Synthesis of Phospholipid by Foam Cells Isolated from Rabbit Atherosclerotic Lesions

Transcription

1 Synthesis f Phsphlipid by Fam Cells Islated frm Rabbit Athersclertic Lesins By Allen J. Day, M.D., D.Phil., H. A. I. Newman, Ph.D., and D. B. Zilversmit, Ph.D. ABSTRACT In a preliminary study, fam cells frm athersclertic lesins have been islated by incubatin f the intima with cllagenase and elastase. The cmpsitin and metablism f hmgeneus preparatins f fam cells btained in this way have been cmpared with ther parts f the athersclertic intima. Abut 1% f the chlesterl and phsphlipid f the intima was present in the cells btained. The phsphlipids f the fam cells cntained a lwer percentage f lecithin and f sphingmyelin and a higher percentage f "phsphatidyl ethanlamine" than the ther parts f the intima. The islated fam cells incubated in vitr incrprated P Jf -phsphate int phsphlipid at a rate f abut.5 m/ijnles/1 cells/hr. The P 31 was incrprated mainly int lecithin and phsphatidyl insitl with smaller amunts incrprated int phsphatidyl ethanlamine, sphingmyelin and lyslecithin. The amunt f P 3t incrprated int phsphlipid by the fam cells was cmpared with that incrprated int phsphlipid by ther prtins f the intima when the arta was incubated in vitr prir t disruptin. Up t 6.8% f the phsphlipid synthesis was brught abut by islated cells. The significance f the findings in relatin t phsphlipid synthesis in the athersclertic lesin was discussed. ADDITIONAL KEY WORDS phsphlipid insitl artic atherma chlesterl lecithin sphingmyelin phsphatidyl ethanlamine artic intima The presence f large numbers f lipidcntaining fam cells in the early athersclertic lesin has led t the suggestin that these cells cntribute in part t the metablic changes in the early lesin. 1 ' 2 These cells have been investigated in situ by histchemical and ultramicrscpic methds and such studies 1 ' s ' * have indicated that metablism and synthesis f lipid by fam cells may be assciated with the develpment f the athersclertic lesin. These studies have bvius Frm the Department f Physilgy and Biphysics, University f Tennessee Medical Units, Memphis, Tennessee. Supprted by Grant HE-2181 frm the U. S. Public Health Service. Dr. Day is n study leave frm the University f Adelaide, supprted by U. S. Public Health Service Fellwship Award FO5-TW Dr. Zilversmit is a Career Investigatr f the American Heart Assciatin. Dr. Newman's present address is Lipid Research Labratry, Akrn City Hspital, Akrn, Ohi. Accepted fr publicatin February 15, limitatins, hwever, in investigatins f bichemical changes taking place in the cells. The metablism f macrphages btained frm the peritneal cavity r lung f rabbits has been studied previusly, 2 ' B ~ 8 but there are difficulties in extraplating the findings t fam cells in the arterial wall. The separatin f fam cells frm the arta fr metablic studies in vitr, hwever, wuld prvide a means f investigating in detail their bichemical prperties and f determining what part these cells play in the lipid changes ccurring in the early athersclertic lesin. Recently Rdbell" has develped a technique in which fat cells were islated frm adipse tissue with the aid f cllagenase. In the present paper a similar methd has been described fr islating artic intimal fam cells frm rabbit athersclertic lesins and sme data are presented cmparing the lipid cmpsitin and phsphlipid synthesis 122 Circulatin Research, Vl. XDC July 1966

