Lakshmy R Dept of Radiation Biology Amala Cancer Research Centre

Free radicals are atoms or molecules with unpaired electron(s). Free

radicals are generally highly reactive but some of them may be stable for long time. Reactive free radicals : superoxide, hydroxyl radical, nitrogen dioxide etc Stable free radicals: Vitamin E (tocopheroxyl) and vitamin C (dehydroascorbate) etc.

Reactive oxygen species

-refers to oxygen containing, free radicals/non-free radical active molecules. Reactive oxygen species that are not free radicals include hydrogen peroxide, singlet oxygen, and lipid hydroperoxide.

Free radicals and other reactive oxygen species are produced in all living organisms and have biological advantage.

important roles in signal transduction, involved in boosting immune system and so on Side effects: When free radicals and other reactive oxygen species accumulate in the body they cause damage to cells, DNA, lipid, sugar, and protein.  In plants and animals, this could lead to cell membrane dysfunction, protein modification, enzyme inactivation, break of DNA strands.  Free-radical induced oxidative damages may be precursors to aging and diseases such as cancer, heart diseases, diabetes mellitus, atherosclerosis, hypertension, brain damage and dementia related diseases such as Alzheimers and Parkinsons.

Antioxidants
Combat oxidation by reacting with free radicals and other reactive oxygen species (ROS) within the body and thereby protect molecules of the body from damaging oxidation reactions. During this reaction the antioxidant sacrifices itself by becoming oxidized. The body has developed several endogenous antioxidant systems to deal with the production of ROS. These systems can be divided into: Enzymatic antioxidants : superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) etc. Nonenzymatic antioxidants : lipid-soluble vitamins:- vitamin E and vitamin A or provitamin A (betacarotene), water-soluble :- vitamin C and Glutathione (GSH). They are intimately linked to one another and appear to interact with one another.

Endogenous sources of ROS ROS Source Defense

O2 .-

Leakage of electrons from electron transport chain/ NADPH oxidase (activated phagocytes) / Xanthine oxidase/ Flavoenzymes

Superoxide dismutase

H2O2

From O2 .-

via superoxide dismutase

Glutathioneperoxidase, Catalases, Peroxiredoxins Glucose-6phosphate dehydrogenase

OH-

From H2O2 via transition metals (Fe and Cu)

Antioxidants have multiple family members

Enzyme/Protein
Superoxide dismutase

Function
dismutates superoxide anion to hydrogen peroxide and molecular oxygen reduces hydrogen peroxide to water and oxygen

Catalase

Glutathione peroxidase

reduces hydrogen peroxide and lipid hydroperoxides using GSH as a substrate reduces oxidized form of glutathione

Glutathione reductase

Peroxiredoxin

reduces peroxides to corresponding alcohol

Thioredoxin reductase

catalyzes the reduction of oxidized thioredoxin

Estimation of Glutathione peroxidase (GPx)

Hafemann et al 1974 Reagents :
       GPx buffer, pH 7 - 61ml Na2HPO4 (0.1M) + 39ml NaH2PO4 (0.1M) GSH, 5mM in GPx buffer NaN3, 25mM in GPx buffer H2O2, 1.2mM Na2HPO4, 0.4M H2PO3, 1.67% DTNB, 1mM in GPx buffer

Principle: GPx degrades H2O2 in the presence of Glutathione (GSH) thereby depleting it. Remaining GSH is measured using 5,5'-Dithio-bis(2-nitrobenzoic acid). Thiols react with this compound, cleaving the disulfide bond to give 2-nitro5-thiobenzoate (NTB-), which ionizes to the NTB2- dianion in water at neutral and alkaline pH. This NTB2- ion has a yellow color.

5mM GSH (50 l)+ 25mM NaN3 (50 l)+ 1.2mM H2O2 (250 l)+ Buffer (875 l) + Tissue (25 l)

Incubate @ 37 C for 6 minutes + 1.67% H2PO3- (1 ml)

Centrifuge @ 800 g for 10 minutes

Supernatant (1 ml) + 0.4M Na2HPO4 (1 ml) + 1mM DTNB (0.5ml)

Incubate @ 37 C for 10 minutes

OD @ 412 nm

NaN3- inhibits catalase H2O2- oxidises GSH in the presence of cellular GPx H2PO3- deproteinates the sample and avoids interference due to particulates and sulfhydryl groups. Na2HPO4- to maintain alkaline condition DTNB- for colorimetric detection of GSH @ 412nm

An enzyme unit of activity is defined as a decrease in the log [GSH] of 0.001 per minute after the decrease in log[GSH] per minute of the non-enzymatic reaction was subtracted.

Calculation: