The Effect of Metal Chelators, Hydroxyl Radical Scavengers, and Enzyme Systems on the Lipid Peroxidation of Raw Turkey Meat1

PROCESSING AND PRODUCTS The Effect of Metal Chelators, Hydroxyl Radical Scavengers, and Enzyme Systems on the Lipid Peroxidation of Raw Turkey Meat1 D. U. AHN,2-3 F. H. WOLFE,2 and J. S. SIM4 Departments of Food Science and Animal Science, University of Alberta, Edmonton, Alberta, Canada, T6G 1P5

1993 Poultry Science 72:1972-1980

ground state oxygen molecules are considered as free radicals, but they do not have The chain initiation step of the peroxi- enough reactivity to carry out an abstracdation sequence in a membrane or poly- tion. A reduced form of oxygen radicals unsaturated fatty acid refers to the attack (•OH) and oxygen-iron complexes [ferryl of any free radical species that has suffi- (FeO2*) or perferryl (Fe0 ) compounds] do cient reactivity to abstract a hydrogen have enough reactivity2 to initiate the atom from a methylene group of fatty peroxidation (Gutteridge, 1982; Halliwell acids adjacent to a double bond (Halliwell and Gutteridge, 1990). The oxygen-iron and Gutteridge, 1990). Ionic iron and complexes (ferryl and perferryl) further catalyze the degradation of hydroperoxides, primary lipid oxidation by-products, to secondary oxidation by-products (HalReceived for publication October 26, 1992. liwell and Gutteridge, 1990). The imporAccepted for publication May 19, 1993. 1 This research was supported by the Natural tance of ionic iron on lipid peroxidation is Science and Engineering Research Council of Canada. not only its involvement as a catalyst in 2 Department of Food Science. 3 To whom correspondence should be addressed. the production of superoxide (02~) and 4 hydroxyl radicals, but also its direct inDepartment of Animal Science. INTRODUCTION

1972

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ABSTRACT The effect of iron chelators, free radical scavengers, and enzyme systems on the formation of hydroxyl radicals and the lipid peroxidation of raw turkey meat was determined. Fresh hand-deboned turkey breast and leg meat without skin or mechanically deboned turkey meat (MDTM) was homogenized with 3 vol of .1 M citrate-phosphate buffer, pH 5.5, or distilled water. Samples were prepared by adding iron chelators, free radical scavengers, or enzyme (xanthine oxidase and superoxide dismutase) systems into the meat homogenate. Free ionic iron was the critical component in the formation of 2-thiobarbituric acid reactive substances (TBARS), and chelation with strong or weak iron chelators was effective in preventing the formation of TBARS in meat, except with weak iron chelators in breast meat. Hydroxyl radical scavengers mannitol, glucose, formic acid, and dimethyl sulfoxide were not effective, but free radical terminators, ascorbate and burylated hydroxyanisol, were very effective antioxidants. Tyrosine had stronger antioxidant effects than hydroxyl radical scavengers. Superoxide dismutase (SOD) and SOD plus catalase had a weak antioxidant effect on lipid oxidation of raw turkey meat but xanthine oxidase had a strong antioxidant effect that could not be explained. The formation of TBARS in meat was mainly catalyzed by the ferryl or perferryl complex-dependent free radicals, not by the superoxide-dependent hydroxyl radicals. {Key words: lipid peroxidation, iron chelators, free radicals, superoxide dismutase, xanthine oxidase)

