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LDL-Associated Phospholipase a Does Not Protect LDL Against Lipid Peroxidation In Vitro
Free Radical Biology & Medicine, Vol. 24, Nos. 7/8, pp. 1294 –1303, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/98 $19.00 1 .00
PII S0891-5849(97)00454-1
Original Contribution LDL-ASSOCIATED PHOSPHOLIPASE A DOES NOT PROTECT LDL AGAINST LIPID PEROXIDATION IN VITRO EDIT SCHNITZER,* ILYA PINCHUK,* MENACHEM FAINARU,† DOV LICHTENBERG,*
and
SAUL YEDGAR‡
*Departments of Physiology and Pharmacology, Tel-Aviv University, Sackler Faculty of Medicine, Tel-Aviv 69978, Israel; † Internal Medicine (Rabin Medical Center), Tel-Aviv University, Sackler Faculty of Medicine, Tel-Aviv 69978, Israel; and ‡ Department of Biochemistry, Hebrew University, Hadassah Medical School, Jerusalem, 91120, Israel (Received 13 November 1997; Accepted 26 November 1997)
Abstract—The irreversible proteinase inhibitor Pefabloc (4-[2-aminoethyl] benzenesulfonyl fluoride) inactivates LDLcatalyzed hydrolysis of the short-chain fluorescent phospholipid C6-NBD-PC (1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3diazole)-aminocaproyl phosphatidylcholine). The dose-dependence of this inactivation is similar to that obtained previously for the inhibitory effect of Pefabloc on the hydrolysis of platelet activating factor (PAF) by the LDLassociated PAF acetylhydrolase (PAF-AH), in agreement with the notion that the hydrolysis of C6-NBD-PC and PAF is catalyzed by the same enzyme (LDL-associated phospholipase A; LDL-PLA). This conclusion is also supported by the finding that hydrolysis of C6-NBD-PC by LDL becomes inactivated by LDL oxidation only at late stages of the oxidation, similar to the effect of oxidation on the hydrolysis of PAF by the LDL-associated PAF-AH. Under conditions of complete inactivation of this enzyme towards C6-NBD-PC, the kinetics of lipid peroxidation, induced either by copper ions or by the free radical generator AAPH at varying doses of the prooxidant, was similar to that observed when the PLA was active (i.e., in the absence of Pefabloc). Hence, LDL-associated PLA (PAF-AH) does not protect LDL lipids from peroxidation. Similar results were obtained with fractionated LDL in albumin-containing buffer and for non-fractionated serum, in which copper-induced peroxidation was also not influenced by inactivation of the enzyme responsible for hydrolysis of C6-NBD-PC. Phospholipolysis of short chain phospholipids by LDL-PLA may still play a protective role against the toxic effects of oxidized phospholipids by reducing their internalization into cells ( Schmitt et al. 1995). © 1998 Elsevier Science Inc. Keywords—Free radical, Lipid peroxidation, Low density lipoproteins (LDL), LDL-associated phospholipase A2 (LDL-PLA2 or LDL-PLA) Platelet activating factor (PAF), PAF acetylhydrolase (PAF-AH), Pefabloc (4-[2-aminoethyl]benzenesulfonyl fluoride), 1-Acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidylcholine (C6NBD-PC)
INTRODUCTION
B is no longer recognized by the LDL receptor and the normal internalization of LDL into the cells via the LDL receptor is therefore impaired. The alternative mechanism of LDL internalization, via the scavenger receptor, becomes the major pathway, and since this mechanism is not down-regulated it may result in excessive accumulation of lipids and in consequent cell death and formation of atherotic plaques.4,6 –7. Decomposition of the conjugated dienic hydroperoxides derived from oxidized PUFA results in phospholipids with fragmented acyl chains.8 These phospholipids can be hydrolyzed by the LDL-associated phospholipase A (PLA), which catalyses the hydrolysis of short chain (and possibly oxidized) phospholipids,9,10 independent of the position of the fatty acid on the glycerol moiety of
Oxidative modification of Low Density Lipoprotein (LDL) plays a major role in the unregulated cellular uptake of LDL responsible for the formation of foam cells, hence in the genesis of atherosclerosis.1– 4 A common modification results from oxidation of the LDL’s polyunsaturated fatty acids (PUFA) into hydroperoxides.4 The latter, unstable products of oxidation decompose, yielding various aldehydes, and these products subsequently react with amino acid residues of apolipoprotein B (apo B) in the LDL particle, to form Schiff bases.5 As a consequence, Apo Address correspondence to: D. Lichtenberg, Fax: 972-3-640-9113; E-Mail: [email protected]. 1294
Interrelationship between LDL oxidation and PLA
the phospholipid.11 Several authors attributed this PLA activity to Apo B as it appeared to be also exhibited by isolated apolipoprotein B (Apo B).11–12 However, other authors13–14 demonstrated that hydrolysis of oxidized phospholipids is catalyzed by the LDL-associated platelet-activating factor acetylhydrolase (PAF-AH), which is not a fragmentation product of Apo B.13–14 Furthermore, the enzyme responsible for the hydrolysis of PAF was purified from LDL and was shown to be also responsible for the hydrolysis of the short chain phospholipid analogue DNGP (1-decanoyl-2-(p-nitrophenyl glutaryl) phosphatidylcholine),15 indicating that ‘PAF-AH and PLA2 are one and the same‘.16 In other words, the activity of the LDL-PLA is not limited to PAF. Accordingly, we refer to it by the abbreviation LDL-PLA (although it is more commonly refered to as PAF-AH or LDL-PLA2).16 The cDNA of this enzyme has been cloned in Escherichia coli and it encodes a 441-amino acid protein of a molecular weight of 47 KDa.14,17 Hydrolysis of short chain phospholipids by LDLassociated PLA results in the formation of lysolecithins and short-chain fatty acids. These products are likely to leave the LDL particle and partition between the aqueous media, albumin and other lipoprotein particles in the plasma and interstitial fluids.18 This, in turn, is likely to influence the propagation of free radical-mediated peroxidation processes in several ways. On one hand, depletion of oxidation products from those LDL particles that became partly oxidized may reduce the rate of propagation. On the other hand, redistribution of oxidized lipids into other LDL particles may enhance oxidation in these particles.19 Hence, the oxidation-dependent, LDL-associated PLA activity may either accelerate, inhibit or have no effect on the overall process of lipid oxidation, depending on the experimental conditions. In fact, the published data on the role of LDL-associated PLA (PAF-AH) in lipid peroxidation is quite confusing, because several different lines of indirect evidence suggest that phospholipolysis accelerates, inhibits or have no effect on the kinetics of oxidation. Specifically, Steinbrecher et al.9 found that inhibition of the PLA activity by the serine esterase inhibitor diisopropylfluorophosphate (DFP) had no effect on lipid oxidation, indicating that PLA does not play a role in the oxidation. This conclusion agrees with the more recent data of Liapikos et al.20 who reported that inhibition of PAF-AH by phenylmethylsulfonylfluoride (PMSF) or/ and by acidification (pH 5 3.5) had no effect on lipid oxidation. This conclusion is inconsistent with the results of both Stafforini et al.,21 who have shown that phospholipolysis by externally-added PAF-AH inhibits oxidative modification of LDL, and Parthasarathy et al.,12, 22 who reported that inhibition of phospholipolysis by the
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site-specific inhibitor of PLA2 para-bromophenacyl bromide (pBPB) inhibits the LDL-catalyzed phospholipolysis and at the same time inhibits LDL lipid oxidation. Based on the latter findings, the authors concluded that inhibition of phospholipolysis reduces the oxidizability, namely that phospholipolysis enhances oxidation. This conclusion is consistent with the finding of Deigner et al.23 that PAF (at high concentrations), which is likely to be formed when PAF-AH is inactivated20, inhibits LDL oxidation. Nonetheless, the validity of this conclusion and of its possible significance is quite questionable. These apparently contradictory results may possibly be explained on the basis of the recent results of Fyrnys et al.,24 that in the presence of albumin in the reaction mixture, PAF-AH protects LDL against oxidation, whereas in the absence of albumin in the reaction mixture, PAF-AH enhances LDL oxidation. In interpreting these results, the authors imply that if the hydrolysis products remain in the LDL particles they enhance the oxidative modification of Apo B, whereas in the presence of albumin oxidatively-fragmented lipids may bind to albumin, and by that reduce the rate of propagation and render protective effect to the PAF-AH activity. Whether or not the contradictions between the results obtained by different groups can be explained as being due to different albumin concentrations (or any other differences in the experimental conditions) remains to be investigated. In view of the complexity of the role of LDL-associated PLA activity in the processes leading to lipid oxidation, we found it of interest to study both the effects of oxidation-induced phospholipolysis on the propagation of oxidation and the effect of lipid oxidation on phospholipolysis. In the present study, we have evaluated the role of LDL-associated PLA in LDL oxidation by investigating the oxidation kinetics in the presence of the non-toxic irreversible serine proteinase inhibitor Pefabloc (4-[2-aminoethyl] benzenesulfonyl fluoride), which has been recently shown to inactivate lipoprotein-associated PAF-AH at relatively low concentrations.25 The results of this study show that inactivation of LDL-PLA does not influence the oxidation of either isolated LDL in the absence or presence of albumin or in unfractionated plasma. This suggests that the enzyme does not play a protective role against lipid peroxidation in vitro. MATERIALS AND METHODS
Bovine serum albumin (essentially fatty acid-free), CuCl2, a-tocopherol, BHT (butylated hydroxytoluene) and EDTA ((ethylenediamine) tetraacetic acid) were purchased from Sigma (St. Louis, MO). AAPH (2,2’azo-bis(2-amidinopropane)hydrochloride) was purchased from Poly Sciences (Warrington, PA). C6-NBD-PC (1-
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acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminocaproylphosphatidylcholine) and NBD-caproic acid were purchased from Avanti (Birmingham, Alabama). Pefabloc (4-[2-aminoethyl] benzene-sulfonyl fluoride) was purchased from Boehringer, Mannheim. LDL was prepared by flotation at the limiting density and assayed for proteins and cholesterol as in our previous studies.19 LDL enrichment by Vitamin E was done as previously described.26 Lipid oxidation was monitored spectrophotometrically, following addition of the oxidant (CuCl2 or AAPH) to an aqueous dispersion of either fractionated LDL or unfractionated serum. LDL oxidation was monitored by continuous recording of the absorption at three wavelengths (245, 250 and 268 nm) and subsequent evaluation of the time dependencies of the major contributors to the spectra (hydroperoxides, dienals and 7 ketocholesterol), using a slight modification of our previously published method.27 Briefly, the concentrations of hydroperoxides, 7-ketocholesterol and dienals (CLOOH, C7-keto and Cdienals in mM, respectively) were evaluated from the measured absorbance using the following three equations, based on the known molar absorbance of the three absorbing components at the three wavelengths27: OD245 z 103 5 CLOOH z 14.85 1 C7-keto z 10.50 1 Cdienals z 5.82 OD250 z 103 5 CLOOH z 4.82 1 C7-keto z 7.07 1 Cdienals z 11.22 OD268 z 103 5 CLOOH z 0.03 1 C7-keto z 0.88 1 Cdienals z 28.98 For unfractionated serum, we have monitored the accumulation of oxidation products at 245 nm using a slight modification of our recently developed optimized assay28. Briefly, oxidation was monitored following 50 fold dilution of the serum in a PBS solution containing 0.72 mM sodium citrate and 100 mM CuCl2. Lipolysis of the short-chain fluorescent phospholipid C6-NBD-PC by native and oxidized LDL was assayed by continuous monitoring of its fluorescence (at 530; ex. at 470 nm).9,29 –30 The affinity of this probe to lipidic particles (lipid/water/interfaces) is high and the quantum yield of the lipid-associated NBD is much higher than that of NBD in aqueous solutions. Hydrolysis of the C6-NBD-PC in LDL particles results in formation of the water soluble NBD-caproic acid, and in a consequent reduction of the fluorescence intensity. To be able to use this decrease of fluorescence to assay phospholipolysis, we had to establish conditions where essentially all the C6-NBD-PC in the system resides in the LDL particles, and the concen-
