- Email: [email protected]
Pyruvate but not lactate prevents NADH-induced myoglobin oxidation
Free Radical Biology & Medicine 38 (2005) 1484 – 1490 www.elsevier.com/locate/freeradbiomed
Original Contribution
Pyruvate but not lactate prevents NADH-induced myoglobin oxidation Robert A. Oleka, Jedrzej Antosiewicza, Jerzy Popinigisa, Rosita Gabbianellib, Donatella Fedelib, Giancarlo Falcionib,T a
Department of Bioenergetics, Jedrzej Sniadecki University School of Physical Education, Wiejska 1, 80-336 Gdansk, Poland b Department of Biology MCA, University of Camerino, Via F. Camerini 2, I-62032 Camerino, Italy Received 15 June 2004; revised 8 February 2004; accepted 8 February 2004 Available online 25 March 2005
Abstract In this work, we investigated the influence of NADH on the redox state of myoglobin and the roles of pyruvate and lactate in this process. NADH increased the autoxidation rate of myoglobin. Both a drop in pH and partial deoxygenation markedly stimulated the autoxidation process and the influence of NADH. A correlation between met-Mb formation rate and NADH oxidation rate was always observed. The increased rate of Mb autoxidation caused by NADH was inhibited by catalase and pyruvate but not by l-lactate. The antioxidant activity versus H2O2 of both pyruvate and lactate was evidenced by chemiluminescence experiments. The antioxidant activity of lactate disappeared completely in the presence of myoglobin or apo-myoglobin, whereas it was only reduced for pyruvate. These results could be of interest in preventing autoxidation of myoglobin that can contribute to ischemia–reperfusion injury during infarction or high–intensity exercise. D 2005 Elsevier Inc. All rights reserved. Keywords: Myoglobin; NADH; Pyruvate; l-lactate; Catalase; ROS; Luminol; Chemiluminescence; Free radicals
Introduction Myoglobin, found primarily in cardiac and red skeletal muscles and particularly abundant in diving mammals, is a respiratory hemoprotein able (in the ferrous form) to reversibly bind molecular oxygen. The two purported functions of myoglobin (Mb) are that of an O2 reserve and of facilitating O2 diffusion from capillaries to mitochondria where O2 is used for cellular respiration. The ferrous form of Mb is required for reversible oxygenation to occur. Recently, it was proposed that ferrous myoglobin is also an intracellular scavenger of bioactive nitric oxide, regulating its level in the skeletal and cardiac muscles [1,2]. Although oxy and deoxy-Mb can turn, during turnover time that exceeds several months, in to the inactive ferric form, the level of the oxidized form of the protein is in vivo kept low by enzymatic and nonenzymatic processes involving
T Corresponding author. Fax: +39 0737 636216. E-mail address: [email protected] (G. Falcioni). 0891-5849/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2005.02.018
reduced pyridine nucleotides. NADH can reduce met-Mb to its oxy form in the presence of metmyoglobin reductase or electron carriers. NADH can also act as an electron donor for some reactive oxygen species at a relatively high rate. Myoglobin has been implicated in several types of muscle oxidative damage [3] through a mechanism entailing the high oxidation state of the hemoprotein. The autoxidation of myoglobin to metmyoglobin is associated with the formation of superoxide anion radical and thereby of products such as hydrogen peroxide or hydroxyl radicals, which can be derived from O2 itself [4,5]. This process may have a significant importance especially in the heart where myoglobin is present at elevated concentrations (up to 0.5 mM) [6]. In this ambit, the study of the mechanisms involved to prevent enhancement of the autoxidation of myoglobin could be of certain interest. Recently, we reported that the autoxidation rate of human hemoglobin (Hb) is increased by NADH [7] and the destabilization of HbA is accompanied by the oxidation of the cofactor. Now we have reinvestigated the system using myoglobin. In the muscle the ratio between NADH and hemoprotein is higher
R.A. Olek et al. / Free Radical Biology & Medicine 38 (2005) 1484–1490
with respect to erythrocytes; in addition the monomeric protein (Mb) is less stable than tetrameric HbA. NADH in vivo is continuously regenerated by a variety of enzymatic reactions and its concentration changes under different physiological and pathological conditions. An increase in the cytosolic NADH content has been reported in cardiac muscle as a consequence of ischemia and in skeletal muscle after intensive exercise [8,9]. Strenuous exercise or infarction is associated with myoglobin deoxygenation [10,11]. The impacts of pyruvate and l-lactate on cytotoxicity due to cardiac ischemia–reperfusion reactive oxygen species (ROS) have been reported to be different [12–14]. Bearing this in mind, we investigated the influence of NADH on the redox state of myoglobin and the role of pyruvate and lactate in this process.
1485
in our laboratory for the determination of the oxygendissociation curves. Apo-myoglobin was prepared using the acid–acetone method [15]. NADH oxidation was monitored spectrophotometrically at 340 nm. Chemiluminescence experiments to study the interactions of pyruvate and lactate with H2O2 were performed using an Autolumat Berthold LB953 (Berthold Co., Wilbard, Germany). A reaction mixture containing 0.4 mg/ml globin, different concentrations of pyruvate or lactate, 100 AM luminol, in 1 ml of 50 mM phosphate buffer, pH 7.0, was prepared. The reaction was initiated by injecting 0.05 ml of H2O2 (final concentration 50 mM). When the experiment was done in the presence of 0.4 mg/ml myoglobin the reaction mixture did not contain luminol because the hemoprotein gives a high luminescence efficiency [16].
Materials and methods Horse heart myoglobin, pyruvate, lactate, ascorbate, and NADH as well as the enzyme catalase (CAT; EC 1.11.1.6) were purchased from Sigma–Aldrich. Myoglobin was reduced by the addition of dithionite and the excess was removed by the passage of the solution through a Sephadex G-25 column equilibrated with 50 mM phosphate buffer, pH 7.0. The rate of met-Mb formation was followed during incubation at 378C with a Cary 219 spectrophotometer in the visible region. Changes in the absorption spectrum ranging from 500 to 700 nm were recorded as a function of time, and reference value for complete oxidation was estimated by addition of potassium ferricyanide. Partial deoxygenation of myoglobin was obtained by removing oxygen due to the exposure of oxymyoglobin to the vacuum. To perform this last experiment we used a tonometer (a modified Thunberg tube) routinely employed
Results The effects of increasing amounts of NADH on the kinetics of oxymyoglobin autoxidation were investigated. The time course of myoglobin autoxidation during incubation at 378C and pH 7.0 is reported in Fig. 1. It appears that NADH has a concentration-dependent accelerating effect on the autoxidation. After 30 min of incubation metmyoglobin reaches 7.2% in the control and 22.7% in the presence of 0.5 mM NADH. Ferryl (Fe4+) species were not detectable using the Whitburn equation [17] during the experimental time. Oxy-Mb was monitored according to Whitburn and its decrease correlated with the met-Mb formation. Both a drop in pH and partial deoxygenation markedly stimulate the autoxidation process and the influence of NADH. At lower pH values the effects of NADH on the
Fig. 1. Effects of NADH on the time course of myoglobin autoxidation. (x) Control, (o) 0.1 mM NADH, (D) 0.3 mM NADH, (5) 0.5 mM NADH. Myoglobin (50 AM) was incubated in 50 mM phosphate buffer, pH 7.0, at 378C. Data are from one representative set of tracings from a total of three experiments.
