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Horseradish peroxidase degrades lipid hydroperoxides and suppresses lipid peroxidation of polyunsaturated fatty acids in the presence of phenolic antioxidants
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 100, No. 6, 653–656. 2005 DOI: 10.1263/jbb.100.653
© 2005, The Society for Biotechnology, Japan
Horseradish Peroxidase Degrades Lipid Hydroperoxides and Suppresses Lipid Peroxidation of Polyunsaturated Fatty Acids in the Presence of Phenolic Antioxidants Norifumi Shirasaka,1* Hiromichi Ohnishi,2 Keiko Sato,2 Rie Miyamoto,2 Takao Terashita,1 and Hajime Yoshizumi2 Department of Applied Biological Chemistry, Faculty of Agriculture, Kin-ki University, 3327-204 Nakamachi, Nara 631-8505, Japan1 and Department of Food and Nutrition, Faculty of Agriculture, Kin-ki University, 3327-204 Nakamachi, Nara 631-8505, Japan2 Received 29 July 2005/Accepted 26 August 2005
Linoleic acid hydroperoxide (LAOOH) was effectively degraded by horseradish peroxidase (HRP) in the presence of quercetin. Several natural phenolic antioxidants, such as quercetin, capsaicin, and α-tocopherol, acted as good hydrogen donors in the peroxidase reaction that occurs during lipid hydroperoxide degradation. However, glutathione, which is a non-phenolic antioxidant that acts as a hydrogen donor for glutathione peroxidase, could not suppress lipid peroxidation in the presence of HRP. Lipid hydroperoxides generated from eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were also degraded with HRP in the presence of quercetin, and oxidative decomposition of DHA was suppressed by this reaction. [Key words: peroxidase, phenolic antioxidant, quercetin, lipid hydroperoxide]
Lipid peroxidation in food and body cells causes decomposition of food and cell toxicity, respectively. In body cells, lipid peroxidation occurs because of active oxygen species (AOS), such as superoxide anion (O2–), hydrogen peroxide (H2O2), hydroxyl radical (•OH), among others; AOS scavenging enzymes and low-molecular-weight antioxidants prevent lipid peroxidation by scavenging AOS. α-Tocopherol is one of the most popular natural antioxidants used in food additives, and it can suppress the generation of lipid hydroperoxides. On the other hand, it is difficult to decompose previously generated lipid hydroperoxides with antioxidants. It has been reported that lipid hydroperoxides generated in body cells are neutralized by glutathione peroxidase (GPx; EC 1.11.1.9) in the presence of glutathione (1, 2). However, it is difficult to use GPx for this purpose because of the high cost of the enzyme. Therefore, we decided to determine whether any of the several plant or fungal peroxidases (donor: H2O2 oxidoreductase; EC 1.11.1.7) (3–12) would be suitable for the enzymatic reaction mentioned above, because these enzymes can be easily obtained at commercially low prices. Peroxidases are clinically important enzymes that are used for colorimetric determination of biological materials by oxidative coupling of H2O2 with 4-aminoantipyrine and phenol. The enzyme from horseradish roots (3) has been primarily used for this purpose. In recent years, fungal peroxidases have received considerable attention for the following reasons. Peroxidases from lignin-degrading Basido-
mycetes (4) are useful for the utilization of biomass, whereas those from the Coprinus strain (5) and Arthromyces ramosus (6, 7) are useful as substitutes for horseradish peroxidase (HRP) during the measurement of H2O2. In most cases, H2O2 has been used as the oxidant for the reaction. Because of the low reactivity of lipid hydroperoxides in comparison with that of H2O2, a peroxidase reaction with a lipid hydroperoxide as the oxidant has rarely been reported. In this study, we examined the enzymatic degradation of lipid hydroperoxides by peroxidase reaction using HRP with phenolic antioxidants