10-Apr-20
• It has been found that the primary oxidation products are further
MEASURING OXIDATIVE
STRESS
Prof. Dr. M. Tariq Javed
Chairman
Department of Pathology,
Faculty of Veterinary Science,
University of Agriculture, Faisalabad, Pakistan.
http://www.geocities.ws/mtjaved/
mtjaved@uaf.edu.pk
• Due to the reactivity of TBA with several reactive substances in the
biological sample, a more widely accepted terminology called
thiobarbituric acid reactive substances (TBARS) is now commonly used
• TBARS is now considered as a standard marker for the lipid peroxidation
induced oxidative stress.
• Several HPLC methods are available regarding the TBARS assay However,
due to the high cost and long operation timing of the HPLC procedures, a
more simple method is therefore required.
• Botsoglou et al. developed a spectrometric method for the TBARS as
marker for lipid oxidation in animal tissues, food, and feed substances.
• However, the method involves 1,1,3,3-tetraethoxypropane (TEP) as MDA
precursor, the hydrolysis of which would need a trained analytical chemist
oxidized to form secondary and tertiary oxidation products.
• These products include a high amount of aldehydes with small to
large chain structures
• The aldehydes and other reactive substances are one of the main
causes of rancidity in foods during preparation and storage
• One of the important oxidation products is known as
malondialdehyde (MDA), which is considered as the main marker in
lipid peroxidation.
• Thiobarbituric acid (TBA) is reacted with MDA, which is resulting in a
colour compound, which can be determined spectrophotometrically,
chromatographically, or through image processing techniques
• Recently Papastergiadis et al. developed a spectrophotometric method for
analysis of MDA in oxidized foods.
• Their method was comparable with HPLC method
• Here we are going to learn about a simple and highly sensitive
spectrometric method for the analysis of TBA reactive substances in the
tissues.
• GSH is intracellular antioxidant defense participating in the neutralization
of free radicals and is regenerated using glutathione reductase.
• However, in severe oxidative stress this compensatory mechanism to
restore the redox status of the cell fails as evidenced by a decrease in GSH
levels.
• A significant increase in left-ventricular TBARS and SAG and reduction in
GSH indicate oxidative stress
1

Preparation of TBA Reagent:
• The standard solution of 4.0 mM of TBA is prepared in glacial acetic
acid.
• For this purpose, 57.66 mg of TBA is dissolved in 100 mL of glacial
acetic acid.
• Fresh solution of TBA is needed.
Preparation of MDA and Calibration Standards.
• Standard stock solution of MDA (1 mM) was prepared in glacial acetic
acid.
Extraction of TBARS in Fried Samples.
• One gram of each fried grinded sample is taken in 25 mL test tube
and 5 mL of the solvent.
• The solvent is either 100% glacial acetic acid (AA) or 50% glacial acetic
acid in water (AW).
• BHT (0.01%) is used to prevent further oxidation of the medium.
• The samples were shaken for 1 h and filtered.
• The filtrate was centrifuged, when required, and was used for
analyses
10-Apr-20
• MDA (31.35 mg) is weighed and dissolved in 100 mL solvent.
• From the stock solution, different concentrations of 0.1, 0.2, 0.4, 0.6,
and 0.8 mM are prepared.
• The calibration curve is constructed in the concentration range of 0.1
to 1.0 mM.
Analytical Procedure.
• The standard MDA solution (1 mL) is taken in a 10 mL test tube and
mixed with TBA (1 mL).
• The mixture is heated in a boiling water bath at 95∘C for 60 minutes.
• The test tubes are cooled at room temperature and absorbance is
measured at 532 nm using UV-visible spectrophotometer
• Each standard for the calibration is repeated (𝑛 = 3) according to the
above procedure.
• A blank sample is repeated (𝑛 = 5) replacing standard or sample by
acetic acid or water
2

