ANTIOXIDANTSMETHODS OF IN-VIVO ANALYSIS

BY KAMAL SIKANDAR

ANTIOXIDANT
An antioxidant is a molecule capable of inhibiting the oxidation of other molecules.

SIGNIFICANCE---Exposure of biological systems to

xenobiotics, pollutants, ionizing radiation orU.V. light and development of certain pathological conditions lead to oxidative stress, consequently increase production of oxy radicals[2]. Cell damage caused by free radicals appears to be a major contributor in aging and degenerative diseases of aging such as cancer, cardiovascular disease, cataracts, compromised immune system, rheumatoid arthritis and brain dysfunction.

 Free radicals have been implicated in the pathogenesis of at least 50 diseases. Fortunately, free radical formation is controlled naturally by various beneficial compounds known as antioxidants. Antioxidants are capable of stabilize, deactivate or scavenge free radicals before they attack cells.

MECHANISM OF ACTION
Antioxidant action 1-Includes free radical scavenging capacity

2-Inhibition of lipid peroxidation

3-Metal ion chelating ability and reducing capacity.

NATURAL ANTIOXIDANTS
a) Enzymes-Antioxidant enzymes such as reduced glutathione (GSH), superoxide dismutase(SOD), catalase(CAT), glutathione Peroxidase (GPx) b)High molecular weight compounds (proteins): Albumin, ceruplasmin, transferin, haptoglobin bind to redox active metals and limits the production of metal catalyzed free radicals.

 c)Low molecular weight compounds: These are subdivided into lipid soluble,carotenoids, quinine and some polyphenols) and water soluble antioxidants(ascorbic acid, uric acid and some polyphenols) d)Minerals: Selenium, manganese, copper, and zinc are recognized as versatile antioxidants. e)Vitamins: Vitamin A, C, and E are proved to have significant role in preventing or minimizing peroxidation damage in biological system f)Plant antioxidants

IN-VIVO ANTIOXIDANT SCREENING METHODS

Antioxidant activity can be measured using invitro as well as in-vivo methods. The chemistry responsible for these effects is ready donation of electrons to reactive oxygen species (ROS) by antioxidants. METHODS Lipid peroxidation (LPO) : LPO is an autocatalytic process, which is a common consequence of cell death. MDA (Malondialdehyde) is one of the end products in the lipid per-oxidation process which is accepted as an indicator of lipid peroxidation.

 Method-I-The tissues are homogenized in 0.1 M

buffer pH 7.4 with a Teflon-glass homogenizer. LPO in this homogenate was determined by measuring the amounts of Malondialdehyde (MDA) produced. 0.2 ml of tissue homogenate, 0.2 ml of 8.1%sodium dodecyl sulphate (SDS), 1.5 ml of 20% acetic acid and 1.5 ml of 8% TBA were added. The volume of the mixture was made up to 4 ml with distilled water and then heated at 95C on a water bath for 60 min using glass balls as condenser.

After incubation the tubes were cooled to room temperature and final volume was made to 5 ml in each tube. 5.0 ml of butanol: pyridine (15:1) mixture was added and the contents were vortexed thoroughly for 2 min. After centrifugation at 3000rpm for 10 min, the upper organic layer was taken and its OD read at 532 nm against an appropriate blank without the sample. The levels of lipid peroxides were expressed as n moles of thiobarbituric acid reactive substances (TBARS)/ mg protein using an extinction coefficient of 1.56 105 M1cm-1

 Method-II-Three sets of tubes were used

for the assay. The first set had 22ml of buffer and 02ml of FeSO4 (07 mM final concentration); another set had 22 ml of buffer and02 ml of H2O2 (075 mM final concentration); the third set contained 22ml of buffer and 02 ml of distilled water. Each set was divided into two batches. To each tube of one batch 06 ml of the tissue homogenate was added.

 The tubes were incubated on a mechanical shaker at 100120 oscillation/min (for aeration) for 60 min. After the incubation 05 ml of 40%trichloroacetic acid (TCA) was added. To each tube of the second batch 05 ml of 40 % TCA was added at zero time immediately after the addition of 06ml of the homogenate .To all the tubes 025 ml of 5 HCl was added and the Contents mixed thoroughly. This was followed by the addition of 05ml of 2%thiobarbituric acid. The tubes were shaken and incubated in a water bath at 90C for 20 min.

