Biochemical Markers

1. OSI Protocol
Oxidative stress index (OSI) is a measurement of Total Antioxidant Capacity (TAC) and
Total Peroxides (TP). For determination of OSI, plasma was divided into 2 parts. One was
subjected for Total Anti-oxidant Capacity (TAC) measurement and other was used for Total
Peroxides (TP) measurement.
Total Anti-oxidant Capacity Measurement
Principle
Total antioxidant capacity was measured through FRAP assay. FRAP assay is based on
principle that at low pH, Ferric Tripyridyl Triazine (Fe III TPTZ) get reduced to ferrous
Tripyridyl Triazine and develop intense blue colour (Benzie & Strain, 1996).
Table 3.2 Reagents for FRAP Assay
Sr. # Case Number Reagent Company
1 7782-63-0 Ferrous Sulphate Merk K GaA
2 10025-77-1 Ferric Chloride Sigma Aldrich
3 64-19-7 Glacial Acetic Acid Merk K GaA
4 7647-01-0 Hydrochloric Acid BDH-Chem
5 127-09-3 Sodium Acetate Sigma Aldrich
6 3682-35-7 2,4,6-tri-pyridyl-s-triazine Glentham Life Sciences

Procedure
Preparation of 300mM Acetate Buffer
16ml of glacial acetic acid (C₂H₄O₂) and 3.1 gram of sodium acetate (CH₃COONa)
were mixed in 1000ml of distilled water. Solution was stored at 4 centigrade after adjusting pH
to 3.6.
Preparation of 40mM dilute HCl
1.46ml of conc. HCl was dissolved in 1000ml of distil water. Solution was stored at room
temperature.
Preparation of 10mM TPTZ
0.031 grams of TPTZ (2, 4, 6-tri-pyridyl-s-triazine) was mixed in 10ml of HCl in
corning tube. Suspension was placed in water bath at 50 centigrade.
Preparation of 20mM Ferric chloride
0.054 grams of ferric chloride (FeCl₃.6H₂O) was dissolved in 10ml of distilled water.
Solution was made on the day of assay.
Standard solution of 1mM Ferrous Sulphate
For preparation of 1mM of Ferrous sulphate (FeSO₄.7H₂O), 0.278 grams were mixed in
1 litre of distilled water and used as standard solution.
By mixing 1 ml of 10mM TPTZ in 4omM HCl, 10 ml of 300mM acetate buffer and 1ml
of 20mM ferric chloride, FRAP reagent was prepared. Before starting reaction, 0.1ml of plasma
and 0.9ml of FRAP reagent were incubated at 30 centigrade for 5 minutes. Reaction mixture
(1ml) contain;
0.1mlof plasma
0.9ml of FRAP reagent
Standard solution of ferrous sulphate was used as blank and absorbance was recorded at 630 nm
wavelength through spectrophotometer.
Calculation
Total Antioxidant Enzyme (µmol/L) = Test Absorbance /Standard Absorbance×STD conc.
(1000)
Total Peroxides Measurement
Principle
Total peroxides were measured by FOX 2 method. Principle depends upon the ability of
ferrous ion get oxidized to ferric ion due to presence of peroxides in plasma and form ferric-
xylenol complex and produce characteristic orange colour (Nourooz-Zadeh, 1999).
Table 3.3 Reagents for Total peroxides
Sr. # Case Number Reagent Company
1 7783-85-9 Ammonium Ferrous Sulphate Sigma Aldrich
2 7664-93-9 Sulphuric Acid Riedel-de-haen
3 128-37-0 Butylated Hydroxyl Toulene Sigma Aldrich
4 3618-43-7 Xylenol Orange Sigma Aldrich