2 PHOSPHOLIPID SYNTHESIS BY FOAM CELLS 123 f such fam cells with that f ther fractins f the arterial wall. Methds ISOLATION OF FOAM CELLS BY ENZYMIC DISRUPTION OF THE AORTIC INTIMA New Zealand albin rabbits, fed daily 1 g Purina chw, cntaining 1 g f chlesterl and 2.8 g f cttnseed il fr perids ranging frm 8 t 123 days, were used in these studies. The rabbits were exsanguinated frm the abdminal arta under ether anesthesia and the thracic arta was remved, washed with saline, and bathed in nrmal rabbit serum. The superficial fat was remved and the intima stripped fr subsequent incubatin as set ut in the accmpanying flw diagram (fig. 1). The intima was incubated with Krebs Ringer phsphate slutin (ph 7.4) cntaining 4% bvine albumin (fatty acid pr, Pentex, Kankakee, Illinis) 1 mg cuagenase (Wrthingtn Bichemical Crp., Freehld, New Jersey), 4 mg elastase (Nutritinal Bichemicals Crp., Cleveland, Ohi) and 15 pjnles f glucse in a vlume f 5 ml. Incubatin was dne in 2 ml plastic screw tp cunting vials at 37 C, the digest being shaken gently every 5 min. After 1 hr the partially disrupted intima was teased apart with frceps and incubatin allwed t prceed fr an additinal 1 hr at 37 C. The digest was then filtered thrugh gauze in a 1 ml plastic syringe, the residue washed with 1 t 2 ml f 4% bvine albumin in Krebs Ringer phsphate and the washings expressed frm the residue (fractin I, fig. 1) by means f the syringe plunger. The filtrate THORACIC AORTA tripped I WTIM TOW CUXS (fractin III) Incubatin 1 hr with cllaganaa«, alait-ata. Taaaa, furthar 1 hr incubatin. filter thrugh 9aux«Cantrifuga 22O g - 3 sin Wath 2 tljmi with alina and centrlfuga cspand in O.W albu»in in HaaXa and int glaaa vaiaal. Incubat* 6O ain at 3?'. Hash adbarant calls with aalina. FIGURE 1 RES1PUE (Fractin I) BUPEWATAJTF (fractin II) I.'^3 (Fractin IV) Flw diagram illustrating separatin f fam cells frm the athersclertic artic intima. CircnUtin Remrch. VL XDC July 1966 was centrifuged at 22 g fr 3 min and the depsited cells were washed twice with.9% sdium chlride slutin and recentrifuged. The initial supernatant (fractin II, fig. 1) was reserved fr study while the washings were discarded. The washed cells were taken up in Hanks slutin 1 fr cunting in a hemcytmeter chamber and then suspended in Hanks slutin cntaining.5% bvine albumin, dispensed int glass cntainers (Leightn tubes fr metablic studies r stppered cnical flasks fr chemical assay) and incubated fr 6 min at 37 C. Mst f the fam cells adhered t the glass while ther cells and particles remained in suspensin. The adhering fam cells were washed with warm saline and the cells which remained in suspensin r were washed ff were cunted t determine lss f cells. The cmbined slutin cntaining the cell washings was then centrifuged (125 g fr 1 min) and the depsit, washed twice with saline, was reserved fr subsequent study (fractin IV, fig. 1). All prcedures except the final stage in which the cells were stuck t glass vessels were dne in plastic cntainers. Fur fractins were btained by the abve prcedure, viz. residue (I), supernatant (II), fam cells (III) and washings (IV). These fractins were used fr subsequent chemical r istpe assay r fr metablic studies. The fractins were extracted with 2:1 chlrfrm methanl as described by Flch et al. 11 and fractinated int phsphlipid and neutral lipid n silicic acid clumns. 12 Lipid phsphrus was determined by the methd f Bartlett 18 and distributin f individual phsphlipids by thin layer chrmatgraphy and elutin by the methd f Skipski et al. 14 Ttal chlesterl was determined as described by Zlatkis et al. 16 in the neutral lipid fractin fllwing sapnificatin by the methd f Abell et al. 1 Free and ester chlesterl were determined by the same methd fllwing separatin f free frm esterified chlesterl n silicic acid clumns 37 and subsequent sapnificatin. Purity f clumn fractins was checked by thin layer chrmatgraphy. 18 SYNTHESIS OF PHOSPHOLIPID BY ISOLATED FOAM CELLS Tw series f metablic experiments were perfrmed. In the first series the incrpratin f P 3 '-labeled phsphate int phsphlipid by islated fam cells was investigated. Apprximately 1.5 X 1 fam cells were dispensed int Leightn tubes, allwed t adhere t the glass, and washed. The cells which did nt adhere t the glass were cunted. One ml f medium (1:1 v/v Hanks:nrmal rabbit serum with.1 mg/ml f streptmycin and penicillin) cntaining up t 3 fie f P"-labeled phsphate (Abbtt Labratries) was added. Duplicate batches f cells were

