Article pubs.acs.org/JAFC
Oxidative and Nitrosative Stress Induced in Myofibrillar Proteins by a Hydroxyl-Radical-Generating System: Impact of Nitrite and Ascorbate Adriana Villaverde, Vita Parra, and Mario Estévez* Department of Animal Production and Food Science, Food Technology, University of Extremadura, 10003 Cáceres, Spain ABSTRACT: Understanding the chemistry behind the redox properties of nitrite and ascorbate is essential to identify the impact of curing agents on food quality and optimize the formulation of cured meat products. This study was designed to gain insight into the interactions between curing agents and myofibrillar proteins (MPs) during in vitro oxidation by a hydroxylradical-generating system. MPs (4 mg/mL) were oxidized for 4 days at 37 °C under constant stirring with 25 μM iron(III) and 2.5 mM hydrogen peroxide. Dependent upon the addition of nitrite (0, 75, and 150 mg/L) and ascorbate (0, 250, and 500 mg/ L), nine different reaction units were prepared in triplicate (n = 3) according to a total factorial design. Upon completion of the oxidation assay, samples were analyzed for the concentration of tryptophan (TRP), α-aminoadipic semialdehyde (AAS), Schiff bases (SBs), and 3-nitrotyrosine (3NT). Ascorbate at 250 mg/L significantly inhibited the depletion of TRP (∼20% inhibition) and the formation of AAS and SBs (>90% inhibition) in MP suspensions. Nitrite, alone, had a negligible effect on protein oxidation but induced the formation of a specific marker of nitrosative stress, namely, 3NT. Ascorbate was also efficient at inhibiting the formation of 3NT by a dose-dependent anti-nitrosative effect and enabled the antioxidant action of nitrite. KEYWORDS: nitrite, ascorbate, tryptophan oxidation, carbonylation, nitrosation, 3-nitrotyrosine
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(reaction 3), that eventually scavenge lipid radicals (LOO•) and, as a result, inhibit lipid oxidation (reaction 4).
INTRODUCTION Preservation of meat with nitrite or nitrate has become essential for controlling meat spoilage and producing safe and appetizing meat products at ambient temperature.1 Nitrite offers protection against microbial growth and the production of toxins (i.e., from Clostridium botulinum strains), contributes to the development and stability of color in cured products, and inhibits rancidity.2 However, the ingoing and residual amounts of nitrite are restricted by law, owing to its intrinsic toxicity and the potential formation of nitrosamines.2 Ascorbate is typically combined with nitrite because it contributes to the prevention of toxin and nitrosamine formation and displays intense reducing abilities.2 In fact, some of the positive effects of curing agents are related to the redox properties of nitrite and ascorbate. However, the chemistry behind these redox properties is considerably complex and sometimes not well-understood. For instance, nitrite (NO2), as such, is a clear pro-oxidant compound, which is able to initiate oxidative reactions by abstracting an electron from a sensitive molecule (reaction 1). NO2− + H 2O + e− → NO + OH− (1)
(3)
NO + LOO• → LOONO + LONO2
(4)
These antioxidant mechanisms are usually described for lipid oxidation, while the effect of these curing agents on the oxidative stability of food proteins is scarcely documented.3,4 The oxidation of muscle proteins as initiated by reactive oxygen species (ROS) leads to manifold chemical manifestations, including the loss of functional groups and the formation of oxidation derivatives, such as cross-links [i.e., disulfide bonds and Schiff bases (SBs)] and carbonyl compounds.5 The depletion of protein components, such as thiols and tryptophan (TRP) residues, involves the degradation of essential amino acids and the loss of nutritional value.5 Protein carbonylation has been described as one of the most remarkable expressions of the oxidative damage to proteins in biological6 and food systems.7 Protein carbonyls and cross-links have been linked to the loss of functionality in myofibrillar proteins,8 the impaired digestibility of meat proteins,9 and an increased toughness in pork and beef.10,11 Interestingly, in a pro-oxidative environment, nitrite is able to yield reactive nitrogen species (RNS), such as nitric oxide and peroxynitrite, which may be able to initiate both oxidation and nitration of proteins.12 The nitrosative stress on proteins leads to the formation of specific products, such as 3-nitrotyrosine
