shown to be distinct from LCAT activity (10, 11), no studies a 1-acyl analog ofpaf. These results thus indicate a function

Transcription

1 Proc. Natd. Acad. Sci. USA Vol. 91, pp , June 1994 Biochemistry Hydrolysis and transesterification of platelet-activating factor by lecithin-cholesterol acyltransferase ysopbosatdyl coe/ l /W d perodaton/phosphoipase A) MING LiU AND PAPASANI V. SUBBAIAH* Departments of Medicine and Biochemistry, Rush Medical College, Chicago, IL 6612 Communicated by John A. Glomset, March 14, 1994 AMBSRACT Purified lecithin-cholesterol acyltrnsferase (, EC ) from human plaa was found to hydrolyze platlet-activating factor (PAF) to Iyso-PAF and acetate. In addition, it catalyzed the transer of the acetate group from PAF to lysophosphatidyicholine, fr i lyso-paf and a 1-acyl analog of PAF. In contrast to the cholesterolesterication reaction carried out by the enzyme, the hydrolysis and t etlation of PAF by did not require an apoprotein activator and were not inhibited by suhydryl inhibitors but were inhibited by serum albumin. When added to a proteoliposome substrate of or to whole plasma, PAF inhibited Cholesterol esterification by competitively. PAF acetylhydrolase (EC ), purified from human plasma, also catalyzed the transfer of acetate from PAF to lysophosphatidylcholine. However, the -catalyzed reactions of PAF were not due to ontnation with PAF acetylhydrolase, since the ratio of acetyl iraer to acetyl hydrolysis was 3 times greater for, when compared with PAF acetylhydrolase under identical conditions. Furthermore, recombinant human secreted by baby hamser kidney cells also catalyzed the hydrolysis and t ylation of PAF. These results demonstrate that can inactivate PAF in plasma by tracetlatio and suggest that it may have a role in the meta of PAF, and possibly of oxidized phospholipids, in plasa. Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycerol-3-phosphocholine) is a highly bioactive phospholipid present in various tissues and plasma and has a broad range of biological effects, including stimulation of platelet aggregation, neutrophil activation, smooth muscle contraction, vascular permeability, and hepatic glycogenolysis (1, 2). The half-life of PAF in plasma is very short (6-1 min), probably because it is rapidly hydrolyzed to lyso-paf by a lipoproteinassociated enzyme called PAF acetylhydrolase (PAF-AH; EC ). The activity of this enzyme is increased in essential hypertension (3, and other pathological conditions (2). PAF-AH has also been shown to hydrolyze shortchain analogs of phosphatidylcholine (PC) generated during the oxidation of lipoproteins (5). Another enzyme that can potentially hydrolyze PAF as well as the oxidized phospholipids in the plasma is lecithin-cholesterol acyltransferase (; PC-sterol O-acyltransferase, EC ), whose primary function is to synthesize cholesteryl esters by transferring a fatty acid from PC to a free cholesterol molecule (6). This enzyme also exhibits phospholipase A activity in the absence of an acyl acceptor (7) and can hydrolyze watersoluble esters such as p-nitrophenyl esters of short-chain fatty acids (8). Earlier studies showed that can also transfer an acyl group from PC to Iyso-PC, forming another PC molecule (9). Although the PAF-AH activity has been The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact. shown to be distinct from activity (1, 11), no studies have been conducted to determine whether itself can hydrolyze PAF. We have investigated the possible role of in the hydrolysis of PAF in plasma. Our results show that not only can hydrolyze PAF to lyso-paf but also can transfer the acetyl group from PAF to lyso-pc, forming a 1-acyl analog ofpaf. These results thus indicate a function for in the metabolism of PAF and identify a pathway for the inactivation of PAF in plasma. MATERIALS AND METHODS Materials. 