LIPID PEROXIDATION OF RAW TURKEY MEAT

and hydroxyl radicals when tissues are reoxygenated after severe hypoxic conditions (Granger et al, 1981; McCord, 1987). Under normal conditions the dehydrogenase form predominates, but following periods of ischemia this form can be converted to the oxidase form (Sussman and Buckley, 1990), which transfers the electron to molecular oxygen and thereby generates a superoxide anion as a byproduct of the oxidation. During ischemia, the breakdown of high-energy phosphate compounds results in the accumulation of the purine metabolites hypoxanthine or xanthine, which can serve as substrates for XOD. The tissue injury observed by the free radicals generated by XOD have been observed in many organs including skeletal muscle, and the damage occurring with reperfusion could be inhibited by SOD, hydroxyl radical scavengers, or allopurinol [an inhibitor of XOD (Grisham et al, 1986)]. In postmortem tissues, the amount of xanthine and hypoxanthine, the substrates from which XOD generates hydrogen peroxide and superoxide radical, increases with time due to the degradation of adenosine triphosphate (ATP). It is assumed that subsequent processing steps such as cutting and grinding of meat could replenish oxygen and promote the generation of free radicals by the xanthine-XOD reaction. Although the involvement of XOD and SOD systems on the formation and removal of free radicals in living tissue have been reported (McCord and Fridovich, 1969; McCord, 1987), their effect on lipid oxidation in post-mortem tissues is unknown. The objective of this study was to determine the effect of iron chelators, XOD and SOD enzyme systems, and free radical scavengers on the formation of hydroxyl radicals and the lipid peroxidation of raw turkey meat. MATERIALS AND METHODS Chemicals 2-Deoxy-D-ribose, desferrioxamine mesylate, mannitol, glucose, formic acid, DMSO, ascorbate, BHA, allopurinol, SOD, xanthine, XOD, catalase, DTPA, ferrozine (3-(2-pyridyl)-5,6-bis (4-phenyl sulfonic

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volvement in the decomposition of hydroperoxides, alkoxyl, and peroxyl radicals (Gutteridge, 1984). Metal chelating agents such as catechol, EDTA, and polyphosphates substantially lower fat oxidation and improve sensory quality of cooked pork (Shahidi et al, 1986). Conalbumen, many amino acids, and citric acid also have iron binding capabilities and could reduce or eliminate the catalytic effects of iron (Stadelman and Cotterill, 1977; Ladikos and Lougovois, 1990). Desferoxamine, a specific ferric iron chelator, has been reported to inhibit the formation of free radicals when the reaction is iron-dependent (Gutteridge et al, 1979) and in most cases was as effective as diethylenetriaminepentaacetic acid (DTPA) and EDTA. Mannitol, glucose, formate, and DMSO (dimethyl sulfoxide) are known as hydroxy! radical scavengers (Halliwell and Gutteridge, 1990). Most of the synthetic phenolic antioxidants are reported to have very strong antioxidant effects by terminating free radicals (Yun et al, 1987), but information on many of the natural free radical scavengers in meat is minimal. Superoxide and hydrogen peroxide (H202), combined with the catalytic effect of iron, react together to form the highly reactive hydroxyl radical, which can destroy almost all known biomolecules (Fridovich, 1975). In biological systems, however, all of the iron ions under normal physiological conditions are bound to iron storage or transport proteins (Halliwell and Gutteridge, 1986). Furthermore, the superoxide dismutase (SOD) system removes superoxide and hydrogen peroxide to protect the organism from damage (detoxification). However, after the animal is processed, the pH of muscle drops to 5.6 or lower, and iron can be released from iron carriers (Halliwell and Gutteridge, 1990). The predominant forms of ironcontaining proteins in muscle tissue are heme pigments, ferritin, and transferrin. They act as powerful lipid oxidation catalysts when the heme pigments are activated by hydrogen peroxide or iron is released from the iron-containing molecules (Kanner and Harel, 1985; Kanner and Doll, 1991; Kanner et al, 1991). The xanthine and xanthine oxidase (XOD) system can generate superoxide

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AHN ET AL.

acid)-l,2,4-triazine, neocuproine, trichloroacetic acid, and turkey hemoglobin were obtained from Sigma Chemical Co.5 2-Thiobarbituric acid (TBA) was obtained from Eastman Organic Chemicals;6 Ltyrosine was from Merck & Co.,7 and Chelex-100 (50-100 mesh, sodium form) was from Bio-Rad.8 All chemicals were reagent grade. Reagents

Sample Preparation

Fresh hand-deboned turkey breast and leg meat without skin, and mechanically deboned turkey meat (MDTM, frames and necks combined) were obtained from a local poultry processing plant. Individual breast

sSigma Chemical Co., St. Louis, MO 63178-9916. 6 Eastman Organic Chemicals, Rochester, NY 14652-3512. TMerck & Co., Rahway, NJ 07065. 8 Bio Rad Laboratories, Richmond, CA 94804. 'Hobart Corp., Troy, OH 45374. 10 Brinkman Instruments Inc., Westbury, NY 11590-0207. n Becton Dickinson Labware, Lincoln Park, NJ 07035. 12 Bamstad, Dubuque, IA 52001.

