Identification and Quantification of the Regioisomeric Cholesteryl

A Perspective on Free Radical Autoxidation: The Physical Organic Chemistry of Polyunsaturated Fatty Acid and Sterol Peroxidation. Ned A. Porter. The J...
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Chem. Res. Toxicol. 1996, 9, 737-744

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Identification and Quantification of the Regioisomeric Cholesteryl Linoleate Hydroperoxides in Oxidized Human Low Density Lipoprotein and High Density Lipoprotein James A. Kenar,† Christine M. Havrilla,† Ned A. Porter,*,† John R. Guyton,‡ Spencer A. Brown,‡ Keith F. Klemp,‡ and Elizabeth Selinger‡ Department of Chemistry, Paul M. Gross Laboratories, Duke University, and Department of Medicine, Duke University Medical Center, Durham, North Carolina 27708 Received January 19, 1996X

Oxidation of human LDL is implicated as an initiatior of atherosclerosis. Isolated low density lipoprotein (LDL) and high density lipoprotein (HDL2) were exposed to aqueous radicals generated from the thermolabile azo compound 2,2′-azobis(2-amidinopropane) dihydrochloride. The primary nonpolar lipid products formed from the autoxidation of LDL and HDL were the regioisomeric cholesteryl linoleate hydroperoxides. In LDL oxidations, 9- and 13-hydroperoxides with trans,cis conjugated diene were formed as the major oxidation products if endogenous R-tocopherol was present in the LDL. After extended oxidation of LDL, at the time when endogenous R-tocopherol was consumed, the two trans,cis conjugated diene hydroperoxides began to disappear and the 9- and 13-hydroperoxides with trans,trans conjugated diene appeared. At very long oxidation times, none of the primary products, the conjugated diene hydroperoxides, were present. In HDL2, which has only very low levels of antioxidants, both the 9- and 13-hydroperoxides with trans,cis conjugated diene and the 9- and 13-hydroperoxides with trans,trans conjugated diene were formed at early stages of oxidation. The corresponding alcohols were also formed in the HDL2 oxidations. A mechanistic hypothesis consistent with these observations is presented.

Introduction Low density lipoprotein (LDL)1 is the major carrier of cholesteryl esters in human blood plasma (1). Free radical-mediated modification of LDL may play a crucial role in the development of atherosclerosis, and much effort has been devoted to the study of lipoprotein oxidation and its prevention over the last decade (2, 3). Peroxidation of the LDL’s lipids affords hydroperoxides mainly from cholesteryl linoleate (Ch18:2), the predominant lipid component in the lipoprotein core (4). Hydroperoxide formation is generally thought to precede and result in the modification of apoprotein B-100, the single protein associated with LDL (5-7). These hydroperoxides can undergo decomposition and give a variety of secondary breakdown products, including reactive aldehydes that are known to react with the lysine residues of the apoprotein (8). Once modified, the apoprotein is taken up by scavenger receptors of monocyte-derived macrophages (9-12). Internalization of lipids in this manner is uncontrolled and leads to intracellular lipid accumulation and the appearance of lipid-laden foam cells similar to those found in early atherosclerotic lesions (13, 14). * Please address correspondence to this author. Phone: (919) 6601550; email: [email protected]. † Department of Chemistry, Duke University. ‡ Department of Medicine, Duke University Medical Center. X Abstract published in Advance ACS Abstracts, May 1, 1996. 1 Abbreviations: LDL, low density lipoproteins; HDL, high density lipoproteins; CoQ10H2, ubiquinol-10; PBS, phosphate buffered saline; AAPH, 2,2′-azobis(2-amidinopropane) dihydrochloride; DTBN, di-tertbutyl hyponitrite; 18:2, linoleic acid; 20:4, arachidonic acid; Ch18:2, cholesteryl linoleate; Ch18:2-OH, cholesteryl linoleate alcohol; Ch18: 2-OOH, cholesteryl linoleate hydroperoxide; Ch20:4, cholesteryl arachidonate.