acting as hydrogen donors. The protective effect of this reaction on docosahexaenoic acid (DHA) was also examined. MATERIALS AND METHODS Chemicals HRP (RZ>3.0; EIA grade) was purchased from Wako Pure Chemical Industries (Osaka). Fatty acids (99% purity) were purchased from Funakoshi (Tokyo). All other reagents were of analytical grade and purchased from Wako Pure Chemical Industries (Osaka). Preparation of lipid hydroperoxide In this study, linoleic acid hydroperoxide (LAOOH) was used as a representative lipid hydroperoxide, and it was prepared by autoxidation of linoleic acid. Linoleic acid was spread on a petri dish, which was allowed to stand at room temperature for 5 d. The peroxide content of the treated linoleic acid was approximately 60 µmol/g. Lipid hydroperoxides of eicosapentaenoic acid (EPA) and DHA were prepared by similarly and the peroxide content of the treated fatty acids was also approximately 60 µmol/g. The resultant lipid hydroperoxides that contained fatty acids were dissolved in ethanol, and the peroxide content of the solutions was determined using a commercial lipid hydroperoxide assay kit (Cayman Chemicals, Ann Arbor, MI,
* Corresponding author. e-mail: [email protected] phone: +81-(0)742-43-8147 fax: +81-(0)742-43-1445 653
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USA). Measurement of amount of lipid hydroperoxide On the Basis of the amount of lipid hydroperoxide, the hydroperoxide scavenging activity was estimated spectrophotometrically by measuring the absorbance at 500 nm (13). One milliliter of the reaction mixture contained 250 nmol quercetin, 1 mg of treated linoleic acid (that contained approximately 60 nmol of LAOOH), 100 µmol potassium phosphate (pH 7.4), and 10 units HRP. The reaction mixture was incubated at 37°C for 2 h, followed by extraction with a mixture of chloroform (CHCl3) and methanol (CH3OH) (2/1, v/v). Five hundred microliters of the resultant CHCl3 layer (lower layer) was diluted with 450 µl of CHCl3/CH3OH (2/1, v/v). The diluted CHCl3 layer was mixed with 50 µl of colorimetric reagents, and 5 min later, the absorbance was measured at 500 nm. The colorimetric reagent was prepared by mixing equal volumes of 4.5 mM FeCl2 ⋅4H2O in 0.2 M HCl and 3% methanol solution of ammonium thiocyanate. HPLC analysis The quantative analysis of quercetin was performed by HPLC on the Shimadzu LC-6A system (Shimadzu, Kyoto) using a Cosmosil 5C18-AR II column (3×250 mm; Nacalai Tesque, Kyoto; mobile phase: CH3CN/H2O/HCOOH, 20/80/0.1, v/v/v; flow rate: 0.5 ml/min; wavelength: 254 nm). Quercetin was eluted at 10.2 min under these conditions. Fatty acid analysis Fatty acid was extracted with n-hexane, followed by evaporation under a stream of nitrogen and the extracted fatty acids were transmethylated with diazomethane. The resultant fatty acid methyl esters were dissolved in 0.5 ml of n-hexane and were later subjected to gas-liquid chromatography (GLC). The analytical conditions were as follows: apparatus, GC-14B (Shimadzu) equipped with a flame ionization detector (FID) with a split injector; column, ULBON HR-SS-10 capillary column (0.25 mm ×50 m; Shimadzu) and column temperature, 200°C; injection port temperature, 250°C; carrier gas, He (inlet pressure 200 kPa); make-up gas, N2 (60 ml/min); air and H2, 60 kPa; and split ratio, 25: 1.
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FIG. 1. Effects of quercetin and HRP on lipid peroxidation of linoleic acid. The reaction was performed in the reaction mixture described in the Materials and Methods. The reaction mixtures were incubated as follows: without quercetin and HRP (blank); with quercetin (quercetin); with HRP (HRP); and with quercetin and HRP (quercetin, HRP) at 37°C for 2 h. Open bars indicate results before incubation; closed bars, after incubation. All data are presented as mean ± SD for three independent experiments.