Lipid Peroxidation and Nonheme Iron Determinations

The measurement of lipid peroxidation was achieved by the method of Halliwell and Gutteridge (1981) modified for use in meat samples (Ann et ah, 1993). Test tubes containing 3.5 mL meat mixture, prepared as described above, were incubated for 2 h in a 37 C water bath. Immediately after

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Deoxyribose solution (50 mM), DTPA (20 mM), EDTA (20 mM), desferoxamine mesylate (20 mM), mannitol (.4 M), glucose (.4 M), formic acid (.4 M), DMSO (20 mM), ascorbate (20 mM), SOD (1,428 U/mL), xanthine (32 mM), XOD (.256 U/mL), catalase (2,487 U/mL) were prepared by dissolving appropriate amount of each chemical directly in distilled water (DW) (Halliwell and Gutteridge, 1981; Gutteridge, 1982). Seventy-five milligrams of ferrozine and 75 mg neocuproine were dissolved in 25 mL DW containing 1 drop of concentrated HC1 to make mixed ferroin color reagent (Carter, 1971). Tyrosine and allopurinol were dissolved in DW by adding NaOH (6 N) until fully dissolved (pH 7.4 for tyrosine and pH 9.6 for allopurinol). Butylated hydroxyanisol (BHA, 45 mg/mL) was dissolved in 97% ethanol. The iron solution was prepared by dissolving .356 g FeCl2-4H20 in 1 L of .1 N HC1, resulting in 100 jig Fe2+/mL solution.

and leg meat were ground twice in a Hobart meat grinder9 (Model 84185) through a 8-mm and a 3-mm plate and used in an experiment within 24 h. A 10-g meat sample was placed in a mortar and macerated by continuous mixing with a pestle while .1 M citrate-phosphate buffer, pH 5.5, or DW was gradually added (total 30 mL). The macerate was transferred to a 50-mL test tube using rubber policemen and further homogenized with a Brinkman Polytron (Type PT 10/35)™ for 2 s at top speed to break up the large meat particles. Citrate and phosphate are weak iron chelators. The use of citrate-phosphate buffer, pH 5.5, would act as a weak iron chelator on the lipid oxidation of meat but would also reduce the effect of the pH differences of the three different meat types. The use of citrate-phosphate buffer would also allow a comparison of the effect of antioxidants in the presence or absence of weak iron chelators used in meat processing. From each meat type, eight meat homogenates were prepared (four with DW; four with citrate-phosphate buffer). One of each of the muscle homogenates was allocated to each antioxidant group (total of four antioxidant groups). Four milliliters of meat homogenate were transferred to disposable Falcon polyethylene test tubes (17 x 100 mm)." Deoxyribose (.1 mL), .1 mL antioxidant or enzyme, .1 mL of iron solution, .4 mL NaCl (12.5%), and .2 and .3 mL DW were added to 4 mL meat homogenate to give a total volume of 5 mL. Control samples were prepared with no added antioxidant, enzyme, or iron solution. The resulting sample mixture was mixed in a vortex,12 and 1.5 mL of the mixture was transferred to a glass test tube (13 x 100 mm) for nonheme iron analysis; the rest was used for the lipid peroxidation study.

LIPID PEROXIDATION OF RAW TURKEY MEAT

Statistical Analysis Each of the three meat types (breast, leg, and MDTM) had 19 treatments, each of the 19 treatments was homogenized with two homogenizing solutions (DW and citratephosphate buffer) and the experiment was replicated four times. For the analysis of the results, the 19 treatments were allocated into four groups according to their antioxidant characteristics (metal chelators, antioxidants, SOD, and XOD systems), and each treatment was subdivided by homogenizing solution. The data for each meat type were analyzed independently by SAS® software (SAS Institute, 1986). The treatment effects were compared for each homogenizing solution, and the effect of homogenizing solution for the treatment within the same meat type was also compared. Analyses of variance were conducted to test for treatment effects within a homogenizing solution, and Student's t test for homogenizing solution within a meat type. After finding no difference in nonheme iron content within a meat type across the two homogenizing solutions, the nonheme iron data from DW and citrate-

13

Whatman Inc., Clifton, NJ 07014.