3 124 incubated fr 1/2, 1, 2, 4 r 18 hurs after additin f the labeled medium. After the apprpriate incubatin time the medium was remved and the adhering cells washed twice with.9% sdium chlride slutin. The medium was centrifuged and the small amunt f depsit washed twice with.9% sdium chlride slutin and extracted tgether with the adhering cells. The centrifuged medium was als extracted. Aliquts f the Flch-washed extracts were assayed fr P3* with the scintillatr described by Grdn and Wlfe19 and fr lipid phsphrus. In the secnd series f experiments the rabbit athersclertic thracic arta was incubated in vitr with P " as described elsewhere.2 After incubatin the arta was washed with.9% sdium chlride slutin, the intima remved by stripping and incubated with cllagenase and elastase in rder t free and islate the fam cells as set ut abve. The fur fractins were extracted and assayed fr P " and lipid phsphrus. In sme f these experiments the supernatant (fractin II) was filtered thrugh a 1.2 fi millipre filter in rder t separate the large numbers f particles present. The particles (which varied up t 3 t 4 fi in diameter) were washed twice with.9% sdium chlride slutin and extracted. IDENTIFICATION OF LABELED PHOSPHOLIPID Separatin f the lipid extract t identify and quantitate the individual phsphlipids labeled by the fam cells was dne by thin layer chrmatgraphy by the methd f Skipski et al.14 In view f the small amunt f lipid phsphrus present in the individual extracts in these metablic experiments, the cell extract was taken up in a lipid extract f artic intima as carrier. The phsphlipid spts were lcated by spraying with idine vapr r by the methd f Dittmer and Lester21 and then scraped int cunting vials fr cunting by the methd f Snyder.22 Identificatin f spts in artic extracts by staining reactins has been described elsewhere.2 P"-labeled phsphlipid was identified by cmparisn f R values with standards and by cchrmatgraphy using three systems as fllws. The labeled spts, lcated by radiautgraphy, were eluted and taken up with the apprpriate standard fr rechrmatgraphy(i) by the methd f Skipski et al., (ii) with silica gel G and the slvent system chlrfrm :methanl: water (14/5/9 v/v/v) and (iii) in the case f the phsphatidyl insitl and phsphatidyl ethanlamine spts n an alkaline plate prepared as described by Skipski et al., but using the slvent system di-isbutyl ketne : acetic acid : water ( 4 : 2 5 : 5 v / v / v ). Satisfactry separatins f phsphatidyl insitl and phsphatidyl serine DAY, NEWMAN, ZILVERSMIT were attained by this methd. Labeled spts were lcated by radiautgraphy. In the cchrmatgraphy experiments butylated hydrxytluene was added t all slvents t minimize xidatin f labile cmpunds.2'1 Standards used were phsphatidyl ethanlamine, phsphatidyl serine and sphingmyelin btained frm Applied Science Labratries (State Cllege, Pennsylvania), lecithin (cntaining lyslecithin) frm Mann Research Labratries (New Yrk, New Yrk) and phsphatidyl insitl kindly supplied by Dr. Faure (Pasteur Institute, Paris). These standards were checked by staining reactins as described elsewhere.2 Remits The number f cells btained frm a single intima varied in 18 experiments frm 4. t 16.8 X 1 (mean 7.8 X 1 ±.68 SEM). Mst f these cells stuck t glass, but 1 t 15% f cells did wash ff and were