Hemoglobin-mediated oxidation of washed minced cod muscle phospholipids: effect of pH and hemoglobin source

Lipid pro-oxidative properties and deoxygenation/autoxidation patterns of hemoglobins from nonmigratory white-fleshed fish (winter flounder and Atlantic pollock) and migratory dark-fleshed fish (Atlantic mackerel and menhaden) were compared during ice storage at pH 7.2 and 6. A washed cod mince model system and a buffer model system were used for studying lipid changes and hemoglobin changes, respectively. TBARS and painty odor were followed as markers for lipid oxidation. At pH 6, all four hemoglobins were highly and equally active as pro-oxidants. At pH 7.2, pro-oxidation by all hemoglobins except that from pollock was slowed down, and activity ranked as pollock > mackerel > menhaden > flounder. The higher catalytic activities of the hemoglobins at pH 6 than at pH 7.2 corresponded with higher formation of deoxyhemoglobin and methemoglobin. Pollock had the most extensive formation of deoxy- and methemoglobin at both pH values, which could explain its high catalytic activity. The pro-oxidative differences among the other hemoglobins at pH 7.2 did not correlate with deoxygenation and autoxidation reactions. This indicates involvement of other structural differences between the hemoglobins such as differences in the heme-crevice volume. It is suggested that a biological reason for the species differences was their adaptations to different depths/water temperatures.     

Effect of pH on hemoglobin-catalyzed lipid oxidation in cod muscle membranes in vitro and in situ

The effect of pH and hemoglobin on oxidation of the microsomal lipids of cod was determined in isolated microsomes and in washed cod muscle. An increase of hemoglobin concentration from 0.5 to 15 microM accelerated lipid oxidation in both systems. In cod microsomes the rate of lipid oxidation increased in the order pH 6.8 > pH 7.6 > pH 8.4 > pH 6.0 > pH 3.5. However, in washed cod muscle a decrease of pH from 7.8 to 6.8 greatly increased the lag phase and decreased the rate of lipid oxidation. A further decrease in pH to 3.5 decreased the lag phase and increased the rate of lipid oxidation further. A decrease of pH from 7.6 to 6.4 greatly reduced the affinity of hemoglobin for oxygen. Formation of methemoglobin due to autoxidation occurred more rapidly at pH 6.0 than at pH 7.5. Structural changes of the isolated microsomal membranes could be the reason for the unexpected slow lipid oxidation in microsomes at pH 6.0 and below.     

Ability of lipid hydroperoxides to partition into surfactant micelles and alter lipid oxidation rates in emulsions

Lipid hydroperoxides are important factors in lipid oxidation due to their ability to decompose into free radicals. In oil-in-water emulsions, the physical location of lipid hydroperoxides could impact their ability to interact with prooxidants such as iron. Interfacial tension measurements show that linoleic acid, methyl linoleate, and trilinolein hydroperoxides are more surface-active than their non-peroxidized counterparts. In oil-in-water emulsion containing surfactant (Brij 76) micelles in the continuous phase, linoleic acid, methyl linoleate, and trilinolein hydroperoxides were solubilized out of the lipid droplets into the aqueous phase. Brij 76 solubilization of the different hydroperoxides was in the order of linoleic acid > trilinolein > or = methyl linoleate. Brij 76 micelles inhibited lipid oxidation of corn oil-in-water emulsions with greater inhibition of oxidation occurring in emulsions containing linoleic acid hydroperoxides. Surfactant solubilization of lipid hydroperoxides could be responsible for the ability of surfactant micelles to inhibit lipid oxidation in oil-in-water emulsions.     

