Changes in membrane properties of rat red blood cells induced by cadmium accumulating in the membrane fraction

When rat red blood cells were incubated in a cadmium (Cd)-free medium following 1-h pretreatment with 0.5 mM CdCl2, incorporated Cd was retained in the cell during 14-h incubation and progressively accumulated in the membrane fraction, especially in the cytoskeleton fraction. In parallel to this accumulation, red cell filterability decreased and echinocytic cells increased, although intracellular ATP was maintained at the control level. The echinocytic shape was maintained in ghosts and cytoskeletons prepared from the Cd-loaded cells. In addition, the association of bands 2.1, 3, 4.2, and 4.5 with cytoskeletons increased and dissociation of cytoskeletal networks at low ionic strength decreased as the incubation time increased. Pretreatment of red blood cells with Cd also induced a release of small vesicles. These vesicles contained hemoglobin but were depleted of spectrin and actin, showing a phospholipid composition similar to that of red cell ghosts. These results suggest that the organization of cell membranes, especially cytoskeletal networks, is altered by Cd accumulation in the cytoskeleton fraction, which results in acceleration of age-related changes of red blood cells such as shape change and decreased filterability.     

Altered potassium permeability in vitamin E-deficient rat erythrocytes

Erythrocytes of vitamin E-deficient rats were investigated as an in vivo model of oxidant stress and cellular aging. To measure possible membrane damage related to the enhanced oxidant stress, the permeability of the erythrocyte membrane to potassium was determined. Rates of non-hemolytic potassium loss were calculated from comparison of total potassium loss and hemolysis rates. The non-hemolytic potassium loss rates for erythrocytes of vitamin E-deficient rats were as much as 2.5-fold higher than controls. The abnormally high permeability of vitamin E-deficient rat erythrocytes indicates molecular damage at the membrane level, and may be significant to our understanding of the normal aging process in erythrocytes and other cells.     

Antioxidant and Antihemolytic Activity of a New Isoflavone, “Factor 2” Isolated from Tempeh

A new isoflavone assigned the trivial name “Factor 2” which was first isolated from tempeh, fermented soybean product, and identified as 6,7,4′-trihydroxyisoflavone was chemically synthesized and tested for antioxidant activity by several methods. Factor 2 was proved to be a potent antioxidant in aqueous solution at pH 7.4. However it was not effective in preventing autoxidation of soybean oil and soybean powder. Factor 2 given orally to rats fed vitamin E-deficient diet was also negative in hemolysis preventing activity. Biological activity of tempeh and the isolated Factor 2 to prevent hemolysis of red blood cells of rats fed vitamin E-deficient diet was discussed.     

Role of vitamin E in glutathione-induced oxidant stress: methemoglobin, lipid peroxidation, and hemolysis.

Red blood cells (RBC) from normal and vitamin E-deficient rats were incubated in a hypertonic solution of reduced glutathione adjusted to pH 8. Methemoglobin formation occurred in intact RBC from both normal and vitamin E-deficient rats. Hemolysis was significantly greater in RBC from vitamin E-deficient rats. Experiments with catalase, superoxide dismutase, and methional showed that H(2)O(2) was the primary extracellular source of oxidant stress. Extracellular superoxide and hydroxyl radical were not involved in oxidant stress. Experiments with dimethyl sulfoxide showed that intracellular hydroxyl radical, generated from H(2)O(2), was the hemolytic agent. Neither methemoglobin formation nor lipid peroxidation involved hydroxyl radical. Indeed, lipid peroxidation and hemolysis in RBC from vitamin E-deficient rats were concurrent rather than consecutive events. Phase contrast microscopy showed that rigid, crenated RBC with a precipitate around the interior periphery formed during glutathione-induced oxidant stress. The precipitate dissolved slowly as the crenated RBC were converted to smooth ghosts. It appeared that protein precipitates involving mixed disulfide bonds were reduced and solubilized when extracellular glutathione penetrated the ruptured cell. Comparisons between normal RBC and vitamin E-deficient RBC suggest that vitamin E has little effect on the inward diffusion of extra-cellular H(2)O(2). Vitamin E apparently interacts with different oxidant species derived from intracellular H(2)O(2) in preventing lipid peroxidation and the sulfhydryl group oxidation leading to hemolysis.     

Atrial natriuretic peptide: direct effects on human red blood cell dynamics.