phosphate buffer homogenates were pooled. The Student-Newman-Keuls multiple range test was used to compare differences among mean values. Mean values and SEM were reported, and replication was used as the error term for the calculations. RESULTS AND DISCUSSION Iron Chelators and the Formation of Thiobarbituric Acid Reactive Substances The effect of three strong iron chelators on the formation of TBARS in meat homogenized with citrate-phosphate buffer (a weak iron chelator, .1M, pH 5.5) and DW is shown in Table 1. The TBARS values of breast meat with strong iron chelators plus Fe2+ were lower (P < .05) than those with Fe2+ only and were similar to that of the control mat contained no added iron. The TBARS value of desferoxamine plus Fe2+ meat was higher (P < .05) than that of the control, but the differences were small. Some differences in TBARS values of breast meat between the citrate-phosphate buffer and DW diluents were also observed but they were too small to be of practical importance. In leg meat, all of the strong iron chelators were very effective in reducing TBARS in DW homogenates (Table 1). The weak iron chelators, citrate and phosphate (used as a citrate-phosphate buffer to homogenize the meat) were especially effective (P < .01) in preventing the formation of TBARS in leg meat. The decreases in the TBARS values of leg meat by strong iron chelators in citrate-phosphate buffer homogenates are small. However, this is not because the strong iron chelators have a low antioxidant effect but is attributed to the very strong antioxidant effect of the weak iron chelator (citrate-phosphate buffer), which greatly reduced the TBARS values of meat. In MDTM, both strong and weak chelators were effective in reducing TBARS, but the TBARS values of the homogenates were reduced further by the synergistic effect of the weak iron chelators (citrate-phosphate buffer). These results indicated that the use of both weak and strong iron chelators would

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incubation, 3.5 mL of perchloric acid (7.72%) was added to the meat homogenate to stop the peroxidation reaction and to extract the 2-thiobarbituric acid reactive substances (TBARS) formed by hydroxyl radical reactions. The mixture was mixed in a vortex and then filtered through Whatman Number 1 filter paper.13 Two milliliters of filtrate were mixed with 2 mL 20 mM TBA in DW and incubated at room temperature in the dark for 17 h to determine the amount of TBA reactive materials that are formed. The absorbance of the resulting solution was determined at 531 nm against a blank containing 2 mL DW and 2 mL 20 mM TBA solution. The TBARS numbers were expressed as milligrams malondialdehyde (MA) per kilogram meat. The ferrozine iron analysis method of Carter (1971) was modified for the use in meat samples as described previously (Ahn et al, 1993).

1975

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AHN ET AL.

TABLE 1. Effect of iron chelators on the formation of 2-thiobarbiruric acid reactive substances (TBARS) in breast, leg, and mechanically deboned turkey meat with 10 /tg iron added/g of meat1 Breast Treatments

Buffer

None .14cy Fe2* only 1.36" EDTA + Fe .21= DTPA + Fe .17cy Desferoxamine + Fe .40b SEM of treatment .04

2

Leg

DW3

SEM

Buffer

DW

Mb* 1.447' .37b .30b" .36b .02

.05 .12 .06 .05 .09

lme .13bX .26"y .18"b .12b .19* .02

MA/kg .20b* 1.59"* .21b .14b .25b .05

MDTM SEM

Buffer

DW

SEM

.06 .13 .08 .04 .05

.10by .53"y .10by .10by .I6by .02

,29
.06 .09 .04 .08 .04

Means (n = 4) within a column with no common superscripts differ significantly (P < .05). "•yMeans (n = 4) within a row of same meat type with no common superscripts differ significantly (P < .05). 1 DW = distilled water; MA = malondialdehyde; EDTA = ethylenediaminetetraacetic acid; MDTM = mechanically deboned turkey meat; DTPA = diethylenetriaminopentaacetic acid. 2 Citrate-phosphate buffer, .1 M, pH 5.5, was used. 3pH of DW treatments: Breast, 5.90; Leg, 6.05; MDTM, 6.20.