Preparation of 25mM H₂SO₄

 25mM H₂SO₄ was prepared by dissolving 12.5ml H₂SO₄, making volume of 100ml by
adding distilled water.
Preparation of 250mM Ammonium ferrous sulphate and
9.8mg of ammonium ferrous sulphate [(NH₄)₂Fe(SO₄).6H₂O] was added in 10ml of
25mM H₂SO₄ making a final concentration of 250mM ferrous iron in acid.
Preparation of 4nM BHT in 90% methanol
For making solution of ion of 4nM BHT [(CH₃)₃C] ₂C₆H₂ (CH₃) OH, 79.2 mg of BHT
in 90ml HPLC grade methanol.
FOX reagent was prepared by 250µM ammonium ferrous sulphate in to 250 mM H2SO4.
4nM BHT solution this solution and stirred well. Then 100µM xylene orange (7.3mg) was added
making a final volume of 100ml.
1800µL FOX 2 reagent and 200μL of plasma were incubated in cuvette for 30 minutes
and centrifuged at the speed of 12000 rpm for 10 minutes. FOX2 reagent excluding ferrous
sulphate was used as blank. Absorbance of supernatant was measured at 560 nm wavelength.
Calculation
Absorbance of standard solution of H₂O₂ having concentration of 5, 10, 15, 20, 25 and
30 µmol/L was measured through spectrophotometer at wavelength of 560nm. Slop equation was
calculated by plotting graph against y-intercept.
Total peroxides = sample Absorbance – Y intercept/Slope
Where Y-intercept is 0.0153 and slope is 0.023
Calculation of OSI
Oxidative stress index is calculated as follow;
OSI= [Total Peroxides (TP) × 100]/Total Antioxidant Capacity (TAC)
 2. Superoxide Dismutase
Principal
Principle is based on inhibition of Nitro blue tetrazole (NBT) by superoxide dismutase
(Beauchamp & Fridovich, 1971).

Table 3.4 Reagents for SOD

Sr. # Case Number Reagent Company
1 7778-77-0 Potassium Dihydrogen Phosphate Sigma Aldrich
2 143-33-9 Sodium Cyanide Sigma Aldrich
3 215300662-2 Nitro Blue Tetrazolium (NBT) Bio PLUS
4 83-88-5 Riboflavin Sigma Aldrich
5 16788-57-1 Dipotassium Hydrogen Phosphate Sigma Aldrich
6 6381-92-6 Ethylene Diamine tetra Acetic Acid Sigma Aldrich

Procedure
Preparation of 1.5mM NBT solution
3.23mg of NBT was taken in flask and volume was raised up to 2.64ml by adding
distilled water. Solution was stored in dark cold bottle.
Preparation of 0.12Mm riboflavin solution
0.06mg of riboflavin was put in flask and volume was raised up to 1.3ml by adding
distilled water. Solution was stored in cold dark bottle.
Preparation of 0.1M NaCN/EDTA solution
0.08 mg of NaCN and 0.16g of EDTA were taken in flask and volume was raised up to
5.4ml by adding distilled water.
Preparation of 0.067 M potassium phosphate buffer (pH 7.8)
0.74g of dibasic salt (K2HPO4) and 0.14g of monobasic salt (KH2PO4) were taken in
flask. Volume was raised up to 80ml by adding distilled water and pH was adjusted to 7.8.
3 ml of buffer was added in cuvette as a blank and its absorbance was noted after placing
it in spectrophotometer. Spectrophotometer was adjusted at zero at wavelength of 560nm after
taking blank reading. 5-6 cuvette were placed in light box with internally mounted 30 watt light
bulb.0.016ml of riboflavin and 0.05 ml enzyme extract was added in each tube. For 12 minutes,
all cuvettes were incubated in light box. After that 0.033 ml of NBT and 0.067ml of
EDTA/NaCN solution was added in illuminated reaction mixture and cuvettes were transferred
to spectrophotometer.
After 20 seconds of reaction, absorbance was noted. Superoxide Dismutase activity was
determined by measuring %age inhibition of NBT (Figure 3.6).

Figure. 3.6 Incubation of Reaction Mixture

Calculation
%age Inhibition = Blank Absorbance – Sample Absorbamce×100 / Blank Absorbance

SOD (U) = 1/50×Percentage

SOD (U/L) = SOD (U) ×Total volume of reaction mixture/plasma volume
 3. Catalase
Principal
Principle is based on ability of Catalase to lower the H₂O₂ concentration at wavelength
of 240 nm (Chance & Maehly, 1955).

Table 3.5 Reagents for Catalase

Sr. # Case Number Reagent Company
1 10028-24-7 Disodium Hydrogen Phosphate Sigma Aldrich
2 13472-35-0 Sodium Dihydrongen Phosphate Sigma Aldrich

Procedure
10mM Hydrogen peroxide and 50mM sodium phosphate buffer (pH = 7.0) are required
for reaction mixture
Preparation of phosphate buffer (50mM)
0.270 gram of dibasic salt (Na2HPO4) and 0.371 gram of monobasic salt (NaH2PO4)
was taken in flask. Distilled water was added, making a total volume of 100ml and pH was 7.0.
Preparation buffer substrate solution
0.442ml of 10mM H2O2 was dissolved in 50mM phosphate buffer solution making a
buffer substrate solution. Reaction mixture (2ml) contains;
 Enzyme extract 0.05ml
 Buffered substrate solution 1.95ml
 Blank 60mM phosphate buffer used as blank
2ml of blank solution was taken in cuvette and placed in spectrophotometer, setting it to
zero at 240nm wavelength. 1.95ml of buffered substrate and 0.05ml of enzyme extract were
taken in cuvette and placed the cuvette in spectrophotometer. Reaction time was 3 minutes and
absorbance was noted after 1 minute interval (Figure 3.7).
 Figure. 3.7 Catalase determination through UV Spectrophotometer

Calculation
Activity (U/ml) = ΔA / min × Dilution × 2ml / 0.04mM1‫ ־‬cm-1 × 0.05ml

4. Lipid peroxidation
Principle
MDA, a lipid peroxidation product, forms orange to pink colour product when heated
with Thiobarbituric acid (TBA), whose absorbance is measured at 535nm (Buege & Aust, 1978).