cunted in the washings. Over the time perid studied (8 t 123 days) there was n bvius crrelatin between the duratin f feeding and the yield f fam cells althugh the lwest figure was in fact frm the 8 day rabbit. The fam cells are shwn, in figures 2 and 3, adhered t glass cver slips. All f the cells bserved were mnnuclear, varying in size frm 15 t 5 fi, and mrphlgically resembling macrphages. They cntained a large number f granules which stained psitively fr lipid with il red O. In sme prep- FIGURE 2 Fam cells islated frm athersclertic intima. Viewed unstained adhering t cver slips. Phase cntrast. Circulatin Reran*, VL XIX, July 1966

4 125 PHOSPHOLIPID SYNTHESIS BY FOAM CELLS aratins the granules were absent frm parts f the cell. In the final cell preparatin shwn in figures 2 and 3 there were practically n cntaminating particles r ther cells. Figure 4 is a free suspensin f washed cells [Cells (2) in fig. 1] btained prir t plating in glass vessels. Cntaminating cells were present tgether with relatively large numbers f particles ranging in size up t 3 t 4 //,. These particles resembled mrphlgically the granules in the fam cells and, in sme areas, clusters f them resembling parts f cells can be seen. Cells present in the supernatant FIGURE 3 Fam cells islated frm athersclertic intima. Viewed unstained adhering t cver dips. Phase cntrast. (fractin II) were cunted in sme experiments and accunted fr abut 1% f the cells present in the fam cell fractin (fractin III). Hwever, the supernatant cntained large numbers f particles bth singly and in clusters (fig. 5). These are remved by filtratin f the supernatant thrugh millipre filters mst being remved by a 1.2 fi filter. They are nt appreciably sedimented hwever, at 18 g fr 15 min. Sme cells culd be btained with cllagenase r with elastase alne, but better yields were fund when bth enzymes were used. When serum was used as incubatin medium, the yield was lwer than with 4% albumin in Krebs Ringer phsphate. The distributin f lipid P and f chlesterl between the fur fractins btained is shwn in table 1. Mst f the lipid P and f the chlesterl was present in the supernatant and residue fractins; nly abut US f the ttal lipid P and chlesterl was fund in the-islated fam cells. The-chlesterl: lipid P ratis f the varius fractins were similar with the pssible exceptin f the residue in which the chlesterl:lipid P rati was smewhat higher. The percentages f chlesterl ester present in the fam cells and washings were significantly greater (P <.1) than the percentages in the supernatant r the residue. FIGURE 4 FIGURE 5 Suspensin f cells btained frm athersclertic intima fllwing incubatin with cuagenase and elastase. Phase cntrast. Supernatant slutin btained frm athersclertic intima fllwing incubatin with cuagenase and elastase and centrifugatin t remve fam cells. Phase cntrast. OicuUrin Reward], VL XIX, July 1966

5 126 DAY, NEWMAN, ZILVERSMIT The per cent distributin f individual phsphlipids in the fur fractins is shwn in table 2. In the fam cells and the washings the percentages f the phsphlipid, present as "phsphatidyl ethanlamine" and as the fractin which travels at the slvent frnt, were greater than the percentages in the supernatant and residue. The percentages f sphingmyelin and lecithin present in the cells and washings were lwer than thse f the supernatant and residue. The incrpratin f phsphate-p" int phsphlipid by islated fam cells is shwn in table 3. P"-phsphh'pid increased in the cells fairly unifrmly ver the time perid studied s that after 4 hr incubatin almst.2$ and after 18 hr almst.7$ f the P" added t the medium has been incrprated TABLE 1 Distributin f Lipids Between Fractins f Artic Intima (means f 4 ± SEM) Fam cells (III) Washings (IV) Supernatant (II) Residue (I) Ttal in intima, mg Llpid (% f ttal in whle intima).92 ± ± ± ± ±.18 Chlesterl ( /, f ttal in whle intima).86 ± ± ± ± ± 7.94 Chlesterl (% presentt as ester in eeacht) / ± ± ± 2.2 Chlesterl/ lipid P rati* 61.5± ± ± 2.16 *78.2 ± 3.25 Analysis f variance shws significant differences at the 5% level. tanalysis f variance shws significant differences at the X% level, tmean f 5. mean f 6. TABLE 2 Per Cent Distributin f PhsphUpid Fractins f Artic Intima (means f 4 fr cells and supernatant and f 2 fr washings and residue) Origin Lyslecithln Sphingmyelint Lecithin* Phsph. insitl "Phsph. ethanlamine"tf Slvent frntt Fam cells (IU) 4. ± ± ± ± ± ± ± 1.1 Washings (IV) 2.3 ± ± ± ± ± ± ±.2 Supernatant (II).5 ± ± ± ± ± ± ±.6 Residue (I).2 ±. 5.2 ± ± ± ± ± ±.28 Analysis f variance shws significant differences at the 5% level. tanalysis f variance shws significant differences at the \% level. TABLE 3 Incrpratin f P"-labeled Phsphate int Phsphlipid by Islated Fam Cells Incubatin time hr I Eip. 1 Nci. f cells, 1.38 X 1 s Phsphate-P" added, 41 :X 1«cpm p -phsphliphlipid Lipid P ( /. f addled P") Cell* Cells Medium 3^ Exp. 2 N. f cells, 1.11 X 1«Phsphate-P «added, 49 X 1 cpm P"-phl[)hlipid Lipid P (% f miided P") Cells Cells Medium 2% Grclatin Research, Vl. XIX, July 1966

6 127 PHOSPHOLIPID SYNTHESIS BY FOAM CELLS int cellular phsphlipid. Table 3 shws that the medium cntained very little P " labeled phsphlipid at the earlier time intervals. Amunts less than.1% prbably represented traces f P"-labeled phsphate present in the lipid extracts. Hwever, the amunts abve this were phsphlipid P " which appeared in the medium in significant, althugh small, amunts relative t thse in the cell extract. The individual phsphlipids labeled by the islated fam cells are shwn in figure 6. Five spts were labeled crrespnding in RF value t lyslecithin, sphingmyelin, lecithin, phsphatidyl insitl and phsphatidyl ethanlamine. The latter fur spts were eluted and cchrmatgraphed with individual standards as shwn in figure 7A, B, C. Sphingmyelin, lecithin and phsphatidyl insitl traveled as single spts tgether with the standard preparatins n bth the alkaline plate (fig. 7A) and the silica gel G plate (fig. 7B). In rder t determine whether phsphatidyl serine cntributed t spt 5, this spt was rechrmatgraphed n an alkaline plate (fig. 7C) with the slvent system di-isbutyl ketne: acetic acid: water (4:25:5) mixed with either phsphatidyl serine r phsphatidyl insitl r with bth standards. Spt 5 traveled with, and can therefre be tentatively identified as, phsphatidyl insitl. Abut 4.$ f the label traveled with phsphatidyl serine, hwever. Spt 6 which ran with phsphatidyl ethanlamine in the Skipski system rnun i P/ETHANOLAMINE O P/INOSITOL O LECITHIN SPHINGOMYELIN LYSOLECITHIN SPOT 6 t SPOT 5 SPOT 4 SPOT 3 * SPOT 2 ORIGIN FIGURE 6 Identificatin f P3S-ldbeled phsphlipids synthesized by islated fam ceus. Separatin f the labeled fam cell extract by thin layer chrmatgraphy fllwed by autradigraphy. Alkaline plate with slvent system chlrfrm:methanhacetic acid:water (23/15/4/1.9). FRONT A O ORIGIN * S/M LKithin /Ly«* * P.I. RE. SPOT SPOT SPOT SPOT FRONT I * RS. Rl., PE Standard SPOT SPOT Mixture 5 5 SPOT SPOT 5 6 B FIGURE 7 O ORIGIN S/M LKilhln PI /Ly«PE Grculitiii Research. Vl. XIX, July 1966 SPOT SPOT SPOT SPOT Identificatin by cchrmatgraphy f Pst-labeled phsphlipids synthesized by islated fam cells. Badiautgraphy f P3t-labeled spts and chemical lcatin f standards are shwn. A: Alkaline plate, slvent system chlrfrm:methanl:acetic acklwater (25/15/4/1.9). B: Neutral plate, slvent system chlrfrm-.methanh water (14/5/9). C: Alkaline plate, slvent system di-isbutyl ketne-.acetic aculwater (4/25/5). Details in text.

