Covalent binding of hydroxy-alkenals 4-HDDE, 4-HHE, and 4-HNE to ethanolamine phospholipid subclasses
2003; Elsevier BV; Volume: 44; Issue: 5 Linguagem: Inglês
10.1194/jlr.m200450-jlr200
ISSN1539-7262
AutoresSandrine Bacot, Nathalie Bernoud‐Hubac, Naïma Baddas, Bernard Chantegrel, Christian Deshayes, Alain Doutheau, Michel Lagarde, Michel Guichardant,
Tópico(s)Alcohol Consumption and Health Effects
ResumoLipid oxidation is implicated in a wide range of pathophysiogical disorders, and leads to reactive compounds such as fatty aldehydes, of which the most well known is 4-hydroxy-2E-nonenal (4-HNE) issued from 15-hydroperoxyeicosatetraenoic acid (15-HpETE), an arachidonic acid (AA) product. In addition to 15-HpETE, 12(S)-HpETE is synthesized by 12-lipoxygenation of platelet AA. We first show that 12-HpETE can be degraded in vitro into 4-hydroxydodeca-(2E,6Z)-dienal (4-HDDE), a specific aldehyde homologous to 4-HNE. Moreover, 4-HDDE can be detected in human plasma. Second, we compare the ability of 4-HNE, 4-HDDE, and 4-hydroxy-2E-hexenal (4-HHE) from n-3 fatty acids to covalently modify different ethanolamine phospholipids (PEs) chosen for their biological relevance, namely AA- (20: 4n-6) or docosahexaenoic acid- (22:6n-3) containing diacyl-glycerophosphoethanolamine (diacyl-GPE) and alkenylacyl-glycerophosphoethanolamine (alkenylacyl-GPE) molecular species. The most hydrophobic aldehyde used, 4-HDDE, generates more adducts with the PE subclasses than does 4-HNE, which itself appears more reactive than 4-HHE. Moreover, the aldehydes show higher reactivity toward alkenylacyl-GPE compared with diacyl-GPE, because the docosahexaenoyl-containing species are more reactive than those containing arachidonoyl.We conclude that the different PE species are differently targeted by fatty aldehydes: the higher their hydrophobicity, the higher the amount of adducts made. In addition to their antioxidant potential, alkenylacyl-GPEs may efficiently scavenge fatty aldehydes. Lipid oxidation is implicated in a wide range of pathophysiogical disorders, and leads to reactive compounds such as fatty aldehydes, of which the most well known is 4-hydroxy-2E-nonenal (4-HNE) issued from 15-hydroperoxyeicosatetraenoic acid (15-HpETE), an arachidonic acid (AA) product. In addition to 15-HpETE, 12(S)-HpETE is synthesized by 12-lipoxygenation of platelet AA. We first show that 12-HpETE can be degraded in vitro into 4-hydroxydodeca-(2E,6Z)-dienal (4-HDDE), a specific aldehyde homologous to 4-HNE. Moreover, 4-HDDE can be detected in human plasma. Second, we compare the ability of 4-HNE, 4-HDDE, and 4-hydroxy-2E-hexenal (4-HHE) from n-3 fatty acids to covalently modify different ethanolamine phospholipids (PEs) chosen for their biological relevance, namely AA- (20: 4n-6) or docosahexaenoic acid- (22:6n-3) containing diacyl-glycerophosphoethanolamine (diacyl-GPE) and alkenylacyl-glycerophosphoethanolamine (alkenylacyl-GPE) molecular species. The most hydrophobic aldehyde used, 4-HDDE, generates more adducts with the PE subclasses than does 4-HNE, which itself appears more reactive than 4-HHE. Moreover, the aldehydes show higher reactivity toward alkenylacyl-GPE compared with diacyl-GPE, because the docosahexaenoyl-containing species are more reactive than those containing arachidonoyl. We conclude that the different PE species are differently targeted by fatty aldehydes: the higher their hydrophobicity, the higher the amount of adducts made. In addition to their antioxidant potential, alkenylacyl-GPEs may efficiently scavenge fatty aldehydes. Ethanolamine phospholipids (PEs) in biological membranes consist of two main subclasses: diacyl-glycerophosphoethanolamine (diacyl-GPE) and alkenylacyl-glycerophosphoethanolamine (alkenylacyl-GPE). The brain is particularly rich in plasmalogens, a unique class of glycerophospholipids exhibiting antioxidant properties due to the presence of a vinyl ether moiety at the sn-1 position of the glycerol backbone. The sn-2 position is acylated primarily by PUFAs such as arachidonic acid (AA) (20:4n-6) and docosahexaenoic acid (DHA) (22:6n-3). Plasmalogens represent 15–20% of total phospholipids (1Sun G.Y. Horrocks L.A. 