In combination with transition metals, such as iron, ascorbate (A) also exerts pro-oxidant actions by reducing the metal ion and perpetuating the formation of a hydroxyl radical (OH•) through the Fenton reaction (reaction 2).However, the combination of
Received: Revised: Accepted: Published:
both usually leads to overall antioxidant effects through the formation of reaction products, such as nitric oxide (NO) © 2014 American Chemical Society
2NO2− + A → 2NO + 2OH− + dehydro‐A
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December 19, 2013 February 14, 2014 February 18, 2014 February 18, 2014 dx.doi.org/10.1021/jf405705t | J. Agric. Food Chem. 2014, 62, 2158−2164
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system [25 μM Fe(III) and 2.5 mM H2O2, final concentrations]. A previous kinetic study was undertaken to set the concentration of prooxidants and the length of the assay. The headspace of the vials was generously flushed with nitrogen and immediately transferred to an oven to induce oxidation at 37 °C and constant stirring. Dependent upon the addition of sodium nitrite (N; 0, 75, and 150 mg/L) and sodium ascorbate (A; 0, 250, and 500 mg/L), nine different reaction units were prepared in triplicate (n = 3) according to a total factorial design. Upon completion of the oxidation assay, samples were analyzed for the concentration of tryptophan, AAS, SBs, and 3NT. Analysis of AAS. A total of 400 μL of protein suspension were dispensed in Eppendorf tubes and treated with a cold 10% trichloroacetic acid (TCA) solution. Each Eppendorf tube was vortexed and then subjected to centrifugation at 2000g for 30 min at 4 °C. The supernatant was removed, and the pellet was treated with a cold 5% TCA solution. A new centrifugation was performed at 5000g for 5 min at 4 °C. The supernatant was removed, and the pellets were incubated with the following freshly prepared solutions: 0.5 mL of 250 mM 2-(Nmorpholino)ethanesulfonic acid (MES) buffer at pH 6.0 containing 1% sodium dodecyl sulfate (SDS) and 1 mM diethylenetriaminepentaacetic acid (DTPA), 0.5 mL of 50 mM ABA in 250 mM MES buffer at pH 6.0, and 0.25 mL of 100 mM NaBH3CN in 250 mM MES buffer at pH 6.0. The tubes were vortexed and then incubated in an oven at 37 °C for 90 min. The samples were stirred every 15 min. After derivatization, samples were treated with a cold 50% TCA solution and centrifuged at 5000g for 10 min. The pellet was then washed twice with 10% TCA and diethyl ether/ethanol (1:1). Finally, the pellet was treated with 6 N HCl and kept in an oven at 110 °C for 18 h until completion of hydrolysis. The hydrolysates were dried in vacuo in a centrifugal evaporator. The generated residue was reconstituted with 200 μL of Milli-Q water and then filtered through hydrophilic polypropylene GH Polypro (GHP) syringe filters (0.45 μm pore size, Pall Corporation, Port Washington, NY) for HPLC analysis. A Shimadzu “Prominence” HPLC apparatus (Shimadzu Corporation, Kyoto, Japan), equipped with a quaternary solvent delivery system (LC20AD), a DGU-20AS online degasser, a SIL-20A autosampler, a RF-10A XL fluorescence detector, and a CBM-20A system controller, was used. An aliquot (1 μL) from the reconstituted protein hydrolysates was injected and analyzed in the above-mentioned HPLC equipment. AAS− ABA was eluted in a Cosmosil 5C18-AR-II RP-HPLC column (5 μm, 150 × 4.6 mm) equipped with a guard column (10 × 4.6 mm) packed with the same material. The flow rate was kept at 1 mL/min, and the temperature of the column was maintained constant at 30 °C. The eluate was monitored with excitation and emission wavelengths set at 283 and 350 nm, respectively. Standards (0.1 μL) were run and analyzed under the same conditions. Identification of both derivatized semialdehydes in the fluorescence detection (FLD) chromatograms was carried out by comparing their retention times to those from the standard compounds. The peak corresponding to AAS−ABA was manually integrated from FLD chromatograms, and the resulting areas were plotted against an ABA standard curve with known concentrations that ranged from 0.1 to 0.5 mM. Results are expressed as nanomoles of carbonyl compound per milligram of protein. Analysis of TRP. The decrease of TRP fluorescence in MP suspensions was measured on a Perkin-Elmer LS 55 luminescence spectrometer (PerkinElmer, Beaconsfield, U.K.). The emission spectra were recorded from 300 