Oxidative stress

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OXIDATIVE STRESS& ITS ROLE IN ALZHEIMER’S DISEASE

1.INTRODUCTION

An antioxidant is a molecule that inhibits the oxidation of other molecules in human body. Antioxidants protect the body from damage caused by harmful molecules called free radicals. This damage is a factor in the development of blood vessel disease atherosclerosis, cancer and other conditions.

1.1What is a free radical?

Free radical is an atom that has at list one unpaired electron.

Free radical are released are during normal metabolism, as well as by pollution, smoking, radiation & stress ,air pollution, Alcohol intake , Toxins, High blood sugar levels etc.

Under normal circumstance the body keeps them in check.

These are highly reactive chemical entities that have a single unpaired electron in their outer most orbits.

Under certain conditions can be highly toxic to the cells.

Generally unstable and try to become stable, either by accepting or donating an electron.

Fig 1.1.Free Radicals Formation. Human Aging Free Radical Theory

OXIDATIVE STRESS & ESTIMATION OF BIOMARKERS FOR ALZHEIMER’S DISEASE

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1.2 TYPES OF FREE RADICALS

Superoxide radical

Hydroxide radical

Nitric oxide radical

Peroxide radicals

H2O2 (non radical).etc

1.3 What are Reactive oxygen species?

Reactive Oxygen Species (ROS) is a phrase used to describe a number of reactive molecules and free radicals derived from molecular oxygen. The production of oxygen based radicals is the bane to all aerobic species. These molecules, produced as by-products during the mitochondrial electron transport of aerobic respiration or by oxidoreductase enzymes and metal catalyzed oxidation, have the potential to cause a number of deleterious events.

It was originally thought that only phagocytic cells were responsible for ROS production as their part in host cell defense mechanisms. Recent work has demonstrated that ROS have a role in cell signaling, including; apoptosis; gene expression; and the activation of cell signalingcascades [1].

ROS are formed as necessary intermediates of metal catalyzed oxidation reactions. Atomic oxygen has two unpaired electrons in separate orbits in its outer electron shell. This electron


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structure makes oxygen susceptible to radical formation. The sequential reduction of oxygen through the addition of electrons leads to the formation of a number of ROS including: superoxide; hydrogen peroxide; hydroxyl radical; hydroxyl ion; and nitric oxide.

1. Superoxide Superoxide radical (O2¯•) is formed by reduction of oxygen molecule with one electron. In aqueous solution it is a weak oxidant and acts mainly on ascorbic acid and thiol compounds. Superoxide radical is a very strong reducing agent and can reduce certain iron complexes, such as cytochrome C[6]. In vivo, it is decomposed by SOD to hydrogen peroxide and oxygen

2. Hydroxyl radical Particularly, the most reactive hydroxyl radical, when generated in excess, causes cellular damage leading to cell death. Hydroxyl radical is generated via the Fenton reaction from hydrogen peroxide in the presence of ferrous ions or via the Heber- Weiss reaction from hydrogen peroxide and superoxide radical [2].

3. Nitric oxide The free radical NO• is synthesized from amino acid L-arginine by vascular endothelial cells, phagocytes, certain cells in the brain and other cell’s types. Nitric oxide is a vasodilator agent and possibly an important neurotransmitter. The NO• contains an unpaired electron and is paramagnetic, it rapidly reacts with O2¯ to form peroxynitrite anion (ONOO¯) in high yield [3].

4. Hydrogen peroxide Hydrogen peroxide (H2O2) is formed in two ways: indirectly through superoxide anion dismutation, and directly in some oxidative reactions associated with the transfer of two electrons to the oxygen. Hydrogen peroxide is a relatively stable in water and appears as a weak oxidizer and reductant. It is readily diffuses through cell membranes and in the presence of ions with variable valency it is formed the highly toxic for the cell –hydroxyl radicals [4]. Hydrogen peroxide is converted by the glutathione peroxidase enzyme to form water and oxygen, thus preventing the accumulation of precursor to free –radical biosynthesis.

