeneous populations is preceded by lipid peroxidation, K⁺ leak and decreased filtrability of the cells. Hydroxy radical scavengers partially inhibited hemolysis while superoxide dismutase had no effect. It is postulated that the differential sensitivity of young and old erythrocytes to thyroxine-peroxide induced metabolic and morphological alterations may play a role in the recognition and removal of senescent cells from the circulation.     

PH-dependence of detergent-induced hemolysis and vesiculation of erythrocytes

The influence of pH of the medium on the parameters of detergent-induced fast hemolysis and vesiculation of human erythrocytes was studied. In the range of pH 6.3-7.2 neither the extent nor the rate of the vesiculation induced by 25 microM sodium dodecyl sulfate (SDS) changed. However, a decrease of pH from 8.0 to 5.8 strongly modified both the extent and the rate of the hemolysis induced by SDS. Within the range of pH 8.0-6.4, the effect can be ascribed to the increase of the positive charge of the membrane. This could lead to the accumulation of the membrane-bound anion detergent and, hence, to the change of the hemolysis parameters. Non-charged detergent Triton X-100 did not display any pH-dependence. At pH between 6.4 and 5.8 the extent and rate of hemolysis changed in a complicated manner. The kinetic curves of hemolysis could be approximated by a single exponential within the pH range between 8.0 and 7.2. Upon further reduction of pH, a second exponential component, with a larger time constant, appeared in the kinetic curves. At 5.8 < pH < 7.2, the contribution of the "fast" hemolysis dropped virtually to zero, with pK about 6.0. This points to a structural transition of the membrane, possibly involving histidine. We suggest that the parameters of the detergent-induced hemolysis are sensitive to the changes of the charge and structural state of erythrocyte membrane.     

Dextran protection of erythrocytes from low-pH-induced hemolysis

Low-pH-induced hemolysis of erythrocytes is inhibited by dextrans. The protective effect was observed with dextrans larger than 40 kDa. Electron microscopy showed dextrans of 150 kDa in a tight association with the erythrocyte membrane. These results indicate that dextrans stop the low-pH-induced hemolysis by interacting with the acid-induced defects in the erythrocyte membrane [(1989) Biochim. Biophys. Acta, in press.     

Comparison of acid and alkaline hemolysis mechanisms in human erythrocytes

A comparative analysis of the mechanisms of base- and acid-induced hemolysis was performed. The results obtained indicate the transport of base equivalents through the anion exchanger during the initial phase of base-induced hemolysis, followed by oxidative stress on cellular membranes and hemolysis. It was shown that the Ellman's reagent (0.4 mM) did not prevent NaOH-induced hemolysis but fully inhibited HCL-induced hemolysis. The inhibition of acid-induced hemolysis was accompanied by the crosslinking membrane proteins, presumably through their acylation. The addition of SH-reducing reagents (cystein, dithiotreitol and, to a lesser extent, albumin eliminated the crosslinkage of membrane proteins and impaired the permeability barrier. It was found that crosslinkage could not prevent the oxidative damage of membrane proteins but was able to preserve the permeability barrier. Based on these results, it was concluded that the barrier impairments associated with acid-induced hemolysis were due to the aggregation of membrane proteins that underwent oxidative damage.     

Delayed hemolysis of human erythrocytes in solutions of glucose

There has been described a type of hemolysis which occurs under certain defined conditions when erythrocytes are suspended in glucose solution. It consists of a prolytic phase lasting about an hour, followed by a hemolytic phase lasting about 2 hours. The physical factors controlling this delayed hemolysis have been investigated. The system is especially sensitive to changes of pH and of temperature. This type of hemolysis is inhibited by increased osmotic pressure and by phlorhizin, but not, as far as can be ascertained, by fluoride or iodoacetate. It is possible, but not yet proved, that delayed hemolysis in glucose solution is dependent on enzymic activity. Phosphorylation may be the limiting factor. During the prolytic phase the cells are easily permeable to potassium. It is concluded that the development of cation permeability is not a direct cause of hemolysis.     

