Comparison of descriptive sensory analysis and chemical analysis for oxidative changes in milk.

Oxidation in 3 types of bovine milk with different fatty acid profiles obtained through manipulation of feed was evaluated by analytical methods quantifying the content of potential antioxidants, the tendency of formation of free radicals, and the accumulation of primary and secondary oxidation products. The milk samples were evaluated in parallel by descriptive sensory analysis by a trained panel, and the correlation between the chemical analysis and the descriptive sensory analysis was evaluated. The fatty acid composition of the 3 types of milk was found to influence the oxidative and lipolytic changes occurring in the milk during chill storage for 4 d. Sensory analysis and chemical analysis showed high correlation between the typical descriptors for oxidation such as cardboard, metallic taste, and boiled milk and specific chemical markers for oxidation such as hexanal. Notably, primary oxidation products (i.e., lipid hydroperoxides) and even the tendency of formation of radicals as measured by electron spin resonance spectroscopy were also highly correlated to the sensory descriptors for oxidation. Electron spin resonance spectroscopy should accordingly be further explored as a routine method for detection of early events in lipid oxidation in milk to predict shelf-life.

Oxidation of ascorbate in raw milk induced by enzymes and transition metals.

The effect of xanthine oxidase, lactoperoxidase, and transition metals [Fe(III), Cu(II)] on the oxidation of ascorbate in raw milk was investigated. Data clearly showed that iron(III) (200 microM) does not accelerate ascorbate oxidation in raw milk in concentrations relevant for raw milk. In contrast, addition of copper(II) (10 microM) to the raw milk accelerated oxidation of ascorbate. Furthermore, both xanthine oxidase and peroxidase activity were found to accelerate ascorbate oxidation dramatically in raw milk, indicating that xanthine oxidase and lactoperoxidase might be some of the most obvious candidates for mediation of ascorbate oxidation in raw milk. The present data are discussed in relation to using the fate of ascorbate in raw milk as an indicator of the oxidative stability of the milk.

Protein radicals in the reaction between H2O2-activated metmyoglobin and bovine serum albumin.

Hydrogen peroxide activation of MMb with and without the presence of BSA gave rise to rapid formation of hyper-valent myoglobin species, myoglobin ferryl radical (*MbFe(IV) = O) and/or ferrylmyoglobin (MbFe(IV) = O). Reduction of MbFe(IV) = O showed first-order kinetics for a 1-2 times stoichiometric excess of H2O2 to MMb while a 3-10 times stoichiometric excess of H2O2 resulted in a biphasic reaction pattern. Radical species formed in the reaction between MMb, H2O2 and BSA were influenced by [H2O2] as measured by electron spin resonance (ESR) spectroscopy and resulted in the formation of cross-linking between BSA and myoglobin which was confirmed by SDS-PAGE and subsequent amino acid sequencing. Moreover, dityrosine was formed in the initial phases of the reaction for all concentrations of H2O2. However, initially formed dityrosine was subsequently utilized in reactions employing stoichiometric excess of H2O2 to MMb. The observed breakdown of dityrosine was ascribed to additional radical species formed from the interaction between H2O2 and the hyper-valent iron-center of H2O2-activated MMb.

Antioxidative activity of urate in bovine milk.

The antioxidative effects of urate on peroxidase-induced protein oxidation and light-induced riboflavin degradation and lipid oxidation in whole milk were studied. In addition, experiments using ascorbate were conducted to directly compare the antioxidative activity of urate and ascorbate. The presence of urate and/or ascorbate (10-30 mg/L) lowered peroxidase-induced formation of dityrosine by 44-96% in unpasteurized whole milk. No synergistic effect of urate and ascorbate on peroxidase-induced dityrosine formation was registered, but merely an additive effect. Light exposure of pasteurized whole milk showed that ascorbate was oxidized at the expense of urate, which indicated ascorbate-mediated recycling of the urate radical. Moreover, both urate and ascorbate (30 mg/L) retarded light-induced lipid oxidation in pasteurized whole milk as measured by formation of lipid hydroperoxides with urate being the most effective (28% reduction in lipid hydroperoxides) compared with ascorbate (14%). Finally, addition of urate or ascorbate (300 mg/L) to pasteurized whole milk showed a slight protective effect against light-induced degradation of riboflavin with urate being the most effective.

