Isoketals form cytotoxic phosphatidylethanolamine adducts in cells
2009; Elsevier BV; Volume: 51; Issue: 5 Linguagem: Inglês
10.1194/jlr.m001040
ISSN1539-7262
AutoresC. Blake Sullivan, Elena Matafonova, L. Jackson Roberts, Venkataraman Amarnath, Sean S. Davies,
Tópico(s)Metabolomics and Mass Spectrometry Studies
ResumoLevuglandins and their stereo- and regio-isomers (termed isolevuglandins or isoketals) are γ-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 (LC50 2.2 μM). 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. Levuglandins and their stereo- and regio-isomers (termed isolevuglandins or isoketals) are γ-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 (LC50 2.2 μM). 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. Levuglandins and their regio- and stereo-isomers (isolevuglandins or isoketals) are highly reactive γ-ketoaldehydes (IsoK) formed by nonenzymatic rearrangement of prostaglandin H2 and their free radical generated counterparts (H2-isoprostanes) (1Salomon R.G. Miller D.B. Zagorski M.G. Coughlin D.J. Solvent induced fragmentation of prostaglandin endoperoxides. New aldehyde products from PGH2 and novel intramolecular 1,2-hydride shift during endoperoxide fragmentation in aqueous solution.J. Am. Chem. Soc. 1984; 106: 6049-6060Crossref Scopus (90) Google Scholar, 2Salomon R.G. Miller D.B. Levuglandins: isolation, characterization, and total synthesis of new secoprostanoid products from prostaglandin endoperoxides.Adv. Prostaglandin Thromboxane Leukot. Res. 1985; 15: 323-326PubMed Google Scholar, 3Salomon R.G. Sha W. Brame C. Kaur K. Subbanagounder G. O'Neil J. Hoff H.F. Roberts II, L.J. 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One feature of IsoKs that drew immediate interest was their extreme reactivity toward the lysyl residues of proteins, with reaction rates several orders of magnitude faster than the α, β-unsaturated aldehyde 4-hydroxynonenal, perhaps the most widely studied lipid peroxidation product (5Brame C.J. Salomon R.G. Morrow J.D. Roberts II, L.J. Identification of extremely reactive gamma-ketoaldehydes (isolevuglandins) as products of the isoprostane pathway and characterization of their lysyl protein adducts.J. Biol. Chem. 1999; 274: 13139-13146Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 6Iyer R.S. Ghosh S. Salomon R.G. Levuglandin E2 crosslinks proteins.Prostaglandins. 1989; 37: 471-480Crossref PubMed Scopus (77) Google Scholar). This increased reactivity derives from the facile formation of stable pyrrole adducts on primary amines. In the presence of oxygen, these pyrrole adducts convert over time into highly stable lactam adducts, as well as hydroxylactam adducts (5Brame C.J. Salomon R.G. Morrow J.D. Roberts II, L.J. Identification of extremely reactive gamma-ketoaldehydes (isolevuglandins) as products of the isoprostane pathway and characterization of their lysyl protein adducts.J. Biol. Chem. 1999; 274: 13139-13146Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 7DiFranco E. Subbanagounder G. Kim S. Murthi K. Taneda S. Monnier V.M. Salomon R.G. Formation and stability of pyrrole adducts in the reaction of levuglandin E2 with proteins.Chem. Res. Toxicol. 1995; 8: 61-67Crossref PubMed Scopus (34) Google Scholar). 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Med. 2006; 40: 1210-1219Crossref PubMed Scopus (61) Google Scholar, 14Poliakov E. Brennan M.L. Macpherson J. Zhang R. Sha W. Narine L. Salomon R.G. Hazen S.L. Isolevuglandins, a novel class of isoprostenoid derivatives, function as integrated sensors of oxidant stress and are generated by myeloperoxidase in vivo.FASEB J. 2003; 17: 2209-2220Crossref PubMed Scopus (46) Google Scholar). Whether IsoK adducts contribute to pathogenesis of these diseases or are simply byproducts of the disease remains an active area of investigation. Evidence supporting a potential role in pathogenesis comes from treatment of cultured cells with exogenous IsoK, which induces a variety of responses relevant to disease, including increased macrophage uptake of LDL, activation of platelet aggregation via p38-MAP kinase, inhibition of sodium and potassium channels, inhibition of proteasome function, and cytotoxicity (9Fukuda K. Davies