Hyperglycemia can cause membrane lipid peroxidation and osmotic fragility in human red blood cells

The present study has examined the effect of elevated glucose levels on membrane lipid peroxidation and osmotic fragility in human red blood cells (RBC). Defibrinated whole blood or RBC were incubated with varying concentrations of glucose at 37 degrees C for 24 h. RBC incubated with elevated levels of glucose showed a significantly increased membrane lipid peroxidation when compared with control RBC. A significant positive correlation was observed between the extent of glucose-induced membrane lipid peroxidation and the osmotic fragility of treated RBC. Glucose-induced membrane lipid peroxidation and osmotic fragility were blocked when RBC were pretreated with fluoride, an inhibitor of glucose metabolism; with vitamin E, an antioxidant; with para-chloromercurobenzoate and metyrapone, inhibitors of the cytochrome P-450 system; or with dimethylfurane, diphenylamine, and thiourea, scavengers of oxygen radicals. RBC treated with elevated glucose concentrations also showed an increase in NADPH levels. Exogenous addition of NADPH to normal RBC lysate induced membrane lipid peroxidation similar to that observed in the glucose-treated RBC. These data suggest that elevated glucose levels can cause the peroxidation of membrane lipids in human RBC.     

Amelioration of glucose induced hemolysis of human erythrocytes by vitamin E

Cells under aerobic condition are always threatened with the insult of reactive oxygen species, which are efficiently taken care of by the highly powerful antioxidant systems of the cell. The erythrocytes (RBCs) are constantly exposed to oxygen and oxidative stress but their metabolic activity is capable of reversing the injury under normal conditions. In vitro hemolysis of RBCs induced by 5, 10 and 20 mM glucose was used as a model to study the free radical induced damage of biological membranes in hyperglycemic conditions and the protection rendered by vitamin E on the same. RBCs are susceptible to oxidative damage, peroxidation of the membrane lipids, release of hemoglobin (hemolysis) and alteration in activity of antioxidant enzymes catalase and superoxide dismutase. The glucose induced oxidative stress and the protective effect of vitamin E on cellular membrane of human RBCs manifested as inhibition of membrane peroxidation and protein oxidation and restoration of activities of superoxide dismutase and catalase, was investigated.Thiobarbituric acid reactive substances are generated from decomposition of lipid peroxides and their determination gives a reliable estimate of the amount of lipid peroxides present in the membrane. Vitamin E at 18 μg/ml (normal serum level) strongly enhanced the RBC resistance to oxidative lysis leading to only 50–55% hemolysis in 24 h, whereas RBCs treated with 10 and 20 mM glucose without vitamin E leads to 70–80% hemolysis in 24 h. Levels of enzymic antioxidants catalase, superoxide dismutase and nonenzymic antioxidants glutathione showed restoration to normal levels in presence of vitamin E. The study shows that vitamin E can protect the erythrocyte membrane exposed to hyperglycemic conditions and so a superior antioxidant status of a diabetic patient may be helpful in retarding the progressive tissue damage seen in chronic diabetic patients.     

Breath alkanes as a marker of oxidative stress in different clinical conditions

We assessed oxidative stress in three different clinical conditions: smoking, human immunodeficiency virus (HIV) infection, and inflammatory bowel disease, using breath alkane output and other lipid peroxidation parameters such as plasma lipid peroxides (LPO) and malondialdehyde (MDA). Antioxidant micronutrients such as selenium, vitamin E, C, beta-carotene and carotenoids were also measured. Lipid peroxidation was significantly higher and antioxidant vitamins significantly lower in smokers compared to nonsmokers. Beta-carotene or vitamin E supplementation significantly reduced lipid peroxidation in that population. However, vitamin C supplementation had no effect. In HIV-infected subjects, lipid peroxidation parameters were also elevated and antioxidant vitamins reduced compared to seronegative controls. Vitamin E and C supplementation resulted in a significant decrease in lipid peroxidation with a trend toward a reduction in viral load. In patients with inflammatory bowel disease, breath alkane output was also significantly elevated when compared to healthy controls. A trial with vitamin E and C is underway. In conclusion, breath alkane output, plasma LPO and MDA are elevated in certain clinical conditions such as smoking, HIV infection, and inflammatory bowel disease. This is associated with lower levels of antioxidant micronutrients. Supplementation with antioxidant vitamins significantly reduced these lipid peroxidation parameters. The results suggest that these measures are good markers for lipid peroxidation.     

