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Role of deoxyhemoglobin in lipid oxidation of washed cod muscle mediated by trout, poultry and beef hemoglobins
Meat Science 62 (2002) 157–163 www.elsevier.com/locate/meatsci
Role of deoxyhemoglobin in lipid oxidation of washed cod muscle mediated by trout, poultry and beef hemoglobins Mark P. Richards*, Angela M. Modra, Rong Li University of Wisconsin-Madison, Muscle Biology and Meat Science Laboratory, 1805 Linden Dr. West, Madison, WI 53706, USA Received 9 October 2001; received in revised form 20 November 2001; accepted 20 November 2001
Abstract Deoxyhemoglobin content was measured in hemoglobins from trout, chicken and bovine sources between pH 5.5 and 7.5. With decreasing pH, deoxyhemoglobin content of trout was highest, low to intermediate in chicken, and lowest in beef hemoglobin. Each type of hemoglobin was added to washed cod muscle and lipid oxidation assessed during 2 C storage. The lipid oxidation rate was trout > > chicken > beef based on thiobarbituric reactive substances (TBARS) and lipid hydroperoxide formation. There was no significant difference in pro-oxidative activity of chicken compared to turkey hemoglobin. Hemoglobins from trout appeared to oxidize more rapidly compared to chicken hemoglobin in the washed cod muscle model system, as measured by a decrease in redness (a-value) during storage. Loss of red color was slowest in beef samples. These studies suggest that deoxyhemoglobin may be a major catalyst of lipid oxidation at post mortem pH values found in muscle foods, especially in fish and poultry compared to beef. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Deoxyhemoglobin; Hemoglobin; Oxygenation; Blood; Lipid oxidation; Trout; Cod; Beef; Chicken; Turkey
1. Introduction Lipid oxidation is a major cause of quality deterioration in muscle foods, and it results in off-odors, offflavors, color problems and texture defects (Kanner, 1994). Hemoglobin is a possible catalyst of lipid oxidation in muscle foods such as beef, poultry and fish. The protein consists of a globin portion plus a porphyrin heme, the latter containing an iron atom coordinated inside the heme ring. Hemoglobin is made up of four polypeptide chains with each chain containing one heme group. Hemoglobin is a tetramer per se although monomers and dimers can be present (Manning, Dumoulin, Li, & Manning, 1998). Just after death, nearly all the heme iron exists in the ferrous (+2) valence state. Oxygen can be bound to the ferrous iron (oxyhemoglobin) or the iron binding site can be vacant (deoxyhemoglobin). An important reaction related to the ability of hemoglobin to stimulate lipid oxidation is hemoglobin autoxidation. This occurs when oxygen is released from oxyhemoglobin to form ferric (+3) * Corresponding author. Tel.: +1-608-262-1792; fax: +1-608-2653110. E-mail address: mprichards@facstaff.wisc.edu (M.P. Richards).
methemoglobin and the superoxide anion radical (O 2 ). O 2 will readily be converted to H2O2, and the reaction of methemoglobin with H2O2 causes the formation of a ferryl protein radical, an initiator of lipid oxidation (Kanner & Harel, 1985). Detachment of ferric hemin from the globin can follow methemoglobin formation to promote lipid oxidation reactions (Everse & Hsia, 1997). Further, when a critical level of peroxides are present, iron can be released from hemin to participate in lipid oxidation processes (Puppo & Halliwell, 1988). An important consideration as to the role of hemoglobin as a catalyst of lipid oxidation in muscle foods is the residual hemoglobin content in muscle tissue, especially after bleeding. Kranen, van Kuppevelt, Goedhart, Veerkamp, Lambooy, and Veerkamp (1999) found that the only detectable heme pigment in breast muscle from bled broilers was hemoglobin while myoglobin was undetectable. In dark muscle of the broilers, 86% of the total heme protein was hemoglobin on a weight basis. In sockeye salmon, there was no significant difference in hemoglobin levels estimated in whole muscle from bled and unbled fish (Porter, Kennish, & Kramer, 1992) which suggest bleeding removed little hemoglobin from the muscle. Up to 30% and sometimes more of the total heme protein in beef from bled animals is hemoglobin
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(Fox, 1966). Thus, it appears that residual hemoglobin exist in muscle foods and has the potential to contribute to lipid oxidation reactions. There is evidence that deoxyhemoglobin accelerates lipid oxidation (Pietrzak & Miller, 1989; Richards & Hultin, 2000). Although the pathway by which deoxyhemoglobin stimulates lipid oxidation is not clear, trout hemoglobins are useful to study the effect of deoxyhemoglobin. This is because trout hemoglobins are largely deoxygenated in air atmospheres at post mortem pH values (Binotti, Giovenco, Giardina, Antonini, Brunori, & Wyman, 1971). The high content of deoxyhemoglobin at post mortem pH can be explained by the Bohr effect, which is defined as a decrease in oxygenation of hemoglobins with increasing H+ concentration between pH 7.4 and 6.5 (Stryer, 1988). A further decrease in hemoglobin oxygenation below pH 6.5 is termed the Root effect (Manning et al., 1998). There are indications that chicken hemoglobins are somewhat deoxygenated in breast muscle (pH 5.6–5.8) although some variation was observed (Millar, Wilson, Moss, & Ledward, 1994). More data is needed pertaining to the oxygenation of hemoglobins from animals used in food production at post mortem pH values and the effect of deoxyhemoglobin on lipid oxidation reactions. This study was undertaken to investigate the effect of deoxyhemoglobin on lipid oxidation using hemoglobins from aquatic and terrestrial animals as catalysts and washed cod muscle as a lipid substrate.
