Uptake and Metabolism of "C-Labeled Oleic Acid by Atherosclerotic Lesions in Rabbit Aorta

Transcription

1 Uptake an Metabolism of "C-Labele Oleic Aci by Atherosclerotic Lesions in Rabbit Aorta A BIOCHEMICAL AND RADIOAUTOGRAPHIC STUDY By Align J. Day, M.D., D.Phil., an Mark L Wahlqvist, B.Me.Sc, M.B., B.S. ABSTRACT The uptake of 14 C-labele oleic aci an its incorporation into combine lipis by aortic intimas from normal an cholesterol-fe rabbits has been investigate in vitro. More than five times as much oleic aci was taken up by the atherosclerotic intima as by the normal intima. About twice as much oleic aci was incorporate into phospholipi, an twenty times as much into cholesterol ester by the atherosclerotic intima as by the normal. Lecithin was the major phospholipi synthesize from oleic aci in both normal an atherosclerotic intimas. Raioautographs of the atherosclerotic vessels show that the 14 C-labele oleic aci an its metabolic erivatives, principally phospholipi an cholesterol ester, were localize in suanophilic cells in the intima in both early an avance lesions. It is conclue that intimal foam cells are primarily responsible for the lipi synthesis that occurs in the atherosclerotic lesion. ADDITIONAL KEY WORDS fatty acis atheroma foam cells lipi metabolism arterial wall metabolism raioautography phospholipi cholesterol ester Downloae from by on January 24, 2019 In numerous in-vivo an in-vitro stuies, lipi precursors have been taken up by the atherosclerotic arterial wall an incorporate into the lipis of the atherosclerotic lesion (1-6). Such stuies have inicate that the phospholipi accumulates in the lesion as a result of synthesis in situ (7, 8). Recent evience inicates that synthesis an hyrolysis of cholesterol ester may also take place in the arterial lesion (9-11). The iversion of fatty aci synthesize from 1 4 C-labele acetate to cholesterol ester in atherosclerotic lesions in the pigeon (12) an the rabbit (13) has also emphasize the potential role of the arterial wall in the esterification of cholesterol in the atherosclerotic lesion. In view of the evience implicating athero- From the Department of Physiology,. University of Melbourne, Victoria 3052, Australia. This work was supporte in part by grants from the National Heart Founation of Australia an the Australian Research Grants Committee an by U. S. Public Health Service Grant RO5-TW Accepte for publication October 14, sclerotic intimal metabolism in the accumulation of lipi, the question arises as to which portions of the lesion are involve. Geer an Guiry (14) an Smith (15) have emonstrate that the human atherosclerotic lesions which contain large numbers of foam cells have a cholesterol ester fatty aci pattern which iffers markely from that of the serum, being high in oleic aci relative to linoleic aci. On this basis these workers suggest that foam cells synthesize cholesterol ester in the lesion.- Day an Wilkinson (13) have shown that isolate foam cells will take up 14 C-labele acetate an incorporate the fatty aci synthesize into both cholesterol ester an phospholipi. Foam cells isolate from rabbit atherosclerotic lesions have also been shown to incorporate 32 P-labele phosphate into phospholipi an to be responsible in part for the phospholipi synthesis in the atherosclerotic intima (16). Parker et al. (17), in a combine biochemical-morphological stuy, have shown that rabbit atherosclerotic lesions take up an incorporate Circulation Reiettch, Volume XXIII, D«OT

2 780 DAY, WAHLQVIST Downloae from by on January 24, 2019 linoleic aci into phospholipi an that the magnitue of such incorporation is relate to the increasing number of myointimal cell membranous organelles present in the eveloping lesion. All of these stuies inicate that foam cells may be responsible in part for the lipi synthesis which takes place in the atherosclerotic lesion, but irect evience implicating these cells is not available. In the present paper, the in-vitro incubation of atherosclerotic intimas with 1 4 C-labele oleic aci has been compare with that of normal intimas. The cellular localization of the uptake an incorporation of 14 