Mechanism of O2- generation in reduction and oxidation cycle of ubiquinones in a model of mitochondrial electron transport systems

O2- generation in mitochondrial electron transport systems, especially the NADPH-coenzyme Q10 oxidoreductase system, was examined using a model system, NADPH-coenzyme Q1-NADPH-dependent cytochrome P-450 reductase. One electron reduction of coenzyme Q1 produces coenzyme Q1-. and O2- during enzyme-catalyzed reduction and O2+ coenzyme Q1-. are in equilibrium with O2- + coenzyme Q1 in the presence of enough O2. The coenzyme Q1-. produced can be completely eliminated by superoxide dismutase, identical to bound coenzyme Q10 radical produced in a succinate/fumarate couple-KCN-submitochondrial system in the presence of O2. Superoxide dismutase promotes electron transfer from reduced enzyme to coenzyme Q1 by the rapid dismutation of O2- generated, thereby preventing the reduction of coenzyme Q1 by O2-. The enzymatic reduction of coenzyme Q1 to coenzyme Q1H2 via coenzyme Q1-. is smoothly achieved under anaerobic conditions. The rate of coenzyme Q1H2 autoxidation is extremely slow, i.e., second-order constant for [O2][coenzyme Q1H2] = 1.5 M-1.s-1 at 258 microM O2, pH 7.5 and 25 degrees C.     

Specificity of coenzyme Q inhibition of an aging-related cell surface NADH oxidase (ECTO-NOX) that generates superoxide

Our laboratories have described a novel class of ectoproteins at the cell surface with both NADH or hydroquinone oxidase (NOX) and protein disulfide-thiol interchange activities (ECTO-NOX proteins). The two activities exhibited by these proteins alternate to generate characteristic patterns of oscillations where the period length is independent of temperature. The period length for the constitutive ECTO-NOX is 24 min. Here we describe a distinctive age-related ECTO-NOX (arNOX) whose activity is blocked by coenzyme Q10. arNOX occurs exclusively in aged cells and tissues. The period length of the oscillations is 26 min. Rather than reducing 1/2 O2 to H2O, electrons are transferred to O2 to form superoxide. Superoxide formation was demonstrated by superoxide dismutase-sensitive reduction of ferricytochrome c and by reduction of a superoxide-specific tetrazolium salt. Quinone inhibition was given by coenzymes Q8, 9 and Q10 but not by Q0, Q2, Q4, Q6 or 7. The arNOX provides a mechanism to propagate reactive oxygen species generated at the cell surface to surrounding cells and circulating lipoproteins of importance to atherogenesis. Inhibition of arNOX by dietary coenzyme Q10 provides a rational basis for dietary coenzyme 10 use to retard aging-related arterial lesions.     

Effects of combined quercetin and coenzyme Q(10) treatment on oxidative stress in normal and diabetic rats

Reactive oxygen species may be actively involved in the genesis of various pathological states such as ischemia-reperfusion injury, cancer, and diabetes. Our objective was to determine if subacute treatment with combined antioxidants quercetin and coenzyme Q(10) (10 mg/kg/day ip for 14 days) affects the activities of antioxidant enzymes in normal and 30-day streptozotocin-induced diabetic Sprague-Dawley rats. Quercetin treatment raised blood glucose concentrations in normal and diabetic rats, whereas treatment with coenzyme Q(10) did not. Liver, kidney, heart, and brain tissues were excised and the activities of catalase, glutathione reductase, glutathione peroxidase, superoxide dismutase, and concentrations of oxidized and reduced glutathione were determined. In the liver of diabetic rats, superoxide dismutase, glutathione peroxidase, and levels of both oxidized and reduced glutathione were significantly decreased from the nondiabetic control, and these effects were not reversed when antioxidants were administered. In kidney, glutathione peroxidase activity was significantly elevated in the diabetic rats as compared to nondiabetic rats, and antioxidant treatment did not return the enzyme activity to nondiabetic levels. In heart, catalase activity was increased in diabetic animals and restored to normal levels after combined treatment with quercetin and coenzyme Q(10). Cardiac superoxide dismutase was lower than normal in quercetin- and quercetin + coenzyme Q(10)-treated diabetic rats. There were no adverse effects on oxidative stress markers after treatment with quercetin or coenzyme Q(10) singly or in combination. In spite of the elevation of glucose, quercetin may be effective in reversing some effects of diabetes, but the combination of quercetin + coenzyme Q(10) did not increase effectiveness in reversing effects of diabetes.     

