Characterization of Assays of Oxidative Damage
Principal Investigator: S. Mitchell Harman, MD, PhD
Co-investigators: Chris Heward, PhD, Richard Cutler, PhD, Frank Gucciardo, PA, Panayiotis Tsitouras, MD, Taylor J. Marcell, PhD
This multi-stage project characterizes and validates laboratory methods for assessing oxidative stress status in human beings. Over the last few years, research evidence has accumulated that damage to cell components (proteins, cell membrane lipids, and the DNA of both chromosomes and mitochondria) by oxygen free radicals (also known as "reactive oxygen species" or ROS) is an important mechanism of aging. Oxidative damage may also play a role in various age-related diseases, such as Alzheimer's, arthritis, diabetic complications, cancers, and atherosclerosis. Most ROS are generated during the process of energy production in the cell's miniature "furnaces," the mitochondria. While most reactive oxygen produced by the mitochondria is used to oxidize fuel and generate high-energy phosphate compounds, which can be used by the cell for energy requiring processes, some ROS "escape" and can react chemically with various cell components. ROS include ozone, superoxide, peroxide, and the hydroxyl radical. Cells have a number of mechanisms for protecting themselves against oxidative damage. For example, enzymes, such as superoxide dismutase (SOD) and catalase, reduce ROS to harmless or less reactive forms. There are also oxygen scavenger compounds, such as glutathione, which "sacrifice themselves" by being oxidized in place of more critical molecules. Finally, various repair and disposal systems clean up the damage caused by the ROS that get past these defenses.
There are a number of assays that purport to measure the rates at which oxidative damage is occurring in living organisms and their ability to protect against such damage. These tests variously measure rates of excretion of damaged DNA nucleotides, oxidized lipid molecules and oxidized protein components, concentrations of antioxidant vitamins and other molecules, and the reserve capacity to resist oxidation, in the blood or urine. To date, however, only isoprostanes (lipid damage) and dityrosine (protein damage) have been shown to discriminate patients having pathological conditions associated with increased oxidative stress from normal, healthy persons. Without such clinical validation, it is impossible to reliably evaluate interventions designed to decrease the rates of oxidative damage because the interpretation of the assay results is uncertain. The Kronos Science Laboratory carries out a number of oxidative stress and oxidative protection assays. KLRI is conducting studies in human populations in order to validate the relationships of various oxidative stress assays to aging and dieases states. Such assays can then be employed to evaluate promising interventions to reduce oxidative stress.
Collaborators:
Chris Heward, PhD
L. Jackson Roberts, MD; Peter Reaven, MD
Abstract:
Harman SM, Liang L, Heward CB, Reaven P, Ping W, Cutler RL, and Duke R.
Urinary excretion of three nucleic acid oxidation adducts and isoprostane F(2)alpha measured by liquid chromatography-mass spectrometry in smokers, ex-smokers, and nonsmokers. Free Rad Biol Med 35:1301-9 (2003)
To assess novel liquid chromatography/mass spectrometric methods for measuring oxidative damage to nucleic acids and lipids, we compared urinary excretion of 8-hydroxy-2-deoxyguanosine (8-OHdG), 5-hydroxymethyl-2-deoxyuridine (5-OHmU), and 8-hydroxyguanosine (8-OxoG), and an isoprostane, 8-iso-prostaglandin F2<(IsopF<) in 234 healthy men (n = 113) and women (n = 121), 80 current smokers, 96 never-smokers), and 58 ex-smokers (no tobacco use for 3 years). The 8-OHdG and 8-OxoG did not differ significantly by group; 5-OHmU was higher in smokers, compared with ex- (p<0.003) and never- (p<0.0001) smokers and in ex- vs. never-smokers (p=.014) at, respectively, 13.5 ±0.7, 11.3 ±1.0, and 8.7± 0.3 µg/g creatinine. IsopF2< was higher in smokers, compared with ex- (p = 0.007) and never-smokers (p < 0.0001) and in ex- vs. never- smokers (p = .002) at, respectively, 1.1 ± 0.10; 0.74 ± 0.07, and 0.51 ± 0.04 µg/g creatinine. There were significant correlations among all three nucleic acid adducts and between IsopF2< and both 5-OHmU and 8-OHdG. Many smokers and ex-smokers had high levels of either 5-OHmU excretion or IsopF2< excretion, but not both. We concluded that 5-OHmU and IsopF2< are more discriminating of oxidative stress from tobacco smoke than the other two compounds measured. Whether characteristic patterns of excretion of these indicators forecast differential disease risk should be explored in future research.
Link: View it on PubMed
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