1-[1-14C]Palmitoyl lyso-pc, [4-14C]cholesterol, and 2-[3H]acetyl PAF were purchased from DuPont/NEN. Egg PC, unlabeled lyso-pc, and PAF were obtained from Avanti Polar Lipids. Diisopropyl fluorophosphate and 5,5'- dithiobis(nitrobenzoic acid) (DTNB) were purchased from Sigma. and apolipoprotein (apo) A-I were purified from normal human plasma (12, 13). The final preparations gave single bands in SDS/polyacrylamide gels, corresponding to a molecular weight of 67 kda (for ) or 28 kda (for apo A-I). PAF-AH was purified from human plasma by the procedure of Stafforini et al. (1) up to the DEAE-Sepharose column step. The enzyme was purified 122-fold from the starting plasma and showed no activity when assayed with standard proteoliposome substrate (1. The final PAF-AH preparation hydrolyzed 39.8 pmol of PAF per hr per mg of protein. Recombinant human was a generous gift from P. H. Pritchard (University of British Columbia). This enzyme was secreted by stably transfected baby hamster kidney (BHK) cells into serum-free medium and was purified by chromatography on a phenyl-sepharose column (15). It had a specific activity of 9.3 nmol of cholesterol esterified per hr per ug of protein, in the proteoliposome assay (1. Enzyme Assays. PAF hydrolytic activity was determined by the release of labeled acetate from labeled PAF (1). The reaction mixture (.4 ml) contained 8,uM 3H-acetate-labeled PAF (.5 uci; 1 pci = 37 kbq) in 1 mm Tris'HCl (ph 7. and either purified (.2-2 jug) or partially purified PAF-AH (.4-.1 ptg). After incubation at 37C, the reaction mixture was extracted (16) and aliquots of the aqueous layer and the chloroform layer were assayed in a liquid scintillation counter to determine the radioactivity in free acetate and unreacted PAF, respectively. The results obtained with this method corresponded closely to those obtained by the C18 column method of Stafforini et al. (1). The transfer of acetate from PAF to lyso-pc was determined in a reaction mixture (.4 ml) which contained 1-[1- Abbreviations: apo, apoplipoprotein; DTNB, 5,5'-dithiobis(nitrobenzoic acid); LAT, lysolecithin acyltransferase;, lecithincholesterol acyltransferase; PAF, platelet-activating factor; PAF- AH, PAF acetylhydrolase; PC, phosphatidylcholine. *To whom reprint requests should be addressed. 635

2 636 Biochemistry: Liu and Subbaiah 14C]palmitoyl lyso-pc (12 nmol), 32 nmol of unlabeled PAF, and 1 mm Tris HCl (ph 7.. Where indicated, 22 pg of apo A-I or 4 mg of human serum albumin was added. After incubation with purified or PAF-AH for various times at 37C, the reaction was stopped by the addition of 1 ml of methanol, and the lipids were extracted (16). Lyso-PC and PAF were separated on silica-gel TLC plates with the solvent system chloroform/methanol/water, 65:25:4 (vol/vol), and their radioactivity was determined in a liquid scintillation counter. The labeled short-chain PC formed by the acetylation of labeled lyso-pc migrated with authentic PAF (Rf.89), which moved below normal PC (Rf.189) but above lyso-pc (Rf.53) in this solvent system. This reaction was termed as LAT-il to distinguish it from the formation of labeled long-chain PC, which was called simply LAT (lysolecithin acyltransferase). The assays of (cholesterol esterification) and LAT (long-chain PC formation) activities were performed with a proteoliposome substrate containing egg PC, 14C-labeled free cholesterol, 14C-labeled lyso-pc, and apo A-I at molar ratios of 25:12.5:12.5:.8 (13, 1. After incubation with the enzyme in the presence of 5 mm 2-mercaptoethanol for 3 min, the reactions were stopped by the addition of methanol and the lipids were extracted (16). Aliquots of the lipid extracts were spotted on two separate silica-gel TLC plates. One plate was developed in chloroform/methanol/water, 65:25:4 (vol/ vol), to separate lyso-pc and PC, and the second plate was developed in hexane/diethyl ether/acetic acid, 7:3:1 (vol/ vol), to separate free cholesterol and cholesteryl ester. The radioactivity was determined in each of the spots and the percent esterification of free cholesterol or lyso-pc was determined. When PAF was included in the reaction mixture, the acetylation of lyso-pc (formation of labeled short-chain PC) was also determined. Unless otherwise mentioned, all the values presented are averages of at least two experiments, each performed in duplicate. Variation between experiments was <1%. RESULTS Hydrolysis oflabeled PAF by Purified. Incubation of acetate-labeled PAF (8 pm) at 37 C with purified human resulted in a time-dependent release of labeled acetate (Fig. 1). There was no hydrolysis in the absence of the enzyme. Addition of apo A-I at a concentration known to la Q Ṉ 'a a FIG. 1. Hydrolysis of PAF by. The control reaction contained 8 pm 3H-acetate-labeled PAF and 2 pg of purified human in 1 mm Tris Cl (ph 7.. After incubation at 3rC for various times, the reaction was stopped by the addition of methanol. The lipids were extracted (16), and the radioactivity in the aqueous layer was determined. Where indicated, 22 pg ofapo A-I or4 mg of human serum albumin was added. Identical results were obtained when the labeled acetate was determined by the Cig column method (1). fully activate cholesterol esterification by (22 pg/.4 ml) had no effect on PAF hydrolysis by the enzyme. Human serum albumin, another known activator of cholesterol esterification, in contrast, inhibited the hydrolysis of PAF, presumably because PAF binds to albumin (17). These results show that can hydrolyze PAF to lyso-paf and acetate and that this activity of does not require apoprotein activators. Transfer of Acetate from PAF to Lyso-PC and Free Cholesterol by. To determine whether could transfer the acetyl group from PAF to free cholesterol or lyso-pc, we incubated unlabeled PAF (8 pm) with purified and either labeled lyso-pc or labeled cholesterol. There was formation of labeled short-chain PC (LAT-Il activity) (Fig. 2), but not cholesteryl ester (results not shown). The LAT-II reaction was not activated by the presence ofapo A-I but was inhibited by albumin. The inhibition of transacetylation by albumin was greater than that observed for the hydrolysis of PAF, probably because albumin sequesters both PAF and lyso-pc. There was no formation of labeled short-chain PC when labeled lyso-pc was incubated with the enzyme and 8-32 mm sodium acetate instead of PAF (results not shown). These results show that can transfer the acetyl group from PAF to lyso-pc, but not to cholesterol, under the conditions of the assay. Effect of PAF on and LAT Reactions Carried Out by Enzyme. If PAF binds to protein at the same site as PC, then it should inhibit the and LAT reactions because of competition with the PC substrate. To test this hypothesis we added various concentrations of PAF to reaction mixtures containing purified and a proteoliposome substrate consisting of egg PC, 14C-labeled lyso PC, 14C-labeled free cholesterol, and apo A-I at molar ratios of 25:12.5:12.5:.8. Using this system, we simultaneously determined the synthesis of labeled cholesteryl ester ( activity), labeled long-chain PC (LAT activity), and labeled short-chain PC (LAT-II activity) as described in Materials and Methods. With increasing concentrations of PAF in the reaction mixture there was a gradual inhibition of and LAT reactions, with a concomitant increase in the formation of short-chain PC (LAT-II activity) (Fig. 3). These results show that PAF can effectively compete with PC as an acyl donor. When the labeled cholesteryl ester was analyzed by reverse-phase HPLC (18), we could not detect any formation ' 4 & W u, 4 a Proc. Natl. Acad. Sci. USA 91 ( FIG. 2. Acetylation of lyso-pc by in the presence of PAR. The control reaction mixture contained 8 pm unlabeled PAF, 3 pm 1-[1-"4C]palmitoyl lyso-pc, and 2 pg of purified, in a final volume of.4 ml. The reactions were stopped by the addition of methanol, lipids were extracted (16), and the lyso-pc and PAF were separated on silica-gel TLC plates as described in the text. The amount oflyso-pc esterified was calculated from the radioactivity in the PAF spot, which contained the labeled short-chain PC formed by the acetylation of labeled lyso-pc.

