Rate Of Superoxide Generation Research Articles

Kinetic Modeling of Energy Metabolism and Superoxide Generation in Hepatocyte Mitochondria

Direct nonenzymatic oxidation of semiquinone by oxygen is one of the main sources of superoxide radicals \(\left( {{O}_{2}^{\overline \cdot } } \right)\) in mitochondria. Using all the known data on hepatocyte mitochondria, we have revealed the correlation between the rate of superoxide generation by the bc1complex and the transmembrane potential (ΔΨ). Assuming that the main electrogenic stage of the Qcycle is the electron transfer between the cytochrome bhemes, then the rate of superoxide generation sharply increases when ΔΨ grows from 150 to 180 mV. However, this interrelation is ambiguous. Indeed, the increase of the generation rate with the growth of the potential can occur faster when succinate dehydrogenase is inhibited by malonate than when external ADP is exhausted. When the potential is changed by adding phosphate or potassium (K+), the rate of \(\left( {{O}_{2}^{\overline \cdot } } \right)\) production remains constant, although the comparison of the rates at the same ΔΨ reveals the effect of phosphate or potassium. It turned out that the rate of \(\left( {{O}_{2}^{\overline \cdot } } \right)\) generation is a function of \(\Delta \overline {\mu } _{H}\) rather than any of its components. Phosphate and K+have practically no influence on \(\Delta \overline {\mu } _{H}\), since the change in ΔΨ is compensated by ΔpH. The rate of superoxide generation by the bc1complex is a multiple function of the electron-transfer activity of enzymes, the processes determining the membrane potential (e.g., loading), and the oxygen concentration. The kinetic model proposed in this work may serve to understand how the superoxide production is regulated.

Increased generation of superoxide in erythrocytes infected with Babesia gibsoni.

The present study was conducted to clarify the mechanism underlying the oxidative process in erythrocytes infected with Babesia gibsoni. The parasite B. gibsoni was cultured together with erythrocytes from normal dogs for 7 days. When parasitemia reached 12.0-13.4% at Day 7. the production of superoxide in erythrocytes was significantly higher in the parasitized culture than in the control culture (p<0.005). The concentration of thiobarbituric acid reactive substances (TBARS) in erythrocytes in parasitized culture was also significantly increased compared with the control culture (p<0.005), indicating that lipid peroxidation was greater in infected erythrocytes than in non-infected cells. In addition, the rates of superoxide generation in the blood of B. gibsoni-infected dogs were also significantly higher than in non-infected dogs (p<0.001). These results indicate that superoxide anions are increased in erythrocytes parasitized with B. gibsoni. and suggest that oxidative damage, due to lipid peroxidation, might be caused in host erythrocytes by the parasite.

A survey of oysters Crassostrea virginica from Tampa Bay, Florida: associations of internal defense measurements with contaminant burdens

Oysters from 16 sites in Tampa Bay, Florida, were collected during a 6-week period in winter 1993 and analyzed for both biological characteristics and tissue chemical concentrations. Using previous sediment contamination and toxicity data, oyster tissues from the selected sites were expected to exhibit a wide range in both quantity and type of chemicals. Chemical analysis showed tissue concentrations at some of these sites to be greater than national averages, as reported by the National Status and Trends Mussel Watch Program, for total PAH, total PCB, total chlordanes, DDT, Cu, Pb and Zn. Measures of oyster internal defense, including hemocyte density, rate of locomotion and superoxide generation, varied significantly among sites and were generally higher at sites with higher tissue concentrations of xenobiotic chemicals. Potential associations between oyster defense characteristics and accumulated chemical contaminants, either singly or in chemical classes, were explored using correlation analysis and a composited ranking procedure. Positive relationships were found for hemocyte characteristics with certain trace metal (Cu, Sn and Zn) and PAH analytes, whereas negative relationships were found with certain PCB and pesticide analytes. Heightened defenses in contaminated conditions may reflect a hemocyte process for sequestration and detoxification of environmental contaminants. Oysters from four of the 16 sites were additionally collected in June and September 1993 and site-related differences did not closely parallel those obtained in winter. Seasonal environmental factors may have altered contaminant-related differences among sites.