The ability to deform is an important feature of red blood cells (RBCs) for performing their function of oxygen delivery. Little is known about the hormonal regulation of RBC deformability. Here we report that human atrial natriuretic peptide (ANP) acts directly on human RBCs leading to the elevation of local bending fluctuations of the cell membrane. These changes are accompanied by an increase in the filterability of RBCs. These ANP effects were mimicked by cyclic GMP analogues, suggesting modulation of local membrane bending fluctuations and RBC filterability via a cyclic GMP-dependent pathway. The effect of ANP on the mechanical properties of RBCs suggests that ANP may increase the passage red blood cells through capillaries resulting in an improved oxygen delivery to the tissues.     

Vitamin E deficiency and vitamin C supplements: exercise and mitochondrial oxidation

The effects of dietary antioxidant vitamins E and C on exercise endurance capacity and mitochondrial oxidation were investigated in rats. The endurance capacity of both vitamin E-deficient and vitamin C-supplemented, E-deficient rats was significantly (P less than 0.05) lower (38.1 and 33.6%, respectively) than control animals. Compared with the normal and vitamin E-deficient rats, there was a significant (P less than 0.05) increase in the concentration of vitamin C in blood and liver of the vitamin E-deficient, C-supplemented animals. Hence dietary vitamin C supplementation does not prevent the inhibition of exercise endurance capacity or increased hemolysis seen in vitamin E deficiency. The mitochondrial activities for the oxidation of palmitoyl carnitine and alpha-ketoglutarate were significantly (P less than 0.05) decreased by a single bout of exercise in brown adipose tissue but not in muscle, heart, or liver from vitamin C-supplemented, E-deficient groups of rats when compared with the activities in the tissue from the same group of rats killed at rest. Similar results were also seen in brown adipose tissue from vitamin E-deficient rats. The results suggest a tissue-specific role for vitamins E and C in substrate oxidation and show that the poor endurance capacity of vitamin E-deficient rats cannot be attributed to any changes in the mitochondrial activity in skeletal or cardiac muscles. It is also concluded that vitamin C supplementation, at least at the dose employed in the present study, cannot counteract the detrimental effects associated with vitamin E deficiency.     

Use of vitamin E deficient red cells to detect a dialyzable hemolytic factor produced by peroxidizing rat liver microsomes.

The tendency of rat red blood cells to hemolyze in the presence of peroxidizing rat liver microsomes is greatly increased if the red cells are obtained from vitamin E deficient rats. Adequate dietary vitamin E supplementation imparts resistance against hemolysis. Dietary butylated hydroxytoluene or the level of erythrocyte glutathione or total thiols are relatively unimportant factors in determining red cell sensitivity to hemolysis induced by perixiziding microsomes. When separated from peroxidizing microsomes by a dialysis membrane, vitamin E deficient cells are completely hemolyzed. Hemolytically active material can be separated from peroxidized microsomes by dialysis at 0°C.     

Vitamin E affects cell death in adult rat dentate gyrus

We have previously reported the presence of dying cells in the granule cell layer (GCL) of adult rat dentate gyrus (DG), where neurogenesis occurs. In particular, we found that cell death in the GCL increased in vitamin E deficiency and decreased in vitamin E supplementation. These findings were regarded as related to changes in neurogenesis rate, which in turn was influenced by vitamin E availability; a neuroprotective effect of vitamin E on cell death was also proposed. In order to verify this latter hypothesis, we have studied cell death in all layers of DG in vitamin E-deficient and vitamin E-supplemented rats and in control rats at different ages, using TUNEL and nick translation techniques. The phenotype of TUNEL-positive cells was characterized and the existence of dying BrdU-positive cells was investigated. Dying cells with neuronal phenotype were observed throughout the DG in all experimental groups. The number of TUNEL-positive cells decreased from juvenile to adult age. A higher number of TUNEL-positive cells in vitamin E-deficient rats and a lower number in vitamin E-supplemented rats, with respect to age-matched controls, were found; moreover, in these groups, TUNEL-positive cells had a different percentage distribution in the different layers of the DG. Our results confirm the occurrence of cell death in DG, demonstrate that cell death affects neuronal cells and support the hypothesis that the effect of vitamin E on cell death is not related to neurogenesis.     

Zinc,iron, vitamin E,and erythrocyte stability in the rat

Previous studies have shown that deficiencies of zinc and vitamin E, as well as iron excess, contribute to peroxidative damage in several tissues in vivo. The present study reports on the sensitivity of red blood cells from young rats exposed to individual or concurrent imbalances of these three nutrients. For 21 d, rats were fed diets that were either deficient or replete in zinc and with or without excess iron or replete or deficient in vitamin E. When red blood cells from these rats were incubated in vitro, erythrocyte hemolysis, lipid peroxidation (assessed by MDA production), and hemoglobin degradation (assessed by alanine release), did not significantly increase unless vitamin E had been omitted from the diet. These results imply that either adequate tightly-bound zinc exists within the zinc-deficient cell to protect it from oxidative damage, or that other antioxidant defense mechanisms (including vitamin E) present within the plasma membrane and cytosol are sufficient to protect the cell from the otherwise damaging effects of zinc deficiency and/or iron excess.     