be an effective strategy to reduce the formation of TBARS in leg meat and MDTM but not in breast meat. The reason for the limited effect of weak iron chelators in breast meat cannot be explained at this point. McCord and Day (1978) reported that the rate of lipid peroxidation increased linearly with increasing amounts of FeEDTA, and Gutteridge (1984) reported that ferrous iron-EDTA complex can stimulate the peroxidation of phospholipids by a reaction independent of hydroxyl radical when the concentration of Fe2+ in the reaction is greater than that of EDTA. However, Kanner et al. (1988) reported that

a low concentration of EDTA inhibited lipid peroxidation in raw and cooked turkey dark meat. The increases in the formation of TBARS samples with added iron and EDTA were very small in this study and were not different (P > .05) from those samples with no added iron (Table 1). Most of the iron chelated by strong iron binders was detectable by the ferrozine iron assay method (Table 2). The iron binding strength of DTPA and desferrioxamine must be stronger than that of EDTA because more iron was detected with EDTA under the iron assay conditions, especially in breast meat, but little difference in the

TABLE 2. Effect of iron chelators on the content of nonheme iron1 in breast, leg, and mechanically deboned turkey meat with 10 pg iron added/g of meat2 Treatments

Breast

None Fe2* only EDTA + Fe DTPA + Fe Desferrioxamine + Fe SEM of treatment

1.85d 12.21" 12.06" 7.72' 10.83b

a_d

.15

Leg (/«g F e / g meat) — 3.13c 13.13" 13.99" 12.33* 11.34b .29

MDTM 2.49= 12.16"b 12.34" 11.73 ab 11.57b .15

Means (n = 4) within a column with no common superscripts differ significantly (P < .05). Nonheme iron data were pooled from .1 M citrate-phosphate buffer, pH 5.5 and DW. 2 EDTA = ethylenediaminotetraacetic acid; MDTM = mechanically deboned turkey meat; DTPA diethylenetriaminepentaacetic acid. 1

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a_d

1977

LIPID PEROXIDATION OF RAW TURKEY MEAT

TABLE 3. Effect of antioxidants on the formation of 2-thiobarbituric acid reactive substances in breast, leg, and mechanically deboned turkey meat with 10 pg iron added/g of meat1 TBARS Breast

Leg

Buffer2

DW

SEM

Buffer

None Fe 2 * only Mannitol + Fe Glucose + Fe Formic acid + Fe DMSO + Fe Tyrosine + Fe Ascorbate + Fe BHA + Fe

.14^ 1.36" 1.12ab-y 1.12ab 1.08ab
.34dA 1.44ab 1.38ab-x 1.22b 1.34ab,x 1.42a
.05 .12 .08 .11 .11 .09 .11 .11 .06

(.mS .134b-y ab .26 -y .333a-y .36a-y .398a
,

SEM of treatment

DW

MDTM

SEM

MA/kg meat) — .20d* .09 b x .13 1.39 1.92 a ' x .06 99cx .09 .77cx .11 d .21 <* .03 .06

Buffer

DW

.io c -y .53a
3.33 a ' x 3.18a
.03

.29d,x

SEM .05 .09 .07 .11 .10 .11 .16 .13 .05

.08

a d

" Means (n = 4) within a column with no common superscripts differ significantly (P < .05). "-yMeans (n = 4) within a row of same meat type with no common superscripts differ significantly (P < .05). 1 TBARS = 2-thiobarbituric acid reactive substances; DW = distilled water; MA = malondialdehyde; MDTM = mechanically deboned turkey meat; DMSO = dimethyl disulfoxide; BHA = butylated hydroxyanisol. 2 Citrate-phosphate buffer, .1 M, pH 5.5, was used. 3 pH of DW treatments: Breast, 5.90; Leg, 6.05; MDTM, 6.20.

However, the free radical terminators, ascorbate and BHA, reduced (P < .05) the formation of TBARS in all three meat types prepared with DW and in breast meat prepared with citrate-phosphate buffer. The added BHA was so effective that it stopped the formation of TBARS in meat under high iron conditions, and the TBARS values of meat with BHA were as low as those of meat with no iron added. Tyrosine also reduced (P < .05) the amount of TBARS formed in breast meat and MDTM with Hydroxyl Radical Scavengers and buffer and leg and MDTM with DW Free Radical Terminators and preparations. the Formation of Thiobarbituric Acid Igene et al. (1985) reported that ascorbate Reactive Substances had a prooxidant effect at the level of 250 Compared with the TBARS values of Mg/g of meat. The added ascorbate (at a Fe2+ added controls (Fe2+ only), the amount level of 400 jig/g meat) used was effective of TBARS formed in samples with the in inhibiting the formation of TBARS in hydroxyl radical scavengers mannitol, glu- meat, but its effect was generally lower (P < cose, formic acid, and DMSO, were differ- .05) than that of BHA. Tyrosine also showed ent (P < .05) only in MDTM with buffer an antioxidant effect in meat due to the system (Table 3). Gutteridge (1984) phenolic-hydroxyl group considered to reported that in the presence of Fe2+ salts, provide the chain-breaking antioxidant acthe peroxidation of phospholipid was not tivity on such molecules (Halliwell and inhibited by hydroxyl radical scavengers. Gutteridge, 1990). Differences in TBARS The hydroxyl radicals formed reacted im- values of breast meat between the citratemediately with the membrane components phosphate buffer and DW homogenization and were not available for scavenging. were small compared with those of leg meat

