Oxidative Stress Markers

1. Oxidative Stress Markers MunibAbdulmalikIgor GasicJoseph SamuelDan Zangari PHM142 Fall 2012 Coordinator: Dr. Jeffrey Henderson Instructor: Dr. David Hampson

2. What is Oxidative Stress? • Reactive oxygen species (ROS) • Byproducts of physiological, metabolic, and other processes • Highly reactive • Can cause oxidative damage to DNA, lipids, and proteins • Underlying pathology in a variety of human diseases

3. What are its implications in the body? • Oxidative stress can lead to cell damage, apoptosis/cell suicide, and necrosis • Skin is subject to oxidative stress; this can be seen when comparing the wrinkles, breakdown and colour of elderly skin to that of a healthy child • Oxidative stress has been found to play a role in many diseases such as: • Heart Disease• Cancer• Arthritis• Lung Disease• Fibromyalgia• Diabetes• Neurodegenerative Diseases (Parkinson’s, Alzheimer's)• Autoimmune Diseases• Eye Diseases (Macular Degeneration)

4. What are Oxidative Stress Markers? A biomarker (or simply marker) is a chemical entity or cellular event that is an indicator of biological state The job of oxidative stress markers are to measure the levels of reactive oxygen species in the body Reactive Oxygen Species are molecules such as peroxides and free radicals that include oxygen and are highly reactive If these molecules build up in the body, they can cause detrimental effects such as cell death and cancer

5. Antioxidant Assay It is important to be able to measure antioxidant capabilities within individuals This can be done either by hydrogen atom transfer (HAT) or by single electron transfer (SET) methods SET assays can quantify the ability of an antioxidant to reduce any compound (i.e. free radicals, carbonyls, metals) The most common manifestation of the SET assay is the reduction of Cu2+ to Cu+ The [Cu+] can then be measured within 5 minutes of reduction using a fluorescent chromogen

6. Antioxidant Assay Protocol We measure activity of the antioxidant by measuring its absorbance versus a known concentration of uric acid Place uric acid in a well, dilute it, (add antioxidant), add copper, add a chromogen, measure activity (by measuring absorbance of the well)

7. Oxidized DNA Damage Markers DNA is one of the most common targets of ROS Modifications of DNA by ROS associated with cellular transformation and genome instability Can assess oxidative modification of DNA bases using a number of assays on various byproducts of DNA oxidation

8. Oxidized DNA Damage Markers • 8-hydroxy-2’-deoxyguanosine (8-OHdG) • Critical marker of oxidative stress and DNA damage • Hydroxide radicals interact with guanine residues of DNA • Leads to formation of C8-hydroxyguanosine • Becomes 8-hydroxy-2'-deoxyguanosine (the nucleoside form) • 8-OHdG can be measured in urine samples and detected/analyzed by HPLC and GC-MS

9. Lipid Peroxidation Markers Lipid peroxidation involves the oxidative degradation of lipid membranes. It is a 3 step chain reaction in which free radicals acquire electrons from lipid cell membranes leading to cell damage and oxidative stress. Lipid peroxides are unstable and decompose to form reactive by-products. Lipid peroxidation markers are used to detect common by-products of lipid peroxidation. These assays look for molecules such as: malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), and acrolein.

10. Lipid Peroxidation Markers • Malondialdehyde (MDA): • Major aldehyde derived from lipid peroxidation • Malondialdehyde (MDA) antibody is specific for MDA-modified proteins • Suitable for immunohistocheistry and western blotting. • 4-hydroxy-2-nonenal (4-HNE): • Major membrane lipid peroxidation product • Monoclonal antibody against 4-HNE (clone HNE-J2) is highly specific for 4-HNE-His/Lys/Cys adducts. • Acrolein (ACR): • Highly reactive, carcinogenic aldehyde found everywhere in the environment, but also as a result of lipid peroxidation. • Reacts with lysine residues in various proteins. • Acrolein (ACR) antibody is specific for ACR-modified proteins

11. Oxidized Protein Damage Markers Proteins are one of the most oxidized entities in the body Proteins can be oxidized on the backbone by the abstraction of the alpha-hydrogen by a hydroxyl radical which leads to protein fragmentation Oxidation of amino acid side chains, especially the side chain of cysteine can occur Several amino acid side chains that contain carbonyl groups can undergo oxidation that form the basis for biological markers for oxidized protein damage