Lactoperoxidase-induced protein oxidation in milk.

The reaction between lactoperoxidase (LPO) and H(2)O(2) in the presence of bovine serum albumin (BSA), beta-lactoglobulin, or casein was investigated for the formation of protein radicals by freeze-quench electron spin resonance (ESR) and by the formation of the protein oxidation product, dityrosine. The presence of BSA resulted in a dramatic change after 1 min of reaction in the obtained ESR spectrum compared with the spectrum obtained for LPO and H(2)O(2) alone. Furthermore, experiments employing BSA or beta-lactoglobulin resulted in the formation of long-lived protein radicals detectable 10 min after initiation of the reaction. The presence of casein resulted in a minor change in the fine structure of the ESR spectrum after 1 min of reaction compared with LPO and H(2)O(2) alone, but no difference between the two reaction mixtures could be observed after 10 min of reaction. The formation of dityrosine could be detected in reaction mixtures containing LPO and H(2)O(2) after 1 and 10 min of incubation at 25 degrees C both in the absence and in the presence of BSA, beta-lactoglobulin, or casein. The presence of casein resulted in an increased dityrosine concentration compared with the reaction with LPO and H(2)O(2) alone. Endogenous LPO in unpasteurized milk was activated at 25 degrees C by adding 1 mM H(2)O(2). Radical species could be detected directly in the milk by freeze-quench ESR during the initial phase of the reaction, and dityrosine could be measured after 4 h of incubation. The role of LPO activity in the formation of ESR detectable radical species and dityrosine in milk was further verified in ultrahigh temperature (UHT) milk with no endogenous enzyme activity, as the formation of ESR detectable radical species and dityrosine took place in UHT milk only upon the addition of both H(2)O(2) and exogenous LPO.

Myoglobin-induced oxidative damage: evidence for radical transfer from oxidized myoglobin to other proteins and antioxidants.

Reaction of equine Fe(III) myoglobin with H2O2 gives rise to an Fe(IV)-oxo species at the heme center and protein (globin)-derived radicals. Studies have shown that there are two (or more) sites for the protein-derived radical: at tyrosine (Tyr-103) or tryptophan (Trp-14). The latter radical reacts rapidly with oxygen to give a Trp-derived peroxyl radical. The formation of both the tyrosine phenoxyl radical and the tryptophan-derived peroxyl species have been confirmed in the present study; the latter appears to be the major initial radical, with the phenoxyl radical appearing at longer reaction times, possibly via secondary reactions. We have investigated, by EPR spectroscopy, the reactivity of the Trp-14 peroxyl radical with amino acids, peptides, proteins, and antioxidants, with the aim of determining whether this species can damage other targets, i.e., whether intermolecular protein-to-protein radical transfer and hence chain-oxidation occurs, and the factors that control these reactions. Three amino acids show significant reactivity: Tyr, Trp, and Cys, with Cys the least efficient. Evidence has also been obtained for (inefficient) hydrogen abstraction at peptide alpha-carbon sites; this may result in backbone cleavage in the presence of oxygen. The myoglobin Trp-14 peroxyl radical has been shown to react rapidly with a wide range of proteins to give long-lived secondary radicals on the target protein. These reactions appear to mainly involve Tyr residues on the target protein, although evidence for reaction at Trp has also been obtained. Antioxidants (GSH, ascorbate, Trolox C, vitamin E, and urate) react with the myoglobin-derived peroxyl radical; in some cases antioxidant-derived radicals are detected. These reactions are only efficient at high antioxidant concentrations, suggesting that protein-to-protein damage transfer and protein chain-oxidation may occur readily in biological systems.

Formation of long-lived radicals on proteins by radical transfer from heme enzymes--a common process?