A microtiter plate assay for superoxide dismutase using a water-soluble tetrazolium salt (WST-1)

A simple and reproducible microtiter plate assay for measuring superoxide dismutase (SOD) activity is described. Water-soluble tetrazolium, the sodium salt of 4-[3-(4iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate, was used as a detector of superoxide radical generated by xanthine oxidase and hypoxanthine, in the presence of a range of concentrations of superoxide dismutase. A major advantage of the assay is that one reaction mixture is prepared and aliquotted into wells, avoiding pipetting errors and variable xanthine oxidase activity between samples. Inclusion of standardized SOD solution in each run enables inter-assay comparability without requiring a constant superoxide generation rate under all occasions. The assay is applicable for chloroform–ethanol red cell extracts as well as tissue homogenates without high-speed centrifugation. Fifty percent inhibition of formazan formation was achieved at 2.4±0.1 ng of Cu,ZnSOD per well with the coefficient of variation 4.2%.

Alteration of free radical metabolism in the brain of mice infected with scrapie agent

Alteration of free radical metabolism in the mouse brain by scrapie infection was evaluated. The infection of mice with scrapie agent, 87V strain, slightly increased the activities of catalase and glutathione-S-transferase, while it had no effect on glutathione peroxidase, glutathione reductase, and Cu, Zn-superoxide dismutase. Results show that the scrapie infection decreased the activity of mitochondrial Mn-superoxide dismutase by 50% but increased that of monoamine oxidase (p < 0.05). Scrapie infection also increased the rate of mitochondrial superoxide generation (p < 0.05). Following scrapie infection, the level of free-sulfhydryl compounds in brain homogenates slightly decreased, but the content of thiobarbituric-acid-reactive substances and malondialdehyde increased significantly. Electron microscopy indicated that the ultrastructure of mitochondria was destroyed in the brain of scrapie-infected mice.These results suggest that elevated oxygen free radical generation and lowered scavenging activity in mitochondria might cause the free radical damage to the brain. Such deleterious changes in mitochondria may contribute to the development of prion disease.

Fluoroquinolone Antimicrobials: Singlet Oxygen, Superoxide and Phototoxicity

The fluoroquinolone antibacterial agents possess photosensitizing properties that lead to phototoxic responses in both human and animal subjects. The phototoxicity order reported in humans is: fleroxacin > lomefloxacin, pefloxacin >> ciprofloxacin > enoxacin, norfloxacin and ofloxacin. Studies both in vivo and in vitro have related this phototoxicity to the generation of reactive oxygen species including hydrogen peroxide and the hydroxyl radical. We determined the quantum yields of singlet oxygen generation (phi delta) by detection of the singlet oxygen (1O2) luminescence at 1270 nm for several fluoroquinolones, naphthyridines and other structurally related compounds. All the fluoroquinolones examined have low phi delta values ranging from 0.06 to 0.09 in phosphate buffer at pD 7.5. We also determined the 1O2 quenching constants for these compounds and their values were on the order of 10(6) M-1 s-1, except for lomefloxacin whose rate constant was 1.8 x 10(7) M-1 s-1. The phi delta values were significantly decreased in a solvent of lower polarity such as methanol (0.007 < or = phi delta < or = 0.02). The production of 1O2 by these antibiotics did not correlate with the order reported for their phototoxicity. We also measured the photogeneration (lambda > 300 nm) of superoxide by these antibacterials in dimethylsulfoxide using electron paramagnetic resonance and the spin trap 5,5-dimethyl-1-pyrroline N-oxide. Although there is not a one-to-one correspondence between the relative rates of superoxide generation and the phototoxicity ranking of the fluoroquinolones, the more phototoxic compounds tended to produce superoxide at a faster rate. Nevertheless, the magnitudes of the observed differences do not appear sufficient to explain the range of fluoroquinolone phototoxicity potencies in human and animal subjects in general and the high activity of fleroxacin and lomefloxacin in particular. For these latter drugs the photoinduced loss of the F8 atom as fluoride and the concomitant generation of a highly reactive carbene at C-8 provide a more plausible mechanism for their potent phototoxic and photocarcinogenic properties.

Reactive oxygen species generation by human spermatozoa is induced by exogenous NADPH and inhibited by the flavoprotein inhibitors diphenylene iodonium and quinacrine.