• Two different kinds of samples extracts are prepared, that is, with
100% glacial acetic acid (AA) and 50% glacial acetic acid with water
(AW).
• The extract of each sample (1 mL) is mixed with 1 mL TBA reagent and
the above procedure was repeated five times (𝑛 = 5).
• The TBARS is calculated using the formula as 𝜇M/g of the sample:
TBARS (𝜇M/g) = (Ac × 𝑉) /𝑊
where Ac is the amount determined from the calibration curve and 𝑊 is the weight
of the sample taken, while 𝑉 is volume in mL or dilution factor of the total extract
prepared.
• A standard calibration curve is prepared using 1–10 nM 1,1,3,3-
tetramethoxy propane.
• The TBARS value is expressed as nanomoles per milligram of protein.
10-Apr-20
ANOTHER METHOD
• 0.2 ml of homogenate of tissue is pipetted into a test tube,
• Add 0.2 ml of 8.1% sodium dodecyl sulfate, 1.5 ml of 30% acetic acid
(pH 3.5), and 1.5 ml of 0.8% of thiobarbituric acid and the volume is
made up to 4 ml with distilled water.
• The test tubes were incubated for 1 h at 95 °C,
• then cooled, and 1 ml of distilled water is added,
• The add 5 ml of n-butanol:pyridine mixture (15:1, v/v).
• The tubes are centrifuged at 4000 g for 10 min.
• The absorbance of the developed color was measured at 532 nm.
Estimation of superoxide anion generation (SAG)
• The tissue SAG is estimated in terms of reduced nitroblue tetrazolium
(NBT)
• Weighed amounts of tissue is taken in phosphate-buffered saline
containing NBT and incubate at 37 °C for 1.5 h, and NBT reduction is
stopped by adding hydrochloric acid.
• The samples are homogenized in a mixture of 0.1 M sodium
hydroxide and 0.1% sodium dodecyl sulfate in water containing 40
mg/L diethylenetriaminepentaacetic acid and centrifuged at 20,000 g
for 20 min.
• The resultant pellets were suspended in 1.5 ml of pyridine and kept at
80 °C for 1.5 h to extract formazan.
3
 10-Apr-20

Estimation of reduced glutathione (GSH)

• The resultant pellets are suspended in 1.5 ml of pyridine and kept at
80 °C for 1.5 h to extract formazan. • The GSH in tissue is estimated using an established method

• The mixtures are again centrifuged at 10,000 g for 10 min and • The supernatant of the homogenate is mixed with trichloroacetic acid
absorbance of formazan was measured at 540 nm. and centrifuged at 1000 g for 10 min at 4 °C

• The amount of reduced NBT was calculated using the formula
amount of reduced NBT = A × V = T × W × s × l;
• where A is absorbance, V is volume of pyridine (1.5 ml), T is time the tissue was
incubated with NBT (1.5 h), W is blotted wet weight of the tissue (25 mg), ε is the
extinction coefficient (0.72 L/mmol/mm), and l is length of light path (1 cm).
• Results are expressed as reduced NBT picomoles per minute per milligram of wet
tissue.
• The resulting supernatant is mixed with 0.3 M disodium hydrogen
phosphate
• Then add 0.001 M freshly prepared 5,5′-dithiobis-2- nitrobenzoic acid
dissolved in 1% w/v citric acid
• absorbance is noted at 412 nm.
• A standard curve is plotted using 5–50 μM reduced-form glutathione
and results were expressed as micromoles of GSH per milligram of
protein.

Sources
• Alam Zeb and Fareed Ullah, A Simple Spectrophotometric Method for
the Determination of Thiobarbituric Acid Reactive Substances in Fried
Fast Foods. Journal of Analytical Methods in Chemistry Volume 2016,
Article ID 9412767, 5 pages http://dx.doi.org/10.1155/2016/9412767
• Impact of obesity on hypertension-induced cardiac remodeling: role
of oxidative stress and its modulation by gemfibrozil treatment in rats.
Randhir Singh a, Amrit Pal Singh a,b,⁎, Manjeet Singh a,†, Pawan
Krishan, Free Radical Biology & Medicine 50 (2011) 363–370