Synthesis, Quantification, Characterization, and Signaling Properties of Glutathionyl Conjugates of Enals

Abstract

Oxidation of lipids generates large quantities of highly reactive α,β-unsaturated aldehydes (enals). Enals and their protein adducts accumulate in the tissues of several pathologies. In vitro, low concentrations of enals such as HNE (4-hydroxy trans-2-nonenal) affect cell signaling whereas high concentrations of enals are cytotoxic. Direct conjugation of the C2-C3 double bond of enals with the sulfhydryl group of GSH is a major route for the metabolism and detoxification of enals. Recently, we found that glutathionyl conjugate of HNE (GS-HNE) enhances the peritoneal leukocyte infiltration and stimulates the formation of proinflammatory lipid mediators. Moreover, the reduced form of the glutathione conjugate of HNE (GS-DHN) elicits strong mitogenic signaling in smooth muscle cells. In this chapter we discuss the methods to study the metabolism of enals and the redox signaling properties of glutathionyl conjugates of HNE.

Lipid Peroxidation Product 4-Hydroxy-trans-2-nonenal Causes Endothelial Activation by Inducing Endoplasmic Reticulum Stress

“…In addition to atherogenesis, several other disease conditions, such as heart failure, Parkinson and Alzheimer diseases (33), obesity, and insulin resistance (34) are associated with tissue accumulation of HNE-modified proteins, indicating on-going oxidative damage. Significantly, these pathological states are also associated with ER stress (35).…”

Progress in the research of GSH in cells

“…At low concentrations, enals such as 4-hydroxy trans-2-nonenal can affect cell signal transduction while high concentrations of enals are cytotoxic. The GSH thiol can adduct the unsaturated bond of the enals to reduce the cytotoxic effects [16]. Thus, the conjugation of reactive drug metabolites to GSH is considered to be an important detoxification mechanism, GSH can also decrease xenobiotic toxicity in erythrocytes in this manner [17], while the toxicity of polycyclic aromatic hydrocarbons, a known carcinogen, can also be reduced by GSH-mediated detoxification as GSH can adduct the bay-and fjord-region diol epoxides for detoxification [17][18][19].…”

Aldehyde Reduction by Cytochrome P450

“…Note: These conditions achieve resolution of the GSH conjugates, 1,4-dihydroxy-trans-2-nonene (DHN), 4-hydroxy-trans-2-nonenoic acid (HNA), and 4-HNE (Amunom et al, 2007;Srivastava et al, 2010). …”

“…Five hundred μL methanol was added and the samples extracted with 2 mL hexane. The hexane layer (upper layer) was removed, dried under a stream of nitrogen and then derivatized with 20 μL of N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) for 1 hour at 60°C as described previously (Srivastava et al, 2010). …”

“…For experiments measuring reduction of the endogenous aldehyde, 4-HNE, the substrate was incubated with CYP in the presence of oxygen and NADPH and the metabolites were separated by High Pressure Liquid Chromatograpy (HPLC), using an adaptation of the method of Srivastava et al . (Srivastava et al, 2010). For study of 9-AA and 4-HNE reduction, the first step involves incubation of the substrate with the CYP in appropriate media, followed by quantification of metabolites through either spectrofluorimetry or analysis by HPLC coupled with a radiometric assay, respectively.…”