biochemistry practical slides

1. LIPID PEROXIDATION TEST
(THIOBARBITURATE TEST)
SERUM MALONDIALDEHYDE (MDA) ESTIMATION
An Indicator of lipid peroxidation

2. Free Radicals
 highly reactive molecular species with an unpaired
persists only for very short duration(of the order of
electron,
10–9–10–
12 sec)
 Collide with another molecule and either abstract or donate
an electron to achieve stability

3. Types of Free Radicals
Superoxide, O2
-
Hydroxyl radical, OH-
Hydroperoxy radical, HOO-
Lipid peroxide radical, ROO-
Nitric oxide, NO-
peroxynitrite, ONOO-
Hydrogen peroxide, H2O2

4. Sources of ROS
• White blood cells such as neutrophils specialize in
producing oxygen radicals, which are used in host
defense to kill invading pathogens.
• Cells exposed to abnormal environments such as
hypoxia or hyperoxia generate abundant and often
damaging ROS.
• A number of drugs have oxidizing effects on cells and
lead to production of oxygen radicals. Also in the case of
xenobiotics
• Ionizing radiation is well known to generate oxygen
radicals within biological systems.

6. The Effects of free Radicals
Free radicals cause
• cell mutations
• damage immune function
• cause wrinkles and aging
• and contributing cause( ie. Aetiogenesis) behind many
diseases including cancer, CVD, Arthritis, Alzheimer’s
disease, Parkinson’s disease etc

7. Free radicals and diseases
Respiratory diseases: destroy endothelium and
cause lung edema
Cigarette smoke contains free radicals and
promotes the production of more free radicals
Diabetes mellitus: Destruction of islets results in
pathogenesis
Cataract
Male infertility: reduce sperm
Ageing process
Others:
motility and viability
Parkinson’s disease
Alzheimer’s disease
multiple sclerosis,

8. Lipid Peroxidation

9. Lipid Peroxidation
• Lipid peroxidation - process by which free radicals or reactive
oxygen species (ROS) attack lipids in cell membranes
containing carbon-carbon double bond(s), especially
polyunsaturated fatty acids
• Results in damage to cell architecture/structure(s). Hence
oxidative stress (overall effect of oxidative species in causing
tissues injury or disease)

11. Malondialdehyde (MDA)
• MDA is a secondary degradation product of Lipid
hydroperoxides.
• This is produced when Reactive oxygen species react with
or degrade polyunsaturated lipids.
• This compound is a reactive aldehyde and is one of the
many reactive electrophile species that cause toxic stress
in cells and form covalent protein adducts referred to as
advanced lipoxidation end-products.

12. Principle

13. Clinical significance
• High level of MDA is an indication of oxidative stress.
• Evidenced by damage of cellular components
• Involved in the damaging mechanism of several acute and
chronic brain disorders eg Alzheimer's, Parkinson's etc;
atherogenic events and cardiovascular disease
• Normal range is 41 – 261.5 nmol/dL
• ɛ= 1.56 x 105 L/mol/cm.

14. Protocol
1)Into labelled test tube, pipette accurately 0.4 ml of
sample.
2)Add 2.0 ml of 10% trichloroacetic acid (TCA) to the
tube and shake thoroughly to mix.
3) Then, add 1.5ml of 0.67% Thiobarbituric Acid (TBA)
to each.
4)Shake and incubate at 100 ºC for 30 minutes and
cool the tubes under the tap or in some water in
plastic beaker.

15. 5.)To each tube add 4.0 ml of n-butanol (organic solvent)
and vortex or close the tube with a cleaned thumb and
shake vigorously for 30 seconds. Then centrifuge the tubes
for 5 minutes.
6.)With 3 or 5 ml variable pipette remove the supernatant
layer (organic -extract) for the assay of the MDA.
7.)Using the spectrophotometer at 535nm and the
prepared blank, measure the Optical Density (OD) or
Absorbance (A) of the extracts.
Protocol

16. Post Lab Questions
• Using your test absorbance calculate for the concentration
of the MDA present in the sample.
• Compare your result to the Normal range (ie. 41 –
261.5 nmol/dL) (extinction coefficient to be 1.56 x 105
L/mol/cm)
• What is the purpose of the addition of n-butanol and TCA?
• Comment on your results relating it to clinical condition