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Understanding the earliest stages of LDL modification, in particular, lipid peroxidation and the protective role of antioxidants, is of fundamental importance since the clues found here may provide means to increase the resistance of LDL to oxidation and ultimately prevent atherosclerosis. The lipid-soluble antioxidant, R-tocopherol, is the major antioxidant associated with plasma and LDL, although minor amounts of γ-tocopherol, carotenoids, retinol, and ubiquinol-10 (COQ10H2) are also present (4). The protective role that these antioxidants play in lipoprotein oxidation has been extensively studied, although not fully understood (15-17). Lipoprotein oxidation is initiated by transition metals such as copper and iron (18), γ-radiation (19), and cultured cells that produce active oxygen (6, 20). Unfortunately, initiation of free radical chains by these methods is not reproducible, and this leads to ambiguities in the oxidation studies of the lipoproteins. The recent use of thermolabile watersoluble azo initiators as a radical source, although not biologically relevant, generates a constant flux of free radicals with known rate constants, and provides a valuable tool in which to study LDL oxidation (21, 22). In contrast to LDL, there are relatively few studies on the oxidation of high density lipoprotein (HDL). Recent reports suggest that HDL is more prone to radicalmediated oxidation and carries most of the cholesteryl ester hydroperoxides detectable in freshly obtained human plasma (23). Although oxidation studies of LDL and HDL have revealed hydroperoxide formation among the various lipid classes, i.e., cholesteryl esters, phospholipids, and free cholesterol, no studies have identified in detail the molecular species Ch18:2-OOH regioisomers and the role antioxidants may have on their distribution (24, 25). There is one literature report concerning the © 1996 American Chemical Society

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chemo-enzymatic synthesis and identification of cholesteryl 13-hydroperoxyoctadeca-cis-9,trans-11-dienoate, one of the Ch18:2-OOH isomers (24), and analyses of cholesterol ester hydroperoxides by the use of a chiral HPLC column have also been reported (25). In an effort to gain further insight into the formation and breakdown of lipid hydroperoxides in the early stages of LDL and HDL oxidation, we have isolated and completely characterized the major regioisomeric hydroperoxides and alcohols formed in the peroxidation of Ch18:2. In addition, we have established methodology based on high performance liquid chromatography (HPLC) to directly assay for the regioisomeric cholesteryl linoleate hydroperoxides and alcohols in oxidized LDL and HDL, and we have monitored oxidations of LDL and HDL initiated by a watersoluble azo initiator. Herein, we report the details and results of our investigations.

Materials and Methods General Methods. Thin layer chromatography was performed with the use of Whatman 250 µm silica plates (5 × 20 cm). The solvent system used was 15% ethyl acetate in hexane. 1H NMR spectra were recorded on a Varian XL 300 MHz spectrometer in CDCl3. Chemical shifts are reported in ppm (δ) with respect to the residual H signal in CDCl3 (δ ) 7.26 ppm), and coupling constants (J values) are given in hertz. Multiplicities are indicated by s (singlet), d (doublet), t (triplet), and m (multiplet). Analytical HPLC was conducted on a Waters Model 590 HPLC instrument with a Hewlett Packard Multiwavelength 1050 detector and a Hewlett Packard 3396 Series III integrator. The HPLC was equipped with two tandem Beckman 5 µm Ultrasphere columns (4.6 mm × 25 cm), 0.5% 2-propanol in hexane, and a delivery rate of 1 mL/min. Preparative HPLC was conducted on a Waters Model 600E HPLC instrument, with a Waters Model 481 variable wavelength detector operating at 234 nm and connected to a chart recorder. A Dynamax-60A Si 83-141-C column purchased from Rainin Instrument Co. (Woburn, MA), 0.66% 2-propanol in hexane, and a solvent delivery rate of 25 mL/min were used for the separations. Chemicals. Phosphate buffered saline (PBS, pH 7.4, 50 mM) was stored over Chelex-100 at least 24 h to remove transition metal contaminants. All chemicals were purchased from Aldrich Chemical Co. (Milwaukee, WI) and used without further purification, unless otherwise noted. Methyl linoleate was purchased from Nu-Chek Prep (Elysian, MN). The free radical initiator, 2,2′-azobis(2-amidinopropane) dihydrochloride, (AAPH) was obtained from Polyscience, Inc. (Warrington, MA). The free radical initiator di-tert-butyl hyponitrite (DTBN) was synthesized prior to use (26, 27). Organic solvents such as 2-propanol, hexane, benzene, and ethyl acetate were HPLC quality and were purchased from Mallinckrodt, Inc. (St. Louis, MO). The standard, methyl 13-hydroxyoctadeca-trans-9,trans-11-dienoate, was prepared by autoxidation of methyl linoleate followed by reduction with triphenylphosphine, and purification by preparative HPLC as described previously (28). The antioxidants, R-tocopherol and δ-tocopherol, were purchased from Sigma Chemical Co. (St. Louis, MO). Oxidation of Cholesteryl Linoleate. A round bottom flask was charged with 300 mg of cholesteryl linoleate and diluted to 0.20 M with freshly distilled benzene. The sealed flask was heated to 37 °C under an oxygen atmosphere, and after a brief period of equilibration, one crystal of DTBN was added and the solution stirred magnetically. Thin-layer chromatography showed the formation of peroxidic products, and the oxidation was continued until enough peroxide products were generated for convenient analysis. Analytical HPLC indicated that four major peaks were present in the product mixture with the following relative percentages: I, tR 15.0 min, 17.2%; II, tR 17.5 min, 32.0%; III, tR 20.1 min, 11.1%; and IV, tR 20.5 min, 39.7%. Preparative HPLC was used to isolate pure fractions of peaks

