Decreased enzymic protection and increased sensitivity to oxidative damage in erythrocytes as a function of cell and donor aging.

Erythrocytes from young and old rats were separated into four age fractions by density-gradient centrifugation. The specific activities per cell were determined for glucose-6-phosphate dehydrogenase (EC 1.1.1.49), glutathione peroxidase (EC 1.11.1.9), glutathione reductase (EC 1.6.4.2) and catalase (EC 1.11.1.6). Decreased specific activities were observed with increasing cell age for all four enzymes in both young and old animals. In addition, significant differences in the activities of these enzymes were observed between cells of the same age fraction from young and old donors. Susceptibility of fractionated erythrocytes to oxidative attack in vitro generated by incubation with xanthine/xanthine oxidase increased with both cell and animal age. The amount of membrane-lipid peroxidation also increased with cell and animal aging, as measured by both thiobarbituric acid and fluorescent chromolipid assays. Increases of 2-3-fold in the contents of lipid peroxides were observed between the youngest and oldest age fractions of young rats. Lipid peroxide contents in young cells of old animals were equal to those in old erythrocytes from young rats and increased by 30% with cell aging in the old donors. These results suggest that the extent of enzymic protection against oxidative and peroxidative damage decreases with erythrocyte aging. More importantly, enzymic protection in cells of old rats is considerably decreased already in the early stages of their lifespan.     

Glutathione contents of tissues in the aging mouse.

1. Previous results from this laboratory demonstrated that the erythrocyte content of reduced glutathione (GSH) decreased as a function of both increasing cell age and mouse age [Abraham, Taylor & Lang (1978) Biochem. J. 174, 819-825]. In the present investigation glutathione concentrations were determined in other tissues of the C57BL/6J mouse of different ages (6--31 months) throughout the life-span. 2. At all ages the total glutathione and the GSH concentrations in liver were 3 times that in kidney and 10 times that in heart. In the old (31 months) mouse the GSH contents were lower by 30% in the liver, 34% in the kidney and 20% in the heart than in the mature (17--23 months) animals. 3. The oxidized glutathione (GSSG) concentrations of the tissues did not vary with age and constituted less than 3% of the total glutathione. 4. The decreased in GSH concentrations were not due to changes in organ weights, which were constant from 10 to 36 months of age. 5. These findings extend our previous results and indicate that a general characteristic of aging tissues may be a decrease in GSH concentrations. Further, this is consistent with our hypothesis that the reducing potential of tissues decreases in senescence.     

A metabolic signature predicts biological age in mice

Our understanding of the mechanisms by which aging is produced is still very limited. Here, we have determined the sera metabolite profile of 117 wild‐type mice of different genetic backgrounds ranging from 8 to 129 weeks of age. This has allowed us to define a robust metabolomic signature and a derived metabolomic score that reliably/accurately predicts the age of wild‐type mice. In the case of telomerase‐deficient mice, which have a shortened lifespan, their metabolomic score predicts older ages than expected. Conversely, in the case of mice that overexpress telomerase, their metabolic score corresponded to younger ages than expected. Importantly, telomerase reactivation late in life by using a TERT‐based gene therapy recently described by us significantly reverted the metabolic profile of old mice to that of younger mice, further confirming an anti‐aging role for telomerase. Thus, the metabolomic signature associated with natural mouse aging accurately predicts aging produced by telomere shortening, suggesting that natural mouse aging is in part produced by presence of short telomeres. These results indicate that the metabolomic signature is associated with the biological age rather than with the chronological age. This constitutes one of the first aging‐associated metabolomic signatures in a mammalian organism.     

Is red blood cell survival limited by the activity of a critical enzyme?

Erythrocyte glutathione reductase is responsible for generating reduced glutathione, which has been implicated in maintaining the integrity of the red blood cell.Erythrocytes from peripheral blood were separated into fractions of increasing age and the activity of glutathione reductase and aspartate amino transferase determined in each fraction.The age-related decline in activity of both enzymes was confirmed, but with detailed resolution of the cells by age a significant secondary rise in only glutathione reductase activity was found in very old cells. As red blood cells from the same cohort survive in the circulation for varying periods they must vary in some way from one another. It is postulated that glutathione reductase is a critical enzyme which limits erythrocyte survival and that the rate of decline in activity varies from cell to cell. A simple mathematical model based on this postulate accounted quantitatively for both the pattern of glutathione reductase activity and the erythrocyte survival curve. In addition, a simplified model of the passage of erythrocytes through the circulation was designed and run. The predicted erythrocyte survival curve and pattern of glutathione reductase activity were very similar to observed patterns. This model may be useful in other situations where a finite resource is degraded at different rates by random passages through different pathways.     

