The (n-3) PUFAs 20:5 (n-3) (EPA) and 22:6 (n-3) (DHA) are thought to benefit human health. The presence of prooxidant compounds in foods, however, renders them susceptible to oxidation during both storage and digestion. The development of oxidation products during digestion and the potential effects on intestinal PUFA uptake are incompletely understood. In the present studies, we examined: (1) the development and bioaccessibility of lipid oxidation products in the gastrointestinal lumen during active digestion of fatty fish using the in vitro digestive tract TNO Intestinal Model-1 (TIM-1); (2) the mucosal cell uptake and metabolism of oxidized compared with unoxidized PUFAs using Caco-2 intestinal cells; and 3) the potential to limit the development of oxidation products in the intestine by incorporating antioxidant polyphenols in food. We found that during digestion, the development of oxidation products occurs in the stomach compartment, and increased amounts of oxidation products became bioaccessible in the jejunal and ileal compartments. Inclusion of a polyphenol-rich grape seed extract (GSE) during the digestion decreased the amounts of oxidation products in the stomach compartment and intestinal dialysates (P < 0.05). In Caco-2 intestinal cells, the uptake of oxidized (n-3) PUFAs was ~10% of the uptake of unoxidized PUFAs (P < 0.05) and addition of GSE or epigallocatechin gallate protected against the development of oxidation products, resulting in increased uptake of PUFAs (P < 0.05). These results suggest that addition of polyphenols during active digestion can limit the development of (n-3) PUFA oxidation products in the small intestine lumen and thereby promote intestinal uptake of the beneficial, unoxidized, (n-3) PUFAs.

The capacity of Atlantic pollock ( Pollachius pollachius ), seabass ( Dicentrarchus labrax ), and horse mackerel ( Trachurus trachurus ) hemoglobin (Hb) to promote lipid oxidation has been evaluated in liposomes and washed minced horse mackerel muscle. The pro-oxidant ability of fish Hbs was related with their vulnerability to suffer oxidation to metHb and release hemin either in spontaneous situation or in the presence of two representative lipid oxidation products, linolein hydroperoxides and trans-2-pentenal. The results indicated similar effectiveness to promote lipid oxidation in liposomes and washed fish muscle: pollock Hb > horse mackerel Hb > seabass Hb. Pollock Hb showed a more elevated autoxidation rate and spontaneous hemin loss and also faster oxidation to metHb in the presence of hydroperoxides and trans-2-pentenal. The autoxidation and spontaneous hemin loss rates were intermediate for horse mackerel Hb, whereas seabass Hb exhibited the highest stability. The isoelectrofocusing (IEF) pattern of pollock Hb revealed the presence of isoforms with elevated anionic character, which are known to have poor oxygen affinity at the pH values found in fish muscle (pH 7.0-5.5). In agreement with the IEF patterns, pollock Hb was less oxygenated at pH 6.8, and seabass Hb exhibited more oxygenation than did horse mackerel. MetHb forms were significantly more effective in catalyzing lipid oxidation than the corresponding reduced fish Hbs and free hemin. The present investigation highlights a direct correlation between the pro-oxidant capacity of fish Hbs and their susceptibility to undergo metHb formation and hemin loss and also suggests a potential role of lipid oxidation byproducts in activating the pro-oxidative action of hemoglobin.

The efficiency of different reductants (reduced glutathione, ascorbic acid, and catalase) and metal chelators [ethylenediaminetetraacetic acid (EDTA), citric acid, sodium tripolyphosphate (STPP), and adenosine-5'-triphosphate (ATP)] to inhibit lipid oxidation promoted by fish hemoglobin was investigated. The inhibitory activity on hemoglobin-catalyzed lipid oxidation was also evaluated for grape oligomeric catechins (proanthocyanidins), which have both reducing and chelating properties. The antioxidant activity was studied in two different lipid oxidation models, liposomes and washed minced fish muscle. Grape proanthocyanidins were found to be significantly more effective than other reductants to prevent hemoglobin-mediated lipid oxidation in both liposomes and washed fish muscle. Reduced glutathione was also efficient to retard lipid oxidation at the same molarity in washed fish muscle, whereas catalase and ascorbic acid showed a lower antioxidant activity. Metal chelators were less active than reductants, and consequently, the former were necessarily evaluated at much higher concentration than grape proanthocyanidins and reducing compounds. STPP was found to be the iron chelator with the strongest efficiency to delay hemoglobin-mediated lipid oxidation followed by EDTA. Citric acid and ATP were ineffective in retarding lipid oxidation in both systems. Grape proanthocyanidins provided the most extensive protection to preserve hemoglobin at ferrous state in washed fish muscle. Our results draw attention to the greater capacity of reducing compounds to prevent fish hemoglobin-mediated lipid oxidation in comparison with iron chelators, suggesting that the free radical scavenging and/or reduction of ferrylHb species are crucial actions to avoid the pro-oxidant capacity of fish hemoglobin.

