Article Critique Final

Biochemistry 4211 Assignment 2 – Group A

Written appraisal of “Capacity of Reductants and Chelators To Prevent Lipid Oxidation Catalyzed by Fish Hemoglobin” by Rodrigo Maestre et al.

Submitted to: Dr. F.Shahidi Submitted by Alison Pittman 200607147

ABSTRACT

The article “Capacity of Reductants and Chelators To Prevent Lipid Oxidation Catalyzed by Fish Hemoglobin” written by R. Maestre et al. explored the antioxidant abilities of various compounds under drastic oxidative conditions. The authors studied the effect of several reductants and iron chelators against fish hemoglobin-catalyzed lipid oxidation. The effect of grape proanthocyanidins, which have reducing and chelating activity, was also analyzed. It was determined that grape proanthocyanidins were significantly more effective than the other reductants at inhibiting hemoglobin-mediated lipid oxidation in both liposomes and washed fish muscle. Iron chelators were found to be less active than reductants, thus were examined at higher concentrations than grape proanthocyanidins and reductants. In washed fish muscle, grape proanthocyanidins provided the most protection to maintain hemoglobin at the ferrous state. The authors suggested that the increased ability of reductors to inhibit fish hemoglobinmediated lipid oxidation is due to the free radical scavenging action and or/ reduction of ferryl hemoglobin species. The article is fairly well written and the conclusions drawn are supported by the research findings. However, there are several weaknesses within the paper, both with the methodology used and the writing itself.

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The article ‘Capacity of Reductants and Chelators To Prevent Lipid Oxidation Catalyzed by Fish Hemoglobin’ by Rodrigo Maestre et al. investigated the effectiveness of various reductants and metal chelators to inhibit fish haemoglobin-catalyzed lipid oxidation. The inhibitory activity of grape oligomeric catechins, promoted by fish hemoglobin, was also examined as they have both chelating and reducing properties. Lipid oxidation catalyzed by fish hemoglobin was studied because it is of great interest and has practical implications. Lipid oxidation is a main cause of shelf life shortening as well as quality deterioration in fish products. The results of lipid oxidation on the seafood products include negative effects on flavour, nutritive value, texture, and safety during storage and processing (Hultin, 1994). There are many catalytic systems that can oxidize lipids. Most of these reactions, including light, enzymes, metalloproteins and temperature involve a free radical species that is often a singlet oxygen (Korycka-Dahl, 1978). The oxidative degradation of fatty acids frequently proceeds by a free radical chain reaction mechanism, also known as an “autoxidation” process. Autoxidation in food systems normally proceeds through free-radical reactions consisting of three steps: initiation, chain propagation and termination (Simic and Taylor, 1987). Initiation is the step where a fatty acid radical is produced, generally due to a reactive oxygen species (ROS) such as a hydroxyl radical, which combines with a hydrogen atom to produce a fatty acid radical and water. Fatty acid radicals are unstable and react readily with molecular oxygen, producing peroxyl-fatty acid radicals. These unstable species react with other free fatty acids, resulting in the creation of a new fatty acid radical and lipid peroxide. The propagation cycle continues with the new fatty acid radicals reacting similarily. This “chain reaction mechanism” continues until two radicals react to produce a non-radical species. Termination occurs when the concentration of radical species is high and it is likely that two radical species will collide and react. However,