2. Materials and methods 2.1. Chemicals Bovine hemoglobin, tetraethoxypropane, cumene hydroperoxide, streptomycin sulfate, sodium heparin, ferrous sulfate, barium chloride, ammonium thiocyanate and tris [hydroxymethyl] aminomethane (Tris) were obtained from Sigma Chemical A/S (St. Louis, MO). All other chemicals used were analytical grade, and distilled, deionized water was used. 2.2. Blood collection Blood from trout, chickens, turkeys, and beef cows were obtained from campus sources. Approximately 4 parts of blood was drawn via a syringe containing 1 part of 150 mM NaCl and sodium heparin (120 Units/ml). Rainbow trout (Onchorhynchus mykiss) (25–30 cm) were bled from the caudal vein according to Rowley (1990) using aminobenzoic acid ethyl ester as an anesthetic. Chickens (Cornish Rock, 8–10 weeks old) and turkeys (Large Whites, closed flock UW-Madison, 18 weeks old) were bled from the brachial vein. Beef cows (Black Angus, 3–4 years old) were bled from the jugular vein. Hemoglobins were prepared within 24 h of blood collection.
2.3. Preparation of hemoglobins Four volumes of ice cold 1.7% NaCl in 1 mM Tris, pH 8.0, were added to heparinized blood and centrifuged (700 g for 10 min at 4 C) Beckman J-6B centrifuge (Beckman Instruments Inc., Palo Alto, CA). After removal of the plasma, the red blood cells were washed by suspending three times in 10 volumes of the above buffer (Fyhn, Fyhn, Davis, Powers, Fink, & Garlick, 1979). Cells were lysed in 3 volumes of 1 mM Tris, pH 8.0 for 1 h. One-tenth volume of 1 M NaCl was then added to aid in stromal removal before ultracentrifugation (28,000 g for 15 min at 4 C) using a Beckman L8– 70M ultracentrifuge (Beckman Instruments Inc., Palo Alto, CA). Hemolysates were then separated from low molecular weight components ( 6 kDa) using econo-pac DG-10 desalting columns (Bio-Rad, Hercules, CA). Hemoglobin solutions were stored at 80 C prior to use. 2.4. Quantifying hemoglobin levels The method of Brown (1961) was adapted. Concentrated hemoglobin solutions were diluted 500 times with 50 mM Tris pH 8.0 buffer. Around 1 mg of sodium dithionite was added to 1.5 ml of the hemolysate and mixed in a cuvette. Carbon monoxide gas (Badger Welding, Madison, WI) was then bubbled into the samples for 30 s. The sample was then scanned from 440 to 400 nm (Soret band) against a blank that contained only buffer using a Model UV-2401 double-beam spectrophotometer (Shimadzu Scientific Instruments Inc., Columbia, MD). The peak at 420 nm was recorded. Standard curves were constructed using twice-crystallized bovine hemoglobin (Sigma) and a molecular weight of 68,000 daltons. 2.5. Washed cod matrix Cod fish (Gadus morhua) fillets without skins were obtained from a local fish market that had the fillets delivered overnight via air transport from Boston, MA. The fillets were considered good to excellent quality based on appearance and odor. All dark muscle was removed. The rest of the fillets were ground in a Hobart 4612 mincer (Hobart Manufacturing Co., Troy, OH; plate diameter 3 mm). The mince was washed twice in distilled deionized water at a 1:3 mince to water ratio (w:w) by stirring with a plastic rod for 2 min. Subsequently, the mixture was allowed to stand for 15 min before dewatering with two layers of cotton cheesecloth. Mince was then mixed with 50 mM sodium phosphate buffer (pH 6.3) at the same 1:3 ratio and homogenized (setting 1) using a Polytron Type PT 10/35 (Brinkmann Instruments, Westbury, NY). It was allowed to stand for 15 min and finally centrifuged (15,000 g for 20 min at 4 C) using a Beckman L8–70M ultracentrifuge
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(Beckman Instruments Inc., Palo Alto, CA). The resulting pellet was then used as the washed cod muscle. 2.6. Addition of hemoglobins to washed cod muscle An appropriate volume of the hemoglobin stock was added to a final concentration of 5.8 mmol per kg washed cod and stirred with a plastic spatula for 3 min to distribute the heme protein. This level of hemoglobin was selected since it is near the range of hemoglobin levels found in light muscle of trout, sartorius muscle of bled broilers and L. dorsi muscle in bled beef (Gingerich, Pityer, & Rach, 1990; Kranen et al., 1999; Warriss & Rhodes, 1977). Streptomycin sulfate (200 ppm) was added to inhibit microbial growth during storage. The pH of samples was checked just after addition of hemoglobin, periodically during storage and finally at the end of storage. To measure pH, around 0.5 g of sample was diluted in 10 vol of distilled deionized water, homogenized, and readings were recorded using an Accumet AR50 pH meter (Fisher Scientific, Pittsburgh, PA). The final moisture content of the samples stored at 2 C was adjusted to 88%. pH was adjusted if necessary by addition of 1 M NaOH or 1 M HCl.
