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Nitric oxide synthase activity in muscle foods
Meat Science 62 (2002) 229–235 www.elsevier.com/locate/meatsci
Nitric oxide synthase activity in muscle foods Robert G. Brannan, Eric A. Decker* Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA Received 25 September 2001; received in revised form 3 December 2001; accepted 3 December 2001
Abstract Nitric oxide is enzymatically produced in animals by nitric oxide synthase (NOS). Nitric oxide reacts with superoxide to form peroxynitrite, which initiates oxidative reactions. To assess the potential for nitric oxide formation in muscle, NOS activity was determined in fresh muscle (<8 h post-mortem) from several species under conditions expected in muscle foods. Fresh muscle from all species exhibited NOS activity. NOS activity in muscle was reduced during refrigerated storage. Chicken thigh muscle NOS activity was not affected by pH over the range of 4.5–7.4, but was inhibited by 1 and 2% NaCl. Chicken thigh muscle NOS activity was stimulated at internal cooking temperatures up to 55 C but was completely inhibited at higher temperatures. Results of this study indicate that postmortem NOS is only active for the first several days of post-mortem storage. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Nitric oxide synthase; Peroxynitrite; Superoxide; Skeletal muscle; Lipid oxidation; Lipid hydroperoxides
1. Introduction Much research has been conducted to identify catalysts that promote oxidation in skeletal muscle because the breakdown products of lipid oxidation can directly impact the sensory quality and shelf-life of muscle foods (Ladikos & Lougovois, 1990). Skeletal muscle and its associated circulatory system contain numerous prooxidants such as lipoxygenase, transition metals, and heme-proteins that promote oxidation (Kanner, German, & Kinsella, 1987). Most are not very active against unoxidized fatty acids but rather catalyze the breakdown of lipid hydroperoxides to exert their strongest prooxidant activities. For example, preformed lipid hydroperoxides have been reported to activate lipoxygenase (Hemler, Cook, & Lands, 1979) and increase the prooxidant activity of iron and heme proteins in model systems (Baron, Skibsted, & Anderson, 1997; Nuchi, McClements, & Decker, 2001) and muscle (Harel & Kanner, 1985; Kanner, Shegalovich, Harel, & Hazan, 1988; Rhee, Ziprin, & Ordonez, 1987; Richards & Hultin, 2000). Thus, factors that promote the formation of lipid hydroperoxides, especially during the early stages
* Corresponding author. Tel.: +1-413-545-1026; fax: +1-413-5451262. E-mail address: [email protected] (E.A. Decker).
of post-mortem storage, could be important events in the ultimate quality of muscle foods. Peroxynitrite, formed from the diffusion limited termination reaction of nitric oxide with superoxide, can interact with membrane lipids to cause the formation of fatty acid hydroperoxides (Rubbo et al., 1994) that in turn could activate skeletal muscle prooxidants. Peroxynitrite has been shown to produce lipid hydroperoxides in membrane lipids (Radi, Beckman, Bush, & Freeman, 1991), low-density lipoproteins (Hogg & Kalyanaraman, 1998), and trout muscle microsomes and homogenate (Brannan & Decker, 2001). Peroxynitrite also has been shown to increase the prooxidant activity of metmyoglobin (Jourd’heuil, Mills, Miles, & Grisham, 1998). Furthermore, the metal chelators diethylenetriaminepentaacetic acid and bathophenanthrolinedisulfonic acid were not able to completely inhibit peroxynitrite-induced oxidation of a skeletal muscle homogenate (Brannan & Decker, 2001), suggesting that peroxynitrite can activate metal-independent prooxidants. Thus, the formation of peroxynitrite could initiate lipid oxidation directly or cause the formation of lipid hydroperoxides which further increase the activity of other prooxidants in muscle foods. In order for peroxynitrite to be produced in muscle foods, nitric oxide must be present in the post-mortem tissue. Nitric oxide is produced in animals by nitric oxide synthase (NOS). NOS catalyzes a complicated
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5-electron oxidation of l-arginine to form l-citrulline and nitric oxide. The NOS reaction (Fig. 1) requires several cofactors including oxygen, NADPH, tetrahydrobiopterin, and flavin adenine mono- and dinucleotides (FMN and FAD). A number of NOS isoenzymes have been identified, all of which share common cofactor requirements and catalytic mechanisms but vary in molecular size, tissue distribution, calciumdependence, and mode of regulation. NOS is found in skeletal muscle fibers, but the concentration and distribution within the muscle is species-dependent (Frandsen, Lopez-Figueroa, & Hellsten, 1996; Grozdanovic, Gosztonyi, & Gossrau, 1996; Kobzik, Reid, Bredt, & Stamler, 1994). The objective of this research was to investigate the potential for nitric oxide production in skeletal muscle by characterizing the activity of NOS in post-mortem tissue from several species as affected by extrinsic conditions commonly encountered in post-mortem muscle foods such as pH, salting, thermal processing, freeze/ thaw stability, and refrigerated storage.