Effects of amyloid-β peptide Aβ25–35 on glycolytic and antioxidant enzymes in erythrocytes of different ages

Amyloid-β peptide Aβ_25–35 was shown to cause lysis of rat erythrocytes of different ages. The toxicity of Aβ_25–35 positively correlated with both the erythrocyte age and the peptide concentration. The activity of glycolytic, antioxidant, and Na⁺/K⁺-ATPase enzymes decreased with erythrocyte aging in vivo. In vitro Aβ_25–35 reduced the activity of hexokinase, phosphofructokinase, pyruvate kinase, glutathione peroxidase, and glutathione transferase and increased Na⁺/K⁺-ATPase activity in aged erythrocytes to a greater degree than in young cells.     

Effect of cell age on erythrocyte fatty acid composition in rats on different dietary regimes

1. Rat erythrocytes were fractionated into young, mature and old cell fractions by centrifugation. The fatty acid composition of each fraction was determined by gas-liquid chromatography, under four different dietary conditions: with adequate linoleic acid in the diet, with a diet deficient in linoleic acid, and with the deficient diet supplemented with corn oil for 3 and 12 days. 2. Significant differences were observed in the fatty acid composition of cells of different ages on all diets. The pattern of fatty acid distribution depended on the particular acid in question, on its concentration in the total erythrocyte sample and on the nature of the dietary fat. 3. When corn oil was fed to rats that had been fed previously on a deficient diet, the changes in fatty acid composition that occurred depended on the acid and on the cell age. For example, young cells were more active in incorporating palmitic acid and arachidonic acid but incorporated linoleic acid at a slightly lower rate than older cells. 4. These results are believed to indicate the presence in the erythrocyte of transacylases with different specificities, and to show that transacylase activity changes as the cells age.     

Naringin Mitigates Erythrocytes Aging Induced by Paclitaxel: An In Vitro Study

In this study, the protective role of naringin (NAR) against paclitaxel (PTX)‐induced erythrocytes aging has been investigated using human erythrocyte as an in vitro model. Erythrocytes were incubated with PTX in the presence and absence of NAR. Incubation of erythrocytes with PTX resulted in increased protein carbonyl content and malondialdehyde and hemolysis percentage compared with control. Furthermore, a significant increase in the ratios of glutathione peroxidase/glutathione reductase, superoxide dismutase/glutathione peroxidase, and superoxide dismutase/catalase in PTX‐treated cells was observed, compared with control cells. In contrast, reduced glutathione/oxidized glutathione ratio and glucose‐6‐phosphate dehydrogenase activity were decreased upon PTX treatment. The simultaneous incubation of erythrocytes with PTX and NAR restored these variables to values similar to those of control erythrocytes. These results suggest that NAR inhibited PTX‐induced aging by lessening the PTX‐induced oxidative stress.     

Effects of age and caloric restriction on glutathione redox state in mice

The main purpose of this study was to determine whether the aging process in the mouse is associated with a pro-oxidizing shift in the redox state of glutathione and whether restriction of caloric intake, which results in the extension of life span, retards such a shift. Amounts of reduced and oxidized forms of glutathione (GSH and GSSG, respectively) and protein-glutathione mixed disulfides (protein-SSG) were measured in homogenates and mitochondria of liver, kidney, heart, brain, eye, and testis of 4, 10, 22, and 26 month old ad libitum-fed (AL) mice and 22 month old mice fed a diet containing 40% fewer calories than the AL group from the age of 4 months. The concentrations of GSH, GSSG, and protein-SSG vary greatly (approximately 10-, 30-, and 9-fold, respectively) from one tissue to another. During aging, the ratios of GSH:GSSG in mitochondria and tissue homogenates decreased, primarily due to elevations in GSSG content, while the protein-SSG content increased significantly. Glutathione redox potential in mitochondria became less negative, i.e., more pro-oxidizing, as the animal aged. Caloric restriction (CR) lowered the GSSG and protein-SSG content. Results suggest that the aging process in the mouse is associated with a gradual pro-oxidizing shift in the glutathione redox state and that CR attenuates this shift.     