S.S. Nakajima T. Ong B.H. Kupershmidt S. Fessel J. Amarnath V. Anderson M.E. Boyden P.A. Viswanathan P.C. et al.Oxidative mediated lipid peroxidation recapitulates proarrhythmic effects on cardiac sodium channels.Circ. Res. 2005; 97: 1262-1269Crossref PubMed Scopus (111) Google Scholar, 15Hoppe G. Subbanagounder G. O'Neil J. Salomon R.G. Hoff H.F. Macrophage recognition of LDL modified by levuglandin E2, an oxidation product of arachidonic acid.Biochim. Biophys. Acta. 1997; 1344: 1-5Crossref PubMed Scopus (39) Google Scholar, 16Bernoud-Hubac N. Alam D.A. Lefils J. Davies S.S. Amarnath V. Guichardant M. Roberts Ii L.J. Lagarde M. Low concentrations of reactive [gamma]-ketoaldehydes prime thromboxane-dependent human platelet aggregation via p38-MAPK activation.Biochim. Biophys. Acta. 2009; 1791: 307-313Crossref PubMed Scopus (21) Google Scholar, 17Brame C.J. Boutaud O. Davies S.S. Yang T. Oates J.A. Roden D. Roberts II, L.J. Modification of proteins by isoketal-containing oxidized phospholipids.J. Biol. Chem. 2004; 279: 13447-13451Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 18Davies S.S. Amarnath V. Montine K.S. Bernoud-Hubac N. Boutaud O. Montine T.J. Roberts II, L.J. Effects of reactive gamma-ketoaldehydes formed by the isoprostane pathway (isoketals) and cyclooxygenase pathway (levuglandins) on proteasome function.FASEB J. 2002; 16: 715-717Crossref PubMed Scopus (93) Google Scholar, 19Davies S.S. Modulation of protein function by isoketals and levuglandins.Subcell. Biochem. 2008; 49: 49-70Crossref PubMed Scopus (11) Google Scholar). The mechanism by which IsoK induces these effects has been presumed to be its adduction to cellular proteins, because of the high concentration of lysyl residues in cells. However, these lysyl residues are not the only potential target of IsoK present in cells. In vitro, IsoK can form adducts with a variety of other primary amines including the aminophospholipid phosphatidylethanolamine (PE) (20Amarnath V. Amarnath K. Amarnath K. Davies S. Roberts II, L.J. Pyridoxamine: an extremely potent scavenger of 1,4-dicarbonyls.Chem. Res. Toxicol. 2004; 17: 410-415Crossref PubMed Scopus (83) Google Scholar, 21Bernoud-Hubac N. Fay L.B. Armarnath V. Guichardant M. Bacot S. Davies S.S. Roberts II, L.J. Lagarde M. Covalent binding of isoketals to ethanolamine phospholipids.Free Radic. Biol. Med. 2004; 37: 1604-1611Crossref PubMed Scopus (42) Google Scholar, 22Davies S.S. Brantley E.J. Voziyan P.A. Amarnath V. Zagol-Ikapitte I. Boutaud O. Hudson B.G. Oates J.A. Roberts II, L.J. Pyridoxamine analogues scavenge lipid-derived gamma-ketoaldehydes and protect against H(2)O(2)-mediated cytotoxicity.Biochemistry. 2006; 45: 15756-15767Crossref PubMed Scopus (54) Google Scholar). In phosphate buffer, the reaction rate of γ-ketoaldehydes to form pyrrole adducts with ethanolamine, the head group of the PE, is 4.4-fold faster than with lysine (20Amarnath V. Amarnath K. Amarnath K. Davies S. Roberts II, L.J. Pyridoxamine: an extremely potent scavenger of 1,4-dicarbonyls.Chem. Res. Toxicol. 2004; 17: 410-415Crossref PubMed Scopus (83) Google Scholar). Oxidation of multilaminar vesicles containing PE or of isolated high density lipoprotein in vitro produces pyrrolized phospholipids (measured by reactivity with Ehrlich reagent), suggesting the robust modification of PE by pyrrole forming lipoxidation products under these conditions (23Hidalgo F.J. Nogales F. Zamora R. Determination of pyrrolized phospholipids in oxidized phospholipid vesicles and lipoproteins.Anal. Biochem. 2004; 334: 155-163Crossref PubMed Scopus (23) Google Scholar). Although the biological effects of IsoK-modified PE have not been studied, modification of the ethanolamine headgroup of PE by IsoK is likely to dramatically affect the physical properties of PE as it converts the neutral PE to an acidic phospholipid and significantly increases headgroup bulk. PE modified by IsoK might also activate receptor-mediated signaling in a manner analogous to N-acyl-PE, which suppresses food intake via cFOS expression in neuropeptide Y neurons, and suppresses phagocytosis via Rac1 and Cdc42 in macrophages (24Gillum M.P. Zhang D. Zhang X-M. Erion D.M. Jamison R.A. Choi C. Dong J. Shanabrough M. Duenas H.R. Frederick D.W. et al.N-acylphosphatidylethanolamine, a gut- derived circulating factor induced by fat ingestion, inhibits food intake.Cell. 2008; 135: 813-824Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 25Shiratsuchi A. Ichiki M. Okamoto Y. Ueda N. Sugimoto N. Takuwa Y. Nakanishi Y. Inhibitory effect of N-palmitoylphosphatidylethanolamine on macrophage phagocytosis through inhibition of Rac1 and Cdc42.J Biochem. 