2. Materials and methods All solvents were acquired from Fisher Scientific Company (Pittsburgh, PA) and other chemicals were obtained from Sigma (St. Louis, MO). Rainbow trout were obtained from Mohawk Trout Hatchery (Sunderland, MA), pork diaphragm muscle from Stafford Enterprises (Stafford Springs, CT), whole turkeys from Twin Willows Turkey Farm (Belchertown, MA), and whole chickens from Diemand Farms (Millers Falls, MA). The activity of NOS was determined on extracts from skeletal muscle using a NOS Assay Kit (Cayman Chemical Company, Ann Arbor, MI). Muscle extracts were prepared by homogenization of 1 part muscle with 10 parts ice-cold homogenization buffer [25 mM Tris–HCl, pH 7.4, 1 mM EDTA, 1 mM ethyleneglycol-bis(b-aminoethylether)-N,N,N0 ,N0 -tetraacetic acid] using a Polytron homogenizer (Brinkman Instruments, Inc., Westbury NY) on its highest setting for 30 s followed by
centrifugation (13,000 g) for 10 min at 4 C using an Eppendorf microcentrifuge (Brinkman Instruments, Inc., Westbury, NY). Reaction buffer (40 ml) containing 250 mM l-[14C]-arginine, 50 mM Tris–HCl, pH 7.4, 6 mM tetrahydrobiopterin, 2 mM flavin adenine dinucleotide (FMD), and 2 mM flavin adenine mononucleotide (FAD), was added to skeletal muscle extracts (40 ml). The irreversible NOS inhibitor L-NAME (10 ml of 10 mM NG-nitro-l-arginine methyl ester HCL) was used to determine citrulline concentrations produced in the tissue extract from non-NOS catalyzed pathways. All samples were then incubated at 37 C for 30 min. The NOS reaction was stopped by addition of 5 mM EDTA in 50 mM HEPES buffer, pH 5.5 (400 ml). Ion exchange chromatography columns (provided in the NOS Assay Kit) were used to retain arginine while citrulline was eluted and transferred to liquid scintillation vials. Elution buffer (400 ml of 1 mM NH4Cl) was then used to elute the bound arginine from the columns which was then transferred to liquid scintillation vials. After the separations, the radioactivity of the citrulline and arginine fractions was measured using a Delta 300 Model 6891 Liquid Scintillation Counting System (TM Analytic, Elk Grove Village, IL). Blanks using deionized water instead of tissue extracts were used to account for residual radioactive material that elutes with the citrulline fraction. NOS activity determined from cerebellum extract from rat (provided in the NOS Assay Kit) and recombinant NOS (Cayman Chemical Company, Ann Arbor, MI) served as positive controls. Counts per minute (CPM) from the citrulline and arginine fractions of each tissue extract and water blank were converted to NOS activity according to the following formula (total CPM equals sum of citrulline CPM and arginine CPM): NOS Activity ¼ Citrulline CPM ðCPMÞ
ðtissue extractÞ
0 1 Total CPM Citrulline CPM ðtissue extractÞ ðwater blankÞ A @ Total CPM ðwater blankÞ
Dilution
Fig. 1. The nitric oxide synthase (NOS) reaction.
Factor
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NOS activity as determined above was converted to nmoles citrulline produced per minute per g muscle based on a standard curve prepared from l-[14C]arginine, which relates to [14C]-citrulline on a mole to mole basis (l-[14C]-citrulline is not commercially available). The NOS activity was measured as a function of pH, NaCl concentration, cooking temperature, freeze/thaw stability, and refrigerated storage. This was accomplished by measuring NOS activity as described above with the following modifications. For pH experiments, the pH of the reaction buffer was adjusted with HCl to 4.5, 5.5, 6.5, and 7.4. For NaCl experiments, NaCl (0– 2%) was added to the reaction buffer. Cooking experiments were performed by heating minced muscle (1 g in capped 16 125 mm tubes) in a boiling water bath to various internal temperatures (45, 55, 65, 75, 85 C). Temperature was monitored by a thermocouple (Omega Engineering, Inc., Stamford, CT) inserted into the center of the tissue. When final internal temperatures were achieved, tissues were immediately cooled by placing tubes in an ice-water bath followed by addition of icecold homogenization buffer (5 ml) to the cooked muscle. NOS activity of an extract from the cooked tissue was determined using standard NOS activity assay as outlined previously. For freeze/thaw stability experiments, fresh minced muscle (0.5 g) was frozen (20 C, 24 hr), thawed at 4 C for approximately 1 h, and assayed for NOS activity as described above. For refrigerated storage experiments, fresh minced muscle was stored refrigerated (4 C) and NOS activity was determined daily as described previously. Muscle from all species was removed from the carcasses within 4 h of slaughter except for turkey which was within 8 h of slaughter. Muscle from turkey and chicken was separated into light (breast) and dark (thigh) fractions. To determine means and standard deviations for NOS activity, muscle from two animals from each species were obtained from the locations described previously. Chickens were obtained on two separate dates. The muscle from each animal and/or tissue type was minced (i.e. muscle from different animals was not pooled) and NOS activity in the presence and absence of L-NAME (a NOS inhibitor) was determined in triplicate for each batch of minced muscle. Since the value from each tissue extract in the presence of L-NAME represents citrulline (or other radioactive contamination) formed independently of NOS, these values were subtracted from the value obtained in the absence of L-NAME before means and standard deviations were generated. For experiments in which the NOS activity was measured as a function of pH, NaCl concentration, cooking temperature, freeze/thaw stability, and refrigerated storage, triplicate analysis from each of two animals were used to generate means. PROC ANOVA (SAS, 2001) with Duncan’s Multiple
Range test was used to separate these treatment means and significance was set at the 5% level.