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

To investigate the mechanism of theregulation of human red blood cell deformability, we examined thedeformability under mechanical stress. Washed human red blood cellswere rapidly injected through a fine needle, and their filterabilitywas measured using a nickel mesh filter. The decrease in filterabilityshowed a V-shaped curve depending on the extracellularCa²⁺ concentration; the maximumdecrease was achieved at ~50 µM. The decreased filterability wasaccompanied by no change in cell morphology and cell volume, indicatingthat the decrease in filterability can be ascribed to alterations ofthe membrane properties. Ca²⁺entry blockers (nifedipine and felodipine) inhibited the impairment offilterability under mechanical stress. ProstaglandinsE₁ and E₂, epinephrine, andpentoxifylline, which are thought to modulate the intracellularadenosine 3',5'-cyclic monophosphate (cAMP) level of redblood cells, improved or worsened the impaired filterability accordingto their expected actions on the cAMP level of the cells. These resultsstrongly suggest that the membrane properties regulating red blood celldeformability are affected by the signal transduction system, includingCa²⁺-dependent and cAMP-mediatedsignaling pathways.

     

Correlation between local cell membrane displacements and filterability of human red blood cells.

Local mechanical fluctuations of the cell membrane of human erythrocytes were shown to involve MgATP- and Mg(2+)-driven fast membrane displacements. We propose that these local bending deformations of the cell membrane are important for cell passage through capillaries. In order to verify this hypothesis, we examined cell membrane fluctuations and filterability of erythrocytes over a wide range of medium osmolalities (180-675 mosmol/kg H2O). The results indicate the existence of a positive correlation between the amplitude of local cell membrane displacements and cell filterability. We suggest that the occurrence of metabolically driven membrane displacements on the side surface of the red blood cell diminishes its bending stiffness and enables it to fold more efficiently upon entrance into blood capillaries. Thus, local cell membrane displacements seem to play an important role in microcirculation.     

Modulation of Platelet thromboxane A2 and arterial prostacyclin by dietary vitamin E

Platelets from vitamin E-deficient and vitamin E-supplemented rats generate the same amount of thromboxane A2 (TxA2) when they are incubated with unesterified arachidonic acid. Platelets from vitamin E-deficient rats produced more TxA2 than platelets from vitamin E-supplemented rats when the platelets are challenged with collagen. Arterial tissue from vitamin E-deficient rats generates less prostacyclin (PGI2) than arterial tissue from vitamin E- supplemented rats. The vitamin E effect with arterial tissue is observed when the tissue is incubated with and without added unesterified arachidonic acid. These data show that arterial prostacyclin synthesis is diminished in vitamin E-deficient rats. Vitamin E, in vivo, inhibits platelet aggregation both by lowering platelet TxA2 and by raising arterial PGI2.     

Effects of subchronic steroid therapy on vitamin E-deficient rats (38480).

Experiments were carried out to determine the effectiveness of steroid therapy in vitamin E-deficiency, as measured by autohemolysis of isolated RBC's body weight gain, serum creatine phosphokinase activity, and stabilization or labilization of isolated hepatic lysosomes. Results of such experiments would indicate whether triamcinolone acetonide could supplant vitamin E in vitamin E-deficiency states via its ability to stablize various membranes. Autohemolysis induced by vitamin E-deficiency could not be prevented by daily administration of triamcinolone. Daily dosages of 0.1 and 0.4 mg/kg (ip) triamcinolone given concomitantly with replacement vitamin E (at sufficient dosages to reverse the autohemolysis) resulted in an increased autohemolysis. No changes in lysosomal membrane fragility were noted when hepatic lysosomes were obtained from vitamin E-deficient rats with triamcinolone resulted in a greater attenuation of body-weight gain. Creatine phosphokinase levels were not augmented in vitamin E-deficient rats. Vitamin E-deficient rats supplemented with vitamin E and treated with triamcinolone, manifested an increase in creatine, phosphokinase. It was therefore concluded that although triamcinolone and vitamin E possess a common ability to stablize membranes and proteins, their mechanisms must be different since triamcinolone could not substitute for vitamin E in a deficiency state. Indeed, triamcinolone was found to be more toxic in the absence of vitamin E.     