Susceptibility of Eimeria bovis and Toxoplasma gondii to oxygen intermediates and a new mathematical model for parasite killing

Eimeria bovis and Toxoplasma gondii differ in their susceptibility to macrophages activated by lymphokines. Interferon-gamma can activate macrophages to totally inhibit E. bovis sporozoite development, whereas growth of T. gondii tachyzoites in macrophages is not totally affected. The susceptibility of these parasites to oxygen intermediates and their ability to evade the oxidative burst by macrophages were investigated in cell-free systems. Using a logistic model to assess growth inhibition, T. gondii growth was impaired by 50% at 10(-4.25) M (56 microM) H2O2, with 30 min as the optimum time for measuring inhibition. Preliminary results indicate that T. gondii follows mode-one and mode-two killing with relation to time after exposure to H2O2, implying a role for OH. and the induction of a DNA repair mechanism. The same model was used to assess inhibition of E. bovis growth that was more susceptible, being inhibited to 50% by 10(-5) M (10 microM) H2O2. Both parasites were susceptible to the effects of xanthine-xanthine oxidase that releases a full complement of oxygen intermediates (H2O2, OH., (1)O2, and O2-). Adding quenchers or scavengers to the system confirmed that T. gondii was susceptible to products of the interaction of O2- and H2O2 (OH. and (1)O2), and that E. bovis sporozoites were at least partially susceptible to H2O2 and O2-, but extremely susceptible to OH.. These data were supported by studies on scavenging enzymes present in the parasites. Toxoplasma gondii was rich in superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPO), and E. bovis had less catalase and SOD.(ABSTRACT TRUNCATED AT 250 WORDS)     

Forms of Selenium Affect its Transport, Uptake and Glutathione Peroxidase Activity in the Caco-2 Cell Model

The aim of this study was to investigate the possible time- and dose-dependent cytotoxic effects of cobalt chloride on Vero cells. The cultured cells were incubated with different concentrations of cobalt chloride ranging from 0.5 to 1,000 μM, and cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) and resazurin assays. Possible protective effects of vitamin E, coenzyme Q(10), and zinc chloride were also tested in this system. A gradual decrease in cell proliferation was observed at concentrations ~≥ 200 μM in incubation periods of 24, 48, 72, and 96 h with MTT assay. Exposure of cells to 500 and 1,000 μM cobalt chloride caused significant decrease in cell survival. A biphasic survival profile of cells was observed at 1-25 μM concentration range following 96 h of incubation. With resazurin assay, cytotoxicity profile of CoCl(2) was found comparable to the results of MTT assay, particularly at high concentrations and long incubation periods. Dose-dependent cytotoxicity was noted following exposure of cells to ≥ 250 μM of CoCl(2) for 24 h and ≥ 100 μM concentrations of CoCl(2) for 48-96 h. Pretreatment of cells with ZnCl(2) for 4 or 24 h provided significant protection against cobalt chloride-induced cytotoxicity when measured with MTT assay. However, vitamin E or coenzyme Q(10) was not protective. CoCl(2) had dose- and time-dependent cytotoxic effects in Vero cells. Preventive effect of ZnCl(2) against CoCl(2)-induced cytotoxicity should be considered in detail to define exact mechanism of toxicity in Vero cells.     