Deoxyhemoglobin-mediated lipid oxidation in washed fish muscle

Deoxyhemoglobin-mediated lipid oxidation was studied by comparing the pro-oxidative activity of anodic and cathodic hemoglobins from trout in a washed cod muscle model system. At pH 6.3, cathodic hemoglobins were nearly fully oxygenated while anodic hemoglobins were poorly oxygenated. Anodic hemoglobins initiated lipid oxidation in washed cod muscle much more rapidly than cathodic hemoglobins, as measured by thiobarbituric acid reactive substances (TBARS) formation. Moreover, anodic hemoglobins appeared to oxidize more rapidly as compared to cathodic hemoglobins in the washed cod muscle model system, as measured by a decrease in redness (a value). A more pronounced pro-oxidative activity of deoxyhemoglobin as compared to oxyhemoglobin was confirmed by accelerated lipid hydroperoxide and TBARS formation in the washed cod muscle model system upon combined addition of anodic hemoglobins and adenosine triphosphate, which is known to lower the oxygenation of anodic hemoglobins at pH 7.2, as compared to only addition of anodic hemoglobins to the washed cod muscle. These studies suggest that deoxyhemoglobin is more pro-oxidative than its oxygenated counterpart at pH values found in postmortem fish muscle.     

Lipid peroxidation by "free" iron ions and myoglobin as affected by dietary antioxidants in simulated gastric fluids

Grilled red turkey muscle (Doner Kabab) is a real "fast food" containing approximately 200 microM hydroperoxides, homogenized in simulated gastric fluid and oxidized more rapidly at pH 3.0 than at pH 5.0, after 180 min, producing 1200 and 600 microM hydroperoxides, respectively. The effects of "free" iron ions and metmyoglobin, two potential catalyzers of lipid peroxidation in muscle foods, were evaluated for linoleic acid peroxidation at pH 3.0 of simulated gastric fluid. The prooxidant effects of free iron ions on linoleic acid peroxidation in simulated gastric fluid was evaluated in the presence of ascorbic acid. At low concentrations of ascorbic acid, the effects were prooxidative, which was reversed at high concentrations. In the presence of metmyoglobin, ascorbic acid with or without free iron enhanced the antioxidative effect. Lipid peroxidation by an iron-ascorbic acid system was inhibited totally by 250-500 microM catechin at pH 3.0. The catechin antioxidant effect was determined also in the iron-ascorbic acid system containing metmyoglobin. In this system, catechin totally inhibited lipid peroxidation at a concentration 20-fold lower than without metmyoglobin. The ability of catechin to inhibit lipid peroxidation was also determined at a low pH with beta-carotene as a sensitive target molecule for oxidation. The results show that a significant protection was achieved only with almost 100-fold higher antioxidant concentration. Polyphenols from different groups were determined for the antioxidant activity at pH 3.0. The results show a high antioxidant activity of polyphenols with orthodihydroxylated groups at the B ring, unsaturation, and the presence of a 4-oxo group in the heterocyclic ring, as demonstrated by quercetin.     

Interactions between iron, phenolic compounds, emulsifiers, and pH in omega-3-enriched oil-in-water emulsions

The behavior of antioxidants in emulsions is influenced by several factors such as pH and emulsifier type. This study aimed to evaluate the interaction between selected food emulsifiers, phenolic compounds, iron, and pH and their effect on the oxidative stability of n-3 polyunsaturated lipids in a 10% oil-in-water emulsion. The emulsifiers tested were Tween 80 and Citrem, and the phenolic compounds were naringenin, rutin, caffeic acid, and coumaric acid. Lipid oxidation was evaluated at all levels, that is, formation of radicals (ESR), hydroperoxides (PV), and secondary volatile oxidation products. When iron was present, the pH was crucial for the formation of lipid oxidation products. At pH 3 some phenolic compounds, especially caffeic acid, reduced Fe(3+) to Fe(2+), and Fe(2+) increased lipid oxidation at this pH compared to pH 6. Among the evaluated phenols, caffeic acid had the most significant effects, as caffeic acid was found to be prooxidative irrespective of pH, emulsifier type, and presence of iron, although the degrees of lipid oxidation were different at the different experimental conditions. The other evaluated phenols were prooxidative at pH 3 in Citrem-stabilized emulsions and had no significant effect at pH 6 in Citrem- or Tween-stabilized emulsions on the basis of the formation of volatiles. The results indicated that phenol-iron complexes/nanoparticles were formed at pH 6.     