1.4 Whatis oxidative stress?

Oxidative stress is defined as a “state in which oxidation exceeds the antioxidant systems in the

body secondary to a loss of the balance between them.” Disturbance in the balance between the

production of reactive oxygen species (free radicals) and antioxidant defenses.Oxidative stress is

the result of an imbalance in pro-oxidant/antioxidant homeostasis that leads to the generation

of toxic reactive oxygen species [5].oxidative stress has been implicated in the ageing process &

many diseases as shown in below fig.


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Fig 1.2 Role of oxidative stress in various diseases (© 2015 by CorinGreenBioIT Team.)

1.4.2. Sources of oxidative stress and defensive mechanisms


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2. CLASSIFICATION OF ANTIOXIDANTS

-


ANTIOXIDANTS

Enzymatic antioxidants Non enzymatic antioxidants

Primary enzymes SOD, Catalase, Glutathione peroxidase Glutathione reductase

Secondary enzymes Glutathione reductase Glucose 6-phosphate

dehydrogenase

VitaminsVitamin A, Vitamin CVitamin E, Vitamin K

MineralsZinc, Selenium

Organosulfur compoundAllium, allyl sulfide, indoles

Carotenoidsβ-carotene, lycopene,Lutein,

Antioxidant cofactors

Coenzyme Q10

Low molecular weight Antioxidants

Glutathione, uric acid

Polyphenols

Flavonoids Phenolic acids

Hydroxy cinnamic acidsFerulic, p-coumaric

Hydroxy benzoic acidsGallic aid, ellagic acid

Flavonolsquercetin

Isoflavonoids

genistein

Flavonolscatechin

Flavanones

Hesperitin

Anthocyanidins

Cyanidin, pelagonidin

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2.1Antioxidant defense system (ADS).

A biological antioxidant may be defined as a substance (present in low concentrations compared to an oxidizable substrate) that significantly delays or inhibits oxidation of a substrate. Substances that neutralize potential ill effect of free radicals are generally grouped in so called an Antioxidant defense system (ADS)[6, 7].

2.2 Mode of Antioxidant defense system (ADS)

Antioxidant defense systems (ADS) traditionally have been termed.

1. Primary defense system 2. Secondary defense systemPrimary defense systemIncludes antioxidant compounds like a Vitamin A, E, C and Glutathione and uric acid. AO scavenging enzymes such as peroxidasesSecondary defense systemIncludes Lipolytic enzymes, Phospholipases, proteolytic enzymes and DNA repair enzymes

3. THE EFFECT OF OXIDATIVE STRESS: PHYSIOLOGICAL, ANDBIOCHEMICAL MECHANISMS

3.1 Effects of Oxidative Stress on DNA

ROS can lead to DNA modifications in several ways, which involves degradation of bases,

single- or double-stranded DNA breaks, purine, pyrimidine or sugar-bound modifications,

mutations, deletions or translocations, and cross-linking with proteins.

Formation of 8-OH-G is the best-known DNA damage occurring via oxidative stress and is

a potential biomarker for carcinogenesis.. Oxidative stress causes instability of microsatellite

(short tandem repeats) regions. Redox active metal ions, hydroxyl radicals increase

microsatellite instability. Even though single-stranded DNA breaks caused by oxidant

injury can easily be tolerated by cells, double-stranded DNA breaks induced by ionizing

radiation can be a significant threat for the cell survival[8].

3.2 Effects of Oxidative Stress on Lipids

ROS can induce lipid peroxidation and disrupt the membrane lipid bilayer arrangement that

may inactivate membrane-bound receptors and enzymes and increase tissue permeability.


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Products of lipid peroxidation, such as MDA and unsaturated aldehydes, are capable of

inactivating many cellular proteins by forming protein cross-linkages. 4-Hydroxy-2-nonenal

causes depletion of intracellular GSH and induces of peroxide production, activates

epidermal growth factor receptor, and induces fibronectin production[9].

Lipid peroxidation products, such as isoprostanes and thiobarbituric acid reactive

substances, have been used as indirect biomarkers of oxidative stress, and increased levels

were shown in thebroncho alveolar lavage fluid or lung of chronic obstructive pulmonary

disease patients or smokers.

3.3 Effects of Oxidative Stress on Proteins

ROS can cause fragmentation of the peptide chain, alteration of electrical charge of proteins,

cross-linking of proteins and oxidation of specific amino acids and therefore lead to

increased susceptibility to proteolysis by degradation by specific proteases.