Impaired generation of reactive oxygen species during differentiation of dendritic cells (DCs) by Mycobacterium tuberculosis secretory antigen (MTSA) and subsequent activation of MTSA-DCs by mycobacteria results in increased intracellular survival

We investigated the role of reactive oxygen species (ROS) in dendritic cell (DC) differentiation by 10-kDa Mycobacterium tuberculosis secretory Ag (MTSA) and survival of mycobacteria therein. Compared with GM-CSF, MTSA induced lower ROS production during DC differentiation from precursors. This result correlated with higher superoxide dismutase 1 expression in MTSA stimulated precursors as compared with GM-CSF stimulation. Furthermore, a negative regulation of protein kinase C (PKC) activation by ROS was observed during DC differentiation. ROS inhibited the rapid and increased phosphorylation of PKCalpha observed during DC differentiation by MTSA. In contrast, ROS inhibition increased the weak and delayed PKCalpha phosphorylation by GM-CSF. Similar to DC differentiation, upon activation with either M. tuberculosis cell extract (CE) or live Mycobacterium bovis bacillus Calmette-Guérin (BCG), DCs differentiated with MTSA (MTSA-DCs) generated lower ROS levels when compared with DCs differentiated with GM-CSF (GM-CSF-DCs). Likewise, a negative regulation of PKCalpha phosphorylation by ROS was once again observed in DCs activated with either M. tuberculosis CE or live M. bovis BCG. However, a reciprocal positive regulation between ROS and calcium was observed. Compared with MTSA-DCs, stimulation of GM-CSF-DCs with M. tuberculosis CE induced a 2-fold higher ROS-dependent calcium influx. However, pretreatment of MTSA-DCs with H(2)O(2) increased calcium mobilization. Finally, lower ROS levels in MTSA-DCs correlated with increased intracellular survival of M. bovis BCG when compared with survival in GM-CSF-DCs. Although inhibiting ROS in GM-CSF-DCs increased M. bovis BCG survival, H(2)O(2) treatment of MTSA-DCs decreased survival of M. bovis BCG. Overall our results suggest that DCs differentiated with Ags such as MTSA may provide a niche for survival and/or growth of mycobacteria following sequestration of ROS.     

Coenzyme Q10 enrichment decreases oxidative DNA damage in human lymphocytes

Ubiquinol-10, the reduced form of coenzyme Q10, is a powerful antioxidant in plasma and lipoproteins. It has been suggested that endogenous ubiquinol-10 also exerts a protective role even towards DNA oxidation mediated by lipid peroxidation. Even though the antioxidant activity of coenzyme Q10 is mainly ascribed to ubiquinol-10, a role for ubiquinone-10 (the oxidized form), has been suggested not only if appropriate reducing systems are present. To investigate whether the concentration of ubiquinol-10 or ubiquinone-10 affects the extent of DNA damage induced by H2O2, we supplemented in vitro human lymphocytes with both forms of coenzyme Q10 and evaluated the DNA strand breaks by Comet assay. The exposure of lymphocytes to 100 microM H2O2 resulted in rapid decrease of cellular ubiquinol-10 content both in ubiquinol-10-enriched and in control cells, whereas alpha-tocopherol and beta-carotene concentration were unchanged. After 30 min from H2O2 exposure, the amount of DNA strand breaks was lower and cells' viability was significantly higher in ubiquinol-10-enriched cells compared with control cells. A similar trend was observed in ubiquinone-10-enriched lymphocytes when compared with control cells. Our experiments suggest that coenzyme Q10 in vitro supplementation enhances DNA resistance towards H2O2-induced oxidation, but it doesn't inhibit directly DNA strand break formation.     