Added triacylglycerols do not hasten hemoglobin-mediated lipid oxidation in washed minced cod muscle

Hemoglobin-mediated lipid oxidation in washed, minced cod muscle was related to the triacylglycerol to membrane lipid ratio. The same rapid development of thiobarbituric acid reactive substances (TBARS) and painty odor occurred with and without the presence of up to 15% menhaden oil. Without hemoglobin, development of TBARS and painty odor was slow, despite a high amount of hydroperoxides in samples with oil added (1135 micromol/kg muscle). This suggested that hemoglobin reacted by cleaving preformed hydroperoxides into secondary oxidation products. Nearly doubling the hemoglobin concentration approximately doubled the extent of lipid oxidation with and without added oil. This indicated that hemoglobin was limiting for the oxidation reaction. The noneffect of added oil suggests that membrane lipids and/or preformed membrane lipid hydroperoxides provided sufficient substrate in hemoglobin-catalyzed oxidation of washed minced cod muscle. Fe(2+-)ADP did not induce any oxidation of washed minced cod with/without added oil. Results suggest that lipid oxidation in fatty fish may be more related to the quantity and type of the aqueous pro-oxidant and the membrane lipids than to variations in total fat contents.     

Quantitative HPLC determination of the antioxidant activity of capsaicin on the formation of lipid hydroperoxides of linoleic acid: a comparative study against BHT and melatonin.

The antioxidant activity of capsaicin, as compared to BHT and melatonin, was determined by the direct measurement of lipid hydroperoxides formed upon linoleic acid autoxidation initiated by AIBN. The formation of four isomeric lipid hydroperoxides was detected after reverse-phase HPLC separation. Data from three detectors, UV absorption, glassy carbon electrode electrochemical detection, and postcolumn chemiluminescence using luminol, were compared. Capsaicin was more effective than melatonin in suppressing the formation of lipid hydroperoxides but not as effective as BHT. The formation of capsaicin and BHT dimers was observed during oxidation, and the dimers were characterized using APCI MS(n).     

Copper-catalyzed oxidation of a structured lipid-based emulsion containing alpha-tocopherol and citric acid: influence of pH and NaCl

The effects of salt and pH on copper-catalyzed lipid oxidation in structured lipid-based emulsions were evaluated. Ten percent oil-in-water emulsions were formulated with a canola oil/caprylic acid structured lipid and stabilized with 0.5% whey protein isolate. alpha-Tocopherol and citric acid were added to the emulsions to determine how changes in pH or the addition of NaCl affected their antioxidant activity. The peroxide values and anisidine values of emulsions stored at 50 degrees C were measured over an 8-day period. Increased lipid oxidation occurred in the pH 7.0 emulsions and when 0.5 M NaCl was added to the pH 3.0 samples. Adding alpha-tocopherol, citric acid, or a combination of the two compounds slowed the formation of hydroperoxides and their subsequent decomposition products in pH 3.0 emulsions.     

Lipid oxidation in fish oil enriched mayonnaise: calcium disodium ethylenediaminetetraacetate, but not gallic acid, strongly inhibited oxidative deterioration

The antioxidative effects of gallic acid, EDTA, and extra emulsifier Panodan DATEM TR in mayonnaise enriched with 16% fish oil were investigated. EDTA reduced the formation of free radicals, lipid hydroperoxides, volatiles, and fishy and rancid off-flavors. The antioxidative effect of EDTA was attributed to its ability to chelate free metal ions and iron from egg yolk located at the oil-water interface. Gallic acid reduced the levels of both free radicals and lipid hydroperoxides but promoted slightly the oxidative flavor deterioration in mayonnaise and influenced the profile of volatiles. Gallic acid may therefore promote the decomposition of lipid hydroperoxides to volatile oxidation products. Addition of extra emulsifier reduced the lipid hydroperoxide levels but did not influence the level of free radicals or the oxidative flavor deterioration in mayonnaisse; however, it appeared to alter the profile of volatiles. The effect of the emulsifier on the physical structure and rheological properties depended on the presence of antioxidants.     