2009; 145: 43-50Crossref PubMed Scopus (23) Google Scholar). We therefore sought to determine the extent to which IsoK adducts to PE compared with proteins and whether IsoK-PE could mediate IsoK-induced cytotoxicity. L-α-phosphatidylethanolamine (PE), transphosphatidylated from chicken egg phosphatidylcholine was purchased from Avanti Polar lipids (Alabaster, AL). According to the manufacturer and our LC/MS analysis, the major species of PE in this product is 1-palmitoyl-2-oleolyl-sn-glycerophosphotidylethanolamine (C34:1 PE). All solvents were HPLC grade and were purchased from EMD Biosciences. Streptomyces chromofuscus phospholipase D (PLD) was purchased from Enzo Life Sciences International (Plymouth Meeting, PA), and Proteinase K was purchased from Clontech (Mountain View, CA). Human embryonic kidney (HEK293) cells were purchased from ATCC (Manassas, VA). Endothelial cells derived from donated human umbilical cords (HUVEC) were a kind gift from Dr. Matthew Duvernay. Endothelial cell basal medium was purchased from Lonza Walkersville Inc. (Walkersville, MD). [2H4]ethanolamine was purchased from Cambridge Isotopes (Cambridge, MA). All other chemicals were from Sigma-Aldrich. 15-E2-isoketal (IsoK) was prepared from dimethoxyacetal precursor (DMA) in methylene chloride as previously described (26Amarnath V. Amarnath K. Masterson T. Davies S. Roberts L.J. A simplified synthesis of diastereomers of Levuglandin E2.Synth. Commun. 2005; 35: 397-408Crossref Scopus (24) Google Scholar) and a stock of 10 mM IsoK prepared in DMSO by evaporation of methylene chloride under nitrogen. For synthesis of [3H4]15-E2-IsoK methyl ester, oct-1-yn-3-ol in acetone was oxidized with chromic acid (room temperature, 1 h) to oct-1-yn-3-one. The ketone was purified by distillation (20–25°C/1 Torr) and reduced with NaB3H4 to [3-3H4]oct-1-yn-3-ol. The latter was then incorporated into IsoK methyl ester as described (26Amarnath V. Amarnath K. Masterson T. Davies S. Roberts L.J. A simplified synthesis of diastereomers of Levuglandin E2.Synth. Commun. 2005; 35: 397-408Crossref Scopus (24) Google Scholar). HEK293 cells were grown on 100mm dishes to >90% confluence. Cells were then scraped, transferred, washed twice in Dulbecco Phosphate Buffer Saline (DBPS), and then resuspended in 2 ml DPBS. [3H4]15-E2-IsoK methyl ester in DMSO (final concentration 1 μM) was added to the resuspended cells and incubated for 2 h while gently rocking. To separate protein adducts from phospholipid adducts, 10 ml of chloroform/methanol (2:1) was added, and the lower (chloroform) phase removed for counting by liquid scintillation. The protein pellet at the interface between the aqueous and chloroform phase was then also removed and counted. To measure the amount of DNA adducts, replicate HEK293 cells were treated with [3H4]15-E2-IsoK methyl ester, and after the 2 h incubation, the cells were pelleted by centrifugation and resuspended in 0.5 ml lysis buffer (100 mM Tris HCl, pH 8.5, 5 mM EDTA, 0.2% SDS, 200 mM NaCl, 100 ug Proteinase K/ml). The resulting lysate was precipitated with isopropanol, and the supernatant discarded. The DNA precipitant was then counted by liquid scintillation. Synthesis of IsoK-PE was performed by two methods. IsoK-PE was initially synthesized as described previously (21Bernoud-Hubac N. Fay L.B. Armarnath V. Guichardant M. Bacot S. Davies S.S. Roberts II, L.J. Lagarde M. Covalent binding of isoketals to ethanolamine phospholipids.Free Radic. Biol. Med. 2004; 37: 1604-1611Crossref PubMed Scopus (42) Google Scholar). In brief, 1.25 μmol PE in CHCl3 was dried down under nitrogen and resuspended in 1ml of a two phase mixture of ether/PBS (4:1). 