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any reaction that prevents propogation or removes free radicals plays an important role in the termination step (Simic and Taylor, 1987). Antioxidants are therefore very effective at inhibiting chain oxidation reactions (Simic et al., 1992). Polyunsaturated fatty acids (PUFAs) are easily oxidizable due to their numerous double bonds, between which lie methylene groups that contain reactive hydrogens. Foods that contain PUFAs are highly prone to lipid oxidation. The greater the number of double bonds (degree of unsaturation), the more susceptible the food is to oxidation. Lipid oxidation in food products leads to the formation of hydroperoxides that are very unstable compounds. The products of their breakdown negatively affect food flavour and quality. These products include compounds such as ketones, aldehydes and acids, which can result in off-putting odours and flavours (St. Angelo, 1996). Seafood products are very rich in easily oxidiziable PUFAs, and therefore are susceptible to oxidative degradations that can negatively affect the food taste and quality. Fish muscle also contains catalytic amounts of redox-active metals and heme proteins, which are able to induce oxidative degradation of PUFAs (Mozuraityte et al., 2008). Studies by Pazos et al. (2005, 2006) have demonstrated the capacity of fish hemoglobin to catalyze lipid oxidation in fish membranes. Fish hemoglobin was also shown to trigger lipid oxidation in fish liposomes as well as in a matrix that is similar to fish muscle but lacking hemoglobin, for example washed fish muscle (Maestre et al., 2009; Richards and Hultin, 2002; Undeland et al., 2002). There have been multiple pathways proposed that potentially contribute to the ability of hemoglobin to promote lipid oxidation. Pazos et al. (2008) indicated that hemoglobin stimulates the generation of free radicals through cleavage of lipid hydroperoxides. It has also been suggested that hypervalent ferryl Hb radicals that are formed by the reaction of metHb and hydrogen peroxide (or lipid hydroperoxides) can abstract a hydrogen atom from a

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PUFA (Kanner, 1994). Finally, the release of inorganic iron ions and the oxidized form of a heme group (known as hemin) from hemoglobin also contributes to the pro-oxidative activity of hemoglobin (Grunwald, 2006). Ferrous ions are known to promote lipid oxidization through the Fenton reaction, which produces an oxidizing hydroxyl radical in the presence of hydrogen peroxide (Kanner, 1994). Hemin and ferrous ions have also been shown to promote lipid oxidation through the decomposition of preformed lipid hydroperoxides. This reaction produces harmful free radicals (Pazos, 2008; O’Brien 1969). A study by Maestre et al. (2009) indicated that hemoglobin oxidation to metHb, therefore hemin release, is hastened by various lipid oxidation byproducts such as adlehydes and hydroperoxides. The pro-oxidant activity of hemoglobin seems to be very complex, as the production of lipid oxidation byproducts directly influences multiple pro-oxidant mechanisms. The existence of many pro-oxidant mechanisms often leads to the negative results of lipid oxidation such as food spoilage. This has led to the study, and use, of bioactive compounds with antioxidant properties. Antioxidant compounds have the ability to slow or prevent the oxidation of other molecules. Supplementation of these compounds is an approach to inhibit lipid oxidative reactions that are catalyzed by hemoglobin in meat-based food such as fish, as muscle is rich in pro-oxidative hemoglobin. Several compounds from various sources have been found to decrease fish hemoglobin-catalyzed lipid oxidation including white grape pomace (Pazos, 2006). More information is needed, however, to determine which of the multiple pro-oxidative mechanisms related to hemoglobin are major contributors in muscle-based foods such as fish. In order to establish effective antioxidant compounds to prevent hemoglobin-catalyzed lipid oxidation, it is necessary to determine the necessary physiochemical properties involved.