7 128 DAY, NEWMAN, ZILVERSMIT did nt rechrmatgraph as a single spt. In bth fig. 7B and 7C this spt cntained, in additin t a spt running with the standard phsphatidyl ethanlamine, ther unidentified spts. The percentage distributins f individual P'Mabeled phsphlipids fr the cell extracts at different times f incubatin are given in table 4. Mst f the P" has been incrprated int cellular lecithin, phsphatidyl insitl and the cmpsite spt "phsphatidyl ethanlamine." The distributin was essentially similar thrughut the fur-hur time perid althugh in the ne experiment at 18 hurs the sphingmyelin and "phsphatidyl ethanlamine" accunted fr a larger amunt and the phsphatidyl insitl fr a smaller amunt f the P" than at the earlier incubatin times. The P" incrprated int phsphlipid by each f the fur fractins f the artic intima after in vitr incubatin f the intact arta with P"-phsphate is shwn in table 5. In the first three experiments [table 5 (a)] the arta was incubated with P" fr fur hurs. In the last three experiments [table 5 (b)] the incubatin f the arta with P" was limited t tw hurs. In the fur-hur experiments the cells may have deterirated since many f the separated cells did nt adhere t glass and a relatively large amunt f lipid-p 3 * was present in the washings. In bth series f experiments the P Sf -phsphlipid was distributed mainly in the supernatant and residue fractins, the cells accunting fr at best 6.8? f the label. The specific activity f the P**-labeled phsphlipid in TABLE 4 Incrpratin f P" int Individual Phsphlipids by Islated Fam Cells (% distributin at different time intervals) Incubatin time hr % Origin Ly»lecithin Sphlngmyel TABLE 5 Lecithin Phapba. initl "Phepha. ethanlamine" Cntributin f Different Fractins f Artic Intima t Phsphlipid Synthesis in Vitr Slvent frnt (a) Incubatin f arta with P st in vitr fr 4 hr fllwed by separatin int fractins. Mean f 3 ± SEM* P M -ph»phllpid Lipid P Specific activity % attributin AC Cells (HI) 2. ± ± ±147 Washings (IV) 9.3 ± ± ± 67.7 Residue (I) 4.4 ± ± ± 9.14 Supernatant (II) 48.2 ± ± ± 24.2 (b) Incubatin f arta with P" in vitr fr 2 hr fllwed by separatin. Mean f 3 ± Cells Washings (IV) Residue (I) Supernatant Ttal (II) Particles P"-ph»phllpid Lipid P Specific activity % distributin 6.8 ± ± ± ± ± X 1 6 cpm P"-phsphate added. t59. X 1«cpm P"-phsphate added. AC 5.7 ± ± ± ± ±15. cpm/flf 226 ± ±17 33 ± ± ± 83.7 Circulatin Research. VL XIX, July 1966

8 PHOSPHOLIPID SYNTHESIS BY FOAM CELLS 129 the cells, hwever, was six t seven times that in the supernatant and residue. In the three experiments shwn in table 5 (b) the distributin f P" in, and the specific activity f, the particles f the supernatant were als determined. This fractin accunted fr 37% f the Pf in the supernatant with a specific activity almst twice that f the supernatant and residue but cnsiderably less than that f the cells. Discussin The cells btained were free f cntaminating cells and represented a micrscpically hmgeneus preparatin f lipid-cntaining fam cells. It is knwn, that in the rabbit athersclertic lesin sme f the lipid-cntaining cells can be identified ultramicrscpically as smth muscle cells. 25 It is pssible that sme f the cells btained by the methd described may be smth muscle cells, but mrphlgically the preparatin cnsisted f runded mnnuclear cells, indistinguishable frm macrphages. Further, while the ability t adhere t a glass surface is by n means specific, this prperty is ne well develped in macrphages frm ther situatins and used fr their separatin frm ther cell types. 28 ' 27 The lipid cntent f fam cells was high cmpared with that f macrphages btained frm the peritneal cavity f nrmal rabbits. It can be calculated frm the data reprted in table 1, knwing the cell cunt in each preparatin, that the ttal chlesterl cntent amunted t 72 fig/1 6 cells, and the lipid phsphrus t 1.1 /tg/1 6 cells. This cmpared with abut 1 /ig/1 6 cells fr chlesterl (Day and Fidge, unpublished data) and.7 /Ag/1 e cells fr lipid phsphrus fr peritneal cavity macrphages derived frm nrmal rabbits. 