This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and

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2 Food Chemistry 123 (2) Contents lists available at ScienceDirect Food Chemistry journal homepage: Catalytic mechanisms of metmyoglobin on the oxidation of lipids in phospholipid liposome model system B. Min a, K.C. Nam b, D.U. Ahn a,c, * a Department of Animal Science, Iowa State University, Ames, IA 511, USA b Department of Animal Science and Technology, Sunchon National University, , Republic of Korea c Department of Agricultural Biotechnology, Major in Biomodulation, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul , Republic of Korea article info abstract Article history: Received 19 August 29 Received in revised form 7 March 2 Accepted 1 April 2 Keywords: Metmyoglobin Lipid oxidation Liposome system Iron chelating agents The catalytic mechanism of metmyoglobin (metmb) on the development of lipid oxidation in a phospholipid liposome model system was studied. Liposome model system was prepared with metmb solutions (2., 1.,.5, and.25 mg metmb/ml) containing none, diethylenetriamine pentaacetic acid (DTPA), desferrioxamine (DFO), or ferric chloride and lipid oxidation was determined at, 15, 3, 6, and 9 min of incubation at 37 C. Metmyoglobin catalysed lipid oxidation in the liposome system, but the rate of lipid oxidation decreased as the concentration of metmb increased. The amount of free ionic iron in the liposome solution increased as the concentration of metmb increased, but the rate of metmb degradation was increased as the concentration of metmb decreased. The released free ionic iron was not involved in the lipid oxidation of model system because ferric iron has no catalytic effect without reducing agents. Both DFO and DTPA showed antioxidant effects, but DFO was more efficient than DTPA because of its multifunctional antioxidant ability as an iron and haematin chelator and an electron donor. The antioxidant activity of DTPA in liposome solution containing.25 mg metmb/ml was two times greater than that with 2 mg metmb/ml due to the increased prooxidant activity of DTPA-chelatable compounds. It was concluded that ferrylmyoglobin and DTPA-chelatable haematin generated from the interaction of metmb and LOOH, rather than free ionic iron, were the major catalysts in metmb-induced lipid oxidation in phospholipid liposome model system. Ó 2 Elsevier Ltd. All rights reserved. 1. Introduction Myoglobin has been recognised as a major catalyst for lipid oxidation in meat, but its mode of action for catalysing lipid oxidation is controversial. It has been suggested that the interaction of metmyoglobin (metmb) with hydrogen peroxide (H 2 O 2 ) or lipid hydroperoxides (LOOH) results in the formation of ferrylmyoglobin, which can initiate free radical chain reactions (Chan, Faustman, Yin, & Decker, 1997; Davies, 199; Egawa, Shimada, & Ishimura, 2; Kanner & Harel, 1985; Min & Ahn, 25; Rao, Wilks, Hamberg, & Ortiz de Montellano, 1994). In addition, ferrylmyoglobin as well as metmb can degrade LOOH to free radicals such as alkoxyl and peroxyl radicals (Reeder & Wilson, 1998, 21), which can initiate and/or catalyse a series of propagation and termination step in the free radical chain reactions of lipid oxidation (Frankel, 1987; Halliwell & Gutteridge, 199). However, others limited the role of myoglobin as only a source for free ionic iron or haematin (Ahn & Kim, 1998; Kanner, Shegalovich, Harel, & Hazan, 1988; * Corresponding author at: Department of Animal Science, Iowa State University, Ames, IA 511, USA. Tel.: ; fax: address: duahn@iastate.edu (D.U. Ahn). Puppo & Halliwell, 1988). They indicated that free ionic iron and/ or haematin released from myoglobin in the presence of H 2 O 2 or lipid hydroperoxide, rather than ferrylmyoglobin, were the major catalysts for lipid oxidation in meat. The ratio of peroxides