1486
R.A. Olek et al. / Free Radical Biology & Medicine 38 (2005) 1484–1490
Fig. 2. Effects of pH on metmyoglobin formation in the absence and in the presence of 0.1 and 0.3 mM NADH. Myoglobin (50 AM) was incubated for 30 min in 50 mM phosphate buffer at 378C. (n) pH 7.0, (E) pH 6.8, (.) pH 6.6, (x) pH 6.4. Data are from one representative set of tracings from a total of three experiments.
autoxidation of myoglobin increase (Fig. 2). After 30 min of incubation at pH 7.0 we measured 7.2% of met-Mb in the absence of NADH and this parameter reached 17.8% when the experiment was performed in the presence of 0.3 mM NADH. The increasing effect of NADH on met-Mb formation rate was more marked at pH 6.4. At this pH value and always after 30 min of incubation, we obtained 13.4 and 34.9%, respectively, in the absence and in the presence of 0.3 mM NADH. Fig. 3 shows the oxidation process of myoglobin at different oxygen fractional saturations in the presence and in the absence of 0.1 mM NADH. The metmyoglobin formation rate increases with the deoxygenation and the effect of
NADH seems to be more pronounced at lower oxygenation values. The autoxidation of MbO2 in the presence of catalase, the enzyme that metabolyzes hydrogen peroxide, drops below the control level both in the presence and in the absence of NADH. The effect of catalase is ameliorated in the presence of NADH; thus MbO2 autoxidation in the presence of catalase and NADH is slowest (Fig. 4). The presence of 0.1 Amol/L SOD under our experimental conditions does not influence the autoxidation rate of MbO2, but it reduces the effect due to the presence of 0.1 mmol/L NADH (data not shown). Similar experiments were performed in the presence of 5 mM pyruvate or lactate (Fig. 5). Also the well-
Fig. 3. Autoxidation of myoglobin at different fractional oxygen saturations in the presence and absence of NADH. (x) Control, (5) 0.1 mM NADH. Myoglobin (50 AM) was incubated for 30 min in 50 mM phosphate buffer, pH 7.0, at 378C. Data are from one representative set of tracings from a total of three experiments.
R.A. Olek et al. / Free Radical Biology & Medicine 38 (2005) 1484–1490
1487
Fig. 4. Inhibition of met-Mb formation rate by catalase (0.1 AM) in the presence and absence of NADH. (x) Control, (o) 0.1 AM CAT, (D) 0.3 mM NADH, (5) 0.1 AM CAT + 0.3 mM NADH. All other conditions are as for Fig. 1. Data are from one representative set of tracings from a total of three experiments.
known ascorbate was included in these experiments (data not shown). The presence of 0.3 mM ascorbate nullifies the effect due to the presence of NADH on MbO2 oxidation. Under our experimental conditions, the presence of pyruvate markedly reduces the stimulating effect of NADH on MbO2 autoxidation, whereas lactate has no effect (Fig. 5). The autoxidation of MbO2 does not change when we add pyruvate or lactate to the control (data not shown). In all the processes described involving NADH, we followed its oxidation (data not shown). We observed a correlation between the met-Mb formation rate induced by NADH and NADH oxidation rate; obviously catalase and pyruvate, which reduce the effects of NADH on the met-Mb formation rate, inhibit NADH oxidation.
Additional experiments were done to solve issues on the mechanism of pyruvate protection and the role of lactate. By using the chemiluminescence technique we evaluated the antioxidant activity of pyruvate and lactate in the presence and absence of myoglobin. Chemiluminescence (CL) determinations were performed using luminol as the chemiluminogenic probe for H2O2. The luminol-amplified chemiluminescence of hydrogen peroxide was reduced by the presence of both pyruvate and lactate. In Fig. 6 is reported the percentage inhibition in the luminol-CL signal at pH 7.0 when different concentrations of pyruvate or lactate were added. Fifty percent of inhibition was obtained using about 30 and 60 mM for pyruvate and lactate, respectively; the same inhibition was obtained in the
Fig. 5. Effects of 5 mM pyruvate or lactate on metmyoglobin formation in the presence and absence of NADH. (x) Control, (.) 0.5 mM NADH, (5) 0.5 mM NADH + 5 mM pyruvate, (D) 0.5 mM NADH + 5 mM lactate. All other conditions are as for Fig. 1. Data are from one representative set of tracings from a total of three experiments.
1488
R.A. Olek et al. / Free Radical Biology & Medicine 38 (2005) 1484–1490
Fig. 6. Inhibition of luminol-amplified chemiluminescence (%) by pyruvate () or lactate (x) in the reaction mixture containing 100 AM luminol, 50 mM H2O2 in 50 mM phosphate buffer, pH 7.0. Data represent the means F SD of three different experiments performed in triplicate.
presence of only 5 AM ascorbate. A decrease in activity versus H2O2 was present at lower pH values (data not shown). The chemiluminescence data indicate that both pyruvate and lactate (although less marked for lactate) have antioxidant activity. This property does not explain the different effects that they have on the NADH-induced myoglobin oxidation reported in Fig. 5. Therefore, we performed CL experiments in the presence of Mb or apomyoglobin (myoglobin without heme) (Fig. 7). When experiments were performed in the presence of myoglobin or apo-Mb the addition of lactate did not change
the CL signal, whereas the addition of pyruvate produced a reduction of the CL signal (Fig. 7). In other words the antioxidant activity of lactate (under our experimental conditions) versus hydrogen peroxide disappears completely in the presence of myoglobin or apo-Mb, whereas it is only reduced for pyruvate. Fifty percent of inhibition in the presence of myoglobin or apomyoglobin was obtained with 80 and 160 mM of pyruvate, respectively. The antioxidant activity of ascorbate was reduced in the presence of the same Mb concentration (50% was obtained with 1 mM ascorbate).