3 Biochemistry: Liu and Subbaiah 2. o LAT-TI / LAT PAF (pm) FiG. 3. Effect of PAF on and LAT reactions. Various amounts of unlabeled PAF were added to a reaction mixture containing 2 eg of purified, 5 mm 2-mercaptoethanol, and a proteoliposome substrate containing egg PC, 14C-labeled lyso-pc, 14C-labeled cholesterol, and apo A-I at molar ratios of 25:12.5:12.5:.8. The formation of labeled cholesteryl ester ( activity), long-chain PC (LAT activity), and short-chain PC (LAT-il activity) was determined as described in the text. of cholesteryl acetate, showing again that the acetyl group is transferred to lyso-pc, but not to cholesterol. To determine whether PAF could compete with PC in intact plasma, we repeated the above experiment with freshly prepared normal human plasma which has been prelabeled with 14C-labeled cholesterol and "4C-labeled lyso-pc. Here also we found an inhibition of both and LAT reactions with simultaneous stimulation of the LAT-II reaction in the presence of increasing amounts of PAF (Fig.. However, compared with the proteoliposome system, the concentration of PAF required to inhibit activity was considerably higher, probably because of the presence of albumin and other factors in plasma which dampen PAF effects. Hydrolysis and Transesterfication of PAF by Recombinant. Since human plasma contains a highly active PAF- AH, it was important to exclude the possibility that the 5. Proc. Nail. Acad. Sci. USA 91 ( activity observed with purified was due to contamination with small amounts of PAF-AH. For this purpose we obtained a recombinant secreted into serum-free medium by BHK cells which have been stably transfected with human cdna (15) and tested its ability to metabolize PAF. The recombinant human was able to hydrolyze PAF as well as transfer the acetate to lyso-pc (Fig. 5). Similar results were obtained with a recombinant secreted by transiently transfected COS-1 cells (results not shown). In both cases, there was no activity in the medium from control cells which had been transfected with "empty" expression vector (pnut). Comparison of PAF-AH and Activities. To further differentiate between the activities of PAF-AH and, we partially purified PAF-AH from human plasma and tested whether it could transfer acetate from PAF to lyso-pc. PAF-AH was observed to transfer acetate from PAF to lyso-pc, in addition to releasing free acetate (Fig. 6). However, it did not show any cholesterol-esterifying activity in the presence of standard proteoliposome substrate (1 (results not presented), showing that it was not contaminated with. The kinetic constants of purified and PAF-AH were determined in the presence of various concentrations of PAP. The apparent Km values calculated from the Lineweaver-Burk plots were 17.9 AM for PAF-AH and 12.8 pm for. The V. value for pure was.2 janol of PAF hydrolyzed per hr per mg, while for the PAF-AH preparation it was 41 umol per hr per mg. In comparison, the cholesterol esterification rate by under optimal conditions was 12 pmol of cholesterol esterified per hr per mg. The and LAT activities were inhibited 8-9%6 by.5 mm DTNB (Fig. 7). However, the inhibition of LAT-II activity was much lower even at higher DTNB concentrations. Therefore the sulflhydryl groups do not appear to be essential for the hydrolysis or transfer of acetate from PAF. The transfer and hydrolytic reactions of PAF-AH were not inhibited by DTNB, as reported by others (5, 17). Diisopropyl fluorophosphate (1 mm) inhibited all the reactions of, although LAT-il appeared to be less sensitive than other.25._ i Transfer '~~~~~~~/ x-- Hydrolysis /.,.15 o o.1 ~~~~~ PAF (pm) FIG. 4. Effect of PAF on and LAT reactions in whole plasma. Freshly prepared human plasma was labeled with trace amounts of [14C]cholesterol and [14C]lyso-PC by overnight incubation at 4C. Unlabeled PAF was added at the indicated concentrations and the mixture was incubated for 3 min at 37C in the presence of 5 mm 2-mercaptoethanol. Labeled cholesteryl ester and cholesterol were separated by TLC ofan aliquot ofthe lipid extract (solvent system of hexane/diethyl ether/acetic acid, 7:3:1) and their radioactivities were determined, to measure activity. Another aliquot of lipid extract was subjected to chromatography in chloroform/methanol/water, 65:25:4, and the radioactivity in short-chain PC (LAT-II activity) and long-chain PC (LAT activity) was determined..' FIG. 5. Hydrolysis and tranacetylation of PAF by recombinant. Recombinant human, expressed by transfected BHK cells (15), was obtained from P. H. Pritchard. The hydrolysis of PAF was determined with 8 AM [3H]acetate-labeled PAF, whereas the transfer of acetate from PAF was determined in the presence of 8 pm unlabeled PAF and 3 pm 1-[1-14C]palmitoyl lyso-pc. Both reaction mixtures contained 5 mm 2-mercaptoethanol and.22 jg of purified recombinant. The values shown are averages of duplicate samples from one typical experiment. Similar results were obtained with medium from transiently transfected COS-1 cells. Medium from control cells which had been transfected with vector without cdna insert did not show any enzyme activity (results not shown).