Human spermatozoa possess a specialized capacity to generate reactive oxygen species (ROS) that is thought to be of significance in the redox regulation of sperm capacitation (De Lamirande and Gagnon, 1993; Aitken et al., 1995). However, the mechanisms by which ROS are generated by these cells are not understood. In this study we have examined the possible significance of NADPH as a substrate for ROS production by human spermatozoa. Addition of NADPH to viable populations of motile spermatozoa induced a sudden dose-dependent increase in the rate of superoxide generation via mechanisms that could not be disrupted by inhibitors of the mitochondrial electron transport chain (antimycin A, rotenone, carbonyl cyanide m-chlorophenylhydrazone [CCCP], and sodium azide), diaphorase (dicoumarol) xanthine oxidase (allopurinol), or lactic acid dehydrogenase (sodium oxamate). However, NADPH-induced ROS generation could be stimulated by permeabilization and was negatively correlated with sperm function. Both NADH and NADPH were active electron donors in this system, while NAD+ and NADP+ exhibited little activity. Stereo-specificity was evident in the response in that only the beta-isomer of NADPH supported superoxide production. The involvement of a flavoprotein in the electron transfer process was indicated by the high sensitivity of the oxidase to inhibition by diphenylene iodonium and quinacrine. These results indicate that NAD(P)H can serve as an electron donor for superoxide generation by human spermatozoa and present a simple strategy for the production of motile populations of free radical generating cells with which to study the significance of these molecules in the control of normal and pathological sperm function.

Excess formation of lysophosphatidic acid with age inhibits myristic acid-induced superoxide anion generation in intact human neutrophils

A superoxide anion generation rate upon exposure to myristate of 1.93 ± 0.34 nmol/min/10 6 cells in neutrophils from elderly human donors was significantly less than a value of 3.02 ± 0.48 nmol/min/10 6 neutrophils from young donors. Myristate activation resulted in equal increases of AA in both the young and the old indicating no effect of aging on the PLA 2 pathway to response. By contrast, the PLD-induced generation of PA was significantly higher in the old than in the young. In addition, myristate induced a significant age-related enhancement in LPA generation, in the old but not in the young. The mass of LPA generated following activation was 3.5 nmol/ 2.5 × 10 7cells/ml in the young while in the old it averaged 7.0 nmol/2.5 × 10 7 cells/ml. The inhibitory effects of LPA may explain the age-related impaired ability to generate superoxide anion following activation by myristate.

High rates of extracellular superoxide generation by cultured human fibroblasts: involvement of a lipid-metabolizing enzyme.

Expression of NADPH oxidase and low superoxide generation (approx. 0.06 nmol/min per 10(6) cells) by cytokine- or ionophore-stimulated human fibroblasts is known. However, we here show that these cells also contain an ectoplasmic enzyme, distinct from NADPH oxidase, which can generate superoxide (2.19 +/- 0.14 nmol/min per 10(6) cells) at levels similar to phorbol ester-stimulated monocytes on exogenous NADH addition. Superoxide generation was temperature-dependent, insensitive to chelation (desferal), and had a K(m) (app)(NADH) of 11.5 microM. Inhibitor studies showed that there was no involvement of NADPH oxidase (diphenylene iodonium, diphenyl iodonium), prostaglandin H synthase (indomethacin), xanthine oxidase (allopurinol), cytochrome P-450 (metyrapone) or mitochondrial respiration (rotenone, antimycin A). NAD+ was a competitive inhibitor, whereas NADPH supported 40% of the rate seen with NADH. No luminescence was observed after the addition of lactate, malate, pyruvate, GSH or L-cysteine. NADH-stimulated superoxide generation was enhanced by the addition of (3-30 microM) arachidonic acid, linoleic acid or (5S)-hydroxyeicosatetraenoic acid [(5S)-HETE] but not palmitic acid, (15S)-hydroperoxyeicosatetraenoic acid [(15S)-HPETE], (15S)-HETE or (12S)-HETE. Several features suggest involvement of an enzyme related to 15-lipoxygenase, and, in support of this, we show superoxide generation and NADH oxidation by recombinant rabbit reticulocyte 15-lipoxygenase. The large amounts of superoxide measured suggest that the fibroblast extracellular enzyme could be a major source of reactive oxygen species after tissue damage.