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Biochem. 1999; 272: 48-55Google Scholar) and protein lysine residues with tyrosine-derived aldehydes (39Hazen S.L. Gaut J.P. Crowley J.R. Hsu F.F. Heinecke J.W. Elevated levels of protein-bound p-hydroxyphenylacetaldehyde, an amino-acid-derived aldehyde generated by myeloperoxidase, are present in human fatty streaks, intermediate lesions and advanced atherosclerotic lesions.Biochem. J. 2000; 352: 693-699Google Scholar). The first part of the present study was to examine whether 12-HpETE could be degraded into its putative aldehyde 4-HDDE as from 15-HpETE to 4-HNE, and to provide evidence that such a compound is formed in vivo. The second part of the study was to compare the ability of 4-HHE, 4-HNE, and 4-HDDE, which have different hydrophobicity, to covalently modify four molecular species of PE. These species were arachidonoyl- and docosahexaenoyl-containing PEs in both diacyl and plasmalogen subclasses from rat brain, chosen because of their biological relevance. All chemicals and reagents were analytical grade and purchased from Sigma-Fluka-Aldrich Chemical Co. (St. Quentin Fallavier, France). Analytical-grade organic solvents and silica TLC plates were from Merck (Nogent/Marne, France). HPLC columns were from Waters (St. Quentin en Yvelines, France). [3H]AA was from N.E.N. Dupont de Nemours (Les Ulis, France). HDDE was prepared from methyl 4-hydroxydodeca-(2E,6Z)-dienoate. To a solution of methyl 4-chlorophenylsulfinylacetate (1.15 g, 4.94 mmol) and piperidine (0.6 ml, 6.0 mmol) in acetonitrile (10 ml) was added dropwise a solution of (4Z)-decenal (0.95 g, 6.2 mmol) in acetonitrile (2.5 ml). The mixture was stirred overnight at room temperature. The solvent was evaporated under reduced pressure, and the residue was flash chromatographed on silica gel with dichloromethane-ether (19:1; v/v) as eluent to yield pure methyl 4-hydroxydodeca-(2E,6Z)-dienoate (0.8 g, yield 71%). To a stirred solution of methyl 4-hydroxydodeca-(2E,6Z)-dienoate (202 mg, 0.89 mmol) in anhydrous dichloromethane (10 ml) at −90°C under nitrogen was added dropwise a solution of 1.8 ml of 1M diisobutylaluminium hydride (DIBAL) in anhydrous dichloromethane diluted with 10 ml of anhydrous dichloromethane. The temperature was allowed to rise from −75°C to −70°C, and the mixture was stirred for 1 h. A 1 M hydrochloric acid aqueous solution (10 ml) was then added at −70°C, and the mixture allowed to warm to room temperature. The organic layer was separated and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was flash chromatographed on silica gel with pentane-ether (1:1; v/v) as eluent to yield pure 4-HDDE (77 mg, yield 44%). Nuclear magnetic resonance spectra were recorded in CDCl3 with a Bruker AC 200 (200/50 MHz) spectrometer. Chemical shifts are given in ppm and are referenced to CHCl3 resonances (7.26 and 77.0 ppm). Splitting pattern abbreviations are s, singlet; d, doublet; dd, doublet of doublet; ddd, doublet of doublet of doublet; t, triplet; and m, multiplet. Blood platelets were isolated from human volunteers (local blood bank) according to Lagarde et al. (40Lagarde M. Bryon P.A. Guichardant M. Dechavanne M. A simple and efficient method for platelet isolation from their plasma.Thromb. Res. 1980; 17: 581-588Google Scholar). Briefly, blood taken onto anticoagulant citric acid-trisodium citrate-dextrose was centrifuged at 150 g for 10 min. The supernatant platelet-rich plasma was acidified to pH 6.4 with citric acid and centrifuged at 900 g for 10 min, and the pellets were resuspended into Tyrode HEPES buffer, pH 7.35. Platelet suspension was preincubated under gentle stirring with 200 μM diamide to lower reduced glutathione (41Kosower N.S. Kosower E.M. Wertheim B. Correa W.S. Diamide, a new reagent for the intracellular oxidation of glutathione to the disulfide.Biochem. Biophys. Res. Commun. 