formation of TBARS was found among chelators (Table 1). Halliwell and Gutteridge (1990) indicated that DTPA is not a general inhibitor of the iron-dependent radical reaction, but in the current study it was more effective in chelating free iron than EDTA and desferrioxamine in breast meat (Table 2) and was an effective inhibitor of free radical formation in meat with iron (Table 1).

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Treatments

1978

AHN ET AL.

TABLE 4. Effect of superoxide dismutase (SOD) system on the formation of 2-thiobarbituric acid reactive substances (TBARS) in breast, leg, and mechanically deboned turkey meat with 10 fig iron added/g of meat1 TBARS

Treatments

.14c
DW3 .34* 1.04cy

SEM .05 .12 .12 .11 .09

.03

Buffer .I3b
DW

SEM

Buffer

M A / k g meat) — .20b<* .06 .ioc-y 1.59a<* .13 .53a
DW

SEM

.29b<* 3.33a<*

.05 .09 .07 .09 .11

330**

3.19a'* 3.21a-* .03

a_d

Means (n = 4) within a column with no common superscripts differ significantly (P < .05). "-yMeans (n = 4) within a row of same meat type with no common superscripts differ significantly (P < .05). iDW = distilled water; MDTM = mechanically deboned turkey meat; MA = malondialdehyde. 2 Citrate-phosphate buffer, .1 M, pH 5.5, was used. 3 pH of DW treatments: Breast, 5.90; Leg, 6.05; MDTM, 6.20.

1982), and under .1 M ammonium acetate (10% homogenate) conditions, the activity of SOD could be maintained for 24 h (at 25 C incubation) with only 11 to 15% loss (Stein et al, 1982). The low antioxidant effect of the Superoxide Dismutase System and SOD system could be related to the low pH the Formation of Thiobarbituric Acid of the meat system. Perferryl complexes Reactive Substances have a very strong reactivity and can When SOD and catalase were added to abstract hydrogen atoms from fatty acids or meat singly or in combination, the amount lipid hydroperoxides (Waters, 1971; Davis of TBARS formed in meat was not much and Slater, 1987). The reactivity of perferryl lower than those of Fe2+-added controls. complexes is much faster than that of the The effect of catalase and SOD plus catalase Fenton reaction (Garnier-Suillerot et al, was not consistent in breast meat (Table 4). 1984). The addition of catalase alone increased the TBARS value in breast meat with DW. Xanthine Oxidase System and When SOD plus catalase was added, they the Formation of Thiobarbituric Acid increased the TBARS value in breast meat Reactive Substances with citrate-phosphate buffer but decreased it in the meat with DW. The SOD and SOD The effects of allopurinol, xanthine, plus catalase reduced (P < .05) the amount XOD, and XOD plus xanthine on the of TBARS formed in MDTM with citrate- formation of TBARS in meat are shown in phosphate buffer although the amounts Table 5. Although allopurinol is known to were small. inhibit the activity of XOD, which generates The low antioxidant effect of SOD sys- superoxide and urate in biological systems, tem (SOD, catalase, and SOD plus catalase) it did not show any effect on the formation indicates that the major catalyst to the of TBARS in all three meat types. Adding formation of TBARS in meat is not depen- xanthine also had no effect on the formation dent on hydroxyl radicals but is dependent of TBARS in meat. However, XOD and on iron and iron complexes (ferryl or XOD plus xanthine reduced (P < .05) the perferryl complexes). The optimum pH TBARS formed in breast meat and MDTM conditions for SOD are neutral to alkaline homogenation in both citrate-phosphate (McCord and Fridovich, 1969; Stein et al, buffer and DW. Only XOD plus xanthine and MDTM except with the BHA treatment, for which TBARS values were very low in all three meat types and both homogenates.