7 The cells btained were metablically active as indicated by their ready incrpratin f P"-phsphate int phsphlipid and can therefre be used t investigate the part played by the fam cell in the develpment f the early lesin. The incrpratin f P^-phsphate int phsphlipid was high when cmpared with that f peritneal cavity macr- CircuUtin Remrch. Vl. XIX July 1966 phages. It can be calculated that fam cells incubated with P 3? -phsphate incrprated int phsphlipid apprximately.5 /imles f the medium phsphate per hr/1 6 cells. Peritneal macrphages frm nrmal rabbits incrprated apprximately.5 /imles/ hr/1 8 cells which is increased 44% in the presence f a.6% chlesterl suspensin. 7 A further cmparisn with peritneal macrphages can be made with respect t the individual phsphlipids labeled fllwing incubatin. Lecithin represented the majr phsphlipid labeled in bth cell types, but in the case f the fam cells, cnsiderably mre phsphatidyl insitl and less sphingmyelin were labeled than was the case with the peritneal macrphages incubated in the presence r absence f chlesterl. 7 The amunt f phsphlipid present in the fam cells btained was nly abut 1% f that in the intima as a whle. If sme disruptin f cells was ccurring, it is pssible that the amunt f phsphlipid present in the fam cells in the intima may accunt fr mre than this. Hwever, even if disruptin f cells had ccurred during preparatin, it seems unlikely that the fam cells culd cntain intracellularly mre than a small amunt f the phsphlipid present in the athersclertic intima. In the metablic experiments fr example, if ne cnsiders that all the P"- phsphlipid present in the supernatant arse frm disrupted cells, and this cannt f curse be asserted n the data, it can be calculated that the actively metablizing fam cells culd accunt fr 1 t 2% f the phsphlipid in the intima. This is in cntrast t histchemical data presented previusly, 1 in which it was shwn that much f the phsphlipid present in the rabbit athersclertic arta was present intracellularly in fam cells. The data, therefre, with regard t phsphlipid distributin in the arta wuld supprt the cnclusin f Adams et al. wh have reprted histchemical wrk demnstrating that phsphlipid is distributed predminantly extracellularly in the rabbit lesin. It is pssible that fam cells cntribute t the phsphlipid accumulatin in the atherm-

9 13 DAY, NEWMAN, ZILVERSMIT atus arterial wall 29 ' 8 in tw ways. Phsphlipid synthesized by such active metablic cells as have been btained in the present experiments may be transferred t the extracellular space. Hwever, very little P"-labeled phsphlipid appeared in the medium in the in vitr experiments, but cnditins in the intima may differ frm thse in the medium. The secnd pssibility is that fam cells degenerate and becme necrtic fllwing thenactive metablic phase thus leading t the accumulatin f extracellular phsphlipid. On the ther hand, degenerated cells may cntain much phsphlipid, demnstrable histchemically, but fall apart when the arta is disrupted with cllagenase, etc. The incrpratin f P"-phsphate int individual phsphlipids by the islated fam cells was almst identical with the incrpratin f P s t-phsphate int individual phsphlipids by the intact artic intima. 2 This culd be because the fam cells were respnsible fr mst f the incrpratin f P J * int phsphlipid that ccurs in the arta, but this cnclusin cannt be supprted n the basis f the data in table 5, unless ne cnsiders that the yield f cells btained in these experiments was relatively small. In the present paper it can be cncluded that phsphlipid can be synthesized by the fam cells present in the athermatus lesin. The relative cntributin t the synthesis f phsphlipid by the intima as a whle cannt be assessed satisfactrily, hwever, until the questins f yield f cells, pssible disruptin during preparatin, and the synthesis f phsphlipid by ther cmpnents f the intima are determined. Acknwledgment We thank Mrs. P. Thmpsn and Miss R. Ward fr technical assistance. References 1. DAY, A. J.: The relatinship f arterial macrphages t the phsphlipid cntent in rabbit atherma. J. Athersclersis Res. 2: 35, DAY, A. J.: The macrphage system, lipid metablism and athersclersis. J. Athersclersis Res. 4: 117, DUNNICAN, M. G.: The distributin f phsphlipid within macrphages in human athermatus plaques. J. Athersclersis Res. 4: 144, GEEB, J. C.: Fine structure f canine experimental athersclersis. Am. J. Pathl. 