Cysteine and methionine residues in proteins are particularly more susceptible to oxidation.

Oxidation of sulfhydryl groups or methionine residues of proteins cause conformational

changes, protein unfolding, and degradation.

In some cases, specific oxidation of proteins may take place. For example, methionine can

be oxidized methionine sulfoxide and phenylalanine to o-tyrosine sulfhydryl groups can be

oxidized to form disulfide bonds and carbonyl groups may be introduced into the side chains

of proteins[10].

4. ROLE OF OXIDATIVE STRESS IN ALZHEIMER’S DISEASE

4.1 Alzheimer’s disease

Alzheimer’s disease (AD) is a neurodegenerative disorder associated with a decline in cognitive impairments, progressive neurodegeneration and formation of amyloid-β (Aβ) containing plaques and neurofibrillary tangles.

Alzheimer's disease (AD) is a slowly progressive disease of the brain that is characterized by impairment of memory and eventually by disturbances in reasoning, planning, language, and perception [11].

Mutations of amyloid precursor protein or presenilin genes or apolipoprotein E gene polymorphism appear to affect amyloid formation, which in turn causes neuronal death via a


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number of possible mechanisms, including Ca2+ homeostasis disruption, oxidative stress, excitotoxicity, energy depletion, neuro- inflammation and apoptosis.

4.2 RELATION OF OXIDATIVE STRESS WITH ALZHEIMER’S DISEASE

Oxidative stress plays a central role in the pathogenesis of AD leading to neuronal dysfunction and cell death.

Oxidative stress can lead to alterations in cells with an accumulation of oxidized products such as aldehydes and isoprostanes from lipid peroxidation, protein carbonyls from protein oxidation, and base adducts from DNA oxidation, all of which serve as markers of oxidation.

The process of aging is also associated with increased oxidative stress. Through pathological redox reactions ROS can denature biomolecules such as proteins, lipids and nucleic acids. This can initiate tissue damage via apoptosis and necrosis[12].

The increased level of oxidative stress in the AD brain is reflected by

increased protein and DNA oxidation,

Decreased level of cytochrome c oxidase and advanced glycosylation end products.

enhanced lipid peroxidation,

Lipid peroxidation can weaken cell membranes causes ion imbalance and impair metabolism. Oxidative stress can influence DNA methylation which regulates gene expression. Internalized beta-amyloid may play a role in this process.

Mitochondria are essential for the formation and maintenance of synapses. There is evidence that oxidative damage precedes pathological changes. Oxidation of mitochondrial DNA renders it more susceptible to somatic mutation as oxidized bases are frequently misread during replication. These mutations may initiate erroneous beta-amyloid processing[13].

Beta-amyloid peptide has been shown to inhibit cytochrome oxidase leading to disruption of the electron transport chain and production of ROS. Thus a viscous cycle may be initiated that culminates in progressive disease.

Four-hydroxynonenal causes degeneration and death of cultured hippocampal neurons by impairing ion motive adenosine triphosphatase activity and disrupting calcium homeostasis. Exposure of cultured hippocampal neurons to amyloid (A) peptide causes a significant increase in levels of free and protein-bound HNE and increases ROS [14].

4-hydroxy-2,3-nonenal (HNE), acrolein, malondialdehyde (MDA) and F2-isoprostanes are important break down products of lipid peroxidation. Elevated HNE levels have been


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observed in Alzheimer’s disease (AD). Acrolein, thiobarbituric acid-reactive substances (TBARs, the most prevalent substrate of which is malondialdehyde) and F2-isoprostanes are all increasedin AD brains relative to age-matched controls.

Under stressful conditions and in aging, the electron transport system can increase ROS formation considerably. Thus, the mitochondria are both a source and a target of toxic ROS. Mitochondrial dysfunction and oxidative metabolism may play an important role in the pathogenesis of AD and other neurodegenerative diseases.

FIG 1.3Role of oxidative stress and acetylcholinesterase in neurodegeneration .Abbreviations: Aβ, amyloid beta; ACh, acetylcholine; AChE, acetylcholinesterase; GSH, glutathione; GSSG, glutathione disulfide; SOD, superoxide dismutase.