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Westbury, NY). Subsequently, the polytron was rinsed for 30 s with 5 ml solvent. The homogenate and wash solution were then combined. Three ml 0.5% NaCl was added and the mixture was mixed for 30 s with a Vortex before centrifugation for 10 min (4 C and 700 g) to separate the mixture into two phases. Then 1.33 ml of ice cold chloroform / methanol (1:1) was added to 2 ml of the lower phase and mixed briefly. Twenty-five microliters ammonium thiocyanate (4.38 M) and 25 ml iron(II)chloride (18 mM) were added to the assay for lipid hydroperoxides (Shantha & Decker, 1994) and samples were incubated for 20 min at room temperature before the absorbances at 500 nm were determined. A standard curve was prepared using cumene hydroperoxide. The chloroform used contained ethanol as a preservative to eliminate high blank readings (Richards & Feng, 2000). 2.10. Color measurements The ‘‘a’’ values were measured with a Minolta CR-200 chroma meter (Minolta Camera Co., Osaka, Japan). A white calibration plate supplied with the unit was used to calibrate the instrument. 2.11. Statistical evaluations
2.7. Measuring the relative oxygenation of hemoglobin Solutions containing hemoglobin were scanned from 630 to 500 nm using a double-beam spectrophotometer model UV-2401 (PC) (Shimadzu Instruments, Inc., Columbia, MD). The blank contained only buffer. The absorbance at the peak (575 nm) minus the absorbance at the valley (560 nm) was calculated. Larger differences indicated that the hemoglobin was more highly oxygenated (Pelster & Weber, 1991). These experiments were run at atmospheric conditions. 2.8. Determination of thiobarbituric acid reactive substances (TBARS) TBARS were determined according to a modified procedure of Buege and Aust (1978). Fifty percent trichloroacetic acid (TCA) containing 1.3% thiobarbituric acid (TBA) was heated to 65 C on the day of use to dissolve the TBA. Approximately 150 mg of sample was added to 1.1 ml of the TCA-TBA mixture and incubated for 1 h at 65 C. After centrifugation (2500 g for 10 min), the absorbance of the supernatant at 532 nm was determined. A standard curve was constructed using tetraethoxypropane. 2.9. Determination of lipid hydroperoxides Between 0.4 and 0.5 g washed cod muscle was homogenized in 5 ml of chloroform/methanol (1:1) for 30 s using a Polytron Type PT 10/35 (Brinkmann Instruments,
All experiments were done at least in duplicate and in each experiment at least two replicates were done. Analysis of variance with a MIXED procedure of the SAS system was used to evaluate data from storage studies (Cody & Smith, 1997). Means were separated using differences of least squares.
3. Results The oxygenation of beef, chicken, and trout hemoglobins were analyzed in the pH range of 5.5–7.5. This pH range was chosen since it encompasses pH values found in post mortem muscle tissue. At pH 7.3 there was little difference in oxygenation among the species (Fig. 1). However, trout hemoglobins exhibit low oxygenation from pH 5.5 to 6.75 compared to the terrestrial species. A sharp increase in oxygenation of trout hemoglobins occurred from pH 6.75 to 7.25. This is in agreement with the findings of Binotti et al. (1971). Oxygenation of chicken hemoglobin was intermediate between fish and beef at pH 6.3. The beef hemoglobins were highly oxygenated over the entire pH range. Chicken hemoglobin showed a gradual decline in oxygenation as pH was decreased over the entire pH range. No significant difference in hemoglobin oxygenation was detected between turkey and chicken hemoglobins at pH 6.3 (data not shown). Beef, chicken and trout hemoglobins were added separately to washed cod muscle at a final concentration
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Fig. 1. Relative oxygenation of trout, chicken, and beef hemoglobins in 20 mM sodium phosphate buffer at various pH values. The hemoglobin concentration was 3 mM. An increase in oxygenation is indicated by an increase in absorbance.
of 5.8 mmol/kg washed cod muscle and adjusted to pH 6.3 to assess the ability of each hemoglobin type to initiate lipid oxidation. pH 6.3 was chosen since this is a typical post mortem pH found in various muscle foods and because at pH 6.3 there were large differences in hemoglobin oxygenation among beef, chicken and fish (Fig. 1). Fig. 2 shows that trout hemoglobins initiated lipid oxidation most rapidly compared to chicken and beef hemoglobins during 2 C storage, as indicated by increase in TBARS (P < 0.01). Chicken hemoglobins caused TBARS to develop more rapidly than beef hemoglobins (P < 0.05). Formation of lipid hydroperoxides was determined as a second indicator of lipid oxidation induced by trout, chicken, turkey and beef hemoglobins added to washed cod muscle. The same trends were observed as when TBARS were determined. Trout hemoglobins promoted the most rapid lipid hydroperoxide formation while
Fig. 2. TBARS development of washed cod muscle with added trout, chicken, or beef hemoglobins (5.8 mmol/kg washed cod) at pH 6.3.
chicken hemoglobins were intermediate between trout and beef hemoglobins (Fig. 3). There was no significant difference between chicken and turkey hemoglobin mediated lipid hydroperoxide or TBARS development in washed cod muscle (Fig. 4). Change in redness was determined in washed cod muscle containing trout, chicken or beef hemoglobins at pH 6.3. The lower zero-time redness values in trout hemoglobin containing samples is indicative of their lower hemoglobin oxygenation compared to bovine and chicken hemoglobins (Fig. 5). During storage, loss of red color occurred most rapidly in the trout samples, intermediate in the chicken samples, and slowest in samples containing beef hemoglobin.
Fig. 3. Lipid hydroperoxide development of washed cod muscle with added trout, chicken, or beef hemoglobins (5.8 mmol/kg washed cod) at pH 6.3.
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Fig. 4. Lipid hydroperoxides and TBARS of washed cod with added chicken or turkey hemoglobins (5.8 mmol/kg washed cod) at pH 6.3.
Fig. 5. Changes in a-values (redness) during 2 C storage of washed cod muscle containing trout, chicken, or beef hemoglobins at pH 6.3 (5.8 mmol per kg washed cod muscle).