Kenar et al. I and II. Peaks III and IV could not be separated and were isolated as a mixture. Cholesteryl 13-Hydroperoxyoctadeca-cis-9,trans-11-dienoate (1). Analytical HPLC fraction I, tR 15.0 min; TLC Rf 0.48; 1H NMR δ 0.67 (s, 3H), 0.8-2.60 (66H), 4.38 (m, 1H), 4.504.70 (m, 1H), 5.36 (m, 1H), 5.40-5.65 (m, 2H), 6.00 (dd, J ) 10.6, 6.6 Hz, 1H), 6.57 (dd, J ) 15.3, 11.2 Hz, 1H), 7.83 (s, 1H). Cholesteryl 13-Hydroperoxyoctadeca-trans-9,trans-11dienoate (2). Analytical HPLC fraction II, tR 17.5 min; TLC Rf 0.48; 1H NMR δ 0.67 (s, 3H), 0.8-2.60 (66H), 4.33 (m, 1H), 4.50-4.70 (m, 1H), 5.36 (m, 1H), 5.47 (dd, J ) 15.1, 8.4 Hz, 1H), 5.75 (dt, J ) 15.0, 7.0, 7.5 Hz, 1H), 6.05 (dd, J ) 14.9, 10.5 Hz, 1H), 6.27 (dd, J ) 15.2, 10.2 Hz, 1H), 7.71 (s, 1H). Mixture of Cholesteryl 9-Hydroperoxyoctadeca-trans10,cis-12-dienoate (3) and Cholesteryl 9-Hydroperoxyoctadeca-trans-10,trans-12-dienoate (4). Analytical HPLC fraction III, tR 20.1 min: IV, tR 20.5 min; TLC Rf 0.48; 1H NMR δ 0.67 (s, 3H), 0.80-2.50 (66H), 4.28-4.41 (m, 1H), 4.50-4.70 (m, 1H), 5.30-5.60 (m, 2H, vinyl H’s 3; 1H, vinyl H 4; 1H, vinyl ring H), 6.58 (dd, J ) 15.3, 11.7 Hz, 1H, vinyl H 3), 6.27 (dd, J ) 15.2, 10.3 Hz, 1H, vinyl H 4), 6.04 (m, 1H, vinyl H 3; 1H, vinyl H 4), 5.75 (dt, J ) 15.3, 7.7, 6.7 Hz, 1H, vinyl H 4), 7.71 (s, 1H, OOH 4), 7.73 (s, 1H, OOH 3). Reduction of Cholesteryl Linoleate Hydroperoxides. A sample of the purified hydroperoxides (ca. 10-20 mg) was dissolved in 500 µL of diethyl ether along with triphenylphosphine (1.2 equiv) and allowed to stir (28). Thin-layer chromatography showed the disappearance of peroxidic products, and the reaction was continued until no peroxides were detected, approximately 30 min. The crude product was concentrated in vacuo, and preparative HPLC afforded pure samples of the four alcohols. Cholesteryl 13-Hydroxyoctadeca-cis-9,trans-11-dienoate (5). TLC Rf 0.39; Analytical HPLC tR 18.0 min; 1H NMR δ 0.67 (s, 3H), 0.80-2.50 (66H), 4.16 (m, 1H), 4.55-4.65 (m, 1H), 5.305.50 (m, 2H), 5.66 (dd, J ) 15.2, 6.8 Hz, 1H), 5.97 (dd, J ) 11.1, 6.0 Hz, 1H), 6.48 (dd, J ) 15.2, 11.1 Hz, 1H). Cholesteryl 13-Hydroxyoctadeca-trans-9,trans-11-dienoate (6). TLC Rf 0.35; Analytical HPLC tR 21.1 min; 1H NMR δ 0.67 (s, 3H), 0.80-2.5 (66H), 4.10 (m, 1H), 4.55-4.65 (m, 1H), 5.37 (m, 1H), 5.56 (dd, J ) 15.0, 7.1 Hz, 1H), 5.70 (dt, J ) 15.0, 7.5, 6.8 Hz, 1H), 6.02 (dd, J ) 14.8, 10.4 Hz, 1H), 6.17 (dd, J ) 15.0, 10.3 Hz, 1H). Cholesteryl 9-Hydroxyoctadeca-trans-10,cis-12-dienoate (7). TLC Rf 0.32; Analytical HPLC tR 27.0 min; 1H NMR δ 0.67 (s, 3H), 0.80-2.50 (66H), 4.15 (m, 1H), 4.50-4.70 (m, 1H), 5.30-5.50 (m, 2H), 5.66 (dd, J ) 15.2, 6.8 Hz, 1H), 5.97 (dd, J ) 11.1, 6.0 Hz, 1H), 6.48 (dd, J ) 15.3, 11.1 Hz, 1H). Cholesteryl 9-Hydroxyoctadeca-trans-10,trans-12-dienoate (8). TLC Rf 0.32; Analytical HPLC tR 29.5 min; 1H NMR δ 0.67 (s, 3H), 0.80-2.50 (66H), 4.10 (m, 1H), 4.50-4.70 (m, 1H), 5.37 (m, 1H), 5.56 (dd, J ) 15.0, 7.0 Hz, 1H), 5.70 (dt, J ) 15.0, 7.4, 6.0 Hz, 1H), 6.02 (dd, J ) 14.8, 6.0 Hz, 1H), 6.17 (dd, J ) 15.1, 10.3 Hz, 1H). Lipoprotein Isolation. Whole blood from fasting, normolipidemic healthy subjects was collected in a 440 mL ACD blood collection bag (Baxter) containing the following: 2 g of dextrose monhydrate; 1.66 g of sodium citrate dihydrate; 188 mg of anhydrous citric acid; 140 mg of monobasic sodium phosphate monohydrate; and 17.3 mg of adenine. Tubes containing blood were centrifuged at 2000g for 30 min at 10 °C. The lipoproteins, LDL (1.019 < d < 1.063 g/mL) and HDL2 (1.063 < d < 1.125 g/mL), were isolated from plasma over 3 days by density gradient sequential ultracentrifugation at 14 °C using a Beckman L 70 (Optima) centrifuge and a 50.2 Ti rotor. Each spin was performed at 100000g for 18 h. Lipoproteins were dialyzed extensively against 50 mM PBS, sterilized by passage through a Millex-HA 0.45 µM filter, and stored at 4 °C under argon. All protein concentrations of the lipoprotein preparations were determined by the method of Lowry (29). Oxidation and Extraction of Lipoproteins. The lipoprotein concentrations were adjusted to 1.5 mg of protein/mL with PBS, allowed to equilibrate to 37 °C for 5 min in a round bottom

