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Article Biochemical Study of Oxidative Stress Markers in the Liver, ...

Spectrophotometric determination of tissues MDA, PCO, and GSH levels

MDA

This method depends on the formation of MDA as an end product of lipid peroxidation which reacts with thiobarbituric acid producing thiobarbituric acid reactive substance (TBARS), a pink chromogen, which can be measured spectrophotometrically at 532 nm, an MDA standard was used to construct a standard curve against which readings of the samples were plotted [22].

PCO

This method depends on the formation of a Schiff base from the reaction of dinitrophenylhydrazine with protein carbonyls to form protein hydrazones which was measured spectrophotometrically. Briefly, after precipitation of protein with an equal volume of 1% trichloroacetic acid, the pellet was resuspended in 10 mmol/L DNPH plus 2N HCl, or in 2N HCl as a control blank. Next, after the washing procedure with 1:1 ethanol-ethylacetate, the final palette was dissolved in 6 mol/L guanidine. The carbonyl group was determined from the absorbance at 370 nm. The carbonyl content was calculated in terms of nmol/mg protein [23,24].

GSH

The method is based on the reduction of 5,5 dithiobis (2-nitrobenzoic acid) (DTNB) with reduced glutathione (GSH) to produce a yellow compound. The reduced chromogen is directly proportional to GSH concentration and its absorbance can be measured at 405 nm by using a commercial kit was used (Biodiagnostic, Egypt) [25].

Enzymatic Assays

Tissues GST enzyme activity: it measures the conjugation of 1-chloro-2,4-dinitro benzene (CDNB) with reduced glutathione that produces a dinitrophenyl thioether which can be detected by spectrophotometer at 340 nm. One unit of GST activity is defined as the amount of enzyme producing 1 mmol of CDNB-GSH conjugate/min under the conditions of the assay according to the method described by Habig et al. [26].

Tissues GPx enzyme activity: it was measured as IU/gm wet tissue by the reaction between glutathione remaining after the action of GPx and 5, 5-dithiobis-(2-nitrobenzoic acid) to form a complex that absorbs maximally at 412 nm. Glutathione peroxidase activity of 1 U/mg protein was defined as 1 μg of GSH consumed/min/mg protein [27].

Determination of tissues CAT enzyme activity: it assayed by the method of Sinha which based on formation of chromic acetate from dichromate and glacial acetic acid in presence hydrogen peroxide, chromic acetate that produced was measures colorimetrically at 570 nm, one enzyme unit was defined as the amount of enzyme which catalyzed the oxidation of 1 μmole H2O2 per minute under assay conditions [28].

Tissues PON1 enzyme activity: PON1 activity towards paraoxon (O,O-diethyl-O-p-nitrophenyl phosphate) was determined by measuring the initial rate of substrate hydrolysis to p- nitrophenol, whose absorbance was monitored at 405 nm in the assay mixture, A PON1 activity of 1 U/mg protein was defined as 1 μmol p-nitrophenol formed per minute per mg protein.

Article Approaches and Methods to Measure Oxidative Stress in Clinic...

@ Hildegunn Dahl ,

Your choice of TBARS is good. Definitely, it is very strong support when you also include the Protein Carbonyl assay since your oxidative stress site is the liver. I used ELISA kits in my previous OS research. I herein include the file for your easy reference. I hope you find it helpful.

All best luck!

We always use commercial kits for TBARS, they are pretty accurate (Cayman and Sigma is my best). You can also measure MDA levels by HPLC too but you should be careful about your tissue homogenate is suitable. The colon may become blocked.

Additionally you may think about antioxidant enzymes activity such as SOD, CAT or GPx since their activity changes in case of oxidative stress.