5. ESTIMATION OF BIOMARKERS FOR ALZHEIMER’S DISEASE

5.1SCREENING METHODS OF ANTIOXIDANT ACTIVITY


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Various in-vitromethods are used to evaluate the ability of antioxidantto reduce theradical. Antioxidantactivity can be measured using both serum sample as well as tissue homogenate[16].

The different in vitro antioxidantscreening methods

Nitric oxide assay

Lipid peroxidation (LPO)

Superoxide Dismustase (SOD)

Reduce Glutathiol (GSH) assay

Glutathione peroxidase (GPx)

Glutathione-s-transferase (GST)

Catalase (CAT)

Glucose 6 phosphatase (G6P)

Creatinine phosphokinase (CPK) etc.

5.2NITRIC OXIDE RADICAL SCAVENGING (NO) ASSAY

Nitric oxide, because of its unpaired electron, is classified as a free radical and displays important reactivity’s with certain types of proteins and other free radicals.

In vitro inhibition of nitric oxide radical is also a measure of antioxidant activity.

This method is based on the inhibition of nitric oxide radical generated from sodium nitroprusside in buffer saline and measured by Griess reagent.

In presence of scavengers, the absorbance of the chromophore is evaluated at 546nm.

The activity is expressed as % reduction of nitric oxide.

Procedure

3.0 ml of 10mM sodium nitroprusside in phosphate buffer (pH-7.5) is added to 2.0 ml of extract and reference compound in different concentrations (20 – 100 µg/ml).

The resulting solutions are then incubated at 25°C for 60 min

A similar procedure is repeated with methanol as blank, which serves as control.


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To 5.0 ml of the incubated sample, 5.0ml of Griess reagent (1% sulphanilamide, 0.1% naphthyethylenediaminedihydrochloride (NED) in 2% H3PO3) is added and pink chromophore generate during diazotization of nitrite ions with sulphanilamide and subsequent coupling with NED was measured spectrophotometrically at 540nm.

Percent inhibition of the nitrite oxide generated is measured by comparing the absorbance values of control and test preparations.

POSITIVE CONTROL: Curcumin, Caffeic acid, Sodium nitrite, BHA, Ascorbic acid, Rutin, BHT or α-tocopherol

5.3 LIPID PEROXIDATION (LPO) ASSAY

LPO is an autocatalytic process, which is a common cause of cell death.

This process may cause peroxidative tissue damage in inflammation, cancer and toxicity of xenobiotics and aging.

MDA is one of the end products in the lipid per-oxidation process .

Malondialdehyde (MDA) is formed during oxidative degeneration as a product of free oxygen radicalswhich is accepted as an indicator of lipid peroxidation.

PROCEDURE:

The reaction mixture contained 0.1 ml of sample, 0.2 ml of 8.1% sodium dodecylsulfate (SDS), 1.5 ml of 20% acetic acid solution of various pHs, and 1.5 ml of 0.8% aqueous solution of thiobarbituric acid (TBA).

The pH of 20% acetic acid solution was adjusted with NaOH above pH 3.0, and in the pH range of 1.0-3.0, 20% acetic acid containing 0.27 M HClwas adjusted to the specified pH with NaOH.

The mixture was finally made up to 4.0 ml with distilled water, and heated at 95°C for 60 min.

After cooling with tap water, 1.0 ml of distilled water and 5.0 ml of the mixture of n-butanol and pyridine (15: 1, v/v) were added, and the mixture was shaken vigorously.

After centrifugation at 4000 rpm for 10 min, the absorbance of the organic layer (upper layer) was measured at 532 nm against blank without the sample.

The levels of lipid peroxidase were expressed as thiobarbituric acid reactive substance (TBARS)/mg protin.