125 nmol IsoK (0.1 molar equivalents) in 12.5 μl DMSO was added to the sample and incubated for 2 h at 37°C. After incubation, the ether layer was dried off under nitrogen and 1ml chloroform/methanol (2:1) added. The resulting chloroform (lower) phase was transferred, dried under nitrogen, resuspended in 0.5 ml methanol/chloroform (9:1) and stored at −70°C until use. Subsequent experiment used PE in a single phase mixture of 1 M triethylammonium acetate/chloroform/ethanol (1:1:3) in place of the two phase ether/PBS solvent. IsoK-PE synthesized in this manner was used without further purification. The reaction of DMA with PE was performed with PBS/ether in a similar manner as the reaction of IsoK with PE, using 125 nmol DMA (0.1 molar equivalents) in 12.5 μl DMSO instead of IsoK. For the vehicle reaction, 12.5 μl DMSO was added to the PE. To synthesize IsoK-Etn, 20 μl neat ethanolamine was added to 20 nmol IsoK in 200 μl PBS and incubated for 2 h at 37°C. IsoK-[2H4]Etn was synthesized in a similar manner using 20 μl neat [2H4]ethanolamine. Triethylammonium acetate (1M) was used in some reactions in place of PBS. IsoK-Etn was then extracted with 3 vols chloroform/methanol (2:1), and the resulting product analyzed by mass spectrometry. For quantification of resulting pyrrole product, the reaction mixture was treated with Ehrlich reagent and the absorbance at 580 nm measure by spectrometer. Twenty-five nmol each of IsoK-PE reaction mixture was added to microfuge tubes, along with 784 pmol of IsoK-[2H4]Etn internal standard and dried under nitrogen. Additional samples containing internal standard only were also prepared. The lipids were resuspended in 50 μl methanol, vortexed, 450 μl HBSS added, and then sonicated for 5 min. 10 μl of PLD (>50,000 U/ml) was added to the appropriate tubes and all samples were incubated overnight at 37°C. Then 1.5 ml of chloroform/methanol (2:1) was added to extract IsoK-Etn, the chloroform layer dried down under nitrogen, resuspended in 50 μl methanol, and 10 μl injected unto LC/MS/MS for analysis. For limited mass scans of the products of the reaction between IsoK and the PE preparation, samples were injected into the ThermoFinnigan Quantum electrospray ionization triple quadrapole mass spectrometer operating in negative ion mode and scanning between m/z 600 and 1200. For collision induced disassociation (CID) spectrum, product ion scanning was performed on the m/z 1032.7 precursor ion, using argon gas in the collision cell. Collision energy was set at 50 eV. Similar analysis was performed on the PE preparation alone, with scanning performed at m/z 600 to m/z 1200, and a CID spectrum obtained for the precursor ion at m/z 716.5. For LC/MS/MS analysis of IsoK-PE, gradient HPLC was performed using a Agilent Zorbax XDB-C8 2.1 × 50 mm 5 μm column at a flow rate of 250 μl/min starting at 99% Solvent A (methanol/acetonitrile/1 mM ammonium acetate (60:20:20)) for 1 min, then a gradient ramp to 99% Solvent B (1 mM ammonium acetate in ethanol) at 7 min, and then holding at 99% Solvent B for 1 additional min before returning to the starting conditions. The HPLC eluant was directly connected to the mass spectrometer operating in multiple reaction monitoring (MRM) mode for m/z 1032.7→[email protected] and 1032.7→[email protected] for IsoK-C34:1 PE and m/z 716.6→[email protected] for C34:1 PE. For limited mass scans of the products of the reaction between IsoK and Etn or IsoK and [2H4]Etn, samples were injected into the mass spectrometer operating in positive ion mode and scanning between m/z 350 and 430. For CID spectrum, product ion scanning was performed on the m/z 378.3, 382.3, 394.3, 398.3, 410.3, and 414.3 ions using argon gas in the collision cell. Collision energy was set at 25 eV. For LC/MS/MS analysis of IsoK-Etn, gradient HPLC was performed using a XDB-C8 column at a flow rate of 500 μl/min starting at 100% Solvent A (water with 0.1% acetic acid) with a gradient ramp to 100% Solvent B (methanol with 0.1% acetic acid) at 3 min, and then holding at 100% Solvent B for 1 additional min before returning to the starting conditions. The HPLC eluant was directly connected to the mass spectrometer operating in multiple reaction monitoring mode for m/z 378.3→[email protected] (IsoK-Etn pyrrole), m/z 394.3→[email protected] (IsoK-Etn