Changes in antioxidant status of myocardium during oxidative stress under the influence of coenzyme Q<Subscript>10</Subscript>

Changes in myocardium were studied during oxidative stress induced by infusion of hydrogen peroxide in the coronary vessels of isolated rat heart. Moderate concentrations of H2O2 increased the heart rate but decreased the contractile force, whereas higher concentrations of H2O2 decreased both parameters and increased the end diastolic pressure. The effect of H2O2 was stable, cumulative, and was associated with disturbance in respiration of mitochondria, increased production of ROS in them, and decrease in activities of antioxidant enzymes in the myocardium. Changes in the antioxidant status of the myocardium induced by long-term addition of coenzyme Q(10) into food was accompanied by decrease in the negative inotropic effect of H2O2, whereas the levels of superoxide dismutase and glutathione peroxidase after oxidative stress were virtually unchanged. The activities of these enzymes displayed a high positive correlation with the cardiac function. The findings suggest that coenzyme Q(10) should increase resistance of the myocardium to oxidative stress not only by a direct antioxidant mechanism but also indirectly, due to increased protection of antioxidant enzymes.     

Inhibition of lipid peroxidation by ubiquinol in submitochondrial particles in the absence of vitamin E

The relationship between the antioxidant effects of reduced coenzyme Q10 (ubiquinol, UQH2) and vitamin E (alpha-tocopherol) was investigated in beef heart submitochondrial particles in which lipid peroxidation was initiated by incubation with ascorbate + ADP-Fe3+. These effects were examined after extraction of coenzyme Q10 (UQ-10) and vitamin E from the particles and reincorporation of the same components alone or in combination. The results show that UQH2 efficiently inhibits lipid peroxidation even when vitamin E is absent. It is concluded that UQH2 can inhibit lipid peroxidation directly, without the mediation of vitamin E.     

Chemiluminescent assay for detection of viable microorganisms

The redox reaction between quinone and viable microorganisms produces active oxygen species. In this study, the production rates of active oxygen species were determined by a luminol chemiluminescent assay, and the luminescence intensity was found to be proportional to the viable cell number. The high sensitivity of the luminol chemiluminescent assay was achieved with Mo-ethylenediaminetetraacetate complex and menadione or coenzyme Q1. The detectable cell densities of bacteria and yeasts were found to be approximately several thousand colony-forming units (CFU/ml) when assays were performed with a 96-well microplate luminometer. The chemiluminescent assay requires 10 min for incubation of quinone and microorganisms and 2s for photon counting. Single Escherichia coli was detected after 4h of cultivation and centrifugation (5 min x 2). This simple chemiluminescent assay is expected to be useful for the rapid detection of viable bacteria and yeast.     

Menadione-catalyzed luminol chemiluminescent assay for viability of Mycobacterium bovis

Stable luminol chemiluminescence was observed 10 min after the addition of menadione to a suspension of Mycobacterium bovis homogenized in Middlebrook 7H9 broth base including OADC enrichment. The chemiluminescence intensity was proportional to the absorbance of the bacterial suspension at 600 nm in a range of 0.005 to 0.15. Luminol chemiluminescence disappeared after 10 min incubation of M. bovis at over 60% of ethanol or 4 days of cultivation of M. bovis in the presence of 40 microg/ml of streptomycin. The bacterium showing the disappearance of chemiluminescence could not grow after being washed, suggesting that the inhibition concentration of the antimicrobials can be estimated on the basis of the disappearance of chemiluminescence. Menadione-catalyzed luminol chemiluminescent assay was rapid and sensitive in comparison to turbidimetry, tetrazolium (WST-8) reduction assay, and the assay using the Mycobacteria growth indicator tube (MGIT).     

Difference in antioxidant activity between reduced coenzyme Q9 and reduced coenzyme Q10 in the cell: studies with isolated rat and guinea pig hepatocytes treated with a water-soluble radical initiator.