Fig. 7. Inhibition of chemiluminescence (%) by pyruvate or lactate in the absence or presence of 0.4 mg/ml myoglobin or apomyoglobin. The reaction mixture was the same as for Fig. 6. Luminol was not added for the experiments involving Mb. (x) Pyruvate, (x) Mb + pyruvate, (E) apo-Mb + pyruvate, (o) Mb + lactate; (D) apo-Mb + lactate. Data represent the means F SD of three different experiments performed in triplicate.
R.A. Olek et al. / Free Radical Biology & Medicine 38 (2005) 1484–1490
Discussion The reduced blood flow observed in the heart due to arterial occlusion or partial ischemia in skeletal muscle during high-intensity exercise leads to an increase in concentration of both NADH and deoxymyoglobin [8–11]. Under this condition, energy production comes from the anaerobic system. Due to acceleration of glycolysis, lactate begins to accumulate and the pH in the cells decreases [18]. Several sources of ROS associated with myocardial ischemia–reperfusion may exist. Autoxidation of various hemoproteins such as myoglobin is one of the possible sources of ROS during ischemia–reperfusion [3]. Autoxidation of myoglobin has been shown to result in the formation of superoxide and hydrogen peroxide. Because the concentration of myoglobin is high in heart (up to 0.5 mM) the autoxidation process should be considered an important source of ROS. Therefore factors that stimulate the process may have substantial influence on heart damage during ischemia and reperfusion. It is known that partial deoxygenation and low pH strongly stimulate the autoxidation process of MbO2. In this study, we observed that NADH stimulates met-Mb formation and that this process is accelerated at low pH. Despite being a coenzyme in many enzymatic reactions, NADH can also react with various reactive oxygen species and the reactivity increases when it is bound to proteins (i.e., lactate dehydrogenase [19] or glyceraldehyde-3-phosphate dehydrogenase [20]) and recently it was claimed to be directly operating as an antioxidant [21]. However, it should be mentioned that oneelectron oxidation of NADH leads to the formation of NAD radical, which at a diffusion-controlled rate reacts with oxygen yielding superoxide [19]. If we consider that under certain conditions, like strenuous exercise and myocardial infarct, the pH drops to 6.3 or less and NADH concentration increases, the level of metmyoglobin could increase. In parallel we observed that NADH is oxidized in the presence of MbO2, thus the superoxide anion radical produced is due to both oxymyoglobin and NADH oxidation. Catalase inhibits met-Mb formation both in the presence and in the absence of NADH, suggesting that hydrogen peroxide stimulates this process. These results are in agreement with a previously published report in which it was observed that catalase inhibits the autoxidation process of myoglobin [3]. In addition, we reported [7] a similar behavior with hemoglobin A; the presence of NADH accelerates the met-Hb formation, the presence of SOD reduces the met-Hb formation rate only in the presence of NADH, and finally the autoxidation of HbA in the presence of catalase drops below the control level both in the presence and in the absence of NADH. Obviously the oxidation rate of tetrameric HbA is less with respect to monomeric Mb. Our data indicate that hydrogen peroxide substantially contributes to heme (Fe2+) oxidation and NADH stimulates the autoxidation process with the involvment of hydrogen peroxide. It is worth noting that protection of Mb
1489
autoxidation by catalase (Fig. 4) is much stronger in the presence of NADH, hence this process may be of physiological importance. It is well documented that catalase activity in heart or red muscle is much higher than in white muscle and that it increases in muscle after strenuous exercise [22–24]. Therefore it may be expected that a rise in the intracellular concentration of NADH accompanied by high catalase activity may protect Mb autoxidation and cell damage. It may also be possible that acceleration of Mb autoxidation by high NADH concentration and drop in pH can overlap catalase protection. Significant attention has been devoted to the production of protein-derived radicals as a result of the reaction of hydrogen peroxide with hemoproteins. The reaction of H2O2 with metmyoglobin results in the formation of a shortlived intermediate with one oxidizing equivalent above that in metmyoglobin (the iron is converted to the iron(IV)–oxo state), and one oxidizing equivalent is on a tyrosine residue on the surface of the globin protein [25–27]. These radicals, or further species arising from them, have been shown to undergo a number of reactions [25–28] that may have considerable pathological consequences. Therefore the absence of ferryl (Fe4+) during incubation of myoglobin with NADH as reported in this study could be significant. There are several reports demonstrating that pyruvate infusion during heart reperfusion has a strong protective effect on heart muscle function and structure [12–14]. It has been also reported that pyruvate reduces the in vitro toxicity of myoglobin in renal cortical slices [29] with a mechanism in part due to its antioxidant capacity. In our experiments, inhibition of MbO2 autoxidation by pyruvate was observed in the presence of NADH. This effect of pyruvate depends on its ability to react nonenzymatically with H2O2 yielding CO2 as a by-product. However, under in vivo conditions, the effect of pyruvate may be more complex. The redox state of cytoplasmic NADH closely correlates with the tissue pyruvate to llactate concentration ratio. In reperfused heart, pyruvate may be expected to inhibit myoglobin autoxidation by lowering hydrogen peroxide and NADH concentrations, the latter via the reaction catalyzed by LDH. Lactate under our conditions has no effect on MbO2 autoxidation, but certainly in vivo it should stimulate the process by increasing NADH concentration. In fact it was shown that the formation of ROS in reperfused hearts is augmented by lactate [30] despite some claims that it can have a direct antioxidant activity [31,32]. Chemiluminescence studies reported here (Fig. 7) show that the antioxidant activity versus hydrogen peroxide of lactate and pyruvate is influenced in a different manner by the presence of Mb or apo-Mb. The antioxidant activity of lactate disappears completely in the presence of the protein; the comparison of experiments in the presence of Mb or apo-Mb suggests that the presence of heme increases the antioxidant activity of pyruvate. In summary, the increased rate of Mb autoxidation caused by a low pH, an increase in
1490
R.A. Olek et al. / Free Radical Biology & Medicine 38 (2005) 1484–1490
NADH concentration, and Mb deoxygenation correlates with an increase in ROS formation that can be inhibited by catalase and pyruvate but not by l-lactate. The data reported here could be of interest from the biochemical point of view because the autoxidation of myoglobin can contribute to ischemia–reperfusion injury during infarction or high-intensity exercise. To prevent enhancement of the autoxidation of myoglobin reaction may prove useful.