C-labele oleic aci into phospholipi an cholesterol ester has then been investigate irectly by raioautographic techniques to etermine which portions of the atherosclerotic arterial lesion are responsible for the lipi synthesis. Materials an Methos Oleic aci 1-1 4C, specific activity 32.5, 43.2, or 57.2 mc/mm (Raiochemical Centre, Amersham, Englan), was issolve in 0.05N soium hyroxie an taken up either in an excess of bovine albumin (Armour) or normal rabbit serum. Raiochemical purity was verifie by thinlayer chromatography an by gas liqui raiochromatography as escribe below. Atherosclerotic aortas were obtaine from male New Zealan white rabbits fe 1 g cholesterol an 3 ml of peanut oil in 100 g rabbit chow aily for 4 months. Normal aortas were obtaine from rabbits of the same stock fe a normal rabbit chow iet for the same perio. In the first series of experiments, the rabbits were kille by ether anesthesia. The thoracic aorta was remove an ivie longituinally into halves, an each half was incubate separately in 5 ml of meium (50 : 50 Hank's solution : normal rabbit serum) containing a known amount of soium l- 14 C-oleate : albumin. After incubation for 4 hours at 37 C in an atmosphere of air, the aortic halves were washe in saline an the atherosclerotic or normal intimas strippe from the meia an aventitia an extracte with chloroform : methanol (2 : 1 v/v) accoring to the metho of Folch et al. (18). In some experiments the atherosclerotic thoracic aorta was longituinally ivie into thirs, each thir being incubate for 1, 2, or 4 hours. To compare irectly the incorporation of relabele oleic aci into cholesterol ester, phospholipi, an triglycerie, paire experiments were set up. Whole aortas obtaine as above from either normal or cholesterol-fe rabbits were incubate together for 4 hours at 37 C in 10 ml of meium containing 50 : 50 Hank's : normal rabbit serum to which ha been ae a known amount (8.2 /xc) of 14 C-labele soium oleate. No aitional albumin was ae; the excess serum albumin present was use to bin the tracer amount of 14 C-labele soium oleate ae. The unesterifie fatty aci content of the serum use in the meium was etermine by the metho of Dole (19), an its oleic aci proportion was measure by gas liqui chromatography following separation by thin-layer chromatography as escribe below. These eterminations were use to calculate the amount of oleic aci incorporate into cholesterol ester, phospholipi, an triglycerie by the normal an atherosclerotic intimas in this secon series of experiments. Following incubation, the intima was separate from the meia an aventitia an extracte as for the first series. The ry efatte weight of the intima was recore. In the raioautographic stuies, the atherosclerotic aortic halves were incubate for 3 hours in the raioactive meium an then for a further hour in nonraioactive meium. The artery was washe in saline an finally in a solution of un- Iabele soium oleate in 15% albumin (5 mg oleate/ml 15% albumin solution) for 1 hour at 4 C. Both halves were fixe in 1% calcium chlorie an 4% formalehye in physiological saline for 4 ays. The intima from one aortic half was then strippe from the remaining meia an aventitia an extracte with chloroform : methanol accoring to Folch et al. (18), in orer to etermine the uptake an incorporation of the 1 4 C- labele oleic aci. Sections (6^) were cut from the remaining half without prior embeing, using an International Cryostat Moel CTI, an raioautographs were prepare with Koak AR10 Stripping Film. After exposure the raioautographs were evelope an staine through the film with Suan IV an hematoxylin. THIN-LAYER CHROMATOGRAPHY After counting the total lipi 14 C, the lipi extracts from the intimas, normal or atherosclerotic, were separate into phospholipi, cholesterol/ iglycerie, unesterifie fatty aci, triglycerie, an cholesterol ester by thin-layer chromatography on Silica Gel G (Merck) using iethyl ether : acetic aci : n-hexane (38 : 3 : 100 v/v/v) as the eveloping solvent. The istribution of "C-labele oleic aci in the iniviual phospholipis of the intimas was also etermine by thin-layer chromatography, using the metho of Skipski et al. (20). For this purpose the total phospholipi moiety was first separate by thinlayer chromatography as above. Ientification of Circulation Research, Volume XXIll, December 1968

