Oxidative stress and healthy life style.

1. Mrs Harsha Goyal
M.Phil, M.Sc Biochemistry
Govt. Holkar Science College, Indore.

2.  Introduction
 Reactive oxygen species (ROS)
 Antioxidant defense system
 Oxidative stress
 Markers of Oxidative stress
 Detrimental effects of oxidative stress on
Human Health
 Exogenous Antioxidants and Human Health

3.  Reactive oxygen species are highly reactive oxygen molecules
with one or more unpaired electrons.
 ROS are like robbers which are deficient in energy. These
molecules attack and snatch energy from the other cells to
satisfy themselves.

4. Two sources of Reactive oxygen species.
 Endogenous (inside body)
 Exogenous (outside body)

5.  Electron transport reactions
 About 90% of free radicals produced in mitochondria because
of premature or incomplete reduction of Oxygen into
superoxide radical (O2
•−).

6.  Metabolic reactions
Superoxide radical also formed in reactions catalyse by
NADPH oxidase, xanthine oxidase, cytochrome P450 system,
and peroxidase.
 Liver detoxification reactions
 Immune reactions
 Superoxide radical in turn triggers synthesis of other free
radicals like hydrogen peroxide, hydroxyl radical (OH•),
peroxynitrite (ONOO−), hypochlorous acid (HOCl).

7. Figure : Generation of reactive oxygen species, where MPO is myeloperoxidase and
SOD is superoxide dismutase.

8.  Formation of Hydroxyl radical (•OH)
 Haber-Weiss reaction
Fe3+ + O2•−⟶Fe2+ + O2
 Fenton reaction
Fe2+ +H2O2⟶Fe3+ + OH− + •OH
 Overall reaction is
O2•− + H2O2 ⟶ •OH + OH− + O2

9.  Ionizing radiations
 Nitrous oxide
 Heavy metals
 Cigarette smoking
 Alcohol
 Insecticides and pesticides
 Drugs

10.  Reactive oxygen species
 Cause lipid peroxidation of cell membrane,
 Distort protein conformation,
 Cause pernicious DNA lesion (8-oxo-2′-deoxyguanosine).

11.  Enzymatic antioxidants
 Superoxide dismutase (SODs)
It catalyzes conversion of superoxide to peroxide and molecular oxygen.
 Glutathione (γ-Glu-Cys-Gly) and GSH peroxidase (GPX)

12.  Peroxidase (POD)
 Non enzymatic antioxidants.
 Ascorbic acid
 α-tocopherol
 Carotenoids
 Flavonoids.

14. Targets of Antioxidants

15. Schematic representation of reactive oxygen species (ROS) generation
and antioxidant defence system

16.  Oxidative stress defines a disequilibrium between the levels of
reactive oxygen species (ROS) produced.
 Cause cellular damage.

17.  Malondialdehyde
 Oxidation of PUFA yields lipid peroxyl radicals and
hydroperoxides which on further degradation generate
Malondialdehyde.
 Level of lipid peroxidation measured indirectly by product
Malondialdehyde.
 Superoxide dismutase (SOD)
 Glutathione peroxidase (GSH-PX)
 Catalase
 Peroxidase
 Guaicol peroxidase
 Proline (plants only)

19.  Cancer
 Oxidative stress cause modification of DNA structure.
 Base and sugar lesions, DNA-protein cross-links, strand breaks.
 Chromosomal abnormalities and oncogene activation.
 Cardiovascular diseases
Circulating LDL oxidized by reactive oxygen species leads to formation of an
atherosclerotic plaque.
 Respiratory diseases
Asthma and chronic obstructive pulmonary disease (COPD)
Oxidants enhance inflammation via the activation of different kinases involving
pathways and transcription factors like NF-kappa B and AP-1.

20.  Kidney disease
 Free radicals initially cause inflammation and form abundant fibrotic tissue that
impairs organ function, leads to renal failure.
 Neurodegenerative diseases
 Parkinson’s disease and Alzheimer’s disease
 Free radicals generate β-amyloid toxin protein which cause neurogeneration.
 Male infertility
 Sperm DNA fragmentation caused by oxidative stress.
 Rheumatoid Arthritis
 Ageing

21.  The various dietary constituents containing antioxidants are
useful in lowering oxidative stress.
 Vitamin C (Ascorbic Acid)
 Oranges, Bell peppers, broccoli, cabbage, sprouts, papaya,
strawberries, kiwi, mango, pineapple.
 Vitamin E (α- Tocopherol)
 Spinach, tomato sauce, red peppers, avocados, grain
products, eggs, almonds, sunflower seeds, peanuts,
vegetable oil.