1969; 37: 593-596Google Scholar) and then slow down GPx, and with 200 μM acetylsalicylic acid as a cyclooxygenase inhibitor (42Roth G.J. Majerus P.W. The mechanism of the effect of aspirin on human platelets. I. Acetylation of a particulate fraction protein.J. Clin. Invest. 1975; 56: 624-632Google Scholar), for 5 min at 37°C. Platelets were then incubated with 300 μM AA labeled with 37 kBq of [5,6,8, 9,11,12,14,15-3H]AA (7.77 TBq/mmol) for 5 min in the presence of oxygen. Platelet suspension was then acidified to pH 3 with 3 M HCl and treated three times with 3 vol of diethyl ether to extract AA derivatives. The organic phase was evaporated to dryness under vacuum. The 12-HpETE formation was checked with an aliquot of the lipid extract separated by TLC using the solvent mixture hexane-diethylether-acetic acid (60:40:1; v/v/v). Labeled compounds were detected by a Berthold TLC linear analyser. The dry lipid extract, which contained the radioactive 12-HpETE, was treated for 22 h at room temperature with 5 ml of 0.1 M HCl containing 0.5 M ascorbate and 0.02 M FeSO4 according to the procedure previously used by Lang et al. (43Lang J. Celotto C. Esterbauer H. Quantitative determination of the lipid peroxidation product 4-hydroxynonenal by high-performance liquid chromatography.Anal. Biochem. 1985; 150: 369-378Google Scholar) to synthesize 4-hydroxynonenal from 15-HpETE. The incubate was treated with 45 ml of the mixture chloroform-ethanol (2:1; v/v). The organic phase was removed, dried under vacuum, and the residue purified by TLC using the solvent mixture pentane-diethylether (50:50; v/v) as eluent. Standard 4-HDDE (30 μg), used as a reference, was spotted on the same plate, the radioactive band corresponding to the standard was scraped off, and the aldehyde was extracted three times with chloroform-ethanol (2:1; v/v). The dry purified aldehyde or crude plasma was treated with 200 μl of O-2,3,4,5,6-pentafluorobenzyl hydroxylamine hydrochloride (50 mM in 0.1 M PIPES buffer, pH 6.5) for 30 min at room temperature, according to the procedure described by Van Kuijk et al. (44Van Kuijk F.J. Siakotos A.N. Fong L.G. Stephens R.J. Thomas D.W. Quantitative measurement of 4-hydroxyalkenals in oxidized low-density lipoprotein by gas chromatography-mass spectrometry.Anal. Biochem. 1995; 224: 420-424Google Scholar). After acidification with 100 μl of 98% H2SO4, pentafluorobenzyloxime derivatives were extracted with 500 μl methanol and 2 ml hexane, and then purified by TLC using pentane-diethylether (70:30; v/v) as eluent. The radioactive band, corresponding to standard pentafluorobenzyl oxime derivative of 4-HDDE, was scraped off and extracted three times with chloroform-ethanol (2:1; v/v). The solvent was removed under nitrogen, and the hydroxyl group was converted into trimethylsilylether after an overnight treatment with N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) at room temperature. The pentafluorobenzyl oxime, trimethylsilylether-4-HDDE derivative (O-PFB-TMS-4-HDDE) was then analyzed by gas chromatography-mass spectrometry (GC-MS). These two aldehydes have been synthesized similarly to 4-HDDE. GC-MS was carried out on a Hewlett-Packard quadripole mass spectrometer interfaced with a Hewlett-Packard gas chromatograph (Les Ullis, France). The gas chromatograph was equipped with an HP-1MS fused-silica capillary column (30 m × 0.25 mm i.d., 0.25 μm film