Antioxidant effect of dipyridamole (DIP) and its derivative RA 25 upon lipid peroxidation and hemolysis in red blood cells

The antioxidant effects of dipyridamol (DIP), a coronary vasodilator, and its derivative RA-25 were compared in intact red blood cells (RBC) and in isolated ghost membranes. Both compounds are quite effective antioxidants in cumene hydroperoxide-induced lipid peroxidation of RBC, showing a much smaller effect for hydrogen peroxide oxidation. The antioxidant effect of DIP was considerably higher than that of RA25. For isolated ghost membranes, the apparent IC50 (the drug concentration that produces 50% inhibition of lipid peroxidation) in cumene hydroperoxide-induced peroxidation was 25 microM, while the maximum protective effect of RA-25 was around 30% in the drug concentration range of 50-100 microM. The drugs can protect the oxidative hemolysis induced by cumene hydroperoxide with a lower effect when the hemolysis is induced by H2O2. The significant antioxidant effect against damages induced by cumene hydroperoxide suggests that DIP, due to its lipophilic character, can interact with RBC membranes, and the protective effect is associated with the binding of the drug to the membrane. On the other hand, RA-25 is more hydrophilic than DIP, binds to the membrane to a smaller extent, and, for this reason, has a lower antioxidant effect.     

Changes observed in antioxidant system in the blood of postmenopausal women with breast cancer.

From experimental studies and epidemiological data, it can be inferred that lipid peroxidation is increased in cancer patients. Cases of post-menopausal, untreated women with benign and malignant breast tumours, were compared with their age matched controls in their serum lipid peroxides, antioxidant vitamins (E and C), serum selenium and serum ceruloplasmin. Erythrocyte and its membrane lipid peroxidation and antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase and glutathione-S-transferase) levels were also analyzed. Significant increase in circulating lipid peroxides, ceruloplasmin and significant decrease in antioxidant vitamins and selenium were observed in breast cancer women. The erythrocyte and its membrane lipid peroxidation was increased significantly and severe impairment of antioxidant potential was observed in breast cancer women.     

Mechanisms of Hemolysis Induced by Copper

An excess of copper is the cause of hemolysis in a number of clinical conditions. Incubation of human erythrocyte (RBC) suspensions with copper (II) causes the formation of methemoglobin, lipid peroxidation and hemolysis.

A new variant of the thiobarbituric acid (TBA) method, which minimizes the formation of interfering chromophores, was used to detect lipid peroxidation. Lipid peroxidation precedes hemolysis and the antioxidant vitamins C and E, which inhibit lipid peroxidation, also inhibit hemolysis. Consequently lipid peroxidation appears to be the cause of RBC destruction. Lipid peroxidation arises mostly from the oxidation of oxyhemoglobin by copper as it is inhibited in RBCs with carbon monoxyhemoglobin or methemoglobin. A direct interaction of copper with the red cell membrane seems to play only a minor role. Copper effects depend on the presence of free SH groups. Lipid peroxidation is probably initiated by activated forms of oxygen as it is increased by an inhibitor of catalase and reduced by hydroxyl radical scavengers. With higher copper concentrations hemolysis is greater: its mechanism appears different as lipid peroxidation is smaller but hemoglobin alterations, namely precipitation, are more pronounced.     

Mechanisms of Hemolysis Induced by Copper

An excess of copper is the cause of hemolysis in a number of clinical conditions. Incubation of human erythrocyte (RBC) suspensions with copper (II) causes the formation of methemoglobin, lipid peroxidation and hemolysis.