RESULTS AND DISCUSSION Effect of quercetin and HRP on LAOOH degradation As shown in Fig. 1, LAOOH concentration in the reaction mixture increased after 2 h of incubation. In this study, both LAOOH and linoleic acid were present in the reaction mixture. A increase in LAOOH concentration was not observed when pure linoleic acid, which did not contain lipid hydroperoxides, was added to the reaction mixture (data not shown). These results suggested that the increase in LAOOH concentration was caused by the simultaneous presence of linoleic acid and LAOOH. However, the increase in LAOOH concentration was suppressed in the presence of quercetin. Furthermore, the lipid hydroperoxide content was reduced when both HRP and quercetin were present in the reaction mixture. Under these reaction conditions, quercetin slightly decomposed in the presence of peroxide containing linoleic acid or HRP (Fig. 2). Nakayama et al. reported that aqueous phenolic compounds generate small amounts of H2O2 (14). It is suggested that this H2O2 acts as an oxidant for HRP and slight decomposition of quercetin occurs. The marked decomposition of quercetin was observed when both peroxide containing linoleic acid and HRP were present in the reaction mixture (Fig. 2). These results suggest that quercetin acted not only as a phenolic antioxidant, which suppresses autoxidation of linoleic acid and the increase in LAOOH concentration, but also as a hydrogen donor during the per-
FIG. 2. Effect of HRP and peroxide containing linoleic acid on degradation of quercetin. The reaction conditions were the same as listed in Fig. 1. The reaction mixtures were incubated as follows: without HRP and linoleic acid (blank); with HRP (HRP); with linoleic acid (LA); and with HRP and linoleic acid (HRP, LA) at 37°C for 2 h. All data are presented as mean ± SD for three independent experiments.
oxidase reaction for scavenging LAOOH. On the other hand, an increase in LAOOH concentration was observed when only HRP was added to the reaction mixture (Fig. 1). It has been reported that HRP causes oxidation of linoleic acid in the presence of H2O2 and molecular oxygen (15). Furthermore, it has also been reported that H2O2 is generated in an aqueous solution of lipid hydroperoxide (16). It was suggested that HRP used such H2O2 molecules for linoleic acid peroxidation under this condition. The time course associated with the changes in LAOOH concentration is shown in Fig. 3. Although LAOOH slightly degraded spontaneously after 4 h of incubation, its concentration was effectively decreased to approximately 20% in the presence of HRP and quercetin. The degradation efficiency did not change even when the quercetin concentration was increased to surplus
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FIG. 3. Time course of changes in lipid hydroperoxide concentration following incubation with (circles) and without (triangles) HRP. The reaction was performed in the presence of quercetin. All other conditions were identical to those described in Materials and Methods. All data are presented as mean ± SD for three independent experiments.
quantities. The decrease in HRP concentration resulted in slow degradation of lipid hydroperoxide. These results suggest that HRP may be used as a supplementary factor for enhancing the activity of phenolic antioxidants. Effects of various phenolic and low-molecular-weight antioxidants on LAOOH degradation by HRP The availability of several phenolic compounds and the effects of low-molecular-weight antioxidants in decreasing LAOOH concentration were investigated (Fig. 4). To date, many phenolic compounds have been reported to act as hydrogen donors in the peroxidase reaction; however, in most cases, the substrate specificities of phenylpropanoids, compounds which originate from plants, have not yet been well investigated. Among the tested phenylpropanoid compounds listed in Fig. 4, the following exhibited very good activity in terms of decreasing LAOOH concentration: quercetin, a flavonoid found in fruits, flowers, and vegetables, and capsaicin, the principal pungent component of red peppers. α-Tocopherol, a commonly used natural antioxidant in food, also sup-
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FIG. 4. Substrate specificities of various antioxidants for lipid hydroperoxide scavenging activity of peroxidase. The reaction was performed in the reaction mixture described in Materials and Methods. The reaction mixtures were incubated with low-molecular-weight antioxidants at 37°C for 2 h with (closed bars) and without HRP (open bars). All data are presented as mean ± SD for three independent experiments.
pressed the increase in LAOOH concentration. These compounds dissolved in water at low concentrations, and it is believed that HRP used these compounds in the aqueous phase as hydrogen donors. Phenol, the conventional substrate of peroxidase, exhibited a very weak activity in terms of decreasing LAOOH concentration. All other phenolic compounds also suppressed lipid peroxidation, but their effects were moderate. The water solubility of these phenolic compounds was sufficient for them to be dissolved at the concentrations used in this experiment, and these compounds were used by HRP as hydrogen donors in the aqueous phase. It is suggested that the weak activity of HRP in terms of LAOOH degradation, when compared with these of quercetin and capsaicin was due to the low substrate specificities of phenolic compounds except for quercetin and capsaicin. On the other hand, glutathione suppressed lipid peroxidation in the absence of HRP, but the LAOOH concentration markedly increased when both glutathione and HRP were
FIG. 5. Hydroperoxide scavenging activity of HRP for lipid hydroperoxides. The reaction mixtures that contained lipid hydroperoxides generated from eicosapentaenoic acid (a) and docosahexaenoic acid (b) were incubated as follows: without quercetin and HRP (blank); with quercetin (quercetin); and with quercetin and HRP (quercetin, HRP) at 37°C. Open bars indicate results before incubation. All data are presented as mean±SD for three independent experiments.
