Prueba de Muestras Independientes

Method: Misra and Fredovich (1972) as described by Akinduko et al., (2014) with little modifications.

101 Principle

The ability of superoxide dismutase (SOD) to inhibit the auto oxidation of adrenaline at pH 10.2 makes this reaction a basis for the SOD assay. Superoxide anion (O2) generated by the xanthine oxidase reaction is known to cause the oxidation of adrenaline to adrenochrome. The yield of adrenochrome produced per superoxide anion introduced increased with increasing pH (Valerino and McCormack, 1971) and also with increasing concentration of adrenaline. These led to the proposal that auto oxidation of adrenaline proceeds by at least two distinct pathways, one of which is a free radical chain reaction involving superoxide radical and hence could be inhibited by SOD.

Procedure

Four test tubes were set up and labeled test, standard, blank and control respectively. 20 microlitre (µl) of serum, standard, distill water and control were added into the test tubes respectively.250µl of 0.05M carbonate buffer (pH 10.2) was added to all the test tubes and allowed to equilibrate at 37 ⁰C for five minutes. 30µl of 0.3mM freshly prepared epinephrine was added to all the test tubes. Absorbance of the tests were read after 30s at 30s interval for 150s at 480 nm against the reagent blank.

Calculation:

Actual OD reading (R) = OD150 – OD30/2

% inhibition = (Rblank – Rtest / Rblank) X 100

Enzyme Unit (U/ml) = (% inhibition/50) X dilution factor ii. Determination of Catalase Activity

Catalase activity was estimated by the method of (Sinha, 1972).

Principle

This method is based on the fact that dichromate in acetic acid is reduced to chromic acetate when heated in the presence of H2O2 with the formation of perchromic acid as an unstable intermediate. The chromic acetate then produced is measured colorimetrically at 570 to 610nm.

Since dichromate has no absorbance in this region, the presence of the compound in the assay mixture does not interfere at all with the colorimetric determination of chromic acetate. The catalase preparation is allowed to split H2O2 for different periods of time. The reaction is stopped at a particular time by the addition of dichromate acetic acid mixture and the remaining H2O2 is determined by measuring chromic acetate colorimetrically after heating the reaction mixture.

102 Procedure

Four test tubes were set up and labeled test, standard, blank and control respectively.1.2ml of 0.2M hydrogen peroxide and 3ml of 0.01M phosphate buffer (pH 7.0) were added into the test tubes respectively. Then 0.3ml of serum, standard, distill water and control were added to the test tubes respectively. The reagents were gently mixed and incubated at RT, 1 ml portion of the reaction mixture was withdrawn and blown into 2 ml dichromate/acetic acid reagent at one minute interval. Mixed and incubated in a boiling water for 10 minutes, cooled and tests absorbance were read at 570 nm.

Calculation:

Change in OD (R) = OD1 – OD2or3/t

The amount of H2O2 degraded in (umol) is extrapolated from the already prepared H2O2 standard curve using the value of R..

Procedure for the Preparation of H2O2 Standard Curve

Six test tubes were set up and labeled 1-6 respectively. 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 millilitres (mls) of 0.019 M H2O2 were added respectively to the test tubes while 0.9, 0.8, 0.7,0.6,0.5 and 0.4 mls of water (H2O) were added respectively to the test tubes, 2mls of potassium dichromate (K2Cr2O7) was added to all the test tubes and H2O2 concentrations of 20,40,60,80,100,and 120 in the test tubes were obtained respectively. The absorbances of the tests were read at 570nm against the reagent blank and recorded. The H2O2 Standard Curve was obtained by plotting a graph of the absorbances against the concentrations.

iii. Determination of Glutathione peroxidase (GPX, E.C. 1.11.1.9).

The activity of glutathione peroxidase was determined by the method of Rotruck et al., (1973) with slight modifications.

Principle

A known amount of enzyme preparation was allowed to react with H2O2 in the presence of GSH for a specific time period. The GSH content remaining after the reaction was measured by the methods of Beutler et al., (1963). 2GSH + H2O2 = GSSG + 2 H2O

Procedure

The reaction mixture in a total volume of 1 ml contained 0.2 ml of phosphate buffer, 0.2 ml EDTA, 0.1 ml sodium azide and 0.5 ml of the enzyme preparation (tissue homogenate/ plasma/

hemolysate). 0.2 ml of glutathione and 0.1 ml of H2O2 were added to this mixture and incubated

103

at 37°C for 10 minutes. The reaction was arrested by the addition of 0.5 ml of 10% TCA. The tubes were centrifuged and the supernatant was assayed for GSH by the method of Beutler and Kelley. A blank was treated similarly to which 0.2 ml of enzyme was added after incubation.

The activity of glutathione peroxidase was expressed as U/l of plasma (U- mmoles of GSH utilised/ minute) and recorded.

iv. Evaluation of Glutathione Reductase

Glutathione Reductase activity is evaluated by colorimetric method as described by Anderson and Davis, (2004).

Principle

Glutathione Reductase catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH), which plays an important role in the GSH redox cycle that maintains adequate levels of reduced GSH. A high GSH/GSSG ratio is essential for protection against oxidative stress.

Procedure

All reagents, standard solutions, and samples were prepared as instructed and brought to room temperature before use. Samples were treated to destroy GSH before the assay. 100 µl sample and 5 µl 3% H2O2 were added, mixed and incubated at 25°C for 5 min. Then 5 µl of catalase was added, mixed and incubated at 25°C for another 5 min. 2 -50 µl of the pretreated samples were added into a 96-well plate, the volume was brought to 50 µl with Assay Buffer. Several doses of the sample was added to make sure the readings were within the standard curve range. 10 µl /well Positive Control was used (optional) and adjusted to 50 µl with Assay Buffer.

TNB standard curve: 0, 2, 4, 6, 8, 10 µl of the TNB Standard was added into 96-well plate in duplicate to generate 0, 10, 20, 30, 40, 50 nmol/well standard. The final volume was brought to 100 µl with Assay Buffer. 50 µl of the Reaction Mix was added to each test samples. Mixed well. OD was measured at 405 nm at T1 (reading A1). The reaction was incubated at 25°C for 10 min (or incubated longer time if the GR activity was low), protected from light, OD was measured at 405 nm again at T2 (reading A2). ΔA405 nm = A2 – A1. Note: A1 and A2 were essentially read in the reaction linear range. Calculation: TNB standard Curve was plotted.

ΔA405nm was applied to the TNB standard curve to get ΔB nmol of TNB (TNB amount generated between T1 and T2 in the reaction wells).

GR Activity = ( ) sample dilution factor = nmol/min/ml = mU/mL

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Where: ΔB is the TNB amount from TNB standard Curve (in nmol).

T1 is the time of the first reading (A1) (in min).

T2 is the time of the second reading (A2) (in min).

V is the pretreated sample volume added into the reaction well (in ml).

0.9 is the sample volume change factor during sample pre-treatment procedure.

Unit definition: One unit is defined as the amount of enzyme that generates 1.0 µmol of TNB per minute at 25°C. The oxidation of 1 mole of NADPH to NADP+ generated 2 mole TNB finally, therefore, 1 TNB unit equals 0.5 NADP unit.