[15]
[16]
[17]
[18]
References [19] [1] Brunori, M. Nitric oxide moves myoglobin centre stage. Trends Biochem. Sci. 26:209 – 210; 2001. [2] Brunori, M. Nitric oxide, cytochrome-c oxidase and myoglobin. Trends Biochem. Sci. 26:21 – 23; 2001. [3] Gunther, M. R.; Sampath, V.; Caughey, W. S. Potential roles of myoglobin autoxidation in myocardial ischemia–reperfusion injury. Free Radic. Biol. Med. 26:1388 – 1395; 1999. [4] Misra, H. P.; Fridovich, I. The generation of superoxide radical during the autoxidation of hemoglobin. J. Biol. Chem. 247:6960 – 6962; 1972. [5] Brantley, R. E., Jr.; Smerdon, S. J.; Wilkinson, A. J.; Singleton, E. W.; Olson, J. S. The mechanism of autooxidation of myoglobin. J. Biol. Chem. 268:6995 – 7010; 1993. [6] Sylven, C.; Jansson, E.; Book, K. Myoglobin content in human skeletal muscle and myocardium: relation to fibre size and oxidative capacity. Cardiovasc. Res. 18:443 – 446; 1984. [7] Olek, R. A.; Antosiewicz, J.; Caulini, G. C.; Falcioni, G. Effect of NADH on the redox state of human hemoglobin. Clin. Chim. Acta 324:129 – 134; 2002. [8] Neely, J. R.; Grotyohann, L. W. Role of glycolytic products in damage to ischemic myocardium: dissociation of adenosine triphosphate levels and recovery of function of reperfused ischemic hearts. Circ. Res. 55:816 – 824; 1984. [9] Sahlin, K.; Katz, A.; Henriksson, J. Redox state and lactate accumulation in human skeletal muscle during dynamic exercise. Biochem. J. 245:551 – 556; 1987. [10] Richardson, R. S.; Newcomer, S. C.; Noyszewski, E. A. Skeletal muscle intracellular PO2 assessed by myoglobin desaturation: response to graded exercise. J. Appl. Physiol. 91:2679 – 2685; 2001. [11] Murakami, Y.; Zhang, Y.; Cho, Y. K.; Mansoor, A. M.; Chung, J. K.; Chu, C.; Francis, G.; Ugurbil, K.; Bache, R. J.; From, A. H.; Jerosch– Herold, M.; Wilke, N.; Zhang, J. Myocardial oxygenation during high work states in hearts with postinfarction remodeling. Circulation 99:942 – 948; 1999. [12] Mallet, R. T. Pyruvate: metabolic protector of cardiac performance. Proc. Soc. Exp. Biol. Med. 223:136 – 148; 2000. [13] Mallet, R. T.; Squires, J. E.; Bhatia, S.; Sun, J. Pyruvate restores contractile function and antioxidant defenses of hydrogen peroxide– challenged myocardium. J. Mol. Cell Cardiol. 34:1173 – 1184; 2002. [14] Bassenge, E.; Sommer, O.; Schwemmer, M.; Bqnger, R. Antioxidant pyruvate inhibits cardiac formation of reactive oxygen species through
changes in redox state. Am. J. Physiol. Heart Circ. Physiol. 279: H2431 – H2438; 2000. Ascoli, F.; Fanelli, M. R.; Antonini, E. Preparation and properties of apohemoglobin and reconstituted hemoglobins. Methods Enzymol. 76:72 – 87; 1981. Gabbianelli, R.; Falcioni, G.; Santroni, A. M.; Fiorini, R.; Coppa, G. V.; Kantar, A. Interaction of trout hemoglobin with H2O2: a chemiluminescence study. J. Biolumin. Chemilumin. 12:79 – 85; 1997. Whitburn, K. D.; Shieh, J. J.; Sellers, R. M.; Hoffman, M. Z. Redox transformations in ferrimyoglobin induced by radiation–generated free radicals in aqueous solution. J. Biol. Chem. 257:1860 – 1869; 1982. Sahlin, K.; Harris, R. C.; Nylind, B.; Hultman, E. Lactate content and pH in muscle obtained after dynamic exercise. Pflugers Archiv. 367:143 – 149; 1976. Bielski, B. H. J.; Chan, P. C. Kinetic study by pulse radiolysis of the lactate dehydrogenase–catalyzed chain oxidation of nicotinamide adenine dinucleotide by HO2 and O2 radicals. J. Biol. Chem. 250:318 – 321; 1975. Chan, P. C.; Bielski, B. H. J. Glyceraldehyde–3–phosphate deydrogenase–catalyzed chain oxidation of reduced nicotinamide adenine dinucleotide by perhydroxyl radicals. J. Biol. Chem. 255:874 – 876; 1980. Kirsch, M.; De Groot, H. NAD(P)H, a directly operating antioxidant? FASEB J. 15:1569 – 1574; 2001. Ji, L. L.; Fu, R.; Mitchell, E. W. Glutathione and antioxidant enzymes in skeletal muscle: effect of fiber type and exercise intensity. J. Appl. Physiol. 73:1854 – 1859; 1992. Ji, L. L.; Fu, R. Responses of glutathione system and antioxidant enzymes to exhaustive exercise and hydroperoxide. J. Appl. Physiol. 72:549 – 554; 1992. Ji, L. L. Oxidative stress during exercise: implication of antioxidant nutrients. Free Radic. Biol. Med. 18:1079 – 1086; 1995. Tew, D.; Ortiz de Montellano, P. R. The myoglobin protein radical: coupling of Tyr–103 to Tyr–151 in the H2O2–mediated cross–linking of sperm whale myoglobin. J. Biol. Chem. 263:17880 – 17886; 1988. Davies, M. J. Identification of a globin free radical in equine myoglobin treated with peroxides. Biochim. Biophys. Acta 1077: 86 – 90; 1991. Newman, E. S.; Rice–Evans, C. A.; Davies, M. J. Identification of initiating agents in myoglobin–induced lipid peroxidation. Biochem. Biophys. Res. Commun. 179:1414 – 1419; 1991. Catalano, C. E.; Choe, Y. S.; Ortiz de Montellano, P. R. Reactions of the protein radical in peroxide–treated myoglobin: formation of a heme–protein cross–link. J. Biol. Chem. 264:10534 – 10541; 1989. Valentovic, M. A.; Minigh, J. Pyruvate attenuates myoglobin in vitro toxicity. Toxicol. Sci. 74:345 – 351; 2003. Mohazzab-H, K. M.; Kaminski, P. M.; Wolin, M. S. Lactate and PO2 modulate superoxide anion production in bovine cardiac myocytes: potential role of NADH oxidase. Circulation 96:614 – 620; 1997. Yanagida, S.; Luo, C. S.; Doyle, M.; Pohost, G. M.; Pike, M. M. Nuclear magnetic resonance studies of cationic and energetic alterations with oxidant stress in the perfused heart. Modulation with pyruvate and lactate. Circ. Res. 77:773 – 783; 1995. Groussard, C.; Morel, I.; Chevanne, M.; Monnier, M.; Cillard, J.; Delamarche, A. Free radical scavenging and antioxidant effects of lactate ion: an in vitro study. J. Appl. Physiol. 89:169 – 175; 2000.