3. Results and discussion 3.1. Differences in NOS activity between and within species NOS has been previously identified in skeletal muscles from humans, rats, mice, gerbils, guinea pigs, hamsters, turtles, chicken, pigeons, goldfish, and rabbit (Blottner & Luck, 1998; Frandsen et al., 1996; Gath et al., 1996; Gossrau, 1998; Grozdanovic & Gossrau, 1998; Grozdanovic, Nakos, Dahrmann, Mayer, & Gossrau, 1995; Sutherland et al., 2001) . As shown in Table 1, NOS was found to be active in skeletal muscle from chicken, pork, turkey, and trout. NOS activity in skeletal muscle could be genetically controlled or be influenced by muscle fiber type, animal age, and muscle exercise regimes. For example, NOS is found in both type I and II fibers in humans (Brenman, Chao, Xia, Aldape, & Bredt, 1995) but only in type II fibers in rats, mice, guinea pigs, hamsters (Brenman et al., 1996; Chang et al., 1996; Grozdanovic et al., 1998). In the muscles in which NOS is only found in type II fibers, type IIB fibers appear to have higher concentrations of NOS than type IIA fibers (Grozdanovic & Baumgarten, 1999). NOS Table 1 Nitric oxide synthase (NOS) activity (nmoles l-[14C]-citrulline per min per g tissue) in tissue extracts from fresh muscle of animals from different species Muscle Chicken dark—April 2001 Animal 1 Animal 2 Chicken dark—May 2001 Animal 1 Animal 2 Chicken light—April 2001 Animal 1 Animal 2 Chicken light—May 2001 Animal 1 Animal 2 Turkey dark Animal 1 Animal 2 Turkey light Animal 1 Animal 2 Pork Animal 1 Animal 2 Trout Animal 1 Animal 2
Mean NOS activity
Standard deviation
117 166
1 19
153 161
15 31
43 26
10 15
3 5
3 1
29 2
30 3
126 61
15 2
183 86
15 24
76 145
13 18
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concentration in skeletal muscle also increases with increasing animal age (Capanni et al., 1998; Chang et al., 1996), an observation that may be related to the increase in the concentration of type II fibers that occurs with age. For example, Ono et al. (Ono, Iwamoto, & Takahara, 1993) report that 1-week-old chicken dark meat contains a type IIA to type IIB ratio of 1:4, while the ratio at 10 weeks, which is closer to the slaughter age of the animal, is 1:2. Furthermore, it has been shown that physical exercise increases the proportion of type IIA fibers at the expense of type IIB fibers (Brackenbury & Holloway, 1991), which may have an effect on NOS activity since type IIB fibers are reported to contain more NOS than type IIA fibers (Grozdanovic et al., 1999). Thus, the level of NOS activity in muscle may be due to many interrelated factors but it may be expected that light muscle from poultry would have higher NOS activity than dark muscle. In this study, it is unclear why light and dark meat from chicken did not fit this predicted pattern. 3.2. Effect of extrinsic factors on NOS activity NOS activity could also be influenced by extrinsic factors encountered during muscle food processing such as pH, salting, cooking temperature, freeze/thaw stability, and refrigerated storage. Chicken dark muscle was used to determine the effects of most of these factors, since chicken dark muscle exhibited high initial NOS activity and fresh chickens were more readily available than the other species. 3.2.1. Refrigerated and frozen storage The activity of NOS was tested as a function of refrigerated storage to determine the stability of NOS and whether nitric oxide production and potential peroxynitrite formation could be long-term post-mortem events. Minced muscle from fresh pork diaphragm, trout, and chicken dark muscle were refrigerated and tested daily for NOS activity. As shown in Table 2, NOS activity was lost within 24 h in chicken dark muscle and in trout muscle, and within 48 h in pork muscle. The duration of NOS activity during refrigerated storage did not depend on the initial level of NOS activity in the tissue on a gram tissue basis. In refrigerated muscle foods, the NOS catalyzed production of nitric oxide would be influenced by the availability of its substrate (l-arginine) and cofactors (calcium, tetrahydrobiopterin, NADPH, FMD, FAD). However sufficient levels of these substrates and cofactors were added to the trout and pork muscle extracts in our in vitro experimental protocol. Thus, our data may represent the maximum NOS activity that could be expected in situ during refrigerated storage for each species. Therefore, the rapid loss of NOS activity during refrigerated storage indicates that the enzyme is not very
stable. In addition, it is likely that NOS cofactors such as NADPH, FMD, FAD would decrease post-mortem and cause an additional decline in NOS activity. For example, red hake muscle stored at 0 and 5 C, loses 30–60% of its FMN and FAD and 25–40% of its NADH within two days of storage (Phillippy & Hultin, 1993). The combination of loss of NOS activity and likely loss of endogenous cofactors suggest that nitric oxide production will decline rapidly during the refrigerated storage of muscle foods. The NOS activity of chicken dark muscle was reduced by more than 70% (from 188 to 48 nmoles citrulline produced per minute per g muscle) by a single freeze/thaw cycle following short term (24 h) frozen storage. This suggests that NOS is more stable in frozen than refrigerated muscle since NOS activity of chicken dark muscle was completely lost within 24 h during refrigerated storage. 3.2.2. pH NOS activity in fresh chicken dark muscle was not affected over the pH range of 4.5–7.4 (Fig. 2). These results agree with previous research reporting that the optimal pH for purified NOS is 7.0–7.5 and that purified NOS was still active at pH 6 (Gorren, Schrammel, Schmidt, & Mayer, 1998). Thus, NOS is still active at pH values commonly encountered in muscle foods and post-mortem pH decline alone should not significantly alter the rate of nitric oxide formation. 3.2.3. NaCl Sodium chloride is another extrinsic factor that can affect enzyme activity in muscle food by causing protein denaturation or altering catalytic activity (Belitz & Grosch, 1999). In our study, we observed that NaCl (1 and 2%) completely inhibited NOS activity in fresh chicken (Fig. 3), since a significant difference (P < 0.05) in [14C]-citrulline production was not observed between
Table 2 Nitric oxide synthase (NOS) activity (nmoles l-[14C]-citrulline per min per mg protein) in tissue extracts from different species stored at 4 C Muscle
Pork muscle Animal 1 Animal 2 Trout muscle Animal 1 Animal 2 Chicken dark muscle Animal 1 Animal 2
NOS activity 0h
24 h
48 h
183 15 8624
11821 9023
NSa NS
7613 145 18
NS NS
NS NS
153 15 161 31
NS NS
NS NS
a NS: Citrulline concentrations in samples with uninhibited and inhibited (10 mM NG-nitro-l-arginine methyl ester HCL) NOS were not significantly different (P <0.05).