Efflux of Glutathione and Glutathione Complexes from Human Erythrocytes in Response to Inorganic Arsenic Exposure

The objective of the present study was to investigate if arsenic exposure results in glutathione efflux from human erythrocytes. Arsenite significantly depleted intracellular nonprotein thiol level in a time- and concentration-dependent manner. The intracellular nonprotein thiol level was decreased to 0.767?±?0.0017???mol/ml erythrocyte following exposure to 10?mM of arsenite for 4?h. Extracellular nonprotein thiol level was increased concomitantly with the intracellular decrease and reached to 0.481?±?0.0005???mol/ml erythrocyte in 4?h. In parallel with the change in extracellular nonprotein thiol levels, significant increases in extracellular glutathione levels were detected. Extracellular glutathione levels reached to 0.122?±?0.0013, 0.226?±?0.003, and 0.274?±?0.004???mol/ml erythrocyte with 1, 5, and 10?mM of arsenite, respectively. Dimercaptosuccinic acid treatment of supernatants significantly increased the glutathione levels measured in the extracellular media. Utilization of MK571 and verapamil, multidrug resistance-associated protein 1 and Pgp inhibitors, decreased the rate of glutathione efflux from erythrocytes suggesting a role for these membrane transporters in the process. The results of the present study indicate that human erythrocytes efflux glutathione in reduced free form and in conjugated form or forms that can be recovered with dimercaptosuccinic acid when exposed to arsenite.     

Age related toxic effects of endosulfan on certain enzymes of rat erythrocytes

The effect of oral administration of endosulfan (12.5 mg/kg body weight), daily for 4 days was investigated on erythrocytes of female rats of 4 different age groups i.e. 15, 30, 70 and 365 days old. Erythrocyte membrane Na+, K(+)-ATPase and Mg2(+)-ATPase activities were significantly inhibited in all the age groups of rats. However, percent inhibition was maximum in the youngest animals. A significant decrease in the activity of erythrocyte glutathione reductase was observed in 30 and 70 days old rats whereas a significant increase in the activity of glucose-6-phosphate dehydrogenase (G-6-PD) was observed in these groups. The increase in G-6-PD activity may be a physiological response to compensate for decrease in the reduced glutathione level which results from decrease in the activity of glutathione reductase.     

Biological age versus physical fitness age

A population of healthy middle-aged (n = 69) and elderly men (n = 12), who participated in a health promotion program, was studied to determine whether really physically fit individuals are in good biological condition, and also whether improvement of physical fitness in the middle-aged and the elderly reduces their "rate of aging". Biological and physical fitness ages of the individuals studied were estimated from the data for 18 physiological function tests and 5 physical fitness tests, respectively, by a principal component model. The correlation coefficient between the estimated biological and physical fitness ages was 0.72 (p less than 0.01). Detailed analyses of the relationship between the estimated biological and physical fitness ages revealed that those who manifested a higher ("older") physical fitness age did not necessarily have a higher biological age, but those who manifested a lower ("younger") physical fitness age were also found to have a lower biological age. These results suggested that there were considerable individual variations in the relationship between biological condition and physical fitness among individuals with an old physical fitness age, but those who were in a state of high physical fitness maintained a relatively good biological condition. The data regarding the elderly men who had maintained a regular exercise program indicated that their estimated biological ages were considerably younger than the expected values. This might suggest that in older individuals regular physical activity may provide physiological improvements which in turn might reduce "the rate of aging".     

Interactions of the antitumor drug, etoposide, with reduced thiols in vitro and in vivo

The interaction of activated etoposide, 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucopyra noside) (VP-16), with thiols has been studied both in vitro and in vivo in mice. We have found that both glutathione (GSH) and cysteine rapidly reduce the VP-16 free radical, which results in the regeneration of the parent drug and the oxidation of the thiol. Using spin-trapping and electron spin resonance (ESR) techniques, we have shown that this one-electron/hydrogen donation by thiols forms thiyl radicals (RS.) which are intermediates for the formation of the oxidized thiols. The administration of VP-16 in vivo to mice decreased the total thiol levels in liver and concomitantly increased the formation of oxidized thiols. Furthermore, VP-16 stimulated glutathione reductase in liver. While administration of VP-16 also increased the total thiol pools in kidney, in contrast, no significant effects were observed on lung and heart thiol pools.     