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None Fe 2+ only SOD + Fe Catalase + Fe SOD + catalase + Fe SEM of treatment

Buffer

MDTM

Leg

Breast 2

1979

LIPID PEROXIDATION OF RAW TURKEY MEAT

TABLE 5. Effect of allopurinol and XOD system on the formation of 2-thiobarbituric acid reactive substances (TBARS) in turkey breast, leg, and mechanically deboned turkey meat with 10 /tg iron/g of meat added 1 TBARS Breast

MDTM

Leg

Buffer2

DW 3

SEM

Buffer

None Fe 2 * only Allopurinol + Fe XOD + Fe Xanthine + Fe XOD + xanthine + Fe SEM of treatment

.14b.y 1.36a 1.19a
.34='" 1.44' 1.47*-* 1.03b'* 1.36a .93b-x .07

.05 .12 .11 .11 .14 .12

(mg MA/kg meat) .13b
DW

SEM

Buffer

DW

SEM

.10c-y .53a
29cx 3.33a-x 3.39a-x 1.26b-x 3.21 a ' x 1.24b-x .07

.05 .09 .09 .05 .10 .15

a_d

Means (n = 4) within a column with no common superscripts differ significantly (P < .05). -yMeans (n = 4) within a row of same meat type with no common superscripts differ significantly (P < .05). *XOD = xanthine oxidase; DW = distilled water; MA = malondialdehyde; MDTM = mechanically deboned turkey meat. 2 Citrate-phosphate buffer, .1 M, p H 5.5, was used. 3pH of DW treatments: Breast, 5.90; Leg, 6.05; MDTM, 6.20. x

reduced (P < .05) the amount of TBARS formed in leg meat in the DW preparation. The XOD-dependent TBARS formation in meat samples was expected to be high because the oxygen replenishment during the meat sample preparation steps is very similar to that of reperfusion of ischemic tissues. Furthermore, XOD was expected to be active under the low meat pH conditions of the meat because XOD is active under the conditions of ischemic tissue, where pH is very low due to the lactic acid formation by anaerobic glycolysis. However, XOD provided an antioxidant effect in homogenized meat samples. The antioxidant effect of XOD could not be explained by the free radical generation mechanism suggested by Granger et al. (1981), but one good explanation for this phenomenon would be the chelation of hydroxyl radicals and Fe2+ by urate, which is a product of the xanthine XOD reaction. These results indicate that iron chelators were more effective than hydroxyl radical scavengers except for BHA in inhibiting the formation of TBARS in meat. Ascorbate (at a level of 400 /Jg/g meat) and tyrosine were quite effective in inhibiting the formation of TBARS in meat, but their effects were lower (P < .05) than that of BHA. The catalytic effect of free ionic iron on the formation of

TBARS was not caused by the superoxidedependent hydroxyl radicals, but catalyzed by ferryl (Fe02+) or perferryl complex (Fe02)-dependent free radicals. Superoxide dismutase and SOD plus catalase had only a limited effect in reducing the amount of TBARS formed in meat.

REFERENCES Ahn, D. U., F. H. Wolfe, and J. S. Sim, 1993. The effect of free and bound iron on lipid peroxidation in turkey meat. Poultry Sci. 72:209-215. Carter, P., 1971. Spectrophotometric determination of serum iron at the submicrogram level with a new reagent (ferrozine). Anal. Biochem. 40:450-458. Davis, M. J., and T. F. Slater, 1987. Studies on the metalion and lipoxygenase-catalysed breakdown of hydroperoxides using electron-spin-resonance spectroscopy. Biochem. J. 245:167-173. Fridovich, I., 1975. Superoxide dismutases. Ann. Rev. Biochem. 44:147-159. Garnier-Suillerot, A., L. Tosi, and E. Panoage, 1984. Kinetic and mechanism of vesicle lipoperoxide decomposition by Fe (II). Biochim. Biophys. Acta 794:307-312. Granger, D. N„ G. Rutili, and J. M. McCord, 1981. Superoxide in feline intestinal ischemia. Gastroenterology 81:22-29. Grisham, M. B., L. A. Hernandez, and D. N. Granger, 1986. Xanthine oxidase and neutrophile infiltration in intestinal ischemia. Am. J. Physiol. 251: G567-G574. Gutteridge, J.M.C., 1982. The role of superoxide and hydroxyl radicals in phospholipid peroxidation