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the tissue extracts in the absence and presence of LNAME, an irreversible NOS inhibitor. Schrammel, Gorren, Stuehr, Schmidt, and Mayer (1998) studied the activity of purified NOS as a function of NaCl concentration and reported NaCl had no impact on the structure of NOS but that increasing NaCl concentration from 0–3% resulted in a 6-fold increase in the km for arginine. This suggests that the inhibition of NOS by NaCl in our study could be due to decreased affinity of NOS for arginine or, as first suggested by Schrammel et al. (1998) with respect to purified NOS, non-specific changes in protein solvation that affect ion binding to the enzyme. The fact that NaCl (1 and 2%) completely inhibited NOS activity in our study suggests that the potential for peroxynitrite formation in high salt environments is
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negligible and peroxynitrite is probably not a factor in the decreased oxidative stability observed in stored, salted muscle foods. 3.2.4. Internal cooking temperature Most enzymes exhibit differences in activity as a function of food processing temperature (Belitz et al., 1999). In our study fresh chicken dark muscle was cooked to different internal temperatures and NOS activity was determined under standard conditions. Cooking the muscle prior to performing the NOS activity assay showed cooking to 55 C increased NOS activity by nearly 60% (Fig. 5). This type of increase in enzyme activity with increasing temperature is not unprecedented, as an increase in enzyme activity with increasing internal cooking temperature up to 75 C has been observed for superoxide dismutase in both beef and pork (Mei, Crum, & Decker, 1994). Cooking to internal temperatures greater than 55 C resulted in total inactivation of NOS (Fig. 4). Since most thermally processed meat and poultry products are cooked to temperatures higher than 55 C, this suggests that peroxynitrite formation may not be a factor in lipid oxidation and/or subsequent warmed-over flavor development in cooked meats during storage. 3.3. Summary
Fig. 2. Effect of pH on nitric oxide synthase (NOS) activity of chicken dark muscle extracts. Means for each pH with different letters are significantly different (P <0.05).
Although fresh muscle from all species tested exhibited NOS activity, the results of this study indicate that post-mortem NOS activity is likely to be of relatively short duration for the following reasons. Under conditions where ample substrates and cofactors are provided, NOS activity remained in muscle for only 24 h, indicating that NOS is unstable in post-mortem muscle. In addition, the cofactors required for NOS (e.g.
Fig. 3. Effect of NaCl concentration on nitric oxide synthase (NOS) activity of chicken dark muscle extracts. Means for each NaCl concentration with different letters are significantly different (P<0.05).
Fig. 4. Nitric oxide synthase (NOS) activity of extracts of chicken dark muscle cooked to different internal temperatures. Means for each temperature with different letters are significantly different (P <0.05).
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NADPH, FMN, FAD, tetrahydrobiopterin) are likely to be lost during post-mortem storage resulting in an additional loss of NOS activity. Thus, the ability of skeletal muscle to produce nitric oxide appears to be an event of relatively short post-mortem duration. With respect to lipid oxidation in skeletal muscle, production of nitric oxide may be important because of its nearly diffusion controlled reaction with superoxide to form peroxynitrite. The potential for nitric oxide formation in the early stages of post-mortem muscle suggests that unless superoxide dismutase can control superoxide levels, peroxynitrite could be formed. Since peroxynitrite can catalyze the formation of lipid hydroperoxides (Brannan & Decker, 2001; Rubbo et al., 1994) and increased levels of preformed lipid hydroperoxides are associated with increased oxidative susceptibility (Frei & Gaziano, 1993), the catalytic activity of NOS and potential formation of peroxynitrite as early postmortem events in muscle may contribute to the formation of reactive compounds that can reduce the longterm oxidative stability of the muscle. The reduced oxidative stability of muscle caused by early post-mortem peroxynitrite formation will not be affected by postmortem pH reduction because pH’s common to postmortem muscle neither inhibited NOS activity, i.e. nitric oxide production, nor the ability of peroxynitrite to initiate lipid oxidation in a skeletal muscle homogenate (Brannan & Decker, 2001). Since NOS-mediated nitric oxide production in muscle is expected to be a postmortem event of short duration, future research on strategies to control peroxynitrite-induced oxidation in muscle should focus on ante-mortem muscle biochemistry, handling during slaughter, and pre-rigor events in the muscle. References Baron, C. P., Skibsted, L. H., & Anderson, H. J. (1997). Proxidative activity of myoglobin species in linoleic acid emulsions. Journal of Agricultural and Food Chemistry, 45, 1704–1710. Belitz, H. D., & Grosch, W. (1999). Food chemistry. Berlin: Springer. Blottner, D., & Luck, G. (1998). Nitric oxide synthase (NOS) in mouse skeletal muscle development and differentiated myoblasts. Cell & Tissue Research, 292, 293–302. Brackenbury, J. H., & Holloway, S. A. (1991). Age and exercise effects on mitochondrial density and capillary fiber ratio in bird leg muscle. British Poultry Science, 332, 645–653. Brannan, R. G., & Decker, E. A. (2001). Peroxynitrite-induced oxidation of lipids: implications for muscle foods. Journal of Agricultural and Food Chemistry, 49, 3074–3079. Brenman, J. E., Chao, D. S., Gee, S. H., McGee, A. W., Craven, S. E., Santillano, D. R., Wu, Z., Huang, F., Xia, H., Peters, M. F., Froehner, S. C., & Bredt, D. S. (1996). Interaction of nitric oxide synthase with the postsynaptic density protein PSD-95 and alpha1syntrophin mediated by PDZ domains. Cell, 84, 757–767. Brenman, J. E., Chao, D. S., Xia, H., Aldape, K., & Bredt, D. S. (1995). Nitric oxide synthase complexed with dystrophin and absent from skeletal muscle sarcolemma in Duchenne muscular dystrophy. Cell, 82, 743–752.