Ca2+-activated Protease Activity in Vitamin E-Deficient Rats

The mechanism and control of protein degradation in cells are quite mysterious. We investigated the change of protease activities in animals fed a vitamin E-deficient diet. The Ca²⁺-activated protease activity was not significantly changed in vitamin E-deficient rats during the 45 weeks of the experiment. The cathepsin B activity was increased in those animals. Electron microscopic observation on the muscle of the vitamin E-deficient rats showed destruction of myofibrils at the Z-line, narrowness of myofibrils, and dispersed myofibrils. The M-line, which is known to disappear with cathepsin L treatment, was clearly observed. The phagocytosis of muscle cells by macrophages was also observed. These results show that the abnormal myofibril protein degradation in muscle tissue of vitamin E-deficient rats is not only due to the activation of macrophages and the increment of lysosomes in muscle cells, but also due to the protease which can destroy the myofibril at the Z-line. It may be a Ca²⁺-activated protease.     

The effect of vitamin E on the intracellular distribution of the different oxidation states of selenium in rat liver

1. The liver intracellular distribution of (75)Se, (75)Se(2-) and (75)SeO(3) (2-) formed from orally administered Na(2) (75)SeO(3) was studied in rats given four different dietary treatments. 2. Subcellular fractionation was done by using sucrose density gradients in a B XIV zonal centrifuge rotor, and conditions were established so that separation of lysosomal, mitochondrial, smooth- and rough-surfaced endoplasmic reticulum, and soluble fractions was achieved. 3. Marker enzymes acid phosphatase, succinate-2 - p - iodophenyl - 3 - p -nitrophenyl - 5 - phenyltetrazolium reductase and glucose 6-phosphatase were used, together with electron microscopy, to establish the identity of the fractions. 4. The dietary treatments investigated were: (a) vitamin E-deficient diet for 3 months, re-fed with vitamin E during the terminal 5 days; (b) vitamin E-deficient diet; (c) adequate diet; (d) vitamin E- and selenium-deficient diet, re-fed with vitamin E during the terminal 5 days. 5. In adequately fed rats, selenide was particularly associated with the mitochondrial fractions; in vitamin E-deficient rats, little selenide was found and the buoyant density of the mitochondria was increased, whereas re-feeding with vitamin E showed a restoration of the normal pattern. In vitamin E- and selenium-deficient rats, re-fed with vitamin E, there was no tendency for selenide to be localized in the mitochondria. 6. In the microsomal regions of the gradients, adequately fed rats showed a concentration of selenide, particularly in the smooth endoplasmic reticulum fractions, and to a lesser extent in the rough endoplasmic reticulum fractions. This was not observed in vitamin E-deficient rats, and the normal pattern was restored on re-feeding with vitamin E, both in rats given the vitamin E-deficient diet and the vitamin E- and selenium-deficient diet. 7. Some selenide was also found in the soluble fractions, when vitamin E was present, and a substantial proportion of this selenide was found to pass through a dialysis membrane. 8. These results are taken to support our hypothesis that the active form of selenium may be selenide located in non-haem iron-containing proteins, and that the function of vitamin E may be to protect the selenide from oxidation.     

Modulation of platelet thromboxane A2 and arterial prostacyclin by dietary vitamin E

Platelets from vitamin E-deficient and vitamin E-supplemented rats generate the same amount fo thromboxane A₂ (TxA₂) when they are incubated with unesterified arachidonic acid. Platelets from vitamin E-deficient rats produce more TxA₂ than platelets from vitamin E-supplemented rats when the platelets are challenged with collagen. Arterial tissue from vitamin E-deficient rats generates less prostacyclin (PGI₂) than arterial tissue from vitamin E-supplemented rats. The vitamin E effect with arterial tissue is observed when the tissue is incubated with and without added unesterified arachidonic acid. These data show that arterial prostacyclin synthesis is diminished in vitamin E-deficient rats. Vitamin E, $\text{in}$$\text{vivo}$, inhibits platelet aggregation both by lowering platelet TxA₂ and by raising arterial PGI₂.     