Lipoprotein Autoxidation

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Figure 1. Products formed in the autoxidation of cholesteryl linoleate. flask. To the magnetically stirred solution was added AAPH (100 mM stock solution) to give a final AAPH concentration of 1 mM. Immediately after the addition of AAPH (time zero), and at various time intervals, two 200 µL and a 400 µL (LDL only) aliquots were removed for analysis. To both 200 µL aliquots were added 20 µL of BHT (25.5 mM in ethanol) and a known amount of the internal standard, methyl 13-hydroxyoctadecatrans-9,trans-11-dienoate (10). One of the 200 µL aliquots was reduced with triphenylphosphine and analyzed for C18:2-OH after extraction. The 400 µL aliquots taken for the R-tocopherol analyses had 50 µL of BHT (25.5 mM in ethanol) and a known amount of the internal standard δ-tocopherol added. Extraction of all the aliquots was performed immediately with (5 volumes of) ice-cold methanol and (25 volumes of) hexane in sequence, vortexed vigorously after the addition of each solvent (ca. 15 s) and then centrifuged at 1700 rpm for approximately 1 min with the use of an Adams analytical centrifuge. The hexane phase was removed by pipet and concentrated under argon, and the resulting organic residue was resuspended in 50 µL of HPLC solvent and immediately analyzed by analytical HPLC for Ch18: 2-OOH and Ch18:2-OH. The 400 µL aliquots were stored in dilute hexane solutions at -120 °C until analysis could be performed (usually within 4 days of sample preparation). The hexane was removed under a stream of argon, and the organic residue was resuspended in 50 µL of hexane and immediately assayed for R-tocopherol by analytical HPLC (30).