tration of the probe in the LDL particles is sufficiently low so that its fluorescence will not be self-quenched. To establish such conditions, we had to conduct control experiments under conditions where no hydrolysis of C6-NBD-PC occurs. For these experiments we have used LDL whose PLA activity was inhibited by pretreatment with Pefabloc (0.1 mM; see Results). The results of these control experiments, described in the appendix, show that in mixtures containing 0.5 mM C6-NBD-PC (or less) and 0.05 mM LDL (or more), all the probe resides in LDL particles and its fluorescence is not quenched. Under such conditions, the timedependent reduction of fluorescence observed for C6NBD-PC in the absence of Pefabloc (e.g., Fig. 1A, curve a) can be attributed to hydrolysis of C6-NBD-PC and can be used as a measure of PLA activity. Inhibition of this decrease, by Pefabloc (0.1 mM in the experiment depicted by curve b in Fig. 1A) reflects inhibition of the LDL-associated PLA. Evaluation of PLA activity in albumin-containing media and in non-fractionated serum required consideration of the fluorescence of lipoprotein-associated C6-NBD-PC in comparison to that of albumin-bound probe (see appendix). In control experiments (Appendix), we have shown that in 50-fold diluted serum containing up to 2 mM C6-NBD-PC the fluorescence was not self-quenched. We have therefore chosen to assay the PLA activity of non-fractionated serum by monitoring the time-dependent decrease of the fluorescence under these conditions (2 mM C6-NBD-PC in samples of 50-fold diluted serum). An example is depicted in Fig. 1B. As is obvious from this figure, the decrease of fluorescence is quite similar to that observed for 0.5 mM C6-NBD-PC in the presence of 0.1 mM LDL. Since a large fraction of C6-NBD-PC in the serum was initially bound to albumin (see Appendix) the marked decrease of fluorescence observed upon hydrolysis (curve a in Fig. 1B) therefore indicates that albumin-bound C6-NBD-PC must have also been hydrolyzed. For this hydrolysis to occur and result in a reduction of fluorescence (as observed), the C6NBD-PC must distribute rapidly between albumin and lipoproteins and the binding-affinity of albumin to the resultant, water soluble NBD-caproic acid must be low. Similar to the results obtained with fractionated LDL, Pefabloc (at concentration above 0.1 mM) prevented the time-dependent decrease of fluorescence (curve b in Fig. 1B). In several representative cases, we have determined the hydrolysis of C6-NBD-PC by thin layer chromatographic separation of the reaction mixtures, using TLC plates and a chloroform/methanol/water mixture (65: 35:5) as an eluant.11 Under these conditions, the Rf of non-hydrolyzed C6-NBD-PC was 0.77 whereas that of
Interrelationship between LDL oxidation and PLA
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Fig. 1. Time dependence of the fluorescence intensity (% of the initial intensity) of C6-NBD-PC contained either in LDL (0.1 mM, A) or in serum (diluted 50-fold, B). Each panel depicts two time-dependencies: curve a) obtained for the native lipid or serum; curve b) obtained for the respective mixtures after pretreatment with Pefabloc (0.1 mM), added to the LDL or serum 15 min prior the addition of C6-NBD-PC. The inset of panel A describes the dependence of the relative activity of the LDL-PLA. The relative activity is given in terms of the ratio k/k0 where k is the pseudo-first order rate constant obtained in the presence of Pefabloc and K0 is the rate constant in the absence of Pefabloc (native LDL) and is described as a function of the concentration of Pefabloc used for pretreatment.
the NBD-caproic acid, formed upon phospholipolysis, was 0.93. The results obtained by this method (not shown) were consistent with those obtained by continuous monitoring of fluorescence.
RESULTS
As discussed above, addition of LDL to a solution containing micellar C6-NBD-PC resulted in a marked
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Fig. 2. Time courses of lipid peroxidation of LDL (solid lines) and of LDL pre-treated with 0.1 mM Pefabloc (broken lines). The absorbance of oxidation products at 245 nm is depicted as a function of time after addition of the oxidant (as indicated in the figure). Panel A presents the kinetics of copper-induced oxidation of native LDL (with and without Pefabloc). Panel C presents the kinetics of AAPH-induced oxidation of the same preparation. Panel B depicts the time courses of copper-induced oxidation (with and without Pefabloc) of an LDL preparation that was enriched with tocopherol before being exposed to copper (see methods). In all the experiments the LDL concentration was 0.1 mM in PBS.
immediate increase of the fluorescence intensity and a subsequent time-dependent decrease of the fluorescence as the phospholipid became hydrolyzed by the LDLassociated phospholipase (curve a in Fig. 1A). As shown by curve b in Fig. 1A, Pefabloc (at a concentration of 0.1 mM) blocked the hydrolysis, as evident from the finding that the fluorescence remained almost constant. In interpreting the results of these experiments, it is important to note that only a small fraction of the LDL particles possess PAF-AH, (ca. 1%;21 and C. Macphee, personal communication). The finding that a large fraction of C6-NBD-PC becomes hydrolyzed by native LDL must mean that most if not all of the C6-NBD-PC is accessible to LDL-PLA (PAF-AH). This may result either from ‘hopping‘ of the enzyme from one LDL particle to another (as indicated by the results of Stafforini et al.21 and Tselepis et al.,31) and/or due to rapid redistribution of C6-NBD-PC between LDL particles. The latter possibility is supported by the findings of our control experiments (not shown), in which we have titrated C6-NBD-PC-containning LDL with additional LDL. In these experiments, the initial fluorescence of the probe was partially self-quenched and titration with LDL resulted in increased fluorescence due to reduction in self-quenching. Since the increase of fluorescence after each step in the titration occurred instantaneously, it follows that the redistribution of C6-NBD-PC between LDL particles is rapid. This conclusion is also consistent
with the finding (not shown) that the fluorescence intensity observed after mixing C6-NBD-PC and LDL in one step was very similar to that obtained upon stepwise addition of LDL to a C6-NBD-PC solution (with the same final C6-NBD-PC /LDL ratio). The enzyme responsible for the hydrolysis of C6NBD-PC must bind to the LDL surface. However, since the exchange of both the enzyme and the substrate between particles is rapid, hydrolysis can be interpreted in terms of a pseudo-first order kinetics. The kinetics can therefore be described by a first order rate constant that can be derived from the time-dependence of the logarithm of fluorescence. Partial inactivation of the phospholipase activity during oxidation of LDL32 or of Lp(a)33 has been shown to correspond to complete inactivation of a fraction of the phospholipase molecules, as it reduces the maximal velocity of hydrolysis (Vmax) without alteration of Km. For the sake of simplicity, we assume that inactivation by Pefabloc is similar. Under this assumption the fraction of inactivated enzyme can be deduced from the apparent rate constant of fluorescencedecrease. This rate constant, evaluated as described above, was a decreasing function of the concentration of Pefabloc (Fig. 1A, inset), indicating that increasing concentrations of Pefabloc cause inactivation of an increasing fraction of LDL-PLA. Interestingly, the latter dosedependence was similar to that observed for the effect of Pefabloc on LDL-catalyzed hydrolysis of PAF,25 in
Interrelationship between LDL oxidation and PLA
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Fig. 3. Time courses of lipid peroxidation in the absence and presence of Pefabloc. In both panels, the solid lines depict the kinetics observed in the absence of Pefabloc whereas the broken lines were observed for samples that were preincubated with Pefabloc (100 mM in A, and 50 –250 mM in B). In Panel A the lipid substrate was LDL (0.1 mM) and the PBS medium contained 15 mM albumin and 45 mM CuCl2. In panel B, the lipid substrate was serum, diluted 50-fold in PBS containing 0.720 mM sodium citrate and 100 mM CuCl2.
agreement with the view that the LDL-associated PLA activity towards PAF and C6-NBD-PC is indeed due to the same enzyme.9,16 Pre-incubation of LDL with 0.1 mM Pefabloc (or more) inactivated this enzyme towards both PAF and C6-NBD-PC almost completely (Ref. 25 and Fig. 1A, respectively). As shown in Fig. 2A, inactivation of the LDL-associated PLA (probably, PAF-AH) had no effect on the kinetics of LDL oxidation induced either by copper ions or AAPH (Fig. 2C), at any studied concentration of the oxidizing agent. Interestingly, at low copper concentration (i.e., when the rate of production of free radicals is slow) the oxidation kinetics exhibited an initial phase of relatively rapid oxidation, which has been previously shown to be a result of tocopherol-mediated peroxidation (TMP).34 Peroxidation through this mechanism is particularly pronounced in Vitamin E-enriched LDL (Fig. 2B). This is consistent with the finding that the initial rate of peroxidation of the tocopherol-enriched LDL at low copper concentrations was higher than at higher concentration of copper (Fig. 2B). Inactivation of PLA by Pefabloc did not affect this peroxidation (Fig. 2B), indicating that phospholipolysis plays no role in peroxidation via the TMP mechanism. As discussed above, the effect of phospholipolysis on the propagation of free radical peroxidation may depend on removal of the hydrolysis products (lysolecithin and
free fatty acids) from the oxidizing LDL. It was therefore conceivable that inactivation of the LDL-PLA in albumin-containing media will be different than in the absence of albumin, because albumin ‘extracts’ hydrolysis products from the oxidizing LDL. Notably, in the presence of albumin, copper-induced oxidation occurs only when the copper concentration is sufficiently high to overcome the effect of copper binding to albumin.26 In fact, under such conditions, the kinetics of lipid oxidation was not affected by Pefabloc-inactivation of the PLA (Fig. 3A). Furthermore, Pefabloc (up to 250 mM) did not affect the oxidation of lipids in unfractionated serum (Fig. 3B), in spite of the almost complete inactivation of the enzyme responsible for hydrolysis of C6NBD-PC (Fig. 1B). The most simple interpretation of all these results is that hydrolysis of oxidized and/or short chain phospholipids does not affect the kinetics of oxidation. However, we had to consider the possibility that the lack of effect of phospholipolysis on oxidation might be a result of oxidation-induced inactivation of the PLA. In other words, we had to consider the possibility that PLA becomes inactivated at early stages of the oxidation of native LDL, and that the lack of effect of Pefabloc on the rate of peroxidation resulted from such inactivation. This possibility is quite unlikely in view of the results of previous studies,33,35 that showed that inactivation of
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and BHT to these solutions. We then divided each of these aliquots into two samples of equal volume, added Pefabloc to one sample of each such pair, and subsequently added C6-NBD-PC to both samples and recorded their fluorescence as a function of time. The results of the latter experiments (e.g., Fig. 5) were interpreted in terms of the fraction of LDL-PLA that became inactivated after any given time of exposure to copper (computed as described in the legend to Fig. 5). The results of these computations are depicted in Fig. 4D in terms of the time-dependence of the inactivated fraction of the enzyme. As obvious from this figure, oxidation-induced inactivation of LDL-PLA lags behind the accumulation
Fig. 4. Time-dependencies of the accumulation of peroxidation products and of the inactivation of PLA-LDL. Accumulation of hydroperoxides (A), 7-keto cholesterol (B) and dienals (C) during copperinduced oxidation (5 mM CuCl2) of LDL (0.1 mM), was computed from continuous monitoring of absorbance at 3 wavelengths (see Methods). Panel D depicts the time dependence of oxidation-induced inactivation of LDL-PLA, as evaluated from the reduction of the rate of hydrolysis of C6-NBD-PC following exposure to copper induced oxidation (see legend to Fig. 5).