A new variant of the thiobarbituric acid (TBA) method, which minimizes the formation of interfering chromophores, was used to detect lipid peroxidation. Lipid peroxidation precedes hemolysis and the antioxidant vitamins C and E, which inhibit lipid peroxidation, also inhibit hemolysis. Consequently lipid peroxidation appears to be the cause of RBC destruction. Lipid peroxidation arises mostly from the oxidation of oxyhemoglobin by copper as it is inhibited in RBCs with carbon monoxyhemoglobin or methemoglobin. A direct interaction of copper with the red cell membrane seems to play only a minor role. Copper effects depend on the presence of free SH groups. Lipid peroxidation is probably initiated by activated forms of oxygen as it is increased by an inhibitor of catalase and reduced by hydroxyl radical scavengers. With higher copper concentrations hemolysis is greater: its mechanism appears different as lipid peroxidation is smaller but hemoglobin alterations, namely precipitation, are more pronounced.     

Effect of <Emphasis Type="Italic">Gymnema montanum</Emphasis> leaves on red blood cell resistance to oxidative stress in experimental diabetes

The aim of the present study was to evaluate the protective effect of Gymnema montanum on red blood cell (RBC) membrane in diabetic rats during lipid peroxidation. Ethanol extract of G. montanum leaves (GLEt) was administered orally to alloxan-induced diabetic rats for 3 weeks, and the effects on blood glucose, insulin, lipid peroxidation markers, thiobarbituric acid reactive substances, hydroperoxides in plasma and antioxidant enzymes including superoxide dismutase, catalase and glutathione peroxidase activities in erythrocytes were studied. Administration of GLEt to diabetic animals at doses of 50, 100, and 200 mg/kg body weight lowered elevated blood glucose levels by 24, 35, and 66%, respectively, relative to untreated diabetic rats. In comparison, treatment with the known antidiabetic drug, glibenclamide (600 μg/kg body weight) decreased blood glucose concentrations by 51%. Plasma insulin concentrations were increased in the diabetic rat by 73% with GLEt (200 mg/kg body weight) and 45% with glibenclamide (600 μg/kg body weight). Although a significant decrease in the lipid peroxidation markers was observed in plasma on treatment with GLEt and glibenclamide, the RBC antioxidant levels were increased significantly in diabetic rats. Furthermore, erythrocytes from the GLEt-treated animals were found to be more resistant to H₂O₂-induced peroxidation than that of untreated diabetic animals. The chemical characterization of the polyphenolics of the extract showed the presence of gallic acid (5.29% w/w), resveratrol (2.2% w/w), and quercetin (16.6% w/w). The results of this study suggest that G. montanum may be useful for the control, management, and prevention of oxidative stress associated with diabetes.     

Effect of elevated glucose concentrations on cellular lipid peroxidation and growth of cultured human kidney proximal tubule cells

This study has examined whether elevated glucose can induce lipid peroxidation and contribute to the inhibition of cell growth in human kidney proximal tubule(HPT) cells. HPT cells were cultured in media containing glucose concentrations of 8 mM (control), 25 mM, and 50 mM. Lipid peroxidation was assessed by the thiobarbituric acid reactivity and cell growth was assessed by ³H-thymidine uptake. Results show decreased (59%, p < 0.01) growth of HPT cells cultured in 50 mM glucose. Cells cultured in 50 mM mannitol did not show any growth inhibition, suggesting that the decreased cell growth associated with glucose is not due to osmolarity changes. There was an increase (108%, p < 0.02) in lipid peroxidation in cells cultured with high levels of glucose (50 mM) compared with controls and cells cultured with 50 mM mannitol. To examine if membrane lipid peroxidation or malondialdehyde (MDA, an end product of lipid peroxidation) has any role in the inhibition of cell growth, we examined the effect of tertiary butylhydroperoxide (TBH, known to cause lipid peroxidation and generate MDA) on the growth of HPT cells. TBH decreased cell growth (49, 17 and 3% of controls at 0.1, 0.25, and 0.5 [mole TBH/ml medium). Similarly, a marked reduction in the growth was observed with exogenous MDA (72, 69 and 34% of controls at 0.1, 0.25, and 0.5 mole MDA/ml medium). This suggests that elevated glucose can induce membrane lipid peroxidation and accumulation of MDA, which in turn can inhibit cellular growth and contribute to the altered structure and function of HPT cells in diabetes.     