Hypoosmotic hemolysis of erythrocytes by active carbonyl forms

Low-molecular-weight dicarbonyls formed during free radical peroxidation of polyene lipids (malondialdehyde) and autooxidation (glyoxal) or other oxidative transformations of glucose (methylglyoxal) are able to modify the structure of lipid-protein supramolecular complexes of cells. We investigated changes in the erythrocyte membrane structure after an 18-h exposure of human red blood cells in the presence of various natural dicarbonyls. The changes in the mechanical properties of the membrane after membrane modification by carbonyls were evaluated by the susceptibility of erythrocytes to hypoosmotic hemolysis. It has been shown that treatment of red blood cells with malondialdehyde increases the resistance of these cells to hypoosmotic hemolysis, whereas the malondialdehyde isomer, methylglyoxal, in contrast, makes red blood cells more sensitive to the action of hypoosmotic solutions. Paradoxically, a homologue of malondialdehyde, glyoxal, has no effect on hemolysis of red blood cells in hypoosmotic solutions. The findings point to the possibility of the multidirectional effect of low-molecular-weight dicarbonyls with similar structures on the structure and function of biological membranes.     

Induction and decay of thermotolerance in human erythrocytes determined by hemolysis

Hemolysis was used as an endpoint for the measurement of damage to the plasma membrane in human erythrocytes after a single or a double heat shock. The thermotolerance of erythrocytes is a transitional phenomenon, reaching its maximum at a 3-hour incubation at 37 degrees C between the heat shocks.     

Differential resistance to calcium-induced bilirubin-dependent hemolysis in mammalian erythrocytes

Washed erythrocytes from human, buffalo, sheep and goat preincubated with different concentrations of calcium chloride (16.7–1830 μM) showed significantly different rates of hemolysis (up to 62%) after addition of bilirubin (72 μM). Goat erythrocytes displayed marked resistance to hemolysis with only 11% hemolysis observed at the highest calcium concentration. Similar trend in hemolysis was also observed when the concentration of CaCl₂ was fixed (330 μM) and bilirubin concentration varied (0–72 μM). (Ca²⁺–Mg²⁺)-ATPase levels were found significantly lower in goat and sheep erythrocyte membranes compared to human and buffalo erythrocyte membranes. This was correlated well with the observed hemolysis in various mammalian erythrocytes.     

Hemin-mediated Hemolysis in Erythrocytes： Effects of Ascorbic Acid and Glutathione

In the present work,we investigated the effect of ascorbic acid and glutathione on hemolysisinduced by hemin in erythrocytes.Ascorbic acid not only enhanced hemolysis,but also induced formationof thiobarbituric acid-reactive substances in the presence of hemin.It has been shown that glutathioneinhibits hemin-induced hemolysis by mediating hemin degradation.Erythrocytes depleted of glutathionebecame very sensitive to oxidative stress induced by hemin and ascorbic acid.H_2O_2 was involved in hemin-mediated hemolysis in the presence of ascorbic acid.However,a combination of glutathione and ascorbicacid was more effective in inhibiting hemolysis induced by hemin than glutathione alone.Extracellular andintracellular ascorbic acid exhibited a similar effect on hemin-induced hemolysis or inhibition of hemin-induced hemolysis by glutathione.The current study indicates that ascorbic acid might function as anantioxidant or prooxidant in hemin-mediated hemolysis,depending on whether glutathione is available.     

Comparative study of the effect of phenazine methosulfate and vitamin K3 on human erythrocytes by hemolysis curves]

Hemolysis curves were used for comparative study of phenazine methylsulfate (PMS) and vitamin K3 action on human erythrocytes. Some differences in PMS and vitamin K3 action were revealed while the concentration of studied compounds and incubation time with them were changed. It is considered that the observed differences in PMS and vitamin K3 action are caused by different degree of oxidation of intracellular hemoglobin.     

The susceptibility of cholesterol-depleted erythrocytes to saponin and sapogenin hemolysis

The assumption that complex formation between erythrocyte membrane cholesterol and saponins or sapogenins is the cause for their hemolytic activity, was tested by measuring the susceptibility of cholesterol-depleted erythrocytes towards these hemolysins. For some of the hemolysins cholesterol depletion caused inhibition of hemolysis, for others an augmentation. The results suggest that cholesterol does not serve as a specific binding site for these hemolysins.     

Comparison of the survival of desialylated erythrocytes and demonstration of the presence of T-agglutinin in rabbits and ducks]

Rabbit as well duck erythrocytes desialization leads to a dramatic decrease of their survival time. Rabbit erythrocytes are trapped transiently by the liver ; in contrast splenic uptake is gradual. Serum from the two species contains a T-agglutinin directed against autologous desialyated erythrocytes. In the two cases, this T-agglutinin is from the IgM class and its immunodominant sugar is galactose in the beta configuration.