to 400 nm with the excitation wavelength established at 283 nm. Emission spectra of the buffer were recorded under the same conditions and used as background spectra. Excitation and emission slit widths were set at 10 nm, and the speed of data collection while scanning was of 500 nm/min. The TRP content was calculated from a standard curve of the analogous N-acetyl-Ltryptophanamide (NATA). The linearity (R2 = 0.9955; p < 0.05) between the NATA concentration (ranging from 0.1 to 0.5 μM) and fluorescence intensity was statistically significant. Results were expressed as milligrams of NATA equivalents per gram of protein. The percent loss of TRP was calculated as [(T0 − T4)/T0] × 100, where T0 is the concentration of TRP in samples at day 0 and T4 is the remaining concentration at the end of the oxidation assay (4 days). The effect of nitrite and ascorbate against TRP oxidation was calculated as [(TC −
(3NT).13 Skibsted1 recently postulated the potential facile conversion among ROS and RNS in cured meat systems assisted by other additives, such as ascorbate. However, the connection between oxidative and nitrosative reactions and the impact of protein nitration on the quality of cured muscle foods are poorly understood. Already 35 years ago, Cassens et al.14 reported that almost half of the inorganic nitrite added to a meat product is bound to proteins, while only 10% corresponds to the addition of nitrite to myoglobin to form nitrosylmyoglobin. A full comprehension of the fate of reactive nitrite and its implication in the formation of specific protein oxidation and nitration products requires a second look to the chemistry of curing agents using innovative methodologies. The understanding of the redox chemistry of nitrite and ascorbate will enable, in turn, the identification of the real impact of these reactions on meat proteins and cured muscle foods. This study was planned to elucidate the impact of nitrite and ascorbate on the chemical changes suffered by myofibrillar proteins in a pro-oxidative environment.
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MATERIALS AND METHODS
Chemicals and Raw Material. All chemicals, reagents, and highperformance liquid chromatography (HPLC)-grade solvents were ́ purchased from Panreac (Panreac Quimica S.A., Barcelona, Spain), Merck (Merk, Darmstadt, Germany), and Sigma Chemicals (SigmaAldrich, Steinheim, Germany). The water used was purified by passage through a Milli-Q system (Millipore Corporation, Bedford, MA). Porcine meat (muscle longissimus dorsi) was obtained from a local slaughterhouse. Synthesis of α-Aminoadipic Semialdehyde (AAS) Standard Compound. N-Acetyl-L-AAS was synthesized from Nα-acetyl-L-lysine using lysyl oxidase activity from an egg shell membrane following the procedure described by Akagawa et al.15 Briefly, 10 mM Nα-acetyl-Llysine was incubated with constant stirring with 5 g of egg shell membrane in 50 mL of 20 mM sodium phosphate buffer at pH 9.0 at 37 °C for 24 h. The egg shell membrane then was removed by centrifugation, and the pH of the solution was adjusted to 6.0 using 1 M HCl. The resulting aldehydes were reductively aminated with 3 mmol of p-aminobenzoic acid (ABA) in the presence of 4.5 mmol sodium cyanoborohydride (NaBH3CN) at 37 °C for 2 h with stirring. Then, ABA derivatives were hydrolyzed by 50 mL of 12 M HCl at 110 °C for 10 h. The hydrolysates were evaporated at 40 °C in vacuo to dryness. The resulting AAS−ABA was purified using silica gel column chromatography and ethyl acetate/acetic acid/water (20:2:1, v/v/v) as the elution solvent. The purity of the resulting solution and authenticity of the standard compounds obtained following the aforementioned procedures have been checked using mass spectrometry (MS) and proton nuclear magnetic resonance (1H NMR).15,16 Extraction of Myofibrillar Proteins (MPs). MPs were extracted from 10 g of porcine meat according to the method described by Utrera and Estévez,8 with slight modifications. Meat was weighed, chopped finely with a knife, and treated with 4 volumes of 10 mM potassium phosphate buffer at pH 7. The mixture was homogenized in the ULTRA-TURRAX for 30 s and then centrifuged at 670g for 15 min. Then, the supernatant was carefully removed, and 4 volumes of 10 mM potassium phosphate buffer at pH 7 was added again. The vials were shaken vigorously and centrifuged under the aforementioned conditions, and the supernatant was removed. A total of 4 volumes of 0.1 M NaCl was added, and the samples were stirred and centrifuged as in the previous steps. This procedure was repeated 3 times. Before the last centrifugation, the solution was filtered by passing through a gauze. The pH of this solution was adjusted to 6 with 0.1 N HCl, and the vials were centrifuged. Finally, the supernatant was carefully withdrawn and added to 200 mL of 100 mM sodium phosphate buffer at pH 7 with 0.6 M NaCl and 8 M urea to obtain a concentration of MP of 4 mg/mL. Experimental Setting. MP suspensions (4 mg/mL) were oxidized for 4 days in screw-capped vials using a hydroxyl radical generating 2159