Effect of alpha-tocopherol on peroxidative membrane damage caused by doxorubicin: an in vitro study in human erythrocytes

Level of lipid peroxidation in doxorubicin treated human erythrocytes was studied and compared with that of cells pretreated with alpha-tocopherol. Erythrocytes treated with alpha-tocopherol had reduced level of lipid peroxidation with concomitantly lowered membrane damage. The membrane damage was monitored by the levels of conjugated diene absorption, lipid hydroperoxides and lipid peroxides. alpha-tocopherol was not effective in inhibiting the conjugated diene formation, but the lipid hydroperoxides and the lipid peroxide levels were significantly decreased. Methemoglobin level was found to be increased in alpha-tocopherol pretreated cells, which protects the membrane from damage. Erythrocyte membrane lipids were found to be decreased during doxorubicin treatment and alpha-tocopherol significantly reduced the membrane lipid breakdown. Level of reduced glutathione was maintained in alpha-tocopherol pretreated cells. These results are discussed with reference to the antioxidant property of alpha-tocopherol.     

Mitochondria induce oxidative stress, generation of reactive oxygen species and redox state unbalance of the eye lens leading to human cataract formation: disruption of redox lens organization by phospholipid hydroperoxides as a common basis for cataract disease

The aging eye appears to be at considerable risk from oxidative stress. Lipid peroxidation (LPO) is one of the mechanisms of cataractogenesis, initiated by enhanced promotion of oxygen free radicals in the eye fluids and tissues and impaired enzymatic and non-enzymatic antioxidant defenses of the crystalline lens. The present study proposes that mitochondria are one of the major sources of reactive oxygen species (ROS) in mammalian and human lens epithelial cells and that therapies that protect mitochondria in lens epithelial cells from damage and reduce damaging ROS generation may potentially ameliorate the effects of free radical-induced oxidation that occur in aging ocular tissues and in human cataract diseases. It has been found that rather than complete removal of oxidants by the high levels of protective enzyme activities such as superoxide dismutase (SOD), catalase, lipid peroxidases in transparent lenses, the lens conversely, possess a balance between peroxidants and antioxidants in a way that normal lens tends to generate oxidants diffusing from lenticular tissues, shifting the redox status of the lens to become more oxidizing during both morphogenesis and aging. Release of the oxidants (O(2)(-)·, H(2)O(2) , OH·, and lipid hydroperoxides) by the intact lenses in the absence of respiratory inhibitors indicates that these metabolites are normal physiological products inversely related to the lens life-span potential (maturity of cataract) generated through the metal-ion catalyzed redox-coupled pro-oxidant activation of the lens reductants (ascorbic acid, glutathione). The membrane-bound phospholipid (PL) hydroperoxides escape detoxification by the lens enzymatic reduction. The lens cells containing these species would be vulnerable to peroxidative attack which trigger the PL hydroperoxide-dependent chain propagation of LPO and other damages in membrane (lipid and protein alterations). The increased concentrations of primary LPO products (diene conjugates, lipid hydroperoxides) and end fluorescent LPO products were detected in the lipid moiety of the aqueous humor samples obtained from patients with cataract as compared to normal donors. Since LPO is clinically important in many of the pathological effects and aging, new therapeutic modalities, such as patented N-acetylcarnosine prodrug lubricant eye drops, should treat the incessant infliction of damage to the lens cells and biomolecules by reactive lipid peroxides and oxygen species and "refashion" the affected lens membranes in the lack of important metabolic detoxification of PL peroxides. Combined in ophthalmic formulations with N-acetylcarnosine, mitochondria-targeted antioxidants are promising to become investigated as a potential tool for treating a number of ROS-related ocular diseases, including human cataracts.     