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Reagents preparation: 1. 8.1% Sodium Dodecyl Sulphate

gently heat the solution at 37o C in water bath if recrystalization occurs

2. 20% Glacial acetic acid

Adjust pH to 3.5 with 10 mM Sodium hydroxide

3. 0.8% Thiobarbituric acid

Dissolve 0.8g of TBA in 100 ml of phosphate buffer

4. MDA standards

+ 100mM

+ 10mM

+ 1mM

Use this solution C for preparation of standard as below


17µl of Tetramethoxypropane (TMP)

983µl of Distilled water

Solution A

100µl of above solution A 900µl of Distilled water

Solution B

100µl of above solution A 900µl of Distilled water

Solution C

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125μl of solution C + 875μl of distilled water = 125μM STOCK

SOLUTION TUBE NUMBER VOLUME OF

WATER VOLUME OF 125 μM STOCK SOLUTION

MDA STANDARD CONC.

A 1000 0 0B 995 50 0.625C 990 10 1.25D 980 20 2.5E 960 40 5F 920 80 10G 800 200 25H 600 400 50

Preparation of homogenate:

+

MDA Estimation procedure:


0.2 g of tissue 1ml of phosphate buffer (Ph7.4)

Add 4µl of 1 Mm EDTA Homogenate

100µl of above homogenateCentrifuge 16000g, 10minutes, 40C

Supernatant

Protein estimation MDA estimation procedure

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REAGENTS FOR 2ML FOR 1ML8.1% SDS 100µl 50µl0.8% TBA 750µl 375µl

20% Glacial acetic acid 750µl 375µlSupernatant sample/standard 100µ 50µl

Mix and make up the volume with distilled water

Make up to 2ml Make up to 1ml

5.4 SUPEROXIDE DISMUTASE (SOD) ASSAY

SOD is a metalloprotein and is the first enzyme involved in theantioxidant defence by lowering the steady-state level of O2.

Superoxide dismutasesare enzymes that catalyze the dismutation of superoxide into oxygen and hydrogen peroxide. Thus, they are an important antioxidant defense in nearly all cells exposed to oxygen[17]. One of the exceedingly rare exceptions is Lactobacillus plantarum and related lactobacilli, which use a different mechanism

Three forms of superoxide dismutase are present in humans, in all other mammals, and most chordates. SOD1 is located in the cytoplasm, SOD2 in the mitochondria, and SOD3 is extracellular. SOD1 and SOD3 contain copper and zinc, whereas SOD2, the mitochondrial enzyme, has manganese in its reactive centre. The genes are located on chromosomes 21, 6, and 4, respectively

Mutations in the first SOD enzyme (SOD1) can cause familial amyotrophic lateral sclerosis (ALS, a form of motor neuron disease) [18].The most common mutation in the U.S. is A4V, while the most intensely studied isG93A. The other two isoforms of SOD have not been linked to any human diseases, however, in mice inactivation ofSOD2 causes perinatal lethality [86] and inactivation of SOD1 causes hepatocellular carcinoma. Mutations inSOD1 can cause familial ALS (several pieces of evidence also show that wild-type SOD1, under conditions ofcellular stress, is implicated in a significant fraction of sporadic ALS cases, which represent 90% of ALSpatients [19] by a mechanism that is presently not understood, but not due to loss of enzymatic activity or a decrease in the conformational stability of the SOD1 protein. Over expression of SOD1 has been linked to the neural disorders seen in Down syndrome.


Incubate for 45-60 minutes in 950C in water bath

Cool the system Pink colour will develop

Centrifuge at 10000 g for 10min

Micro titer plate Absorbance at 530nm

Calculation by standard curve method

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Procedure:

Assay mixture contained 0.1ml of sample, 1.2ml of sodium pyrophosphate buffer (pH 8.3, 0.052 M), 0.1 ml phenazinemethosulphate (186 μM), 0.3 ml of 300 μM nitrobluetetrazolium, 0.2 ml NADH (750 μM).

Reaction was started by addition of NADH.

After incubation at 30°C for 90 s, then addition of 0.1ml glacial acetic acid to stop reaction.

Reaction mixture was stirred vigorously with 4.0ml of n-butanol.

Mixture was allowed to stand for 10min, centrifuged and butanol layer was separated.

Colour intensity of the chromogen in the butanol layer was measured at 560 nm spectrophotometrically and concentration of SOD was expressed as units/mg.

5.5 REDUCE GLUTATHIOL (GSH) ASSAY

If deficiency of GSH within living organisms can lead to tissue disorder and injury.