4. Discussion At post mortem pH values, trout hemoglobin was largely deoxygenated compared to bovine hemoglobin, while chicken hemoglobin had an intermediate level of deoxyhemoglobin content (Fig. 1). Varying oxygenation of hemoglobins among animal species is relevant since
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there is evidence that deoxyhemoglobin accelerates lipid oxidation reactions (Pietrzak & Miller, 1989). If in fact deoxyhemoglobin accelerates lipid oxidation, the poorly oxygenated trout hemoglobin would be expected to stimulate lipid oxidation more readily than chicken hemoglobin, and chicken hemoglobin would be a better catalyst than the highly oxygenated bovine hemoglobin. Indeed, rates of lipid oxidation increased as deoxyhemoglobin content increased when trout, chicken and bovine hemoglobins were added to washed cod muscle (Fig. 2). Hemoglobin from trout is largely deoxygenated at pH 6.3 due to a strong Bohr effect (a decrease in hemoglobin oxygenation with decreasing pH). Note the higher oxygenation of trout hemoglobin at pH 7.25. Trout possess anodic and cathodic types of hemoglobin. The anodic hemoglobins have low oxygen affinity at pH 6.3 whereas cathodic hemoglobins bind oxygen strongly independent of pH (Harrington, 1986). Anodic hemoglobins were found to promote lipid oxidation more rapidly than cathodic hemoglobins (Richards, Østdal, & Andersen, 2002). The need for trout to have these different hemoglobins is attributed to the variety of environmental oxygen pressures that these fish inhabit (Zolese, Gabbianelli, Caulini, Bertoli, & Falconi, 1999). Highly active pelagic species of fish, such as mackerel, are noted for having very strong Bohr effects (Riggs, 1970). Thus, the likelihood that substantial levels of deoxyhemoglobin exist in muscle of mackerel may be one reason that rapid rates of lipid oxidation occur during refrigerated and frozen storage (Hwang & Regenstein, 1996). The rapid lipid oxidation that occurs in mackerel muscle is a major reason these fish remain underutilized. Mackerel hemoglobins were found to promote lipid oxidation in washed cod more rapidly than trout hemoglobins (Richards & Hultin, 2002). Chicken hemoglobin had intermediate levels of deoxyhemoglobin compared to trout and beef hemoglobin at pH 6.3. Tetramer-tetramer hemoglobin associations in birds have been described which causes decreased affinity for oxygen (Riggs, 1998). A possible explanation for the low oxygen affinity was that it would increase the amount of oxygen available to tissues to sustain flight. At times of intense activity, acidification of the muscle is common (Bonafe, Matsukuma, & Matsuura, 1999). Thus, the decline in pH that occurs post mortem may establish conditions that cause tetramers of chicken hemoglobin to associate and thereby increase the content of deoxyhemoglobin in the muscle. To compare trout, chicken and beef hemoglobins, each heme protein was added to washed cod muscle. Washing removes endogenous antioxidants and the high degree of polyunsaturated fatty acids (Sigurgisladottir & Palmadottir, 1993) provide a lipid substrate that oxidizes in a reasonable amount of time. Fatty acids from chicken and beef muscle are more saturated and
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therefore likely less susceptible to oxidation than the lipids in washed cod muscle. Cod muscle also has low levels of iron and heme proteins that are further reduced by washing and, thereby, do not interfere with the effect of added hemoglobins. The presence of deoxyhemoglobin has been shown to accelerate methemoglobin formation compared to fully oxygenated hemoglobin (Shikama & Matsuoka, 1986). To inhibit lipid oxidation, it is likely imperative to prevent methemoglobin formation since the reaction of methemoglobin with peroxide results in the formation of a ferryl hemoglobin radical that is capable of initiating lipid oxidation (Kanner, German, & Kinsella, 1987). In addition, methemoglobin is at least 60 times more likely to release its heme group compared to oxyhemoglobin or deoxyhemoglobin (Hargrove, Wilkinson, & Olson, 1996); released heme is a possible catalyst of lipid oxidation (Ryter & Tyrrell, 2000). One explanation for deoxyhemoglobin-accelerated methemoglobin formation involves access of oxidants to the heme crevice. H2O2 can oxidize deoxyhemoglobin around 100 times faster than oxyhemoglobin, which leads to formation of methemoglobin (Shikama, 1998). Oxyhemoglobin is more compact than deoxyhemoglobin which may hinder access of H2O2 to the oxygenated molecule (Stryer, 1988). In the absence of H2O2, it has been proposed that methemoglobin and O 2 formation occurs in tissues due to the oxidation of deoxyhemoglobin by free O2 (Brown & Mebine, 1969). Loss of red color in the washed cod muscle can be used to estimate the conversion of oxyhemoglobin (red) to methemoglobin (brown). The fact that the loss in red color occurred most rapidly in samples containing trout hemoglobins compared to bovine or chicken (Fig. 4) suggest that the elevated level of deoxyhemoglobin present in trout hemoglobins accelerated methemoglobin formation in the washed cod muscle. Just after death, molecular oxygen (O2) in muscle is quickly consumed in an attempt to maintain cells in a charged energy state. This causes the interior of intact muscle to become anaerobic during post mortem storage but not at the surfaces. O2 from the atmosphere penetrates 1–4 mm into the muscle (Lawrie, 1974). The surfaces of the muscle are more prone to lipid oxidation than the interior since O2 is required for the lipid oxidation process to be manifested (Labuza, 1971). Apparently O2 is pulled off of heme proteins in the interior of muscle; the purple color of the interior portion of beef muscle that has just been cut into indicates that most of the heme protein is in the deoxygenated form. Therefore when lipids in the interior of muscle are less oxidized than the exterior, it is likely that the lack of molecular O2 in the interior prevents lipid oxidation rather than deoxyhemoglobin being an inadequate catalyst. Similarly, anaerobic packaging generally decelerates rates of lipid oxidation by preventing O2 penetration. When mincing muscle tissue in air, the reintroduction of O2 into the
interior of the muscle not only provides O2 substrate to oxidize fatty acids but also an opportunity for any deoxyhemoglobin that is present (via a Bohr effect) to accelerate lipid oxidation. In conclusion, these studies suggest that the elevated levels of deoxyhemoglobin determined in fish and poultry hemoglobins compared to bovine hemoglobin causes hemoglobins of fish and poultry to be better catalysts of lipid oxidation. This is based not only on the increased rates of lipid oxidation mediated by the fish and poultry hemoglobins compared to bovine hemoglobin in these studies but also previous evidence that deoxyhemoglobin accelerates lipid oxidation compared to more oxygenated hemoglobins (Richards et al., submitted for publication). Future work should continue to investigate the role of deoxyhemoglobin in lipid oxidation processes that occur in muscle foods. Further, the oxygenation of trout myoglobin and its ability to promote lipid oxidation should be determined at post mortem pH values in relation to the rapid rate of lipid oxidation caused by the poorly oxygenated trout hemoglobins.