12. Oxidized Protein Damage Markers Detecting specifically oxidized proteins using Western Blots Kits Most common way of detecting specifically oxidized proteins is by using 2,4-Dinitrophenylhydrazine (DNPH) kits The DNPH kit detects DNPH carbonyl conjugates that are formed by binding to a anti-DNP antibody and are analyzed using a Western Blot. Detection of protein carbonyls using immunohistochemical staining Oxidized carbonyl groups in tissue are conjugated by reaction with DNPH and using anti-DNPH antibodies are used for immunohistochemical analyses.

13. Oxidized Protein Damage Markers A new biomarker caused by Neutrophils Myeloperoxidase (found in neutrophils) and eosinophilperoxidase catalyze the formation hypochlorous acid (HOCl) and hypobromous acid (HOBr). These reactive metabolites are important in the formation of 3,5 dibromotyrosine which can be used in immunohistochemical analysis of oxidative stress

14. Conclusion Oxidative stress markers are used to measure the amount of reactive oxygen species in the body Antioxidant assays can be used to measure the effectiveness of antioxidants Markers can be of DNA damage, lipid peroxide, or protein damage types in order to assess amount of ROS in the body

15. Summary Oxidative stress can lead to cell damage, apoptosis/cell suicide, and necrosis Antioxidant assays include hydrogen atom transfer (HAT) and single electron transfer (SET) Hydroxide radicals interact with guanosine bases in DNA to form 8-hydroxy-2’-deoxyguanosine (8-OHdG); a critical marker of oxidative stress and DNA damage Lipid Peroxidation involves the oxidative degradation of lipid membranes and markers include: malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), and acrolein Proteins can be oxidized at the backbone or the side chain (esp. cysteine) and markers include: 2,4-Dinitrophenylhydrazine (DNPH) and myeloperoxidase

16. References • "Exploring Oxidative Stress And Nitrosative Stress With OxisResearch®." Oxis International Inc.N.p., n.d. Web. 19 Nov. 2012. <http://oxisresearch.com/oxidative_stress_completen.html>. • Guichardant, M. "Specific Markers of Lipid Peroxidation Issued from N-3 and N-6 Fatty Acids." National Center for Biotechnology Information. U.S. National Library of Medicine, n.d. Web. 19 Nov. 2012. <http://www.ncbi.nlm.nih.gov/pubmed/14748733>. • "Lipid Peroxidation." Life Science Research Products & Solutions. Cell Bioloabs Inc., n.d. Web. 19 Nov. 2012. <http://www.cellbiolabs.com/lipid-peroxidation>. • “Oxidative Stress Markers.” Cosmo Bio Co, LTD. Web. 19 Nov. 2012. < https://docs.google.com/viewer?a=v&q=cache:Ry7_ZonvwmoJ:www.cosmobio.co.jp/export_e/products/kits/products_NNS_20060421/Oxidative_Stress_Marker-JPN.pdf+oxidative+stress+markers&hl=en&gl=ca&pid=bl&srcid=ADGEEShM9Y2eLHr3o4yowz79mS2zXfFevLpcAHAdGgH2DbcKIvxvbVbMQMHMDVxZ8jUg9ABFmaEzHeB3QECyIndQ9Ri_cxkTQfDKAPoP2WN1EEI1U28Ro6oW411sC9UBVRiLbTr2mE3S&sig=AHIEtbRsyS6UlUWjtX4WiBvlmJCQZAMiBA

17. References Berlett, Barbera S., and Earl R. Stadtman. "Protein Oxidation in Aging, Disease, and Oxidative Stress." The Journal of Biological Chemistry 272 (n.d.): 20313-0316. Print. "What Is Oxidative Stress?" What Is Oxidative Stress? N.p., n.d. Web. 21 Nov. 2012. http://www.news-medical.net/health/What-is-Oxidative-Stress.aspx Di Domenico, Fabio, RaffaellaCoccia, D. Allan Butterfield, and MarziaPerluigi. "Circulating Biomarkers of Protein Oxidation for Alzheimer Disease: Expectations within Limits." Biochimica Et BiophysicaActa (BBA) - Proteins & Proteomics 1814.12 (2011): 1785-795. Print.