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TT)/TC] × 100, where TC is the concentration of TRP in the control sample and TT is the percent of TRP loss for each treated sample. Analysis of SBs. The formation of SBs was assessed in myofibrillar protein isolates (MPIs) using a LS-55 Perkin-Elmer fluorescence spectrometer (PerkinElmer, Beaconsfield, U.K.). Prior to the analysis, reaction mixtures were diluted (1:20) with 8 M urea in 100 mM sodium phosphate buffer at pH 7. SBs were excited at 350 nm, and the emitted fluorescence was recorded at 450 nm. The excitation and emission slit widths were set at 10 nm, and the speed of data collection while scanning was of 500 nm/min. The height of the peaks corresponding to SB spectra was recorded. After the applied dilutions were taken into consideration, the results were expressed as fluorescence units. Analysis of 3NT. The extent of protein nitration was evaluated by calculating the nitrosation degree (ND), which is defined as the average number of 3NT residues divided by the total number of tyrosine residues in a protein molecule. 3NT was quantified by spectrophotometry according to the procedure recently described by Yang et al.17 Briefly, 5 mL of MP suspension was treated with 10% cold TCA and then centrifuged at 670g for 5 min. Protein pellets were reconstituted with 1 mL of 8 M urea in 100 mM sodium phosphate buffer at pH 3.5. Tyrosine and 3NT were assessed at 280 and 357 nm, respectively. Statistical Analysis. Data from the analysis (n = 3) were collected and subjected to statistical analyses. To assess the effect of the different concentrations of nitrite and ascorbate, an analysis of variance (ANOVA) was applied (SPSS, version 15.5). A Tukey test was applied when ANOVA found significant differences between treatments. The statistical significance was set at p < 0.05. Response surface analysis was also carried out using the Unscrambler program (version 10.1).
The addition of nitrite had no influence on the residual amount of TRP in the experimental units. In opposition to our initial hypothesis, nitrite displayed neither antioxidant nor prooxidant actions on TRP residues. Among the antioxidant properties attributed to nitrite, authors have reported the ability of nitrite to sequester oxygen and to chelate metals and, hence, block their pro-oxidant actions.1,2 Oxygen was removed from the headspace of the experimental units, and its role in the oxidation events described in the present study may have been negligible. On the other hand, the chelation of iron, given its role in the Fenton chemistry and the metal-catalyzed oxidation of proteins,6 would have been a hypothetical mechanism for nitrite to inhibit TRP oxidation. In fact, the chelation of transition metals by phenolic compounds has been reported to be behind the antioxidant action of various phytochemicals on TRP residues.19,21,24 Whereas the ability of nitrite to chelate iron in the conditions of the present experiment remains indefinite, it is obvious that this mechanism was not effective to inhibit TRP oxidation. The potential pro-oxidant effect of nitrite on TRP residues was not found, even though RNS, such as peroxynitrite, were actually found in the present samples (data not shown). TRP residues are known to be sensitive to both ROS20 and RNS.25 Because TRP depletion was independent of the nitrite concentration, it is reasonable to conclude that RNS contributed to neither the oxidation nor the nitration of TRP, because both nitrite-mediated modifications would have led to the loss of TRP fluorescence. Consistently, Ito et al.26 reported that, even at the most favorable conditions of acidic pH and protein conformation, nitrite weakly reacts with TRP in myosin to form nitrosated species. Additional insight on the nitration of protein