PAF-AH during peroxidation lags considerably behind the accumulation of dienic hydroperoxides whereas inactivation by Pefabloc is rapid. Furthermore, we have evaluated the kinetics of oxidation-induced inactivation of the LDL-associated enzyme responsible for the hydrolysis of C6-NBD-PC and obtained similar results to those of Karabina et al.33,35 for the hydrolysis of PAF. Specifically, we have exposed LDL to copper-induced oxidation, and monitored the oxidation at three different wavelengths, to evaluate the time-dependencies of accumulation of hydroperoxides, 7-ketocholesterol and dienals (Fig. 4, A, B and C, respectively). At several time points during oxidation, we have terminated the oxidation in aliquots of the oxidizing LDL by adding EDTA
Fig. 5. Kinetics of the hydrolysis of C6-NBD-PC (as monitored by its fluorescence intensity) upon exposure to partially oxidized LDL (0.1 mM). The figure depicts the time-dependent decrease of the fluorescence of C6-NBD-PC (0.5 mM) added to partially oxidized LDL after 42 min (squares) or 190 min (circles) of exposure to CuCl2 (as in Fig. 4). The experimental conditions were as follows: at several time points during oxidation (including 42 min and 190 min) we have withdrawn two aliquots of the reaction mixture into solutions containing EDTA and BHT (final concentrations 50 mM and 30 mM, respectively). Pefabloc was then added to one of each such pair of samples and the samples were incubated at room temperature for 15 min. Each of these mixtures was then assayed for PLA activity by continuous monitoring of the fluorescence of C6-NBD-PC (empty symbols represent the fluorescence of the probe in the presence of Pefabloc; bold symbols, in its absence). The apparent rate constants of phospholipolysis (obtained from the time-dependencies of the logarithm of fluorescence) were corrected for reduction of fluorescence caused by bleaching of the fluorescence of C6-NBD-PC by oxidation products. This was done by subtracting the apparent first order rate constant of the (quite limited) decrease of fluorescence obtained in each LDL sample in the presence of Pefabloc from that obtained in the absence of Pefabloc. The resultant values of the normalized rate constants were used to compute the ratio between the normalized rate constant, as observed at any given time during oxidation, and the corresponding value obtained for non-oxidized LDL. The normalized rate constants were then used to compute the % of inactivation presented in Fig. 4D as a function of time.
Interrelationship between LDL oxidation and PLA
of all the lipidic oxidation products (Fig. 4A–C). Inactivation of LDL-PLA in the time course of oxidation can therefore not explain the lack of effect of Pefabloc on the rate of oxidation. Hence, hydrolysis of oxidized and/or fragmented phospholipids by LDL-associated PLA indeed does not protect LDL (in vitro) against continuing oxidation.
DISCUSSION
The obvious physiological role of PAF-AH is to hydrolyze PAF. Notably, LDL oxidation in vitro does not produce detectable amounts of PAF unless the LDLassociated PAF-AH is inactivated,20 indicating that this enzyme is capable of hydrolyzing the formed PAF, thus modulating its concentration. This is also accompanied by enhanced production of lysophosphatidylcholine during oxidation, suggesting that PAF-AH is also responsible for hydrolysis of oxidized phospholipids.20,33,35–36 Copper-induced peroxidation results in a time-dependent partial inactivation of PAF-AH.32–33,35 Nonetheless, since this inactivation is not accompanied by accumulation of PAF, it follows that under normal conditions PAF-AH is not the rate limiting step in PAF hydrolysis.20 The results of our studies on the hydrolysis of the fluorescent phospholipid C6-NBD-PC lend further support to the hypothesis that PAF-AH is in fact a phospholipase A responsible for the hydrolysis of other short chain phospholipids, besides PAF.16 Such phospholipids are produced by fragmentation of the hydroperoxidized phospholipids that are formed upon oxidation of PUFAbearing phospholipids. Similar to PAF-hydrolysis, the hydrolysis of C6NBD-PC is also inhibited by Pefabloc (with a similar dose-dependence) and by oxidation (with a similar timedependence). The products of phospholipolysis of both PAF and short-chain phospholipids (lyso-PAF, lysolecithin and short-chain free fatty acids) are more soluble in water than the respective phospholipids and are therefore likely to leave the oxidized LDL, especially in the presence of albumin in the solution. This could have possibly resulted in reduced rate of propagation, i.e., protect the LDL-associated lipid from continuing oxidation.24 Therefore, it has been previously hypothesized that PAF-AH plays a protective role against oxidation.21 In our studies of lipid oxidation in vitro, inactivation of the LDL-PLA had no effect on LDL oxidation by either Cu21 or AAPH, either in the absence or in the presence of albumin. This means that PLA-catalyzed phospholipolysis does not protect LDL against continuing peroxidation. Moreover, since similar results were observed for unfractionated serum, it follows that the phospholipoly-
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sis does not play a role in the in vitro oxidation of all the lipoproteins. Yet, phospholipolysis may play a protective role against lipid oxidation and/or against the consequences of oxidation in vivo. The recent findings of Schmitt et al.37 indicated that phospholipolysis of minimally oxidized LDL reduces its toxicity towards endothelial cells. In interpreting these results, the authors propose that the products of phospholipolysis (lysolecithin and oxidized or short-chain fatty acids) partition into the medium, thus reducing the internalization of the cytotoxic oxidized phospholipids into the cells upon endocytosis of oxidized LDL. Evaluation of this (and other) possibilities requires further investigations. Acknowledgements—Financial support of this work by the chief scientist of Israel Ministry of Health (to D.L. and S.Y.), the Lady Davis Fund (to E.S.) and the Ministry of Absorbtion (to I.P) is acknowledged. The authors thank Drs. Edgar Pick of Tel-Aviv University, Alenexandros Tselepis and Theodoros Liapikos of the University of Ioaninna, and Colin H. Macphee of SmithKline Beecham Pharmaceuticals for helpful discussions.
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13. Yamada, Y.; Stafforini, D. M.; Imaizumi, T. A.; Zimmerman, G. A., McIntyre, T. M.; Prescott, S. M. Characterization of the platelet-activating factor acetylhydrolase from human plasma by heterologous expression in Xenopus laevis oocytes. Proc. Natl. Acad. Sci. USA 91:10320 –10324; 1994. 14. Tjoelker, L. W.; Wilder, C.; Eberhardt, C.; Stafforini, D. M.; Dietsch, G.; Schimpf, B.; Hooper, S.; Trong, H. L.; Cousens, L. S.; Zimmerman, G. A.; Yamada, Y.; McIntyre, T. M.; Prescott, S. M.; Gray, P. W. Anti- inflammatory properties of a platelet-activating factor. Nature 374:549 – 552; 1995. 15. Washburn, W. N.; Dennis, E. A. Novel general approach for the assay and inhibition of hydrolytic enzymes utilizing suicide-inhibitory bifunctionally linked substrates (SIBLINKS): exemplified by a phospholipase A2 assay. J. Am. Chem. Soc. 112:2040 –2041; 1990. 16. Tew, D. G.; Southan, C.; Rice, S. Q. J.; Lawrence, G. M. P.; Li, H.; Boyd, H. F.; Moores, K.; Gloger, I. S.; Macphee, C. H. Purification, properties, sequencing, and cloning of a lipoprotein-associated, serine-dependent phospholipase involved in the oxidative modification of low-density lipoproteins. Arterioscler. Thromb. Vasc. Biol. 16:591–599; 1996. 17. Tjoelker, L. W.; Eberhardt, C.; Unger, J.; Trong, H. L.; Zimmerman, G. A.; McIntyre, T. M.; Stafforini, D. M.; Prescott, S. M.; Gray, P. W. PlasmapPlatelet-activating factor acetylhydrolase is a secreted phospholipase A2 with catalytic triad. J. Biol. Chem. 270:25481–25487; 1995. 18. Deigner, H. P.; Friedrich E.; Sinn, H.; Dresel, H. A. Scavenging of lipid peroxidation products from oxidizing LDL by albumin alters the plasma half-life of a fraction of oxidized LDL particles. Free Radical Res. Commun. 16:239 –246; 1992. 19. Schnitzer, E.; Fainaru, M.; Lichtenberg, D. Oxidation of low density lipoprotein upon sequential exposure to copper ions. Free Radical Res. 23:137–149; 1994. 20. Liapikos, T. A.; Antonopoulou, S.; Karabina, S. A.; Tsoukatos, D. C.; Demopoulos, C. A.; Tselepis, A. D. Platelet-activating factor formation during oxidative modification of low-density lipoprotein when PAF-acetylhydrolase has been inactivated. Biochim. Biophys. Acta 1212:353–360; 1994. 21. Stafforini, D. M.; Zimmerman, G. A.; McIntyre, T. M.; Prescott, S. M. The platelet-activating factor acetylhydrolase from human plasma prevents oxidative modification of low-density lipoprotein. Trans. Assoc. Am. Physicians 105:44 – 63; 1992. 22. Parthasarathy, S.; Steinbrecher, U. P.; Barnett, J.; Witztum, J. L.; Steinberg, D. Essential role of phospholipase A2 activity in endothelial cell-induced modification of low density lipoprotein. Proc. Natl. Acad. Sci. USA 82:3000 –3004; 1985. 23. Deigner, H. P.; Dresal, H. A. Effect of platelet activating factor on the kinetics of LDL oxidation in vitro. FEBS Lett. 317:202–206; 1993. 24. Fyrnys, B.; Blencowe, C.; Diegner, H. P. Susceptibility of phospholipids of oxidizing LDL to enzymatic hydrolysis modulates uptake by P388D1 macrophage-like cells. FEBS Lett. 357:7–12; 1995. 25. Danten, C.; Tselepis, A. D.; Chapman, M. J.; Ninibo, E. Pefabloc, 4-[2-aminoethyl]benzenesulfonyl fluoride, is a new, potent non-
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
