- Email: [email protected]
Antioxidant mechanism: Electron vs. H-atom transfer
10 2.5
AntoxidantCompounds PHOSPHOLIPID HYDROPEROXlDE ACCUMULATION IN LIVER OF RATS INTOXICATED CARBONTETRACHLORIDE AND ITS PREVENTION BY a-TOCOPHEROL Teruo Miyazawa, Toshihide Suzuki and Kenshiro Fujimoto Department of Food Chemistry, Tohoku University, Tsutsumidori I - I , Sendai 980, Japan Formation and accumulation of phospholipid hydroperoxide in l i v e r of CCl4-intoxicated rats was investigated. Phosphatidylcholine hydroperoxide (PCOOH) was measured by a chemiluminescence-HPLC method (T.Miyazawa; Free radical Biol. Med., 7, 209, 1989). Male SD rats (120g, 5 wk age) were used. The l i v e r PCOOHof control rats (CCl4-untreated) was 160 ± 20 pmol/lOOmg protein and the PCOOH/PCmolar r a t i o was I . I ± O.IxlO-5. In CCl 4 (0.I ml/lOOg rat)-dosed r a t s , l i v e r PCOOH was 289 ± 65 pmol/lOOmg protein (PCOOH/PC = 2.4 ± 0.4xi0-5), 764 ± 271 (5.2 ~ 1.7xi0-5), and 856 ± 165 (6.0 ± 0.8xi0-5) at 6, 24 hr and 1 week a f t e r the dose, respectively. The plasma PCOOH was less than I0 nM in control and CCl4treated rats even at 6 and 24 hr a f t e r the dose, but 1 week a f t e r the PCOOH in plasma evidently increased to 135 ~ 9nM. The increments of l i v e r PCOOH were suppressed 56% by the oral supplementation of dl-a-tocopherol (5mg/lOOg rat/day) for a week before the CC14 dose. The results c l e a r l y demonstrate that phosphatidylcholine is the most susceptible l i p i d class to l i p i d hydroperoxidation in vivo, and dietary e-tocopherol e f f e c t i v e l y prevents the PC hydroperoxidation.
2.7
ANTIOXIDANT~C}[ANISM: ELECTRON VS. H-ATOM TRANSFER Slobodan V. Jovanovi~ Laboratory 030, The Boris Kidric Institute Beograd, Yugoslavia
Despite extensive research the mechanism of action of antioxidants has not been fully resolved. At present there are two mechanisms, one involving electron transfer and the other one H-atom transfer from phenolic antioxidants, PhOH, to oxidizing intermediates, such as peroxy radicals, ROO.. Since simple bond strengths considerations are not adequate and kinetics parameters not sufficient, activation parameters of antioxidant reactions involving these two types of reactions were measured. The activation parameters of antioxidant action of well known repair agents glutathione (GSH), vitamin E (Chr-OH), and vitamin C are compared. The activation parameters of the reaction of thymine OH-adducts, .T-OH, with GSH AH + = 15±2 kcal/mol and AS = 26±5 cal mol -l K -I indicate bond breaking and formation, in full support of the H-atom transfer mechanism. In contrast, considerably smaller activation enthalpies (< i0 kcal mol -I) are measured for electron transfer reactions of vitamin E and C. On the basis of these data, the significance of H-atom transfer versus electron transfer in chemical repair and inhibition of chain oxidations for different types of repair agents and free radicals will be presented.
AN ANTIOXIDANT ROLE FOR HEMOGLOBIN Cecilia Giulivi and Kelvin J. A. Davies Institute for Toxicology & Department of Biochemistry, University of Southern California, Los Angeles, California 90033, U.S.A.
2.6
Ferryl hemoglobin (ferrylHb or Hb-FelV-oH) is formed during reaction of oxyHb or metHb with H 2 0 2. We have quantified ferrylHb production, using a continuous flux of H202 (generated by glucose oxidase + glucose), both with purified Hb in vitro and with intact red blood cells (RBC). Although ferrylHb can cause significant oxidative damage in RBC we now show that such problems are minimized by an, antioxidant, comproportionation reaction between oxyHb and ferrylHb, that harmlessly generates two molecules of met Hb. The met Hb so produced is re-reduced by methemoglobin reductase, providing for an efficient antioxidant cycle. The comproportionation reaction appears to involve electron transfer between tyrosyl residues, and is prevented by tyrosine blocking agents. Although the comproportionation reaction is quite efficient, a significant percentage of ferrylHb escapes reduction by oxyHb and undergoes oxidative degeneration. Initial damage to Hb includes increased hydrophobicity due to a partial unfolding and exposure of hydrophobic amino acids. The exposed hydrophobic amino acids are selectively recognized by a 670 kDa multicatalytic proteinase complex, which we call Macroxyproteinase or M.O.P., that rapidly degrades the damaged Hb. Selective degradation of oxidatively damaged Hb by M.O.P. can be measured by a number of techniques in vitro and by the proteoltyic release of dityrosine and tyrosine oxidation products in intact RBC. Continued exposure of Hb to H202 causes damage to exposed amino acids, an overall increase in hydrophillicity, aggregation and covalent cross-linking, and decreased selective degradation by M.O.P. Thus, RBC appear to use glutathione peroxidase and catalase to minimize H202 exposure and ferryl Hb production, a comproportionation reaction between oxyHb and ferryl Hb to remove most of the ferryl Hb formed, and selective proteolysis by M.O.P. to remove the small percentage of oxidatively damaged Hb molecules that are still produced.