Effect of selenium deficiency and vitamin E deficiency on glutathione metabolism in isolated rat hepatocytes

Selenium deficiency and vitamin E deficiency both affect xenobiotic metabolism and toxicity. In addition, selenium deficiency causes changes in the activity of some glutathione-requiring enzymes. We have studied glutathione metabolism in isolated hepatocytes from selenium-deficient, vitamin E-deficient, and control rats. Cell viability, as measured by trypan blue exclusion, was comparable for all groups during the 5-h incubation. Freshly isolated hepatocytes had the same glutathione concentration regardless of diet group. During the incubation, however, the glutathione concentration in selenium-deficient hepatocytes rose to 1.4 times that in control hepatocytes. The selenium-deficient cells also released twice as much glutathione into the incubation medium as did the control cells. Total glutathione (intracellular plus extracellular) in the incubation flask increased from 47.7 +/- 8.9 to 152 +/- 16.5 nmol/10(6) selenium-deficient cells over 5 h compared with an increase from 46.7 +/- 7.1 to 92.0 +/- 17.4 nmol/10(6) control cells and from 47.7 +/- 11.7 to 79.5 +/- 24.9 nmol/10(6) vitamin E-deficient cells. This overall increase in glutathione concentration suggested that glutathione synthesis was accelerated by selenium deficiency. The activity of gamma-glutamylcysteine synthetase was twice as great in selenium-deficient liver supernatant (105,000 X g) as in vitamin E-deficient or control liver supernatant (105,000 X g). Hemoglobin-free perfused livers were used to determine the form of glutathione released and its route. Selenium-deficient livers released 4 times as much GSH into the caval perfusate as did control livers. Plasma glutathione concentration in selenium-deficient rats was found to be 2-fold that in control rats, suggesting that increased GSH synthesis and release is an in vivo phenomenon associated with selenium deficiency.     

Studies on the role of vitamin E in the oxidation of blood components by fatty hydroperoxides.

Studies are reported on the oxidation of vitamin E and changes in lipid and fatty acid composition of rat blood components incubated in vitro with hydroperoxides prepared from autoxidized methyl linoleate. Red blood cells, plasma, serum, and hemoglobin free stroma were incubated at 37 °C with suspensions of linoleate hydroperoxide in Tris buffer at pH 7.4. The RBC were destroyed and substances with excitation-fluorescent properties were produced. Phosphatidylethanolamine, vitamin E and unsaturated fatty acids were oxidized in the reaction. Among the reaction products were substances that gave a positive thiobarbituric acid value, tocoquinone, and an unidentified substance isolated in the nonsaponifiable fraction of the lipid extract of the hemolyzed red cells. The reaction of linoleate hydroperoxide with stroma was similar to that with red blood cells and the same products were observed. In contrast there was little reaction of linoleate hydroperoxide with vitamin E or lipids of the serum or plasma in the absence of red blood cells. The destruction of the red blood cells appeared to be closely related to the oxidation of vitamin E indicating that the strong antioxygenic action of vitamin E in vivo was due to its particular form or structural orientation in the red cell membrane.     

Changes in blood viscosity after administration of vitamin E in the rabbit

Vitamin E is an essential factor to maintain biological membranes stability and its lack may affect membranes structures and reduce erythrocyte life-span. Vitamin E also play a role in the maintenance of a normal platelet aggregation. A.A. studied the effects of a ten days supply of d-1-alpha tocopherol acetate (50 mg/Kg/die) on blood viscosity in 8 rabbits. Results obtained show a significant reduction of blood viscosity on 6th day of treatment in the male rabbits and a progressive reduction of values from the 6th till the 10th day in female rabbits. The most significant decrease of blood viscosity were obtained at the lowest shear-rates, due to an increased red cells deformability to the antioxidative action of vitamin E on the erythrocytes membrane and to a reduced red cells aggregation. Such modifications on the red blood cells caratheristics can be determined by vitamin E through different mechanism: a) inhibiting red cell membrane's polyunsaturable fatty acids oxidation; b) by removal of abnormal lipids from erythrocyte membrane; c) physical and chemical stabilization of membrane's surface.     

Complement-component-C3-opsonized immunoglobulin G anti-DNA antibodies do not bind effectively to red blood cells unless aggregated on a high-Mr DNA matrix.

The significance of microsomal vitamin E in protecting against the free-radical process of lipid peroxidation was evaluated with the low-level-chemiluminescence technique in microsomal fractions from vitamin E-deficient and control rats. The induction period that normally precedes the ascorbate/ADP/Fe3+-induced lipid peroxidation was taken as reflecting the microsomal vitamin E content and was found to be 5-6-fold decreased in microsomal fractions from vitamin E-deficient rats. Supplementation of microsomal fractions from vitamin E-deficient rats with exogenous vitamin E partially restores the induction period observed in that from control rats. The decrease in chemiluminescence intensity and the increase in the induction period both correlate linearly with the amount of vitamin E added. However, the efficiency of exogenous vitamin E is about 50-fold lower than that exerted by the naturally occurring vitamin E in microsomal membranes. These observations are discussed in terms of the process of re-incorporation of vitamin E into membranes, the experimental model for lipid peroxidation selected, and the method to evaluate lipid peroxidation, namely low-level chemiluminescence.