toxic and irreversible inhibitor of PAF-degrading acetylhydrolase. Biochem. Biophys. Acta 1299:353–357; 1996. Schnitzer, E.; Pinchuk, I.; Bor, A.; Fainaru, M.; Lichtenberg, D. The effect of albumin on copper-induced oxidation. Biochim. Biophys. Acta 1344:300 –311; 1997. Pinchuk, I.; Lichtenberg, D. Continuous monitoring of intermediates and final products of oxidation of low density lipoprotein by means of UV-spectroscopy. Free Radical Res. 24:351–360; 1996. Schnitzer, E.; Pinchuk, I.; Fainaru, M.; Schafer, Z.; Lichtenberg, D. Copper-induced lipid oxidation in unfractionated plasma:the lag preceding oxidation as a measure of oxidation-resistance. Biochem. Biophys. Res. Comm. 216:854 – 861; 1995. Wittenauer, L. A.; Shirai, K.; Jackson, R. L.; Johnson, D. Hydrolysis of a fluorescent phospholipid substrate by phospholipase A2 and lipoprotein lipase. Biochem. Biophys. Res. Commun. 118:894 – 901; 1984. Meyuhas, D.; Yedgar, S.; Rotenberg, M.; Reisfeld, N.; Lichtenberg, D. The use of C6-NBD-PC for assaying phospholipase A2activity: Scope and limitations. Biochim. Biophys. Acta 1124:223– 232; 1992. Tselepis, A. D.; Dentan, C.; Karabina, S. A. P.; Chapman, M. J.; Ninio, E. PAF-degrading acetylhydrolase is preferentially associated with dense LDL and VHDL-1 in human plasma. Arterioscler. Thromb. Vasc. Biol. 15:1764 –1773; 1995. Dentan, C.; Lesnik, P.; Chapman, M. J.; Ninio, E. PAF-acetherdegrading acetylhydrolase in plasma LDL is inactivated by copperand cell-mediated oxidation. Arterioscler. Thromb. 14:353–360; 1994. Karabina, S. A. P.; Elisaf, M. C.; Goudevenos, J.; Siamopoulos, K. C.; Sideris, D.; Tselepis, A. D. PAF-acetylhydrolase activity on Lp(a) before and during Cu21-induced oxidative modification in vitro. Atherosclerosis 125:121–134; 1996. Bowry, V. W.; Stocker, R. Tocopherol-mediated peroxidation the prooxidant effect of Vitamin E on the radical-initiated oxidation of human low-density lipoprotein. J. Am. Chem. Soc. 115:6029 – 6044; 1993. Karabina, S. A. P.; Liapikos, T. A.; Grekas, G.; Goudevenos, J.; Tselepis, A. D. Distribution of PAF-acetylhydrolase activity in human plasma low-density lipoprotein subfractions. Biochim. Biophys. Acta 1213:34 –38; 1994. Karabina, S. A. P.; Elisaf, M.; Bairaktari, E.; Tziallas, C.; Siamopoulos, K. C.; Tselepis, A. D. Increased activity of platelet-activating factor acetylhydrolase in low-density lipoprotein subfractions induces enhanced lysophosphatidylcholine production during oxidation in patients with heterozygous familial hypercholesterolamia. Europ. J. Clin. Inves. 27:595– 602; 1997. Schmitt, A.; Negre–Salvayre, A.; Troly, M.; Valdiguie, P.; Salvare, R. Phospholipid hydrolysis of mildly oxidized LDL reduces their cytotoxicity to cultured endothelial cells. Potential protective role against atherogenesis. Biochem. Biophys. Acta 1256:284 –292; 1995. Reisfeld, N.; Lichtenberg, D.; Yedgar, S. Inhibition of LDL-associated phospholipase A activity in human plasma by albumin. J. Basic Clin. Physiol. Pharm. 5:107–115; 1994.
APPENDIX
Determination of lipoprotein-associated phospholipase A (PLA) activity by PLA-catalyzed hydrolysis of C6NBD-PC The fluorescence intensity of C6-NBD-PC depends on: (i) its concentration in the solution (ii) the environment of the medium where it resides, which determines the quantum yield of fluorescence and (iii) occurrence of self-quenching at high concentrations of the probe in the compartment of its residence. In aqueous media, the quantum yield of the NBD fluorescence is very low. Moreover, in the absence of lipids in the aqueous system, C6-NBD-PC self-assembles into micelles and its
fluorescence is further reduced by self-quenching and is therefore very low. In the presence of lipoproteins, the probe partitions between the lipidic cores (and interfaces) of the lipoprotein particles and the aqueous medium. The high affinity of the C6-NBD-PC to lipids at the lipid/water interface and the high quantum yield of the probe when it resides in a lipidic environment result in a marked increase of the fluorescence intensity upon addition of LDL to a micellar solution of C6-NBD-PC30. Hydrolysis of the probe results in the formation of the water soluble NBD-caproic acid and in a consequent decrease of the fluorescence intensity. This can be used to monitor the hydrolysis of LDL-associated C6-NBD-PC only under conditions where: (i) prior to hydrolysis, all the
Interrelationship between LDL oxidation and PLA probe resides in LDL particles, so that its hydrolysis will not be accompanied by redistribution of C6-NBD-PC from the aqueous solution into LDL particles (which might attenuate the reduction of fluorescence due to hydrolysis), and (ii) the fluorescence intensity of the LDL-associated probe is not self-quenched. This appendix describes the results of control experiments conducted with LDL in the absence and presence of albumin and with 50-fold diluted serum under conditions of complete inactivation of the lipoprotein-associated PLA by Pefabloc. In all the experiments described below, the fluorescence intensity of C6-NBD-PC depended on the concentration of the probe and on the probe /lipid ratio but was independent of the time after mixing the probe with the lipoprotein(s). This implies that equilibration of the system (i.e., partitioning of the probe between the aqueous phase and lipidic compartments) is rapid and that following equilibration the probe is indeed not hydrolyzed in the time course of the experiments. For fractionated LDL, we have conducted the following two experiments: first, we have titrated LDL (0.05 mM) with C6-NBD-PC, and monitored the increase of fluorescence as a function of the concentration of the probe. In this experiment the fluorescence increased linearly with the concentration of the probe (i.e., the molar fluorescence remained constant) up to 0.5 mM C6-NBD-PC. At higher concentrations of the probe, the molar fluorescence decreased. This may either be a result of ‘saturation‘ of the LDL with C6-NBD-PC (i.e., partitioning of the added probe into water, where its quantum yield is low) or of self-quenching of fluorescence in LDL particles when the concentration of the probe is high (i.e., at C6-NBD-PC/LDL ratios higher than 10). The former possibility can be ruled out on the basis of our second control experiment, in which we have measured the change in fluorescence of a mixture of 4 mM C6-NBD-PC and 0.3 mM LDL upon an 8-fold dilution. In this experiment, the (partially quenched) fluorescence intensity decreased linearly with the concentration of the probe (i.e., the molar fluorescence remained constant), indicating that under these experimental conditions, essentially all the C6-NBD-PC resides in LDL particles and not in the aqueous medium. In other words, the fluorescence of C6-NBD-PC is not a function of the LDL/water partitioning but only of the probe/LDL ratio. When the latter ratio was lower than 10 (as in all the experiments described in the present study) the fluorescence was not self-quenched. Since each LDL particle contains 700 –1000 phospholipid molecules, a C6-NBD-PC/LDL ratio of 10
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implies that the ratio of C6-NBD-PC to phospholipid molecules of the outer monolayer of LDL is about 1/100 –1/70. Interestingly, a similar threshold of self-quenching was observed for emulsion particles composed of PC and triglycerides,30 indicating that in both these systems, which are different with respect to the core/interface ratio, the C6-NBD-PC resides predominantly, if not exclusively in the outer phospholipid monolayer. In albumin-containing media and in non-fractionated serum, part of the C6-NBD-PC is likely to be bound to albumin.38 To evaluate the fraction of LDL-associated C6-NBD-PC, we have first determined the fluorescence of albumin-bound C6-NBD-PC in comparison to that of the lipoprotein-associated probe. This was done by monitoring the fluorescence intensity of a constant C6-NBD-PC concentration (1 mM) upon stepwise addition of increasing concentrations of either LDL, HDL, albumin or unfractionated serum. In each of these experiments, the fluorescence increased with the concentration of the additive until it became constant. The highest fluorescence was observed for LDL. In comparison to LDL, the maximal fluorescence in HDL was 75%, in albumin it was 50% whereas in serum the maximal fluorescence was about 72% of that observed in LDL. In this study we examined the PLA activity of 50-fold diluted serum. In serum, the level of fluorescence intensity is of course a function of the (rather complex) partitioning of C6-NBD-PC between albumin, LDL and HDL. Accordingly, the fluorescence intensity observed for any given concentration of C6-NBD-PC below 5 mM in media containing 12 mM albumin (as in 50-fold diluted serum) was lower than in 50-fold diluted serum, where part of C6-NBD-PC resides in lipoproteins in which the fluorescence is higher than that of albuminbound probe. Stepwise addition of C6-NBD-PC to 50-fold diluted serum resulted in a linear increase of the fluorescence intensity (i.e., in constant molar fluorescence) up to a C6-NBD-PC concentration of 2 mM. Only at higher C6-NBD-PC concentrations, the molar fluorescence decreased, probably due to self-quenching of lipoprotein-associated C6-NBD-PC. One important implication of this finding is that at 2 mM C6-NBD-PC the time-dependent decrease of fluorescence can be used as a measure of PLA activity of serum lipoproteins. Secondly, since self-quenching occurred only above 2 mM C6-NBD-PC, as compared to 0.5 mM in LDL of similar lipid concentration, we estimate that in 50-fold diluted serum only about a fourth of the probe (2 mM) is associated with LDL.