3 LOCALIZATION OF LIPID SYNTHESIS IN ATHEROMA 781 phospholipis was mae by comparison of Rf values with stanars as escribe previously (21). Following separation of the lipi extracts into the various lipi components or the iniviual phospholipis, the proportion of the 14 C-label in each moiety was etermine by scraping the plates an counting irectly, using the metho of Snyer (22). All counting was one with a Packar Tricarb Spectrometer. GAS LIQUID RADIOCHROMATOGRAPHY To etermine an compare the specific activity of the oleic aci incorporate into cholesterol ester an phospholipi in the normal an atherosclerotic intimas, the respective fractions were separate by thin-layer chromatography as above, an the fatty aci present in each was converte to methyl esters by heating at 65 C with 5% sulphuric aci in methanol in seale ampoules. The labele methyl esters were extracte with petroleum ether an then run on iethylene glycol succinate columns at 185 C in an F & M Moel 5750 Gas Chromatograph, using argon as the carrier gas. The gas stream was split, an fractions of the eluant were combuste to 14 CC> 2 an its 14 C content was monitore with a Pye Raiochromatography unit. All of the 14 C-labele fatty aci in the cholesterol ester an phospholipi moieties was present as 14 C-labele oleate, an its specific activity was etermine by comparison with 14 C-labele methyl palmitate of known specific activity. The specific activity of the 14 C-labele oleic aci present in the cholesterol ester an phospholipi moieties of both the normal an atherosclerotic intimas was expresse as counts/min 14 C/m^u.mole oleic aci. Results The uptake of oleic aci after incubation for 4 hours in a meium containing 14 C-labele soium oleate is shown for the first TABLE 1 Uptake of U C-Labele Oleic Aci an Its Incorporation into Combine Lipi by Normal an Atherosclerotic Intimas Downloae from by on January 24, 2019 Normal* Atherosclerotict Atherosclerotict "C Present initially in Inciibation incubation meium time (counts/min X 10") Uptiake 4 hr 4 hr 1 hr 2 hr 4 hr ± ± ± %»C I:nitially present u rate int lipi ± ± *Mean of four experiments (uplicate aortic halves) with SE of mean; tmean of six experiments (five with uplicate aortic halves) with SE of mean; ttwo experiments as shown. TABLE 2 Percentage Distribution of "ic-labele Oleic Aci Among, Lipi Fractions After Incubation with Normal or Atherosclerotic Intimas Incu >atiori ti Phospholipi Diglycerie Fatty aci Triglycerie Cholesierol este Normal* 4 hr 48.1 ± ± ± : ± ± 0.6 Atherosclerotict 4 hr 32.2 ± ± ± : ± ± 3.3 Atherosclerotict 1 hr 2 hr 4 hr *Mean of four experiments (uplicate aortic halves) with SE of mean; t mean of six experiments (five with uplicate aortic halves) with SE of mean; tmean of two experiments (both with single aortic thirs). Circulation Research, Volume XXIII, December 1968

4 1 782 DAY, WAHLQVIST Downloae from by on January 24, 2019 series of experiments in Table 1. In the normal intima, a mean of 0.830% of the oleic aci was taken up by the intima an most of this (0.702%) was incorporate into combine lipis. In the atherosclerotic intima more than five times as much 1 4 C-labele oleic aci (4.48%) was taken up, an again most of this (3.51%) was incorporate into other lipis in the intima. The uptake an incorporation of the 1 4 C-labele oleic aci into lipi was approximately linear over the 4-hour perio (Table 1). The percentage istribution of the relabele oleic aci among phospholipi, iglycerie, free fatty aci, triglycerie, an cholesterol ester is shown for the first series in Table 2. In the normal artery very little cholesterol ester (2.4%) was labele; most of the oleic aci taken up was incorporate into phospholipi, an to a lesser extent into triglycerie. In the atherosclerotic artery, however, a mean of 29.4% of the 14 C-labele fatty aci was incorporate into the cholesterol ester fraction. The pattern of incorporation of oleic aci into cholesterol ester an phospholipi is similar at 