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Bhatia et al. Hepatoma Res 2018;4:9 I http://dx.doi.org/10.20517/2394-5079.2018.04 Page 5 of 16

Assessment of inflammatory markers
The quantitative estimation of various inflammatory markers including tumor necrosis factor (TNF)-α,
interleukin (IL)-1β and IL-6 was carried out in serum using a commercially available kit by solid phase enzyme
linked immunosorbent assay according to the instruction provided by the manufacturer (RayBiotech, Inc., USA).

Assessment of antioxidant defense system
Preparation of blood plasma
At the end of various treatments, blood was withdrawn from the retro-orbital plexus of a mouse eye in an
eppendorf containing EDTA as an anticoagulant. This was followed by the centrifugation of blood samples
at 3000 rpm for 15 min at 4 °C. The supernatant (plasma) thus obtained was used for the estimation of
various oxidative stress markers.

Lipid peroxidation
Lipid peroxidation (LPO) levels were measured in plasma as per the method described by Trush et al. . It is
[27]
based on the reaction of malondialdehyde (MDA) and thiobarbituric acid (TBA) to form pink colored MDA-
TBA complex which has its maximum absorption intensity at 532 nm. The amount of chromophore thus
obtained was measured as an index of lipid peroxidation using an extinction coefficient of 1.56 × 10 M cm
5
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and expressed as nanomoles of MDA-TBA chromophore formed/min/mg protein.
Reduced glutathione
The plasma levels of reduced glutathione (GSH) were estimated as a total non-protein sulphydryl compound
according to the method of Moron et al. . It involves the reduction of a 5,5’-dithiobis-2-nitrobenzoic acid
[28]
by the -SH group of reduced GSH to produce a yellow colored 2-nitro-5-mercaptobenzoic acid. The optical
density of the compound thus produced was measured spectrophotometrically at 412 nm and expressed as
nanomoles of GSH/mg protein.

Glutathione-S-transferase
Plasma activity of glutathione-S-transferase (GST) was estimated using a method of Habig et al. . GST
[29]
aids in the coupling of GSH with a substrate, i.e., 1-chloro-2, 4 dinitrobenzene (CDNB). The absorbance
of chromophore thus formed was read at 340 nm and described as micromoles of GSH-CDNB conjugates
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formed/min/mg protein using an extinction coefficient of 9.6 mM cm .
GSH peroxidase
Plasma GSH-peroxidase (Px) activity was assayed as per the method given by Paglia and Valentine . It
[30]
catalyzes the production of GSSG from GSH with the simultaneous oxidation of NADPH. The change in
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optical density was read at 340 nm based on an extinction coefficient of 6.22 mM cm and expressed as
nanomoles of NADPH consumed/min/mg protein.
Glutathione reductase
Plasma glutathione reductase (GR) activity was estimated according to the method of Williams and
[31]
Arscott . GR causes the reduction of GSSG to GSH using NADPH as a reducing agent with the simultaneous
conversion of FAD to FADH . The change in optical density was monitored at 340 nm and calculated as
-
nanomoles of NADPH consumed/min/mg protein using an extinction coefficient of 6.22 mM cm .
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Catalase
Catalase (CAT) activity was determined in plasma using the method of Luck . CAT assists in the breakdown
[32]
of hydrogen peroxide to produce water and molecular oxygen. The activity was assayed at 240 nm and
measured as international units (IU)/mg protein based on the extinction coefficient of 0.0394 mM cm .
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