PII S0891-5849(97)00454-1
Original Contribution LDL-ASSOCIATED PHOSPHOLIPASE A DOES NOT PROTECT LDL AGAINST LIPID PEROXIDATION IN VITRO EDIT SCHNITZER,* ILYA PINCHUK,* MENACHEM FAINARU,† DOV LICHTENBERG,*
and
SAUL YEDGAR‡
*Departments of Physiology and Pharmacology, Tel-Aviv University, Sackler Faculty of Medicine, Tel-Aviv 69978, Israel; † Internal Medicine (Rabin Medical Center), Tel-Aviv University, Sackler Faculty of Medicine, Tel-Aviv 69978, Israel; and ‡ Department of Biochemistry, Hebrew University, Hadassah Medical School, Jerusalem, 91120, Israel (Received 13 November 1997; Accepted 26 November 1997)
Abstract—The irreversible proteinase inhibitor Pefabloc (4-[2-aminoethyl] benzenesulfonyl fluoride) inactivates LDLcatalyzed hydrolysis of the short-chain fluorescent phospholipid C6-NBD-PC (1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3diazole)-aminocaproyl phosphatidylcholine). The dose-dependence of this inactivation is similar to that obtained previously for the inhibitory effect of Pefabloc on the hydrolysis of platelet activating factor (PAF) by the LDLassociated PAF acetylhydrolase (PAF-AH), in agreement with the notion that the hydrolysis of C6-NBD-PC and PAF is catalyzed by the same enzyme (LDL-associated phospholipase A; LDL-PLA). This conclusion is also supported by the finding that hydrolysis of C6-NBD-PC by LDL becomes inactivated by LDL oxidation only at late stages of the oxidation, similar to the effect of oxidation on the hydrolysis of PAF by the LDL-associated PAF-AH. Under conditions of complete inactivation of this enzyme towards C6-NBD-PC, the kinetics of lipid peroxidation, induced either by copper ions or by the free radical generator AAPH at varying doses of the prooxidant, was similar to that observed when the PLA was active (i.e., in the absence of Pefabloc). Hence, LDL-associated PLA (PAF-AH) does not protect LDL lipids from peroxidation. Similar results were obtained with fractionated LDL in albumin-containing buffer and for non-fractionated serum, in which copper-induced peroxidation was also not influenced by inactivation of the enzyme responsible for hydrolysis of C6-NBD-PC. Phospholipolysis of short chain phospholipids by LDL-PLA may still play a protective role against the toxic effects of oxidized phospholipids by reducing their internalization into cells ( Schmitt et al. 1995). © 1998 Elsevier Science Inc. Keywords—Free radical, Lipid peroxidation, Low density lipoproteins (LDL), LDL-associated phospholipase A2 (LDL-PLA2 or LDL-PLA) Platelet activating factor (PAF), PAF acetylhydrolase (PAF-AH), Pefabloc (4-[2-aminoethyl]benzenesulfonyl fluoride), 1-Acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidylcholine (C6NBD-PC)
INTRODUCTION
B is no longer recognized by the LDL receptor and the normal internalization of LDL into the cells via the LDL receptor is therefore impaired. The alternative mechanism of LDL internalization, via the scavenger receptor, becomes the major pathway, and since this mechanism is not down-regulated it may result in excessive accumulation of lipids and in consequent cell death and formation of atherotic plaques.4,6 –7. Decomposition of the conjugated dienic hydroperoxides derived from oxidized PUFA results in phospholipids with fragmented acyl chains.8 These phospholipids can be hydrolyzed by the LDL-associated phospholipase A (PLA), which catalyses the hydrolysis of short chain (and possibly oxidized) phospholipids,9,10 independent of the position of the fatty acid on the glycerol moiety of
Oxidative modification of Low Density Lipoprotein (LDL) plays a major role in the unregulated cellular uptake of LDL responsible for the formation of foam cells, hence in the genesis of atherosclerosis.1– 4 A common modification results from oxidation of the LDL’s polyunsaturated fatty acids (PUFA) into hydroperoxides.4 The latter, unstable products of oxidation decompose, yielding various aldehydes, and these products subsequently react with amino acid residues of apolipoprotein B (apo B) in the LDL particle, to form Schiff bases.5 As a consequence, Apo Address correspondence to: D. Lichtenberg, Fax: 972-3-640-9113; E-Mail: [email protected]. 1294
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the phospholipid.11 Several authors attributed this PLA activity to Apo B as it appeared to be also exhibited by isolated apolipoprotein B (Apo B).11–12 However, other authors13–14 demonstrated that hydrolysis of oxidized phospholipids is catalyzed by the LDL-associated platelet-activating factor acetylhydrolase (PAF-AH), which is not a fragmentation product of Apo B.13–14 Furthermore, the enzyme responsible for the hydrolysis of PAF was purified from LDL and was shown to be also responsible for the hydrolysis of the short chain phospholipid analogue DNGP (1-decanoyl-2-(p-nitrophenyl glutaryl) phosphatidylcholine),15 indicating that ‘PAF-AH and PLA2 are one and the same‘.16 In other words, the activity of the LDL-PLA is not limited to PAF. Accordingly, we refer to it by the abbreviation LDL-PLA (although it is more commonly refered to as PAF-AH or LDL-PLA2).16 The cDNA of this enzyme has been cloned in Escherichia coli and it encodes a 441-amino acid protein of a molecular weight of 47 KDa.14,17 Hydrolysis of short chain phospholipids by LDLassociated PLA results in the formation of lysolecithins and short-chain fatty acids. These products are likely to leave the LDL particle and partition between the aqueous media, albumin and other lipoprotein particles in the plasma and interstitial fluids.18 This, in turn, is likely to influence the propagation of free radical-mediated peroxidation processes in several ways. On one hand, depletion of oxidation products from those LDL particles that became partly oxidized may reduce the rate of propagation. On the other hand, redistribution of oxidized lipids into other LDL particles may enhance oxidation in these particles.19 Hence, the oxidation-dependent, LDL-associated PLA activity may either accelerate, inhibit or have no effect on the overall process of lipid oxidation, depending on the experimental conditions. In fact, the published data on the role of LDL-associated PLA (PAF-AH) in lipid peroxidation is quite confusing, because several different lines of indirect evidence suggest that phospholipolysis accelerates, inhibits or have no effect on the kinetics of oxidation. Specifically, Steinbrecher et al.9 found that inhibition of the PLA activity by the serine esterase inhibitor diisopropylfluorophosphate (DFP) had no effect on lipid oxidation, indicating that PLA does not play a role in the oxidation. This conclusion agrees with the more recent data of Liapikos et al.20 who reported that inhibition of PAF-AH by phenylmethylsulfonylfluoride (PMSF) or/ and by acidification (pH 5 3.5) had no effect on lipid oxidation. This conclusion is inconsistent with the results of both Stafforini et al.,21 who have shown that phospholipolysis by externally-added PAF-AH inhibits oxidative modification of LDL, and Parthasarathy et al.,12, 22 who reported that inhibition of phospholipolysis by the
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site-specific inhibitor of PLA2 para-bromophenacyl bromide (pBPB) inhibits the LDL-catalyzed phospholipolysis and at the same time inhibits LDL lipid oxidation. Based on the latter findings, the authors concluded that inhibition of phospholipolysis reduces the oxidizability, namely that phospholipolysis enhances oxidation. This conclusion is consistent with the finding of Deigner et al.23 that PAF (at high concentrations), which is likely to be formed when PAF-AH is inactivated20, inhibits LDL oxidation. Nonetheless, the validity of this conclusion and of its possible significance is quite questionable. These apparently contradictory results may possibly be explained on the basis of the recent results of Fyrnys et al.,24 that in the presence of albumin in the reaction mixture, PAF-AH protects LDL against oxidation, whereas in the absence of albumin in the reaction mixture, PAF-AH enhances LDL oxidation. In interpreting these results, the authors imply that if the hydrolysis products remain in the LDL particles they enhance the oxidative modification of Apo B, whereas in the presence of albumin oxidatively-fragmented lipids may bind to albumin, and by that reduce the rate of propagation and render protective effect to the PAF-AH activity. Whether or not the contradictions between the results obtained by different groups can be explained as being due to different albumin concentrations (or any other differences in the experimental conditions) remains to be investigated. In view of the complexity of the role of LDL-associated PLA activity in the processes leading to lipid oxidation, we found it of interest to study both the effects of oxidation-induced phospholipolysis on the propagation of oxidation and the effect of lipid oxidation on phospholipolysis. In the present study, we have evaluated the role of LDL-associated PLA in LDL oxidation by investigating the oxidation kinetics in the presence of the non-toxic irreversible serine proteinase inhibitor Pefabloc (4-[2-aminoethyl] benzenesulfonyl fluoride), which has been recently shown to inactivate lipoprotein-associated PAF-AH at relatively low concentrations.25 The results of this study show that inactivation of LDL-PLA does not influence the oxidation of either isolated LDL in the absence or presence of albumin or in unfractionated plasma. This suggests that the enzyme does not play a protective role against lipid peroxidation in vitro. MATERIALS AND METHODS
Bovine serum albumin (essentially fatty acid-free), CuCl2, a-tocopherol, BHT (butylated hydroxytoluene) and EDTA ((ethylenediamine) tetraacetic acid) were purchased from Sigma (St. Louis, MO). AAPH (2,2’azo-bis(2-amidinopropane)hydrochloride) was purchased from Poly Sciences (Warrington, PA). C6-NBD-PC (1-
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acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminocaproylphosphatidylcholine) and NBD-caproic acid were purchased from Avanti (Birmingham, Alabama). Pefabloc (4-[2-aminoethyl] benzene-sulfonyl fluoride) was purchased from Boehringer, Mannheim. LDL was prepared by flotation at the limiting density and assayed for proteins and cholesterol as in our previous studies.19 LDL enrichment by Vitamin E was done as previously described.26 Lipid oxidation was monitored spectrophotometrically, following addition of the oxidant (CuCl2 or AAPH) to an aqueous dispersion of either fractionated LDL or unfractionated serum. LDL oxidation was monitored by continuous recording of the absorption at three wavelengths (245, 250 and 268 nm) and subsequent evaluation of the time dependencies of the major contributors to the spectra (hydroperoxides, dienals and 7 ketocholesterol), using a slight modification of our previously published method.27 Briefly, the concentrations of hydroperoxides, 7-ketocholesterol and dienals (CLOOH, C7-keto and Cdienals in mM, respectively) were evaluated from the measured absorbance using the following three equations, based on the known molar absorbance of the three absorbing components at the three wavelengths27: OD245 z 103 5 CLOOH z 14.85 1 C7-keto z 10.50 1 Cdienals z 5.82 OD250 z 103 5 CLOOH z 4.82 1 C7-keto z 7.07 1 Cdienals z 11.22 OD268 z 103 5 CLOOH z 0.03 1 C7-keto z 0.88 1 Cdienals z 28.98 For unfractionated serum, we have monitored the accumulation of oxidation products at 245 nm using a slight modification of our recently developed optimized assay28. Briefly, oxidation was monitored following 50 fold dilution of the serum in a PBS solution containing 0.72 mM sodium citrate and 100 mM CuCl2. Lipolysis of the short-chain fluorescent phospholipid C6-NBD-PC by native and oxidized LDL was assayed by continuous monitoring of its fluorescence (at 530; ex. at 470 nm).9,29 –30 The affinity of this probe to lipidic particles (lipid/water/interfaces) is high and the quantum yield of the lipid-associated NBD is much higher than that of NBD in aqueous solutions. Hydrolysis of the C6-NBD-PC in LDL particles results in formation of the water soluble NBD-caproic acid, and in a consequent reduction of the fluorescence intensity. To be able to use this decrease of fluorescence to assay phospholipolysis, we had to establish conditions where essentially all the C6-NBD-PC in the system resides in the LDL particles, and the concen-
tration of the probe in the LDL particles is sufficiently low so that its fluorescence will not be self-quenched. To establish such conditions, we had to conduct control experiments under conditions where no hydrolysis of C6-NBD-PC occurs. For these experiments we have used LDL whose PLA activity was inhibited by pretreatment with Pefabloc (0.1 mM; see Results). The results of these control experiments, described in the appendix, show that in mixtures containing 0.5 mM C6-NBD-PC (or less) and 0.05 mM LDL (or more), all the probe resides in LDL particles and its fluorescence is not quenched. Under such conditions, the timedependent reduction of fluorescence observed for C6NBD-PC in the absence of Pefabloc (e.g., Fig. 1A, curve a) can be attributed to hydrolysis of C6-NBD-PC and can be used as a measure of PLA activity. Inhibition of this decrease, by Pefabloc (0.1 mM in the experiment depicted by curve b in Fig. 1A) reflects inhibition of the LDL-associated PLA. Evaluation of PLA activity in albumin-containing media and in non-fractionated serum required consideration of the fluorescence of lipoprotein-associated C6-NBD-PC in comparison to that of albumin-bound probe (see appendix). In control experiments (Appendix), we have shown that in 50-fold diluted serum containing up to 2 mM C6-NBD-PC the fluorescence was not self-quenched. We have therefore chosen to assay the PLA activity of non-fractionated serum by monitoring the time-dependent decrease of the fluorescence under these conditions (2 mM C6-NBD-PC in samples of 50-fold diluted serum). An example is depicted in Fig. 1B. As is obvious from this figure, the decrease of fluorescence is quite similar to that observed for 0.5 mM C6-NBD-PC in the presence of 0.1 mM LDL. Since a large fraction of C6-NBD-PC in the serum was initially bound to albumin (see Appendix) the marked decrease of fluorescence observed upon hydrolysis (curve a in Fig. 1B) therefore indicates that albumin-bound C6-NBD-PC must have also been hydrolyzed. For this hydrolysis to occur and result in a reduction of fluorescence (as observed), the C6NBD-PC must distribute rapidly between albumin and lipoproteins and the binding-affinity of albumin to the resultant, water soluble NBD-caproic acid must be low. Similar to the results obtained with fractionated LDL, Pefabloc (at concentration above 0.1 mM) prevented the time-dependent decrease of fluorescence (curve b in Fig. 1B). In several representative cases, we have determined the hydrolysis of C6-NBD-PC by thin layer chromatographic separation of the reaction mixtures, using TLC plates and a chloroform/methanol/water mixture (65: 35:5) as an eluant.11 Under these conditions, the Rf of non-hydrolyzed C6-NBD-PC was 0.77 whereas that of
Interrelationship between LDL oxidation and PLA
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Fig. 1. Time dependence of the fluorescence intensity (% of the initial intensity) of C6-NBD-PC contained either in LDL (0.1 mM, A) or in serum (diluted 50-fold, B). Each panel depicts two time-dependencies: curve a) obtained for the native lipid or serum; curve b) obtained for the respective mixtures after pretreatment with Pefabloc (0.1 mM), added to the LDL or serum 15 min prior the addition of C6-NBD-PC. The inset of panel A describes the dependence of the relative activity of the LDL-PLA. The relative activity is given in terms of the ratio k/k0 where k is the pseudo-first order rate constant obtained in the presence of Pefabloc and K0 is the rate constant in the absence of Pefabloc (native LDL) and is described as a function of the concentration of Pefabloc used for pretreatment.