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The main goal of the paper ‘Capacity of Reductants and Chelators To Prevent Lipid Oxidation Catalyzed by Fish Hemoglobin’ by Maestre et al. was to explore the effects of antioxidant compounds to prevent hemoglobin-promoted lipid oxidation. Multiple compounds with reducing ability or iron-chelating capability, as well as both reducing and chelating ability, were tested for antioxidant activity against fish hemoglobin-mediated lipid oxidation. Two models were used to analyze the activity of the antioxidant compounds. The models, liposomes and washed minced fish muscle, were deemed sufficient to study the pro-oxidant capacity of hemoglobin. Both of the models offer high concentrations of membrane lipids, primary substrates of lipid oxidative reactions and contain no hemoglobin to begin with. Hemoglobin from the Atlantic Pollock fish species was used to activate lipid oxidation because it demonstrates pro-oxidative activity in both of the models (Maestre, 2009). The effectiveness of each antioxidant compound was tested under these severe pro-oxidative conditions. Loss of redness in washed fish muscle was measured in order to determine the effect of reductants and iron chelators on hemoglobin redox stability. The capacity, in vitro, of grape proanthocyanidins (compounds with both reducing and chelating properties) to avoid hemoglobin autoxidation was also studied. In this study, experiments were carried out to examine the effects of the various compounds with antioxidant properties. Compounds with reducing capacity [reduced glutathione (GSH), ascorbic acid, and catalase] and compounds with iron-chelating ability [ethylenediaminetetraacetic acid (EDTA), citric acid, sodium tripolyphosphate (STPP) and adenosine-5’-triphosphate (ATP)] were purchased, as well as other buffers and reagents necessary for the experiments. A grape fraction rich in grape proanthocyanins was prepared by size exclusion chromatography of a commercial grape seed extract.

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Hemoglobin was extracted from caudal vein blood that was obtained from fresh Atlantic pollock. Hemoglobin was also extracted from the blood of horse mackerel because the experiments performed to determine in vitro effect of grape proanthocyanidins on fish hemoglobin redox stability required more stable hemoglobin. Absorption peaks at approximately 540 and 570 nm indicated that the hemoglobin was mainly oxygenated (OxyHb). The concentration of hemoglobin was determined according to a method used by Brown (1961). The effect of grape proanthocyanidins on in vitro redox stability of fish hemoglobin was analyzed by incubating horse mackerel HB with grape proanthocyanidins in a L-histine buffer. The hemoglobin redox stability was determined by monitoring the spectral changes experienced by hemoglobin in both the presence and absence of grape proanthocyanidins. MetHb formation was calculated using an adaptation of the Winterbourn’s equation (Winterbourn, 1990), estimating the concentration of metHb. Liposomes were prepared by adapting a method used by Huand and Frankel (1997). The effects of reductants and grape proanthocyanidins on hemoglobin-mediated lipid oxidation were analyzed using the liposomes and a phosphate buffer. The effects of the iron chelating compounds were analyzed using a separate buffer solution of L-histidine and KCl because the chelating ability of the phosphate buffer could potentially affect results. The iron chelators were studied at a much higher concentration than the reductants and grape proanthocyanidins due to their decreased inhibitory activity. To monitor the progression of lipid oxidation in the presence of the various antioxidants, the formation of conjugated dienes as well as thiobarbituric acid reactive substances (TBARS) were examined. The inhibition of hemoglobin-mediated lipid oxidation was evaluated in washed minced fish muscle. Once again, a substitute buffer was used for the iron-chelating compounds to avoid

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the phosphate buffer chelating ability. The washed fish muscle was supplemented with either reductors, grape proanthocyanidins or chelating agents. The addition of pollock hemoglobin initiated lipid oxidation in the washed fish muscle, and during storage at 4˚C, oxidation was observed by TBARS and peroxide value (PV). Loss of muscle redness was also analyzed, as it indicates hemoglobin redox stability. A colorimeter was used to measure changes in the redness of the washed fish muscle that had been supplemented with hemoglobin. The results of the experiments showed that grape proanthocyanidins were more effective at inhibiting hemoglobin-catalyzed production of conjugated dienes in liposomes during lipid oxidation propagation stages than the reductors that were analyzed at the same molar concentration. The examined grape proanthocyanidins also demonstrated a significant reduction of TBARS production, while liposome supplementation with GSH and catalase did not decrease the formation of TBARS. The grape proanthocyanidins also showed the strongest inhibitory capacity on hemoglobin-catalyzed lipid oxidation in washed fish muscle. Induction periods of approximately four days were exhibited for the formation of TBARS and lipid peroxides, and control muscle devoid of reducing compounds did not demonstrate induction periods for lipid oxidation products. The only other reductant that increased the induction period was GSH, which increased the period for the formation of TBARS up to one day. GSH also showed some activity in decreasing production of peroxides and TBARS during lipid oxidation propagation stages. Fish muscle with no added reductants showed a dramatic loss of redness at day one, but fish muscle with grape proanthocyanidins supplementation preserved initial redness values for three days of storage. This indicates a decrease in hemoglobin oxidation. GSH, ascorbic acid and catalase did not slow the loss of redness in the fish muscle.