GSH is an intra-cellular reductant and plays major role in catalysis, metabolism and transport.

It protects cells against free radicals, peroxides and other toxic compounds. .

Glutathione also plays an important role in the kidney and takes part in a transport system involved in the reabsorption of amino acids.

Thus GSH monoesters are also useful in the treatment of aminoaciduria.

PROCEDURE

To measure the GSH level, the tissue homogenate (in 0.1 M phosphate buffer pH 7.4) was taken.

The homogenate was added with equal volume of 20% trichloroacetic acid (TCA) containing 1 mM EDTA to precipitate the tissue proteins.

The mixture was centrifugation for 10 min at 200 rpm allowed to stand for 5 min.

The supernatant (200 μl) was then transferred to a new set of test tubes and added 1.8ml of the 5, 5'-dithiobis-2-nitrobenzoic acid (0.1mM) was prepared in 0.3 M phosphate buffer with 1% of sodium citrate solution).

Then all the test tubes make up to the volume of 2ml with double distilled water.


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After completion of the total reaction, solutions were measured at 412 nm against blank.

Absorbance values were compared with a standard curve generated from standard curve from known GSH[20, 21].


Reagents preparation:

1. GSH Standard Preparation

+ 20mM

+ 400µM


6.14 mg GSH 1 ml of phosphate buffer (pH7.4)

Solution A

40 µl of solution A 1960µl of phosphate buffer (pH7.4)

Solution B

Use for standard preparation for GSH

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STANDARDNAME

SOLUTION B (μl) PHOSPHATE BUFFER (μl)

FINALCONC. (μM)

BLANK 0 500 0A 5 495 4B 10 490 8C 20 480 16D 40 460 32E 80 420 64F 160 340 128G 320 180 256

2. Ellman’s Reagent (0.1 mM)

Dissolve 3.9636mg of DTNB in 100 ml phosphate buffer (pH 7.4)

2. 5% Sulfosalicylic acid


+


350µl of homogenate from MDA

Centrifuge at 700 g for 10min at 40C

250 µl of supernatant 250 µl of 5% chilled SSA

Centrifuge at 1200 g for 10min at 40C

Supernatant

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GSH estimation procedure:


Protein estimation GSH estimation procedure

Sample Blank Standard

25µl sample + 475µl of phosphate buffer

25µl of solution C + 475µl of phosphate buffer

25µl of solution (A-G) +475µl of phosphate buffer

Vortex

Add 1500µl of Ellman’s reagent

Vortex

Incubation at 370C for 10 minutes

Absorbance at 412 nm

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5.6 GLUTATHIONE PEROXIDASE (GPx) ASSAY

Low levels of GPx have been correlated with free radical related disease

Principle: indirect determination method, kinetic assay GPx is the sample (supernatant of homogenate) reduces H2O2 while oxidizing GSH (reduced) to GSSG (oxidized). The generated GSSG is reduced to GSH with consumption of NADPH by GR. The rate of decrease of NADPH (whose λmax is 340nm) is proportional to GPx activity. H2O2 & GSH react spontaneously at a slow rate.

Sample: plasma, erythrocyte lysates, tissue homogenates, cell lysates.

R-OOH + 2GSH GPx in sample R-OH + GSSG+H2O

GSSG +NADPH + H+ GR in assay buffer 2GSH + NADP+

Where GPx = glutathione peroxidase

GR = glutathione reductase

GSH = glutathione reductase – a major intracellular antioxidant

GSSG = glutathione oxidase – glutathione disulfide

GSH = Glutamyl cysteinyl glycine

R = tert-butyl/cumene/H

ROOH = R-hydroperoxide

1. Homogenization bufferPhosphate buffer saline (pH7.2) 100mg tissue + 1ml homogenizing buffer

3 × 8 strokes (8strokes each time & 30-90sec gap between each time

Centrifuge at 10,000 g for 15min at 40C

Use the supernatant for the assay

Assay reagents:

1) Assay buffer 50mM phosphate buffer, pH7.0

Prepare 0.2M solutions of NaH2PO4(A), NaH2PO4 (B)


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39ml of A + 61ml of B + 100ml H2O = 0.1M sodium phosphate buffer pH7.0