4 638 Biochemistry: Liu and Subbaiah 1 U O FiG. 6. Hydrolysis and transacetylation of PAF by PAF-AH. Hydrolysis was determined by the release of labeled acetate from [3H]acetate-labeled PAF (8 AM), and tranacetylation was measured by the formation of labeled short chain-pc in the presence of 1-[1-14C]palmitoyl lyso-pc and unlabeled PAF. All reaction mixtures contained.94 p&g of partially purified PAF-AH. Values shown are averages of duplicate samples from a typical experiment. reactions (results not shown). Diisopropyl fluorophosphate also inhibited the PAF-AH activities, in agreement with previous studies (5, 19). To compare the relative activities of acetyl transfer and acetyl hydrolysis by and PAF-AH, we studied the two reactions of each enzyme by employing identical amounts of enzyme and PAF for both reactions. As expected, the ratio of acetyl transfer to acetyl hydrolysis increased for both enzymes with increasing, concentrations of lyso-pc (Fig. 8). However, the ratio was about 3-fold higher for than for PAF-AH at all'lyso-pc concentrations. The ratio obtained for recombinant was similar to that of purified plasma (results not shown). These data indicate that it is unlikely that the transferase and hydrolase activities of are due to contamination with PAF-AH. DISCUSSION The results provide evidence for a function of in the metabolism of PAF. Although the major function of in 12 1' I- a 8 t 6.* DTNB (mm) FiG. 7. Effect of DTNB on the reactions carried out by and PAF-AH. Various amounts of DTNB were added to reaction mixtures containing either 8 pm [3H]acetate-labeled PAF alone or 8 pm unlabeled PAF and 3 pm labeled lyso-pc. Each reaction mixture contained either 1.5 pig of or.5 pg of PA-AH. a, Cholesterol esterification by ; o, LAT reaction of (formation of long-chain PC);,, LAT-I1 reaction by (formation of short-chain PC);, hydrolysis of PAF by PAF-AH; A, formation of short-chain PC by PAF-AH. 2. *._ Cu : 1.2., ~. >b Z.8 n.4. Proc. Nad. Acad Sci. USA 91 ( LPC (pm) - /, PAF-AH ~~~~~~~~~1 o lo o o.,b FIG. 8. Ratio of acetyl transfer to acetyl hydrolysis reactions by and PAF-AH. Acetyl transfer (LAT-il reaction) was determined in the presence of 15 IAM unlabeled PAF and the indicated amounts of labeled lyso-pc (LPC), either with 1.5 pg of or with.6 pg of PAF-AH. Hydrolysis of PAF was determined under identical conditions, except that acetate-labeled PAF and unlabeled lyso-pc were employed. plasma is to catalyze the esterification of free cholesterol, it is essentially a specialized phospholipase A which utilizes cholesterol, instead of water, as the acyl acceptor. The enzyme in fact exhibits phospholipase A activity in the absence of an acyl acceptor (7). It has also been shown to hydrolyze short-chain acyl esters in vitro, and this reaction is neither apo A-I-dependent nor inhibited by sulflhydryl inhibitors (8). The hydrolysis ofpaf by is therefore similar to its hydrolysis of other water-soluble esters, because it is also not dependent upon the apoprotein activator and is not inhibited by DTNB. The apoprotein activatoris required only for the interfacial activation of the enzyme, and DTNB inhibits the hydrolysis of only long-chainfatty acids from PC (8), presumably by steric interference at the active site (2). The'involvement of the same active site for cholesterol esterification and PAF hydrolysis is indicated because PAF inhibits cholesterol esterification competitively and PAF hydrolysis is inhibited by diisopropyl fluorophosphate. Although we have not been able to selectively inhibit PAF-AH activity, it appears very unlikely that the hydrolysis of PAF by is due to a contamination with PAF-AH because of the following. (i) All the PAF-AH in plasma is associated with low density and high density lipoproteins (1), whereas our starting material for purification was lipoprotein-free plasma (13). (ii) The purified gave a single protein band at 67 kda in SDS/polyacrylamide gels, whereas PAF-AH has a molecular mass of 43 kda (1). (iii) Recombinant secreted by two different cell lines also carried out PAF hydrolysis. (iv) The ratio of acetyl transfer to acetyl hydrolysis by was much higher than the ratio obtained for PAF-AH. In addition to the hydrolysis of PAF to lyso-paf and free acetate, can transfer the acetate from PAF to lyso-pc. While Malone et al. (21) have reported that the acetate from PAF is not directly transferred to other lipids in platelets, our data show that such a transfer does occur in plasma and that it is carried out by both and PAF-AH. Since there is no acetyl-coa-generating mechanism in plasma, the formation of labeled short-chain PC from labeled lyso-pc could have taken place only by the direct transfer of acetate from PAF. Lee et al. (22) reported the presence ofa similar enzyme activity in HL-6 human promyelocytic leukemia cells', with broad specificity toward acyl acceptors. However, this enzyme activity differs from activity, because it is membrane-bound, is activated by phenylmethanesulfonyl fluoride, and does not transfer long-chain acyl groups.