22.  β-carotene (provitamin A)
Carrots, squash, spinach, sweet potatoes, strawberries,
broccoli.
 Minerals- Copper, magnesium and selenium
Green leafy vegetables, apples, cherries, beetroot, strawberries.
Zinc - Pumpkin seeds.
 Amino acids- Glutathione (GSH)
Asparagus.
 Essential fatty acids
Raw nuts, dark fish and avocado.

23.  Drink plenty of water
 A regular, moderate exercise routine.
 Avoid smoking.
 Use caution with chemicals.
 Be environmentally conscious.
 Wear sunscreen.
 Decrease your alcohol intake.
 Get plenty of sleep.
 Avoid overeating.

24.  Principle
 The level of lipid peroxidation was measured by estimating MDA content
using Thiobarbituric acid (TBA) as reactive agent.
 Reagents
 TCA, TBA reagent.
 Method
 The tissues were homogenized with 5% TCA and 1 ml of homogenate was
mixed with 4 ml of TBA reagent.
 The reaction mixture were heated at 95oC for 30 min in a water bath and
then quickly cooled in an ice bath and centrifuged at 1900g for 10 min.
 The absorbance of the colored supernatant was measured at 532 nm.
 For the reference blank 1ml of 5% TCA was mixed with 4 ml TBA reagent.

25.  Calculation
Concentration of MDA was measured colorimetrically at 532 nm .
A = 1εc
Where
A = Absorbance at specific wavelength
ε = Extinction coefficient (155 nM-1cm-1 )
I = length of cell (1cm)
C = Concentration
Results expressed in mM/g

26.  Principle
The enzyme activity is assayed using o-dianisidine as hydrogen donor and
H2O2 as electron acceptor. The rate of formation of yellow orange colored
dianisidine dehydrogenation product is assayed spectrophotometrically at
430 nm.
 Reagents
Potassium phosphate buffer, pH 6.0, o-Dianisidine in methanol, 0.02M
H2O2, H2SO4.
 Extraction
Homogenize the material in ice cold 0.1M phosphate buffer, pH 6.0 in a
chilled pestle and mortar.

27.  Strain through two folds of muslin cloth and centrifuge the
homogenate at 16,000g for 20 min at 4oC. Use the supernatant as
enzyme source.
 Enzyme assay
 Pipette out 1ml of 0-dianisidine, 0.5ml of H2O2 1 ml of phosphate
buffer and 2.4 ml of distilled water into a test tube.
 For blank, exclude H2O2 but add additional volume of water.
 Incubate at 30oC and start the reaction by adding 0.2 ml of enzyme.
 After 5 min, stop the reaction by adding 1ml of 2N H2SO4.
 Read the absorbance at 430nm.
 Calculation
Express the specific activity of enzyme as units/min/mg protein.

28.  Principle
The extracted proline is made to react with ninhydrin under
acidic conditions to form a red colour which is measured
colorimetrically at 520nm.
 Reagents
Acid ninhydrin reagent, 3% aqueous sulphosalicylic acid,
Glacial acetic acid, Toluene, Standard proline solution.
 Method
 Homogenize 0.5 gm of tissue in a pestle and mortar with 10
ml of 3% aqueous sulphosalicylic acid and filter through
whatman no.2 filter paper.
 Repeat the extraction and pool the filtrate.

29.  To 2ml of filterate, add 2ml glacial acetic and ninhydrin and mix.
 Keep in boiling water bath for 1 hour and then terminate reaction by
placing on ice bath.
 Add 4ml of toluene, mix vigorously for 20-30 sec.
 Aspirate the chromophore (toluene) layer and warm to room
temperature.
 Measure the absorbance of red color at 520nm.
 Calculate the amount of proline in sample using standard curve
prepared from pure proline (range 0.1-36 µmole) and express on
fresh weight basis of sample.
 Calculation
µmoles of Proline/g tissue = µg proline/ ml × toluene (ml) × 5
g sample 115.5
Where, 115.5 is the molecular weight of proline.
Results expressed in µmoles/g.