Synthesis, quantification, characterization, and signaling properties of glutathionyl conjugates of enals

Abstract

Oxidation of lipids generates large quantities of highly reactive alpha,beta-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.

Figure 1

Purification and characterization of radiolabeled GS-HNE and GS-DHN. GS-HNE is purified by reverse-phase HPLC on an ODS C₁₈ column. One milliliter fractions are collected and the radioactivity is quantified on a scintillation counter (A). The HPLC-purified peak I is analyzed by ESI⁺-MS (B). Ions with m/z values of 464.3 and 446.3 represent, [M + H]⁺ of GS-HNE and its dehydrated daughter ion, respectively. Panel C shows the ESI⁺-MS analysis of reagent GS-DHN. Retention time of the reagent GS-DHN on HPLC is identical to that of reagent GS-HNE. ESI⁺-MS of the HPLC peak corresponding to reagent GS-DHN shows the molecular ion with m/z 466.3 corresponds to [M + H]⁺ of GS-DHN.

Figure 2

Purification and characterization of GS-HNE-ester and GS-DHN-ester. Reagentesterified glutathionyl conjugates of HNE are separated by reverse-phase HPLC on an ODS C₁₈ column (A). Chemical identities of GS-HNE-ester and GS-DHN-ester are established by ESI⁺-MS. ESI⁺-MS of the HPLC peak I shows strong molecular ion with m/z value of 494.3 corresponding to the [M + H]⁺ of GS-DHN-ester (B). ESI⁺-MS of the HPLC- peak II shows strong ions with m/z values of 492.2 and 474.2; consistent with the [M + H]⁺ of GS-HNE-ester and its dehydrated daughter ion, respectively (C).

Figure 3

Metabolism of HNE in PC12 Cells. PC12 Cells are incubated with 4-(³H)-HNE (5 μM) in 2.5 ml HBSS for 30 min at 37 °C. After the incubation, radioactivity in the incubation medium is separated by HPLC on an ODS C₁₈ reverse-phase HPLC column. Panel A shows the HPLC profile of radiolabeled metabolites of [³H]-HNE. Peaks I–IV correspond to the retention time of reagent glutathione conjugates of HNE (GS-HNE and GS-DHN), DHN, HNA, and unmetabolized HNE, respectively. Chemical identities of peaks V and VI have yet not been established. Panel B shows the ESI⁺-MS chromatogram of HPLC peak I. Molecular ions with m/z value of 464.3 and 466.3 correspond to [M + H]⁺ of GS-HNE and GS-DHN, respectively. Ion with m/z ratio of 446.3 corresponds to the dehydrated daughter ion of GS-HNE.

Figure 4

Systemic metabolism of HNE. Male C57 are injected with 4-(³H)-HNE (1 mg/kg in PBS) in the tail vein and urine is collect for 8 h. Fifty microliters urine was mixed with an equal volume of trichloroacetic acid (20%, w/v) and the proteins that are precipitated are removed by centrifugation. Radioactivity in the supernatant is separated by HPLC on an ODS C₁₈ reverse-phase HPLC column as described in Fig. 1. Panel A shows the HPLC profile of radiolabeled metabolites of [³H]-HNE in the urine. Chemical identity of the radiolabeled peak eluting at 26 min, corresponding to the retention time of reagent NAC-DHN, is established by ESI⁻-MS. As shown in Panel B, ESI⁻-MS of these peak shows a strong molecular ion with m/z value of 320.1, consistent with [M - H]⁻ of NAC-DHN.

Figure 5. Effect of HNE and its glutathionyl conjugates on redox signaling