Results Oxidation of Cholesteryl Linoleate. The bond dissociation energy of a bis allylic carbon-hydrogen bond (CdCCH2CdC) is substanitially lower than that of an allylic carbon-hydrogen bond (CdCCH2) (31). For this reason, autoxidation of cholesteryl linoleate should give peroxidic products predominantly from linoleate side chain oxidation. Indeed, the azo-initiated bulk phase oxidation of 0.2 M cholesteryl linoleate, in benzene at 37 °C, generated four major peroxidic products, 1-4, in the ratio 1.5:2.9:1.0:3.6 (Figure 1). A typical HPLC analysis (λ ) 234 nm detection) of the Ch18:2-OOH isomeric product mixture is shown in Figure 2 (panel A). As can be seen, the two later eluting peaks III and IV coelute. Preparative HPLC cleanly isolated peaks I and II, while peaks III and IV were isolated as a mixture. Because the hydroperoxides readily decompose, the isolated peroxide fractions were immediately reduced with the use of triphenylphosphine to give their corresponding alcohols, 5-8 (Figure 1). By HPLC, the alcohols obtained from the isolated hydroperoxides are resolved from one another, Figure 2 (panel B). Preparative HPLC provided purified alcohol samples from peaks V-VIII, which were subsequently identified by 1H NMR and chemical transformations.

Figure 2. HPLC chromatograms (0.5% 2-propanol in hexane, λ ) 234 nm) from the autoxidation of cholesteryl linoleate with DTBN at 37 °C. (A) Isomeric Ch18:2-OOH’s: I ) 1; II ) 2; III ) 3; IV ) 4. (B) Isomeric Ch18:2-OHs obtained from Ch18:2OOH’s reduction with the use of PPh3: V ) 5; VI ) 6; VII ) 7; VIII ) 8.

The four alcohols gave two sets of essentially identical H NMR spectra; presumably, the cis,trans isomers 1 and 3 formed one set, while the trans,trans isomers 2 and 4 formed the other set. The geometry of the conjugated double bonds in the linoleate side chain was determined from the vinyl H’s coupling constants (Figure 3). Thus, the vinyl region of spectrum A is consistent with the cis,trans double bond geometry found in 1 and 3 and consists of signals at δ 5.54 [d,t, HD, (buried in the vinyl H of the cholesterol ring)], 5.66 [d,d, HA, JA,B ) 15.2 Hz, JA,CH ) 6.8 Hz)], 5.97 [d,d, HC, JC,D ) 11.1 Hz (cis, C, D)], and 6.48 [d,d, HB, JB,A ) 14.8 Hz (trans B, A)]. Likewise, the vinyl region of spectrum B is consistent with the trans,trans double bond geometry in 2 and 4 and consists of signals at δ 5.56 [d,d, HA, JA,B ) 15.0 Hz, JA,CH ) 7.0 Hz], 5.70 [d,t, HD, JD,C ) 15.0 Hz, JD,CH2 ) 7.4 and 6.0 Hz], 6.02 [d,d, HC, JC,D ) 14.8 Hz (trans C, D)], and 6.17 [d,d, HB, JB,A ) 15.1 Hz (trans B, A)]. Unfortunately, it was not possible to distinguish between 1

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Figure 3. Vinyl region of 1H NMR spectra of (A) cholesteryl 13-hydroxyoctadeca-cis-9,trans-11-dienoate (5) and cholesteryl 9-hydroxyoctadeca-trans-10,cis-12-dienoate (7); and (B) cholesteryl 13-hydroxyoctadeca-trans-9,trans-11-dienoate (6) and cholesteryl 9-hydroxyoctadeca-trans-10,trans-12-dienoate (8). R ) cholesterol.

the 9 and 13 position of the hydroxyl functionality by NMR; therefore, the position of the hydroxyl group was determined through chemical transformations. The isolated Ch18:2-OHs, 5-8, were transesterified with the use of NaOMe in MeOH to afford the hydroxy linoleate fatty acid Me esters 9-12. These Me esters have been previously characterized (28), and their HPLC elution order is known. Comparison of the Me esters obtained from the transesterification reaction to an authentic mixture allowed assignment of the -OH position in each Ch18:2-OH isomer. The elution order of alcohols 5-8 is shown in Figure 2 (panel B): peak V, 5; peak VI, 6; peak VII, 7; peak VIII, 8. The identities of the hydroperoxides 1-4, Figure 2 (panel A), were assigned by reduction to their alcohol derivatives: peak I, 1; peak II, 2; peak III, 3; peak IV, 4. Oxidation of LDL. With the ability to identify the isomeric Ch18:2-OOH and Ch18:2-OHs by HPLC, analysis of freshly extracted LDL samples by HPLC indicated no detectable traces of Ch18:2-OOH or Ch18:2-OH (UV detection at 234 nm). Exposure of isolated LDL to aqueous radicals, generated from the water-soluble azo initiator AAPH, results in the time-dependent formation of Ch18:2-OOH’s 1-4, and Ch18:2-OH’s 5-8, as shown from a representative oxidation experiment (Figure 4). Panel A in Figure 4 shows the concomitant formation of Ch18:2-OOH and Ch18:2-OH’s during the early stages of LDL oxidation. As can be seen, there is an initial 1 h lag time in which hydroperoxide formation is suppressed, after which time substantial amounts of Ch18:2-OOH begin to form, although approximately 90% of the R-tocopherol remains. Small amounts of isomeric Ch18:2OH’s are also produced during the initial stages of LDL oxidation, which are represented by the 9-trans,cis and trans,trans Ch18:2-OH’s in Figure 4. Unfortunately, the chromatographic region where the 13-Ch18:2-OH isomers elute has adjacent peaks that make accurate analysis difficult; therefore, we do not report the 13-Ch18:2-OHs (5 and 6) in Figure 4, although we assume that they are formed with equal proclivity as the 9-Ch18:2-OH’s (7 and 8).

Since the 9-trans,cis and 9-trans,trans Ch18:2-OOH isomers (3 and 4) coelute, analysis of their corresponding alcohols derived from Ch18:2-OOH reduction gives a more accurate picture of the hydroperoxide product distributions. Accordingly, aliquots from various times of the LDL oxidation were immediately reduced with the use of triphenylphosphine to give their corresponding isomeric Ch18:2-OH’s, which were then analyzed by HPLC. A typical HPLC analysis presented in Figure 4 (panel C) shows that, in the initial stages of oxidation, the cis,trans alcohols 5 and 7 predominate. For example, after 120 min of oxidation with 1 mM AAPH, the cis,trans/trans,trans [(5 + 7)/(6 + 8)] ratio is 14.7:1.0. Formation of 5 and 7 maximizes after approximately 8 h of oxidation under these conditions of intiation and then begins to decline as the oxidation continues. Meanwhile, production of the trans,trans Ch18:2-OHs, 6 and 8, increases and eventually exceeds 5 and 7 after 16 h of oxidation. It is of interest to note that by 16 h nearly all of the R-tocopherol has been consumed. At extended LDL oxidation times, there is a substantial time-dependent decrease in the amount of all Ch18:2OOH formed, presumably from the various hydroperoxide decomposition pathways. Interestingly, Ch18:2-OH was not produced as a result of these processes. The cholesteryl linoleate hydroperoxides obtained from oxidized LDL were also analyzed by reverse phase HPLC with 50% acetonitrile in 2-propanol and UV detection at 210 nm (data not shown) (32). The general lipid classes, cholesterol, Ch20:4, Ch18:2, and Ch18:1, were observed under these conditions in freshly extracted samples of LDL. No detectable traces of Ch18:2-OOH and Ch18:2OH were observed. As the LDL was oxidized, peaks corresponding to the formation of Ch18:2-OOH, Ch18:2OH, and Ch20:4-OOH were observed along with the simultaneous depletion of the Ch20:4 and Ch18:2 peaks. Oxidation of HDL2. An HPLC analysis for Ch18:2OOH and Ch18:2-OH similar to that used to monitor the LDL oxidation reported above was performed with the use of HDL2 as the lipid substrate. Freshly extracted

Lipoprotein Autoxidation

Figure 4. Free radical oxidation of LDL (1.5 mg of protein/ mL) initiated by 1 mM AAPH at 37 °C. At the time points indicated, samples were withdrawn, extracted, and analyzed by HPLC with UV detection (λ ) 234 nm) as described in the Materials and Methods. trans,cis-13-OOH Ch18:2 (1, 9); trans,trans-13-OOH Ch18:2 (2, 2); cis,trans-9-OOH Ch18:2 and trans,trans-9-OOH Ch18:2 (3 and 4, O); trans,cis-13-OH Ch18:2 (5, *); trans,trans-13-OH Ch18:2 (6, b); cis,trans-9-OH Ch18:2 (7, )); trans,trans-9-OH Ch18:2 (8, 3); R-tocopherol (+). A and B, analysis before PPh3 reduction; C, analysis after PPh3 reduction.

samples of HDL2 showed no detectable amounts of Ch18: 2-OOH or Ch18:2-OH isomers by HPLC (UV detection at 234 nm). Oxidation of HDL2 with aqueous radicals generated from AAPH results in the time-dependent formation of Ch18:2-OOH’s 1-4 and Ch18:2-OH’s 5-8 as shown from a typical experiment (Figure 5). As in the case of LDL, triphenylphosphine reduction of the isomeric Ch18:2-OOH’s gave their corresponding alcohols, which were then analyzed by HPLC to accurately determine hydroperoxide product distributions,

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Figure 5. Free radical oxidation of HDL2 (1.5 mg of protein/ mL) initiated by 1 mM AAPH at 37 °C. At the time points indicated, samples were withdrawn, extracted, and analyzed by HPLC with UV detection (λ ) 234 nm) as described in the Materials and Methods. trans,cis-13-OOH Ch18:2 (1, 9); trans,trans-13-OOH Ch18:2 (2, 2); cis,trans-9-OOH Ch18:2 and trans,trans-9-OOH Ch18:2 (3 and 4, O); trans,cis-13-OH Ch18:2 (5, *); trans,trans-13-OH Ch18:2 (6, b); cis,trans-9-OH Ch18:2 (7, )); trans,trans-9-OH Ch18:2 (8, 3). A and B, analysis before PPh3 reduction; C, analysis after PPh3 reduction.