the NBD-caproic acid, formed upon phospholipolysis, was 0.93. The results obtained by this method (not shown) were consistent with those obtained by continuous monitoring of fluorescence.
RESULTS
As discussed above, addition of LDL to a solution containing micellar C6-NBD-PC resulted in a marked
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Fig. 2. Time courses of lipid peroxidation of LDL (solid lines) and of LDL pre-treated with 0.1 mM Pefabloc (broken lines). The absorbance of oxidation products at 245 nm is depicted as a function of time after addition of the oxidant (as indicated in the figure). Panel A presents the kinetics of copper-induced oxidation of native LDL (with and without Pefabloc). Panel C presents the kinetics of AAPH-induced oxidation of the same preparation. Panel B depicts the time courses of copper-induced oxidation (with and without Pefabloc) of an LDL preparation that was enriched with tocopherol before being exposed to copper (see methods). In all the experiments the LDL concentration was 0.1 mM in PBS.
immediate increase of the fluorescence intensity and a subsequent time-dependent decrease of the fluorescence as the phospholipid became hydrolyzed by the LDLassociated phospholipase (curve a in Fig. 1A). As shown by curve b in Fig. 1A, Pefabloc (at a concentration of 0.1 mM) blocked the hydrolysis, as evident from the finding that the fluorescence remained almost constant. In interpreting the results of these experiments, it is important to note that only a small fraction of the LDL particles possess PAF-AH, (ca. 1%;21 and C. Macphee, personal communication). The finding that a large fraction of C6-NBD-PC becomes hydrolyzed by native LDL must mean that most if not all of the C6-NBD-PC is accessible to LDL-PLA (PAF-AH). This may result either from ‘hopping‘ of the enzyme from one LDL particle to another (as indicated by the results of Stafforini et al.21 and Tselepis et al.,31) and/or due to rapid redistribution of C6-NBD-PC between LDL particles. The latter possibility is supported by the findings of our control experiments (not shown), in which we have titrated C6-NBD-PC-containning LDL with additional LDL. In these experiments, the initial fluorescence of the probe was partially self-quenched and titration with LDL resulted in increased fluorescence due to reduction in self-quenching. Since the increase of fluorescence after each step in the titration occurred instantaneously, it follows that the redistribution of C6-NBD-PC between LDL particles is rapid. This conclusion is also consistent
with the finding (not shown) that the fluorescence intensity observed after mixing C6-NBD-PC and LDL in one step was very similar to that obtained upon stepwise addition of LDL to a C6-NBD-PC solution (with the same final C6-NBD-PC /LDL ratio). The enzyme responsible for the hydrolysis of C6NBD-PC must bind to the LDL surface. However, since the exchange of both the enzyme and the substrate between particles is rapid, hydrolysis can be interpreted in terms of a pseudo-first order kinetics. The kinetics can therefore be described by a first order rate constant that can be derived from the time-dependence of the logarithm of fluorescence. Partial inactivation of the phospholipase activity during oxidation of LDL32 or of Lp(a)33 has been shown to correspond to complete inactivation of a fraction of the phospholipase molecules, as it reduces the maximal velocity of hydrolysis (Vmax) without alteration of Km. For the sake of simplicity, we assume that inactivation by Pefabloc is similar. Under this assumption the fraction of inactivated enzyme can be deduced from the apparent rate constant of fluorescencedecrease. This rate constant, evaluated as described above, was a decreasing function of the concentration of Pefabloc (Fig. 1A, inset), indicating that increasing concentrations of Pefabloc cause inactivation of an increasing fraction of LDL-PLA. Interestingly, the latter dosedependence was similar to that observed for the effect of Pefabloc on LDL-catalyzed hydrolysis of PAF,25 in
Interrelationship between LDL oxidation and PLA
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Fig. 3. Time courses of lipid peroxidation in the absence and presence of Pefabloc. In both panels, the solid lines depict the kinetics observed in the absence of Pefabloc whereas the broken lines were observed for samples that were preincubated with Pefabloc (100 mM in A, and 50 –250 mM in B). In Panel A the lipid substrate was LDL (0.1 mM) and the PBS medium contained 15 mM albumin and 45 mM CuCl2. In panel B, the lipid substrate was serum, diluted 50-fold in PBS containing 0.720 mM sodium citrate and 100 mM CuCl2.
agreement with the view that the LDL-associated PLA activity towards PAF and C6-NBD-PC is indeed due to the same enzyme.9,16 Pre-incubation of LDL with 0.1 mM Pefabloc (or more) inactivated this enzyme towards both PAF and C6-NBD-PC almost completely (Ref. 25 and Fig. 1A, respectively). As shown in Fig. 2A, inactivation of the LDL-associated PLA (probably, PAF-AH) had no effect on the kinetics of LDL oxidation induced either by copper ions or AAPH (Fig. 2C), at any studied concentration of the oxidizing agent. Interestingly, at low copper concentration (i.e., when the rate of production of free radicals is slow) the oxidation kinetics exhibited an initial phase of relatively rapid oxidation, which has been previously shown to be a result of tocopherol-mediated peroxidation (TMP).34 Peroxidation through this mechanism is particularly pronounced in Vitamin E-enriched LDL (Fig. 2B). This is consistent with the finding that the initial rate of peroxidation of the tocopherol-enriched LDL at low copper concentrations was higher than at higher concentration of copper (Fig. 2B). Inactivation of PLA by Pefabloc did not affect this peroxidation (Fig. 2B), indicating that phospholipolysis plays no role in peroxidation via the TMP mechanism. As discussed above, the effect of phospholipolysis on the propagation of free radical peroxidation may depend on removal of the hydrolysis products (lysolecithin and
free fatty acids) from the oxidizing LDL. It was therefore conceivable that inactivation of the LDL-PLA in albumin-containing media will be different than in the absence of albumin, because albumin ‘extracts’ hydrolysis products from the oxidizing LDL. Notably, in the presence of albumin, copper-induced oxidation occurs only when the copper concentration is sufficiently high to overcome the effect of copper binding to albumin.26 In fact, under such conditions, the kinetics of lipid oxidation was not affected by Pefabloc-inactivation of the PLA (Fig. 3A). Furthermore, Pefabloc (up to 250 mM) did not affect the oxidation of lipids in unfractionated serum (Fig. 3B), in spite of the almost complete inactivation of the enzyme responsible for hydrolysis of C6NBD-PC (Fig. 1B). The most simple interpretation of all these results is that hydrolysis of oxidized and/or short chain phospholipids does not affect the kinetics of oxidation. However, we had to consider the possibility that the lack of effect of phospholipolysis on oxidation might be a result of oxidation-induced inactivation of the PLA. In other words, we had to consider the possibility that PLA becomes inactivated at early stages of the oxidation of native LDL, and that the lack of effect of Pefabloc on the rate of peroxidation resulted from such inactivation. This possibility is quite unlikely in view of the results of previous studies,33,35 that showed that inactivation of
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and BHT to these solutions. We then divided each of these aliquots into two samples of equal volume, added Pefabloc to one sample of each such pair, and subsequently added C6-NBD-PC to both samples and recorded their fluorescence as a function of time. The results of the latter experiments (e.g., Fig. 5) were interpreted in terms of the fraction of LDL-PLA that became inactivated after any given time of exposure to copper (computed as described in the legend to Fig. 5). The results of these computations are depicted in Fig. 4D in terms of the time-dependence of the inactivated fraction of the enzyme. As obvious from this figure, oxidation-induced inactivation of LDL-PLA lags behind the accumulation
Fig. 4. Time-dependencies of the accumulation of peroxidation products and of the inactivation of PLA-LDL. Accumulation of hydroperoxides (A), 7-keto cholesterol (B) and dienals (C) during copperinduced oxidation (5 mM CuCl2) of LDL (0.1 mM), was computed from continuous monitoring of absorbance at 3 wavelengths (see Methods). Panel D depicts the time dependence of oxidation-induced inactivation of LDL-PLA, as evaluated from the reduction of the rate of hydrolysis of C6-NBD-PC following exposure to copper induced oxidation (see legend to Fig. 5).
