Measurement of Lipid Peroxidation in Plasma

A persistent problem plaguing the development and use of fats and oils in cooking has been
oxidative damage leading to rancidity. Polyunsaturated fatty acids (PUFAs) are particularly
vulnerable leading to the development of techniques such as hydrogenation to increase their
shelf life. Unfortunately this process leads to the formation of trans-fatty acids (TFAs) with
their associated implications for health. Lipids are also an important component of cell
membranes. Oxidative damage to the lipids present in cell membranes can cause changes in
membrane structure and fluidity resulting in changes to cell permeability. For example, an
extremely good indicator of the amount of damage that has been sustained by the cardiac
muscle during an insult is the concentration of intracellular proteins that leak into the plasma.
In addition, peroxidation of low density lipoproteins following their entry into the tunica
intima is a fundamental step contributing to the development of atherosclerosis. The trouble
is once the process of lipid peroxidation has begun it can be self-propagating. This is
illustrated in the diagram below:

The TBARs Assay
The amount of lipid peroxidation in a sample can be determined using malondialdehyde
(MDA), which is formed from the breakdown of polyunsaturated fatty acids. MDA reacts
with thiobarbituric acid (TBA) to form a pinky red coloured TBA-MDA complex. The
absorbance of the TBA-MDA complex can be measured at 535 nm against a blank containing
all of the reagents except plasma and the concentration of MDA calculated using the Beer
Lambert equation:
Abs = cl
Where  is the molar extinction coefficient of MDA, which is 1.56 x 10
5
M
-1
; c is
concentration and l is the path length (1 cm).

Aim
The aim of this practical will be to use the TBARs assay to investigate the effect that
different diets have on lipid peroxidation in various subjects.

Methods
1) A sample of plasma was collected from the following subjects:
i. A 42-year-old Caucasian male who consumes a normal varied diet
ii. A 42-year-old Caucasian female who consumes a normal varied diet
iii. A 42-year-old African male who consumes a normal varied diet
iv. A 42-year-old Caucasian male who is a vegan
v. A 42-year-old Caucasian female who is on a strict Calorie reducing diet
vi. A 42-year-old Caucasian female who readily acknowledges that she is an
alcoholic drinking at least 10 stubbies of beer each night

2) Measurements were carried out in duplicate

3) 250µl of each plasma sample from the different subjects were pipetted into 2 separate
pre-labelled eppendorff tubes

4) To each tube was added 250µl of 50 mM 2,2’-azidobis-(2-amidinopropane)dihydrochloride (AAPH) followed by mixing

5) To each tube was added 1ml of a solution containing 15% (w/v) trichloroacetic acid
(TCA) and 0.375% (w/v) thiobarbituric acid (TBA) in 0.25M HCl followed by mixing

6) The tubes were placed into a boiling water bath for 15 minutes

7) Upon removal from the water bath, the tubes were cooled on ice for 10 minutes

8) Following cooling, the tubes were centrifuged at 10,000 rpm (12,000 x g) for 5 minutes

9) 250µl AAPH and 1ml of the TCA/TBA mixture were pipetted into a cuvette to use as a
blank for the spectrophotometer

10) The supernatant from the centrifugation step above (step 8) was pipetted into a cuvette
and its absorbance measured at 535nm (against the blank prepared in step 9)

11) The amount of lipid peroxidation in each sample was calculated using the Beer Lambert
law as described in the section entitled The TBARs assay above.

Results
The table below shows the absorbance measured for each of the different subjects (remember
measurements were made in duplicate):