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FIG. 6. Effect of quercetin and HRP on the oxidative decomposition of docosahexaenoic acid. The reaction was performed in the reaction mixture as described in Materials and Methods. The reaction mixtures were incubated with quercetin and the indicated concentration of horseradish peroxidase at 37°C for 24 h (open bars) and 48 h (closed bars). All data are presented as mean ± SD for three independent experiments.
added to the reaction mixture. Although glutathione is known to be a water soluble non-phenolic antioxidant and a hydrogen donor for GPx, HRP cannot use it as a hydrogen donor due to its non-phenolic nature. Furthermore, the LAOOH concentration increased following the addition of HRP, because lipid peroxidation that was enhanced by HRP exceeded the antioxidative activity of glutathione. These results indicated that a phenolic structure is necessary for the LAOOH degradation by HRP. Suppression of lipid peroxidations of EPA and DHA by HRP We investigated the suppression of lipid peroxidations of EPA and DHA by HRP. In both fatty acids, the amount of lipid hydroperoxides increased when the reaction mixtures were incubated in the absence of quercetin (Fig. 5). The addition of quercetin suppressed the increase in the amounts of lipid hydroperoxides, and the amount of lipid hydroperoxides was effectively decreased when both quercetin and HRP were added to the reaction mixture in a manner similar to linoleic acid. Furthermore, the suppressive effect on the oxidative decomposition of DHA by HRP was investigated. The amount of DHA markedly decreased, and approximately 60% of DHA during 24 h, and 90% during 48 h of incubation, had decomposed in the absence of an antioxidant. The addition of quercetin effectively suppressed the decomposition of DHA during 24 h of incubation; however, the residual DHA was less than 80% after 48 h (Fig. 6). On the other hand, when both quercetin and HRP were added to the reaction mixture, the decomposition of DHA was effectively suppressed until 48 h. Marked differences were not observed in terms of the protective effects on DHA between different concentrations of HRP. In conclusion, the addition of HRP alone cannot suppress the decomposition of DHA.
1. Ursin, F., Maiorino, M., and Gregolin, C.: Phospholipid hydroperoxide glutathione peroxidase. Int. J. Tissue Reac., 8, 99–103 (1986). 2. Miwa, T., Adachi, T., Hirano, K., and Sugiura, M.: Decomposition of hydroperoxides derived from microsomes or lipoprotein by glutathione peroxidase and glutathione S-transferase. J. Pharmabio. Dyn., 6, 459–465 (1983). 3. Kinae, N., Masuda, H., Shin, I. S., Furugori, M., and Shimoi, K.: Functional properties of wasabi and horseradish. BioFactors, 13, 265–269 (2000). 4. Leisola, M. S., Kozulic, B., Meussdoerffer, F., and Fiechter, A.: Homology among multiple extracellular peroxidases from Phanerochaete chrysosporium. J. Biol. Chem., 262, 419–424 (1987). 5. DePillis, G. D. and Ortiz de Montellano, P. R.: Substrate oxidation by the heme edge of fungal peroxidases. Reaction of Coprinus macrorhizus peroxidase with hydrazines and sodium azide. Biochemistry, 19, 7947–7952 (1989). 6. Shinmen, Y., Asami, S., Amachi, T., Shimizu, S., and Yamada, H.: Crystallization and characterization of an extracellular fungal peroxidase. Agric. Biol. Chem., 50, 247–249 (1986). 7. Akimoto, K., Shinmen, Y., Sumida, M., Asami, S., Amachi, T., Yoshizumi, H., Saeki, Y., Shimizu, S., and Yamada, H.: Luminol chemiluminescence reaction catalyzed by a microbial peroxidase. Anal. Biochem., 189, 182–185 (1990). 8. Vamos-Vigyazo, L.: Polyphenol oxidase and peroxidase in fruits and vegetables. Crit. Rev. Food Sci. Nutr., 15, 49–127 (1981). 9. Nair, A. R. and Showalter, A. M.: Purification and characterization of a wound-inducible cell wall cationic peroxidase from carrot roots. Biochem. Biophys. Res. Commun., 226, 254–260 (1996). 10. Sung, H. Y., Yu, R. H., and Chang, C. T.: Purification and some properties of peroxidase isozymes from pineapple stem. Biochem. Mol. Biol. Int., 29, 185–195 (1993). 