Acknowledgements This work was supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison, HATCH project WIS04512.
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Role of deoxyhemoglobin in lipid oxidation of washed cod muscle mediated by trout, poultry and beef hemoglobins Mark P. Richards*, Angela M. Modra, Rong Li University of Wisconsin-Madison, Muscle Biology and Meat Science Laboratory, 1805 Linden Dr. West, Madison, WI 53706, USA Received 9 October 2001; received in revised form 20 November 2001; accepted 20 November 2001
Abstract Deoxyhemoglobin content was measured in hemoglobins from trout, chicken and bovine sources between pH 5.5 and 7.5. With decreasing pH, deoxyhemoglobin content of trout was highest, low to intermediate in chicken, and lowest in beef hemoglobin. Each type of hemoglobin was added to washed cod muscle and lipid oxidation assessed during 2 C storage. The lipid oxidation rate was trout > > chicken > beef based on thiobarbituric reactive substances (TBARS) and lipid hydroperoxide formation. There was no significant difference in pro-oxidative activity of chicken compared to turkey hemoglobin. Hemoglobins from trout appeared to oxidize more rapidly compared to chicken hemoglobin in the washed cod muscle model system, as measured by a decrease in redness (a-value) during storage. Loss of red color was slowest in beef samples. These studies suggest that deoxyhemoglobin may be a major catalyst of lipid oxidation at post mortem pH values found in muscle foods, especially in fish and poultry compared to beef. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Deoxyhemoglobin; Hemoglobin; Oxygenation; Blood; Lipid oxidation; Trout; Cod; Beef; Chicken; Turkey
1. Introduction Lipid oxidation is a major cause of quality deterioration in muscle foods, and it results in off-odors, offflavors, color problems and texture defects (Kanner, 1994). Hemoglobin is a possible catalyst of lipid oxidation in muscle foods such as beef, poultry and fish. The protein consists of a globin portion plus a porphyrin heme, the latter containing an iron atom coordinated inside the heme ring. Hemoglobin is made up of four polypeptide chains with each chain containing one heme group. Hemoglobin is a tetramer per se although monomers and dimers can be present (Manning, Dumoulin, Li, & Manning, 1998). Just after death, nearly all the heme iron exists in the ferrous (+2) valence state. Oxygen can be bound to the ferrous iron (oxyhemoglobin) or the iron binding site can be vacant (deoxyhemoglobin). An important reaction related to the ability of hemoglobin to stimulate lipid oxidation is hemoglobin autoxidation. This occurs when oxygen is released from oxyhemoglobin to form ferric (+3) * Corresponding author. Tel.: +1-608-262-1792; fax: +1-608-2653110. E-mail address: mprichards@facstaff.wisc.edu (M.P. Richards).
methemoglobin and the superoxide anion radical (O 2 ). O 2 will readily be converted to H2O2, and the reaction of methemoglobin with H2O2 causes the formation of a ferryl protein radical, an initiator of lipid oxidation (Kanner & Harel, 1985). Detachment of ferric hemin from the globin can follow methemoglobin formation to promote lipid oxidation reactions (Everse & Hsia, 1997). Further, when a critical level of peroxides are present, iron can be released from hemin to participate in lipid oxidation processes (Puppo & Halliwell, 1988). An important consideration as to the role of hemoglobin as a catalyst of lipid oxidation in muscle foods is the residual hemoglobin content in muscle tissue, especially after bleeding. Kranen, van Kuppevelt, Goedhart, Veerkamp, Lambooy, and Veerkamp (1999) found that the only detectable heme pigment in breast muscle from bled broilers was hemoglobin while myoglobin was undetectable. In dark muscle of the broilers, 86% of the total heme protein was hemoglobin on a weight basis. In sockeye salmon, there was no significant difference in hemoglobin levels estimated in whole muscle from bled and unbled fish (Porter, Kennish, & Kramer, 1992) which suggest bleeding removed little hemoglobin from the muscle. Up to 30% and sometimes more of the total heme protein in beef from bled animals is hemoglobin
0309-1740/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0309-1740(01)00242-X
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(Fox, 1966). Thus, it appears that residual hemoglobin exist in muscle foods and has the potential to contribute to lipid oxidation reactions. There is evidence that deoxyhemoglobin accelerates lipid oxidation (Pietrzak & Miller, 1989; Richards & Hultin, 2000). Although the pathway by which deoxyhemoglobin stimulates lipid oxidation is not clear, trout hemoglobins are useful to study the effect of deoxyhemoglobin. This is because trout hemoglobins are largely deoxygenated in air atmospheres at post mortem pH values (Binotti, Giovenco, Giardina, Antonini, Brunori, & Wyman, 1971). The high content of deoxyhemoglobin at post mortem pH can be explained by the Bohr effect, which is defined as a decrease in oxygenation of hemoglobins with increasing H+ concentration between pH 7.4 and 6.5 (Stryer, 1988). A further decrease in hemoglobin oxygenation below pH 6.5 is termed the Root effect (Manning et al., 1998). There are indications that chicken hemoglobins are somewhat deoxygenated in breast muscle (pH 5.6–5.8) although some variation was observed (Millar, Wilson, Moss, & Ledward, 1994). More data is needed pertaining to the oxygenation of hemoglobins from animals used in food production at post mortem pH values and the effect of deoxyhemoglobin on lipid oxidation reactions. This study was undertaken to investigate the effect of deoxyhemoglobin on lipid oxidation using hemoglobins from aquatic and terrestrial animals as catalysts and washed cod muscle as a lipid substrate.