PAF-AH during peroxidation lags considerably behind the accumulation of dienic hydroperoxides whereas inactivation by Pefabloc is rapid. Furthermore, we have evaluated the kinetics of oxidation-induced inactivation of the LDL-associated enzyme responsible for the hydrolysis of C6-NBD-PC and obtained similar results to those of Karabina et al.33,35 for the hydrolysis of PAF. Specifically, we have exposed LDL to copper-induced oxidation, and monitored the oxidation at three different wavelengths, to evaluate the time-dependencies of accumulation of hydroperoxides, 7-ketocholesterol and dienals (Fig. 4, A, B and C, respectively). At several time points during oxidation, we have terminated the oxidation in aliquots of the oxidizing LDL by adding EDTA
Fig. 5. Kinetics of the hydrolysis of C6-NBD-PC (as monitored by its fluorescence intensity) upon exposure to partially oxidized LDL (0.1 mM). The figure depicts the time-dependent decrease of the fluorescence of C6-NBD-PC (0.5 mM) added to partially oxidized LDL after 42 min (squares) or 190 min (circles) of exposure to CuCl2 (as in Fig. 4). The experimental conditions were as follows: at several time points during oxidation (including 42 min and 190 min) we have withdrawn two aliquots of the reaction mixture into solutions containing EDTA and BHT (final concentrations 50 mM and 30 mM, respectively). Pefabloc was then added to one of each such pair of samples and the samples were incubated at room temperature for 15 min. Each of these mixtures was then assayed for PLA activity by continuous monitoring of the fluorescence of C6-NBD-PC (empty symbols represent the fluorescence of the probe in the presence of Pefabloc; bold symbols, in its absence). The apparent rate constants of phospholipolysis (obtained from the time-dependencies of the logarithm of fluorescence) were corrected for reduction of fluorescence caused by bleaching of the fluorescence of C6-NBD-PC by oxidation products. This was done by subtracting the apparent first order rate constant of the (quite limited) decrease of fluorescence obtained in each LDL sample in the presence of Pefabloc from that obtained in the absence of Pefabloc. The resultant values of the normalized rate constants were used to compute the ratio between the normalized rate constant, as observed at any given time during oxidation, and the corresponding value obtained for non-oxidized LDL. The normalized rate constants were then used to compute the % of inactivation presented in Fig. 4D as a function of time.
Interrelationship between LDL oxidation and PLA
of all the lipidic oxidation products (Fig. 4A–C). Inactivation of LDL-PLA in the time course of oxidation can therefore not explain the lack of effect of Pefabloc on the rate of oxidation. Hence, hydrolysis of oxidized and/or fragmented phospholipids by LDL-associated PLA indeed does not protect LDL (in vitro) against continuing oxidation.
DISCUSSION
The obvious physiological role of PAF-AH is to hydrolyze PAF. Notably, LDL oxidation in vitro does not produce detectable amounts of PAF unless the LDLassociated PAF-AH is inactivated,20 indicating that this enzyme is capable of hydrolyzing the formed PAF, thus modulating its concentration. This is also accompanied by enhanced production of lysophosphatidylcholine during oxidation, suggesting that PAF-AH is also responsible for hydrolysis of oxidized phospholipids.20,33,35–36 Copper-induced peroxidation results in a time-dependent partial inactivation of PAF-AH.32–33,35 Nonetheless, since this inactivation is not accompanied by accumulation of PAF, it follows that under normal conditions PAF-AH is not the rate limiting step in PAF hydrolysis.20 The results of our studies on the hydrolysis of the fluorescent phospholipid C6-NBD-PC lend further support to the hypothesis that PAF-AH is in fact a phospholipase A responsible for the hydrolysis of other short chain phospholipids, besides PAF.16 Such phospholipids are produced by fragmentation of the hydroperoxidized phospholipids that are formed upon oxidation of PUFAbearing phospholipids. Similar to PAF-hydrolysis, the hydrolysis of C6NBD-PC is also inhibited by Pefabloc (with a similar dose-dependence) and by oxidation (with a similar timedependence). The products of phospholipolysis of both PAF and short-chain phospholipids (lyso-PAF, lysolecithin and short-chain free fatty acids) are more soluble in water than the respective phospholipids and are therefore likely to leave the oxidized LDL, especially in the presence of albumin in the solution. This could have possibly resulted in reduced rate of propagation, i.e., protect the LDL-associated lipid from continuing oxidation.24 Therefore, it has been previously hypothesized that PAF-AH plays a protective role against oxidation.21 In our studies of lipid oxidation in vitro, inactivation of the LDL-PLA had no effect on LDL oxidation by either Cu21 or AAPH, either in the absence or in the presence of albumin. This means that PLA-catalyzed phospholipolysis does not protect LDL against continuing peroxidation. Moreover, since similar results were observed for unfractionated serum, it follows that the phospholipoly-
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sis does not play a role in the in vitro oxidation of all the lipoproteins. Yet, phospholipolysis may play a protective role against lipid oxidation and/or against the consequences of oxidation in vivo. The recent findings of Schmitt et al.37 indicated that phospholipolysis of minimally oxidized LDL reduces its toxicity towards endothelial cells. In interpreting these results, the authors propose that the products of phospholipolysis (lysolecithin and oxidized or short-chain fatty acids) partition into the medium, thus reducing the internalization of the cytotoxic oxidized phospholipids into the cells upon endocytosis of oxidized LDL. Evaluation of this (and other) possibilities requires further investigations. Acknowledgements—Financial support of this work by the chief scientist of Israel Ministry of Health (to D.L. and S.Y.), the Lady Davis Fund (to E.S.) and the Ministry of Absorbtion (to I.P) is acknowledged. The authors thank Drs. Edgar Pick of Tel-Aviv University, Alenexandros Tselepis and Theodoros Liapikos of the University of Ioaninna, and Colin H. Macphee of SmithKline Beecham Pharmaceuticals for helpful discussions.
REFERENCES 1. Steinberg, D.; Parthasarathy, S.; Carew, T. E.; Khoo, J. C.; Witzum, J. L. Beyond cholesterol: Modifications of low-density lipoprotein that increase its atherogenicity. N. Eng. J. Med. 320: 915–924; 1989. 2. Parthasarathy, S.; Steinberg, D.; Witzum, J. L. The role of oxidized low-density lipoprotein in the pathogenesis of atherosclerosis. Annu. Rev. Med. 43:219 –225; 1992. 3. Steinbrecher, U. P.; Zhang, H.; Lougheed, M. The role of oxidatively modified LDL in atherosclerosis. Free Radical Biol. Med. 9:155–168;1990. 4. Esterbauer, H.; Gebicki, J.; Puhl, H.; Jurgens, G. The role of lipid peroxidation and antioxidants in oxidative modification of LDL. Free Radical Biol. Med. 13:341–390;1992. 5. Requena, J. R.; Fu, M. X.; Ahmed, M. U.; Jenkins, A. J.; Lyons, T. J.; Baynes, J. W.; Thorpe, S. R. Quantification of malondialdehyde and 4-hydroxynonenal adducts to lysine residues in native and oxidized human low-density lipoprotein. Biochem. J. 322: 317–325;1997. 6. Steinberg, D. and workshop participants. Summary of the proceedings of a National Heart, Lung and Blood Institute Workshop: September 5– 6, 1991, Bethesda, Maryland. Circulation 85:2337– 2344; 1992. 7. Hoffman, R. M.; Garewall, H. S. Antioxidants and the prevention of coronary heart disease. Arch. Intern. Med. 155:241–246; 1995. 8. Esterbauer, H.; Ramos, P. Chemistry and pathophysiology of oxidation of LDL. Rev. Physiol. Biochem. Pharmacol. 127:31– 64; 1995. 9. Steinbrecher, U. P.; Pritchard, P. H. Hydrolysis of phosphatidylcholine during LDL oxidation is mediated by platelet-activating factor acetylhydrolase. J. Lip. Res. 30:305–314; 1989. 10. Stremler, K. E.; Stafforini, D. M.; Prescott, S. M.; McIntyre, T. Human plasma platelet-activating factor acetylhydrolase: oxidatively fragmented phospholipids as substrates. J. Biol. Chem. 266: 11095–11103; 1991. 11. Reisfeld, N.; Lichtenberg, D.; Dagan, A.; Yedgar, S. Apolipoprotein B exhibits phospholipase A1 and phospholipase A2 activities. FEBS Lett. 315:267–270; 1993. 12. Parthasarathy, S.; Barnett, J. Phospholipase A2 activity of low density lipoprotein: evidence for intrinsic phospholipase A activity of apolipoprotein B-100. Proc. Natl. Acad. Sci. USA 87:9741– 9745; 1990.