Figure 5 (panel C). In HDL2, the cis,trans/trans,trans [(5 + 7)/(6 + 8)] ratio is only 2.6:1.0 after 120 min of oxidation with 1 mM AAPH (in LDL oxidations, this ratio was 14.7:1.0). As the oxidation proceeds, 6 and 8 formed concomitantly with alcohols 5 and 7, although at slightly lower levels. After 6 h, 6 and 8 production surpassed 5 and 7 formation (6 and 8 surpassed 5 and 7 after 16 h of LDL oxidation). Since fewer antioxidants are associated with HDL2 relative to LDL, any antioxidants present are probably consumed sooner in HDL2, which allows 6 and 8 to form earlier. HDL2 also shows a time-dependent

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Kenar et al. Scheme 1

decrease in the amount of all hydroperoxides without the formation of Ch18:2-OH, presumably from various hydroperoxide decomposition pathways.

Discussion Mechanistically, the four hydroperoxide products, 1-4, from cholesteryl linoleate autoxidation are formed as shown in Scheme 1 (33-35). First, hydrogen atom abstraction from C-11 of the linoleate side chain leads to the pentadienyl radical 13. Subsequent oxygen addition to 13 generates one of two trans,cis peroxyl radicals, 14 or 15, either of which has three possible options. Considering just the 9-substituted radical 14, hydrogen atom abstraction from a donor such as an antioxidant or another linoleate side chain generates 9-cis,trans hydroperoxide 3, a kinetic product. The other two mechanistic options involve β-fragmentation of the intermediate peroxyl radical. Nonproductive β-fragmentation gives the original pentadienyl radical 13. Alternatively, if bond rotation about C9-C10 precedes β-fragmentation, then a new pentadienyl radical 16 is formed. This radical has trans rather than cis geometry about the partial double bond ∆-9,10. Oxygen addition at C-13 then leads to a peroxyl radical that gives the 13trans,trans hydroperoxide 2, the thermodynamic product. The same processes can occur with the initially formed 13-substituted peroxyl radical 15. Lipid peroxidation product ratios are, therefore, determined by the competition between hydrogen atom abstraction from hydrogen donor molecules, like antioxidants, R-tocopherol, and ubiquinol-10, to give the cis,trans kinetic products and β-fragmentation to give the trans,trans thermodynamic products. In addition to the role antioxidants may play in preventing lipid peroxidation products, their presence should also efficiently trap any peroxyl radicals formed during the initial stages of oxidation as the kinetic 13and 9-cis,trans Ch18:2-OOH products, 1 and 3.

The results from this study demonstrate that peroxidation of isolated LDL and HDL2 induced by chemically generated aqueous radicals results in the formation of significant amounts of Ch18:2-OOH and lower levels of Ch18:2-OH after an initial 1 h lag time. After the lag time, substantial amounts of hydroperoxides begin to form, although approximately 90% of the R-tocopherol is still present in LDL. Recent results by Stocker et al. have shown ubiquinol-10, a minor endogenous antioxidant present in LDL and HDL, is a more effective antioxidant than R-tocopherol, and no hydroperoxides form until the ubiquinol-10 is consumed (4). Notably, in the early phases of LDL oxidation, we observe the exclusive formation of 13- and 9-cis,trans Ch18:2-OOH, 1 and 3. Apparently, the antioxidants present in LDL, and to a lesser extent in HDL2, effectively trap the initially formed cis,trans peroxyl radicals 14 and 15 to give the kinetic Ch18:2-OOH’s 1 and 3. Only after depletion of R-tocopherol, the main antioxidant present in LDL, do β-fragmentation and readdition reactions occur to give 2 and 4, the thermodynamic 13- and 9-trans,trans Ch18:2-OOH’s. The kinetic/thermodynamic (cis,trans/trans,trans) Ch18: 2-OOH product ratio in oxidized LDL and HDL2 was determined from the Ch18:2-OH’s obtained from Ch18: 2-OOH reduction. After 120 min of oxidation with 1 mM AAPH, there was approximately 14.7 times as much cis,trans Ch18:2-OH as trans,trans Ch18:2-OH in LDL, while in HDL2, only 2.6 times as much of the cis,trans Ch18:2-OH formed relative to the 13-trans,trans Ch18: 2-OH. In the absence of antioxidants, as in the LDL case when R-tocopherol is depleted after 16 h, β-fragmentation of the initially formed radicals (14 and 15) that lead to 13- and 9-cis,trans Ch18:2-OOH 1 and 3 can occur, and readdition of molecular oxygen leads to the 13- and 9-trans,trans Ch18:2-OOH, 2 and 4. It is also possible