to met- Mb is a determining factor for the formation of ferrylmyoglobin or the release of free ionic iron or haematin (Rhee, Ziprin, & Ordonez, 1987). Haematin is released from myoglobin in the presence of H 2 O 2, followed by the liberation of free ionic iron from haematin (Prasad, Engelman, Jones, & Das, 1989). Haematin reacts with H 2 O 2 or lipid hydroperoxide to form haematin with higher oxidation state (Ferrylhaematin, Fe(IV = O)), which can initiate and propagate lipid oxidation (Kim & Sevanian, 1991). Dix and Marnett (1985) indicated that LOOH such as linoleic acid hydroperoxide were more efficient for haematin-catalysed lipid oxidation than H 2 O 2, and ferrylhaematin and alkoxyl radical (LO) generated from the interaction of haematin with LOOH were responsible for the haematin-catalysed lipid oxidation. Haematin can be easily intercalated into membrane due to its hydrophobicity and catalyse lipid oxidation (Schmitt, Frezzatti, & Schreier, 1993). The concentration of metmb is a determining factor for its prooxidative activity in the presence of fatty acid or LOOH (Baron, Skibsted, & Andersen, 22; Lapidot, Granit, & Kanner, 25). In /$ - see front matter Ó 2 Elsevier Ltd. All rights reserved. doi:.16/j.foodchem

3 232 B. Min et al. / Food Chemistry 123 (2) addition, myoglobin shows a pseudo-hydroperoxidase activity in the presence of reducing agents such as ascorbic acid and phenolic antioxidants to remove lipid hydroperoxides (Gorelik & Kanner, 21; Harel & Kanner, 1989). Iron chelators such as diethylenetriamine pentaacetic acid (DTPA) and desferrioxamine (DFO) have been widely used to elucidate the mechanism of iron compounds on lipid oxidation (Ahn, Wolfe, & Sim, 1993; Harel, Salan, & Kanner, 1988). DFO has been known as an excellent chelating agent for ferric ion and DTPA for ferrous and ferric ions (Kanner & Harel, 1987; Rahhal & Richter, 1989). Both DFO and DTPA have chelating ability to haematin (Radi, Turrens, & Freeman, 1991). DFO can also act as an electron donor to ferrylmyoglobin to suppress the prooxidant activity of ferrylmyoglobin and release free ionic iron from metmb as well as to free radicals to break down the free radical chain reaction of lipid oxidation (Rice-Evans, Okunade, & Khan, 1989). The objectives of this study were to determine the concentration effect of metmb and the effect of ferric ion and chelators such as DFO and DTPA on the metmb-induced lipid oxidation in the phospholipid liposome model system. 2. Materials and methods 2.1. Chemicals and reagents Metmyoglobin (from equine skeletal muscle), linoleic acid, 2- thiobarbituric acid (TBA), ferrozine (3-(2-pyridyl)-5,6-bis (4-phenyl sulphonic acid)-1,2,4-triazine), neocuproine (2,9-dimethyl- 1,-phenanthroline), ferric chloride, diethylenetriamine pentaacetic acid (DTPA), desferrioxamine (DFO), chelex- chelating resin (5 dry mesh, sodium form), butylated hydroxytoluene (BHT), and Tween-2 were purchased from Sigma (St. Louis, MO). All other chemicals and reagents used were of reagent grade. Deionised distilled water (DDW) by Nanopure infinity ultrapure water system with ultraviolet (UV) (Barnstead, Dubuque, IA) was used for the preparation of all reagents and buffers. All DDW and buffers were treated with the chelex- chelating resin to remove any free metal ion before use Preparation of metmyoglobin solution An appropriate amount of metmb was dissolved in 5 mm acetate buffer (ph 5.6) at 4 C. The metmb solution was centrifuged at 3g at 4 C for 6 min to remove undissolved impurities. The concentration of metmb and percentages of metmb in the solution were calculated according to Krzywicki (1982). The metmb concentration of solution was adjusted to 2., 1.,.5, and.25 mg/ml with 5 mm acetate buffer (ph 5.6). The average concentration of metmb and percentages of metmb were 2.2 ±.2, 1.2 ±.1,.5 ±.1, and.25 ±. mg/ml and.82 ±.19%,.9 ±.24%,.84 ±.12%,.28 ±.86%, respectively. DTPA (2 mm; final concentration), DFO (2 mm; final concentration), and ferric chloride (5 lg/ml; final concentration) were added to the metmb solutions. The metmb solution was treated with Chelex- chelating resin to remove any free ironic ion present before use Lipid oxidation potential in metmyoglobin liposome model system The metmb liposome model system was prepared using egg phospholipids. The fatty acid composition of the phospholipids (Table 1) was determined by the method of Ahn, Wolfe, and Sim (1995). An aliquot of phospholipids dissolved in chloroform was placed in a scintillation vial and evaporated under nitrogen gas to make thin film on the wall. The metmb solution containing none, DTPA, DFO, or ferric chloride was added to a phospholipidcoated vial and shaken vigorously for 2 min to make metmb liposome solution with final concentration of 3 mg phospholipids per ml. The solution was incubated at 37 C for 9 min to accelerate lipid oxidation. Lipid oxidation was determined at, 15, 3, 6, and 9 min. An aliquot (.5 ml) of the solution was mixed with ll BHT solution (6% BHT in ethanol), added with 1 ml TBA/TCA solution (15 mm TBA/15% trichloroacetic acid (TCA; w/v)), and incubated in boiling water bath for 15 min. After cooling, the mixture was centrifuged at 15,g for min. The absorbance of the supernatant was determined at 531 nm against a reagent blank. Lipid oxidation was expressed as mmol malondialdehyde (MDA) equivalents (eq.) per kg phospholipids, calculated from the molar extinction coefficient of M 1 cm 1. In addition, the generation of nonheme iron during the incubation was measured at, 15, 3, 6, and 9 min using the ferrozine method of Min and Ahn (29) with modification. In brief, sample (.6 ml) and ascorbic acid (.2 ml, 1% in.2 M HCl, w/v) were thoroughly mixed with 11.3% TCA solution (w/v,.4 ml). After 5 min at room temperature, the mixture was centrifuged at 3g for 15 min at 2 C. The supernatant (1 ml) was mixed with.4 ml of % ammonium acetate (w/v) and.1 ml of the ferrozine colour reagent. After colour development at room temperature for min, the absorbance was determined at 562 nm against a reagent blank. The concentration of nonheme iron released from metmb during reaction was expressed as lg iron/ml metmb liposome solution. All measurements were quadruplicated Lipoxygenase-like activity of metmyoglobin Lipoxygenase-like (LOX-like) activity of metmb (1 mg/ml) was measured by the method of Gata, Pinto, and Macias (1996) with some modifications. Linoleic acid ( mm) in.2 M NaOH solution emulsified with Tween-2 was used as a substrate solution, which was flushed with and kept under nitrogen. The reaction mixture was composed of 8 ll of the substrate solution, 8 ll of each metmb solution as an enzyme solution, and 5 mm acetate buffer (ph 5.6) to a final volume of 1 ml. Lipoxygenase-like activity was assessed by the increase of absorbance at 234 nm due to the generation of conjugated dienes from linoleic acid at 27 C. The results were expressed as units of activity (U) per ml, calculated from the molar extinction coefficient of hydroperoxyl linoleic acid (e = 25, M 1 cm 1 ). One unit of lipoxygenase-like activity was defined as the amount of enzyme catalysing the formation of Table 1 Fatty acid composition of phospholipids used in model system. Fatty acid Content (%) Myristic acid.19 ±.3 Palmitic acid 28.7 ±.22 Palmitoleic acid 1.28 ±.18 Margaric acid.28 ±.1 Margaroleic acid.12 ±.3 Stearic acid ±.14 Oleic acid 27.1 ±.2 trans-vaccenic acid 1.59 ±.15 Linoleic acid ±.14 c-linolenic acid.17 ±.2 Gondoic acid.21 ±.3 Arachidonic acid 6.68 ±.9 DTA.41 ±.8 DPA.14 ±.2 DHA 1.59 ±.4 Means was expressed with the standard deviation. n = 4. Abbreviations: DTA, all cis-7,,13,16-docosatetraenoic acid; DPA, all-cis- 7,,13,16,19-docosapentaenoic acid, DHA, all cis-4,7,,13,16,19-docosahexaenoic acid.

