Sushil K. Jain

Oxidative stress and apoptosis.

Apoptosis or programmed cell death, is essential for the normal functioning and survival of most multi-cellular organisms. The morphological and biochemical characteristics of apoptosis, however, are highly conserved during the evolution. It is currently believed that apoptosis can be divided into at least three functionally distinct phases, i.e. induction, effector and execution phase. Recent studies have demonstrated that reactive oxygen species (ROS) and the resulting oxidative stress play a pivotal role in apoptosis. Antioxidants and thiol reductants, such as N-acetylcysteine, and overexpression of manganese superoxide (MnSOD) can block or delay apoptosis. Bcl-2, an endogenously produced protein, has been shown to prevent cells from dying of apoptosis apparently by an antioxidative mechanism. Taken together ROS, and the resulting cellular redox change, can be part of signal transduction pathway during apoptosis. It is now established that mitochondria play a prominent role in apoptosis. During mitochondrial dysfunction, several essential players of apoptosis, including pro-caspases, cytochrome C, apoptosis-inducing factor (AIF), and apoptotic protease-activating factor-1 (APAF-1) are released into the cytosol. The multimeric complex formation of cytochrome C, APAF-1 and caspase 9 activates downstream caspases leading to apoptotic cell death. All the three functional phases of apoptosis are under the influence of regulatory controls. Thus, increasing evidences provide support that oxidative stress and apoptosis are closely linked physiological phenomena and are implicated in pathophysiology of some of the chronic diseases including AIDS, autoimmunity, cancer, diabetes mellitus, Alzheimers and Parkinsons and ischemia of heart and brain.

Erythrocyte Membrane Lipid Peroxidation and Glycosylated Hemoglobin in Diabetes

Erythrocytes of diabetic patients have abnormal membrane properties. We examined in vivo membrane lipid peroxidation in erythrocytes of diabetic subjects and its possible relationship with hyperglycemia. Lipid peroxidation was assessed in fresh, untreated erythrocytes by quantitating thiobarbituric acid reactivity and an adduct of phospholipids and malonyldialdehyde (MDA), an end product of lipid peroxidation, with thin-layer chromatography of lipid extract of diabetic erythrocytes. There was a significantly increased membrane lipid peroxidation in diabetic erythrocytes compared with nondiabetic erythrocytes. The degree of membrane lipid peroxidative damage in erythrocytes was significantly correlated with the level of glycosylated hemoglobin, an index of mean glucose level for the preceding 3–4mo. This suggests that peroxidation of membrane lipids and accumulation of MDA occurs in erythrocytes of diabetic patients.

Evidence for membrane lipid peroxidation during the in vivo aging of human erythrocytes

This study has examined the occurrence of lipid peroxidation in in vivo aged human erythrocyte membranes. Erythrocytes of various ages were separated on discontinuous stractan density gradients. Three erythrocyte fractions were analyzed: (I) Light--erythrocytes staying between stractan densities 1.053 and 1.043 g/ml, (II) predensest--erythrocytes staying between stractan densities 1.081 and 1.111, and (III) densest--erythrocytes passing stractan density 1.111. Peroxidative lipid damage of erythrocytes was assessed by measuring lipid extract fluorescence, by lipid thin-layer chromatography for the presence of adduct of phosphatidylserine (PS), phosphatidylethanolamine (PE) and malondialdehyde, and by thiobarbituric acid-reactivity. Fractions I, II and III contained, respectively, 0.2 +/- 0.1 (S.E.), 1.1 +/- 0.1 and 1.5 +/- 0.1 of phospholipid-malondialdehyde adduct (percent of total phospholipids), and relative lipid fluorescence 22.5 +/- 0.8, 29.3 +/- 0.5, and 33.4 +/- 0.8 per ml packed cells, respectively. Thiobarbituric acid-reactivity of erythrocytes in various fractions was similar. Untreated densest erythrocytes contained significantly reduced PS (12.9 +/- 0.5%), in contrast to light erythrocytes (16.1 +/- 0.1%) and increased PC (31.2 +/- 0.3 versus 27.8 +/- 0.8% of the total phospholipid). This study provides evidence for significant lipid peroxidative damage in the erythrocyte membrane during aging in vivo.