2. Materials and methods 2.1. Chemicals Bovine hemoglobin, tetraethoxypropane, cumene hydroperoxide, streptomycin sulfate, sodium heparin, ferrous sulfate, barium chloride, ammonium thiocyanate and tris [hydroxymethyl] aminomethane (Tris) were obtained from Sigma Chemical A/S (St. Louis, MO). All other chemicals used were analytical grade, and distilled, deionized water was used. 2.2. Blood collection Blood from trout, chickens, turkeys, and beef cows were obtained from campus sources. Approximately 4 parts of blood was drawn via a syringe containing 1 part of 150 mM NaCl and sodium heparin (120 Units/ml). Rainbow trout (Onchorhynchus mykiss) (25–30 cm) were bled from the caudal vein according to Rowley (1990) using aminobenzoic acid ethyl ester as an anesthetic. Chickens (Cornish Rock, 8–10 weeks old) and turkeys (Large Whites, closed flock UW-Madison, 18 weeks old) were bled from the brachial vein. Beef cows (Black Angus, 3–4 years old) were bled from the jugular vein. Hemoglobins were prepared within 24 h of blood collection.
2.3. Preparation of hemoglobins Four volumes of ice cold 1.7% NaCl in 1 mM Tris, pH 8.0, were added to heparinized blood and centrifuged (700 g for 10 min at 4 C) Beckman J-6B centrifuge (Beckman Instruments Inc., Palo Alto, CA). After removal of the plasma, the red blood cells were washed by suspending three times in 10 volumes of the above buffer (Fyhn, Fyhn, Davis, Powers, Fink, & Garlick, 1979). Cells were lysed in 3 volumes of 1 mM Tris, pH 8.0 for 1 h. One-tenth volume of 1 M NaCl was then added to aid in stromal removal before ultracentrifugation (28,000 g for 15 min at 4 C) using a Beckman L8– 70M ultracentrifuge (Beckman Instruments Inc., Palo Alto, CA). Hemolysates were then separated from low molecular weight components ( 6 kDa) using econo-pac DG-10 desalting columns (Bio-Rad, Hercules, CA). Hemoglobin solutions were stored at 80 C prior to use. 2.4. Quantifying hemoglobin levels The method of Brown (1961) was adapted. Concentrated hemoglobin solutions were diluted 500 times with 50 mM Tris pH 8.0 buffer. Around 1 mg of sodium dithionite was added to 1.5 ml of the hemolysate and mixed in a cuvette. Carbon monoxide gas (Badger Welding, Madison, WI) was then bubbled into the samples for 30 s. The sample was then scanned from 440 to 400 nm (Soret band) against a blank that contained only buffer using a Model UV-2401 double-beam spectrophotometer (Shimadzu Scientific Instruments Inc., Columbia, MD). The peak at 420 nm was recorded. Standard curves were constructed using twice-crystallized bovine hemoglobin (Sigma) and a molecular weight of 68,000 daltons. 2.5. Washed cod matrix Cod fish (Gadus morhua) fillets without skins were obtained from a local fish market that had the fillets delivered overnight via air transport from Boston, MA. The fillets were considered good to excellent quality based on appearance and odor. All dark muscle was removed. The rest of the fillets were ground in a Hobart 4612 mincer (Hobart Manufacturing Co., Troy, OH; plate diameter 3 mm). The mince was washed twice in distilled deionized water at a 1:3 mince to water ratio (w:w) by stirring with a plastic rod for 2 min. Subsequently, the mixture was allowed to stand for 15 min before dewatering with two layers of cotton cheesecloth. Mince was then mixed with 50 mM sodium phosphate buffer (pH 6.3) at the same 1:3 ratio and homogenized (setting 1) using a Polytron Type PT 10/35 (Brinkmann Instruments, Westbury, NY). It was allowed to stand for 15 min and finally centrifuged (15,000 g for 20 min at 4 C) using a Beckman L8–70M ultracentrifuge
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(Beckman Instruments Inc., Palo Alto, CA). The resulting pellet was then used as the washed cod muscle. 2.6. Addition of hemoglobins to washed cod muscle An appropriate volume of the hemoglobin stock was added to a final concentration of 5.8 mmol per kg washed cod and stirred with a plastic spatula for 3 min to distribute the heme protein. This level of hemoglobin was selected since it is near the range of hemoglobin levels found in light muscle of trout, sartorius muscle of bled broilers and L. dorsi muscle in bled beef (Gingerich, Pityer, & Rach, 1990; Kranen et al., 1999; Warriss & Rhodes, 1977). Streptomycin sulfate (200 ppm) was added to inhibit microbial growth during storage. The pH of samples was checked just after addition of hemoglobin, periodically during storage and finally at the end of storage. To measure pH, around 0.5 g of sample was diluted in 10 vol of distilled deionized water, homogenized, and readings were recorded using an Accumet AR50 pH meter (Fisher Scientific, Pittsburgh, PA). The final moisture content of the samples stored at 2 C was adjusted to 88%. pH was adjusted if necessary by addition of 1 M NaOH or 1 M HCl.