S
[20]
[21] [22]
[23]
[24] [25]
[26]
[27]
[28]
[29] [30]
[31]
[32]
Original Contribution
Pyruvate but not lactate prevents NADH-induced myoglobin oxidation Robert A. Oleka, Jedrzej Antosiewicza, Jerzy Popinigisa, Rosita Gabbianellib, Donatella Fedelib, Giancarlo Falcionib,T a
Department of Bioenergetics, Jedrzej Sniadecki University School of Physical Education, Wiejska 1, 80-336 Gdansk, Poland b Department of Biology MCA, University of Camerino, Via F. Camerini 2, I-62032 Camerino, Italy Received 15 June 2004; revised 8 February 2004; accepted 8 February 2004 Available online 25 March 2005
Abstract In this work, we investigated the influence of NADH on the redox state of myoglobin and the roles of pyruvate and lactate in this process. NADH increased the autoxidation rate of myoglobin. Both a drop in pH and partial deoxygenation markedly stimulated the autoxidation process and the influence of NADH. A correlation between met-Mb formation rate and NADH oxidation rate was always observed. The increased rate of Mb autoxidation caused by NADH was inhibited by catalase and pyruvate but not by l-lactate. The antioxidant activity versus H2O2 of both pyruvate and lactate was evidenced by chemiluminescence experiments. The antioxidant activity of lactate disappeared completely in the presence of myoglobin or apo-myoglobin, whereas it was only reduced for pyruvate. These results could be of interest in preventing autoxidation of myoglobin that can contribute to ischemia–reperfusion injury during infarction or high–intensity exercise. D 2005 Elsevier Inc. All rights reserved. Keywords: Myoglobin; NADH; Pyruvate; l-lactate; Catalase; ROS; Luminol; Chemiluminescence; Free radicals
Introduction Myoglobin, found primarily in cardiac and red skeletal muscles and particularly abundant in diving mammals, is a respiratory hemoprotein able (in the ferrous form) to reversibly bind molecular oxygen. The two purported functions of myoglobin (Mb) are that of an O2 reserve and of facilitating O2 diffusion from capillaries to mitochondria where O2 is used for cellular respiration. The ferrous form of Mb is required for reversible oxygenation to occur. Recently, it was proposed that ferrous myoglobin is also an intracellular scavenger of bioactive nitric oxide, regulating its level in the skeletal and cardiac muscles [1,2]. Although oxy and deoxy-Mb can turn, during turnover time that exceeds several months, in to the inactive ferric form, the level of the oxidized form of the protein is in vivo kept low by enzymatic and nonenzymatic processes involving
T Corresponding author. Fax: +39 0737 636216. E-mail address: [email protected] (G. Falcioni). 0891-5849/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2005.02.018
reduced pyridine nucleotides. NADH can reduce met-Mb to its oxy form in the presence of metmyoglobin reductase or electron carriers. NADH can also act as an electron donor for some reactive oxygen species at a relatively high rate. Myoglobin has been implicated in several types of muscle oxidative damage [3] through a mechanism entailing the high oxidation state of the hemoprotein. The autoxidation of myoglobin to metmyoglobin is associated with the formation of superoxide anion radical and thereby of products such as hydrogen peroxide or hydroxyl radicals, which can be derived from O2 itself [4,5]. This process may have a significant importance especially in the heart where myoglobin is present at elevated concentrations (up to 0.5 mM) [6]. In this ambit, the study of the mechanisms involved to prevent enhancement of the autoxidation of myoglobin could be of certain interest. Recently, we reported that the autoxidation rate of human hemoglobin (Hb) is increased by NADH [7] and the destabilization of HbA is accompanied by the oxidation of the cofactor. Now we have reinvestigated the system using myoglobin. In the muscle the ratio between NADH and hemoprotein is higher
R.A. Olek et al. / Free Radical Biology & Medicine 38 (2005) 1484–1490
with respect to erythrocytes; in addition the monomeric protein (Mb) is less stable than tetrameric HbA. NADH in vivo is continuously regenerated by a variety of enzymatic reactions and its concentration changes under different physiological and pathological conditions. An increase in the cytosolic NADH content has been reported in cardiac muscle as a consequence of ischemia and in skeletal muscle after intensive exercise [8,9]. Strenuous exercise or infarction is associated with myoglobin deoxygenation [10,11]. The impacts of pyruvate and l-lactate on cytotoxicity due to cardiac ischemia–reperfusion reactive oxygen species (ROS) have been reported to be different [12–14]. Bearing this in mind, we investigated the influence of NADH on the redox state of myoglobin and the role of pyruvate and lactate in this process.
1485
in our laboratory for the determination of the oxygendissociation curves. Apo-myoglobin was prepared using the acid–acetone method [15]. NADH oxidation was monitored spectrophotometrically at 340 nm. Chemiluminescence experiments to study the interactions of pyruvate and lactate with H2O2 were performed using an Autolumat Berthold LB953 (Berthold Co., Wilbard, Germany). A reaction mixture containing 0.4 mg/ml globin, different concentrations of pyruvate or lactate, 100 AM luminol, in 1 ml of 50 mM phosphate buffer, pH 7.0, was prepared. The reaction was initiated by injecting 0.05 ml of H2O2 (final concentration 50 mM). When the experiment was done in the presence of 0.4 mg/ml myoglobin the reaction mixture did not contain luminol because the hemoprotein gives a high luminescence efficiency [16].
Materials and methods Horse heart myoglobin, pyruvate, lactate, ascorbate, and NADH as well as the enzyme catalase (CAT; EC 1.11.1.6) were purchased from Sigma–Aldrich. Myoglobin was reduced by the addition of dithionite and the excess was removed by the passage of the solution through a Sephadex G-25 column equilibrated with 50 mM phosphate buffer, pH 7.0. The rate of met-Mb formation was followed during incubation at 378C with a Cary 219 spectrophotometer in the visible region. Changes in the absorption spectrum ranging from 500 to 700 nm were recorded as a function of time, and reference value for complete oxidation was estimated by addition of potassium ferricyanide. Partial deoxygenation of myoglobin was obtained by removing oxygen due to the exposure of oxymyoglobin to the vacuum. To perform this last experiment we used a tonometer (a modified Thunberg tube) routinely employed
Results The effects of increasing amounts of NADH on the kinetics of oxymyoglobin autoxidation were investigated. The time course of myoglobin autoxidation during incubation at 378C and pH 7.0 is reported in Fig. 1. It appears that NADH has a concentration-dependent accelerating effect on the autoxidation. After 30 min of incubation metmyoglobin reaches 7.2% in the control and 22.7% in the presence of 0.5 mM NADH. Ferryl (Fe4+) species were not detectable using the Whitburn equation [17] during the experimental time. Oxy-Mb was monitored according to Whitburn and its decrease correlated with the met-Mb formation. Both a drop in pH and partial deoxygenation markedly stimulate the autoxidation process and the influence of NADH. At lower pH values the effects of NADH on the
Fig. 1. Effects of NADH on the time course of myoglobin autoxidation. (x) Control, (o) 0.1 mM NADH, (D) 0.3 mM NADH, (5) 0.5 mM NADH. Myoglobin (50 AM) was incubated in 50 mM phosphate buffer, pH 7.0, at 378C. Data are from one representative set of tracings from a total of three experiments.