4 B. Min et al. / Food Chemistry 123 (2) lmol of hydroperoxide per minute. All measurements were quadruplicated Statistical analysis All the analyses were performed on the samples with four replications. Data were analysed using the JMP software (version 5.1.1; SAS Institute Inc., Cary, NC). Differences among mean values were determined by the Student-Newman Keuls multiple range test (P <.5) (Kuehl, 2). 3. Results and discussion Metmyoglobin, at all concentrations, induced lipid oxidation and increased the TBARS values linearly in phospholipid liposome model system during the 9 min-incubation (Fig. 1). However, the increasing rate of TBARS values significantly decreased with the increase of metmb concentration (P <.5): the highest rates at lower metmb concentrations (.199 and.194 mmol MDA eq./kg phospholipid per min at.25 and.5 mg/ml, respectively), followed by.177 mmol MDA eq./kg phospholipid per min at 1. mg/ml and.157 mmol MDA eq./kg phospholipid per min at the highest metmb concentration (2. mg/ml) (P <.5). Especially, after 6 and 9 min of incubation, the TBARS values at the highest metmb concentration (2 mg/ml) (11.43 and mmol MDA eq./kg phospholipids, respectively) was significantly lower than those at the lowest concentration (.25 mg/ml) (12.5 and mmol MDA eq./kg phospholipid) (P <.5). The presence of LOOH was detected right after the preparation of the liposome model system (data not shown). Trace amount of LOOH during the preparation of liposome solution have been widely recognised (Halliwell & Gutteridge, 199; Kim & Sevanian, 1991). This result indicates that the concentration of metmb is a critical factor for determining prooxidant activity of myoglobin in the presence of LOOH and/or fatty acid: at low concentrations, metmb acts as a prooxidant (Baron et al., 22; Lapidot et al., 25). The amount of free ionic iron significantly increased during incubation, and was proportional to the concentration of metmb (Fig. 2). The concentrations of free ionic iron after 9 min of incubation were 15.93, 11.82, 11., and 7.88 lm at 2., 1.,.5, and.25 mg metmb per ml metmb liposome solution, respectively, indicating that 13.94%, 2.69%, 38.85%, and 55.14% of metmb in 2., 1.,.5, and.25 mg/ml, respectively, were decomposed and liberated free ionic iron. These results agreed with many previous reports (Baron & Andersen, 22; Lapidot et al., 25), which suggested that the interaction of H 2 O 2 or LOOH with metmb caused the liberation of free ionic iron as well as haematin. Thus, the LOOH preexisted or generated during the incubation should be the major catalysts to release free ionic irons from metmb because H 2 O 2 was not added in this study. Prasad et al. (1989) suggested that haematin was released from myoglobin before free ionic iron release in the presence of H 2 O 2 and the amount of haematin released from metmb during incubation was greater than that of free ionic iron released. Thus, the amount of haematin and free ionic iron produced during incubation should be proportional to the concentration of metmb in the liposome system. The release of haematin was confirmed by Chiu et al. (1996) but it was readily decomposed by LOOH to release free ionic iron (Kim & Sevanian, 1991). Haematin-catalysed lipid oxidation more efficiently than ionic iron because of its hydrophobicity that allowed it to permeate into membrane (Schmitt et al., 1993). Although haematin was more active than other hemeproteins and ferrous ion (Chiu et al., 1996; Kaschnitz & Hatefi, 1975), the ratio of haematin to lipids was the determining factor for its prooxidant activity (Schmitt et al., 1993). They suggested that haematin formed either dimer at low ratio or aggregated at high ratio in aqueous solution: a dimer was less effective than a monomer for lipid oxidation but could permeate to membrane where it was degraded to monomer, and aggregates were inactive. The haematin monomer within membrane interacted with LOOH to form alkoxyl radical and haematin-containing hypervalent iron (Fe(IV) = O) both of which were regarded as initiators and catalysts for the haematin-catalysed lipid oxidation (Dix & Marnett, 1985; Kim & Sevanian, 1991). Therefore, a high amount of haematin at a high concentration of metmb in a liposome system (2 