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Westbury, NY). Subsequently, the polytron was rinsed for 30 s with 5 ml solvent. The homogenate and wash solution were then combined. Three ml 0.5% NaCl was added and the mixture was mixed for 30 s with a Vortex before centrifugation for 10 min (4 C and 700 g) to separate the mixture into two phases. Then 1.33 ml of ice cold chloroform / methanol (1:1) was added to 2 ml of the lower phase and mixed briefly. Twenty-five microliters ammonium thiocyanate (4.38 M) and 25 ml iron(II)chloride (18 mM) were added to the assay for lipid hydroperoxides (Shantha & Decker, 1994) and samples were incubated for 20 min at room temperature before the absorbances at 500 nm were determined. A standard curve was prepared using cumene hydroperoxide. The chloroform used contained ethanol as a preservative to eliminate high blank readings (Richards & Feng, 2000). 2.10. Color measurements The ‘‘a’’ values were measured with a Minolta CR-200 chroma meter (Minolta Camera Co., Osaka, Japan). A white calibration plate supplied with the unit was used to calibrate the instrument. 2.11. Statistical evaluations
2.7. Measuring the relative oxygenation of hemoglobin Solutions containing hemoglobin were scanned from 630 to 500 nm using a double-beam spectrophotometer model UV-2401 (PC) (Shimadzu Instruments, Inc., Columbia, MD). The blank contained only buffer. The absorbance at the peak (575 nm) minus the absorbance at the valley (560 nm) was calculated. Larger differences indicated that the hemoglobin was more highly oxygenated (Pelster & Weber, 1991). These experiments were run at atmospheric conditions. 2.8. Determination of thiobarbituric acid reactive substances (TBARS) TBARS were determined according to a modified procedure of Buege and Aust (1978). Fifty percent trichloroacetic acid (TCA) containing 1.3% thiobarbituric acid (TBA) was heated to 65 C on the day of use to dissolve the TBA. Approximately 150 mg of sample was added to 1.1 ml of the TCA-TBA mixture and incubated for 1 h at 65 C. After centrifugation (2500 g for 10 min), the absorbance of the supernatant at 532 nm was determined. A standard curve was constructed using tetraethoxypropane. 2.9. Determination of lipid hydroperoxides Between 0.4 and 0.5 g washed cod muscle was homogenized in 5 ml of chloroform/methanol (1:1) for 30 s using a Polytron Type PT 10/35 (Brinkmann Instruments,
All experiments were done at least in duplicate and in each experiment at least two replicates were done. Analysis of variance with a MIXED procedure of the SAS system was used to evaluate data from storage studies (Cody & Smith, 1997). Means were separated using differences of least squares.
3. Results The oxygenation of beef, chicken, and trout hemoglobins were analyzed in the pH range of 5.5–7.5. This pH range was chosen since it encompasses pH values found in post mortem muscle tissue. At pH 7.3 there was little difference in oxygenation among the species (Fig. 1). However, trout hemoglobins exhibit low oxygenation from pH 5.5 to 6.75 compared to the terrestrial species. A sharp increase in oxygenation of trout hemoglobins occurred from pH 6.75 to 7.25. This is in agreement with the findings of Binotti et al. (1971). Oxygenation of chicken hemoglobin was intermediate between fish and beef at pH 6.3. The beef hemoglobins were highly oxygenated over the entire pH range. Chicken hemoglobin showed a gradual decline in oxygenation as pH was decreased over the entire pH range. No significant difference in hemoglobin oxygenation was detected between turkey and chicken hemoglobins at pH 6.3 (data not shown). Beef, chicken and trout hemoglobins were added separately to washed cod muscle at a final concentration
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Fig. 1. Relative oxygenation of trout, chicken, and beef hemoglobins in 20 mM sodium phosphate buffer at various pH values. The hemoglobin concentration was 3 mM. An increase in oxygenation is indicated by an increase in absorbance.
of 5.8 mmol/kg washed cod muscle and adjusted to pH 6.3 to assess the ability of each hemoglobin type to initiate lipid oxidation. pH 6.3 was chosen since this is a typical post mortem pH found in various muscle foods and because at pH 6.3 there were large differences in hemoglobin oxygenation among beef, chicken and fish (Fig. 1). Fig. 2 shows that trout hemoglobins initiated lipid oxidation most rapidly compared to chicken and beef hemoglobins during 2 C storage, as indicated by increase in TBARS (P < 0.01). Chicken hemoglobins caused TBARS to develop more rapidly than beef hemoglobins (P < 0.05). Formation of lipid hydroperoxides was determined as a second indicator of lipid oxidation induced by trout, chicken, turkey and beef hemoglobins added to washed cod muscle. The same trends were observed as when TBARS were determined. Trout hemoglobins promoted the most rapid lipid hydroperoxide formation while
Fig. 2. TBARS development of washed cod muscle with added trout, chicken, or beef hemoglobins (5.8 mmol/kg washed cod) at pH 6.3.
chicken hemoglobins were intermediate between trout and beef hemoglobins (Fig. 3). There was no significant difference between chicken and turkey hemoglobin mediated lipid hydroperoxide or TBARS development in washed cod muscle (Fig. 4). Change in redness was determined in washed cod muscle containing trout, chicken or beef hemoglobins at pH 6.3. The lower zero-time redness values in trout hemoglobin containing samples is indicative of their lower hemoglobin oxygenation compared to bovine and chicken hemoglobins (Fig. 5). During storage, loss of red color occurred most rapidly in the trout samples, intermediate in the chicken samples, and slowest in samples containing beef hemoglobin.
Fig. 3. Lipid hydroperoxide development of washed cod muscle with added trout, chicken, or beef hemoglobins (5.8 mmol/kg washed cod) at pH 6.3.
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Fig. 4. Lipid hydroperoxides and TBARS of washed cod with added chicken or turkey hemoglobins (5.8 mmol/kg washed cod) at pH 6.3.
Fig. 5. Changes in a-values (redness) during 2 C storage of washed cod muscle containing trout, chicken, or beef hemoglobins at pH 6.3 (5.8 mmol per kg washed cod muscle).