マレイン酸プログルメタシンによるヒト好中球スーパーオキシド産生阻害

Polymorphonuclear leukocytes (PMN) play an important role in development of inflammation. In the clinical field, non-steroidal anti-inflammation drugs (NSAID), such as proglumethacin maleate, were used to theraphy with arthritis. In this study, we investigated the effects of NSAIDs on superoxide generation of human PMN from synovial fluid.On the PMNs from synovial fluid of rheumatoid arthritis (RA) patients, proglumethacin maleate significantly inhibited the superoxide generation by phorbol 12-myristate 13-acetate (PMA) . The rate and maximum amount of superoxide generation of PMNs were reduced to 12.72±6.32% and 24.23±9.44%, respectively. The other NSAIDs, Indomethacin farnesil, diclofenac sodium and loxoprofen sodium, were also tended to inhibit the superoxide generation by PMA, but not significantly. Indomethacin farnesil, diclofenac sodium and loxoprofen sodium reduced the rate of superoxide generation to 55.88±5.58%, 41.17±2.36% and 45.24±1.77%, respectively. Proglumethacin maleate also strongly inhibited the superoxide generation of PMNs from patients of osteoarthritis (OA), but other NSAIDs did not. The rate and maximum amount of superoxide generation of PMNs from OA patients were reduced by proglumethacin maleate to 21.20±2.79% and 32.36±10.09%, respectively. Indomethacin which is active metabolite of proglumethacin maleate for NSAID did not inhibit the superoxide generation of PMNs from RA and OA patients.These result indicated that proglumethacin maleate have inhibitory action to superoxide generation of PMNs which is not NSAID's action. This inhibition of superoxide generation of PMNs may be one of the anti-inflammatory mechanisms of proglumethacin maleate.

Reactions of nitric oxide and peroxynitrite with organic molecules and ferrihorseradish peroxidase: Interference with the determination of hydrogen peroxide

Endothelial, inflammatory, and other cell types, in addition to forming reactive oxygen species, under proper stimulation release free radicals such as nitric oxide ('NO) and strong oxidants such as peroxynitrite (ONOO -), which is the product of the reaction of nitric oxide with superoxide. Several methods for the detection of H 2O 2 are based on the ferrihorseradish peroxidase catalyzed oxidation of organic molecules. We investigated the reactions of nitric oxide and peroxynitrite with organic molecules as well as with ferrihorseradish peroxidase and examined the potential interference with the detection of H 2O 2. Peroxynitrite at low concentrations (010 μM) induced a concentration-dependent oxidation of 1,2 phenylenediamine, 3-3′ dimethoxybenzidine (odianisidine) and para-hydroxyphenylacetic acid (pHPA). With the exception of pHPA, the oxidation of the above compounds by peroxynitrite was not affected by the presence of ferrihorseradish peroxidase. The yield of HPA-dimmer, the oxidation product of pHPA by peroxynitrite, was decreased because ferrihorseradish peroxidase catalyzed the formation of a different product, 3-nitro-HPA. Nitrogen oxides, formed by the reaction of nitric oxide with oxygen, oxidized the aromatic amines o-phenylenediamine and o-dianisidine. A 10-fold excess of nitric oxide over H 2O 2 decreased the yield of pHPA and dihydrorhodamine 123 (DHR123) by 58 and 72%, respectively, as compared to H 2O 2 plus ferrihorseradish peroxidase. The inhibition of pHPA oxidation by nitric oxide was due in part to the reaction of nitric oxide with compound I and compound II and in part due to the reaction with the phenoxyl radical. These data suggest that the simultaneous generation of nitric oxide and peroxynitrite can interfere with the detection of H 2O 2. The overestimation or underestimation of the H 2O 2 detected is dependent upon the organic molecule utilized for detection and by the relative rate of nitric oxide, superoxide, and peroxynitrite generation.

Membrane association of Rac is required for high activity of the respiratory burst oxidase.

