A Guide to Oxidative Stress Markers

Reactive oxygen and nitrogen species (ROS and RNS) are generated through both normal physiological functions and pathological processes. Antioxidant defense mechanisms, in turn, work to neutralize their harmful effects. Oxidative stress arises when there is an imbalance between the formation of reactive species and the ability of antioxidant defense mechanisms to counteract their effects. This state leads to the disruption of redox pathways and physiological functions. Elevated ROS and RNS also lead to irreversible and damaging modifications of carbohydrates, lipids, proteins, nucleic acids, and other metabolic compounds. Cumulatively, the process is known to contribute to the risk factors and outcomes of a variety of disorders such as metabolic, cardiovascular, neurological, renal diseases, and cancer. Biomarkers of oxidative stress are thus of particular importance in clinical assessments, therapeutic approaches, and investigative research into affected biological pathways. Here, we review recent literature to identify a list of commonly referenced chemical and protein markers of oxidative stress.

Markers of ROS and RNS production

Certain endogenous proteins and compounds directly involved in biochemical pathways for ROS and RNS generation are suggested markers for oxidative stress. Among the most common examples are NADPH oxidase, myeloperoxidase, and nitric oxide synthase, which generate radical superoxide anions (O₂^•-), hypochlorous acid (HOCl), and nitric oxide (NO), respectively. 

The superoxide-generating enzyme xanthine oxidase (XO) produces uric acid and allantoin, which can also be used as markers for oxidative activity. Other endogenous sources of reactive species include lipoxygenase, lysyl oxidase, and angiotensin II. 

Markers for enzymatic ROS production are valuable in the evaluation of therapeutic responses and in developing novel drugs that target ROS generation, such as NADPH oxidase and MPO inhibitors. These protein markers can be detected using immunodetection techniques, enzyme assays, or gene expression analysis using molecular methods. 

This diagram highlights key pathways in oxidative stress and common markers.

Markers of nucleic acid oxidation

Reactive species can cause oxidative modifications in DNA and RNA, including oxidized nucleotides, strand breaks, base conversions, and the formation of adducts. DNA damage from oxidation is known to have mutagenic potential, resulting in transcriptional arrest, replication errors, and genomic instability. Elevated levels of DNA and RNA oxidation have been implicated in the increased risk of breast and lung cancer, as well as diseases like atherosclerosis and diabetes. 

The most commonly used DNA markers of oxidative stress are 8-hydroxydeoxyguanosine (8-oxodG; 8-OHdG) and 8-hydroxyguanine (8-oxoG). Additional nucleic acid markers include isoguanine, 8-oxoadenine, 5-hydroxycytosine, 5-chlorocytosine, and 5-chlorouracil. These markers can be detected using HPLC, HPLC/GC-MS, and antibody-based immunodetection, including ELISAs. 

Markers of lipid peroxidation

Markers of lipid peroxidation are key indicators of oxidative damage and are increasingly studied for their role in important cellular processes. A well-studied mechanism of lipid peroxidation (LPO) involves the reaction of ROS with polyunsaturated fatty acids, such as linoleic and arachidonic acid, that triggers autocatalyzed chain reactions. These produce lipid hydroperoxides and reactive aldehyde end products that not only cause cellular damage, but can also play important roles in cell signaling, stress response, and metabolic regulation. 

Common markers of lipid peroxidation include malondialdehyde (MDA), 4-Hydroxynonenal (4-HNE), F2-isoprostanes, isolevuglandins, and acrolein. These products can also react with proteins, forming advanced lipoxidation end products (ALEs), which include modified amino acid moieties like cysteine, histidine, and arginine. LPO markers can be measured analytically with HPLC or GC-MS-based methods, or with immunological assays, such as ELISAs. Dedicated lipid peroxidation assay kits often utilize a reaction with MDA to produce a colorimetric signal for quantitative measurement. 

Markers of protein oxidation

Investigating oxidative protein modifications by ROS and RNS holds significant biological and clinical relevance. The accumulation of these modifications results in altered protein structures and biochemical properties, leading to loss of function or toxicity. Protein oxidation profiles, which can be determined from a variety of samples including blood and urine, may be used to implicate different oxidative stress pathways or pathological conditions. 