4. Discussion At post mortem pH values, trout hemoglobin was largely deoxygenated compared to bovine hemoglobin, while chicken hemoglobin had an intermediate level of deoxyhemoglobin content (Fig. 1). Varying oxygenation of hemoglobins among animal species is relevant since
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there is evidence that deoxyhemoglobin accelerates lipid oxidation reactions (Pietrzak & Miller, 1989). If in fact deoxyhemoglobin accelerates lipid oxidation, the poorly oxygenated trout hemoglobin would be expected to stimulate lipid oxidation more readily than chicken hemoglobin, and chicken hemoglobin would be a better catalyst than the highly oxygenated bovine hemoglobin. Indeed, rates of lipid oxidation increased as deoxyhemoglobin content increased when trout, chicken and bovine hemoglobins were added to washed cod muscle (Fig. 2). Hemoglobin from trout is largely deoxygenated at pH 6.3 due to a strong Bohr effect (a decrease in hemoglobin oxygenation with decreasing pH). Note the higher oxygenation of trout hemoglobin at pH 7.25. Trout possess anodic and cathodic types of hemoglobin. The anodic hemoglobins have low oxygen affinity at pH 6.3 whereas cathodic hemoglobins bind oxygen strongly independent of pH (Harrington, 1986). Anodic hemoglobins were found to promote lipid oxidation more rapidly than cathodic hemoglobins (Richards, Østdal, & Andersen, 2002). The need for trout to have these different hemoglobins is attributed to the variety of environmental oxygen pressures that these fish inhabit (Zolese, Gabbianelli, Caulini, Bertoli, & Falconi, 1999). Highly active pelagic species of fish, such as mackerel, are noted for having very strong Bohr effects (Riggs, 1970). Thus, the likelihood that substantial levels of deoxyhemoglobin exist in muscle of mackerel may be one reason that rapid rates of lipid oxidation occur during refrigerated and frozen storage (Hwang & Regenstein, 1996). The rapid lipid oxidation that occurs in mackerel muscle is a major reason these fish remain underutilized. Mackerel hemoglobins were found to promote lipid oxidation in washed cod more rapidly than trout hemoglobins (Richards & Hultin, 2002). Chicken hemoglobin had intermediate levels of deoxyhemoglobin compared to trout and beef hemoglobin at pH 6.3. Tetramer-tetramer hemoglobin associations in birds have been described which causes decreased affinity for oxygen (Riggs, 1998). A possible explanation for the low oxygen affinity was that it would increase the amount of oxygen available to tissues to sustain flight. At times of intense activity, acidification of the muscle is common (Bonafe, Matsukuma, & Matsuura, 1999). Thus, the decline in pH that occurs post mortem may establish conditions that cause tetramers of chicken hemoglobin to associate and thereby increase the content of deoxyhemoglobin in the muscle. To compare trout, chicken and beef hemoglobins, each heme protein was added to washed cod muscle. Washing removes endogenous antioxidants and the high degree of polyunsaturated fatty acids (Sigurgisladottir & Palmadottir, 1993) provide a lipid substrate that oxidizes in a reasonable amount of time. Fatty acids from chicken and beef muscle are more saturated and
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therefore likely less susceptible to oxidation than the lipids in washed cod muscle. Cod muscle also has low levels of iron and heme proteins that are further reduced by washing and, thereby, do not interfere with the effect of added hemoglobins. The presence of deoxyhemoglobin has been shown to accelerate methemoglobin formation compared to fully oxygenated hemoglobin (Shikama & Matsuoka, 1986). To inhibit lipid oxidation, it is likely imperative to prevent methemoglobin formation since the reaction of methemoglobin with peroxide results in the formation of a ferryl hemoglobin radical that is capable of initiating lipid oxidation (Kanner, German, & Kinsella, 1987). In addition, methemoglobin is at least 60 times more likely to release its heme group compared to oxyhemoglobin or deoxyhemoglobin (Hargrove, Wilkinson, & Olson, 1996); released heme is a possible catalyst of lipid oxidation (Ryter & Tyrrell, 2000). One explanation for deoxyhemoglobin-accelerated methemoglobin formation involves access of oxidants to the heme crevice. H2O2 can oxidize deoxyhemoglobin around 100 times faster than oxyhemoglobin, which leads to formation of methemoglobin (Shikama, 1998). Oxyhemoglobin is more compact than deoxyhemoglobin which may hinder access of H2O2 to the oxygenated molecule (Stryer, 1988). In the absence of H2O2, it has been proposed that methemoglobin and O 2 formation occurs in tissues due to the oxidation of deoxyhemoglobin by free O2 (Brown & Mebine, 1969). Loss of red color in the washed cod muscle can be used to estimate the conversion of oxyhemoglobin (red) to methemoglobin (brown). The fact that the loss in red color occurred most rapidly in samples containing trout hemoglobins compared to bovine or chicken (Fig. 4) suggest that the elevated level of deoxyhemoglobin present in trout hemoglobins accelerated methemoglobin formation in the washed cod muscle. Just after death, molecular oxygen (O2) in muscle is quickly consumed in an attempt to maintain cells in a charged energy state. This causes the interior of intact muscle to become anaerobic during post mortem storage but not at the surfaces. O2 from the atmosphere penetrates 1–4 mm into the muscle (Lawrie, 1974). The surfaces of the muscle are more prone to lipid oxidation than the interior since O2 is required for the lipid oxidation process to be manifested (Labuza, 1971). Apparently O2 is pulled off of heme proteins in the interior of muscle; the purple color of the interior portion of beef muscle that has just been cut into indicates that most of the heme protein is in the deoxygenated form. Therefore when lipids in the interior of muscle are less oxidized than the exterior, it is likely that the lack of molecular O2 in the interior prevents lipid oxidation rather than deoxyhemoglobin being an inadequate catalyst. Similarly, anaerobic packaging generally decelerates rates of lipid oxidation by preventing O2 penetration. When mincing muscle tissue in air, the reintroduction of O2 into the
interior of the muscle not only provides O2 substrate to oxidize fatty acids but also an opportunity for any deoxyhemoglobin that is present (via a Bohr effect) to accelerate lipid oxidation. In conclusion, these studies suggest that the elevated levels of deoxyhemoglobin determined in fish and poultry hemoglobins compared to bovine hemoglobin causes hemoglobins of fish and poultry to be better catalysts of lipid oxidation. This is based not only on the increased rates of lipid oxidation mediated by the fish and poultry hemoglobins compared to bovine hemoglobin in these studies but also previous evidence that deoxyhemoglobin accelerates lipid oxidation compared to more oxygenated hemoglobins (Richards et al., submitted for publication). Future work should continue to investigate the role of deoxyhemoglobin in lipid oxidation processes that occur in muscle foods. Further, the oxygenation of trout myoglobin and its ability to promote lipid oxidation should be determined at post mortem pH values in relation to the rapid rate of lipid oxidation caused by the poorly oxygenated trout hemoglobins.
Acknowledgements This work was supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison, HATCH project WIS04512.
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