A possible difference in antioxidant activity between reduced coenzyme Q9 (CoQ9H2) and reduced coenzyme Q10 (CoQ10H2) in animal cells was studied by incubation of hepatocytes with a hydrophilic radical initiator, 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH). Two kinds of hepatocytes differing in their content of CoQ homologs were used: rat, total (oxidized plus reduced) CoQ9: total CoQ10 6:1, guinea pig, 1:5. The sum of total CoQ9 and CoQ10 in rat and guinea-pig hepatocytes was about 780 and 400 pmol/mg protein, respectively. The concentration of CoQ9H2 in rat hepatocytes decreased linearly after the addition of AAPH, whereas that of oxidized CoQ9 showed a reciprocal increase. No loss of cell viability or increase of lipid peroxidation was observed until most of the CoQ9H2 had been consumed. Cellular CoQ9H2 was consumed probably through scavenging of lipid peroxyl radicals produced by incubation with AAPH. On the other hand, CoQ10H2 was not significantly consumed in the AAPH-treated rat hepatocytes during incubation compared with the control cells. In guinea-pig hepatocytes, cellular CoQ10H2 as well as CoQ9H2 was consumed by addition of AAPH. alpha-Tocopherol also showed linear consumption with incubation time regardless of the cell types used. It is concluded that CoQ9H2, together with alpha-tocopherol, constantly acts as a potential antioxidant in hepatocytes when incubated with AAPH, whereas CoQ10H2 mainly exhibits its antioxidant activity in cells containing CoQ10 as the predominant CoQ homolog.     

The reaction of nitric oxide with ubiquinol: kinetic properties and biological significance

The reaction of nitric oxide (*NO) with ubiquinol-0 and ubiquinol-2, short-chain analogs of coenzyme Q, was examined in anaerobic and aerobic conditions in terms of formation of intermediates and stable molecular products. The chemical reactivity of ubiquinol-0 and ubiquinol-2 towards *NO differed only quantitatively, the reactions of ubiquinol-2 being slightly faster than those of ubiquinol-0. The ubiquinol/*NO reaction entailed oxidation of ubiquinol to ubiquinone and reduction of *NO to NO-, the latter identified by its reaction with metmyoglobin to form nitroxylmyoglobin and indirectly by measurement of nitrous oxide (N2O) by gas chromatography. Both the rate of ubiquinone accumulation and *NO consumption were linearly dependent on ubiquinol and *NO concentrations. The stoichiometry of *NO consumed per either ubiquinone formed or ubiquinol oxidized was 1.86 A 0.34. The reaction of *NO with ubiquinols proceeded with intermediate formation of ubisemiquinones that were detected by direct EPR. The second order rate constants of the reactions of ubiquinol-0 and ubiquinol-2 with *NO were 0.49 and 1.6 x 10(4) M(-1)s(-1), respectively. Studies in aerobic conditions revealed that the reaction of *NO with ubiquinols was associated with O2 consumption. The formation of oxyradicals - identified by spin trapping EPR- during ubiquinol autoxidation was inhibited by *NO, thus indicating that the O2 consumption triggered by *NO could not be directly accounted for in terms of oxyradical formation or H2O2 accumulation. It is suggested that oxyradical formation is inhibited by the rapid removal of superoxide anion by *NO to yield peroxynitrite, which subsequently may be involved in the propagation of ubiquinol oxidation. The biological significance of the reaction of ubiquinols with *NO is discussed in terms of the cellular O2 gradients, the steady-state levels of ubiquinols and *NO, and the distribution of ubiquinone (largely in its reduced form) in biological membranes with emphasis on the inner mitochondrial membrane.     

Effect of atorvastatin withdrawal on circulating coenzyme Q10 concentration in patients with hypercholesterolemia

Statin therapy can reduce the biosynthesis of both cholesterol and coenzyme Q10 by blocking the common upstream mevalonate pathway. Coenzyme Q10 depletion has been speculated to play a potential role in statin-related adverse events, and withdrawal of statin is the choice in patients developing myotoxicity or liver toxicity. However, the effect of statin withdrawal on circulating levels of coenzyme Q10 remains unknown. Twenty-six patients with hypercholesterolemia received atorvastatin at 10 mg/day for 3 months. Serum lipid profiles and coenzyme Q10 were assessed before and immediately after 3 months and were also measured 2 and 3 days after the last day on the statin. After 3 months' atorvastatin therapy, serum levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and coenzyme Q10 (0.43 +/- 0.23 to 0.16 +/- 0.10 microg/mL) were all significantly reduced (all p<0.001). On day 2 after the last atorvastatin, the coenzyme Q10 level was significantly elevated (0.37 +/- 0.16 microg/mL) and maintained the same levels on day 3 (0.39 +/- 0.18 microg/mL) compared with those on month 3 (both p< 0.001), while TC and LDL-C did not significantly change within the same 3 days. These results suggest that statin inhibition of coenzyme Q10 synthesis is less strict than inhibition of cholesterol biosynthesis.     

