Identification and quantitation of 4-hydroxy-2-nonenal and 4-oxo-2-nonenal metabolites in vivo as biomarkers of oxidative stress

Abstract

Oxidative stress occurs when the balance between reactive oxygen species and antioxidant defense mechanisms in the body becomes tipped heavily in favor of the reactive oxygen species. Reactive oxygen species can be present in excess due to elevated O2 levels, radiation, infection, smoking, or even excessive exercise. It is also possible that a reduction in antioxidant levels could result in oxidative stress. When this occurs, reactive oxygen species can cause damage by reacting with DNA, proteins, and lipids. Lipid peroxidation products are breakdown products of polyunsaturated fatty acids, formed under conditions of oxidative stress. These lipid peroxidation products, such as 4-hydroxy-2-nonenal, 4-oxo-2-nonenal, and their metabolites, have been demonstrated to be cytotoxic and genotoxic. They have also been shown to play a role in the development and progression of age related diseases such as Alzheimer’s and atherosclerosis. 4-Hydroxy-2-nonenal levels have even been shown to increase with disease progression. Conjugation with glutathione, followed by further metabolism to mercapturic acid conjugates, can mitigate the effects of these lipid peroxidation products in disease development by facilitating their excretion from the body. The increase of lipid peroxidation products in disease states suggests utility for the mercapturic acid conjugates of these metabolites as biomarkers of oxidative stress in vivo. In order to assess the utility of lipid peroxidation product-mercapturic acid conjugates as biomarker of oxidative stress, we first developed liquid chromatography-tandem mass spectrometry methods by which to analyze the conjugates. Then, using CCl4 treatment of rats, a widely accepted model of acute oxidative stress, we were able to discover the first in vivo evidence for 4-oxo-2-nonenal-mercapturic acid and its phase one metabolites 4-oxo-2-nonen-1-ol-mercapturic acid and 4-oxo-2-nonenoic acid-mercapturic acid. This proved to be non-trivial since 4-hydroxy-2-nonenal-mercapturic acid and 4-oxo-2-nonen-1-ol-mercapturic acid are isomers with similar retention times and fragmentation patterns. The distinction between 4-hydroxy-2-nonenal-mercapturic acid and 4-oxo-2-nonen-1-ol-mercapturic acid is an important one because previous analyses have likely attributed the effects of 4-oxo-2-nonen-1-ol to 4-hydroxy-2-nonenal. These metabolites also form by different pathways, so being able to distinguish between the two could provide insight into the mechanisms of oxidative stress in biological systems. We were also able to show a significant increase in urinary levels of 1,4-dihydroxy-2-nonene-mercapturic acid, 4-hydroxy-2-nonenoic acid lactone-mercapturic acid, 4-oxo-2-nonenal-mercapturic acid, and 4-oxo-2-nonenoic acid-mercapturic acid in the CCl4 rat model of oxidative stress by semi-quantitative analysis. These results suggest that conjugates of 4-hydroxy-2-nonenal and 4-oxo-2-nonenal metabolites have value as markers of in vivo oxidative stress and lipid peroxidation. The next step was to develop quantitative methods for the analysis of lipid peroxidation product conjugates and to assess their levels in humans. We developed a quantitative method to simultaneously analyze the levels of 4-oxo-2-nonen-1-ol-mercapturic acid, 4-hydroxy-2-nonenal-mercapturic acid, and 1,4-dihydroxy-2-nonene-mercapturic acid in human urine samples utilizing isotope-dilution mass spectrometry. We were also able to detect 4-hydroxy-2-nonenoic acid-mercapturic acid, 4-hydroxy-2-nonenoic acid lactone-mercapturic acid, and 4-oxo-2-nonenoic acid-mercapturic acid with this method. The detection of 4-oxo-2-nonen-1-ol-mercapturic acid and 4-oxo-2-nonenoic acid-mercapturic acid in humans is significant because it demonstrates that 4-hydroxy-2-nonenal/4-oxo-2-nonenal branching occurs in the breakdown of polyunsaturated fatty acids and suggests that 4-oxo-2-nonen-1-ol may contribute to the harmful effects currently associated with 4-hydroxy-2-nonenal. We were able to show significant decreases in 4-hydroxy-2-nonenal-mercapturic acid, 1,4-dihydroxy-2-nonene-mercapturic acid, and total lipid peroxidation product-mercapturic acid conjugates in a group of seven smokers upon smoking cessation. This data demonstrates the value of 4-hydroxy-2-nonenal and 4-oxo-2-nonenal metabolites as in vivo markers of oxidative stress.