residues will be provided in the 3NT section. Ascorbate had a significant effect on the residual amounts of TRP (Table 1). Regardless of the concentration of nitrite, MP suspensions treated with 500 mg/L of ascorbate had significantly higher concentrations of TRP than the control counterparts. The effect of the combination of both curing agents as displayed in the response surface (Figure 1A) is similar to that of the ascorbate alone. The potential pro-oxidant effect of ascorbate in the presence of transition metals via the Fenton reaction (reaction 2) was obviously not observed in the present study. On the contrary, these results reflect an antioxidant effect of ascorbate against the oxidation of TRP residues. Ascorbate has been highlighted as an efficient scavenger of hydroxyl radicals,27 and this ability may explain the present results. The ascorbyl radical formed as a result of the electron donation finds stability in the form of the dehydroascorbic acid, which does not yield further reaction species.27 In medical research, Frei et al.28 already described as “outstanding” the antioxidant activity of ascorbate in human plasma. Unfortunately, the authors reported the antioxidant effect on plasma lipids, while the effect on other plasma components, such as proteins, was not assessed. While the food science literature on this topic is scarce, studies on medical research often varied and, at times, provided contradictory results on the effect of ascorbate on TRP. Some authors highlight the pro-oxidant action of ascorbate under physiological conditions,29,30 while others described the ability of ascorbate to inhibit and even revert oxidation and nitrosation of TRP residues.31 The present results show that ascorbate offers antioxidant protection to TRP in MP as a likely consequence of scavenging hydroxyl radicals. Effect of Nitrite and Ascorbate on AAS Formation. As expected, the hydroxyl-radical-generating system induced efficiently the formation of AAS in MP (Table 2). The
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RESULTS AND DISCUSSION Effect of Nitrite and Ascorbate against TRP Oxidation. Table 1 shows the residual amounts of TRP in the suspensions of Table 1. Concentration of TRP (mg of NATA/g of Protein) in MPIs with Added Nitrite (N) and Ascorbate (A) (Mean ± Standard Deviation)a 0 mg/L A 250 mg/L A 500 mg/L A pc
0 mg/L N
75 mg/L N
150 mg/L N
pb
3.32 y ± 0.37 4.01 y ± 0.49 6.35 x ± 1.46 0.013
2.63 y ± 0.70 4.45 x ± 0.58 4.85 x ± 0.71 0.004
2.59 y ± 0.22 4.47 x ± 0.44 5.58 x ± 0.99 0.003
0.369 0.500 0.986
a Means with different letters (x and y) within a column were significantly different (p < 0.05) in a Tukey’s test subsequent to ANOVA. bSignificance value of ANOVA for the effect of added nitrite. c Significance value of ANOVA for the effect of added ascorbate.
MP treated with curing agents after the in vitro oxidation assay. The initial concentration of TRP in the protein suspensions (∼13 mg/g of protein; no significant differences between experimental units) significantly decreased as a result of the prooxidant action of the hydroxyl-radical-generating system. The loss of TRP ranged from 51 to 80% depending upon the treatment applied to the MP suspensions. These results are consistent with previous studies aimed to assess the oxidation of TRP by systems emulating the Fenton chemistry.18,19 A decrease of the TRP fluorescence denotes chemical changes on the TRP residue typically attributed to oxidative deterioration.5 Simat and Steinhart,20 Salminen and Heinonen,21 and Utrera and Estévez19 attributed the oxidative degradation of TRP to the action of hydroxyl radicals and the subsequent reaction between these radicals and the pyrrole ring or the phenyl moiety of the TRP residue. The depletion of TRP in processed muscle foods has also been ascribed to oxidative stress and regarded as a relevant nutritional loss.22,23 2160
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Figure 1. Response surfaces corresponding to the effects of “ascorbate concentration” and “nitrite concentration” on the concentration of (A) TRP, (B) AAS, (C) SBs, and (D) ND in MP suspensions subjected to a hydroxyl radical generation system.
Table 2. Concentration of AAS (nmol of AAS/mg of Protein) in MPIs with Added Nitrite (N) and Ascorbate (A) (Mean ± Standard Deviation)a 0 mg/L A 250 mg/L A 500 mg/L A pc
0 mg/L N
75 mg/L N
150 mg/L N
pb
19.29 x ± 0.67 1.56 y ± 0.34 1.52 a,y ± 0.25