5 The possible physiological importance of in PAF metabolism can only be speculative at present. Although the in vitro activity of PAF-AH (as assayed under optimal conditions) is in large excess relative to the PAF concentration in the plasma (1), in vivo this activity is about 6 times lower than expected (1, 23). It appears to be increased in certain pathological conditions (2) and is regulated by estrogen (11). It is therefore possible that the PAF-AH activity is limiting in vivo and that may have a supplementary role in the degradation of PAF, especially in high density lipoprotein and in the lipoprotein-free fraction of plasma, which contain most of the PAF (17) and (2 but little PAF-AH (17). It is of interest that in the presence of physiological concentrations of lyso-pc, carries out predominantly the transacetylation of PAF, whereas PAF-AH catalyzes the hydrolysis (Fig. 8). It therefore appears that PAF is converted to lyso-paf by two different pathways in the plasma; one by PAF-AH, which releases free acetate from PAF, and the other by, which predominantly transfers the acetate to a lyso-pc acceptor. Since is also capable of esterifying lyso-paf with a long-chain fatty acid (25), the complete inactivation cycle of PAF (hydrolysis followed by reacylation), which occurs intracellularly in several tissues (1, 2), can also take place in the plasma (Fig. 9). Our preliminary results (not shown) suggest that can transfer the acetate group from 1-palmitoyl 2-acetyl PC to lyso-paf, thus regenerating PAF. Another possible function ofthe -mediated reactions described here is in the metabolism ofoxidized phospholipids in plasma. The oxidation ofphospholipids is one of the initial events in the oxidative modification of low density lipoproteins, which leads to rapid uptake of low density lipoproteins through the scavenger-receptor pathway and the formation of foam cells (26). There is strong evidence that PAF-AH can hydrolyze the oxidatively modified short-chain residues (up to six carbons) from PC (5, 27), and this might be part of a repair mechanism following the oxidative damage. However, the oxidative cleavage of linoleate (9, 12-octadecadienoate), the major unsaturated fatty acid in plasma, leads to the formation of 9- or 12-carbon residues at the sn-2 position of PC, which cannot be hydrolyzed by PAF-AH (5). Similarly, the hydroxy and hydroperoxy derivatives of the long-chain fatty acids cannot be cleaved from PC by PAF-AH (27). Our recent studies have shown that purified can hydrolyze most of the oxidatively modified PCs, including 1-palmitoyl 2-linoleoyl PC (unpublished results). Similar phospholipase A activity associated with low density lipoproteins was reported by Parthasarathy and Barnett (28), who suggested PAF-AH 1-alkyl-2-acetyl GPC PAF 1-acyl-2-lyso GPC PAF-AH acetateic 1-alkyl-2-lyso GPC 1-acyl-2-acetyl GPC 1-alkyl-2-Iyso 1,2-diacyl GPC ` 1-acyl-2-lyso FIG. 9. Biochemistry: Liu and Subbaiah GPCO* 1-alkyl-2-acyl GPO GPC Possible pathways of PAF metabolism in plasma. GPC, sn-u1vceronhosnhochoine. Proc. Nat!. Acad. Sci. USA 91 ( that this activity was intrinsic to apo B. Our results show that purified not only can hydrolyze the oxidized PCs but also can transfer the modified acyl group to a lyso-pc acceptor. Further, we found that oxidation of whole plasma by Cu2+ in the presence of labeled lyso-pc resulted in the formation of labeled "modified PC" which was more polar than the normal PC. At the same time, the oxidation resulted in an inhibition of esterification of cholesterol and lyso-pc with normal fatty acids. 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