1486
R.A. Olek et al. / Free Radical Biology & Medicine 38 (2005) 1484–1490
Fig. 2. Effects of pH on metmyoglobin formation in the absence and in the presence of 0.1 and 0.3 mM NADH. Myoglobin (50 AM) was incubated for 30 min in 50 mM phosphate buffer at 378C. (n) pH 7.0, (E) pH 6.8, (.) pH 6.6, (x) pH 6.4. Data are from one representative set of tracings from a total of three experiments.
autoxidation of myoglobin increase (Fig. 2). After 30 min of incubation at pH 7.0 we measured 7.2% of met-Mb in the absence of NADH and this parameter reached 17.8% when the experiment was performed in the presence of 0.3 mM NADH. The increasing effect of NADH on met-Mb formation rate was more marked at pH 6.4. At this pH value and always after 30 min of incubation, we obtained 13.4 and 34.9%, respectively, in the absence and in the presence of 0.3 mM NADH. Fig. 3 shows the oxidation process of myoglobin at different oxygen fractional saturations in the presence and in the absence of 0.1 mM NADH. The metmyoglobin formation rate increases with the deoxygenation and the effect of
NADH seems to be more pronounced at lower oxygenation values. The autoxidation of MbO2 in the presence of catalase, the enzyme that metabolyzes hydrogen peroxide, drops below the control level both in the presence and in the absence of NADH. The effect of catalase is ameliorated in the presence of NADH; thus MbO2 autoxidation in the presence of catalase and NADH is slowest (Fig. 4). The presence of 0.1 Amol/L SOD under our experimental conditions does not influence the autoxidation rate of MbO2, but it reduces the effect due to the presence of 0.1 mmol/L NADH (data not shown). Similar experiments were performed in the presence of 5 mM pyruvate or lactate (Fig. 5). Also the well-
Fig. 3. Autoxidation of myoglobin at different fractional oxygen saturations in the presence and absence of NADH. (x) Control, (5) 0.1 mM NADH. Myoglobin (50 AM) was incubated for 30 min in 50 mM phosphate buffer, pH 7.0, at 378C. Data are from one representative set of tracings from a total of three experiments.
R.A. Olek et al. / Free Radical Biology & Medicine 38 (2005) 1484–1490
1487
Fig. 4. Inhibition of met-Mb formation rate by catalase (0.1 AM) in the presence and absence of NADH. (x) Control, (o) 0.1 AM CAT, (D) 0.3 mM NADH, (5) 0.1 AM CAT + 0.3 mM NADH. All other conditions are as for Fig. 1. Data are from one representative set of tracings from a total of three experiments.
known ascorbate was included in these experiments (data not shown). The presence of 0.3 mM ascorbate nullifies the effect due to the presence of NADH on MbO2 oxidation. Under our experimental conditions, the presence of pyruvate markedly reduces the stimulating effect of NADH on MbO2 autoxidation, whereas lactate has no effect (Fig. 5). The autoxidation of MbO2 does not change when we add pyruvate or lactate to the control (data not shown). In all the processes described involving NADH, we followed its oxidation (data not shown). We observed a correlation between the met-Mb formation rate induced by NADH and NADH oxidation rate; obviously catalase and pyruvate, which reduce the effects of NADH on the met-Mb formation rate, inhibit NADH oxidation.
Additional experiments were done to solve issues on the mechanism of pyruvate protection and the role of lactate. By using the chemiluminescence technique we evaluated the antioxidant activity of pyruvate and lactate in the presence and absence of myoglobin. Chemiluminescence (CL) determinations were performed using luminol as the chemiluminogenic probe for H2O2. The luminol-amplified chemiluminescence of hydrogen peroxide was reduced by the presence of both pyruvate and lactate. In Fig. 6 is reported the percentage inhibition in the luminol-CL signal at pH 7.0 when different concentrations of pyruvate or lactate were added. Fifty percent of inhibition was obtained using about 30 and 60 mM for pyruvate and lactate, respectively; the same inhibition was obtained in the
Fig. 5. Effects of 5 mM pyruvate or lactate on metmyoglobin formation in the presence and absence of NADH. (x) Control, (.) 0.5 mM NADH, (5) 0.5 mM NADH + 5 mM pyruvate, (D) 0.5 mM NADH + 5 mM lactate. All other conditions are as for Fig. 1. Data are from one representative set of tracings from a total of three experiments.
1488
R.A. Olek et al. / Free Radical Biology & Medicine 38 (2005) 1484–1490
Fig. 6. Inhibition of luminol-amplified chemiluminescence (%) by pyruvate () or lactate (x) in the reaction mixture containing 100 AM luminol, 50 mM H2O2 in 50 mM phosphate buffer, pH 7.0. Data represent the means F SD of three different experiments performed in triplicate.
presence of only 5 AM ascorbate. A decrease in activity versus H2O2 was present at lower pH values (data not shown). The chemiluminescence data indicate that both pyruvate and lactate (although less marked for lactate) have antioxidant activity. This property does not explain the different effects that they have on the NADH-induced myoglobin oxidation reported in Fig. 5. Therefore, we performed CL experiments in the presence of Mb or apomyoglobin (myoglobin without heme) (Fig. 7). When experiments were performed in the presence of myoglobin or apo-Mb the addition of lactate did not change
the CL signal, whereas the addition of pyruvate produced a reduction of the CL signal (Fig. 7). In other words the antioxidant activity of lactate (under our experimental conditions) versus hydrogen peroxide disappears completely in the presence of myoglobin or apo-Mb, whereas it is only reduced for pyruvate. Fifty percent of inhibition in the presence of myoglobin or apomyoglobin was obtained with 80 and 160 mM of pyruvate, respectively. The antioxidant activity of ascorbate was reduced in the presence of the same Mb concentration (50% was obtained with 1 mM ascorbate).
Fig. 7. Inhibition of chemiluminescence (%) by pyruvate or lactate in the absence or presence of 0.4 mg/ml myoglobin or apomyoglobin. The reaction mixture was the same as for Fig. 6. Luminol was not added for the experiments involving Mb. (x) Pyruvate, (x) Mb + pyruvate, (E) apo-Mb + pyruvate, (o) Mb + lactate; (D) apo-Mb + lactate. Data represent the means F SD of three different experiments performed in triplicate.