Membrane protein carboxyl methylation increases with human erythrocyte age. Evidence for an increase in the number of methylatable sites

The level of carboxyl methylation of membrane proteins has been measured in intact human erythrocyte populations of different ages separated by density gradient centrifugation. Age separation was confirmed by measurement of cytosolic pyruvate kinase specific activity in each fraction. When cells of different ages were incubated with L-[methyl-3H]methionine, the steady state level of 3H radioactivity covalently bound to membrane proteins is observed to be at least 3-fold higher in older erythrocytes. Because the specific radioactivity of the methyl group donor S-adenosyl-L-[methyl-3H]methionine was identical in all age fractions, this represents an increase in the extent of modification of membrane proteins by carboxyl methylation. Of the three major methylated erythrocyte membrane proteins, this increase in carboxyl methylation with age is 4 to 7-fold for bands 2.1 and 3, while the increase in band 4.1 is 3 to 4-fold. This increase in the steady state level of methylation with age cannot be explained by changes in either the intrinsic rate of methyl transfer or by changes in the rate constant of methyl turnover. We, therefore, propose that the age-dependent change in carboxyl methylation is due to an increase in the number of available acceptor sites as the erythrocyte ages in vivo. Since methylation of acidic residues on erythrocyte membrane proteins has been detected exclusively on D-aspartic acid residues (McFadden, P. N., and Clarke, S. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2460-2464), these results are consistent with an accumulation of D-aspartic acid in membrane protein due to spontaneous racemization a the cell ages. The relationship of these observations to possible functions of erythrocyte membrane protein carboxyl methylation is discussed.     

Age-dependent increase in green autofluorescence of blood erythrocytes

Green auto-fluorescence (GAF) of different age groups of mouse blood erythrocytes was determined by using a double in vivo biotinylation (DIB) technique that enables delineation of circulating erythrocytes of different age groups. A significant increase in GAF was seen for erythrocytes of old age group (age in circulation more than 40 days) as compared to young erythrocytes (age less than 15 days). Erythrocytes are removed from blood circulation by macrophages in the reticulo-endothelial system and depletion of macrophages results in an increased proportion of aged erythrocytes in the blood. When mice were depleted of macrophages for 7 days by administration of clodronate loaded liposomes, the overall GAF of erythrocytes increased significantly and this increase could be ascribed to an increase in GAF of the oldest population of erythrocytes. Using the DIB technique, the GAF of a cohort of blood erythrocyte generated during a 5 day window was tracked in vivo. GAF of the defined cohort of erythrocytes remained low till 40 days of age in circulation and then increased steeply till the end of the life span of erythrocytes. Taken together our results provide evidence for an age dependent increase in the GAF of blood erythrocytes that is accentuated by depletion of macrophages. Kinetics of changes in GAF of circulating erythrocytes with age has also been defined.     

Mitochondrial membrane potential in density-separated trout erythrocytes exposed to oxidative stress in vitro

Previous literature reports have demonstrated that nucleated trout erythrocytes in condition of oxidative stress are subjected to DNA and membrane damage, and inactivation of glutathione peroxidase. The present study was undertaken to investigate if mitochondrial membrane potential in stressed conditions was also influenced. Density-separated trout erythrocyte fractions, obtained using a discontinuous Percoll gradient, were submitted to stress conditions and the mitochondrial membrane potential was determined by means of cytofluorimetric analysis after incubation of each subfraction with JC-1, a mitochondrial specific fluorescent probe. The results clearly show that the mitochondrial membrane potential decreased significantly in all erythrocyte fractions, also if the oxidative effect on mitochondria is more severe with increased density (age) of the cell. Ebselen was very effective in preventing mitochondrial depolarization in young as well as in old erythrocytes.     