thickness) (Hewlett-Packard), which was held at 57°C. The following oven temperature program was used: 2 min at 57°C, then increased to 180°C at 20°C/min, followed by an increase to 280°C at 4°C/min. Samples were injected with a splitless injector with a head pressure of 7.9 psi. The interface, injector, and ion source were kept at 280°C, 260°C, and 130°C, respectively. Electron energy was set at 70 eV. Helium and methane were used as carrier and reagent gases, respectively. Mass spectra were acquired from 50 to 800 Da using both the electron ionization (EI) and the negative ion chemical ionization (NICI) modes. The electron multiplier voltage was usually set at 1,400 V. Rat brain lipids were extracted twice with a solvent mixture of chloroform-ethanol (2:1; v/v). Nonphosphorus lipids and phospholipids were separated according to Juaneda and Rocquelin (45Juaneda P. Rocquelin G. Rapid and convenient separation of phospholipids and non phosphorus lipids from rat heart using silica cartridges.Lipids. 1985; 20: 40-41Scopus (554) Google Scholar). Briefly, the lipid residue was dissolved in chloroform, and the mixture was loaded onto a Silica-Sep-Pak cartridge (solid-phase extraction) equilibrated with chloroform. Neutral lipids were washed through with chloroform, and total phospholipids were subsequently eluted with methanol. The alcoholic fraction was taken to dryness by rotary evaporation, and 2 ml of chloroform-ethanol (2:1; v/v) was added to each residue. Aliquots (500 μl) were taken for further TLC analysis. The different phospholipid classes were then separated by TLC using the solvent mixture chloroform-methanol-aqueous methylamine solution (14%) (60:20:5; v/v/v) as eluent. PE was detected by spraying the silica gel plate with 0.2% dichlofluorescein in ethanol. Silica gel was scraped off, and PE was extracted by a mixture of chloroform-ethanol (2:1; v/v). PE subclasses were then fractionated by reverse-phase HPLC (RP-HPLC) using an Agilent Technologies instrument model 1100 according to the procedure described by Khaselev and Murphy (46Khaselev N. Murphy R.C. Susceptibility of plasmenyl glycerophosphoethanolamine lipids containing arachidonate to oxidative degradation.Free Radic. Biol. Med. 1999; 26: 275-284Google Scholar) and modified as follows. PE was loaded onto a 3.9 × 300 mm NovaPak column packed with C18 silica (particle size, 4 μm). A flow rate of 1 ml/min was used, and the detection was achieved by monitoring UV absorbance of the effluent at 205 nm. The mobile phase consisted of a linear gradient elution with two eluents, A and B. Eluent A was a mixture of methanol-water-acetonitrile (100:14:2.5; v/v/v) containing 1 mM ammonium acetate adjusted to pH 7.4, and Eluent B contained the same solvents as A but in different proportions (70:4:2.5; v/v/v). Eluent A was pumped from 0 to 10 min at 100%, then was replaced by a linearly increasing percentage of solvent B to reach 100% at 155 min. Eluent B was held for 15 min, and then the system returned to the initial conditions within 5 min. Only four fractions were collected and taken to dryness under nitrogen. They were 1-stearoyl,2-arachidonoyl-GPE (18:0/20:4-GPE), 1-stearoyl,2-docosahexaenoyl-GPE (18:0/22:6-GPE), 1-O-stearyl-1′-enyl,2-arachidonoyl-GPE (18:0p/20:4-GPE), and 1-O-stearyl-1′-enyl,2-docosahexaenoyl-GPE (18:0p/22:6-GPE). Their purity was checked by measuring their fatty acyl content by gas chromatography (GC). Each fraction was treated separately with 500 μl of toluene-methanol (2:3; v/v) and 500 μl of 14% boron trifluoride in methanol. After 90 min at 100°C, the tubes were cooled to 0°C, and 1.5 ml K2CO3 in 10% water was added. The resulting fatty acid methyl esters (FAMEs) from diacyl-GPE, and FAME and dimethylacetals from alkenylacyl-GPE were extracted by 2 ml of isooctane and analyzed by GC with