R.A. Olek et al. / Free Radical Biology & Medicine 38 (2005) 1484–1490
Discussion The reduced blood flow observed in the heart due to arterial occlusion or partial ischemia in skeletal muscle during high-intensity exercise leads to an increase in concentration of both NADH and deoxymyoglobin [8–11]. Under this condition, energy production comes from the anaerobic system. Due to acceleration of glycolysis, lactate begins to accumulate and the pH in the cells decreases [18]. Several sources of ROS associated with myocardial ischemia–reperfusion may exist. Autoxidation of various hemoproteins such as myoglobin is one of the possible sources of ROS during ischemia–reperfusion [3]. Autoxidation of myoglobin has been shown to result in the formation of superoxide and hydrogen peroxide. Because the concentration of myoglobin is high in heart (up to 0.5 mM) the autoxidation process should be considered an important source of ROS. Therefore factors that stimulate the process may have substantial influence on heart damage during ischemia and reperfusion. It is known that partial deoxygenation and low pH strongly stimulate the autoxidation process of MbO2. In this study, we observed that NADH stimulates met-Mb formation and that this process is accelerated at low pH. Despite being a coenzyme in many enzymatic reactions, NADH can also react with various reactive oxygen species and the reactivity increases when it is bound to proteins (i.e., lactate dehydrogenase [19] or glyceraldehyde-3-phosphate dehydrogenase [20]) and recently it was claimed to be directly operating as an antioxidant [21]. However, it should be mentioned that oneelectron oxidation of NADH leads to the formation of NAD radical, which at a diffusion-controlled rate reacts with oxygen yielding superoxide [19]. If we consider that under certain conditions, like strenuous exercise and myocardial infarct, the pH drops to 6.3 or less and NADH concentration increases, the level of metmyoglobin could increase. In parallel we observed that NADH is oxidized in the presence of MbO2, thus the superoxide anion radical produced is due to both oxymyoglobin and NADH oxidation. Catalase inhibits met-Mb formation both in the presence and in the absence of NADH, suggesting that hydrogen peroxide stimulates this process. These results are in agreement with a previously published report in which it was observed that catalase inhibits the autoxidation process of myoglobin [3]. In addition, we reported [7] a similar behavior with hemoglobin A; the presence of NADH accelerates the met-Hb formation, the presence of SOD reduces the met-Hb formation rate only in the presence of NADH, and finally the autoxidation of HbA in the presence of catalase drops below the control level both in the presence and in the absence of NADH. Obviously the oxidation rate of tetrameric HbA is less with respect to monomeric Mb. Our data indicate that hydrogen peroxide substantially contributes to heme (Fe2+) oxidation and NADH stimulates the autoxidation process with the involvment of hydrogen peroxide. It is worth noting that protection of Mb
1489
autoxidation by catalase (Fig. 4) is much stronger in the presence of NADH, hence this process may be of physiological importance. It is well documented that catalase activity in heart or red muscle is much higher than in white muscle and that it increases in muscle after strenuous exercise [22–24]. Therefore it may be expected that a rise in the intracellular concentration of NADH accompanied by high catalase activity may protect Mb autoxidation and cell damage. It may also be possible that acceleration of Mb autoxidation by high NADH concentration and drop in pH can overlap catalase protection. Significant attention has been devoted to the production of protein-derived radicals as a result of the reaction of hydrogen peroxide with hemoproteins. The reaction of H2O2 with metmyoglobin results in the formation of a shortlived intermediate with one oxidizing equivalent above that in metmyoglobin (the iron is converted to the iron(IV)–oxo state), and one oxidizing equivalent is on a tyrosine residue on the surface of the globin protein [25–27]. These radicals, or further species arising from them, have been shown to undergo a number of reactions [25–28] that may have considerable pathological consequences. Therefore the absence of ferryl (Fe4+) during incubation of myoglobin with NADH as reported in this study could be significant. There are several reports demonstrating that pyruvate infusion during heart reperfusion has a strong protective effect on heart muscle function and structure [12–14]. It has been also reported that pyruvate reduces the in vitro toxicity of myoglobin in renal cortical slices [29] with a mechanism in part due to its antioxidant capacity. In our experiments, inhibition of MbO2 autoxidation by pyruvate was observed in the presence of NADH. This effect of pyruvate depends on its ability to react nonenzymatically with H2O2 yielding CO2 as a by-product. However, under in vivo conditions, the effect of pyruvate may be more complex. The redox state of cytoplasmic NADH closely correlates with the tissue pyruvate to llactate concentration ratio. In reperfused heart, pyruvate may be expected to inhibit myoglobin autoxidation by lowering hydrogen peroxide and NADH concentrations, the latter via the reaction catalyzed by LDH. Lactate under our conditions has no effect on MbO2 autoxidation, but certainly in vivo it should stimulate the process by increasing NADH concentration. In fact it was shown that the formation of ROS in reperfused hearts is augmented by lactate [30] despite some claims that it can have a direct antioxidant activity [31,32]. Chemiluminescence studies reported here (Fig. 7) show that the antioxidant activity versus hydrogen peroxide of lactate and pyruvate is influenced in a different manner by the presence of Mb or apo-Mb. The antioxidant activity of lactate disappears completely in the presence of the protein; the comparison of experiments in the presence of Mb or apo-Mb suggests that the presence of heme increases the antioxidant activity of pyruvate. In summary, the increased rate of Mb autoxidation caused by a low pH, an increase in
1490
R.A. Olek et al. / Free Radical Biology & Medicine 38 (2005) 1484–1490
NADH concentration, and Mb deoxygenation correlates with an increase in ROS formation that can be inhibited by catalase and pyruvate but not by l-lactate. The data reported here could be of interest from the biochemical point of view because the autoxidation of myoglobin can contribute to ischemia–reperfusion injury during infarction or high-intensity exercise. To prevent enhancement of the autoxidation of myoglobin reaction may prove useful.