Number and distribution density of ABH and MN antigen sites on young and old human erythrocyte surfaces

There were no differences in the number of A and M antigen sites between young and old human erythrocyte surfaces. No essential differences in the number of A1, N and Vicia graminea N antigen sites could be observed between young and old erythrocytes. The number of B and H antigen sites on cell surface was significantly higher in young erythrocytes than in old ones. The distribution density of A and M antigen sites on young erythrocyte was remarkably higher than that on old ones. Compared with young erythrocytes, significant increases in the distribution density of A1, B, H, N and Vicia graminea N antigen sites were observed in aged erythrocytes. It is suggested from these and other observations that human erythrocyte aging is accompanied by elimination of a small amount of B and H antigens from cell membranes, while A, A1, M, N and Vicia graminea N antigens are not released from cell membranes during in vivo aging.     

Effects of cold stress on glutathione and related enzymes in rat erythrocytes

Effects of acute and chronic cold stress on glutathione and related enzymes in rat erythrocytes were investigated. Blood from both cold-acclimated (CA) and cold-adapted (CG) rats had significantly lower concentrations of glutathione than blood from control animals. Superoxide dismutase activity was increased significantly in CA rats and tended to rise in CG rats. Activity of glutathione peroxidase in erythrocytes was inconsistent in that it tended to increase in CA rats but decreased significantly in CG rats. The results may imply that CG rats suffered deleterious effects of hydrogen peroxide. On the other hand, there were marked decreases in glutathione peroxidase and glutathione reductase activities in acutely cold-exposed rats in conjunction with unchanged levels of glutathione. In all treatments the state of riboflavin metabolism was estimated to be adequate, since no increases were observed in the erythrocyte glutathione reductase activity coefficient.     

The decline in catalytic enzyme activity concentration with aging of the rabbit erythrocyte

Rabbit erythrocytes were separated by centrifugation on a discontinuous Percoll gradient into fractions of progressively increasing cell age to measure the in vivo decline in catalytic activity of eleven enzymes during the erythrocyte life span. Erythrocyte enzymes decline exponentially at different rates. The maximal and minimal catalytic activities (erythrocyte catalytic activity at the beginning and at the end of the erythrocyte life span), the intracellular half-life of enzymes and the daily loss of catalytic activity of total body erythrocytes were estimated.     

Enzymatic changes in rat erythrocytes with increasing cell and donor age: Loss of superoxide dismutase activity associated with increases in catalytically defective forms

Erythrocytes from young (6–8 month old) and old (28–31 month old) WF rats were separated into four age fractions by centrifugation on a discontinuous, isotonic, arabinogalactan density gradient. Specific activities of super-oxide dismutase (SOD) with respect to activity per unit hemoglobin (Hb) and activity per cell were determined for each cell age group. SOD activity was found to diminish with respect to erythrocyte age. More significantly, it was found that young erythrocytes of old animals already contain considerably reduced SOD activity as compared to cells of young animals. The level of SOD catalytic activity per unit enzyme antigen was also found to decrease with both increasing cell and animal age. Young cells of old animals contain significant amounts of catalytically altered molecules.     

Free radical generation by skeletal muscle of adult and old mice: effect of contractile activity

Oxidative modification of cellular components may contribute to tissue dysfunction during aging. In skeletal muscle, contractile activity increases the generation of reactive oxygen and nitrogen species (ROS). The question of whether contraction-induced ROS generation is further increased in skeletal muscle of the elderly is important since this influences recommendations on their exercise participation. Three different approaches were used to examine whether aging influences contraction-induced ROS generation. Hind limb muscles of adult and old mice underwent a 15-min period of isometric contractions and we examined ROS generation by isolated skeletal muscle mitochondria, ROS release into the muscle extracellular fluid using microdialysis techniques, and the muscle glutathione and protein thiol contents. Resting skeletal muscle of old mice compared with adult mice showed increased ROS release from isolated mitochondria, but no changes in the extracellular levels of superoxide, nitric oxide, hydrogen peroxide, hydroxyl radical activity or muscle glutathione and protein thiol contents. Skeletal muscle mitochondria isolated from both adult and old mice after contractile activity showed significant increases in hydrogen peroxide release compared with pre-contraction values. Contractions increased extracellular hydroxyl radical activity in adult and old mice, but had no significant effect on extracellular hydrogen peroxide or nitric oxide in either group. In adult mice only, contractile activity increased the skeletal muscle release of superoxide. A similar decrease in muscle glutathione and protein thiol contents was seen in adult and old mice following contractions. Thus, contractile activity increased skeletal muscle ROS generation in both adult and old mice with no evidence for an age-related exacerbation of ROS generation.