a DELSI instrument model DI 200 equipped with a fused silica capillary SP-2380 column (60 × 0.22 mm). Helium was used as the carrier gas at 1 ml/min. Temperatures of the Ross injector and the flame ionization detector were set at 230°C and 250°C, respectively. Diheptadecanoyl-GPE was used as internal standard and added to each fraction before derivatization. One equivalent of each molecular species of PE collected by HPLC was incubated under nitrogen with two equivalents of either 4-HHE, 4-HNE, or 4-HDDE in a biphasic system containing 200 μl buffer, pH 8, (0.75 M NaCl and 1 mM HEPES) and 800 μl of diethylether. The aldehyde was replaced by ethanol in the control. The incubation was performed for 2 h at room temperature under continuous vigorous stirring. The resulting adducts and the unreacted PE were then extracted with a mixture of chloroform-ethanol (2:1; v/v) and separated by TLC using the solvent mixture chloroform-methanol-aqueous methylamine solution (14%) (60:20:5; v/v/v) as eluent. Michael and Schiff base adducts were extracted all together from the silica, and were quantified by measuring their fatty acyl content by GC as previously described in Experimental Procedures. For this study, in which conventional mass spectrometry was used, racemic mixtures of 4-HDDE were elaborated in two steps from commercially available (4Z)-decenal (Ester 1) through the key intermediate ester (Ester 2), according to Scheme 1. Reaction of Ester 1 with methyl 4-chlorophenylsulfinylacetate [SPAC reaction (47Tanikaga R. Nozaki Y. Tamura T. Kaji A. Facile synthesis of 4-hydroxy-(E)-2-alkenoic esters from aldehydes.Synthesis. 1983; 2: 134-135Google Scholar, 48Burgess K. Henderson I. Lipase-mediated resolutions of SPAC reaction products.Tetrahedron Asymmetry. 1990; 1: 57-60Google Scholar)] yielded methyl 4-hydroxydodeca-(2E,6Z)-dienoate 2 (49Gunn B.P. Brooks D.W. Total synthesis of (+/−)-12-hydroxy-5(Z),8(Z),10(E),14(Z)-eicosatetraenoic acid (12-HETE).J. Org. Chem. 1985; 50: 4417-4418Google Scholar) (yield: 71%). 4-HDDE was easily obtained by reduction of Ester 2 with DIBAL (50Uchida K. Watanabe H. Kitahara T. Synthesis of (+/−)- (3R*, 4S*, 4aR*)-4,8-dihydroxy-3-methyl-3,4,4a,5-tetrahydro-1H–2-benzopyran-1-one.Heterocycles. 2000; 53: 539-542Google Scholar) (yield: 44%). NMR data and resonance attributions corresponding to 4-HDDE are as follows: [1H]NMR: 9.59 (d, J = 7.8 Hz, H-1); 6.85 (dd, J = 15.7, 4.3 Hz, H-3); 6.35 (ddd, J = 15.7, 7.8, 1.4 Hz, H-2); 5.73–5.60 (m, H-6); 5.45–5.31 (m, H-7); 4.60–4.40 (m, H-4); 2.43 (t, J = 7.0 Hz, 2H-5); 2.08–2.00 (m, 2H-8); 1.45–1.20 (m, 2H-9, 2H-10, 2H-11); 0.89 (t, J = 7 Hz, 3H-12). [13C]NMR: 193.61 (C-1); 158.40 (C-3); 135.10 (C-7); 130.92 (C-6); 122.90 (C-2); 70.54 (C-4); 34.57 (C-5); 31.50 (C-10); 29.21 (C-9); 27.44 (C-8); 22.54 (C-11); 14.04 (C-12). Chemically synthesized 4-HDDE was derivatized as previously described in Experimental Procedures and analyzed by GC-MS in the EI mode to confirm its chemical structure. Two syn- and antiisomers were eluted at 18.19 and 18.86 min, respectively (Fig. 1A). Electron impact mass spectra of both syn- and antiisomers show the same fragmentation pattern, except for the relative abundance of the different ions; the spectrum of the isomer eluted at 18.19 min is shown as an example (Fig. 1B). The base peak at m/z 352 corresponds to the loss of the alkyl chain (C8H15) from the molecular ion, and two other important ions at m/z 73 and 181 correspond to the trimethylsilyl (TMS) and pentafluorobenzyl (PFB) groups, respectively. Two minor characteristic ions were also detected: one at m/z 156 corresponds to a loss of pentafluorobenzaldehyde from the fragment at m/z 352, and one at m/z 129 derives from the m/z 156 ion, with a loss of cyanhydric acid. In ord
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