11. González, E. M., de Ancos, B., and Cano, M. P.: Partial characterization of peroxidase and polyphenol oxidase activities in blackberry fruits. J. Agric. Food Chem., 48, 5459–5464 (2000). 12. Ye, X. Y. and Ng, T. B.: Isolation of a novel peroxidase from French bean legumes and first demonstration of antifungal activity of a non-milk peroxidase. Life Sci., 71, 1667–1680 (2002). 13. Mihaljevic, B., Katusin-Razem, B., and Razem, D.: The reevaluation of the ferric thiocyanate assay for lipid hydroperoxide with special considerations of the mechanistic aspects of the response. Free Radic. Biol. Med., 21, 53–63 (1996). 14. Nakayama, T., Kodama, M., and Nagata, C.: Generation of hydrogen peroxide and superoxide anion radical from cigarette smoke. Gann, 75, 95–98 (1984). 15. Garner, C. W.: Peroxidation of free and esterified fatty acids by horseradish peroxidase. Lipids, 19, 863–868 (1984). 16. Santo, H., Hashizume, T., and Yokoyama, T.: Influence of oxidized fats on the microdetermination of residual hydrogen peroxide in foods by 4-aminoantipyrine colorimetry and the removal of such fats. J. Food Hyg. Soc. Jpn., 23, 325–330 (1982).
© 2005, The Society for Biotechnology, Japan
Horseradish Peroxidase Degrades Lipid Hydroperoxides and Suppresses Lipid Peroxidation of Polyunsaturated Fatty Acids in the Presence of Phenolic Antioxidants Norifumi Shirasaka,1* Hiromichi Ohnishi,2 Keiko Sato,2 Rie Miyamoto,2 Takao Terashita,1 and Hajime Yoshizumi2 Department of Applied Biological Chemistry, Faculty of Agriculture, Kin-ki University, 3327-204 Nakamachi, Nara 631-8505, Japan1 and Department of Food and Nutrition, Faculty of Agriculture, Kin-ki University, 3327-204 Nakamachi, Nara 631-8505, Japan2 Received 29 July 2005/Accepted 26 August 2005
Linoleic acid hydroperoxide (LAOOH) was effectively degraded by horseradish peroxidase (HRP) in the presence of quercetin. Several natural phenolic antioxidants, such as quercetin, capsaicin, and α-tocopherol, acted as good hydrogen donors in the peroxidase reaction that occurs during lipid hydroperoxide degradation. However, glutathione, which is a non-phenolic antioxidant that acts as a hydrogen donor for glutathione peroxidase, could not suppress lipid peroxidation in the presence of HRP. Lipid hydroperoxides generated from eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were also degraded with HRP in the presence of quercetin, and oxidative decomposition of DHA was suppressed by this reaction. [Key words: peroxidase, phenolic antioxidant, quercetin, lipid hydroperoxide]
Lipid peroxidation in food and body cells causes decomposition of food and cell toxicity, respectively. In body cells, lipid peroxidation occurs because of active oxygen species (AOS), such as superoxide anion (O2–), hydrogen peroxide (H2O2), hydroxyl radical (•OH), among others; AOS scavenging enzymes and low-molecular-weight antioxidants prevent lipid peroxidation by scavenging AOS. α-Tocopherol is one of the most popular natural antioxidants used in food additives, and it can suppress the generation of lipid hydroperoxides. On the other hand, it is difficult to decompose previously generated lipid hydroperoxides with antioxidants. It has been reported that lipid hydroperoxides generated in body cells are neutralized by glutathione peroxidase (GPx; EC 1.11.1.9) in the presence of glutathione (1, 2). However, it is difficult to use GPx for this purpose because of the high cost of the enzyme. Therefore, we decided to determine whether any of the several plant or fungal peroxidases (donor: H2O2 oxidoreductase; EC 1.11.1.7) (3–12) would be suitable for the enzymatic reaction mentioned above, because these enzymes can be easily obtained at commercially low prices. Peroxidases are clinically important enzymes that are used for colorimetric determination of biological materials by oxidative coupling of H2O2 with 4-aminoantipyrine and phenol. The enzyme from horseradish roots (3) has been primarily used for this purpose. In recent years, fungal peroxidases have received considerable attention for the following reasons. Peroxidases from lignin-degrading Basido-
