All tags Metabolism Oxidative stress assays and oxidative stress markers

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Oxidative stress assays and oxidative stress markers

Learn about markers including ROS, MDA, GSH:GSSG, and more. Review assays for use with tissues, cells, and biofluids.

Oxidative stress reflects the toxic side of oxygen and metabolism. Oxidative stress has been defined as “An imbalance between oxidants and antioxidants in favor of the oxidants, leading to a disruption of redox signaling and control and/or molecular damage” (Sies and Jones, 2007).

Oxidative stress markers fall into three main classes:
- Reactive oxygen species (ROS)
- DNA/RNA, lipids and proteins that have been damaged by oxidation
- Antioxidants

For these three classes:
- ROS represent the agents that transmit oxidative stress and damage components of the cell
- DNA/RNA damage, lipid peroxidation, and protein oxidation/nitration represent the damage caused by oxidation
- antioxidants represent the systems for managing oxidative stress

Reactive oxygen species (ROS)

Reactive oxygen species (ROS) are reactive chemical species containing oxygen. They include peroxides, superoxide, hydroxyl radicals, singlet oxygen, and alpha-oxygen.

Due to their transient nature, they are easily measured in live cells using fluorescent dye-based assays, such as with DCFDA. They are harder to measure in tissue and biofluid samples.


Assay

Readout

Assay kits

DCFDA – cellular reactive oxygen species

Flow cytometry, plate reader

ab113851

Cellular superoxides

Cellular ROS/Superoxide

Microscope, flow cytometry

ab139477

ab139476

Cellular reactive oxygen species

Plate reader

ab186027, ab186028, ab186029

Cellular ROS/RNS

Microscope

ab139473

Mitochondrial hydroxyl radical

Mitochondrial superoxide

Hydrogen peroxide

Microscope, plate reader

ab219931

ab219943

ab138874, ab138886, ab102500

DCFDA web v1

"We are currently using the product [ab113851] to measure microglial activation after 24 hours in response to activating stimuli. The product has been giving us very consistent results and is very easy to use.” 
- Dr Neal Bennett


DNA/RNA damage, lipid peroxidation, and protein oxidation/nitration

Oxidative stress can be measured indirectly by measuring the levels of DNA/RNA damage, lipid peroxidation, and protein oxidation/nitration, rather than a direct measurement of reactive oxygen species. These oxidative stress markers are more enduring than reactive oxygen species.

DNA/RNA damage

There are several types of DNA/RNA damage that can be measured as oxidative stress markers. 8-hydroxydeoxyguanosine (8-OHdG) is probably the most commonly used DNA damage marker for oxidative stress. Comet assays, assays for apurinic/apyrimidinic sites, and assays for aldehyde-induced damage can be used as less direct measures of DNA damage which is potentially related to oxidative stress.

Lipid peroxidation

Malondialdehyde (MDA) is the most commonly used lipid marker of oxidative stress. It is formed via peroxidation of polyunsaturated fatty acids and is typically quantified using the TBARS assay. The TBARS assay is not entirely specific for MDA, as other aldehydes also generate a signal with the assay, however, the TBARS assay is generally more convenient than using HPLC to measure MDA. Competitive ELISA assays for MDA are also available.

Other lipid peroxidation markers include 4-HNA, 8-isoprostane, lipid hydroperoxides, and oxidized LDL.

Protein oxidation / nitration

Oxidative damage to proteins can take the form of protein carbonylation and protein nitration (3-nitrotyrosines). Reactive oxygen species can also cause the formation of advanced glycation end products (AGE) and advanced oxidation protein Products (AOPP). All of these markers can be measured by standard assays.


Assay

Readout

Assay kits

8-OHdG

Plate reader

ab201734

Lipid hydroperoxide (LPO)

Lipid peroxidation (MDA)

Protein carbonyl content

DNA damage –  apurinic/apyrimidinic  sites

Plate reader

ab133085

ab118970

ab126287

ab211154

Oxidized proteins

Western blot

ab178020











Antioxidants

Antioxidant enzymes and other redox molecules counteract the ROS that cause oxidative damage. There are three classes of antioxidants used as oxidative stress markers: small molecules, enzymes, and proteins (such as albumin).

A number of assays exist to measure the total antioxidant capacity of a sample, including small molecule and protein antioxidant based capacity. One of the most common total antioxidant capacity assays is the Trolox equivalent antioxidant capacity assay (TEAC). The oxygen radical antioxidant capacity (ORAC) assay is another common oxidative stress assay that measures antioxidant capacity by measuring the ability of antioxidants to reduce the quenching of a fluorescent dye by ROS.

Antioxidant activity can also be measured at the level of specific analytes. For instance by looking at the relative levels of GSH and GSSG. Glutathione (GSH) is considered the most abundant molecule among endogenous antioxidants, forming GSSG in its oxidized form. It is recycled by glutathione reductase.

Otherwise, the levels of activity of antioxidant enzymes, such as GST and Superoxide dismutase can be measured in relation to the levels of oxidative stress.

Assay

Readout

Assay kits

Total antioxidant capacity: copper-based

Total antioxidant capacity - FRAP assay (Ferric Reducing Antioxidant Power Assay

Plate reader

ab65329


ab234626

Ascorbic acid

NAD/NADH

NADP/NADPH

GSH/GSSG ratio

Thiol

Plate reader

ab65656

ab65348, ab176723

ab65349, ab176724

ab138881, ab205811

ab112158, ab219272

Intracellular glutathione (GSH)

Flow cytometry, plate reader

ab112132

GST

Superoxide dismutase

Glutathione reductase

Xanthine oxidase

Glutathione peroxidase

Aconitase

Catalase

Thioredoxin reductase

NQO1

Peroxidase

Plate reader

ab65325, ab65326

ab65354

ab83461

ab102522

ab102530

ab109712

ab118184, ab83464

ab190804

ab184867

ab155895

Oxidative stress defense cocktail (catalase, SOD1, TRX, smActin)

Western blot

ab179843


Learn about:

Assays for metabolites and enzymes of sugar, lipid, alcohol, and amino acid metabolism, glycolysis, and the citric acid cycle

Metabolic activity analysis in live cells with your microplate reader: respiration, glycolysis, and fatty acid metabolism

Assays for ATP, NADH, and similar molecules

Assays for mitochondria and mitochondrial function


References

Sies H., Jones D. Oxidative stress. In: Fink G., editor. 2nd ed. Vol. 3. Elsevier; Amsterdam: 2007. pp. 45–48. (Encyclopedia of Stress)


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