Lipoprotein Autoxidation

that the trans,trans Ch18:2-OOH isomers observed in the early oxidation time points are formed by rearrangement of the cis,trans isomers even in the presence of antioxidants (35). In HDL2, 2 and 4 formed concomitantly, although at slightly lower levels, with 1 and 3. Since fewer antioxidants are associated with HDL2 relative to LDL, the antioxidants present in HDL2 are probably consumed sooner in the oxidation, allowing β-fragmentation to occur, and generating trans,trans Ch18:2-OOHs. In any case, one expects the product distibution of isomeric hydroperoxides to depend on the H atom donating propensity of the medium of oxidation, and the antioxidant-rich LDL should give more kinetic products (the 13and 9-cis,trans Ch18:2-OOH 1 and 3) than the antioxidant-poor HDL. Extended oxidation times showed a time-dependent decrease in the amount of Ch18:2-OOH in LDL and HDL2, presumably from various hydroperoxide decomposition pathways. Large amounts of the cholesteryl linoleate alcohols were not produced as a result of these processes. Recently, Stocker et al. reported Ch18:2-OH were formed simultaneously with Ch18:2-OOH in the early stages of LDL and HDL oxidation (36). After 180 min of oxidation with 2 mM AAPH, these workers found that the Ch18:2-OOH/Ch18:2-OH ratio ranged from 2.7 to 9 for LDL and from 1.0 to 3.5 for HDL. In our experiments, low levels of alcohols were also produced. For example, after 120 min of oxidation with 1 mM AAPH, the overall hydroperoxide/alcohol ratio was approximately 3.8:1.0 for LDL, while we determine a ratio of 2.6:1.0 for HDL2, in agreement with Stocker’s results. In addition, we observed only the formation of the 13- and 9-cis,trans Ch18: 2-OH, 5 and 7, in the early stages of the oxidation. As the oxidation continued, products 6 and 8 were formed in greater amounts than 5 and 7 (approximately 22 h for LDL, and 11 h for HDL2). The thermolabile AAPH used in this study as a radical generator initiates lipid peroxidation along the conventional chain pathway, which does not include significant formation of lipid hydroxides. In addition, we note that Ch18:2-OOH decomposition during the course of oxidation does not form significant amounts of Ch18:2-OH. Therefore, Ch18:2-OH formation was probably not produced via these pathways, which suggests an alternative pathway for their production. Stocker et al. have implied the involvement of phospholipid hydroperoxide glutathione peroxidase (PxGSH) which can use Ch18:2-OOH as a substrate (37). The role, if any, the cis,trans Ch18:2-OOH and Ch18: 2-OH play in the development of atherosclerotic plaques relative to their trans,trans isomers is presently unclear and warrants further investigation (38). Recently, 9-hydroxy Ch18:2 and free 9-hydroxy 18:2 were found to stimulate the release of interleukin 1, an inflammatory cytokine, from cultured human macrophages (39). Cholesteryl esters in atherosclerotic lesions are subject to enzymatic cleavage of the ester bond, producing free fatty acids that diffuse readily into cells. Enhancement of cell proliferation induced by epidermal growth factor was shown after administration of linoleic acid derivatives, 13-OOH and 13-OH, to cultured cells (40). The free 18: 2-OOH’s were also found to injure endothelial cells, stimulate collagen production, modulate production of matrix metalloproteinases by vascular cells, and activate endothelial phospholipase D (41, 42). These and other

Chem. Res. Toxicol., Vol. 9, No. 4, 1996 743

effects, acting over decades in concert with other cellular and molecular processes, may eventually lead to overt atherosclerotic disease. In addition, the oxidation products derived from arachidonic acid are known to generate prostaglandins, leukotrienes, and hydroxyeicosatetraenoic acids, which themselves initiate various cellular signaling cascades (43). Therefore, since cholesteryl arachidonate (Ch20:4) is also present in LDL and HDL, we have undertaken the identification and quantification of Ch20:4 oxidation products found in oxidized LDL and HDL. The results of this study will be reported in due course.

Acknowledgment. Support for this research by NIH HL17921 is gratefully acknowledged. C.M.H. acknowledges support from an NIEHS Toxicology Training Grant.

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