BIOL2537 Laboratory in Nutritional Science

Name: Hung Wing TungUID: 2009053464Group: 3Date: 8/4/2010

Practical 6: Lipid peroxidation measurement and FRAP test

Objectives: 1. To compare the level of lipid peroxidation of different roadside Chinese snacks cooked in reused oil and fresh uncooked food by TBARS test.2. To determine the total antioxidant capacity of popular drinks and compared to that of Vitamin C by FRAP assay.

Introduction:Part A: Lipid Peroxidation measurementFormation of conjugated dienes is caused by free radical attack of polyunsaturated fatty acids. These conjugated dienes take up oxygen to produce peroxyl radicals. Meanwhile, lipid hydroperoxides are formed with the abstraction of a hydrogen atom from surrounding fatty acids, which decompose alkoxyl and peroxyl radicals. Under -cleavage, the alkoxyl radicals form aldehydes such as malonaldehyde (MDA). The amount of MDA formed, which reflects the extent of lipid peroxidation in food system and biological samples, can be measured by a simple method called the thiobarbituric acid reacting substances (TBARS) test.

In this part of the practical, the lipid peroxidation level of different Chinese snacks cooked in reused oil and fresh uncooked food will be examined by TBARS test, in which MDA produced from these foods will undergo nucleophilic addition reaction with 2-thiobarbituric acid (TBA) and produce red, fluorescent 1:2 MDA:TBA adducts, which show a high molar absorptivity in 532 nm.

Part B: Ferric Reducing Ability of Plasma (FRAP)Reactive oxygen species (ROS) are normal byproducts of metabolism but the oxidative stress-induced tissue damage can cause degenerative diseases like cancers and coronary heart diseases. Therefore, from the nutrition point of view, ROS should be minimized.

Since ROS can be neutralized by antioxidants, the antioxidant capacities of foods are worth to examine. The FRAP assay is one of the methods available for assessment of antioxidant capacity, which capitalizes the concept that antioxidants are reductants that mediate redox reactions.

In this part of the practical, the antioxidant capacity of different drinks and vitamin C will be investigated using FRAP assay, where ferric tripyridyltriazine (FeIII-TPTZ) is added as oxidants. Under low pH, FeIII-TPTZ will be reduced to the ferrous form with the development of intense blue color with an absorption maximum at 593 nm. Procedures (Materials and Methods): As shown in lab manual.

Results:Part A: Lipid Peroxidation measurement

Table a. Absorbance at 532 nm of different standard MDA solutionsConcentrations of MDA solutions (g/mL)Absorbance

1.0930.092

4.370.350

8.740.693

17.481.291

Table b. Absorbance at 532 nm of different food samplesFood SamplesAbsorbance at 532 nm

TestRecovery

Raw Green Bell Pepper0.2630.352

Raw Bean Curd0.3060.324

Raw Eggplant0.2020.287

Fried Green Bell Pepper0.2820.762

Fried Bean Curd0.5290.771

Fried Eggplant0.9311.463

From the equation y (Absorbance at 532 nm) = 0.073 x (Concentration of MDA) + 0.0286 of the standard curve, the amount of MDA in both TEST and RECOVERY can be obtained.

Table c. Amount of MDA in different food samplesFood SamplesAmount of MDA

TestRecovery

Raw Green Bell Pepper3.214.43

Raw Bean Curd3.804.05

Raw Eggplant2.383.54

Fried Green Bell Pepper3.4710.05

Fried Bean Curd6.8510.17

Fried Eggplant12.3619.65

Table d. Percentage of recovery, total amount of MDAtest and total amount of MDA per gram of different food samplesFood SamplesRecovery (%)*Total amount of MDAtest (g)**Total amount of MDA per gram of food (g/g)***

Raw Green Bell Pepper27.911.50211.502

Raw Bean Curd5.7066.42466.424

Raw Eggplant26.39.0269.026

Fried Green Bell Pepper150.62.3052.305

Fried Bean Curd75.889.0279.027

Fried Eggplant166.87.4107.410

* Recovery (%) = Amount of MDArecovery Amount of MDAtest / Amount of MDA added to the recovery x 100% [Amount of MDA added to the recovery = 4.37 g]** Total amount of MDAtest (g) = Amount of MDAtest / Recovery (%)*** Total amount of MDA per gram of food (g/g) = Total amount of MDAtest / Amount of food sample in gram [Amount of food sample in gram = 1 g]

Part B: Ferric Reducing Ability of Plasma (FRAP)

Table e. Absorbance at 593 nm of blank and standard FeSO4 solutionsSamplesAbsorbance at 593 nm*

Blank0.063

Standard 0.125 mM0.070

Standard 0.625 mM0.400

Standard 1.25 mM0.830

Standard 2.5 mM1.697

*Absorbance of standards was subtracted with that of blank.