Protective effect of vitamin E on ischaemia-reperfusion injury in ovarian grafts

Ovarian cortical tissue cryopreservation with subsequent autografting is a potential strategy for the preservation of fertility in patients undergoing systemic chemotherapy and pelvic radiotherapy. Non-vascular implants are first subjected to a period of ischaemia before revascularization and are, therefore, vulnerable to ischaemia-reperfusion injury from reactive oxygen species. Ischaemia-reperfusion injury was investigated during the first week after surgery in murine ovarian grafts and human ovarian xenografts in mice with severe combined immune deficiency (SCID) by measuring total lipid peroxides and malondialdehyde concentrations with a colorometric assay. The effects of administering an antioxidant, vitamin E, on these concentrations were also tested. Products of lipid peroxidation were higher in non-supplemented murine autografts compared with control ovaries (P < 0.05), and were significantly reduced on day 3 by vitamin E administration (P < 0.05). Similarly, in human xenografts, there was a significant reduction in lipid peroxidation with vitamin E administration. These results correspond to a significantly greater total follicle survival in the murine grafts of the supplemented group (45 versus 72%; P < 0.05). They suggest that antioxidant treatment improves the survival of follicles in ovarian grafts by reducing ischaemia-reperfusion injury.     

Lipoic acid decreases lipid peroxidation and protein glycosylation and increases (Na(+) + K(+))- and Ca(++)-ATPase activities in high glucose-treated human erythrocytes

Lipoic acid supplementation has been found to be beneficial in preventing neurovascular abnormalities in diabetic neuropathy. Insufficient (Na(+) + K(+))-ATPase activity has been suggested as a contributing factor in the development of diabetic neuropathy. This study was undertaken to test the hypothesis that lipoic acid reduces lipid peroxidation and glycosylation and can increase the (Na(+) + K(+))- and Ca(++)-ATPase activities in high glucose-exposed red blood cells (RBC). Washed normal human RBC were treated with normal (6 mM) and high glucose concentrations (45 mM) with 0-0.2 mM lipoic acid (mixture of S and R sterioisomers) in a shaking water bath at 37 degrees C for 24 h. There was a significant stimulation of glucose consumption by RBC in the presence of lipoic acid both in normal and high glucose-treated RBC. Lipoic acid significantly lowered the level of glycated hemoglobin (GHb) and lipid peroxidation in RBC exposed to high glucose concentrations. High glucose treatment significantly lowered the activities of (Na(+) + K(+))- and Ca(++)-ATPases of RBC membranes. Lipoic acid addition significantly blocked the reduction in activities of (Na(+) + K(+))- and Ca(++)-ATPases in high glucose- treated RBC. There were no differences in lipid peroxidation, GHb and (Na(+) + K(+))- and Ca(++)-ATPase activity levels in normal glucose-treated RBC with and without lipoic acid. Thus, lipoic acid can lower lipid peroxidation and protein glycosylation, and increase (Na(+) + K(+))- and Ca(++)-ATPase activities in high-glucose exposed RBC, which provides a potential mechanism by which lipoic acid may delay or inhibit the development of neuropathy in diabetes.     

Exposure to the fern Onychium contiguum causes increase in lipid peroxidation and alters antioxidant status in urinary bladder

