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Molecular Journal

In this assay, a water soluble tetrazolium salt WST is converted to a formazan dye WST formazan by O, which is measurable at nm. SOD reduces the level of O by dismutation of it into HO and O, and thereby lowers the rate of WST formazan.In contrast, any reduction in SOD activity will result in an increase in the signal of WST formazan.This assay has been used to measure ROS activity under many different conditions, such as in amyloid precursor protein transgenic mice and diabetic mouse models. Glutathione reductase assay is an approach to determine activity of GR.GR is a ubiquitous enzyme that catalyzes the reduction of oxidized glutathione, which is essential for maintenance of adequate buy Adapalene levels of reduced cellular GSH for antioxidant reaction.This assay utilizes enzymaticrecycling method for measurement of activity of glutathione reductase.The yield of glutathione interacts with DTNB and generated a yellowcoloreed thionitrobenzoic acid, which is absorbed at nm.The activity of glutathione reductase could be suppressed in certain abnormal conditions, such as from side effects of therapeutic drugs. It reduces lipid hydroperoxides to their corresponding alcohols and catalyzes the reduction of HO to HO while converting GSH to GSSG.GSSG is reduced back to GSH by glutathione reductase.In this process, NADPH is oxidized to NADP, which has absorbency at nm with a spectrophotometer. Thus, this assay is used to indirectly measure the activity of GP.However, the activity of GR theoretically affects the outcome of this assay since it is involved in the reactions.Catalase assay is a way to measure activity of CAT.Catalase is a ubiquitous antioxidant enzyme that is present in nearly all living organisms.It functions to catalyze the decomposition of HO to HO and O.The peroxidatic function of CAT can be used for determination of its enzyme activity.It becomes colorless or pale yellow when neutralized with antioxidants.In the cellfree system, this method is readily used to distinguish whether an agent that suppresses ROS in an in vitro or an in vivo system is a free radical scavenger or indirectly affect ROS level in the cells. The assay has been used for determination of NO scavenging activity of plant. Immunocytochemistry assay is a convenient way to determine the existence of HNE under oxidative stress conditions. HPLC is also utilized for determination of protein oxidation products generated by direct oxidation of amino acid residues or nitration of tyrosine. HPLC with electrochemical detection is the most widely used method for quantitative analysis of these molecules. RNA oxidation products oxo,dihydroguanosine can be also determined using HPLC coupled to electrochemical detection. Both nitrotyrosine and proteinassociated nitrotyrosine can be quantified with gas chromatographytandem mass spectrometry. ROS are generated at different locations in the cell, especially in the mitochondria.There are both enzymatic and nonenzymatic systems to maintain constant ROS levels.In contrast, high levels of ROS, either through over production or restricted elimination, puts a living organism under an oxidative stress condition.Under oxidative stress, excessive ROS abnormally modify all biomacromolecules, causing lipid peroxidation, protein misfolding and aggregation, RNA mistranscription, and DNA damage and mutations.Among these damages, the role of ROS in the oxidation of hallmark proteins of neurodegenerative disease is of special interest, since this episode bridges nonspecific cytotoxicity of ROS to a given neurodegenerative disorder.

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