Myoglobin oxidation in a model system as affected by nonheme iron and iron chelating agents

A model system was used to study the effect of nonheme iron on myoglobin oxidation at pH 5.6 and pH 7.2 at 23 degrees C. The addition of ferrous iron significantly (p < 0.05) increased the rate of myoglobin oxidation in the absence of lipid, demonstrating that iron promoted myoglobin oxidation independent of the effect of lipid oxidation. The addition of the type II, iron chelating antioxidants sodium tripolyphosphate (at pH 7.2) or milk mineral (at pH 5.6) negated the effect of added iron, slowing oxidation of myoglobin. A clear concentration dependence was seen for iron-stimulated myoglobin oxidation, based on both spectral and visual evidence. Further investigation is needed to determine the possible role for nonheme ferrous iron on myoglobin oxidation in vivo or in meat.     

Lipid peroxidation and coupled vitamin oxidation in simulated and human gastric fluid inhibited by dietary polyphenols: health implications

The Western diet contains large quantities of oxidized lipids, because a large proportion of the food in the diet is consumed in a fried, heated, processed, or stored form. We investigated the reaction that could occur in the acidic pH of the stomach and accelerate the generation of lipid hydroperoxides and cooxidation of dietary vitamins. To estimate the oxygen content in the stomach after food consumption, oxygen released from masticated bread (20 g) into deoxygenated water (100 mL) was measured. Under these conditions, the oxygen concentration rose by 250 microM and reached a full oxygen saturation. The present study demonstrated that heated red meat homogenized in human gastric fluid, at pH 3.0, generated hydroperoxides and malondialdehyde. The cross-reaction between free radicals produced during this reaction cooxidized vitamin E, beta-carotene, and vitamin C. Both lipid peroxidation and cooxidation of vitamin E and beta-carotene were inhibited at pH 3.0 by red wine polyphenols. Ascorbic acid (44 mg) at a concentration that represented the amount that could be ingested during a meal inhibited lipid peroxidation only slightly. Red wine polyphenols failed to prevent ascorbic acid oxidation significantly but, in conjunction with ascorbic acid, did inhibit lipid peroxidation. In the presence of catechin, a well-known polyphenol found in red wine, ascorbic acid at pH 3.0 works in a synergistic manner preventing lipid peroxidation and beta-carotene cooxidation. The present data may explain the major benefits to our health and the crucial role of consuming food products rich in dietary antioxidants such as fruits, vegetables, red wines, or green tea during the meal.     

Comparative analysis of different hemoglobins: autoxidation,reaction with peroxide,and lipid oxidation

Beef hemoglobin (Hb) had lower levels of deoxyHb and autoxidized much slower as compared to trout Hb at pH 6.3. Chicken Hb autoxidized at a rate intermediate between beef and trout Hb. In the presence of hydrogen peroxide, metHb formed rapidly from trout Hb whereas beef Hb was essentially nonreactive with hydrogen peroxide. The autoxidation rate of perch Hb was more rapid than trout Hb despite the low deoxyHb content of perch Hb. Perch Hb was a better catalyst of lipid oxidation than trout Hb when added to washed cod muscle based on formation of lipid hydroperoxides and thiobarbituric acid reactive substances. These studies indicate that autoxidation rate does not always increase with increasing deoxyHb content. The role of heme crevice volume in heme protein autoxidation is discussed. Among other factors, these studies suggest that rates of lipid oxidation in various muscle foods may depend on the relative ability of hemoglobins from different animal species to promote lipid oxidation.     

Lipid oxidation in a menhaden oil-in-water emulsion stabilized by sodium caseinate cross-linked with transglutaminase

Transglutaminase-catalyzed cross-linking of interfacial proteins in oil-in-water has been shown to influence physical stability, but little is known about how this reaction impacts lipid oxidation. Therefore, this study evaluated the influence of transglutaminase-induced interfacial protein cross-linking on the oxidative stability of casein-stabilized menhaden oil-in-water emulsions. Interfacial casein in menhaden oil-in-water emulsions cross-linked by transglutaminase (pH 7.0) produced a cohesive interfacial protein layer that could not be removed from the emulsion droplet by Tween 20. Although transglutaminase cross-linked the interfacial casein, these emulsions did not show increased oxidative stability when compared to untreated emulsions as determined by measurement of lipid hydroperoxides and thiobarbituric acid reactive substances. These results indicate that increasing the cohesiveness of proteins at the interface of oil-in-water emulsions does not inhibit lipid oxidation. This could be due to the ability of prooxidative species such as iron to diffuse through the cross-linked protein layer where it could promote the decomposition of lipid hydroperoxides into free radicals that could oxidize unsaturated fatty acids in the emulsion droplet core.     