[15]
[16]
[17]
[18]
References [19] [1] Brunori, M. Nitric oxide moves myoglobin centre stage. Trends Biochem. Sci. 26:209 – 210; 2001. [2] Brunori, M. Nitric oxide, cytochrome-c oxidase and myoglobin. Trends Biochem. Sci. 26:21 – 23; 2001. [3] Gunther, M. R.; Sampath, V.; Caughey, W. S. Potential roles of myoglobin autoxidation in myocardial ischemia–reperfusion injury. Free Radic. Biol. Med. 26:1388 – 1395; 1999. [4] Misra, H. P.; Fridovich, I. The generation of superoxide radical during the autoxidation of hemoglobin. J. Biol. Chem. 247:6960 – 6962; 1972. [5] Brantley, R. E., Jr.; Smerdon, S. J.; Wilkinson, A. J.; Singleton, E. W.; Olson, J. S. The mechanism of autooxidation of myoglobin. J. Biol. Chem. 268:6995 – 7010; 1993. [6] Sylven, C.; Jansson, E.; Book, K. Myoglobin content in human skeletal muscle and myocardium: relation to fibre size and oxidative capacity. Cardiovasc. Res. 18:443 – 446; 1984. [7] Olek, R. A.; Antosiewicz, J.; Caulini, G. C.; Falcioni, G. Effect of NADH on the redox state of human hemoglobin. Clin. Chim. Acta 324:129 – 134; 2002. [8] Neely, J. R.; Grotyohann, L. W. Role of glycolytic products in damage to ischemic myocardium: dissociation of adenosine triphosphate levels and recovery of function of reperfused ischemic hearts. Circ. Res. 55:816 – 824; 1984. [9] Sahlin, K.; Katz, A.; Henriksson, J. Redox state and lactate accumulation in human skeletal muscle during dynamic exercise. Biochem. J. 245:551 – 556; 1987. [10] Richardson, R. S.; Newcomer, S. C.; Noyszewski, E. A. Skeletal muscle intracellular PO2 assessed by myoglobin desaturation: response to graded exercise. J. Appl. Physiol. 91:2679 – 2685; 2001. [11] Murakami, Y.; Zhang, Y.; Cho, Y. K.; Mansoor, A. M.; Chung, J. K.; Chu, C.; Francis, G.; Ugurbil, K.; Bache, R. J.; From, A. H.; Jerosch– Herold, M.; Wilke, N.; Zhang, J. Myocardial oxygenation during high work states in hearts with postinfarction remodeling. Circulation 99:942 – 948; 1999. [12] Mallet, R. T. Pyruvate: metabolic protector of cardiac performance. Proc. Soc. Exp. Biol. Med. 223:136 – 148; 2000. [13] Mallet, R. T.; Squires, J. E.; Bhatia, S.; Sun, J. Pyruvate restores contractile function and antioxidant defenses of hydrogen peroxide– challenged myocardium. J. Mol. Cell Cardiol. 34:1173 – 1184; 2002. [14] Bassenge, E.; Sommer, O.; Schwemmer, M.; Bqnger, R. Antioxidant pyruvate inhibits cardiac formation of reactive oxygen species through
changes in redox state. Am. J. Physiol. Heart Circ. Physiol. 279: H2431 – H2438; 2000. Ascoli, F.; Fanelli, M. R.; Antonini, E. Preparation and properties of apohemoglobin and reconstituted hemoglobins. Methods Enzymol. 76:72 – 87; 1981. Gabbianelli, R.; Falcioni, G.; Santroni, A. M.; Fiorini, R.; Coppa, G. V.; Kantar, A. Interaction of trout hemoglobin with H2O2: a chemiluminescence study. J. Biolumin. Chemilumin. 12:79 – 85; 1997. Whitburn, K. D.; Shieh, J. J.; Sellers, R. M.; Hoffman, M. Z. Redox transformations in ferrimyoglobin induced by radiation–generated free radicals in aqueous solution. J. Biol. Chem. 257:1860 – 1869; 1982. Sahlin, K.; Harris, R. C.; Nylind, B.; Hultman, E. Lactate content and pH in muscle obtained after dynamic exercise. Pflugers Archiv. 367:143 – 149; 1976. Bielski, B. H. J.; Chan, P. C. Kinetic study by pulse radiolysis of the lactate dehydrogenase–catalyzed chain oxidation of nicotinamide adenine dinucleotide by HO2 and O2 radicals. J. Biol. Chem. 250:318 – 321; 1975. Chan, P. C.; Bielski, B. H. J. Glyceraldehyde–3–phosphate deydrogenase–catalyzed chain oxidation of reduced nicotinamide adenine dinucleotide by perhydroxyl radicals. J. Biol. Chem. 255:874 – 876; 1980. Kirsch, M.; De Groot, H. NAD(P)H, a directly operating antioxidant? FASEB J. 15:1569 – 1574; 2001. Ji, L. L.; Fu, R.; Mitchell, E. W. Glutathione and antioxidant enzymes in skeletal muscle: effect of fiber type and exercise intensity. J. Appl. Physiol. 73:1854 – 1859; 1992. Ji, L. L.; Fu, R. Responses of glutathione system and antioxidant enzymes to exhaustive exercise and hydroperoxide. J. Appl. Physiol. 72:549 – 554; 1992. Ji, L. L. Oxidative stress during exercise: implication of antioxidant nutrients. Free Radic. Biol. Med. 18:1079 – 1086; 1995. Tew, D.; Ortiz de Montellano, P. R. The myoglobin protein radical: coupling of Tyr–103 to Tyr–151 in the H2O2–mediated cross–linking of sperm whale myoglobin. J. Biol. Chem. 263:17880 – 17886; 1988. Davies, M. J. Identification of a globin free radical in equine myoglobin treated with peroxides. Biochim. Biophys. Acta 1077: 86 – 90; 1991. Newman, E. S.; Rice–Evans, C. A.; Davies, M. J. Identification of initiating agents in myoglobin–induced lipid peroxidation. Biochem. Biophys. Res. Commun. 179:1414 – 1419; 1991. Catalano, C. E.; Choe, Y. S.; Ortiz de Montellano, P. R. Reactions of the protein radical in peroxide–treated myoglobin: formation of a heme–protein cross–link. J. Biol. Chem. 264:10534 – 10541; 1989. Valentovic, M. A.; Minigh, J. Pyruvate attenuates myoglobin in vitro toxicity. Toxicol. Sci. 74:345 – 351; 2003. Mohazzab-H, K. M.; Kaminski, P. M.; Wolin, M. S. Lactate and PO2 modulate superoxide anion production in bovine cardiac myocytes: potential role of NADH oxidase. Circulation 96:614 – 620; 1997. Yanagida, S.; Luo, C. S.; Doyle, M.; Pohost, G. M.; Pike, M. M. Nuclear magnetic resonance studies of cationic and energetic alterations with oxidant stress in the perfused heart. Modulation with pyruvate and lactate. Circ. Res. 77:773 – 783; 1995. Groussard, C.; Morel, I.; Chevanne, M.; Monnier, M.; Cillard, J.; Delamarche, A. Free radical scavenging and antioxidant effects of lactate ion: an in vitro study. J. Appl. Physiol. 89:169 – 175; 2000.
S
[20]
[21] [22]
[23]
[24] [25]
[26]
[27]
[28]
[29] [30]
[31]
[32]