 They were then cooled and 3 ml of chloroform was added. After thorough mixing they were centrifuged for 15 min at 3000 g. The supernatant was aspirated and its absorbance at 532 nm determined using water blank. Standard malonaldehyde was treated in a similar fashion and the colour developed was measured.

Method-III: one volume of homogenate

was mixed with 0.5 volume of trichloroacetic acid (15%w/v) and centrifuged at 200 rev for 10 min.

 One milliliter of the supernatant was Mixed with 0.5ml TBA(0.7%w/v) and boiled for 10min. After cooling, the absorbance was recorded at 535 by spectrophotometer (Shimadzu UV-3100).MDA concentration was calculated using extinction coefficient of 1.56 105M1 cm1.

Reduced Glutathione (GSH): Method -I: To measure the GSH level, the tissue
homogenate was taken. The homogenate was Added with equal volume of 20%trichloroacetic acid (TBA) containing 1 mM EDTA.

 The mixture was allowed to stand for 5 min prior to centrifugation for 10 min at 200 rpm.The supernatant (200 l) was then transferred to a newest of test tubes and added 1.8ml of the Ellmans reagent (0.1mM).Then all the test tubes make upto the volume of 2ml.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.

 Glutathione Peroxidase (GSHPx): Cytosolic

GPx was assayed via a 3-ml cuvette containing 2.0 ml of 75 mmol/L phosphate buffer, pH 7.0. The following solutions were then added : 50l of 60mmol/L glutathione reductase solution (30 U/mL) , 50L OF 0.12 mol/L NaN3, 0.10 OF 0.15mmol/L Na2EDTA , 100Lof 3.0mmol/L NADPH, and 100Lof cytosolic fraction obtained after centrifugation at 20,000 g for 25 minutes.

 Glutathione-S-transferase ( GSt): The

reaction mixture (1mL) consisted of 0.1 N potassium phosphate (pH 6.5), 1 nmol/LGSH, 1mol/L l-chloro-2, 4-dinitrobenzene as substrate and a suitable amount of cytosol (6 mg protein/mL). The reaction mixture was incubated at 370C for 5 min and the reaction was initiated by the addition of the substrate. The increase in absorbance at 340 nm was measured spectrophotometrically.

 Glutathione Reductase: Method:

Preparation of Liver Supernatant- Livers (about 400 g) were obtained from 40 male SpragueDawley rats (200 to 250 g), which were killed by decapitation. The livers were cut into small pieces and homogenized in 9 ml of 0.25Mice-cold sucrose per g of rat liver in a Turmix blender.The homogenate was centrifuged for 45min at 14,000 rpm. The pellets were suspended in a small volume of 0.25Msucrose and centrifuged. The supernatants were combined with the previous centrifugate. The pooled material was adjusted to pH 5.5 with cold 0.2M acetic acid and centrifuged .

The rate of oxidation of NADPH by GSSG at 300 was used as a standard measure of enzymatic activity. The reaction system of 1ml contained: 1.0mM GSSG, 0.1 mM NADPH, 0.5 mM EDTA, 0.10 M sodium phosphate buffer (pH 7.6), and a suitable amount of the glutathione reductase sample to give a change in absorbance of 0.05 to 0.03/min. The oxidation of 1 mol of NADPH/min under these conditions is used as a unit of glutathione reductase activity. The specific activity is expressed as units per mg of protein.

 Superoxide dismutase (SOD): Method-I: Assay mixture contained 0.1ml of

sample, 1.2ml of sodiumpyrophosphate buffer (pH8.3, 0.052 M), 0.1 ml phenazine methosulphate (186 M), 0.3 ml of 300 M nitroblue tetrazolium, 0.2 ml NADH (750 M). Reaction was started by addition of NADH.After incubation at 30Cfor 90 s, the reaction was stopped by the addition of 0.1ml glacial acetic acid. Reaction mixture was stirred vigorously with 4.0ml of n-butanol. Mixture was allowed to stand for 10min, centrifuged and butanol layer was separated

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

Method-II : Supernatant was assayed for SOD

activity by following inhibition of pyrogallol autooxidation. Pyrogallol (24mmol/l)was prepared in 10mmole/l HCl and kept at 40 C before use.Catalase (30 mol/l stock solution)was prepared in an alkaline buffer pH 9. Aliquots of supernatant (150g protein) were added to this HCL buffer containing 25l pyrogallol and 10 l of catalase.

