Original Contribution
Quantitation of mercapturic acid conjugates of 4-hydroxy-2-nonenal and 4-oxo-2-nonenal metabolites in a smoking cessation study

https://doi.org/10.1016/j.freeradbiomed.2009.10.025Get rights and content

Abstract

The breakdown of polyunsaturated fatty acids (PUFAs) under conditions of oxidative stress results in the formation of lipid peroxidation (LPO) products. These LPO products such as 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) can contribute to the development of cardiovascular and neurodegenerative diseases and cancer. Conjugation with glutathione, followed by further metabolism to mercapturic acid (MA) conjugates, can mitigate the effects of these LPO products in disease development by facilitating their excretion from the body. We have developed a quantitative method to simultaneously assess levels of 4-oxo-2-nonen-1-ol (ONO)-MA, HNE-MA, and 1,4-dihydroxy-2-nonene (DHN)-MA in human urine samples utilizing isotope-dilution mass spectrometry. We are also able to detect 4-hydroxy-2-nonenoic acid (HNA)-MA, 4-hydroxy-2-nonenoic acid lactone (HNAL)-MA, and 4-oxo-2-nonenoic acid (ONA)-MA with this method. The detection of ONO-MA and ONA-MA in humans is significant because it demonstrates that HNE/ONE branching occurs in the breakdown of PUFAs and suggests that ONO may contribute to the harmful effects currently associated with HNE. We were able to show significant decreases in HNE-MA, DHN-MA, and total LPO-MA in a group of seven smokers upon smoking cessation. These data demonstrate the value of HNE and ONE metabolites as in vivo markers of oxidative stress.

Introduction

Oxidative degradation of polyunsaturated fatty acids (PUFAs) occurs under conditions of oxidative stress when the cellular antioxidant defense mechanisms are overwhelmed, leading to the formation of electrophilic lipid peroxidation (LPO) products. 4-Hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) are two of the most thoroughly studied LPO products. These reactive aldehydes have been shown to be cytotoxic and genotoxic [1], [2], as well as to contribute to the development and progression of cancer [3], cardiovascular diseases such as atherosclerosis and chronic obstructive pulmonary disease [4], [5], [6], and neurodegenerative diseases like Alzheimer's [7], [8], [9]. In biological systems, HNE and ONE undergo phase I metabolism, resulting in their respective oxidation products 4-hydroxy-2-nonenoic acid (HNA) [10] and 4-oxo-2-nonenoic acid (ONA) [11] or reduction products 1,4-dihydroxy-2-nonene (DHN) [12] and 4-oxo-2-nonen-1-ol (ONO) [13], [14], [15] (Scheme 1). HNE, ONE, and their phase I metabolites have also been shown to undergo phase II metabolism, forming Michael-type conjugates with glutathione (GSH) [2], a reaction mediated by glutathione S-transferase (GST) [16], [17], [18]. On conjugation, HNA can form a lactone (HNAL) via spontaneous intramolecular condensation [19]. Further metabolism of these LPO-GSH conjugates in the liver and kidney results in LPO-mercapturic acid (MA) conjugates which are excreted in urine.

We have previously reported that HNE and ONE metabolite levels are significantly increased in rats after an acute oxidative stress insult [20]. In that study we were able to differentiate between HNE-MA and its isomer ONO-MA which had not been previously demonstrated. This is an important distinction because previous analyses have likely attributed the effects of ONO to HNE. 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. Previous studies have focused on the quantitation of DHN-MA [21], [22], [23], [24].

Here we report the quantitation of HNE-MA and ONO-MA, as well as DHN-MA in human urine. The phase I metabolites of HNE-MA and ONE-MA represent biologically relevant pathways for the elimination of these LPO products in a rat model of oxidative stress [20]. We have detected HNE-MA, DHN-MA, HNA-MA, HNAL-MA, ONO-MA, and ONA-MA in human samples and are able to quantitate the HNE-MA, ONO-MA, and DHN-MA metabolites in smokers. Twelve weeks of smoking cessation resulted in a significant decrease in the levels of urinary HNE-MA, DHN-MA, and overall LPO-MA. These results demonstrate the potential utility of these metabolites as noninvasive diagnostic tools for assessing oxidative stress in vivo.

Section snippets

Materials

[2H]Chloroform was purchased from Cambridge Isotope Laboratories (Andover, MA). HPLC-grade formic acid (0.1%) in water was purchased from Honeywell Burdick and Jackson (Muskegon, MI). 3-Chloroperoxybenzoic acid and dithiothreitol were purchased from TCI America (Portland, OR). HNE-MA (1 mg in 100 μl ethanol) was purchased from Cayman Chemical (Ann Arbor, MI). Cotinine was purchased from Alfa Aesar (Ward Hill, MA) and cotinine-d3 (99 atom % D, 1 mg/ml in methanol) was from Sigma-Aldrich (St.

Quantitation of LPO products in human urine

We have developed an LC-MS/MS method for the simultaneous quantitation of HNE-MA, ONO-MA, and DHN-MA in human urine. Previous human studies have focused only on the quantitation of DHN-MA [22]. A study in rats by Mally et al. [26] quantified both HNE-MA and DHN-MA; however, they did not account for ONO-MA in their analysis.

Quantitation of HNE-MA

A calibration curve was constructed, using standard solutions containing varying concentrations of HNE-MA and a fixed concentration of HNE-MAd3 (1.0 μM) as the internal

Discussion

A semiquantitative method for analysis of HNE-MA and ONE-MA metabolites in the urine of oxidatively stressed rats was previously reported [20]. While this method allowed for the simultaneous analysis of multiple LPO-MA conjugates, the data were not quantitative. Appropriate internal standards for each of our analytes of interest are necessary in order to perform absolute quantitation. We first synthesized MAd3 following the method of Slatter et al. [25]. MAd3 conjugates of HNE, DHN, HNA, ONE,

Acknowledgments

This work was supported in part by the National Institutes of Health (R01HL081721, S10RR022589, and P30ES000210), an OSU Center for Healthy Aging Research Fellowship, a grant from the John C. Erkkila, MD, Endowment for Health and Human Performance, and by a donation from the estate of Leland J. Gross. We thank pharmacy residents Rachelle Collier and Tiffany Boehland for teaching smoking cessation classes to the smokers enrolled in the study.

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