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13. Yamada, Y.; Stafforini, D. M.; Imaizumi, T. A.; Zimmerman, G. A., McIntyre, T. M.; Prescott, S. M. Characterization of the platelet-activating factor acetylhydrolase from human plasma by heterologous expression in Xenopus laevis oocytes. Proc. Natl. Acad. Sci. USA 91:10320 –10324; 1994. 14. Tjoelker, L. W.; Wilder, C.; Eberhardt, C.; Stafforini, D. M.; Dietsch, G.; Schimpf, B.; Hooper, S.; Trong, H. L.; Cousens, L. S.; Zimmerman, G. A.; Yamada, Y.; McIntyre, T. M.; Prescott, S. M.; Gray, P. W. Anti- inflammatory properties of a platelet-activating factor. Nature 374:549 – 552; 1995. 15. Washburn, W. N.; Dennis, E. A. Novel general approach for the assay and inhibition of hydrolytic enzymes utilizing suicide-inhibitory bifunctionally linked substrates (SIBLINKS): exemplified by a phospholipase A2 assay. J. Am. Chem. Soc. 112:2040 –2041; 1990. 16. Tew, D. G.; Southan, C.; Rice, S. Q. J.; Lawrence, G. M. P.; Li, H.; Boyd, H. F.; Moores, K.; Gloger, I. S.; Macphee, C. H. Purification, properties, sequencing, and cloning of a lipoprotein-associated, serine-dependent phospholipase involved in the oxidative modification of low-density lipoproteins. Arterioscler. Thromb. Vasc. Biol. 16:591–599; 1996. 17. Tjoelker, L. W.; Eberhardt, C.; Unger, J.; Trong, H. L.; Zimmerman, G. A.; McIntyre, T. M.; Stafforini, D. M.; Prescott, S. M.; Gray, P. W. PlasmapPlatelet-activating factor acetylhydrolase is a secreted phospholipase A2 with catalytic triad. J. Biol. Chem. 270:25481–25487; 1995. 18. Deigner, H. P.; Friedrich E.; Sinn, H.; Dresel, H. A. Scavenging of lipid peroxidation products from oxidizing LDL by albumin alters the plasma half-life of a fraction of oxidized LDL particles. Free Radical Res. Commun. 16:239 –246; 1992. 19. Schnitzer, E.; Fainaru, M.; Lichtenberg, D. Oxidation of low density lipoprotein upon sequential exposure to copper ions. Free Radical Res. 23:137–149; 1994. 20. Liapikos, T. A.; Antonopoulou, S.; Karabina, S. A.; Tsoukatos, D. C.; Demopoulos, C. A.; Tselepis, A. D. Platelet-activating factor formation during oxidative modification of low-density lipoprotein when PAF-acetylhydrolase has been inactivated. Biochim. Biophys. Acta 1212:353–360; 1994. 21. Stafforini, D. M.; Zimmerman, G. A.; McIntyre, T. M.; Prescott, S. M. The platelet-activating factor acetylhydrolase from human plasma prevents oxidative modification of low-density lipoprotein. Trans. Assoc. Am. Physicians 105:44 – 63; 1992. 22. Parthasarathy, S.; Steinbrecher, U. P.; Barnett, J.; Witztum, J. L.; Steinberg, D. Essential role of phospholipase A2 activity in endothelial cell-induced modification of low density lipoprotein. Proc. Natl. Acad. Sci. USA 82:3000 –3004; 1985. 23. Deigner, H. P.; Dresal, H. A. Effect of platelet activating factor on the kinetics of LDL oxidation in vitro. FEBS Lett. 317:202–206; 1993. 24. Fyrnys, B.; Blencowe, C.; Diegner, H. P. Susceptibility of phospholipids of oxidizing LDL to enzymatic hydrolysis modulates uptake by P388D1 macrophage-like cells. FEBS Lett. 357:7–12; 1995. 25. Danten, C.; Tselepis, A. D.; Chapman, M. J.; Ninibo, E. Pefabloc, 4-[2-aminoethyl]benzenesulfonyl fluoride, is a new, potent non-
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toxic and irreversible inhibitor of PAF-degrading acetylhydrolase. Biochem. Biophys. Acta 1299:353–357; 1996. Schnitzer, E.; Pinchuk, I.; Bor, A.; Fainaru, M.; Lichtenberg, D. The effect of albumin on copper-induced oxidation. Biochim. Biophys. Acta 1344:300 –311; 1997. Pinchuk, I.; Lichtenberg, D. Continuous monitoring of intermediates and final products of oxidation of low density lipoprotein by means of UV-spectroscopy. Free Radical Res. 24:351–360; 1996. Schnitzer, E.; Pinchuk, I.; Fainaru, M.; Schafer, Z.; Lichtenberg, D. Copper-induced lipid oxidation in unfractionated plasma:the lag preceding oxidation as a measure of oxidation-resistance. Biochem. Biophys. Res. Comm. 216:854 – 861; 1995. Wittenauer, L. A.; Shirai, K.; Jackson, R. L.; Johnson, D. Hydrolysis of a fluorescent phospholipid substrate by phospholipase A2 and lipoprotein lipase. Biochem. Biophys. Res. Commun. 118:894 – 901; 1984. Meyuhas, D.; Yedgar, S.; Rotenberg, M.; Reisfeld, N.; Lichtenberg, D. The use of C6-NBD-PC for assaying phospholipase A2activity: Scope and limitations. Biochim. Biophys. Acta 1124:223– 232; 1992. Tselepis, A. D.; Dentan, C.; Karabina, S. A. P.; Chapman, M. J.; Ninio, E. PAF-degrading acetylhydrolase is preferentially associated with dense LDL and VHDL-1 in human plasma. Arterioscler. Thromb. Vasc. Biol. 15:1764 –1773; 1995. Dentan, C.; Lesnik, P.; Chapman, M. J.; Ninio, E. PAF-acetherdegrading acetylhydrolase in plasma LDL is inactivated by copperand cell-mediated oxidation. Arterioscler. Thromb. 14:353–360; 1994. Karabina, S. A. P.; Elisaf, M. C.; Goudevenos, J.; Siamopoulos, K. C.; Sideris, D.; Tselepis, A. D. PAF-acetylhydrolase activity on Lp(a) before and during Cu21-induced oxidative modification in vitro. Atherosclerosis 125:121–134; 1996. Bowry, V. W.; Stocker, R. Tocopherol-mediated peroxidation the prooxidant effect of Vitamin E on the radical-initiated oxidation of human low-density lipoprotein. J. Am. Chem. Soc. 115:6029 – 6044; 1993. Karabina, S. A. P.; Liapikos, T. A.; Grekas, G.; Goudevenos, J.; Tselepis, A. D. Distribution of PAF-acetylhydrolase activity in human plasma low-density lipoprotein subfractions. Biochim. Biophys. Acta 1213:34 –38; 1994. Karabina, S. A. P.; Elisaf, M.; Bairaktari, E.; Tziallas, C.; Siamopoulos, K. C.; Tselepis, A. D. Increased activity of platelet-activating factor acetylhydrolase in low-density lipoprotein subfractions induces enhanced lysophosphatidylcholine production during oxidation in patients with heterozygous familial hypercholesterolamia. Europ. J. Clin. Inves. 27:595– 602; 1997. Schmitt, A.; Negre–Salvayre, A.; Troly, M.; Valdiguie, P.; Salvare, R. Phospholipid hydrolysis of mildly oxidized LDL reduces their cytotoxicity to cultured endothelial cells. Potential protective role against atherogenesis. Biochem. Biophys. Acta 1256:284 –292; 1995. Reisfeld, N.; Lichtenberg, D.; Yedgar, S. Inhibition of LDL-associated phospholipase A activity in human plasma by albumin. J. Basic Clin. Physiol. Pharm. 5:107–115; 1994.
APPENDIX
Determination of lipoprotein-associated phospholipase A (PLA) activity by PLA-catalyzed hydrolysis of C6NBD-PC The fluorescence intensity of C6-NBD-PC depends on: (i) its concentration in the solution (ii) the environment of the medium where it resides, which determines the quantum yield of fluorescence and (iii) occurrence of self-quenching at high concentrations of the probe in the compartment of its residence. In aqueous media, the quantum yield of the NBD fluorescence is very low. Moreover, in the absence of lipids in the aqueous system, C6-NBD-PC self-assembles into micelles and its
fluorescence is further reduced by self-quenching and is therefore very low. In the presence of lipoproteins, the probe partitions between the lipidic cores (and interfaces) of the lipoprotein particles and the aqueous medium. The high affinity of the C6-NBD-PC to lipids at the lipid/water interface and the high quantum yield of the probe when it resides in a lipidic environment result in a marked increase of the fluorescence intensity upon addition of LDL to a micellar solution of C6-NBD-PC30. Hydrolysis of the probe results in the formation of the water soluble NBD-caproic acid and in a consequent decrease of the fluorescence intensity. This can be used to monitor the hydrolysis of LDL-associated C6-NBD-PC only under conditions where: (i) prior to hydrolysis, all the
Interrelationship between LDL oxidation and PLA probe resides in LDL particles, so that its hydrolysis will not be accompanied by redistribution of C6-NBD-PC from the aqueous solution into LDL particles (which might attenuate the reduction of fluorescence due to hydrolysis), and (ii) the fluorescence intensity of the LDL-associated probe is not self-quenched. This appendix describes the results of control experiments conducted with LDL in the absence and presence of albumin and with 50-fold diluted serum under conditions of complete inactivation of the lipoprotein-associated PLA by Pefabloc. In all the experiments described below, the fluorescence intensity of C6-NBD-PC depended on the concentration of the probe and on the probe /lipid ratio but was independent of the time after mixing the probe with the lipoprotein(s). This implies that equilibration of the system (i.e., partitioning of the probe between the aqueous phase and lipidic compartments) is rapid and that following equilibration the probe is indeed not hydrolyzed in the time course of the experiments. For fractionated LDL, we have conducted the following two experiments: first, we have titrated LDL (0.05 mM) with C6-NBD-PC, and monitored the increase of fluorescence as a function of the concentration of the probe. In this experiment the fluorescence increased linearly with the concentration of the probe (i.e., the molar fluorescence remained constant) up to 0.5 mM C6-NBD-PC. At higher concentrations of the probe, the molar fluorescence decreased. This may either be a result of ‘saturation‘ of the LDL with C6-NBD-PC (i.e., partitioning of the added probe into water, where its quantum yield is low) or of self-quenching of fluorescence in LDL particles when the concentration of the probe is high (i.e., at C6-NBD-PC/LDL ratios higher than 10). The former possibility can be ruled out on the basis of our second control experiment, in which we have measured the change in fluorescence of a mixture of 4 mM C6-NBD-PC and 0.3 mM LDL upon an 8-fold dilution. In this experiment, the (partially quenched) fluorescence intensity decreased linearly with the concentration of the probe (i.e., the molar fluorescence remained constant), indicating that under these experimental conditions, essentially all the C6-NBD-PC resides in LDL particles and not in the aqueous medium. In other words, the fluorescence of C6-NBD-PC is not a function of the LDL/water partitioning but only of the probe/LDL ratio. When the latter ratio was lower than 10 (as in all the experiments described in the present study) the fluorescence was not self-quenched. Since each LDL particle contains 700 –1000 phospholipid molecules, a C6-NBD-PC/LDL ratio of 10
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implies that the ratio of C6-NBD-PC to phospholipid molecules of the outer monolayer of LDL is about 1/100 –1/70. Interestingly, a similar threshold of self-quenching was observed for emulsion particles composed of PC and triglycerides,30 indicating that in both these systems, which are different with respect to the core/interface ratio, the C6-NBD-PC resides predominantly, if not exclusively in the outer phospholipid monolayer. In albumin-containing media and in non-fractionated serum, part of the C6-NBD-PC is likely to be bound to albumin.38 To evaluate the fraction of LDL-associated C6-NBD-PC, we have first determined the fluorescence of albumin-bound C6-NBD-PC in comparison to that of the lipoprotein-associated probe. This was done by monitoring the fluorescence intensity of a constant C6-NBD-PC concentration (1 mM) upon stepwise addition of increasing concentrations of either LDL, HDL, albumin or unfractionated serum. In each of these experiments, the fluorescence increased with the concentration of the additive until it became constant. The highest fluorescence was observed for LDL. In comparison to LDL, the maximal fluorescence in HDL was 75%, in albumin it was 50% whereas in serum the maximal fluorescence was about 72% of that observed in LDL. In this study we examined the PLA activity of 50-fold diluted serum. In serum, the level of fluorescence intensity is of course a function of the (rather complex) partitioning of C6-NBD-PC between albumin, LDL and HDL. Accordingly, the fluorescence intensity observed for any given concentration of C6-NBD-PC below 5 mM in media containing 12 mM albumin (as in 50-fold diluted serum) was lower than in 50-fold diluted serum, where part of C6-NBD-PC resides in lipoproteins in which the fluorescence is higher than that of albuminbound probe. Stepwise addition of C6-NBD-PC to 50-fold diluted serum resulted in a linear increase of the fluorescence intensity (i.e., in constant molar fluorescence) up to a C6-NBD-PC concentration of 2 mM. Only at higher C6-NBD-PC concentrations, the molar fluorescence decreased, probably due to self-quenching of lipoprotein-associated C6-NBD-PC. One important implication of this finding is that at 2 mM C6-NBD-PC the time-dependent decrease of fluorescence can be used as a measure of PLA activity of serum lipoproteins. Secondly, since self-quenching occurred only above 2 mM C6-NBD-PC, as compared to 0.5 mM in LDL of similar lipid concentration, we estimate that in 50-fold diluted serum only about a fourth of the probe (2 mM) is associated with LDL.