Inhibition of linoleic acid oxidation by interaction with a protein-rich oat fraction

A protein-rich fraction from oat was found to protect linoleic acid against oxidation in an aqueous suspension containing soybean lipoxygenase-1 and micellar linoleic acid. In this system the oat fraction reduced the initial oxidation rate of linoleic acid by 50% when the oat fraction/linoleic acid ratio was 5:1 (w/w). The oat fraction did not act on the lipoxygenase enzyme but reduced the concentration of linoleic acid that serves as a substrate for lipoxygenase-1. To achieve the reduction in the oxidation rate a contact between linoleic acid and the oat fraction was required. The efficiency of the protection was dependent on the duration of this contact: the maximum protection was reached after a 5-min incubation period. However, total cessation of oxidation was not reached with any concentration of the oat fraction, indicating that the oxidizible and non-oxidizible forms of linoleic acid are in equilibrium. Because lipoxygenase-1 prefers the monomeric form of the substrate, the present findings agree with the hypothesis that the oat fraction reduces the concentration of monomolecular form of substrate. In most food systems monomolecular free linoleic acid is liberated slowly and at relatively low concentrations, therefore, even a small amount of the oat fraction would guard the system from oxidative deterioration.     

Role of proteins in oil-in-water emulsions on the stability of lipid hydroperoxides

The purpose of this research was to better understand the mechanisms by which proteins affect the rates of lipid oxidation in order to develop protein-stabilized emulsion delivery systems with maximal oxidative stability. This study evaluated the affect of pH and emulsifier concentration on the stability of cumene hydroperoxide in hexadecane-in-water emulsions stabilized by beta-lactoglobulin (beta-Lg). Emulsions prepared with 0.2 wt % beta-Lg (at pH 7.0) showed a 26.9% decrease in hydroperoxide concentrations 5 min after 0.25 mM ferrous ion was added to the emulsion. EDTA, but not continuous phase beta-Lg, could inhibit iron-promoted lipid hydroperoxide decomposition. Lipid hydroperoxides were more stable to iron-promoted degradation at pH values below the pI of beta-Lg, where the emulsion droplet would be cationic and thus able to repel iron away from the lipid hydroperoxides. Heating the beta-Lg-stabilized emulsions to produce a cohesive protein layer on the emulsion droplet surface did not alter the ability of iron to decompose lipid hydroperoxides. These results suggest that proteins at the interface of emulsion droplets primarily stabilize lipid hydroperoxides by electrostatically inhibiting iron-hydroperoxide interactions.     

Evaluation of occasional nonresponse of a washed cod mince model to hemoglobin (Hb)-mediated oxidation

An emerging model to test antioxidants for application in seafoods is washed cod mince fortified with hemoglobin (Hb) as a catalyst. This system has been used to test the antioxidative activity of certain muscle extracts and some pure compounds such as BHA, BHT, TBHQ, and propyl gallate during ice storage. However, the washed cod mince model has occasionally been resistant to Hb-mediated oxidation. This has been in cases when the moisture of the model has been minimized by washes at the protein isoelectric point (pH approximately 5.5) to allow for large additions of potentially antioxidative solutions. In this paper, noncontrollable and controllable factors for this intriguing occasional oxidation resistance were studied. Compositional analyses (lipid content, alpha-tocopherol, and lipid hydroperoxides) and structural analysis of a "normal" oxidizing model and a stable model were done to identify any differences among them. Some controllable factors related to the model preparation that were studied included different washing pH values (5.5-6.6), Hb concentrations (7.2 and 13.5 microM), final model moisture contents (75, 81, and 90%), and light exposure during ice storage (0 h, 3-4 h, or 24 h of light/day). Results revealed a 2-fold higher alpha-tocopherol content in the stable model than in the oxidizing model. Electron microscopy images showed a more and less disrupted myofibrillar structure in the stable and the oxidizing cod model, respectively. This indicated that "cold setting" (i.e., pre-gelation) of the stable model may have occurred and prevented Hb from diffusing freely in the model. Controllable factors that reduced lipid oxidation in the models were less Hb and lower moisture.     