4 hours to that at the earlier intervals stuie. There is, however, relatively more unesterifie oleic aci present at the earlier time intervals. Table 3 shows the istribution of the label between the iniviual phospholipis for both the normal an atherosclerotic intimas. In both groups the major phospholipi labele is lecithin. No significant ifferences are apparent in the istribution of 1 4 C-labele fatty aci in the phospholipi moieties between the normal an the atherosclerotic intimas. Distribution of the label was similar over the perio stuie. In the secon series of experiments, normal an atherosclerotic aortas were incubate together in the same meium to compare irectly the incorporation of 14 C-labele oleic aci into phospholipi, triglycerie, an cholesterol ester. The percentage uptake by the normal or atherosclerotic intima of relabele oleic aci present in the meium, together with the istribution of this oleic aci between the various lipi moieties, is given p.5 osphs anola iyl is II x J G s c ' > 6 > time r-t +1 OJ CD o +1 t~ 15, ^ m as +1 Vi < i II al +1 **" lerot o s S3 S a. -5 Circulation Research, V o lume XXIII, December 1968 II q CD II JS t CO (M in his CO O> CD CO <M 11. -H a CD CD CD CD CD CD <D O i ter lerot Ath , CD JZ B -

5 LOCALIZATION OF LIPID SYNTHESIS IN ATHEROMA 783 TABLE 4 Percentage Distribution of 'tc-labele Oleic Aci Among Lipi Fractions After Incubation with Normal an Atherosclerotic Intimas Normal Atherosclerotic % Uptake of oleic aci Phospholipi Data are means of three paire experiments. TABLE 5 Amount of Oleic Aci Incorporate into an Specific Activities of Oleic Aci as Combine Lipis for Normal an Atherosclerotic Intimas Incubate in Vitro Exp. 1* 2t 3 Normal Atherosclerotic Normal Atherosclerotic Normal Atherosclerotic mjtmoles oleic aci incorpo:ate/g ry efatte intima Phospholipi j2.fi Specifii: activity (counts/min/n/imole oleic aci) Cholesterol ester Cholesterol Triglycerie Choleste Phospholipi *Four-hour incubations carrie out in pairs; normal an atherosclerotic intimas incubate in same meium. tau incubations ha 12.7 X 106 counts/min oleic aci ae. There was a total of 1.94 ^Eq. oleic aci present in 10 ml of meium. Downloae from by on January 24, 2019 for this series of experiments in Table 4. As for the first series of experiments, a higher proportion of the label was incorporate into cholesterol ester an less into phospholipi an triglycerie in the atherosclerotic intima than in the normal. The istribution of the label between the various moieties has been use to calculate the amount of oleic aci incorporate into phospholipi, triglycerie, an cholesterol ester by the normal an atherosclerotic intima (Table 5). The ry efatte weight of the normal intima was similar to that of the atherosclerotic intima, but the amount of oleic aci incorporate into all of the lipi moieties was consierably greater in the atherosclerotic than in the normal. The amount of oleic aci incorporate into phospholipi an triglycerie was about twice that in the normal. The amount of oleic aci incorporate into cholesterol ester was increase some twenty times. Most of the increase incorporation of oleic aci into lipi in the atherosclerotic intima is therefore ac- Circulation Research, Volume XXIII, December 1968 counte for by its increase incorporation into cholesterol ester. Also in Table 5, the specific activities of oleic aci in phospholipi an cholesterol ester of the normal an atherosclerotic intimas are shown. These are expresse as counts/min/m/imole oleic aci present. Biologically occurring phospholipis have iffering fatty acis in the a an j3 positions so that it can be assume that only one fatty aci position of the lecithin (the major phospholipi labele) woul be occupie by oleic aci. The specific activities of the cholesterol ester an phospholipi, as expresse in Table 5, are therefore irectly comparable on a molar basis. For phospholipi, the specific activity is somewhat less, but of the same orer, in the atherosclerotic intima than in the normal. The specific activity of the cholesterol ester fatty aci in the atherosclerotic intima is about one fifth that in the normal intima.