mycetes (4) are useful for the utilization of biomass, whereas those from the Coprinus strain (5) and Arthromyces ramosus (6, 7) are useful as substitutes for horseradish peroxidase (HRP) during the measurement of H2O2. In most cases, H2O2 has been used as the oxidant for the reaction. Because of the low reactivity of lipid hydroperoxides in comparison with that of H2O2, a peroxidase reaction with a lipid hydroperoxide as the oxidant has rarely been reported. In this study, we examined the enzymatic degradation of lipid hydroperoxides by peroxidase reaction using HRP with phenolic antioxidants acting as hydrogen donors. The protective effect of this reaction on docosahexaenoic acid (DHA) was also examined. MATERIALS AND METHODS Chemicals HRP (RZ>3.0; EIA grade) was purchased from Wako Pure Chemical Industries (Osaka). Fatty acids (99% purity) were purchased from Funakoshi (Tokyo). All other reagents were of analytical grade and purchased from Wako Pure Chemical Industries (Osaka). Preparation of lipid hydroperoxide In this study, linoleic acid hydroperoxide (LAOOH) was used as a representative lipid hydroperoxide, and it was prepared by autoxidation of linoleic acid. Linoleic acid was spread on a petri dish, which was allowed to stand at room temperature for 5 d. The peroxide content of the treated linoleic acid was approximately 60 µmol/g. Lipid hydroperoxides of eicosapentaenoic acid (EPA) and DHA were prepared by similarly and the peroxide content of the treated fatty acids was also approximately 60 µmol/g. The resultant lipid hydroperoxides that contained fatty acids were dissolved in ethanol, and the peroxide content of the solutions was determined using a commercial lipid hydroperoxide assay kit (Cayman Chemicals, Ann Arbor, MI,
* Corresponding author. e-mail: [email protected] phone: +81-(0)742-43-8147 fax: +81-(0)742-43-1445 653
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USA). Measurement of amount of lipid hydroperoxide On the Basis of the amount of lipid hydroperoxide, the hydroperoxide scavenging activity was estimated spectrophotometrically by measuring the absorbance at 500 nm (13). One milliliter of the reaction mixture contained 250 nmol quercetin, 1 mg of treated linoleic acid (that contained approximately 60 nmol of LAOOH), 100 µmol potassium phosphate (pH 7.4), and 10 units HRP. The reaction mixture was incubated at 37°C for 2 h, followed by extraction with a mixture of chloroform (CHCl3) and methanol (CH3OH) (2/1, v/v). Five hundred microliters of the resultant CHCl3 layer (lower layer) was diluted with 450 µl of CHCl3/CH3OH (2/1, v/v). The diluted CHCl3 layer was mixed with 50 µl of colorimetric reagents, and 5 min later, the absorbance was measured at 500 nm. The colorimetric reagent was prepared by mixing equal volumes of 4.5 mM FeCl2 ⋅4H2O in 0.2 M HCl and 3% methanol solution of ammonium thiocyanate. HPLC analysis The quantative analysis of quercetin was performed by HPLC on the Shimadzu LC-6A system (Shimadzu, Kyoto) using a Cosmosil 5C18-AR II column (3×250 mm; Nacalai Tesque, Kyoto; mobile phase: CH3CN/H2O/HCOOH, 20/80/0.1, v/v/v; flow rate: 0.5 ml/min; wavelength: 254 nm). Quercetin was eluted at 10.2 min under these conditions. Fatty acid analysis Fatty acid was extracted with n-hexane, followed by evaporation under a stream of nitrogen and the extracted fatty acids were transmethylated with diazomethane. The resultant fatty acid methyl esters were dissolved in 0.5 ml of n-hexane and were later subjected to gas-liquid chromatography (GLC). The analytical conditions were as follows: apparatus, GC-14B (Shimadzu) equipped with a flame ionization detector (FID) with a split injector; column, ULBON HR-SS-10 capillary column (0.25 mm ×50 m; Shimadzu) and column temperature, 200°C; injection port temperature, 250°C; carrier gas, He (inlet pressure 200 kPa); make-up gas, N2 (60 ml/min); air and H2, 60 kPa; and split ratio, 25: 1.