mg/ml) should be partially responsible for the lower lipid oxidation rate, compared to that at lower metmb concentrations (<1 mg/ml) in Fig. 1. The addition of ferric ion did not affect myoglobin-catalysed lipid oxidation in phospholipid liposome model system (Fig. 3), indicating that either ferrylmyoglobin or haematin generated from metmb rather than free ionic iron was the major catalyst for met- Mb-induced lipid oxidation in this system. It has been suggested that the oxidation state of iron is more important than the amount of iron for the development of lipid oxidation in model system TBARS value (mmol MDA eq. / kg ) Mb.25 Mb.5 Mb1. Mb Fig. 1. Lipid oxidation potential of metmb with various concentrations in phospholipid liposome model system during incubation at 37 C for 9 min (TBARS: mmol malondialdehyde (MDA) equivalents/kg phospholipid ()). The concentrations of metmb in 5 mm acetate buffer (ph 5.6) were 2 (Mb2.), 1 (Mb1.),.5 (Mb.5), and.25 (Mb.25) mg per ml, respectively. Phospholipid liposome model system with buffer alone was used as a control (). Means with standard deviation were expressed. n = 4. Nonheme iron content (µg / ml) Mb.25 Mb.5 Mb1. Mb Fig. 2. Formation of nonheme iron in a phospholipid liposome model system with various concentrations of metmb during incubation at 37 C for 9 min (lg nonheme iron/ml metmb liposome solution). The concentrations of metmb in 5 mm acetate buffer (ph 5.6) were 2 (Mb2.), 1 (Mb1.),.5 (Mb.5), and.25 (Mb.25) mg per ml, respectively. Phospholipid liposome model system with buffer alone was used as a control (). Means with standard deviation were expressed. n =4.

5 234 B. Min et al. / Food Chemistry 123 (2) A..25 mg metmyoglobin / ml latable compounds, haematin (Baysal et al., 199; Radi et al., 1991), was partially responsible for the metmb-induced lipid oxidation in the liposome model system. Free ionic iron (ferric form) was already ruled out because it did not show any prooxidant effect in model system (Ahn & Kim, 1998). The antioxidant activity (95.24%) of DFO at low myoglobin concentration (.25 mg/ml) was higher than that (89.43%) at high myoglobin concentration (P <.5). Moreover, the antioxidant activity (36.24%) of DTPA in liposome model system with low concentration of metmb (.25 mg/ml) was twice as high as that (18.8%) with high concentration (>1. mg/ml) (Fig. 3A and B) (P <.5), indicating that DTPA-chelatable compound, probably haematin, was contributed more to the development of lipid oxidation at lower than at higher concentration of metmb. LOX-like activity is related to the generation of conjugated diene at initial stage of lipid oxidation. LOX-like activity of metmb was not changed by ferric ion in the absence of reducing agents (Fig. 4), indicating that free ionic ion released from myoglobin was not involved in the initiation of lipid oxidation in metmb-induced lipid oxidation. The addition of DFO and DTPA to the liposome model system decreased LOX-like activity of metmb, but DTPA (34.98%) suppressed it more effectively than DFO (15.57%). In this study, the catalytic mechanism of metmb on lipid oxidation was investigated in phospholipid liposome solutions incubated at 37 C, which is different from the refrigerated temperature conditions (4 C) for normal meat storage and distribution. In general, the reaction rates increase as the reaction temperature increase. Although temperature at or below 37 C is not likely to change the nature of metmb, it may affect the reactivity and/or solubility of metmb and other compounds such as lipids and haematin. In addition, meat products contain various anti- and prooxidative factors. Therefore, further studies on the effect of low temperature and other factors on metmb-induced lipid oxida- B. 1. mg metmyoglobin / ml TBARS value (mmol MDA eq. / kg ) Mb Fe(III) DTPA DFO TBARS value (mmol MDA eq. / kg ) Mb Fe(III) DTPA DFO Fig. 3. Lipid oxidation potential of metmb treated with desferrioxamine (DFO, 2 mm; final concentration), diethylenetriamine pentaacetic acid (DTPA, 2 mm; final concentration), or ferric chloride (Fe(III), 5 lg/ml; final concentration) in phospholipid liposome model system during incubation at 37 C for 9 min (TBARS value, mmol malondialdehyde (MDA) equivalents/kg phospholipid ()). The