The muscle composition of a pelagic fish species, Atlantic mackerel (Scomber scombrus), has been studied to determine the relationship with its susceptibility to develop lipid oxidation during chilled storage. For such an aim, the initial concentrations of the major components (water, total lipids, protein, and PUFAs) and minor pro-oxidant and antioxidant components (ascorbic acid, α-tocopherol, hemoglobin, total iron, LMW-iron, copper, and zinc) of different batches of mackerel were characterized. For the study, several batches of mackerel were caught during the spring and summer periods. The different batches were subjected to chilled storage, and the onset of lipid oxidation was statistically related with the initial muscle composition. Results showed significant compositional differences among the mackerel lots, especially for the muscle lipid content (2.83-9.50%). In a first step, a Pearson correlation test was used to check the influence of each component on the progress of lipid oxidation. Results showed a significant relationship between shelf life and water and total lipid contents. Multiple regression was performed to reveal the contribution of each component to the susceptibility to lipid oxidation. The model obtained combines the content of PUFAs, total iron, hemoglobin, and ascorbic acid. An accurate prediction of shelf life in terms of rancidity was achieved by the model created (R(2) = 0.9975). These results establish that the levels of endogenous pro-oxidants and antioxidants present in fish muscle together with the polyunsaturated lipids are relevant factors affecting the shelf life of mackerel muscle.

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The influence of polymerization (number of monomers) and galloylation (content of esterified gallates) of oligomeric catechins (proanthocyanidins) on their effectiveness to prevent lipid oxidation in pelagic fish muscle was evaluated. Non-galloylated oligomers of catechin with diverse mean polymerization (1.9-3.4 monomeric units) were extracted from pine (Pinus pinaster) bark. Homologous fractions with galloylation ranging from 0.25 to <1 gallate group per molecule were obtained from grape (Vitis vinifera) and witch hazel (Hamamelis virginiana). The results showed the convenience of proanthocyanidins with medium size (2-3 monomeric units) and low galloylation degree (0.15-0.25 gallate group/molecule) to inhibit lipid oxidation in pelagic fish muscle. These optimal structural characteristics of proanthocyanidins were similar to those lately reported in fish oil-in-water emulsions using phosphatidylcholine as emulsifier. This finding suggests that the antioxidant behavior of polyphenols in muscle-based foods can be mimicked in emulsions prepared with phospholipids as emulsifier agents. The present data give relevant information to achieve an optimum use of polyphenols in pelagic fish muscle.

The interaction and location of phenolic antioxidants in model membranes has been related with their effectiveness for inhibiting lipid oxidation of fish mince with the aim to identify mechanisms involved in the antioxidant effectiveness in muscle foods. For such scope, the effect of grape seed extract and its main components, catechin, epicatechin and procyanidin B(2) to be located and induce changes in phospholipid model membranes was studied by different biophysical techniques and related to their antioxidant efficiency. Grape seed extract showed the highest inhibition of oxidation in chilled minced fish muscle. Antioxidant in- vitro capacities were also studied but they did not show a clear relationship with the antioxidant efficiency found in fish muscle. The phospholipid/water partition coefficients and fluorescence quenching studies showed that procyanidin B(2) was located in a more internal location than monomeric catechin and epicatechin within the phospholipid palisade. Grape seed extract showed strongest effect compared to its main components in the increase of the lipid order at the DMPC fluid phase by fluorescence anisotropy measurements. Grape seed extract also promoted a dehydration effect in DMPC membranes at the phospholipid/water interface and resistance to solubilization by nonionic detergents in DMPC membranes. The presence of molecular linkages, probably by hydrogen bonding, is proposed between procyanidins (or some galloylated catechins) and the polar head groups of the phospholipids to account for the dehydration effect at the phospholipid/water interface and membrane-stabilizing effects. These effects may be directly related to the higher efficacy of grape seed extract to inhibit lipid oxidation in fish muscle, probably by hindering radical propagation.

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.