Formation of Monohydroxy Derivatives of Arachidonic Acid, Linoleic Acid
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Formation of monohydroxy derivatives of arachidonic acid, linoleic acid, and oleic acid during oxidation of low density lipoprotein by copper ions and endothelial cells Tao Wang,' Wen@ Yu, and William S. Powell* MeakinsChristie Laboratories, Respiratory Health Network of Centers of Excellence, Department of Medicine, McGill University, 3626 St. Urbain Street, Montreal, Quebec, Canada H2X 2P2; and the Endocrine Laboratory, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec, Canada, H3A 1Al Abstract An important event in the formation of arachidonic acid, linoleic acid, and oleic acid during oxida- atherosclerotic lesions is the uptake of modified low density tion of low density lipoprotein by copper ions and en- lipoprotein (LDL) by macrophages via scavenger receptors. dothelial cells. J. Lipid Res. 1992. 33: 525-537. Modification of LDL, which results in its recognition by these receptors, can be initiated by peroxidation of LDL Supplementary key words lipid peroxidation unsaturated fatty lipids. The first step in this process is the formation of acids conjugated dienes thiobarbituric acid-reactive substances monohydroperoxy derivatives of fatty acids, which are sub gas chromatography-mass spectrometry sequently degraded to the corresponding monohydroxy compounds, or to a variety of secondary oxidation products. In order to understand this process more completely, we One of the pathological characteristics of have developed a mass spectrometric procedure to measure atherosclerosis is the formation of lipid-laden cells the amounts of specific hydroperoxy/hydroxy fatty acids formed by oxidation of the major unsaturated fatty acids in (foam cells) underneath the endothelium of arteries. human LDL, oleic acid, linoleic acid, and arachidonic acid. Many of these foam cells are present in the early stages Oxidation of human LDL in the presence of a relatively of the lesion and are postulated to be derived from cir- strong stimulus (20 p~ CuSO4) resulted in very large in- culating monocytes which have taken up low density creases in the amounts of the major monohydroxy deriva- lipoprotein (LDL) (1, Although normal LDL is not tives of linoleic acid (9- and 13hydroxy derivatives) and 2). arachidonic acid (5-, 8-, 9-, 11-, 12-, and 15-hydroxy deriva- well taken up by monocytes (3), LDL that has been tives) in LDL lipids in the early stages of the reaction. After 20 h, the amounts of these products declined due to sub strate depletion, but large amounts of monohydroxy deriva- tives of oleic acid (8-, lo-, and 11-hydroxy derivatives) were detected. Although thiobarbituric acid-reactive substances Abbreviations: LDL, lowdensity lipoprotein; TBARS, thiobar- clearly increased under these conditions, the changes were bituric acid-reactive substances; WHHL, Watanabe heritable hyper- not nearly so dramatic as those observed for monohydroxy lipidemic; EDTA, ethylenediamine tetraacetic acid; ETYA, 5,8,11,14 fatty acids. Oxidation of LDL in the presence of endothelial eicosatetraynoic acid; MSTFA, "ethyl-Ntrimethylsilyltrifluorcl cells, a much milder stimulus, resulted in 2.5- to Sfold in- acetamide; HPLC, high pressure liquid chromatography; ODS, oc- creases in the amounts of monohydroxy derivatives of tadecylsilyl GC,gas chromatography; MS, mass spectrometry; HETE, linoleic and arachidonic acids, as well as thiobarbituric acid- hydroxyeicosatetraenoic acid; PUFA, polyunsaturated fatty acids; h- reactive substances, with more modest increases in the 18:0, hydroxyoctadecanoic acid, h-20:0, hydroxyeicosanoic acid; 8h- amounts of hydroxylated derivatives of oleic acid. There was 18:1, 8-hydroxy-9-octadecenoic acid; 10h-18:1, 10-hydroxy-hc- little positional specificity in the oxidation of the above fatty tadecenoic acid; 11h-18:0, 1I-hydroxy-9-octadecenoic acid; 9h- 182, acids in the presence of either stimulus, suggesting that the 9-hydroxy-IO,12-octadecadienoic acid; 13h-18:1, 13-hydroxy-9,l I-oc- formation of these products proceeds primarily by lipid tadecadienoic acid; 5h-20:4, 5-hydroxy-6,8,11,14eicosatetraenoic peroxidation, rather than by catalysis by lipoxygenases. How- acid; 8h-20:4, &hydroxy-5,9,11,14-eicosatetraenoicacid; 9h-20:4, 9- ever, an important role for lipoxygenases in the initiation of hydroxy-5,7,11,14-eicosatetraenoic acid 1lh-20:4, 1 I-hydroxy- these reactions cannot be excluded. In conclusion, 5,8,12,lkicosatetraenoic acid; 12h-204, 12-hydroxy-5,8,10,14 oxidation of LDL in the presence of copper ions or en- eicosatetraenoic acid; 1531-204, 15-hydroxy-5,8,11,13-eicosate- dothelial cells results in the formation of a large number of traenoic acid. monohydroxy derivatives of oleic, linoleic, and arachidonic 'Present address: DCpartement de Pharmacplogie, FacultC de acids. The relative amounts of products formed from each MCdicine, Universitk de MontrCal, 2900, boul Edouard-Montpetit, C.P. 6128, succursale A, Montreal, QuCbec, Canada, H3C 357. of these fatty acids depends on the strength of the stimulus 'To whom correspondence and reprint requests should be ad- as well as the incubation time.