Table 3.6 Reagents for Lipid Peroxidation

Sr. # Case Number Reagent Company
1 76-03-9 Tri Chloroacetic Acid (TCA) SRL-Chem
2 504-17-6 Thiobarbituric Acid (TBA) UMI-Chem
3 7647-01-0 Hydrochloric acid BDH

Procedure
Stock solution of 15 grams TCA, 0.375 grams TBA and 875ul HCl was prepared. 2ml of
stock solution and 0.1 ml of enzyme source were taken in eppendrops and heated at 100
centigrade for 15 minutes in water bath. After heating, solution was allowed to cool and then
centrifuged at 3600 rpm for 10 minutes. Orange to pink coloured supernatant was observed.
 3 ml of stock solution was taken in cuvette, placed in spectrophotometer and set it to zero
at wavelength of 535 nm. Absorbance of orange to pink supernatant was read at 535nm.

Figure 3.8 Sample preparation and centrifugation

Calculation
Concentration of MDA was measured using following formula;
Absorbance = Extinction coefficient × 2 × Activity
Activity (mM) = Absorbance / Ɛ × 2
Ɛ= extinction coefficient (Ɛ = 156mM¹‫־‬cm¹‫)־‬

5. Glutathione Peroxidase
Principle
Principle is based on the activity of glutathione peroxidase to utilize NADPH during
conversion of lipid peroxides and H₂O₂ at wavelength of 340nm (Paglia & Valentine, 1967).
Table 3.7 Reagents for Glutathione peroxidase
Sr. # Case Number Reagent Company
1 7722-84-1 Hydrogen Peroxide Merck K GaA
2 26628-22-8 Sodium Azide Daejung-Chem
3 27025-41-8 Glutathione oxidized Oakwood-Chem
4 6381-92-6 Ethylene diamine tetra acetic acid Sigma Aldrich
(EDTA)
5 16788-57-1 Dipotassium Hydrogen Phosphate Sigma Aldrich
 6 2646-71-1 Nicotinamide adenine dinucleotide Chem-impex int’l
phosphate
7 7778-77-0 Potassium Dihydrogen Phosphate Sigma Aldrich
8 70-18-8 Reduced Glutathione BDH-chem VWR

Preparation of 50mM Potassium Phosphate Buffer

0.570 grams of dibasic potassium phosphate (K₂HPO₄) and 0.342 grams of monobasic
potassium phosphate (KH₂PO₄) was added in 100ml of distilled water and pH was adjusted to
7.0.
Preparation of 1mM EDTA
0.3722 grams of EDTA was added in 100ml of distilled water making a solution of 1mM.
Preparation of 1Mm Sodium Azide (NaN₃)
0.0065 grams of sodium azide was added in 100ml of distilled water.
Preparation of 0.2mM NADPH
For making 0.2mM NADPH solution, 0.0164 grams of NADPH was added in 100ml of
distilled water.
Preparation of 1 EU/ml Glutathione Reductase (GR)
0.2mg glutathione reductase was dissolved in 1litre of distilled water.
Preparation of 1mM Glutathione oxidase (GSH)
For preparation of glutathione oxidase 0.031 grams of respective chemical was added in
100ml of distilled water.
Preparation of 0.25mM H₂O₂
0.025ml of H₂O₂ were dissolved in 100ml of distilled water.
100µl of 0.25mM H₂O₂, 100µl of GSH, 4µl of GR, 100µl of EDTA, 566µl of potassium
phosphate buffer and 10µl of NaN₃ were added in cuvette. This blank solution was placed in
spectrophotometer and its absorbance was measured at wavelength of 340nm.
100µl of plasma and 800µl of solution were added in cuvette and incubate it for 5
minutes at room temperature. After that H₂O₂ was added and absorbance was measured.

Calculation
GPx value was calculated as follow;
 GPx (mU/mL) = A₃₄₀ per minute/ε
Where ε = 6.02×10ˉ³ Mˉcmˉ