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In liposomes, even very high concentrations of iron chelating compounds did not show considerable reduction of lipid TBARS or conjugated dienes. The iron chelator STPP demonstrated significant reduction of TBARS production during propagation of lipid oxidation, however it was not effective in preventing the formation of conjugated dienes. The other iron chelators, EDTA, citric acid and ATP did not inhibit the formation of lipid oxidation products. STPP was significantly efficient to delay lipid peroxide production in washed fish muscle and was the most effective of the iron chelators. TBARS production rates were also lower in fish muscle supplemented with EDTA and STPP, indicating decreased lipid oxidation. None of the iron chelating compounds slowed the loss of redness between days zero and two, indicating low influence on the redox stability on hemoglobin. Fish muscle supplemented with EDTA and STPP demonstrated significantly higher redness values at days two and three than the control samples. A reduction of oxygenated hemoglobin levels was observed in samples supplemented with grape proanthocyanidins. The control hemoglobin showed lower spectral changes during 48 hours of incubation than the hemoglobin with grape proanthocyanidins. Grape proanthocyanidins hastened the formation of metHb from approximately 1.0 to 7.5 uM after the incubation, whereas control hemoglobin did not surpass metHb values greater than 2.0uM during the same incubation. Several pathways exist which have the ability to slow proliferation of hemoglobincatalyzed lipid oxidation, such as the deactivation of hypervalent ferryl hemoglobin and free radical scavenging. The reductants studied in the article may be capable of retarding hemoglobin-mediated lipid oxidation via these pathways, not including catalase. It is possible that catalase inhibits the pro-oxidant activity of hemoglobin through its ability to decompose

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hydrogen peroxide (Kanner and Harel, 1985). This activity is important because the reaction of hydrogen peroxide with metHb produces oxidizing ferryl hemoglobin. The ferric ion reducing antioxidant parameter (FRAP), which “indicates free radical scavenging capacity through electron transfer to reactive free radical species”, is larger for grape proanthocyanidins (3.2 – 6.0 electrons per molecule) than another of another reductant, ascorbic acid, with a FRAP value of 2.0 electrons per molecule. Therefore, the excellent antioxidant activity of grape proanthocyanidins is in agreement with their superior ability to scavenge free radicals by means of electron donation. The reductor GSH exhibited active slowing of the pro-oxidant activity of hemoglobin, and was more effective than ascorbic acid and catalase, though not as efficient as grape proanthocyanidins. GSH is known to donate only one electron per molecule to reactive free radicals, and therefore has lower reducing capacity. Although the FRAP value of ascorbic acid suggests that ascorbic acid should exhibit superior inhibition than GSH, ascorbic acid also has potential pro-oxidant effects. Ascorbic acid can decompose lipid hydroperoxides to reactive aldehyde compounds (Seon et al., 2001). The inefficiency of catalase to slow the propagation of hemoglobin-mediated lipid oxidation may be attributed to the low involvement of hydrogen peroxide in the pro-oxidant effect of fish hemoglobin in liposomes and washed fish muscle. This does not indicate that highly oxidizing ferryl species are not involved in the pro-oxidative activity of hemoglobin. Ferryl species are also produced as a result of the reaction of metHb with preformed lipid hydroperoxides (Reeder and Wilson, 1998) and this reaction may have a greater pro-oxidant effect than the interaction with hydrogen peroxide (Davies, 1988). Overall, the iron chelators studied did not exhibit strong antioxidant activity on hemoglobin-catalyzed lipid oxidation, in comparison to grape proanthocyanidins and the