-Wang, T., W-g. Yu, and W. dressed at: Meakins-Christie Laboratories, 3626 St. Urbain Street, S. Powell. Formation of monohydroxy derivatives of Montreal, Quebec, Canada, H2X 2P2. Journal of Lipid Research Volume 33, 1992 525 chemically modified by acetylation (3) or biologically MATERIALS AND METHODS modified by treatment with endothelial cells (4, 5), arterial smooth muscle cells (6), activated human Materials monocytes (7, 8), activated platelets (9-11) as well as Ham's F10 medium was purchased from Flow by extracts from human atherosclerotic lesions (12), is Laboratories, Inc. 1,1,3,3Tetramethoxypropane, avidly taken up via monocyte scavenger receptors. propyl gallate, and Diazold were obtained from The biological modification of LDL is accompanied Aldrich. Sodium chloride, potassium bromide, by increases in the amounts of thiobarbituric acid-reac- ethylenediamine tetraacetic acid (EDTA, disodium tive substances (TBARS) and other lipid peroxidation salt), ricinoleic acid (12h-18:1), and thiobarbituric products and is inhibited by superoxide dismutase (8, acid were purchased from the Sigma Chemical Com- 13), antioxidants (4), and lipoxygenase inhibitors pany. Anhydrous methanol and other solvents were (14), depending on the cell type. This suggests that from Fisher Scientific. Oleic, linoleic, and arachidonic this process is initiated by lipid peroxidation due acids were purchased from Nu-Chek Prep, Inc. either to superoxide, which is released from smooth muscle cells and monocytes, or to radicals formed Preparation of standards for quantitation of during the lipoxygenase-catalyzed oxidation of fatty monohydroxy fatty acids by mass spectrometry acids by endothelial cells. The importance of lipid Monohydroxy derivatives of oleic acid. Hydroxy deriva- peroxidation in the development of atherosclerosis is tives of oleic acid (h-18:l) were prepared by autooxida- supported by the finding that probucol, due to its an- tion of oleic acid by a procedure similar to the one tioxidant properties, prevented the formation of described in the literature for the preparation of lesions in WHHL rabbits (15-17). hydroxy derivatives of arachidonic acid (20). Oleic Lipid peroxidation is characterized by the abstrac- acid (10 mg; 99% purity) was dissolved in methanol tion of a hydrogen atom from an allylic methylene (40 ml) followed by the addition of 10 ml of 0.2 M Tris group to form an alkyl radical, followed by migration buffer containing cupric sulfate (final concentration of one of the double bonds and insertion of molecular of 100 pM). The reaction mixture was stirred for 5 h at oxygen to give a peroxyl radical. Peroxyl radicals are room temperature in the presence of hydrogen converted to hydroperoxides as a result of abstraction peroxide (0.36 mmol; 400 pl of a 30% solution) which of a hydrogen atom from another molecule of un- was added at 0, 1, and 3 h of the incubation. The reac- saturated fatty acid, initiating a chain reaction. The tion mixture was then diluted with H20 (100 ml) and position at which oxygen is inserted depends on which extracted on an ODSsilica SepPak (21). The hydro- hydrogen is abstracted. In the case of a monoun- peroxy products were reduced with sodium saturated fatty acid, oxygenation occurs at either end borohydride and purified using an open column of of the two possible allylic radicals. Therefore, four silicic acid (22). This was followed by normal-phase- positional isomers, substituted in the 8, 9, 10, and 11 high pressure liquid chromatography (NP-HPLC) positions, will be formed from oleic acid (18). In the purification on a 5 pm RoSil silicic acid column (350 x case of polyunsaturated fatty acids, each 1,4cis,ci*pen- 4.6 mm; Alltech Associates) using a mobile phase con- tadiene group will give rise to two positional isomers. sisting of isopropanol-hexane-acetic acid 0.9:99:0.1. For example, 9- and 13-hydroperoxy isomers will be 8H-18:l had a retention time of 48 min (Fig. 1). Frac- formed from linoleic acid (18),whereas 5-, 8-, 9-, 11-, tions containing either 10h-18:l (27.5-29.5 min) or 12-, and 15-hydroperoxy isomers will be formed from llh-18:l (26-27.5 min) were further purified by arachidonic acid (19). reversed-phase-high pressure liquid chromatography The objectives of the present study were to charac- (RP-HPLC) on a 5-pm Novapak octadecylsilyl silica terize the monohydroxy fatty acids formed by (ODSsilica) column (150 x 3.9 mm; Waters-Millipore) peroxidation of unsaturated fatty acids in LDL, before using a gradient between water-acetonitrile-acetic and after treatment with copper ions or endothelial acid 60:40:0.02 and water-acetonitrile-acetic acid cells. Two distinct patterns for the formation of 35:65:0.02 over 30 min followed by isocratic elution monohydroxy fatty acids in the presence of Cu2+were with the latter mobile phase. 10H-18:l and llh-18:l apparent. Products formed from oleic acid increased had retention times of 33.4 min and 30.1 min, respec- linearly with time, so that after 20 h, they were the tively.