39ml of A + 61ml of B + 200ml H2O = 0.05M = 500mM sodium phosphate buffer pH7.0

Mixing of these solutions yields exactly pH7.0 as determined with pH meter

2) 10mM GSH 307.3232g 1000ml H2O 1M = 1000mM3.0732g 1000mlH2O 10mM

0.0307g = 30.732mg 10ml H2O 10mM

3) 2mM β – NADPH

833.35g 1000ml H2O 1M

83.335mg 10ml H2O 10mM

16.667mg 10mlH2O 2mM

4) 5mM H2O2

5.1µl of 30% H2O2(W/W) 10ml 5mM

5) 1000U/ml Glutathione reductase

6) 1.125M sodium aride

31.362mg 10ml H2O 1.125M

Assay procedure:

For 1ml assay

50mM sodium phosphate buffer, pH7.0 0.63ml

10mM GSH 0.1ml

2mM NADPH 0.1ml

1.125 sodium aride 0.01ml

1000U/ml GR enzyme 0.01ml

Sample/standard 0.1ml


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Mix well & allow equilibrating for 10-15min

Add 50µl of 5mM H2O2

Mix quickly

Read at 340nm for 5min with 30sec interval

Calculation:

λA340nm/min = (sampleA1 – sample A2) – (Reagent/blank A1 – Reagent/blank A2)

T2 (min) – T1 (min)

Where A1 is absorbance at time T1

A2 is absorbance at time T2

Reagent = blank is the one in which, instead of sample phosphate buffer is used

% inhibition of NADPH oxidation = λA340nm/min × 100

5.7 CATALASE (CAT) ASSAY

CAT is a hemeprotein, localized in the peroxisomes or the microperoxisomes.

This enzyme catalyses the decomposition of H2O2 to water and oxygen and thus protecting the cell from oxidative damage by H2O2 and OH.

CAT is a key component of the antioxidant defence system.

Inhibition of these protective mechanisms results in enhanced sensitivity to free radical-induced cellular damage.

PROCEDURE:

0.1 ml of supernatant was added to cuvette containing 1.9 ml of 50 mM phosphate buffer (pH 7.0)

Reaction was started by the addition of 1.0 ml of freshly prepared 30 mM H2O2


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The rate of decomposition of H2O2 was measured spectrophotometrically from changes in absorbance at 240 nm.

Absorbance is calculated was expressed as units/mg protein.

2H2O2 Catalase2H2O + O2

1) homogenizationPhosphate buffer saline (pH7.2) 100mg tissue + 1ml homogenizing buffer

3 × 8 strokes (8strokes each time & 30-90sec gap between each time

Centrifuge at 10,000 g for 15min at 40C

Use the supernatant for the assay

Assay reagents:

1) phosphate buffer: 0.1M sodium phosphate buffer, pH7.0

Prepare 0.2M solutions of NaH2PO4 (A), Na2HPO4 (B)

39ml of A + 61ml of B + 100ml H2O = 0.1M sodium phosphate buffer,pH7.0

2) 65µM H2O2 in PBS

1.3ml of 5mM H2O2 100ml PBS = 65µM H2O2 in PBS

3) 32.4mM Ammonium molybdate (M.W. 1235.86, SIGMA)

1235.86g 1000ml 1M

123.586g 10ml PBS 10mM

2g 50ml PBS 32.4m

Assay procedure:


Sample Blank 1 Blank 2

0.5ml of 65µM H2O2 + 100µl sample

0.5ml of 65µM H2O2 + 100µl PBS

0.5ml of PBS+ 100µl PBS

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Mix and allow to stand for 60sec

Add 0.5ml of 32.4mM Ammonium molybdate

Read at 405nm

Reaction type: end point

Catalase activity (KU/L) = A sample - A blank1 × 271A blank1 – A blank2

Where, blank 1 = H2O2 + buffer (no enzyme control) Blank 2 = buffer + (no enzyme/no substrate)

Sample containing Catalase when added to H2O2, decomposes H2O2 in the buffer exactly after 60sec (a fixed time). Undecomposed H2O2 is made to react with ammonium molybdate to form a yellow complex whose λmax is 405nm.

2H2O2 Catalase2H2O + O2


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Oxidative stress

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