6 784 DAY, WAHLQVIST»/ / Raioautograph of an early atherosclerotic lesion consisting of several closely relate foam cells. Granulation representing 1tC-labele oleic aci an its metabolic erivatives is confine to foam cell areas. Aorta from rabbit age 21 weeks an cholesterol-fe for 15 weeks. Hematoxylin an Suan IV. Exposure time 7 ays. Downloae from by on January 24, 2019 The percentage istribution of 14 C-labele oleic aci between the iniviual lipi fractions of the atherosclerotic intimas use for raioautography was etermine in the half reserve for analysis. In these experiments only 12.2% of "C-labele unesterifie fatty aci was still present, most of the label being incorporate into the cholesterol ester an phospholipi as for the above experiments. RADIOAUTOGRAPHY The localization of 14 C-labele lipi by the atherosclerotic artery is shown in Figures 1 to 3. No significant film backgroun granulation was present in any of the raioautographs. Early suanophilic cellular lesions with associate fragmentation of the internal elastic lamina are shown in Figure 1. Granulation, representing essentially 1 4 C-labele oleic aci an the cholesterol ester an phospholipi forme from it, overlies the foam cell areas. There is little label in intervening relatively normal intima, an little in unerlying meia. The higher magnification of a similar lesion in Figure 2 confirms localization of 1 4 C to intimal foam cells. A more avance lesion with grossly thickene intima is shown in Figure 3. Localization of "C to the foam cells scattere FIGURE 1 Early atherosclerotic lesion from the same rabbit as in Figure 1, viewe uner oil immersion, confirming the relationship of raioautograph granules to foam cells. Hematoxylin an Suan TV. Exposure time 8 ays. Circulation Research, Volume XXIII, December 1968

7 LOCALIZATION OF LIPID SYNTHESIS IN ATHEROMA 785 Downloae from by on January 24, m FIGURE 3 Raioautograph of an avance atherosclerotic lesion icith much extracellular lipi. Uptake an metabolism of 1'iC-labele oleic aci has occurre in the foam cells. Aorta from rabbit age 21 weeks an cholesterol-fe for IS weeks. Hematoxylin an Suan IV. Exposure time 38 ays. throughout the intima is apparent, an there is little evience of label in other areas of the thickene intima or in the,ajacent meia. The foam cells labele are mainly in the more superficial portion of the intima with little localization at the intimo-meial junction. Discussion It has been shown that oleic aci is taken up by both normal an atherosclerotic rabbit intimas an incorporate into various lipis. In the normal intima, fatty aci was incorporate mainly into phospholipi an triglycerie as reporte previously by Stein et al. (2, 3). The iversion of oleic aci to choles- CircuUiion Research, Volume XX11I. December 1968 terol ester in the atherosclerotic intima, however, is in marke contrast to events in the normal. Whether such incorporation inicates hyrolysis an reesterification of cholesterol ester by the intima, however, cannot be asserte. It is possible that enzymic exchange of 1 4 C-labele oleic aci for the fatty acis of the cholesterol ester present in the wall is being brought about. Whatever the mechanism, however, the process of rearrangement of the fatty aci pattern of the cholesterol ester is clearly occurring more in the atherosclerotic vessel than in the normal. The incorporation of 14 C-labele oleic aci into cholesterol ester in the atherosclerotic intima was approximately linear over the 4- hour incubation perio stuie. If the normal intima behave in the same way with respect to time, the relative fractional turnover of the phospholipi an cholesterol ester fatty aci pools in the intima can be estimate. The specific activity of the phospholipi fatty aci after incubation for 4 hours was roughly of the same orer in the atherosclerotic as that in the normal. The small cholesterol ester fatty aci pool in the normal intima ha a specific activity consierably higher than that of the atherosclerotic intima, so that although the total amount of fatty aci incorporate per unit time into cholesterol ester in the atherosclerotic intima was many times that in the normal, the fractional turnover rate was much less. The pathway for incorporation of fatty aci into cholesterol ester in the atherosclerotic intima is not known. The ata presente here may inicate either irect esterification of enogenous cholesterol with fatty aci entering or enzymic exchange with rearrangement of the large cholesterol ester fatty aci pool in the intima. One other possible mechanism may be the acyl transferase mechanism of Glomset (23) which accounts for cholesterol esterification in the serum. In the present experiments the 14 C-labele oleic aci is incorporate preominantly into lecithin, an the transferase mechanism is therefore a possibility. The fact that phospholipi fatty aci has a higher specific activity relative to the