The Effect of Oxygen Radicals Metabolites and Vitamin E on Glycosylation of Proteins

Glycosylation (glycation) of proteins is a major complication of hyperglycemia in diabetes. This study has examined the effect of hydrogen peroxide (H2O2) and tertiary-butylhydroperoxide (TBH) and vitamin E (E) on glycation of hemoglobin (GHb). The RBC (15%) in phosphate-buffered saline were treated with 5-50 mM glucose (G) with and without H2O2 or TBH at 37 degrees C for 1-3 d. Glycation of hemoglobin was assessed by GHb formation using Glyc-affinity columns. There was an increase in the GHb formation with increasing G concentrations. GHb formation increased significantly in the presence of H2O2 at all G concentrations. The increase in GHb was blocked when RBC were pretreated with E. E also inhibited formation of malondialdehyde (MDA), an end product of lipid peroxidation, as assessed by the thiobarbituric acid reactivity. Similar increase in the GHb formation was observed when TBH was used instead of H2O2 to induce oxidant stress to the RBC. To examine any role of MDA per se in increased glycation, RBC were treated ex vivo with and without exogenous standard MDA. GHb formation was significantly higher with G-MDA in contrast to G alone. Thus, increased oxygen radicals activity can initiate per-oxidation of lipids and MDA accumulation, which in turn, can stimulate glycation of proteins in diabetes. E can block the glycation of proteins by inhibiting MDA formation.

Low Levels of Hydrogen Sulfide in the Blood of Diabetes Patients and Streptozotocin-Treated Rats Causes Vascular Inflammation?

Hydrogen sulfide (H(2)S) is emerging as a physiological neuromodulator as well as a smooth muscle relaxant. We submit the first evidence that blood H(2)S levels are significantly lower in fasting blood obtained from type 2 diabetes patients compared with age-matched healthy subjects, and in streptozotocin-treated diabetic rats compared with control Sprague-Dawley rats. We further observed that supplementation with H(2)S or an endogenous precursor of H(2)S (l-cysteine) in culture medium prevents IL-8 and MCP-1 secretion in high-glucose-treated human U937 monocytes. These first observations led to the hypothesis that lower blood H(2)S levels may contribute to the vascular inflammation seen in diabetes.

Curcumin Supplementation Lowers TNF-α, IL-6, IL-8, and MCP-1 Secretion in High Glucose-Treated Cultured Monocytes and Blood Levels of TNF-α, IL-6, MCP-1, Glucose, and Glycosylated Hemoglobin in Diabetic Rats

This study examined the hypothesis that curcumin supplementation decreases blood levels of IL-6, MCP-1, TNF-alpha, hyperglycemia, and oxidative stress by using a cell-culture model and a diabetic rat model. U937 monocytes were cultured with control (7 mM) and high glucose (35 mM) in the absence or presence of curcumin (0.01-1 microM) at 37 degrees C for 24 h. Diabetes was induced in Sprague-Dawley rats by injection of streptozotocin (STZ) (i.p., 65 mg/kg BW). Control buffer, olive oil, or curcumin (100 mg/kg BW) supplementation was administered by gavage daily for 7 weeks. Blood was collected by heart puncture with light anesthesia. Results show that the effect of high glucose on lipid peroxidation, IL-6, IL-8, MCP-1, and TNF-alpha secretion was inhibited by curcumin in cultured monocytes. In the rat model, diabetes caused a significant increase in blood levels of IL-6, MCP-1, TNF-alpha, glucose, HbA(1), and oxidative stress, which was significantly decreased in curcumin-supplemented rats. Thus, curcumin can decrease markers of vascular inflammation and oxidative stress levels in both a cell-culture model and in the blood of diabetic rats. This suggests that curcumin supplementation can reduce glycemia and the risk of vascular inflammation in diabetes.