NADPH-dependent superoxide generation can be reconstituted in a cell-free system using recombinant cytosolic factors (p47-phox, p67-phox, and Rac) plus flavocytochrome b558. Rac1 and Rac2 are closely related small GTPases, differing primarily in the C-terminal 10 residues where Rac1 but not Rac2 contains a polybasic sequence. In their nonisoprenylated forms, Rac1 was highly effective in reconstituting NADPH oxidase activity (low EC50, high Vmax), whereas Rac2 was only minimally effective (high EC50, low Vmax). In contrast, low concentrations of isoprenylated Rac1 and Rac2 both supported high rates of superoxide generation. Like full length Rac2, truncated forms of both Rac1 and Rac2 in which the C-terminal 10 residues were eliminated were poorly activating, pointing to the C terminus of Rac1 as a determinant of activity. Mutation of single positively charged residues in the C terminus of nonisoprenylated Rac1 markedly reduced its ability to support superoxide generation, affecting both its EC50 and the Vmax. In contrast, mutation or truncation of the C terminus failed to affect the activation of PAK, a Rac-regulated protein kinase. The EC50 for Rac1 increased with increasing salt concentrations, whereas that of Rac2 was independent of salt, implicating the involvement of electrostatic forces for the former. Using flavocytochrome b558 reconstituted into phosphatidylcholine vesicles, the EC50 for Rac1 but not Rac2 decreased (increased binding) when an acidic phospholipid (phosphatidylinositol) was present, supporting a role for the Rac1 polybasic C terminus in binding to the membrane. A model in which Rac must associate simultaneously both with p67-phox and with the membrane to activate the NADPH oxidase can account for the above observations.

Lipoxygenase‐mediated glutathione oxidation and superoxide generation

Soybean lipoxygenase-mediated cooxidation of reduced glutathione (GSH) and concomitant superoxide generation was examined. The oxidation of GSH was dependent on the concentration of linoleic acid (LA), GSH, and the enzyme. The optimal conditions to observe maximal enzyme velocity included the presence of 0.42 mM LA, 2 mM GSH, and 50 pmole of enzyme/mL. The GSH oxidation was linear up to 10 minutes and exhibited a pH optimum of 9.0. The reaction displayed a Km of 1.49 mM for GSH and Vmax of 1.35 +/- 0.02 mumoles/min/nmole of enzyme. Besides LA, arachidonic and gamma-linolenic acids also supported the lipoxygenase-mediated GSH oxidation. Hydrogen peroxide and 13-hydroperoxylinoleic acid supported GSH cooxidation, but to a very limited extent. Oxidized glutathione (GSSG) was identified as the major product of the reaction based on the depletion of nicotinamide-adenine dinucleotide 3'-phosphate (NADPH) in the presence of glutathione reductase. The GSH oxidation was accompanied by the reduction of ferricytochrome c, which can be completely abolished by superoxide dismutase (SOD), suggesting the generation of superoxide anion radicals. Under optimal conditions, the rate of superoxide generation (measured as the SOD-inhibitable reduction of ferricytochrome c) was 10 +/- 1.0 nmole/min/nmole of enzyme. These results clearly suggest that lipoxygenase is capable of oxidizing GSH to GSSG and simultaneously generating superoxide anion radicals, which may contribute to oxidative stress in cells under certain conditions.

Catalase- and Superoxide Dismutase-induced Morphological Changes and Growth Inhibition in the Red Tide PhytoplanktonChattonella marina

Chattonella is one of the most toxic red tide phytoplankton and causes severe damage to fish farming. Recent studies demonstrated that Chattonella sp. generates superoxide and hydroxyl radicals, which may be responsible for the toxicity of this plankton. However, little is known about the mechanism of the production of oxygen radicals by Chattonella, and the role of oxygen radicals in Chattonella themselves is also unclear. In this study, we found that superoxide dismutase (SOD) and catalase inhibited the growth of Chattonella marina concomitant with their morphological changes. In the presence of these enzymes, the shape of vegetative C. marina cells changed from spindle to round. Furthermore, the generation of oxygen radicals by C. marina depended on the growth phase ; the rate of superoxide and hydrogen peroxide generation was the highest during exponentially growing phase and subsequently decreased to one-fifth of the maximal level in the stationary growth phase. These results suggest that oxygen radicals generated by C. marina play an essential role in their own survival, especially in cell division.