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Nitric oxide synthase activity in muscle foods Robert G. Brannan, Eric A. Decker* Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA Received 25 September 2001; received in revised form 3 December 2001; accepted 3 December 2001
Abstract Nitric oxide is enzymatically produced in animals by nitric oxide synthase (NOS). Nitric oxide reacts with superoxide to form peroxynitrite, which initiates oxidative reactions. To assess the potential for nitric oxide formation in muscle, NOS activity was determined in fresh muscle (<8 h post-mortem) from several species under conditions expected in muscle foods. Fresh muscle from all species exhibited NOS activity. NOS activity in muscle was reduced during refrigerated storage. Chicken thigh muscle NOS activity was not affected by pH over the range of 4.5–7.4, but was inhibited by 1 and 2% NaCl. Chicken thigh muscle NOS activity was stimulated at internal cooking temperatures up to 55 C but was completely inhibited at higher temperatures. Results of this study indicate that postmortem NOS is only active for the first several days of post-mortem storage. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Nitric oxide synthase; Peroxynitrite; Superoxide; Skeletal muscle; Lipid oxidation; Lipid hydroperoxides
1. Introduction Much research has been conducted to identify catalysts that promote oxidation in skeletal muscle because the breakdown products of lipid oxidation can directly impact the sensory quality and shelf-life of muscle foods (Ladikos & Lougovois, 1990). Skeletal muscle and its associated circulatory system contain numerous prooxidants such as lipoxygenase, transition metals, and heme-proteins that promote oxidation (Kanner, German, & Kinsella, 1987). Most are not very active against unoxidized fatty acids but rather catalyze the breakdown of lipid hydroperoxides to exert their strongest prooxidant activities. For example, preformed lipid hydroperoxides have been reported to activate lipoxygenase (Hemler, Cook, & Lands, 1979) and increase the prooxidant activity of iron and heme proteins in model systems (Baron, Skibsted, & Anderson, 1997; Nuchi, McClements, & Decker, 2001) and muscle (Harel & Kanner, 1985; Kanner, Shegalovich, Harel, & Hazan, 1988; Rhee, Ziprin, & Ordonez, 1987; Richards & Hultin, 2000). Thus, factors that promote the formation of lipid hydroperoxides, especially during the early stages
* Corresponding author. Tel.: +1-413-545-1026; fax: +1-413-5451262. E-mail address: [email protected] (E.A. Decker).
of post-mortem storage, could be important events in the ultimate quality of muscle foods. Peroxynitrite, formed from the diffusion limited termination reaction of nitric oxide with superoxide, can interact with membrane lipids to cause the formation of fatty acid hydroperoxides (Rubbo et al., 1994) that in turn could activate skeletal muscle prooxidants. Peroxynitrite has been shown to produce lipid hydroperoxides in membrane lipids (Radi, Beckman, Bush, & Freeman, 1991), low-density lipoproteins (Hogg & Kalyanaraman, 1998), and trout muscle microsomes and homogenate (Brannan & Decker, 2001). Peroxynitrite also has been shown to increase the prooxidant activity of metmyoglobin (Jourd’heuil, Mills, Miles, & Grisham, 1998). Furthermore, the metal chelators diethylenetriaminepentaacetic acid and bathophenanthrolinedisulfonic acid were not able to completely inhibit peroxynitrite-induced oxidation of a skeletal muscle homogenate (Brannan & Decker, 2001), suggesting that peroxynitrite can activate metal-independent prooxidants. Thus, the formation of peroxynitrite could initiate lipid oxidation directly or cause the formation of lipid hydroperoxides which further increase the activity of other prooxidants in muscle foods. In order for peroxynitrite to be produced in muscle foods, nitric oxide must be present in the post-mortem tissue. Nitric oxide is produced in animals by nitric oxide synthase (NOS). NOS catalyzes a complicated
0309-1740/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0309-1740(01)00251-0
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5-electron oxidation of l-arginine to form l-citrulline and nitric oxide. The NOS reaction (Fig. 1) requires several cofactors including oxygen, NADPH, tetrahydrobiopterin, and flavin adenine mono- and dinucleotides (FMN and FAD). A number of NOS isoenzymes have been identified, all of which share common cofactor requirements and catalytic mechanisms but vary in molecular size, tissue distribution, calciumdependence, and mode of regulation. NOS is found in skeletal muscle fibers, but the concentration and distribution within the muscle is species-dependent (Frandsen, Lopez-Figueroa, & Hellsten, 1996; Grozdanovic, Gosztonyi, & Gossrau, 1996; Kobzik, Reid, Bredt, & Stamler, 1994). The objective of this research was to investigate the potential for nitric oxide production in skeletal muscle by characterizing the activity of NOS in post-mortem tissue from several species as affected by extrinsic conditions commonly encountered in post-mortem muscle foods such as pH, salting, thermal processing, freeze/ thaw stability, and refrigerated storage.
