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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               At the end of various treatments, blood was withdrawn from the retro-orbital plexus of a mouse eye in an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
                                                                                                         -1
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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
                                                                       -1
                                                                          -1
               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
                                                                                      -1
                                                                                          -1
               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 .
                                                                                                  -1
                                                                                              -1
               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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