Abstract: Lipid peroxidation is used as an indicator of oxidative stress in cells and tissues.

Lipid peroxides includes compounds such as aldehydes of which the most abundant is malondialdehyde (MDA). Therefore, MDA measurement is widely used as an indicator of lipid peroxidation. The method is designed to assay MDA in hydrochloric acid. From the experiment, tube 3 gave the highest O.D reading of 0.455nm thereby indicating the most amount of lipid perodixation. The least amount of lipid perodixation came from tube 1 with O.D reading of 0.053nm. This was also the control tube with minute volumes of buffer Tris HCL (0.02M) pH 7.2 and liver homogenate. Aim: This experiment aimed at demonstrating the degradative process of lipid perodixation in the liver and also the potential s of anti-oxidants to prevent such damage. Introduction: Lipid perodixation is the process that occurs during many pathophysiological processes. This process includes the development of atherosclerosis, stroke and ageing. Lipid perodixation process starts when free radicals like .OH (hydroxyl and superoxide (.O2-) are generated. The free radicals are unstable forms of oxygen and have an unpaired electron in their outer orbital. It is important to note that free radical are produced as part of the normal body function and their production are counterbalanced by the production of anti-oxidants which prevent the harmful effects of free radicals. Many diseases are thought to involve oxidative stress where the balance between free radical production and their neutralization is skewed in the favour of the former. The fenton reaction commonly used to generate free radicals (usually OH) and will be used in this experiment to initiate lipid perodixation of liver. The final breakdown of lipids is malonedialdehyde which reacts with thiobarbituric acid to form a pink adduct which can be measured optically. The assay is often referred to as thiobarbituric acid reaction and the products formed are often referred to as TBARS (Thiobarbituric acid reactive substance). Materials and Methods: The materials and methods used were as described in the practical booklet. Series of test tubes were being incubated for 30mins at 30C. Before this, calculated volumes of reagents were pipette and added to the test tubes. Care was taken to add the liver homogenate last to prevent untimely kick-starting of the reaction. After tubes were removed from boiling water, extra care was taken to remove only the clear fluid (brown pink in colour) using the pipette.

Results: The results of the lipid peroxidation are shown on the table and calculations below Test Tube Test Buffer FeCl2 Tris HCl (0.02M) pH 7.2 1.6ml H2O2 Catalase Quercetin Homogenate (Liver) Total Optical Density (O.D)

Control

0.9ml

2.5ml

0.053

2 3

Fe2+ Fe2+ H2O2 Catalase Fe2+ H2O2 Quercetin

1.1ml 0.6ml

0.5ml 0.5ml 0.5ml

0.9ml 0.9ml

2.5ml 2.5ml

0.326 0.455

0.5ml

0.5ml

0.1ml

0.9ml

2.5ml

0.280

4 5

0.5ml

0.5ml

0.5ml

0.1ml

0.9ml

2.5ml

0.370

Table 1. Volumes of the reagents for assay This table above shows the optical density for the individual tube experiments performed. Tube 3 gave the highest reading of 0.455nm. Test tube Final concentration required (mM) 0.1 Dilutions Volume Volume of Total Volume of buffer MDA stock 1mM 0.15 2.85 3ml Optical Density (O.D)

Dilute MDA 1:10

3.059

0.05

Dilute 0.1mM 1 MDA (tube 6) 1:2 Dilute 0.05 mM 0.4 MDA (tube 7) 1:5

2ml

2.365

8 0.01 1.6 2ml 2.795

Table 2. Showing individually obtained optical density reading for use in construction of MDA (Malondialdehyde) standard curve on next page. MDA standard Curve

Fig. 1 Showing corresponding volumes of MDA (nmol.ml) against OD readings on an MDA standard Tube No. Optical Density (532nm)

MDA (nmol/ml)

1. 2. 3. 4. 5.