lactam), m/z 382.3→[email protected] (IsoK-[2H4]Etn pyrrole), and m/z 398.3→[email protected] (IsoK-[2H4]Etn lactam). IsoK (0–10 μM) was incubated with human embryonic kidney (HEK293) plated in duplicate wells of 6 well dishes containing HBSS for two h. The treatment media was then removed carefully to avoid disturbing the cell layer, and any unreacted IsoK in the treatment media extracted with hexane and measured by negative ion electrospray LC/MS/MS by monitoring MRM transitions m/z 351→m/z 271. To measure IsoK adducts in cells, 2 ml HBSS was immediately added to the plated cells after removing incubation media, the cell scraped, and cell and wash media transferred to clean centrifuge tubes. Six ml of chloroform/methanol (2:1) was added to each sample to extract the phospholipids, and the lower chloroform layer containing phospholipids was transferred to a new tube. The protein pellet at the interphase was also transferred to a separate tube. After addition of 1 nmol of IsoK-[2H4]Etn as the internal standard, the chloroform layer was dried down under nitrogen gas and resuspended in 50 μl methanol, vortexed, and then 450 μl HBSS added. The entire mixture was then sonicated for 5 min. Fifteen uL PLD was added to the samples and incubated overnight at 37°C. The samples were extracted with 1.5 ml chloroform/methanol (2:1). The chloroform (lower) layer was transferred, dried down under nitrogen, and resuspended in 50 μl methanol for LC/MS/MS analysis. Analysis of IsoK-protein adducts as IsoK-lysyl-lactam adduct after protease digestion was performed on the protein pellet as previously described after addition of 10 pmol IsoK-[13C615N2]lysyl-lactam the internal standard. Human umbilical vein endothelial cells (HUVEC; passage 6-8) were plated in 6 well dishes coated with 0.2% gelatin and allowed to grow to >90% confluence. The EBM medium was removed and the cells washed three times with HBSS and then duplicate wells treated with 0-1 μM IsoK. Analysis was then performed in similar manner as for HEK293 cells, except that 2 ml 0.25% Trypsin EDTA solution was added to plated cells, allowed to incubate for 3 min, and the cells and wash media removed and analyzed. HUVEC (passage 6-8) in EBM basal medium were plated into 96 well plates coated with 0.2% gelatin and allowed to grow to >90% confluence. The EBM medium was removed and the cells washed three times with HBSS. PE treated with 0.1 molar equivalent IsoK, DMA, or vehicle was prepared as described above, evaporated under nitrogen and resuspended in HBSS containing 0.1% BSA (HBSS/A). To determine the concentration dependence of IsoK-PE induced HUVEC cytotoxicity, the concentration of IsoK, DMA, or vehicle treated PE were calculated from the amount of IsoK or DMA added to PE, respectively. Therefore, for each stated treated PE concentration, the total PE concentration (modified and unmodified PE) was equivalent for each type of treatment and was exactly 10- fold higher than the stated treated PE concentration. IsoK-, DMA-, or vehicle-treated PE (0.1–30 μM final concentration) were each added to six replicate wells of HUVEC. Untreated control wells were used to determine maximum viability. Cells were incubated with their respective treated PE for 22 h, and then 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) added to each well for an additional two h, the culture media replaced with isopropanol and the extent of MTT conversion measured by the absorbance at 570 nm as previously described. Viability was normalized to the HBSS/A only treated cells. To determine the amount of IsoK-PE that entered the cells during the incubation process to that amount of IsoK-PE formed when IsoK was directly incubated with cells, replicate plates of HUVEC cells were treated with 3 μM IsoK-PE for 2 h, the incubation media removed, and IsoK-PE measured after PLD incubation as described for IsoK treated HUVEC cells. To identify significant cellular targets of IsoKs, a radiolabeled methyl ester ([3H4]IsoK-methyl ester) of 15-E2-IsoK, a representative IsoK, was incubated with HEK293 cells and the resulting adduct species separated by chloroform/methanol extraction. HEK293 cells were chosen for initial cellular studies