Lipid oxidation in a chicken muscle model system: oxidative response of lipid classes to iron ascorbate or methemoglobin catalysis

Catalysis by iron ascorbate and activated methemoglobin generated different oxidative responses in chicken muscle model systems. In iron ascorbate systems, large increases in hydroperoxides and thiobarbituric acid-reactive substances (TBARS) occurred during the initial stage of incubation. Thereafter, iron ascorbate catalysis led to a slow increase in the oxidation of triacylglcyerol (TG) and sarcoplasmic reticulum (SR) membrane lipids. By the end of incubation, 24, 36, and 32% of the initial content of n-3 fatty acids in free fatty acids, TG, and SR single-lipid model systems catalyzed by iron ascorbate had been lost. Reduced losses of n-3 fatty acids were observed in the SR and TG fractions (0 and 24%, respectively) when iron ascorbate model systems contained all three lipid fractions (mix). Hydroperoxides and TBARS in model systems catalyzed by activated methemoglobin were characterized by a lag phase during most of the incubation. Consistent with their role as antioxidants, losses of alpha-tocopherol (42-49%), gamma-tocopherol (36-42%), and protein sulfhydryls (41-52%) were observed in model systems catalyzed by activated methemoglobin. SR and mix model systems were 30-50% slower to oxidize than TG model systems when activated methemoglobin served as the catalytic agent.     

Galloylated polyphenols as inhibitors of hemoglobin-catalyzed lipid oxidation in fish muscle

The influence of galloyl residues on the antioxidant mechanism of polyphenols to prevent hemoglobin-promoted lipid oxidation was investigated by using polyphenolic fractions with different degrees of galloylation: nongalloylated structures from pine bark (IVP), medium-galloylated from grape pomace (IVG), and high-galloylated from witch hazel bark (IVH). Hemoglobin (Hb) from the pelagic fish horse mackerel (Trachurus trachurus) was employed as a Hb standard. In vitro experiments showed an important increase in the deoxygenation and autoxidation of horse mackerel Hb at acidic pH values. All polyphenolic fractions significantly reduced the redox stability of Hb in buffer solutions, showing a greater deoxygenation and methemoglobin (metHb) formation in the presence of IVH, followed in decreasing order by IVG and IVP. However, galloylated polyphenols (IVH and IVG) were efficient to inhibit the oxidation of the oxygenated Hb (OxyHb) and the formation of lipid oxidation products in chilled washed fish muscle. This antioxidant activity of galloylated proanthocyanidins showed a positive relationship with the phenolic concentration. Polyphenols devoid of galloyl groups (IVP) were less active to prevent either Hb oxidation or lipid oxidation in fish muscle. The results draw attention to the potential role of galloyl residues to lessen Hb-catalyzed lipid oxidation in muscle and to maintain Hb in reduced and oxygenated states, which exhibit lower pro-oxidant activity as compared to the metHb and deoxyHb species.     

Determination of neutral lipid hydroperoxides by size exclusion HPLC with fluorometric detection. Application to fish oil enriched mayonnaises during storage

A fast (12 min) high-performance size exclusion chromatography (HPSEC) method for the separation of neutral lipid class hydroperoxides in the oil phases from fish oil enriched mayonnaises was developed. Detection and quantification were performed using the postcolumn fluorometric (FL) diphenyl-1-pyrenylphosphine oxidation principle. The reproducibilities judged by intra- and inter-assay variations were 0.64 and 7.2%, respectively. The HPSEC-FL method was applied to assess the effect of supplementations with emulsifier, gallic acid, and EDTA on the oxidative processes in the mayonnaises during storage. Substantial amounts of hydroperoxy triacylglycerols (TAGOOH) and cholesterol esters (CEOOH), together with traces of TAGOOH-dimers, were detected. All supplementations significantly decreased the levels of TAGOOH and to a lesser degree CEOOH. Supplementations with EDTA and gallic acid resulted in constant and slightly increasing levels of TAGOOH, respectively, thus affecting the oxidation mechanisms seen in reference mayonnaise. The emulsifier Panodan TR DATEM reduced the levels of TAGOOH as compared to the appropriate controls.