2. Materials and methods All solvents were acquired from Fisher Scientific Company (Pittsburgh, PA) and other chemicals were obtained from Sigma (St. Louis, MO). Rainbow trout were obtained from Mohawk Trout Hatchery (Sunderland, MA), pork diaphragm muscle from Stafford Enterprises (Stafford Springs, CT), whole turkeys from Twin Willows Turkey Farm (Belchertown, MA), and whole chickens from Diemand Farms (Millers Falls, MA). The activity of NOS was determined on extracts from skeletal muscle using a NOS Assay Kit (Cayman Chemical Company, Ann Arbor, MI). Muscle extracts were prepared by homogenization of 1 part muscle with 10 parts ice-cold homogenization buffer [25 mM Tris–HCl, pH 7.4, 1 mM EDTA, 1 mM ethyleneglycol-bis(b-aminoethylether)-N,N,N0 ,N0 -tetraacetic acid] using a Polytron homogenizer (Brinkman Instruments, Inc., Westbury NY) on its highest setting for 30 s followed by
centrifugation (13,000 g) for 10 min at 4 C using an Eppendorf microcentrifuge (Brinkman Instruments, Inc., Westbury, NY). Reaction buffer (40 ml) containing 250 mM l-[14C]-arginine, 50 mM Tris–HCl, pH 7.4, 6 mM tetrahydrobiopterin, 2 mM flavin adenine dinucleotide (FMD), and 2 mM flavin adenine mononucleotide (FAD), was added to skeletal muscle extracts (40 ml). The irreversible NOS inhibitor L-NAME (10 ml of 10 mM NG-nitro-l-arginine methyl ester HCL) was used to determine citrulline concentrations produced in the tissue extract from non-NOS catalyzed pathways. All samples were then incubated at 37 C for 30 min. The NOS reaction was stopped by addition of 5 mM EDTA in 50 mM HEPES buffer, pH 5.5 (400 ml). Ion exchange chromatography columns (provided in the NOS Assay Kit) were used to retain arginine while citrulline was eluted and transferred to liquid scintillation vials. Elution buffer (400 ml of 1 mM NH4Cl) was then used to elute the bound arginine from the columns which was then transferred to liquid scintillation vials. After the separations, the radioactivity of the citrulline and arginine fractions was measured using a Delta 300 Model 6891 Liquid Scintillation Counting System (TM Analytic, Elk Grove Village, IL). Blanks using deionized water instead of tissue extracts were used to account for residual radioactive material that elutes with the citrulline fraction. NOS activity determined from cerebellum extract from rat (provided in the NOS Assay Kit) and recombinant NOS (Cayman Chemical Company, Ann Arbor, MI) served as positive controls. Counts per minute (CPM) from the citrulline and arginine fractions of each tissue extract and water blank were converted to NOS activity according to the following formula (total CPM equals sum of citrulline CPM and arginine CPM): NOS Activity ¼ Citrulline CPM ðCPMÞ
ðtissue extractÞ
0 1 Total CPM Citrulline CPM ðtissue extractÞ ðwater blankÞ A @ Total CPM ðwater blankÞ
Dilution
Fig. 1. The nitric oxide synthase (NOS) reaction.
Factor
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NOS activity as determined above was converted to nmoles citrulline produced per minute per g muscle based on a standard curve prepared from l-[14C]arginine, which relates to [14C]-citrulline on a mole to mole basis (l-[14C]-citrulline is not commercially available). The NOS activity was measured as a function of pH, NaCl concentration, cooking temperature, freeze/thaw stability, and refrigerated storage. This was accomplished by measuring NOS activity as described above with the following modifications. For pH experiments, the pH of the reaction buffer was adjusted with HCl to 4.5, 5.5, 6.5, and 7.4. For NaCl experiments, NaCl (0– 2%) was added to the reaction buffer. Cooking experiments were performed by heating minced muscle (1 g in capped 16 125 mm tubes) in a boiling water bath to various internal temperatures (45, 55, 65, 75, 85 C). Temperature was monitored by a thermocouple (Omega Engineering, Inc., Stamford, CT) inserted into the center of the tissue. When final internal temperatures were achieved, tissues were immediately cooled by placing tubes in an ice-water bath followed by addition of icecold homogenization buffer (5 ml) to the cooked muscle. NOS activity of an extract from the cooked tissue was determined using standard NOS activity assay as outlined previously. For freeze/thaw stability experiments, fresh minced muscle (0.5 g) was frozen (20 C, 24 hr), thawed at 4 C for approximately 1 h, and assayed for NOS activity as described above. For refrigerated storage experiments, fresh minced muscle was stored refrigerated (4 C) and NOS activity was determined daily as described previously. Muscle from all species was removed from the carcasses within 4 h of slaughter except for turkey which was within 8 h of slaughter. Muscle from turkey and chicken was separated into light (breast) and dark (thigh) fractions. To determine means and standard deviations for NOS activity, muscle from two animals from each species were obtained from the locations described previously. Chickens were obtained on two separate dates. The muscle from each animal and/or tissue type was minced (i.e. muscle from different animals was not pooled) and NOS activity in the presence and absence of L-NAME (a NOS inhibitor) was determined in triplicate for each batch of minced muscle. Since the value from each tissue extract in the presence of L-NAME represents citrulline (or other radioactive contamination) formed independently of NOS, these values were subtracted from the value obtained in the absence of L-NAME before means and standard deviations were generated. For experiments in which the NOS activity was measured as a function of pH, NaCl concentration, cooking temperature, freeze/thaw stability, and refrigerated storage, triplicate analysis from each of two animals were used to generate means. PROC ANOVA (SAS, 2001) with Duncan’s Multiple
Range test was used to separate these treatment means and significance was set at the 5% level.