final concentrations of metmb in liposome solution were.25 (A) and 1. (B) mg per ml, respectively. Phospholipid liposome model system with metmb and buffer were used as a control (Mb) and blank control (), respectively. Means with standard deviation were expressed. n =4. (Ahn & Kim, 1998). However, the released free ionic iron may play a significant role in the acceleration of lipid oxidation in meat where the ferric ion-reducing capacity has been detected (Ahn & Kim, 1998; Kanner, Salan, Harel, & Shegalovich, 1991). Iron chelators, DTPA and DFO, showed different antioxidant effects in the liposome model system (Fig. 3). DFO inhibited myoglobin-catalysed lipid oxidation effectively, but DTPA showed only partial inhibitions. Both DTPA and DFO are known as strong iron chelators and inhibit free ionic iron-catalysed lipid oxidation (Graf, Mahoney, Bryant, & Eaton, 1984). However, DFO showed stronger antioxidant activity than DTPA. The antioxidant activity of DTPA was affected by the ratio of DTPA to free ionic iron, but DFO was not. DFO can act not only as an efficient iron chelator but also an electron donor or hydrogen donor to ferrylmyoglobin, resulting in the suppression of ferrylmyoglobin-catalysed lipid oxidation (Rice-Evans et al., 1989). Rice-Evans et al. (1989) suggested that DFO can prevent the release of free ionic iron from myoglobin by reducing ferrylmyoglobin and breaking free radical chain reactions. In addition, DFO can interact with haematin via the iron moiety to prevent their catalytic and membrane-intercalating activity for lipid oxidation (Baysal, Monteiro, Sullivan, & Stern, 199). On the other hand, DTPA can inhibit iron-catalysed lipid oxidation by occupying all six coordination sites of iron. Also, DTPA can inhibit haematin-catalysed lipid oxidation (Radi et al., 1991). Free haematin may have one or two unoccupied or loosely bound coordination sites. It is assumed that DTPA or DFO may bind to those coordination sites to inactivate the catalytic activity of haematin, but no evidence is available. DTPA did not inhibit lipid oxidation catalysed by ferrylmyoglobin (Harel & Kanner, 1988). Consequently, the high inhibitory effect of DFO was from the synergistic effect of DFO as a chelator for chelatable compounds, probably haematin, and an electron donor to ferrylmyoglobin and free radicals, whereas the partial effect of DTPA was attributed to its chelating ability, indicating that DTPA-che-

6 1 B. Min et al. / Food Chemistry 123 (2) Lipoxygenase-like activity (Unit / ml) tion are needed to strengthen the proposed catalytic mechanism of metmyoglobin on lipid oxidation in this study. 4. Conclusion Lipid oxidation in phospholipid liposome model system was accelerated in the presence of metmb. Increases in metmb concentration in model system decreased lipid oxidation, due to the ratio of myoglobin to LOOH or fatty acid and the ratio of haematin to lipids. The concentration of free ionic iron released from metmb increased during incubation but was not involved in the development of lipid oxidation. The addition of DFO and DTPA inhibited lipid oxidation. DFO was more effective than DTPA because DFO can inactivate haematin and reduce ferrylmyoglobin and free radicals whereas DTPA only binds to haematin. The metmb-induced lipid oxidation was caused by both ferrylmyoglobin and haematin generated from the interaction of metmb with LOOH in phospholipid liposome system, rather than the released free ionic irons. Acknowledgement The work has been supported by the National Integrated Food Safety Initiative/USDA (USDA Grant ), Washington DC, and WCU (World Class University) program (R31-56) through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology. References a b Control DFO DTPA Fe(III) Fig. 4. Lipoxygenase-like activity (Unit/mL) of metmb solution treated with none (control), metmb (1 mg/ml; final concentration) desferrioxamine (DFO, 2 mm; final concentration), diethylenetriamine pentaacetic acid (DTPA, 2 mm; final concentration), or ferric chloride (Fe(III), 5 lg/ml; final concentration) in 5 mm acetate buffer, ph 5.6. Means with different letters (a c) are significantly different (P <.5). n =4. Ahn, D. U., & Kim, S. M. (1998). 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