Vitamin C conjugates of genotoxic lipid peroxidation products: structural characterization and detection in human plasma

Abstract

alpha,beta-Unsaturated aldehydes such as 4-hydroxy-2-nonenal (HNE) and other electrophilic lipid peroxidation (LPO) products may contribute to the pathogenesis of cancer, cardiovascular diseases, and other age-related diseases by cytotoxic, genotoxic, and proinflammatory mechanisms. The notion that vitamin C (ascorbic acid) acts as a biological antioxidant has been challenged recently by an in vitro study showing that ascorbic acid promotes, rather than inhibits, the formation of genotoxic LPO products from the lipid hydroperoxide, hydroperoxy octadecadienoic acid [Lee, S. H., Oe, T. & Blair, I. A. (2001) Science 292, 2083-2086]. Here, we demonstrate that ascorbic acid acts as a nucleophile and forms Michael-type conjugates with electrophilic LPO products. Several ascorbyl-LPO product conjugates, resulting from the interaction of ascorbic acid with hydroperoxy octadecadienoic acid in vitro, were identified by tandem MS, including ascorbyl conjugates of HNE, 4-oxo-2-nonenal, and presumably, 12-oxo-9-hydroxy-10-dodecenoic acid. The same ascorbyl-LPO product conjugates were detected in human plasma. The concentration of the ascorbyl-HNE conjugate in plasma from 11 healthy subjects was found to be 1.30 +/- 0.74 microM (mean +/- SD). Our data identify ascorbylation (vitamin C conjugation) as a previously unrecognized, biologically relevant pathway for the elimination of electrophilic LPO products, and have implications for the prevention and treatment of chronic inflammatory diseases, as well as the development of novel biomarkers of oxidative stress.

Figures

Scheme 1.

Vitamin C as one-electron donor and Michael donor. Vitamin C may function as a one-electron donor to HPODE, thereby inducing formation of the alkoxy radical. The alkoxy radical then undergoes α,β-carbon-carbon bond cleavage, generating HNE as well as other LPO products. As shown in this study, vitamin C may also function as a Michael donor and react with HNE and other LPO products, giving a variety of ascorbyl-LPO product conjugates.

Fig. 1.

LC/MS/MS-MRM analysis of an isotopomeric mixture of 12C and 13C6-ascorbylated HNE. Equal amounts of unlabeled ascorbic acid and isotopically labeled 13C6-ascorbic acid (0.5 mg of each, 5.7 mM) were added to a 1.0-ml solution of HNE (5 mM) in 100 mM chelex-treated phosphate buffer (pH 7.4). The reaction was stirred at 37°C for 2 h. LC system 1 was used for chromatographic separation. (A) Electrospray mass spectrum of the ascorbyl-HNE adduct: ions with m/z 350, m/z 333 and m/z 315 represent the 12C-isotopomer, and ions with m/z 356, m/z 339 and m/z 321 represent the 13C6-isotopomer. (B) MS/MS daughter scan of the m/z 333 [MH]+ ion of the unlabeled ascorbyl-HNE conjugate. (C) MS/MS daughter scan of the m/z 339 [MH]+ ion of the labeled 13C6-ascorbyl-HNE conjugate.

Fig. 2.

LC/MS/MS-MRM analysis of the reaction products between ascorbic acid and HPODE: formation and ascorbylation of HNE. Ascorbic acid (0.53 mg; 3 mM) or 0.053 mg (0.3 mM) (A/B, and C, respectively) was added to a 1.0-ml solution of HPODE (0.2 mM) in 100 mM chelex-treated phosphate buffer (pH 7.4). The reactions were stirred at 37°C. Aliquots (10 μl) were taken at various time points and analyzed by using LC system 1. (A) Vitamin C-mediated conversion of HPODE to HNE and subsequent depletion of HNE. (B) Concomitant formation of the ascorbyl-HNE adduct. (C) At 0.3 mM ascorbic acid, HPODE was converted into HNE without formation of the ascorbyl-HNE adduct, presumably due to vitamin C depletion.

Fig. 4.

LC/MS/MS-MRM analysis of plasma from a healthy 38-year-old male, demonstrating the presence of ascorbylated LPO products other than ascorbylated HNE. Plasma was extracted and analyzed as described in Materials and Methods.

Fig. 3.

LC/MS/MS-MRM analysis of plasma from a healthy 38-year-old male, demonstrating the presence of ascorbylated HNE. Plasma was extracted and analyzed as described in Materials and Methods. Shown are detection of specific fragment ions, i.e., m/z 315 [MH-H2O]+ (A), m/z 297 [MH-2H2O]+ (B), m/z 139 [hydroxynonenal-H2O+H]+ (C), and m/z 177 [ascorbic acid+H]+ (D) arising from collisional fragmentation of the quasimolecular ion with m/z 350 [M+NH4]+.

Fig. 5.

Plots of a calibration curve (curve A) and a standard addition curve (curve B) for ascorbylated HNE. Curve A was derived from analysis of various concentrations of the synthetic ascorbyl-HNE adduct and a fixed concentration of the ascorbyl-octenal adduct (internal standard). The standard addition experiment was conducted by spiking 200-μl aliquots of a plasma sample with internal standard and known amounts of the ascorbyl-HNE adduct to give concentrations of 1.0, 2.0, 4.0, and 6.0 μM (curve B). Extrapolation of curve B to y = 0 gives an endogenous ascorbyl-HNE conjugate concentration of 2.28 μM.