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reductants. EDTA has a greater capacity than grape proanthocyanidins to chelate ferrous iron (Pazos, 2005). In this study, EDTA was not as efficient at preventing hemoglobin-mediated lipid oxidation. The chelating ability of grape proanthocyanidins appears not to play an important role in their antioxidant ability. The data found through these experiments indicates that the reducing ability alone and/or combined with iron-chelating property of the grape proanthocyanidins determines their effectiveness at preventing hemoglobin-mediated lipid oxidation. Generally, the paper demonstrates that the compounds with reducing capacity showed higher antioxidant ability than iron chelators on hemoglobin-mediated lipid oxidation. This indicates that free radical scavenging and/or reduction of ferryl hemoglobin species are more important for preventing the pro-active mechanisms of hemoglobin than iron chelation. The grape proanthocyanidins were the most effective to slow the loss of redness in washed fish muscle, indicating slower hemoglobin oxidation to brown ferric metHb. Oxidation of hemoglobin to metHb has been suggested to involve hydrogen peroxide (Jia and Alayash, 2008), and metHb formation was slowed by catalase and SOD, which promote hydrogen peroxide breakdown. The paper concludes that overall, grape proanthocyanidins have a very strong ability to decrease hemoglobin-mediated lipid oxidation. The authors propose that grape proanthocyanidins can be used as an effective ingredient in foods containing catalytic amounts of fish hemoglobin, in order to preserve beneficial PUFAs. Generally, this paper was straightforward and easily comprehended. The figures presented by the authors were informative and clear. The overall paper was fairly well written, however there were several instances of spelling mistakes and grammar problems. For example, in the materials and methods section of the paper, when referring to absorption peaks, the authors specify “540 and 570 mn” in the ‘Fish Hemoglobin Extraction’ section, and later correctly

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specify “465 nm” in the ‘Determination of Hemoglobin Concentration’ section. The correct units for absorption are generally presented in nano meters, or ‘nm’, indicating a typing error in the final published paper. At the beginning of the discussion, the first sentence includes “by which the reductants here evaluated”, where it seems it should read, “by which the reductants evaluated here”. These, and other, mistakes may be due the fact that the paper was written in Spain, and errors could have occurred during translation. The experiments carried out seemed appropriate to answer the question posed by the authors. The only methods that seems inconsistent are the experiments that were used to determine the inhibition of hemoglobin-catalyzed lipid oxidation in liposomes and washed fish muscle. One buffer was used to analyze the effect of both reductors and grape proanthocyanidins, while a separate buffer was used to analyze the iron chelating compounds. The explanation given for the buffer change was that while testing the iron chelators, the researchers wanted to avoid the chelating ability of the phosphate buffer that had been used for the other compounds. It is not explained why this chelating ability is not a concern for the reductors or proanthocyanidins, even though it could potentially affect the results for all of the groups. The conclusions drawn in the paper were well supported by the research findings. All of the statements made by the authors were clearly supported by experimental data. Unfortunately, the authors did not provide any additional questions or experiments that could be explored in the future. It may perhaps be useful to investigate the effect of combined antioxidants to search for synergic effects. The compounds studied in this paper were evaluated for their independent activity but the authors did not seem to consider testing them in combinations. It is possible that the combined effects of several antioxidant compounds, that affect different points in the lipid

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oxidation pathway, could be even stronger than the most effective independent compound. The authors did not consider this possibility. Overall, the paper did not seem to have any other obvious flaws. Despite the grammatical and spelling errors, the paper was fairly easy to understand. The results were presented using graphs that clearly showed the differences between groups. The reasoning and background provided were sufficient to support the purpose for the study. researchers evaluated that reducing compounds have a greater capacity to prevent fish hemoglobin-mediated lipid oxidation in comparison with iron chelators. They also succeeded in finding a compound that can effectively inhibit fish hemoglobin-promoted lipid oxidation.

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