The status of lipid peroxidation, glutathione, glutathione peroxidase, glutathione reductase, glutathione-S-transferase, superoxide dismutase, catalase, ascorbic acid, and alpha-tocopherol was studied in the urinary bladder of guinea pigs exposed to the carcinogenic fern Onychium contiguum. There was significant increase in the preformed lipid peroxides in the urinary bladders from fern exposed animals. The amount of lipid peroxides produced on incubation of urinary bladder homogenates with or without catalyst was significantly higher in the fern exposed animals. The concentrations of glutathione and alpha-tocopherol and the activities of glutathione reductase and catalase were elevated in the urinary bladders of the animals exposed to the fern. No effect was observed on the concentration of ascorbic acid and the activities of glutathione peroxidase, glutathione-S-transferase, and superoxide dismutase. It is summarized that the fern toxins increased oxidative stress in the urinary bladder and antioxidant status was altered. However, the altered antioxidant status did not provide protection from the toxin induced injury. Histopathology of the urinary bladder in the fern exposed animals revealed oedema, haemorrhages, and congestion. This is the first study to show increase in lipid peroxidation along with altered antioxidant status in the urinary bladder of fern exposed animals.     

Flow cytometric analysis of the oxidative status of normal and thalassemic red blood cells.

BACKGROUND: The oxidative status of cells has been shown to modulate various cell functions and be involved in physiological and pathological conditions, including hereditary chronic anemias, such as thalassemia. It is maintained by the balance between oxidants, such as reactive oxygen species (ROS), and antioxidants, such as reduced glutathione (GSH). METHODS: We studied peripheral RBC derived from normal and thalassemic donors. Flow cytometric methods were used to measure (1) generation of ROS; (2) the content of reduced GSH; and (3) peroxidation of membrane lipids as an indication of membrane damage. RESULTS: ROS and lipid peroxidation were found to be higher, and GSH lower, in thalassemic RBC compared with normal RBC, both at baseline as well as following oxidative stress, such as exposure to hydrogen peroxide. To simulate a state of iron overload, normal RBC were exposed to extracellular ferric ammonium citrate or hemin, or their Hb was denatured by phenylhydrazine. All these treatments increased ROS and lipid peroxidation and decreased GSH. These effects were reversed by N-acetyl cysteine, a known ROS scavenger. CONCLUSIONS: Flow cytometry can be useful for measuring oxidative stress and its effects on RBC in various diseases and for studying various chemical agents as antioxidants.     

Plasma thiobarbituric acid reactivity: reaction conditions and the role of iron, antioxidants and lipid peroxy radicals on the quantitation of plasma lipid peroxides

The effects of Fe3+, lipid peroxy radicals and the antioxidant butylated hydroxytoluene on the 2-thiobarbituric (TBA) acid quantitation of plasma lipid peroxides were investigated. Whole plasma and plasma fractions prepared by trichloroacetic acid (TCA) protein precipitation and lipid extraction, demonstrated markedly differing TBA reactivities in the presence or absence of added Fe3+. Examination of the spectral profiles of the TBA reacted whole plasma and TCA precipitated fractions demonstrated the presence of interfering compounds which gave rise to an artifactual increase in lipid peroxide concentrations. In contrast the TBA reacted lipid extracts had low levels of interfering compounds that could be removed by our previously described high pressure liquid chromatographic method (Wade, Jackson and van Rij (1985) Biochem. Med. 33, 291-296). Further characterization of the TBA reactivity of the lipid extract showed that Fe3+ at an optimal concentration of 0.5 mM was necessary for the quantitative decomposition of the lipid peroxides to the TBA reactive product malondialdehyde (MDA). However the presence of Fe3+ resulted in further peroxidation of any unsaturated lipids present. Butylated hydroxytoluene (BHT) at an optimal concentration of 1.4 mM inhibited Fe3+ stimulated peroxidation without affecting the formation of the MDA-TBA chromogen. Using a standardized TBA test with plasma lipid extracts and the addition of optimal concentrations of Fe3+ and BHT, we have determined the mean concentration of lipid peroxides in 30 healthy human subjects to be 102.7 +/- 20.0 ngm/ml.     