ROLE OF ERGOTHIONEINE IN THE OXIDATION OF OXYFUEMOGLOBIN INDUCED BY NITRITE Arduino Arduinil, Giovanna Mancinellil, Maurizio Belfigliol, Gianluca Radatti I and Giorgio Federici 2. lIstituto di Scienze Biochimiche, Universita' degli Studi "G. D'Annunzio", Chieti and 2Dipartimento di Biologia, Universita' di "Tor Vergata', Roma, Italy. Recently, it has been shown that 2-imidazolthiones possess a remarkable antioxidant activity in the oxidation of oxyhemoglobin induced by nitrite, although 2-imidazolones were not effected. Uric acid, a purine which contains a 2-imidazolone group, shows an antioxidant activity comparable to 2-imidazolthiones. Since hydrogen peroxide is an important and well established intermediate of the oxidation reaction of oxyhemoglobin with nitrite, it has been proposed that uric acid and 2imidazolthiones protects oxyhemoglobin from the oxidation to methemoglobin by nitrite through a scavenging activity toward hydrogen peroxide. On the contrary, we considered the possibility that other reactive intermediates may be more efficently scavenged by such compounds. In fact, during the oxidation of oxyhemoglobin to methemoglobin by nitrite an ESR-detectable ferrylhemoglobin radical has been detected. This higher valence state of hemoglobin is a more powerfull oxidant than hydrogen peroxide. We have demonstrated that ergothioneine, a naturally occuring 2imidazolthione analogue, is able to protect oxyhemoglobin from the oxidation induced by nitrite, and this action is most likely carried out through the reduction of ferrylhemoglobin to methemoglobin. We have also reconciled the discrepancy between the lack of protection of 2imidazolones and the good protection offered by uric acid in the course of oxyhemoglobin oxidation by nitrite. In fact, uric acid, like ergothioneine, reduces the ferryl-derivitave of hemoglobin, and this antioxidant activity has to be ascribed to the pirimidine ring and not to the imidazolone portion of the molecule.
2.8
AntoxidantCompounds PHOSPHOLIPID HYDROPEROXlDE ACCUMULATION IN LIVER OF RATS INTOXICATED CARBONTETRACHLORIDE AND ITS PREVENTION BY a-TOCOPHEROL Teruo Miyazawa, Toshihide Suzuki and Kenshiro Fujimoto Department of Food Chemistry, Tohoku University, Tsutsumidori I - I , Sendai 980, Japan Formation and accumulation of phospholipid hydroperoxide in l i v e r of CCl4-intoxicated rats was investigated. Phosphatidylcholine hydroperoxide (PCOOH) was measured by a chemiluminescence-HPLC method (T.Miyazawa; Free radical Biol. Med., 7, 209, 1989). Male SD rats (120g, 5 wk age) were used. The l i v e r PCOOHof control rats (CCl4-untreated) was 160 ± 20 pmol/lOOmg protein and the PCOOH/PCmolar r a t i o was I . I ± O.IxlO-5. In CCl 4 (0.I ml/lOOg rat)-dosed r a t s , l i v e r PCOOH was 289 ± 65 pmol/lOOmg protein (PCOOH/PC = 2.4 ± 0.4xi0-5), 764 ± 271 (5.2 ~ 1.7xi0-5), and 856 ± 165 (6.0 ± 0.8xi0-5) at 6, 24 hr and 1 week a f t e r the dose, respectively. The plasma PCOOH was less than I0 nM in control and CCl4treated rats even at 6 and 24 hr a f t e r the dose, but 1 week a f t e r the PCOOH in plasma evidently increased to 135 ~ 9nM. The increments of l i v e r PCOOH were suppressed 56% by the oral supplementation of dl-a-tocopherol (5mg/lOOg rat/day) for a week before the CC14 dose. The results c l e a r l y demonstrate that phosphatidylcholine is the most susceptible l i p i d class to l i p i d hydroperoxidation in vivo, and dietary e-tocopherol e f f e c t i v e l y prevents the PC hydroperoxidation.