The Reaction of Superoxide with Reduced Glutathione

Superoxide, generated by a xanthine oxidase/hypoxanthine system, reacts with reduced glutathione (GSH) to cause an increase in oxygen consumption and oxidized glutathione (GSSG) formation, both of which are fully inhibited by superoxide dismutase. In this study we have shown that little, if any, of the additional oxygen consumed is converted to hydrogen peroxide. We have confirmed that approximately 90% of the GSH is oxidized to GSSG, the remainder being converted to the sulfonic acid. Approximately 1.2 mol of GSSG was formed for each additional mole of oxygen consumed in the presence of GSH. The efficiency of the reaction increased with increasing GSH concentration (1-8 mM), pH, and pO2 and with decreasing superoxide generation rate. The results are consistent with a superoxide-dependent chain that does not produce hydrogen peroxide and that is terminated primarily by superoxide dismutation. We propose that this occurs via an initial reaction of superoxide with GSH to produce a sulfinyl radical rather than hydrogen transfer to give the thiyl radical. Our data suggest a rate constant for the superoxide/GSH reaction in the 102-103 M−1 s−1 range. GSH at the millimolar concentrations found intracellularly should react with superoxide, but because superoxide is regenerated, it will not be an effective scavenger. Physiologically, superoxide dismutase is required to prevent chain oxidation of GSH.

Superoxide generation by lipoxygenase in the presence of NADH and NADPH

The ability of soybean lipoxygenase to mediate NAD(P)H oxidation and concomitant Superoxide generation in the presence of linoleic acid was examined. At optimum pH of 8.3, lipoxygenase oxidized both NADH and NADPH in the presence of 700 μM linoleic acid. The oxidation of NAD(P)H was biphasic. The initial rates of NADH and NADPH oxidation were 130 and 140 nmoles/min/nmole of enzyme respectively and the corresponding final rates were 344 and 350 nmoles/min/nmole of enzyme. The apparent K m values calculated for NADH and NADPH oxidation were 13 ju.m and 117 μM respectively. NAD(P)H oxidation was accompanied by the reduction of either ferricytochrome c or nitroblue tetrazolium (NBT) which can be abolished (approx. 85%) by Superoxide dismutase (SOD) suggesting the generation of Superoxide anion radicals. Under optimal conditions, the rates of Superoxide generation, measured as the SOD-inhibitable reduction of ferricytochrome c, were 325 and 235 nmoles/min/nmole of enzyme for NADH and NADPH respectively. Under identical experimental conditions, the SOD-inhibitable NBT reduction rates were 308 and 210 nmoles/min/nmole of enzyme for NADH and NADPH respectively. Both NADH and NADPH could be regenerated after oxidation using the appropriate dehydrogenases. These results strongly suggest that lipoxygenase not only generates lipid hydroperoxides but can also generate Superoxide via oxidation of pyridine nucleotides and may, therefore, significantly contribute to oxidative stress in cells.

Determination of the rate of superoxide generation from biological systems by spin trapping: Use of rapid oxygen depletion to measure the decay rate of spin adducts

A method was developed to measure the superoxide rate from biological systems using the spin trapping method. Kinetic treatment of the decay rate of the superoxide adduct of 5,5-dimethylpyrroline N-oxide (DMPO) revealed that the EPR signal intensity of the system is proportional to the superoxide generation rate. Rapid depletion of oxygen in the sample was utilized to terminate superoxide generation so that the decay rate of t he DMPO superoxide adduct (DMPO-OOH) could be determined. For this decay measurement, a controlled atmosphere EPR cavity was developed and was used with an open-air sample cell. Superoxide generation rates determined with this method for stimulated neutrophils and for the hypoxanthine-xanthine oxidase system were comparable to those obtained with the cytochrome c reduction method. This method is specifically applicable to the system in which dissolved oxygen supplied from the gas phase is utilized as a source of superoxide.

Oxidative damage, mitochondrial oxidant generation and antioxidant defenses during aging and in response to food restriction in the mouse

This study was conducted in order to test the concept that oxidative damage is associated with aging and may be a factor in the modulation of life span in response to variations in caloric intake. Mice fed a diet that was 40% lower in calories (DR) than the ad libitum fed (AL) animals exhibited a 43% extension in average life span and a 61% prolongation in mortality rate doubling time. A comparison of AL and DR mice at 9, 17 and 23 months of age indicated that the protein carbonyl content in the brain, heart and kidney increased with age and was significantly greater in the AL than DR group in each organ at each of the three ages. Mitochondrial state 4 or resting respiratory rate increased with age in the AL, but not the DR group, and was also relatively higher in the former. The rates of mitochondrial superoxide and hydrogen peroxide generation increased with age and were higher in the AL than DR mice in all the three organs at each age. In contrast, there was no clear-cut overall pattern of age-related or dietary-related changes in antioxidant defenses provided by superoxide dismutase, catalase and glutathione peroxidase. Results suggest that mechanisms of aging and life span shortening by enhanced caloric intake are associated with oxidative damage arising from corresponding changes in mitochondrial oxidant production. Protein carbonyl content, and mitochondrial O 2 ·− and H 2O 2 generation may act as indices of aging.