J. BIOSCI. BIOENG.,
FIG. 1. Effects of quercetin and HRP on lipid peroxidation of linoleic acid. The reaction was performed in the reaction mixture described in the Materials and Methods. The reaction mixtures were incubated as follows: without quercetin and HRP (blank); with quercetin (quercetin); with HRP (HRP); and with quercetin and HRP (quercetin, HRP) at 37°C for 2 h. Open bars indicate results before incubation; closed bars, after incubation. All data are presented as mean ± SD for three independent experiments.
RESULTS AND DISCUSSION Effect of quercetin and HRP on LAOOH degradation As shown in Fig. 1, LAOOH concentration in the reaction mixture increased after 2 h of incubation. In this study, both LAOOH and linoleic acid were present in the reaction mixture. A increase in LAOOH concentration was not observed when pure linoleic acid, which did not contain lipid hydroperoxides, was added to the reaction mixture (data not shown). These results suggested that the increase in LAOOH concentration was caused by the simultaneous presence of linoleic acid and LAOOH. However, the increase in LAOOH concentration was suppressed in the presence of quercetin. Furthermore, the lipid hydroperoxide content was reduced when both HRP and quercetin were present in the reaction mixture. Under these reaction conditions, quercetin slightly decomposed in the presence of peroxide containing linoleic acid or HRP (Fig. 2). Nakayama et al. reported that aqueous phenolic compounds generate small amounts of H2O2 (14). It is suggested that this H2O2 acts as an oxidant for HRP and slight decomposition of quercetin occurs. The marked decomposition of quercetin was observed when both peroxide containing linoleic acid and HRP were present in the reaction mixture (Fig. 2). These results suggest that quercetin acted not only as a phenolic antioxidant, which suppresses autoxidation of linoleic acid and the increase in LAOOH concentration, but also as a hydrogen donor during the per-
FIG. 2. Effect of HRP and peroxide containing linoleic acid on degradation of quercetin. The reaction conditions were the same as listed in Fig. 1. The reaction mixtures were incubated as follows: without HRP and linoleic acid (blank); with HRP (HRP); with linoleic acid (LA); and with HRP and linoleic acid (HRP, LA) at 37°C for 2 h. All data are presented as mean ± SD for three independent experiments.
oxidase reaction for scavenging LAOOH. On the other hand, an increase in LAOOH concentration was observed when only HRP was added to the reaction mixture (Fig. 1). It has been reported that HRP causes oxidation of linoleic acid in the presence of H2O2 and molecular oxygen (15). Furthermore, it has also been reported that H2O2 is generated in an aqueous solution of lipid hydroperoxide (16). It was suggested that HRP used such H2O2 molecules for linoleic acid peroxidation under this condition. The time course associated with the changes in LAOOH concentration is shown in Fig. 3. Although LAOOH slightly degraded spontaneously after 4 h of incubation, its concentration was effectively decreased to approximately 20% in the presence of HRP and quercetin. The degradation efficiency did not change even when the quercetin concentration was increased to surplus
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FIG. 3. Time course of changes in lipid hydroperoxide concentration following incubation with (circles) and without (triangles) HRP. The reaction was performed in the presence of quercetin. All other conditions were identical to those described in Materials and Methods. All data are presented as mean ± SD for three independent experiments.