Elevated Lipid Peroxidation Levels in Red Blood Cells of Streptozotocin-Treated Diabetic Rats

This study was performed to determine whether or not hyperglycemia in diabetes results in elevated levels of lipid peroxidation products in red blood cells (RBC). Diabetes was induced in rats by treatment with streptozotocin. The level of lipid peroxidation products was examined in fresh RBC by measuring their thiobarbituric acid (TBA) reactivity after 2 and 4 months of induction of diabetes. Hyperglycemia was assessed by measuring the level of glycosylated hemoglobin and blood glucose. Results show that lipid peroxidation levels were significantly higher (50% to 84%) in RBC of diabetic rats than in controls. The increase in the level of lipid peroxidation was blocked in diabetic rats in which hyperglycemia was controlled by insulin treatment. Among phospholipid classes, relative percentage of sphingomyelin (SM) was significantly reduced in RBC at both 2 and 4 months of diabetes; whereas phosphatidylethanolamine (PE) levels were higher in RBC at 4 months of diabetes only. The level of phosphatidylcholine (PC) did not differ significantly between RBC of control and diabetic rats. This study suggests a significantly altered lipid composition and an accumulation of lipid peroxidation products in RBC of streptozotocin-treated diabetic rats.

The effect of malonyldialdehyde, a product of lipid peroxidation, on the deformability, dehydration and 51Cr‐survival of erythrocytes

Summary. Erythrocyte membrane lipid peroxidation has been reported to occur in various haemolytic anaemias. In the present study, treatment of human erythrocytes with malonyldialdehyde (MDA). a product of fatty acid peroxidation, induced membrane rigidity, cellular dehydration and reduced whole cell deformability. These effects of MDA were blocked by histamine and fluorescamine, which can act as alternate substrates for MDA. Additionally, reduced deformability of MDA‐treated rabbit cells was associated with shortened 51Cr survival in vivo. These findings suggest a biochemical basis for decreased survival of erythrocytes undergoing peroxidative damage of the membrane.

Protein and lipid oxidation of banked human erythrocytes:: Role of glutathione

In banked human erythrocytes (RBCs), biochemical and functional changes are accompanied with vesiculation and reduced in vivo survival. We hypothesized that some of these changes might have resulted from oxidative modification of membrane lipids, proteins, or both as a result of atrophy of the antioxidant defense system(s). In banked RBCs, we observed a time-dependent increase in protein clustering, especially band 3; carbonyl modification of band 4.1; and malondialdehyde, a lipid peroxidation product. Examination of the antioxidative defense system showed a time-dependent decline in glutathione (GSH) concentration and glutathione-peroxidase (GSH-PX) activity, with a concomitant increase in extracellular GSH, cysteine, and homocysteine, and unchanged catalase activity. When subjected to acute oxidant stress by exposure to ferric/ascorbic acid or tert-butylhydroperoxide (tert-BHT), catalase activity showed a steeper decline compared with GSH-PX. The results demonstrate that GSH and GSH-PX appear to provide the primary antioxidant defense in stored RBCs, and their decline, concurrent with an increase in oxidative modifications of membrane lipids and proteins, may destabilize the membrane skeleton, thereby compromising RBC survival.

Effect of glycemic control, race (white versus black), and duration of diabetes on reduced glutathione content in erythrocytes of diabetic patients.

We designed this study to examine whether uncontrolled hyperglycemia, duration of diabetes, or race (black v white) have any effect on glutathione levels in erythrocytes of type I diabetic patients. Hyperglycemia was assessed by measuring the level of hemoglobin A1c (HbA1c). Results show that erythrocytes of diabetic patients have a significantly lower glutathione level compared with those of age-matched normal subjects (P < .004). We found a significant negative correlation (r = -.59, P < .001) between the degree of hyperglycemia and the level of reduced glutathione (GSH) in erythrocytes of diabetic patients. There was no significant relationship (r = -.29, P > .12) between the level of GSH in erythrocytes and the duration of diabetes. Erythrocytes of black diabetic patients had significantly lower levels of GSH (P < .05) than those of white diabetic patients. Using erythrocytes as a model, this study suggests that a lower level of GSH may have a role in the cellular damage and impaired insulin secretion in uncontrolled diabetic patients.