Oxidative modifications of amino acid residues, protein backbones, or functional groups can be identified in several key biomarkers. Protein carbonylation, which can occur on glutamine, lysine, arginine, and threonine side chains, is a common marker detected by HPLC and protein carbonyl assays utilizing 2,4-dinitrophenylhydrazine (DNPH). Additional markers such as nitrotyrosine (3-NO-Tyr), oxidized low-density lipoprotein (oxLDL), and ischemia-modified albumin (IMA) can be detected using HPLC or antibody-based methods such as ELISA. Another marker, 3-Chlorotyrosine (3-Cl-Tyr) generally requires more sensitive detection using LC-MS/MS. 

ROS-induced oxidation can cause protein functional groups to react with carbohydrates, generating advanced glycation end products (AGEs). AGEs accumulate with aging and carbohydrate intake and are linked to diabetes and obesity, among other conditions. These comprise a heterogeneous group of molecules, such as carboxymethyl lysine, carboxymethyl valine, pentosidine, glucosepane, and hydroimidazolone.

Markers of antioxidant defense

Biomarkers involved in antioxidant defense can be used to assess the susceptibility or resistance to oxidative stress. Among these are enzymes that contribute to ROS/RNS decomposition, such as catalase, superoxide dismutase, glutathione peroxidase, glutathione disulfide reductase, thioredoxin reductase, and peroxiredoxin. Other prominent antioxidant molecules include glutathione, bilirubin, vitamin C, vitamin E, β-carotene, and uric acid. Some of these markers can be quantified using dedicated assay kits. 

Considerations in marker detection

In measuring various oxidative stress markers, analytical methods such as HPLC, mass spectrometry, and gas chromatography are often touted due to their sensitivity, accuracy, and low background. However, these approaches may not be feasible in higher throughput applications, such as in clinical analysis or the screening of large samples. Specific antibodies and plate-based immunoassays, which can be adapted to accommodate more samples, are another widely used tool for detecting oxidation products, albeit at the cost of reduced sensitivity. Those who opt for antibody-based methods should take additional precautions, such as verifying the antigen and epitope, accounting for any cross-reactivities, and including appropriate controls. 

Table of oxidative stress markers

The table below highlights notable proteins and molecules involved in oxidative stress as compiled from recent literature. Where applicable, links to relevant antibodies and assay kit product catalogs are provided, which can serve as tools in the detection and measurement of these markers. The product catalogs contain product listings from a variety of life science reagent suppliers. 

References

1. Liguori I, Russo G, Curcio F, et al. Oxidative stress, aging, and diseases. Clin Interv Aging. 2018;13:757-772. Published 2018 Apr 26. doi:10.2147/CIA.S158513

2. Ghezzi P. Environmental risk factors and their footprints in vivo - A proposal for the classification of oxidative stress biomarkers. Redox Biol. 2020;34:101442. doi:10.1016/j.redox.2020.101442

3. Frijhoff J, Winyard PG, Zarkovic N, et al. Clinical Relevance of Biomarkers of Oxidative Stress. Antioxid Redox Signal. 2015;23(14):1144-1170. doi:10.1089/ars.2015.6317

4. Murphy MP, Bayir H, Belousov V, et al. Guidelines for measuring reactive oxygen species and oxidative damage in cells and in vivo. Nat Metab. 2022;4(6):651-662. doi:10.1038/s42255-022-00591-z

5. Marrocco I, Altieri F, Peluso I. Measurement and Clinical Significance of Biomarkers of Oxidative Stress in Humans. Oxid Med Cell Longev. 2017;2017:6501046. doi:10.1155/2017/6501046

6. Bai J, Tan R, An Z, Xu Y. Quantitative estimation of intracellular oxidative stress in human tissues. Brief Bioinform. 2022;23(4):bbac206. doi:10.1093/bib/bbac206

7. Cipak Gasparovic A, Zarkovic N, Zarkovic K, et al. Biomarkers of oxidative and nitro-oxidative stress: conventional and novel approaches. Br J Pharmacol. 2017;174(12):1771-1783. doi:10.1111/bph.13673