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Treatments

1980

AHN ET AL. and free metal ions. J. Agric. Food Chem. 36: 409-412. Ladikos, D., and V. Lougovois, 1990. Lipid oxidation in muscle foods: A review. Food Chem. 35:295-314. McCord, J. M., 1987. Oxygen-derived radicals: a link between reperfusion injury and inflammation. Fed. Proc. 46:2402-2406. McCord, J. M., and D. Day, Jr., 1978. Superoxidedependent production of hydroxyl radical catalyzed by iron-EDTA complex. Fed. Eur. Biol. Soc. Lett. 86:139-142. McCord, J. M., and I. Fridovich, 1969. Superoxide dismutase. An enzymatic function for erythrocuprein (Hemocuprein). J. Biol. Chem. 244:6056-6063. SAS Institute, 1986. SAS® User's Guide. SAS Institute Inc., Cary, NC. Shahidi, F., L. J. Rubin, L. L. Diosady, N. Kassum, J. C. Fong, and D. F. Wood, 1986. Effect of sequestering agents on lipid oxidation in cooked meats. Food Chem. 21:145-152. Stadelman, W. J., and O. J. Cotterill, 1977. Egg Science Technology. 2nd ed. AVI Publishing Inc., Westport, CT. Stein, T. N., C. L. Keen, B. Lonnerdal, and L. S. Hurley, 1982. Effect of sample preparation on analysis of superoxide dismutase activity and isozymes. J. Inorg. Chem. 16:71-77. Sussman, M. S., and G. B. Buckley, 1990. Oxygenderived free radicals in reperfusion injury. Methods Enzymol. 186:711-729. Waters, W. A., 1971. The kinetics and mechanism of metal-catalyzed autoxidation. J. Am. Oil Chem. Soc. 48:427-433. Yun, J., F. Shahidi, L. J. Rubin, and L. L. Diosady, 1987. Oxidative stability and flavor acceptability of nitrite-free meat-curing system. Can. Inst. Food Sri. Technol. J. 20:246-251.

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catalyzed by iron salts. Fed. Eur. Biol. Soc. Lett. 150:454-458. Gutteridge, J.M.C, 1984. Ferrous iron-EDTAstimulated phospholipid peroxidation. A reaction changing from alkoxyl-radical- to hydroxylradical-dependent initiation. Biochem. J. 224: 697-701. Gutteridge, J.M.C, R. Richmond, and B. Halliwell, 1979. Inhibition of the iron-catalyzed formation of hydroxyl radicals from superoxide and lipid peroxidation by desferrioxamine. Biochem. J. 184: 469-472. Halliwell, B., and J.M.C. Gutteridge, 1981. Formation of a thiobarbituric acid reactive substance from deoxyribose in the presence of iron salts. Fed. Eur. Biol. Soc. Lett. 128:347-352. Halliwell, B., and J.M.C. Gutteridge, 1986. Oxygen free radicals and iron in relation to biology and medicine: some problems and concepts. Arch. Biochem. Biophys. 246:501-514. Halliwell, B., and J.M.C. Gutteridge, 1990. Role of free radicals and catalytic metal ions in human diseases: an overview. Methods Enzymol. 186: 1-85. Igene, J. O., K. Yamaguchi, A. M. Pearson, and J. I. Gray, 1985. Mechanisms by which nitrite inhibits the development of warmed-over flavor (WOF) in cured meat. Food Chem. 18:1-18. Kanner, J., and L. Doll, 1991. Ferritin in turkey muscle tissue: a source of catalytic iron ions for lipid peroxidation. J. Agric. Food Chem. 39:247-249. Kanner, J., and S. Harel, 1985. Initiation of lipid peroxidation by activated metmyoglobin and methemoglobin. Arch. Biochem. Biophys. 237: 314-321. Kanner, J., S. Harel, and R. Jaffe, 1991. Lipid peroxidation of muscle food as affected by NaCl. J. Agric. Food Chem. 39:1017-1021. Kanner, J., I. Shegalovich, S. Harel, and B. Hazan, 1988. Muscle lipid peroxidation dependent on oxygen