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 hemoglobin-

mediated 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 hemoglobin-

catalyzed 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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