Incubation of Fe(III)myoglobin (Fe(III)Mb) with H2O2 in the presence of bovine serum albumin (BSA) has been shown previously to give albumin-derived radicals as a result of radical transfer from myoglobin to BSA. In this study the occurrence of similar processes with peroxidases has been investigated using horseradish peroxidase (HRP)/H2O2, in the presence and absence of added tyrosine. Incubation of HRP with H2O2 and bovine or human serum albumins, in the presence and absence of tyrosine, gave long-lived albumin-derived radicals as detected by EPR spectroscopy. Evidence has been obtained for these albumin radicals being located on buried tyrosine residues on the basis of blocking experiments. The effect of protein conformation on radical transfer has been investigated using partial proteolytic digestion prior to protein oxidation. With HRP/H2O2/BSA and Fe(III)Mb/H2O2/BSA increased radical concentrations were observed after limited digestion, although this effect was less marked with the HRP/H2O2/BSA system than with Fe(III)Mb/H2O2/BSA, consistent with different modes of radical transfer. More extensive digestion of BSA decreased the radical concentration to levels below those detected with native albumin, indicating that the tertiary structure of the target protein plays an important role in determining the rate of radical transfer and/or the stability of the resultant species. These results are consistent with a mechanism for the HRP/H2O2/no free tyrosine system involving radical transfer to the albumin via the heme edge of the peroxidase. In contrast, albumin radical formation by the HRP/H2O2/free tyrosine system was only marginally affected by proteolysis, consistent with free tyrosine phenoxyl radicals being the mediators of radical transfer, without significant protein-protein interaction. These protein-to-protein radical transfer reactions may have important consequences for understanding protein oxidation in biological systems.

Formation of long-lived protein radicals in the reaction between H2O2-activated metmyoglobin and other proteins.

Free radicals formed during the reaction of H2O2 and metmyoglobin in the presence of bovine serum albumin (BSA) were investigated using freeze quench and spin-trap ESR spectroscopy. Increasing concentrations of BSA (0-300 microM) resulted in drastic changes in the characteristic freeze quench ESR signal of H2O2-activated metmyoglobin (perferryl protein radical) under physiological conditions (pH = 7.4; I = 0.16). The radical species formed during reaction of 100 microM H2O2, 100 microM metmyoglobin, and 200 microM BSA have half-lives of approximately 13 min at 25 degrees C, in contrast to the perferryl protein radical that has a half-life of approximately 28 s at 25 degrees C. The radical species formed in the presence of BSA were reactive towards ascorbate, glutathione, cysteine, and tyrosine. Substitution of BSA with defatted BSA, gamma-globulin or beta-lactoglobulin also resulted in formation of long-lived free radical species (half-lives: 13-18 min); however, the ability to form these was dependent of the specific protein and decreased in the following order: BSA > defatted BSA > gamma-globulin > beta-lactoglobulin. The spin-trap alpha-phenyl-tert-butylnitrone (PBN) showed the presence of transient protein radical species formed in the reaction between MMb, H2O2, and BSA. Transient radical species that could be proposed as intermediates in the formation of the long-lived protein radicals detected by freeze-quench ESR. Dityrosine was formed in the reaction between MMb, H2O2, and BSA, showing the involvement of tyrosine residues in the present reaction. The described chemical interaction between H2O2-activated myoglobin and other proteins have major consequences on future interpretations of the significance of the perferryl protein radical in biological systems where proteins are abundant.

Reduction of ferrylmyoglobin by beta-lactoglobulin.

Reduction of iron (IV) in ferrylmyoglobin in the presence of beta-lactoglobulin in aqueous solution is the result of two parallel reactions: (i) a so-called autoreduction, and (ii) reduction by beta-lactoglobulin in a second-order-reaction resulting in bityrosine formation in beta- lactoglobulin. In the pH-region investigated (5.4-7.4), the rate of reduction increased for both reactions with decreasing pH. The second order-reaction had for non-denatured beta-lactoglobulin the activation parameters: delta H* = 45 kJ.mol-1 and delta S not equal to = -93 J.mol-1.K-1 at pH = 7.0 and ionic strength 0.16 (NaCl). Reduction of ferrylmyoglobin by beta-lactoglobulin denatured by heat (86 degrees C for 3 min) or by hydrostatic pressure (300 MPa for 15 min) resulted in formation of higher molecular weight species as detected by size-exclusion chromatography and by SDS-PAGE. No molecular weight changes were observed for reduction of ferrylmyoglobin by native beta-lactoglobulin. Detection of bityrosine in the native beta-lactoglobulin fraction after oxidation with ferrylmyoglobin indicated intra-molecular bityrosine formation. In heat-denatured beta-lactoglobulin bityrosine formation could be of intra-molecular and/or of inter-molecular origin, the latter being confirmed by size-exclusion chromatography.