47: 241, DAY, A. J., AND FIDCE, N. H.: The uptake and metablism f ^C-labeled fatty acids by macrphages in vitr. J. Lipid Res. 3: 333, DAY, A. J., AND FIDCE, N. H.: The incrpratin f ^C-labeled acetate int lipids by macrphages in vitr. J. Lipid Res. 5: 163, DAY, A. J., FmcE, N. H., AND WELKTNSON, G. N.: Effect f chlesterl in suspensin n the incrpratin f phsphate int phsphlipid by macrphages in vitr. J, Lipid Res. 7: 132, ELSBACH, P.: Uptake f fat by phagcytic cells. An examinatin f the rle f phagcytsis. II. Rabbit alvelar mactphages. Bichim. Biphys. Acta 98: 42, RODBELL, M.: Metablism f islated fat cells. I. Effects f hrmnes f glucse metablism and liplysis. J. Bil. Chem. 239: 375, HANKS, J. H.: The lngevity f chick tissue cultures withut renewal f media. J. Cellular Cmp. Physil. 31: 235, FOLCH, J., LEES, M., AND SLOANE STANLEY, G. H.: A simple methd fr the islatin and purificatin f ttal lipides frm animal tissue. J. Bil. Chem. 226: 497, NEWMAN, H. A. I., Liu, C. T., AND ZTLVER- SMTT, D. B.: Evidence fr the physilgical ccurrence f lyslecithin in rat plasma. J. Lipid Res. 2: 43, BAHTLETT, G. R.: Phsphrus assay in clumn chrmatgraphy. J. Bil. Chem. 234: 466, SKTPSKL, V. P., PETERSON, R. F., AND BARCLAY, M.: Quantitative analysis f phsphlipids by thin layer chrmatgraphy. Bichem. J. 9: 374, ZLATKIS, A., ZAK, B., AND BOYLE, A. J.: A new methd fr the direct determinatin f serum chlesterl. J. Lab. Clin. Med. 41: 486, ABELL, L. L., LEVY, B. B., BRODIE, B. B., AND KENDALL, F. E.: A simplified methd fr the estimatin f ttal chlesterl in serum and demnstratin f its specificity. J. Bil. Chem. 195: 357, VAN HANDEL, E.: Separatin and chemical assay f lipid classes. J. Am. Oil Chemists' Sc. 36: 294, MANGOLD, H. K., AND MALINS, D. C: Fractinatin f fats, ils and waxes n thin layers f silicic acid. J. Am. Oil Chemists' Sc. 37: 383, GORDON, C. F., AND WOLFE, A. L.: Liquid Circulinn Raeucb, Vl. XTX, July 1966

10 PHOSPHOLIPID SYNTHESIS BY FOAM CELLS 131 scintillatin cunting f aqueus samples. Analyt. Chem. 32: 574, NEWMAN, H. A. I., DAY, A. J., AND ZILVER- SMTT, D. B.: In vitr phsphlipid synthesis in nrmal and athermatus rabbit artas. Circulatin Res. 19: 132, DITTMEB, J. C, AND LESTER, R. L.: A simple, specific spray fr the detectin f phsphlipids n thin layer chrmatgrams. J. Lipid Res. 5: 126, SNYDER, F.: Radi-assay f thin layer chrmatgrams: A high-reslutin znal scraper fr quantitative C 1 * and H 3 scanning f thin layer chrmatgrams. Analyt. Bichem. 9: 183, MARINETTI, G. V.: Chrmatgraphic separatin, identificatin and analysis f phsphatides. J. Lipid Res. 3: 1, WHEN, J. J., AND SZCZEPANOWSKA, A. D.: Chrmatgraphy f lipids in the presence f an antixidant, 4-methyl-2, 6 ditert.-butylphenl. J. Chrmatg. 14: 45, STILL, W. J. S.: An electrn micrscpe study f chlesterl athersclersis in the rabbit. Exptl. Ml. Pathl., suppl. 2: 491, MACKANESS, G. B.: The actin f drugs n intracellular tubercle bacilli. J. Pathl. Bacteril. 64: 429, RABINOWITZ, Y.: Separatin f lymphcytes, plymrphnuclear leuccytes and mncytes n glass clumns including tissue culture bservatins. Bld 23: 811, ADAMS, C. W. M., BAYLJSS, O. B., AND IBABHTM, M. Z.: The distributin f lipids and enzymes in the artic wall in dietary rabbit atherma and human athersclersis. J. Pathl. Bacteril. 86: 421, ZELVERSMIT, D. B., SHORE, M. L., AND ACKER- MAN, R. F.: The rigin f artic phsphlipid in rabbit athermatsis. Circulatin 9: 581, ZLLVERSMTT, D. B., AND MCCANDLESS, E. L.: Independence f arterial phsphlipid synthesis frm alteratins in bld lipids. J. Lipid Res. 1: 118, Circulatin Raewch. VL XIX, July 1966