because of their rapid growth characteristic and ease of maintenance. We found that only 8% of the radiolabel associated with the protein interphase, while 40% of the radiolabel associated with the chloroform lower phase that includes phospholipids (Fig. 2). The remaining radiolabel associated with the upper aqueous phase potentially containing adducts with small peptides, polyamines, nucleic acids, and other polar amines. When DNA was isolated directly from replicate treated cells, 2% of the radiolabel was associated with DNA. The distribution of radiolabel into the lower chloroform layer is consistent with the formation of substantial amounts of adducts with aminophospholipids such as PE; however, [3H4]IsoK methyl ester is itself quite lipophilic, so that the radiolabel in the lower chloroform layer could also represent unreacted IsoK or metabolites. Therefore, we sought definitive evidence for the formation of IsoK-aminophospholipid adducts in treated cells. The amount of PE adduct formed in cells should depend both on the relative amount of PE and the reaction rate of PE versus other primary amines. The model γ-ketoaldehyde, 4-oxopentanal (OPA) reacts with ethanolamine 4.4 times faster than the reaction rate with N-acetyl lysine (20Amarnath V. Amarnath K. Amarnath K. Davies S. Roberts II, L.J. Pyridoxamine: an extremely potent scavenger of 1,4-dicarbonyls.Chem. Res. Toxicol. 2004; 17: 410-415Crossref PubMed Scopus (83) Google Scholar). We determined the second order reaction rate of OPA and PE using methods similar to our previous study, except that we used a more lipophilic solvent system (1 M triethylammonium acetate/chloroform/ethanol 1:1:3 v/v/v) to maintain the solubility of the final product. In this lipophilic system, the reaction rate of OPA with PE was 4.5 × 10−3 M−1 s−1, which was 25 times faster than the reaction rate of OPA with N-acetyl lysine (0.18 × 10−3 M−1 s−1) that we had previously measured. To characterize the IsoK-PE adduct and develop an analytical method to quantify these adducts, we reacted a commercial preparation of PE primarily containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (C34:1 PE) and lesser amounts of C34:2 PE, C36:1 PE, and C36:2 PE (Fig. 3A) with a representative IsoK, 15-E2-IsoK (Fig. 3B) and compared the resulting products (Fig. 3C) by negative ion mass spectrometry. Addition of IsoK decreased the amount of signals at m/z 716.7 (C34:1PE), m/z 714.7 (C34:2 PE), m/z 744.7 (C36:1 PE), and m/z 742.7 (C36:2 PE) and led to the formation of new signals at m/z 1032.7, 1030.7, 1060.7, and 1058.7. The +316 amu mass shift of these novel peaks from the starting PE is consistent with formation of IsoK-pyrrole adducts for C34:1, C34:2, C36:1, and C36:2 PE, respectively. Under our reaction conditions, we did not observe formation of oxidized pyrrole adducts (lactam or hydroxylactam) even after overnight incubations, in contrast to our previous observations with IsoK protein adducts, probably because the organic solvents used for the PE reaction tend to exclude oxygen to a greater extent than aqueous buffers used for protein reactions. However, when the final PE product was dried down and left exposed to room air for several h, we observed a peak at 1048.7, consistent with the formation of a lactam adduct (not shown.) The CID spectrum of m/z 1032.7 includes major product ions at m/z 255, 281, 391, 512, and 750 (Fig. 4A). The product ions at m/z 255 and m/z 281 are consistent with the two fatty acid side chains (Fig. 4B), while the product at m/z 391 is consistent with a dehydrated C16:0 lysoPA product. The product ion at m/z 512 is consistent with the neutral loss of the two acyl chains from IsoK-PE, and the product ion at m/z 750 is consistent with the neutral loss of the sn-2 acyl chain. Given the characteristic fragmentation of IsoK-C34:1 PE, the expected product ions formed by other diacyl PE modified by IsoK can be readily predicted. Therefore, we used IsoK-C34:1 PE to develop an appropriate LC/MS/MS assay to measure the amount of IsoK-PE and PE. Modification of the HPLC method previously described for the separation of N-acyl-PE from PE, achieved sign
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