quantities. The decrease in HRP concentration resulted in slow degradation of lipid hydroperoxide. These results suggest that HRP may be used as a supplementary factor for enhancing the activity of phenolic antioxidants. Effects of various phenolic and low-molecular-weight antioxidants on LAOOH degradation by HRP The availability of several phenolic compounds and the effects of low-molecular-weight antioxidants in decreasing LAOOH concentration were investigated (Fig. 4). To date, many phenolic compounds have been reported to act as hydrogen donors in the peroxidase reaction; however, in most cases, the substrate specificities of phenylpropanoids, compounds which originate from plants, have not yet been well investigated. Among the tested phenylpropanoid compounds listed in Fig. 4, the following exhibited very good activity in terms of decreasing LAOOH concentration: quercetin, a flavonoid found in fruits, flowers, and vegetables, and capsaicin, the principal pungent component of red peppers. α-Tocopherol, a commonly used natural antioxidant in food, also sup-
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FIG. 4. Substrate specificities of various antioxidants for lipid hydroperoxide scavenging activity of peroxidase. The reaction was performed in the reaction mixture described in Materials and Methods. The reaction mixtures were incubated with low-molecular-weight antioxidants at 37°C for 2 h with (closed bars) and without HRP (open bars). All data are presented as mean ± SD for three independent experiments.
pressed the increase in LAOOH concentration. These compounds dissolved in water at low concentrations, and it is believed that HRP used these compounds in the aqueous phase as hydrogen donors. Phenol, the conventional substrate of peroxidase, exhibited a very weak activity in terms of decreasing LAOOH concentration. All other phenolic compounds also suppressed lipid peroxidation, but their effects were moderate. The water solubility of these phenolic compounds was sufficient for them to be dissolved at the concentrations used in this experiment, and these compounds were used by HRP as hydrogen donors in the aqueous phase. It is suggested that the weak activity of HRP in terms of LAOOH degradation, when compared with these of quercetin and capsaicin was due to the low substrate specificities of phenolic compounds except for quercetin and capsaicin. On the other hand, glutathione suppressed lipid peroxidation in the absence of HRP, but the LAOOH concentration markedly increased when both glutathione and HRP were
FIG. 5. Hydroperoxide scavenging activity of HRP for lipid hydroperoxides. The reaction mixtures that contained lipid hydroperoxides generated from eicosapentaenoic acid (a) and docosahexaenoic acid (b) were incubated as follows: without quercetin and HRP (blank); with quercetin (quercetin); and with quercetin and HRP (quercetin, HRP) at 37°C. Open bars indicate results before incubation. All data are presented as mean±SD for three independent experiments.
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REFERENCES
FIG. 6. Effect of quercetin and HRP on the oxidative decomposition of docosahexaenoic acid. The reaction was performed in the reaction mixture as described in Materials and Methods. The reaction mixtures were incubated with quercetin and the indicated concentration of horseradish peroxidase at 37°C for 24 h (open bars) and 48 h (closed bars). All data are presented as mean ± SD for three independent experiments.
added to the reaction mixture. Although glutathione is known to be a water soluble non-phenolic antioxidant and a hydrogen donor for GPx, HRP cannot use it as a hydrogen donor due to its non-phenolic nature. Furthermore, the LAOOH concentration increased following the addition of HRP, because lipid peroxidation that was enhanced by HRP exceeded the antioxidative activity of glutathione. These results indicated that a phenolic structure is necessary for the LAOOH degradation by HRP. Suppression of lipid peroxidations of EPA and DHA by HRP We investigated the suppression of lipid peroxidations of EPA and DHA by HRP. In both fatty acids, the amount of lipid hydroperoxides increased when the reaction mixtures were incubated in the absence of quercetin (Fig. 5). The addition of quercetin suppressed the increase in the amounts of lipid hydroperoxides, and the amount of lipid hydroperoxides was effectively decreased when both quercetin and HRP were added to the reaction mixture in a manner similar to linoleic acid. Furthermore, the suppressive effect on the oxidative decomposition of DHA by HRP was investigated. The amount of DHA markedly decreased, and approximately 60% of DHA during 24 h, and 90% during 48 h of incubation, had decomposed in the absence of an antioxidant. The addition of quercetin effectively suppressed the decomposition of DHA during 24 h of incubation; however, the residual DHA was less than 80% after 48 h (Fig. 6). On the other hand, when both quercetin and HRP were added to the reaction mixture, the decomposition of DHA was effectively suppressed until 48 h. Marked differences were not observed in terms of the protective effects on DHA between different concentrations of HRP. In conclusion, the addition of HRP alone cannot suppress the decomposition of DHA.
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