Isoketals form cytotoxic phosphatidylethanolamine adducts in cells

Abstract

Levuglandins and their stereo- and regio-isomers (termed isolevuglandins or isoketals) are gamma-ketoaldehydes (IsoK) that rapidly react with lysines to form stable protein adducts. IsoK protein adduct levels increase in several pathological conditions including cardiovascular disease. IsoKs can induce ion channel dysfunction and cell death, potentially by adducting to cellular proteins. However, IsoKs also adduct to phosphatidylethanolamine (PE) in vitro, and whether PE adducts form in cells or contribute to the effects of IsoKs is unknown. When radiolabeled IsoK was added to HEK293 cells, 40% of the radiolabel extracted into the chloroform lower phase suggesting the possible formation of PE adducts. We therefore developed methods to measure IsoK-PE adducts in cells. IsoK-PE was quantified by LC/MS/MS after hydrolysis to IsoK-ethanolamine by Streptomyces chromofuscus phospholipase D. In HEK293 and human umbilical vein endothelial cells (HUVEC), IsoK dose-dependently increased PE adduct concentrations to a greater extent than protein adduct. To test the biological significance of IsoK-PE formation, we treated HUVEC with IsoK-PE. IsoK-PE dose dependently induced cytotoxicity (LC(50) 2.2 muM). These results indicate that cellular PE is a significant target of IsoKs, and that formation of PE adducts may mediate some of the biological effects of IsoKs relevant to disease.

Fig. 1.

Pathways leading to the formation of γ-ketoaldehyde isomers and adducts. In vivo, arachidonic acid is converted to bicyclic endoperoxides either enzymatically by the action of cyclooxygenase working on free arachidonic acid (forming prostaglandin H₂) or nonenzymatically by free radical mediated lipid peroxidation of esterified arachidonic acid (forming H₂-isoprostanes). Nonenzymatic rearrangement of these bicyclic endoperoxide generates IsoKs, termed levuglandins and isolevuglandins (alternatively named isoketals). IsoKs react with the lysyl residues of proteins, and potentially other primary amines including the ethanolamine head group of PE, to rapidly form pyrrole adducts which convert to lactam and hydroxylactam adducts in the presence of oxygen. IsoK, γ-ketoaldehyde isomer; PE, phosphatidylethanolamine.

Fig. 2.

Distribution of IsoK adducts in cells. [³H]IsoK methyl ester (1 μM) was added to HEK293 cells for two h, and the distribution of the resulting adducts measured by liquid scintillation counting of the [³H] extracted into lower chloroform organic phase that includes phospholipids, into the protein interphase, and into isolated DNA. IsoK, γ-ketoaldehyde isomer.

Fig. 3.

IsoK reacts with PE to form pyrrole adduct. IsoK was incubated with a commercial preparation of PE and negative ion limited mass scanning from m/z 600 to 1200 performed on the starting reactants and reaction mixture to identify novel ions in the reaction mixture. A: Limited mass scans of the commercial PE preparation containing primarily C34:1 PE (m/z 716.7) as well as some C34:2 PE, C36:1 PE, and C36:2 PE. B: Limited mass scans of the IsoK preparation. C: Limited mass scans of the reaction mixture of PE and IsoK showing novel peaks with +316 amu mass shifts from the PE preparation, consistent with the formation of IsoK-PE pyrrole adducts. IsoK, γ-ketoaldehyde isomer; PE, phosphatidylethanolamine.

Fig. 4.

Development of IsoK-PE LC/MS/MS assay. A: CID spectrum of m/z 1032.7 ion. B: Interpretation of CID spectrum. C: MRM ion chromatographs from LC/MS/MS analysis of IsoK-PE preparation for C34:1 PE and IsoK-34:1 PE pyrrole. IsoK, γ-ketoaldehyde isomer. CID, collision induced disassociation; IsoK, γ-ketoaldehyde isomer; MRM, multiple reaction monitoring; PE, phosphatidylethanolamine.

Fig. 5.

IsoK treatment of HEK293 cells forms IsoK-PE. HEK293 were treated either with vehicle (0 μM IsoK, left panel) or 10 μM IsoK (right panel) for 2 h. MRM ion chromatographs of the IsoK pyrrole adducts for the most abundant diacyl PE species. IsoK, γ-ketoaldehyde isomer; MRM, multiple reaction monitoring; PE, phosphatidylethanolamine.

Fig. 6.

Identification of IsoK ethanolamine (Etn) adducts. IsoK was incubated with Etn and resulting products analyzed by positive ion electrospray ionization mass spectrometry. A: Limited mass scan spectrum of the reaction of IsoK with Etn produces ion consistent with pyrrole (m/z 378), lactam (m/z 394), Schiff base (m/z 396), and hydroxylactam (m/z 410) adducts. B: CID spectrum and interpretation of m/z 378 ion. C: CID spectrum and interpretation of m/z 394 ion. D: CID spectrum and interpretation of m/z 410 ion. CID, collision induced disassociation; IsoK, γ-ketoaldehyde isomer.

Fig. 7.

LC/MS/MS analysis of IsoK-Etn adducts. MRM ion chromatographs for the pyrrole, lactam, and hydroxylactam adducts in reaction mixtures containing Etn only, IsoK only, or both IsoK and Etn. IsoK, γ-ketoaldehyde isomer; MRM, multiple reaction monitoring.

Fig. 8.

Streptomyces chromofuscus PLD hydrolyzes IsoK-PE to IsoK-Etn. IsoK-PE preparation was treated with vehicle or S. chromofuscus PLD for 2 h, IsoK-[²H₄]Etn added as internal standard, and the resulting products analyzed by LC/MS/MS for IsoK-Etn. Left panel: MRM ion chromatographs for reaction with PLD only. Middle panel: MRM ion chromatographs for IsoK-PE treated with vehicle only. Right panel: MRM ion chromatographs for PLD treated IsoK-PE. IsoK, γ-ketoaldehyde isomer; MRM, multiple reaction monitoring; PE, phosphatidylethanolamine; PLD, phospholipase D.

Fig. 9.

PE adducts form to a greater extent than protein adducts in cells. IsoK was added for 2 h at increasing concentrations to HEK293 or HUVEC cells plated in six well plates and the amount of PE and protein adduct quantified by LC/MS/MS and normalized to amount of DNA in each well. A: Levels of PE and protein adduct in HEK293 cells incubated with 0-10 μM IsoK. B: Levels of PE and protein adduct in HUVEC cells incubated with 0-1 μM IsoK. IsoK, γ-ketoaldehyde isomer; PE, phosphatidylethanolamine.

Fig. 10.

IsoK-PE is cytotoxic to HUVEC. PE was treated with either 0.1 molar equivalent of IsoK (IsoK-PE), the inactive dimethoxy acetal synthetic precursor of IsoK (DMA-PE), or the DMSO vehicle (veh-PE) and 0.1 to 30 μM of the resulting PE reaction mixture incubated with HUVEC overnight. Viability was determined by MTT conversion assay and normalized to viability of untreated cells. HUVEC, human umbilical vein endothelial cell; IsoK, γ-ketoaldehyde isomer; PE, phosphatidylethanolamine.