Evidence for a relationship between protein glycation and red blood cell membrane fluidity

This study examines the relationship between protein glycation and membrane fluidity in RBC membranes. Incubation of RBC membranes of healthy subjects with 25mM glucose or galactose at 37 degrees C induced a 38% (p less than 0.02) increase in protein glycation (using furosine determination by HPLC) and higher fluidity (p less than 0.05) in DPH polarization ratio). However, incubation of RBC membranes from diabetic subjects under the same conditions did not modify either membrane fluidity or protein glycation; protein glycation was above normal before incubation because of the high diabetic plasma glucose. There was no difference in the membrane fluidities of 21 healthy subjects and 32 diabetic subjects, despite a significantly elevated protein glycation in diabetics. Furthermore, there was no change with respect to age in either population. We conclude that other in vivo factors, such as membrane lipid changes (increase in CL/PL ratio) or formation of advanced Maillard products and peroxidation in the diabetic subjects, could be responsible for the difference between these in vitro results and the in vivo situation.     

Effects of new antioxidant compounds PNU-104067F and PNU-74389G on antioxidant defense in normal and diabetic rats

Diabetes mellitus and its complications are associated with elevated oxidative stress, leading to much interest in antioxidant compounds as possible therapeutic agents. Two new classes of antioxidant compounds, the pyrrolopyrimidines and the 21-aminosteroids, are known to inhibit lipid peroxidation and other biomolecular oxidation. We hypothesized that in the presence of excess oxidants or the impaired antioxidant defense seen in diabetes mellitus, administration of antioxidants such as these may reverse the effects of diabetes on antioxidant parameters. This study measured the effects of subchronic (14 day) treatment with a pyrrolopyrimidine (PNU-104067F) or a 21-aminosteroid (PNU-74389G) in normal and diabetic Sprague-Dawley rats. Activity levels of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase, concentrations of oxidized and reduced glutathione, and lipid peroxidation were used as measures of antioxidant defense in liver, kidney, heart, and brain tissue. In normal rats, the only effect was a 43% increase in cardiac lipid peroxidation after treatment with PNU-104067F. In diabetic rats, the only reversals of the effects of diabetes were a 30% decrease in hepatic glutathione peroxidase activity after PNU-74389G treatment and a 33% increase in cardiac glutathione disulfide concentration after PNU-104067F treatment. In contrast to these effects, increased cardiac glutathione peroxidase and catalase activities, increased brain glutathione peroxidase activity, increased hepatic lipid peroxidation, decreased hepatic glutathione content, and decreased hepatic catalase activity were seen in diabetic rats, reflecting an exacerbation of the effects of diabetes.     

Functional significance of the pentose phosphate pathway and glutathione reductase in the antioxidant defenses of human sperm

Glutathione peroxidase is one of the principal antioxidant defense enzymes in human spermatozoa, but it requires oxidized glutathione to be reduced by glutathione reductase using NADPH generated in the pentose phosphate pathway. We investigated whether flux through the pentose phosphate pathway would increase in response to oxidative stress and whether glutathione reductase was required to protect sperm from oxidative damage. Isotopic measurements of the pentose phosphate pathway and glycolytic flux, thiobarbituric acid assay of malondialdehyde for lipid peroxidation, and computer-assisted sperm analysis for sperm motility were assessed in a group of normal, healthy semen donors. Applying moderate oxidative stress to human spermatozoa by adding cumene hydroperoxide, H(2)O(2), or xanthine plus xanthine oxidase or by promoting lipid peroxidation with ascorbate increased flux through the pentose phosphate pathway without changing the glycolytic rate. However, adding higher concentrations of oxidants inhibited both the pentose phosphate pathway and glycolytic flux. At concentrations of 50 microg/ml or greater, the glutathione reductase-inhibitor 1,3-bis-(2-chloroethyl) 1-nitrosourea decreased flux through the pentose phosphate pathway and blocked the response to cumene hydroperoxide. It also increased lipid peroxidation and impaired the survival of motility in sperm incubated under 95% O(2). These data show that the pentose phosphate pathway in human spermatozoa can respond dynamically to oxidative stress and that inhibiting glutathione reductase impairs the ability of sperm to resist lipid peroxidation. We conclude that the glutathione peroxidase-glutathione reductase-pentose phosphate pathway system is functional and provides an effective antioxidant defense in normal human spermatozoa.     

Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide. The relative roles of haem- and glutathione-dependent decomposition of t-butyl hydroperoxide and membrane lipid hydroperoxides in lipid peroxidation and haemolysis

Red cells exposed to t-butyl hydroperoxide undergo lipid peroxidation, haemoglobin degradation and hexose monophosphate-shunt stimulation. By using the lipid-soluble antioxidant 2,6-di-t-butyl-p-cresol, the relative contributions of t-butyl hydroperoxide and membrane lipid hydroperoxides to oxidative haemoglobin changes and hexose monophosphate-shunt stimulation were determined. About 90% of the haemoglobin changes and all of the hexose monophosphate-shunt stimulation were caused by t-butyl hydroperoxide. The remainder of the haemoglobin changes appeared to be due to reactions between haemoglobin and lipid hydroperoxides generated during membrane peroxidation. After exposure of red cells to t-butyl hydroperoxide, no lipid hydroperoxides were detected iodimetrically, whether or not glucose was present in the incubation. Concentrations of 2,6-di-t-butyl-p-cresol, which almost totally suppressed lipid peroxidation, significantly inhibited haemoglobin binding to the membrane but had no significant effect on hexose monophosphate shunt stimulation, suggesting that lipid hydroperoxides had been decomposed by a reaction with haem or haem-protein and not enzymically via glutathione peroxidase. The mechanisms of lipid peroxidation and haemoglobin oxidation and the protective role of glucose were also investigated. In time-course studies of red cells containing oxyhaemoglobin, methaemoglobin or carbonmono-oxyhaemoglobin incubated without glucose and exposed to t-butyl hydroperoxide, haemoglobin oxidation paralleled both lipid peroxidation and t-butyl hydroperoxide consumption. Lipid peroxidation ceased when all t-butyl hydroperoxide was consumed, indicating that it was not autocatalytic and was driven by initiation events followed by rapid propagation and termination of chain reactions and rapid non-enzymic decomposition of lipid hydroperoxides. Carbonmono-oxyhaemoglobin and oxyhaemoglobin were good promoters of peroxidation, whereas methaemoglobin relatively spared the membrane from peroxidation. The protective influence of glucose metabolism on the time course of t-butyl hydroperoxide-induced changes was greatest in carbonmono-oxyhaemoglobin-containing red cells followed in order by oxyhaemoglobin- and methaemoglobin-containing red cells. This is the reverse order of the reactivity of the hydroperoxide with haemoglobin, which is greatest with methaemoglobin. In studies exposing red cells to a wide range of t-butyl hydroperoxide concentrations, haemoglobin oxidation and lipid peroxidation did not occur until the cellular glutathione had been oxidized. The amount of lipid peroxidation per increment in added t-butyl hydroperoxide was greatest in red cells containing carbonmono-oxyhaemoglobin, followed in order by oxyhaemoglobin and methaemoglobin. Red cells containing oxyhaemoglobin and carbonmono-oxyhaemoglobin and exposed to increasing concentrations of t-butyl hydroperoxide became increasingly resistant to lipid peroxidation as methaemoglobin accumulated, supporting a relatively protective role for methaemoglobin. In the presence of glucose, higher levels of t-butyl hydroperoxide were required to induce lipid peroxidation and haemoglobin oxidation compared with incubations without glucose. Carbonmono-oxyhaemoglobin-containing red cells exposed to the highest levels of t-butyl hydroperoxide underwent haemolysis after a critical level of lipid peroxidation was reached. Inhibition of lipid peroxidation by 2,6-di-t-butyl-p-cresol below this critical level prevented haemolysis. Oxidative membrane damage appeared to be a more important determinant of haemolysis in vitro than haemoglobin degradation. The effects of various antioxidants and free-radical scavengers on lipid peroxidation in red cells or in ghosts plus methaemoglobin exposed to t-butyl hydroperoxide suggested that red-cell haemoglobin decomposed the hydroperoxide by a homolytic scission mechanism to t-butoxyl radicals.