3. Results and discussion 3.1. Differences in NOS activity between and within species NOS has been previously identified in skeletal muscles from humans, rats, mice, gerbils, guinea pigs, hamsters, turtles, chicken, pigeons, goldfish, and rabbit (Blottner & Luck, 1998; Frandsen et al., 1996; Gath et al., 1996; Gossrau, 1998; Grozdanovic & Gossrau, 1998; Grozdanovic, Nakos, Dahrmann, Mayer, & Gossrau, 1995; Sutherland et al., 2001) . As shown in Table 1, NOS was found to be active in skeletal muscle from chicken, pork, turkey, and trout. NOS activity in skeletal muscle could be genetically controlled or be influenced by muscle fiber type, animal age, and muscle exercise regimes. For example, NOS is found in both type I and II fibers in humans (Brenman, Chao, Xia, Aldape, & Bredt, 1995) but only in type II fibers in rats, mice, guinea pigs, hamsters (Brenman et al., 1996; Chang et al., 1996; Grozdanovic et al., 1998). In the muscles in which NOS is only found in type II fibers, type IIB fibers appear to have higher concentrations of NOS than type IIA fibers (Grozdanovic & Baumgarten, 1999). NOS Table 1 Nitric oxide synthase (NOS) activity (nmoles l-[14C]-citrulline per min per g tissue) in tissue extracts from fresh muscle of animals from different species Muscle Chicken dark—April 2001 Animal 1 Animal 2 Chicken dark—May 2001 Animal 1 Animal 2 Chicken light—April 2001 Animal 1 Animal 2 Chicken light—May 2001 Animal 1 Animal 2 Turkey dark Animal 1 Animal 2 Turkey light Animal 1 Animal 2 Pork Animal 1 Animal 2 Trout Animal 1 Animal 2
Mean NOS activity
Standard deviation
117 166
1 19
153 161
15 31
43 26
10 15
3 5
3 1
29 2
30 3
126 61
15 2
183 86
15 24
76 145
13 18
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concentration in skeletal muscle also increases with increasing animal age (Capanni et al., 1998; Chang et al., 1996), an observation that may be related to the increase in the concentration of type II fibers that occurs with age. For example, Ono et al. (Ono, Iwamoto, & Takahara, 1993) report that 1-week-old chicken dark meat contains a type IIA to type IIB ratio of 1:4, while the ratio at 10 weeks, which is closer to the slaughter age of the animal, is 1:2. Furthermore, it has been shown that physical exercise increases the proportion of type IIA fibers at the expense of type IIB fibers (Brackenbury & Holloway, 1991), which may have an effect on NOS activity since type IIB fibers are reported to contain more NOS than type IIA fibers (Grozdanovic et al., 1999). Thus, the level of NOS activity in muscle may be due to many interrelated factors but it may be expected that light muscle from poultry would have higher NOS activity than dark muscle. In this study, it is unclear why light and dark meat from chicken did not fit this predicted pattern. 3.2. Effect of extrinsic factors on NOS activity NOS activity could also be influenced by extrinsic factors encountered during muscle food processing such as pH, salting, cooking temperature, freeze/thaw stability, and refrigerated storage. Chicken dark muscle was used to determine the effects of most of these factors, since chicken dark muscle exhibited high initial NOS activity and fresh chickens were more readily available than the other species. 3.2.1. Refrigerated and frozen storage The activity of NOS was tested as a function of refrigerated storage to determine the stability of NOS and whether nitric oxide production and potential peroxynitrite formation could be long-term post-mortem events. Minced muscle from fresh pork diaphragm, trout, and chicken dark muscle were refrigerated and tested daily for NOS activity. As shown in Table 2, NOS activity was lost within 24 h in chicken dark muscle and in trout muscle, and within 48 h in pork muscle. The duration of NOS activity during refrigerated storage did not depend on the initial level of NOS activity in the tissue on a gram tissue basis. In refrigerated muscle foods, the NOS catalyzed production of nitric oxide would be influenced by the availability of its substrate (l-arginine) and cofactors (calcium, tetrahydrobiopterin, NADPH, FMD, FAD). However sufficient levels of these substrates and cofactors were added to the trout and pork muscle extracts in our in vitro experimental protocol. Thus, our data may represent the maximum NOS activity that could be expected in situ during refrigerated storage for each species. Therefore, the rapid loss of NOS activity during refrigerated storage indicates that the enzyme is not very
stable. In addition, it is likely that NOS cofactors such as NADPH, FMD, FAD would decrease post-mortem and cause an additional decline in NOS activity. For example, red hake muscle stored at 0 and 5 C, loses 30–60% of its FMN and FAD and 25–40% of its NADH within two days of storage (Phillippy & Hultin, 1993). The combination of loss of NOS activity and likely loss of endogenous cofactors suggest that nitric oxide production will decline rapidly during the refrigerated storage of muscle foods. The NOS activity of chicken dark muscle was reduced by more than 70% (from 188 to 48 nmoles citrulline produced per minute per g muscle) by a single freeze/thaw cycle following short term (24 h) frozen storage. This suggests that NOS is more stable in frozen than refrigerated muscle since NOS activity of chicken dark muscle was completely lost within 24 h during refrigerated storage. 3.2.2. pH NOS activity in fresh chicken dark muscle was not affected over the pH range of 4.5–7.4 (Fig. 2). These results agree with previous research reporting that the optimal pH for purified NOS is 7.0–7.5 and that purified NOS was still active at pH 6 (Gorren, Schrammel, Schmidt, & Mayer, 1998). Thus, NOS is still active at pH values commonly encountered in muscle foods and post-mortem pH decline alone should not significantly alter the rate of nitric oxide formation. 3.2.3. NaCl Sodium chloride is another extrinsic factor that can affect enzyme activity in muscle food by causing protein denaturation or altering catalytic activity (Belitz & Grosch, 1999). In our study, we observed that NaCl (1 and 2%) completely inhibited NOS activity in fresh chicken (Fig. 3), since a significant difference (P < 0.05) in [14C]-citrulline production was not observed between
Table 2 Nitric oxide synthase (NOS) activity (nmoles l-[14C]-citrulline per min per mg protein) in tissue extracts from different species stored at 4 C Muscle
Pork muscle Animal 1 Animal 2 Trout muscle Animal 1 Animal 2 Chicken dark muscle Animal 1 Animal 2
NOS activity 0h
24 h
48 h
183 15 8624
11821 9023
NSa NS
7613 145 18
NS NS
NS NS
153 15 161 31
NS NS
NS NS
a NS: Citrulline concentrations in samples with uninhibited and inhibited (10 mM NG-nitro-l-arginine methyl ester HCL) NOS were not significantly different (P <0.05).