Monocyte Dysfunction in Patients With Gaucher Disease: Evidence for Interference of Glucocerebroside With Superoxide Generation

AbstractGaucher disease patients are occasionally affected by chronic or fulminant infections. Since Gaucher cells originate from tissue phagocytes, we studied the functional implications of glucocerebroside accumulation on phagocytes in Gaucher disease patients. Circulating monocytes and granulocytes from nine type I Gaucher disease patients, and matched controls, were studied. Evaluation of phagocytic activity included (1) maximal superoxide generation rates following stimulation by phorbol 12-myristate 13-acetate (PMA), opsonized zymosan (OZ), or formyl-methionyl-leucyl-phenylalanine (FMLP); (2) nitroblue tetrazolium reduction test (NBT); (3) chemotaxis toward FMLP; (4) phagocytosis of OZ particles; and (5) killing activity against Staphylococcus aureus. Superoxide generation in monocytes following PMA, OZ, and FMLP stimulation was significantly suppressed at 52% ± 15%,39% ±8%,and 51% ± 11%of control, respectively. Superoxide generation in granulocytes was normal. NBT reduction, staphylococcal killing, and phagocytosis were also markedly decreased in monocytes, and normal in granulocytes. Mean chemotaxis rates were normal in both monocytes and granulocytes; however, decreased chemotactic rates were observed in some patients. The abnormality of superoxide generation could be reproduced in a dose- and time-dependent manner in normal circulating monocytes incubated with glucocerebroside. Superoxide generation in glucocerebroside-conditioned normal monocytes in a cell-free system showed normal superoxide generation, reflecting the integrity of the NADPH oxidase complex itself. These results demonstrate markedly compromised phagocytic functions in circulating monocytes in Gaucher disease patients. These abnormalities can be attributed to accumulation of glucocerebroside, since it could be reproduced in normal monocytes incubated with glucocerebroside. Similar abnormalities in Gaucher cells throughout the reticuloendothelial system could impair host defense, and may be of particular importance in the pathogenesis of osteomyelitis in Gaucher disease patients.

Monocyte dysfunction in patients with Gaucher disease: evidence for interference of glucocerebroside with superoxide generation [see comments

Gaucher disease patients are occasionally affected by chronic or fulminant infections. Since Gaucher cells originate from tissue phagocytes, we studied the functional implications of glucocerbroside accumulation on phagocytes in Gaucher disease patients. Circulating monocytes and granulocytes from nine type I Gaucher disease patients, and matched controls, were studied. Evaluation of phagocytic activity included (1) maximal superoxide generation rates following stimulation by phorbol 12-myristate 13-acetate (PMA), opsonized zymosan (OZ), or formyl-methionyl-leucylphenylalanine (FMLP); (2) nitroblue tetrazolium reduction test (NBT); (3) chemotaxis toward FMLP; (4) phagocytosis of OZ particles; and (5) killing activity against Staphylococcus aureus. Superoxide generation in monocytes following PMA, OZ, and FMLP stimulation was significantly suppressed at 52% +/- 15%, 39% +/- 8%, and 51% +/- 11% of control, respectively. Superoxide generation in granulocytes was normal. NBT reduction, staphylococcal killing, and phagocytosis were also markedly decreased in monocytes, and normal in granulocytes. Mean chemotaxis rates were normal in both monocytes and granulocytes; however, decreased chemotactic rates were observed in some patients. The abnormality of superoxide generation could be reproduced in a dose- and time-dependent manner in normal circulating monocytes incubated with glucocerebroside. Superoxide generation in glucocerebroside-conditioned normal monocytes in a cell-free system showed normal superoxide generation, reflecting the integrity of the NADPH oxidase complex itself. These results demonstrate markedly compromised phagocytic functions in circulating monocytes in Gaucher disease patients. These abnormalities can be attributed to accumulation of glucocerebroside, since it could be reproduced in normal monocytes incubated with glucocerebroside. Similar abnormalities in Gaucher cells throughout the reticuloendothelial system could impair host defense, and may be of particular importance in the pathogenesis of osteomyelitis in Gaucher disease patients.