2.7
ANTIOXIDANT~C}[ANISM: ELECTRON VS. H-ATOM TRANSFER Slobodan V. Jovanovi~ Laboratory 030, The Boris Kidric Institute Beograd, Yugoslavia
Despite extensive research the mechanism of action of antioxidants has not been fully resolved. At present there are two mechanisms, one involving electron transfer and the other one H-atom transfer from phenolic antioxidants, PhOH, to oxidizing intermediates, such as peroxy radicals, ROO.. Since simple bond strengths considerations are not adequate and kinetics parameters not sufficient, activation parameters of antioxidant reactions involving these two types of reactions were measured. The activation parameters of antioxidant action of well known repair agents glutathione (GSH), vitamin E (Chr-OH), and vitamin C are compared. The activation parameters of the reaction of thymine OH-adducts, .T-OH, with GSH AH + = 15±2 kcal/mol and AS = 26±5 cal mol -l K -I indicate bond breaking and formation, in full support of the H-atom transfer mechanism. In contrast, considerably smaller activation enthalpies (< i0 kcal mol -I) are measured for electron transfer reactions of vitamin E and C. On the basis of these data, the significance of H-atom transfer versus electron transfer in chemical repair and inhibition of chain oxidations for different types of repair agents and free radicals will be presented.
AN ANTIOXIDANT ROLE FOR HEMOGLOBIN Cecilia Giulivi and Kelvin J. A. Davies Institute for Toxicology & Department of Biochemistry, University of Southern California, Los Angeles, California 90033, U.S.A.
2.6
Ferryl hemoglobin (ferrylHb or Hb-FelV-oH) is formed during reaction of oxyHb or metHb with H 2 0 2. We have quantified ferrylHb production, using a continuous flux of H202 (generated by glucose oxidase + glucose), both with purified Hb in vitro and with intact red blood cells (RBC). Although ferrylHb can cause significant oxidative damage in RBC we now show that such problems are minimized by an, antioxidant, comproportionation reaction between oxyHb and ferrylHb, that harmlessly generates two molecules of met Hb. The met Hb so produced is re-reduced by methemoglobin reductase, providing for an efficient antioxidant cycle. The comproportionation reaction appears to involve electron transfer between tyrosyl residues, and is prevented by tyrosine blocking agents. Although the comproportionation reaction is quite efficient, a significant percentage of ferrylHb escapes reduction by oxyHb and undergoes oxidative degeneration. Initial damage to Hb includes increased hydrophobicity due to a partial unfolding and exposure of hydrophobic amino acids. The exposed hydrophobic amino acids are selectively recognized by a 670 kDa multicatalytic proteinase complex, which we call Macroxyproteinase or M.O.P., that rapidly degrades the damaged Hb. Selective degradation of oxidatively damaged Hb by M.O.P. can be measured by a number of techniques in vitro and by the proteoltyic release of dityrosine and tyrosine oxidation products in intact RBC. Continued exposure of Hb to H202 causes damage to exposed amino acids, an overall increase in hydrophillicity, aggregation and covalent cross-linking, and decreased selective degradation by M.O.P. Thus, RBC appear to use glutathione peroxidase and catalase to minimize H202 exposure and ferryl Hb production, a comproportionation reaction between oxyHb and ferryl Hb to remove most of the ferryl Hb formed, and selective proteolysis by M.O.P. to remove the small percentage of oxidatively damaged Hb molecules that are still produced.
ROLE OF ERGOTHIONEINE IN THE OXIDATION OF OXYFUEMOGLOBIN INDUCED BY NITRITE Arduino Arduinil, Giovanna Mancinellil, Maurizio Belfigliol, Gianluca Radatti I and Giorgio Federici 2. lIstituto di Scienze Biochimiche, Universita' degli Studi "G. D'Annunzio", Chieti and 2Dipartimento di Biologia, Universita' di "Tor Vergata', Roma, Italy. Recently, it has been shown that 2-imidazolthiones possess a remarkable antioxidant activity in the oxidation of oxyhemoglobin induced by nitrite, although 2-imidazolones were not effected. Uric acid, a purine which contains a 2-imidazolone group, shows an antioxidant activity comparable to 2-imidazolthiones. Since hydrogen peroxide is an important and well established intermediate of the oxidation reaction of oxyhemoglobin with nitrite, it has been proposed that uric acid and 2imidazolthiones protects oxyhemoglobin from the oxidation to methemoglobin by nitrite through a scavenging activity toward hydrogen peroxide. On the contrary, we considered the possibility that other reactive intermediates may be more efficently scavenged by such compounds. In fact, during the oxidation of oxyhemoglobin to methemoglobin by nitrite an ESR-detectable ferrylhemoglobin radical has been detected. This higher valence state of hemoglobin is a more powerfull oxidant than hydrogen peroxide. We have demonstrated that ergothioneine, a naturally occuring 2imidazolthione analogue, is able to protect oxyhemoglobin from the oxidation induced by nitrite, and this action is most likely carried out through the reduction of ferrylhemoglobin to methemoglobin. We have also reconciled the discrepancy between the lack of protection of 2imidazolones and the good protection offered by uric acid in the course of oxyhemoglobin oxidation by nitrite. In fact, uric acid, like ergothioneine, reduces the ferryl-derivitave of hemoglobin, and this antioxidant activity has to be ascribed to the pirimidine ring and not to the imidazolone portion of the molecule.
2.8