Protection of Rat Myocardium by Coenzyme Q during Oxidative Stress Induced by Hydrogen Peroxide

Ubiquinone Q(10) (coenzyme Q) is an important component of the mitochondrial electron transport chain and an antioxidant. The purpose of this work was to find out whether an increase in the level of coenzyme Q in the heart changes its maximal working capacity and resistance to oxidative stress. Male Wistar rats were treated with coenzyme Q (10 mg/kg body weight per day) for six weeks, and this increased its content in the myocardium by 63%. The myocardial content of malonic dialdehyde and activities of key antioxidant enzymes were unchanged, except nearly 2.5-fold decrease in the activity of superoxide dismutase. The maximal working capacity of the isolated isovolumic heart did not change, but under conditions of oxidative stress induced by 45-min infusion of hydrogen peroxide (70 micro M) into coronary vessels the contractile function of these hearts decreased significantly more slowly. This was associated with less pronounced lesions in the ultrastructure of cardiomyocytes and lesser disorders in the oxidative metabolism of mitochondria that suggested increased antioxidant protection of the myocardium.     

Suppression of monocyte oxidative response by phenolic glycolipid I of Mycobacterium leprae

Mycobacterium leprae synthesizes a unique phenolic glycolipid (PGL-I) in abundant quantities. We studied the effect of PGL-I on the generation of superoxide anion (O2-) by stimulated human monocytes. Peripheral blood monocytes pretreated with PGL-I released less O2- when stimulated with M. leprae than did control monocytes. Monocytes pretreated with dimycocerosyl phthiocerol, mycoside A of Mycobacterium kansasii, or mycoside B of Mycobacterium microti, on the other hand, released O2- in quantities comparable to control monocytes in response to M. leprae stimulation. Monocyte O2- release in response to other stimuli of the oxidative metabolic burst, such as PMA, zymosan, Mycobacterium bovis Bacille Calmette-Guérin, or M. kansasii, was unaffected by lipid pretreatment. These findings demonstrate that PGL-I has a direct effect on monocyte O2- generation in response to M. leprae and suggest that PGL-I is a modulator of phagocytic cell function.     

Tumor necrosis factor alpha stimulates mycobactericidal/mycobacteriostatic activity in human macrophages by a protein kinase C-independent pathway.

Tumor necrosis factor (TNF) is a 17-kDa protein produced by endotoxin-stimulated macrophages. We have demonstrated that recombinant human TNF activates human macrophages to kill intracellular bacteria of the Mycobacterium avium complex (MAC) in a dose-related manner. TNF also primed macrophages to produce superoxide anion (O2-) following treatment with phorbol esther PMA (0.1 micrograms/ml). To investigate the intracellular pathway involved in the TNF-mediated activation of mycobacteriostatic/mycobactericidal activity in macrophages, we used two different protein kinase C (PKC) inhibitors: H7 (10(-5)-10(7) M) and staurosporine (10(-7)-10(-9) M). Mellitin (1 and 100 mM) was used as a calmodulin inhibitor. Human peripheral blood-derived macrophages cultured for 7 days were treated with H7, mellitin, or staurosporine for 1 hr prior to incubation with TNF (10(3) U/ml). Twenty-four hours after treatment with TNF the O2- release was measured spectrophotometrically following exposure to PMA. Macrophages were infected with MAC and the viable intracellular bacilli were quantitated following 4 days of treatment with TNF. All PKC inhibitors suppressed O2- production after incubation with PMA. However, treatment with either PKC or calmodulin inhibitors did not influence the intracellular killing of M. avium by TNF-stimulated macrophages. Exposure of the macrophages to cGMP inhibitor but not to cAMP inhibitor significantly impaired the response to the stimulation with TNF. In contrast, incubation of macrophages with protein kinase A (PKA) had no effect on TNF-mediated mycobacteriostatic/mycobactericidal activity. These results suggest that the TNF-mediated mycobactericidal activity in cultured macrophages probably occurs by a PKC-independent mechanism.     