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the tissue extracts in the absence and presence of LNAME, an irreversible NOS inhibitor. Schrammel, Gorren, Stuehr, Schmidt, and Mayer (1998) studied the activity of purified NOS as a function of NaCl concentration and reported NaCl had no impact on the structure of NOS but that increasing NaCl concentration from 0–3% resulted in a 6-fold increase in the km for arginine. This suggests that the inhibition of NOS by NaCl in our study could be due to decreased affinity of NOS for arginine or, as first suggested by Schrammel et al. (1998) with respect to purified NOS, non-specific changes in protein solvation that affect ion binding to the enzyme. The fact that NaCl (1 and 2%) completely inhibited NOS activity in our study suggests that the potential for peroxynitrite formation in high salt environments is
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negligible and peroxynitrite is probably not a factor in the decreased oxidative stability observed in stored, salted muscle foods. 3.2.4. Internal cooking temperature Most enzymes exhibit differences in activity as a function of food processing temperature (Belitz et al., 1999). In our study fresh chicken dark muscle was cooked to different internal temperatures and NOS activity was determined under standard conditions. Cooking the muscle prior to performing the NOS activity assay showed cooking to 55 C increased NOS activity by nearly 60% (Fig. 5). This type of increase in enzyme activity with increasing temperature is not unprecedented, as an increase in enzyme activity with increasing internal cooking temperature up to 75 C has been observed for superoxide dismutase in both beef and pork (Mei, Crum, & Decker, 1994). Cooking to internal temperatures greater than 55 C resulted in total inactivation of NOS (Fig. 4). Since most thermally processed meat and poultry products are cooked to temperatures higher than 55 C, this suggests that peroxynitrite formation may not be a factor in lipid oxidation and/or subsequent warmed-over flavor development in cooked meats during storage. 3.3. Summary
Fig. 2. Effect of pH on nitric oxide synthase (NOS) activity of chicken dark muscle extracts. Means for each pH with different letters are significantly different (P <0.05).
Although fresh muscle from all species tested exhibited NOS activity, the results of this study indicate that post-mortem NOS activity is likely to be of relatively short duration for the following reasons. Under conditions where ample substrates and cofactors are provided, NOS activity remained in muscle for only 24 h, indicating that NOS is unstable in post-mortem muscle. In addition, the cofactors required for NOS (e.g.
Fig. 3. Effect of NaCl concentration on nitric oxide synthase (NOS) activity of chicken dark muscle extracts. Means for each NaCl concentration with different letters are significantly different (P<0.05).
Fig. 4. Nitric oxide synthase (NOS) activity of extracts of chicken dark muscle cooked to different internal temperatures. Means for each temperature with different letters are significantly different (P <0.05).
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NADPH, FMN, FAD, tetrahydrobiopterin) are likely to be lost during post-mortem storage resulting in an additional loss of NOS activity. Thus, the ability of skeletal muscle to produce nitric oxide appears to be an event of relatively short post-mortem duration. With respect to lipid oxidation in skeletal muscle, production of nitric oxide may be important because of its nearly diffusion controlled reaction with superoxide to form peroxynitrite. The potential for nitric oxide formation in the early stages of post-mortem muscle suggests that unless superoxide dismutase can control superoxide levels, peroxynitrite could be formed. Since peroxynitrite can catalyze the formation of lipid hydroperoxides (Brannan & Decker, 2001; Rubbo et al., 1994) and increased levels of preformed lipid hydroperoxides are associated with increased oxidative susceptibility (Frei & Gaziano, 1993), the catalytic activity of NOS and potential formation of peroxynitrite as early postmortem events in muscle may contribute to the formation of reactive compounds that can reduce the longterm oxidative stability of the muscle. The reduced oxidative stability of muscle caused by early post-mortem peroxynitrite formation will not be affected by postmortem pH reduction because pH’s common to postmortem muscle neither inhibited NOS activity, i.e. nitric oxide production, nor the ability of peroxynitrite to initiate lipid oxidation in a skeletal muscle homogenate (Brannan & Decker, 2001). Since NOS-mediated nitric oxide production in muscle is expected to be a postmortem event of short duration, future research on strategies to control peroxynitrite-induced oxidation in muscle should focus on ante-mortem muscle biochemistry, handling during slaughter, and pre-rigor events in the muscle. References Baron, C. P., Skibsted, L. H., & Anderson, H. J. (1997). Proxidative activity of myoglobin species in linoleic acid emulsions. Journal of Agricultural and Food Chemistry, 45, 1704–1710. Belitz, H. D., & Grosch, W. (1999). Food chemistry. Berlin: Springer. Blottner, D., & Luck, G. (1998). Nitric oxide synthase (NOS) in mouse skeletal muscle development and differentiated myoblasts. Cell & Tissue Research, 292, 293–302. Brackenbury, J. H., & Holloway, S. A. (1991). Age and exercise effects on mitochondrial density and capillary fiber ratio in bird leg muscle. British Poultry Science, 332, 645–653. Brannan, R. G., & Decker, E. A. (2001). Peroxynitrite-induced oxidation of lipids: implications for muscle foods. Journal of Agricultural and Food Chemistry, 49, 3074–3079. Brenman, J. E., Chao, D. S., Gee, S. H., McGee, A. W., Craven, S. E., Santillano, D. R., Wu, Z., Huang, F., Xia, H., Peters, M. F., Froehner, S. C., & Bredt, D. S. (1996). Interaction of nitric oxide synthase with the postsynaptic density protein PSD-95 and alpha1syntrophin mediated by PDZ domains. Cell, 84, 757–767. Brenman, J. E., Chao, D. S., Xia, H., Aldape, K., & Bredt, D. S. (1995). Nitric oxide synthase complexed with dystrophin and absent from skeletal muscle sarcolemma in Duchenne muscular dystrophy. Cell, 82, 743–752.
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