 The final volume of 3ml was made up of the same buffer.Changes in absorbance at 420 nm were recorded at 1min interval for 5min. SOD activity was determined from a std. curve of % inhibition of pyrogallol autooxidation with known SOD activity. This assay was highly reproducible, and the standard curve was linear upto 250 g protein with a correlation coefficient of 0.998. Data are expressed as SOD units/mg proteins compared with standard.

 Method-III : The SOD activity was estimated after

3 h of reperfusion by using NADH oxidation method.Thus, an aliquot (100 L) of brain supernatant or phosphate buffer (50mM) for the blank was added to a reaction mixture containing triethanolamine-diethanolamine buffer (800 L, pH 7.4), NADH (40L of 7.5 mM) and EDTA-MnCl2 (25 Lof 10050mM, pH7).After thorough mixing the reaction was then, initiated by adding 100 L of10mM mercaptoethanol and it was monitored at 340 nm using a spectrophotometer.

 The rate of the nucleotide oxidation (in the control) and inhibition of the same by the supernatant was calculated. One unit of the SOD activity was defined as the amount of the enzyme required to inhibit the rate of NADH oxidation of the control by 50%. Method-IV: The supernatant (50 l) was added to 0.75ml of carbonate buffer (100mM, pH 10.2)AND10 l OF epinephrine (3mM). The change in absorbance of each sample was then recorded at 480 nm in spectrophotometer for 2 min at an interval of 15 sec. Parallel blank and standard were run for determination SOD activity.

 One unit of SOD is defined as the amount of enzyme required to produce 50% inhibition of epinephrine autooxidation.

Catalase (CAT): Method-I: 0.1 ml of supernatant was added to

cuvette containing 1.9ml of 50mMphosphate buffer (pH 7.0).Reaction was started by the addition of 1.0 ml of freshly prepared 30 mM H2O2. The rate of decomposition ofH2O2 was measured spectrophotometrically from changes in absorbance at 240 nm.Activity of catalase was expressed as units/ mg protein.

 Method-II: The estimation was done

spectrophotometrically following the decrease in absorbance at 230 nm. The tissue was homogenized in M/150 phosphate buffer (pH 7.0) AT 40C and centrifuged at 50000 rpm . The reaction mixture contained 0.01 M phosphate buffer (pH 7.0), 2Mm H2O2 and the enzyme extract. The specific activity of catalase is expressed in terms of units/mg protein. A unit is defined as the velocity constant per Second.

 Glutamyl transpeptidase activity (GGT): Method: The serum sample was added to a
substrate solution containing glycylglycine, MgCl2 and Glutamyl-p-nitroanilide in 0.05Mtris (free base), pH 8.2. The mixture was incubated at 370C for 1 min and the absorbance read at 405 nm at 1 min interval for 5min. The activity of GGT was calculated from the absorbance values.

Lactate Dehydrogenase (LDH): 10% (w/v)

homogenates of the myocardium was prepared in distilled water using glass homogenizer.

(0.1 M sodium lactate), 1.0 ml of 0.66M phosphate buffer pH 7.4, and 0.5 ml of 0.3 % triphenyl tetrazoniumchloride and 1.0ml of 10%homogenate. The incubation was carried out at 370C for 45 min in a water bath. After incubation, the reaction was stopped by the addition of 6 ml glacial acetic acid. The formazon liberated was extracted in 6 ml of toluene by keeping mixture over night in a refrigerator. The toluene layer was then separated and its optical density was read at 505 nm in DU-2 Beckmanns spectrophotometer. Blood was collected by cardiac puncture and serum is separated.

LDH activity of the serum was estimated calorimetrically.

Method -II: The assay mixture consisted of 1.84

ml 0f water, 1.o ml of phosphate buffer (Ph 7.4), NADPH to give an absorbance value of 0.5, 0.05 ml 0f supernatant ,and 0.1 ml of pyruate (0.33 nM final concentration). The total volume was 3.o ml; the Reaction was started by adding pyruate last and noting the decrease in A340 at intervals of 30 sec in a BeackmanDBmodel spectrophotometer.The activity of lactate dehydrogenase of each tissue was expressed as units per g of tissue, wet weight .

In this compilation various in vivo antioxidant screening methods have been described which are simple and require only use of spectrophotometer and chemicals. The procedures are very popular and have been widely used to evaluate antioxidant properties.

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THANKS