From the equation y (Absorbance at 593 nm) = 0.687 x (Concentration of FeSO4) 0.0232 of the standard curve, the amount of ferrous ions in different drinks can be calculated.

Table f. Absorbance at 593 nm and amount of FeSO4 of different drink samplesSamplesAbsorbance at 593 nm*Amount of FeSO4 (mM)

Green tea**1.91312.49

Apple Juice1.6552.75

Vitamin C1.9143.13

V8 vegetable juice**1.2308.52

Sprite0.0290.38

*Absorbance of samples was subtracted with that of blank.** Green tea and V8 vegetable juice are having a dilution factor of 4, hence the amount of ferrous ions should be multiply by 4.

Discussion:Comparison of MDA level in different food samples and that in uncooked and fried foods

Referring to Fig (c), the results obtained were abnormal. First, the amount of MDA in raw bean curd was extremely high (~66 g/g). Second, fried food samples had lower amounts of MDA than the raw ones. These might be attributed to the limitations of the TBARS test and some experimental errors, which will be discussed in later sections.

Theoretically, amounts of MDA produced in fried foods should be higher than those in uncooked foods. It is because during firing, some cooking oil is absorbed by the food. The lipids from the oil will undergo peroxidation. This is favored when the oil is heated at a high temperature as a rich supply of oxygen is provided for production of free radicals. The extent of lipid peroxidation is even more significant when reused oil is used since the lipids have undergone peroxidation and more free radicals are present. Hence, more byproducts, such as MDA, will be produced. It is also noted that fried eggplant should produce the highest amount of MDA among all food samples since it has the greatest ability to absorb oil during the firing process.

For uncooked or raw foods, lipid peroxidation is mainly caused by the oil or fats originally present. The amount of lipid is less than that of fried foods. Also, in the absence of high temperatures, lipid peroxidation is less favored. Therefore, lipid peroxidation occurs in a smaller extent and less MDA will be produced.

Comparison of the antioxidant capacity of different drinks and that relative to Vitamin C

From Fig (d), the concentration of FeSO4 of Tao Ti Green Tea was the highest, indicating that it had the highest ability to reduce Fe3+ to Fe2+. Thus, it had the greatest antioxidant capacity. The high antioxidant capacity of green tea can be attributed to the presence of phenolic compounds called catechins, among which epigallocatechin gallate (EGCG) is the most powerful antioxidant. Followed by green tea was V8 vegetable juice, since it is rich in vitamin C, vitamin E, beta-carotene, etc., which are all known to have high antioxidant capacities. Thus, these 2 drink samples could produce more Fe2+ than vitamin C, which is a well-known antioxidant.

Apple juice had a comparable antioxidant capacity with vitamin C as it also contains some vitamin C from the apple extract, and polyphenols (a type of phenolic compounds which had a high antioxidizing power) are also present. Sprite had the lowest antioxidant capacity since it does not contain any antioxidants.

Relationship between antioxidant and lipid peroxidationLipid peroxidation produces free radicals and reactive oxygen species (ROS) which attack and damage tissues and cells, while antioxidants can scavenge these free radicals and ROS by themselves becoming radicals. The radicals of antioxidants, however, are not harmful to our body. They are either used to regenerate the antioxidants back or excreted.One good example of antioxidant is vitamin C (ascorbic acid, AH2). It donates electrons to reduce superoxide radicals to form hydrogen peroxide (H2O2) and dehydroascorbic acid (DHAA). Vitamin C can also react with H2O2 to produce water, which is a harmless product. Besides, vitamin C also scavenges hydroxyl and peroxyl radicals to form water and lipid peroxide respectively.

Vitamin E is another well-known antioxidant. Due to its lipophilic nature, it can effectively react with carbon-centered free radicals and those initiate lipid peroxidation. For example, it donates its phenolic hydrogen to carbon-centered free radicals from polyunsaturated fatty acids and hence terminates lipid peroxidation.