Effect of coenzyme Q(10) and alpha-tocopherol content of mitochondria on the production of superoxide anion radicals.

The effects of coenzyme Q(10) (CoQ(10)) and alpha-tocopherol on the rate of mitochondrial superoxide anion radical (O2(./-)) generation were examined in skeletal muscle, liver, and kidney of 24-month-old mice. Mice were orally administered alpha-tocopherol (200 mg.kg(-1).day(-1)) alone, CoQ(10) (123 mg.kg(-1).day(-1)) alone, or the two together for 13 wk. Administration of alpha-tocopherol resulted in an approximately sevenfold elevation of mitochondrial alpha-tocopherol content. Intake of CoQ(10) alone caused an approximately fivefold increase in CoQ content (CoQ(9) and/or CoQ(10)) and alpha-tocopherol of mitochondria. The rate of (O2(./-)) generation by submitochondrial particles (SMPs) was inversely related to their alpha-tocopherol content but unrelated to CoQ content. Experimental in vitro augmentation of SMPs with varying amounts of alpha-tocopherol caused an up to approximately 50% decrease in the rate of O2(./-) generation. Similar in vitro augmentations of SMPs with CoQ(10) had previously been found to have no effect on the rate of O2(./-) generation The CoQ(10)-induced elevation of alpha-tocopherol in the present study was inferred to be due to a 'sparing/regeneration' by CoQ. Results indicate the involvement of alpha-tocopherol in the elimination of mitochondrially generated O2(./-)     

Superoxide anion and hydrogen peroxide metabolism in soybean embryonic axes during germination.

The total rate of mitochondrial O2- production in the presence of NADH as substrate increased from 200 to 1340 pmol/min per axis between 2 and 30 h of imbibition. The activities of the enzymes involved in hydroperoxide metabolism, e.g., superoxide dismutase, catalase, peroxidase and glutathione and ascorbate peroxidases, markedly changed during the germination of soybean embryonic axes. Superoxide dismutase was the enzymatic activity affected the most during the initial stages of germination. Intracellular O2- steady-state concentration, calculated from the rate of O2- production and superoxide dismutase activity, showed a 2-fold increase from 2 x 10(-8) M to 4 x 10(-8) M in germination phase I, declined in phase II to 2 x 10(-8) M and remained constant over the rest of the incubation period. The reaction of H2O2 and luminol catalyzed by Co2+ was utilized to measure H2O2 diffused out of the soybean axes after 5 to 10 min of incubation. The catalase-sensitive luminol emission of diffusates prepared from axes previously imbibed from 2 to 30 h corresponded to a H2O2 intracellular steady-state concentration in the range of 0.3 to 0.9 microM. The activity of metal-containing antioxidant enzymes was determined in the extracellular fluid. Cell wall peroxidase activity increased from 10 to 300 mumol/min per mg protein and appears as a potentially important pathway for H2O2 utilization. Hydrogen peroxide metabolism in soybean embryonic axes during early inhibition appears to have the following main features: (a) mitochondrial membranes are the most important source of cytosolic O2- and H2O2; (b) H2O2 is regulated at a steady-state concentration of 0.3-0.9 microM; (c) catalase is the main enzyme in terms of H2O2 utilization; (d) H2O2 exo-diffusion is quantitatively important destiny of intracellular H2O2; and (e) extracellular peroxidase located at the cell wall affords an enzymatic system able to use diffused H2O2.