8 786 DAY, WAHLQVIST Downloae from by on January 24, 2019 cholesterol ester fatty aci inicates that this transferase mechanism of cholesterol ester formation cannot be exclue in either the normal or atherosclerotic intima. The specific activity of phospholipi relative to cholesterol ester is consierably higher in the atherosclerotic intima, however. Whether this can be interprete to inicate transferase enzyme activity or simply a reflection of an inert cholesterol ester pool cannot be asserte without further evience. The incorporation of 1 4 C-labele oleic aci into iniviual phospholipis is similar in the normal an atherosclerotic artery. This is in contrast to previous in-vitro stuies (21) in which it has been shown that after 4 hours of incubation in vitro 32 P-labele phosphate is incorporate preominantly into phosphatiyl inositol in the normal artery an into lecithin in the atherosclerotic artery. In the present experiments, oleic aci is incorporate preominantly into lecithin by both normal an atherosclerotic intimas. In the normal artery, fatty aci may be incorporate into phospholipi by the Lans pathway, lysolecithin serving as a fatty aci acceptor (24, 25). It is therefore likely that some, possibly most, of the fatty aci in the present experiments is taken up an incorporate irectly into phospholipi via the lysolecithin pathway. In this case, the major phospholipi labele woul be lecithin as observe. For atherosclerotic arteries, however, more information is require before the importance of this pathway can be etermine. In the arteries examine by raioautography, the possibility that some of the raiographically emonstrable material was not lipi was exclue by etermining, in some of the experiments, the 14 C present in both the Folch wash an the tissue resiue following lipi extraction of the "C-labele intima. More than 97% of the 1 4 C present in the intima was recovere in the lipi extract. Phospholipi an cholesterol ester present in approximately equal amounts together accounte for over 70% of this lipi 1 4 C present in the intima. Despite the high amount of combine oleic aci in the intima as a whole, however, the presence of up to 30% free oleic aci in some intimas presente the possibility that the 14 C localize over foam cells may represent preominantly uptake of oleic aci an have no implications with respect to localization of synthesis of phospholipi an cholesterol ester from this oleic aci. This possibilty can be ismisse on experimental evience. It has been shown (Day an Tume, unpublishe observations) that isolate foam cells incubate in vitro take up an incorporate oleic aci into both phospholipi an cholesterol ester, little free fatty aci remaining unchange. Further, it has been shown that foam cells isolate from atherosclerotic intimas incubate in vitro with relabele oleic aci, as for the present experiments, contain between 85% an 90% of their 14 C-labele lipi as cholesterol ester an phospholipi, about 1% only being present as free fatty aci (Day, unpublishe observations). Further, in the latter experiments, the specific activity of oleic aci in both cholesterol ester an phospholipi in the isolate foam cells was seven to eight times that in the original intima, so that the 14 C-labeIe cholesterol ester an phospholipi present in the foam cells are not likely to be synthesize elsewhere an transferre preforme to the foam cell. In the present experiments, oleic aci was use as a precursor for cholesterol ester an phospholipi synthesis so that raioautographic examination coul be meaningful, that is, with no nonlipi "C an little I4 C lipi precursor present. However, it has been shown previously that 14 C-labele acetate is incorporate into fatty acis by both atherosclerotic rabbit intima an by isolate foam cells (13), so that the provision of fatty aci, by synthesis in the foam cell, for incorporation into cholesterol ester an phospholipi presents no problem. When the above information is consiere with the raioautographic evience of localization of 1 4 C by the intimal foam cell, it can be reasonably conclue that, in the Circulation Research, Volume XXIII, December 1968

9 LOCALIZATION OF LIPID SYNTHESIS IN ATHEROMA 787 Downloae from by on January 24, 2019 atherosclerotic lesion, most of the incorporation of fatty aci into phospholipi an cholesterol ester takes place in the foam cells present. This possibility has been suggeste by inirect evience in human lesions (14, 15) an by stuies using isolate rabbit foam cells (13, 16), but the present ata provies irect evience for this possibility. The lack of localization of the 14 C in the meial cells is striking. Since aortas were incubate suspene freely in the incubation meium an some areas of meia covere only by a thin layer of normal intima, this ifference is presume not to be accounte for by reuce access of labele oleic aci to the meial cells. Clearly uptake an synthesis of