0.053 0.326 0.455 0.280 0.370

9.58 59.21 82.67 50.85 67.21

Table 3. Showing calculated amount of MDA produced from individual experiment. MDA values calculated from the excel derived equation (y=0.0055x + 0.0003)

CLASS DATA

Group/Date of Practical : Practical :

Group A (06 October 2011) PP1: Lipid Peroxidation

Standard Quercetin Deviatio data Absorbance at 532nm Mean n Tube 1 0.06 0.05 0.10 0.13 0.15 0.03 0.05 0.05 0.08 0.042792 Tube 2 0.18 0.21 0.13 0.18 0.43 0.09 0.41 0.33 0.24 0.129679 Tube 3 0.14 0.07 0.19 0.17 0.51 0.30 0.24 0.46 0.26 0.154584 Tube 4 0.12 0.16 0.15 0.17 0.14 0.09 0.14 0.28 0.16 0.055902 Tube 5 0.08 0.06 0.10 0.11 0.07 0.05 0.08 0.37 0.12 0.104263 Table 4. Illustrating general lipid peroxidation class data at optical density format and also indicating the mean and standard deviation values.

Fig. 2. Standard deviation bar-chart showing mean +/- sd OD values for class data for tubes 1-5 (Quercetin). Discussion: Test tube 1 was used as the control tube and gave a final O.D (Optical density) reading of 0.053nm. This was the lowest reading compared to the rest of the tubes. The highest reading was gotten from test tube 3 with a final O.D reading of 0.455nm. 0.1ml of Quercetin was added to test tube 5 and that addition brought an O.D reading of 0.370nm. Most metals catalyze reactions that produce reactive radicals. Fenton reaction is a notable one in which hydroxyl radical is produced from reduced hydrogen and iron peroxide. This hydroxyl radical then leads to alteration of lipid peroxidation. Anti-oxidants are substances which in small amounts; interfere with the normal oxidation process in oils and fats so as to delay the time when oxidation would have proceeded far enough to produce

noticeable odours (Gunstone & Norris, 1983). Anti-oxidants function as free radical acceptors, thus terminating oxidation at the initiation step. In this experiment, Quercetin was used as the anti-oxidant and it was applied in tube 5. Tube 3 gave the highest reading of 0.455nm. This is due to reduced hydrogen and iron peroxide. This modification changes the amount of lipid perodixation produced by increasing it. There was a notable stable increase in OD readings upto tube 4 were catalase was applied. This brought down the OD reading to 0.280nm. The effect of the anti-oxidant (Quercetin) applied to the subsequent tube would be to delay the oxidation process and bring down the O.D reading to a value lower than that of tube 4 as Quercetin retards peroxidation damage. But in this experiment, tube 5 O.D value went higher. This could be attributed to experimental errors such as wrong pipetting. The limitations of this experiment include the unsuitability of homolyzed, lipepic plasma samples in TBARs analysis. Secondly, a more specific test for lipid peroxidation such as use of HPLC is recommended as non-lipid TBARs sample may be present in the sample. Another limitation is the fact that normal tissues contain low level of free malondialdehyde. They are slight similarities between the individual and class data. There is a notable difference between individual O.D reading for tubes 3 and 5 were 0.455nm and 0.370nm against the class data of 0.260nm and 0.120nm respectively. This huge variance could be attributed to wrong pipetting techniques, failure of zeroing spectrophotometer and care should be taken on repeat of experiment. References: Enzo Life Sciences. (2008). Aldetect (MDASpecific) Lipid Peroxidation Assay Kit BMLAK171. Retrieved October 26, 2011, from http://www.enzolifesciences.com/fileadmin/enzo/BML/AK171.pdf. Gunstone, F. & Norris F. (1983). Lipids in foods: Chemistry, biochemistry and technology. Oxford: Pergamon Press. Gurr, M. & James, A. (1975). Lipid biochemistry: An introduction. (2nd ed.) . Kent: Whitstable Litho. Oxford Biomedical Research. (2003). Colorimetric Assay for Lipid Peroxidation Product No: FR 12. Retrieved October 28, 2011, from http://www.funakoshi.co.jp/data/datasheet/OBR/fr12.pdf.