Health risks of lipid peroxidation (cell/body)In normal metabolism, free radicals are produced and they initiate lipid peroxidation in the polyunsaturated fatty acids in cell membranes and the membranes of intracellular organelles, such as nucleus, mitochondria and endoplasmic reticulum. This leads to degradation of lipids. For example, hemolysis of red blood cells may occur due to extensive damage. Aqueous peroxyl and peroxy nitrite radicals may also induce oxidation of low-density lipoproteins (LDLs), increasing the chances of atherosclerosis, which may cause heart diseases and cataracts.

When consumed foods high in oxidized lipids, such as reused oil, more lipid peroxidation will take place in our body. Free radicals can also be formed from lipid peroxidation besides normal metabolism, and they will again attack polyunsaturated fatty acids. They will also take electrons from amino acids (especially tryptophan, tyrosine, histidine, arginine, cysteine and proline) and cause oxidative damage in proteins, which alters the secondary or tertiary structures of proteins and lead to premature degradation of proteins. Besides, hydroxyl radical-induced changes in purine and pyrimidine bases in DNA may also cause mutations and breakages. This may result in cancers.

Limitations of MDA and FRAP assaysThough TBARS test is a simple and quick method to estimate the extent of lipid peroxidation in foods, it is non-specific and subjected to interference. Chromogens, which react with TBA and have an absorbance at 532 nm, can be generated from other aldehydes apart from MDA. Other constituents in foods, such as sugars, amino acids and esters, can also react with TBA. Therefore, using MDA as markers might cause overestimation of the extent of lipid peroxidation in foods.

For FRAP assay, presence of other substances which can reduce Fe3+ to Fe2+ and ingredients that have an absorbance at 593 nm might lead to overestimation of the antioxidant capacity of the drink samples. Oppositely, if the antioxidants in samples were not able to react with Fe3+ readily, less Fe2+ would be produced at the time of measurement. This leads to underestimation of antioxidant capacity of the samples. Besides, low pH conditions required in this assay would actually inhibit the electron transfer from the antioxidants to Fe3+.

Possible experimental errorsIn Part A (TBARS test), errors must be present, causing abnormal readings in the absorbance of food samples and hence the calculated total amount of MDA. The following are some possible errors: The food samples were not completely homogenized. This lead to uneven distribution of food component. Some samples might contain a very low amount of food extract which was responsible for lipid peroxidation. It was assumed that the amount of MDA in both TEST and RECOVERY was the same before the addition of standard MDA solutions in the RECOVERY. In fact, the RECOVERY series might contain much more or much less MDA than the TEST series. Presence of other ingredients (e.g. esters, sugars) in food samples which can react with TBA, as previously mentioned, might also cause overestimation of total MDA produced. Poor experimental techniques also accounted for the errors. The experimenters might pour insufficient or excess solutions into some food samples, leading to abnormal readings. Also, some tubes might not be capped tightly during the experiment, thereby increasing the oxygen supply for lipid peroxidation.In Part B (FRAP test), errors might also exist. Here are some possible errors: The time of measurement had to be caught very accurately since reactions were continuously proceeding and the absorbance was continuously increasing. Any missing of exact timing would cause variation in the results obtained. As mentioned before, presence of other ingredients in the drink samples might increase the absorbance readings as these ingredients might show absorbance at 593 nm too. Again as mentioned, some antioxidants in the drink samples might react with Fe3+ slowly. Less Fe2+ was produced and this underestimated the antioxidant capacity of the drinks.

Conclusions:1. The levels of lipid peroxidation of different Chinese snacks foods were compared using TBARS test. The results were abnormal, in which raw bean curd contained the highest MDA per gram, while fried green bell pepper contained the lowest amount of MDA per gram. This violated the theory that fried foods should contain higher MDA level than raw foods.2. The antioxidant capacity of different drink samples and vitamin C were examined by FRAP test. Tao Ti Green Tea had the highest antioxidant capacity since it can reduce the greatest amount of Fe3+, while that of sprite was the lowest.

References:1. J.L. Groff, J.L. Smith, S.S. Gropper. Advanced Nutrition and Human Metabolism. 5th Edition. 2009. Wadsworth. 2. E. A. Meagher, G.A. Fitzgerald. Indices of Lipid Peroxidaiton in vivo: strengths and limitations. Free Radical Biology & Medicine, 2000, 28 (12), pp 1745-1750. 3. B. Ou, et al. Analysis of Antioxidant Activities of Common Vegetables Employing Oxygen Radical Absorbance Capacity (ORAC) and Ferric Reducing Antioxidant Power (FRAP) Assays: A Comparative Study. J. Agric. Food Chem., 2002, 50 (11), pp 31223128.

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