lipi is a particular property of the intimal foam cells so that if these arise from smooth muscle cells (26, 27), the metabolism of the ifferentiate cells in the meia is quite ifferent from those synthesizing lipi in the intima. Further, there is little 1 4 C localize at the intimo-meial junction. There is evience that cells in this area are preominantly smooth muscle cells, an again little synthesis can be attribute to them uner the circumstances of the present experiments. However, ientification of the more typical foam cells in the lesion is not possible at the light microscope level so that the foam cells which take up the label in the more superficial portions of the intima may represent either smooth muscle or macrophage foam cells, or both. Although localization of 14 C-labele oleic aci is relate to its uptake an conversion to, principally, phospholipi an cholesterol ester by intimal foam cells, there may be other factors which encourage its retention within the cells. The low rate of hyrolysis of cholesterol oleate relative to other cholesterol esters shown by atherosclerotic intima (11) an the slow catabolism of unsaturate fatty acis by this tissue (28) coul both be foam cell phenomena an partly explain the localization of label. Foam cells synthesize polyunsaturate fatty acis from 1 4 C-labele acetate (13) which may facilitate removal of cholesterol esters OrculMim Rcmarch, Volume XXIII, December 1968 forme from such fatty acis more than removal of the cholesterol oleate investigate in the present work. However, the role of foam cells in this an other atherosclerotic processes, an their relevance in terms of accumulation an removal of lipi in the atherosclerotic lesion must await further experimental assessment. Acknowlegments We wish to thank Misses J. Anerson, J. Dare, an J. Colwell, Mrs. G. M. Neill, an Mr. D. M. Vickery for technical assistance. References 1. CHERNICK, S., SRERE, P. A., AND CHAIKOFF, I. L.: Metabolism of arterial tissue. II. Lipie syntheses: The formation in vitro of fatty acis an phospholipis by rat artery with C 1 * an P 3 2 as inicators. J. Biol. Chem. 179: 113, STEIN, Y., AND STEIN, O.: Incorporation of fatty acis into lipis of aortic slices of rabbits, ogs, rats an baboons. J. Atherosclerosis Res. 2: 400, STEIN, O., SELINGER, Z., AND STEIN, Y.: Incorporation of [l- 14 C]-linoleic aci into lipis of human umbilical arteries. J. Atherosclerosis Res. 3: 189, PARKER, F., SCHIMMELBUSCH, W., AND WIL- LIAMS, R. H,: The enzymatic nature of phospholipi synthesis in normal rabbit an human aorta. Results of in vitro stuies. Diabetes 13: 182, LOFLAND, H. B., JR., AND CLAHKSON, T. B.: Certain metabolic patterns of atheromatous pigeon aortas. Arch. Pathol. 80: 291, CHOBANIAN, A. V., AND HOLLANDER, W.: Phospholipi synthesis in the human arterial intima. J. Clin. Invest. 45: 932, ZILVERSMIT, D. B., SHORE, M. L., AND ACKER- MAN, R. F.: The origin of aortic phospholipi in rabbit atheromatosis. Circulation 9: 581, ZILVERSMIT, D. B., AND MCCANDLESS, E. L.: Inepenence of arterial phospholipi synthesis from alterations in bloo lipis. J. Lipi Res. 1: 118, NEWMAN, H. A. I., GRAY, G. W., AND ZILVER- SMIT, D. B.: Cholesterol ester formation in aortas of cholesterol-fe rabbits. J. Atherosclerosis Res., in press. 10. DAY, A. J., AND GOULD-HURST, P. R. S.: Cholesterol esterase activity of normal an atherosclerotic rabbit aorta. Biochim. Biophys. Acta 116: 169, 1966.

10 788 DAY, WAHLQVIST Downloae from by on January 24, HOWARD, A. N., BOWYER, D. E., AND GRESHAM, G. A.: Cholesterol metabolism in normal an atherosclerotic aortas. Circulation 36 (suppl. II): 11-18, LOFLAND, H. B., JR., MOUHY, D. M., HOFFMAN, C. W., AND CLARKSON, T. B.: Lipi metabolism in pigeon aorta uring atherogenesis. J. Lipi Res. 6: 112, DAY, A. J., AND WILKINSON, G. K.: Incorporation of 14 C-labele acetate into lipi by isolate foam cells an by atherosclerotic arterial intima. Circulation Res. 21: 593, GEER, J. C, AND GUIDRY, M. A.: Cholesteryl ester composition an morphology of human normal intima an fatty streaks. Exptl. Mol. Pathol. 3: 485, SMITH, E. B.: Influence of age an atherosclerosis on the chemistry of aortic intima. Part I. The lipis. J. Atherosclerosis Res. 5: 224, DAY, A. J., NEWMAN, H. A. I., AND ZILVER- SMIT, D. B.: Synthesis of phospholipi by foam cells isolate from rabbit atherosclerotic lesions. Circulation Res. 19: 122, PARKER, F., ORMSBY, J. W., PETERSON, N. F., ODLAND, G. F., AND WILLIAMS, R. H.: In vitro stuies of phospholipi synthesis in experimental atherosclerosis. Possible role of myointimal cells. Circulation Res. 19: 700, FOLCH, J., LEES, M., AND SLOANE STANLEY, G. H.: A simple metho for the isolation an purification of total lipies from animal tissues. J. Biol. Chem. 226: 497, DOLE, V. P.: A relation between non-esterifie fatty acis in plasma an the metabolism of glucose. J. Clin. Invest. 35: 150, SKIPSKI, V. P., PETERSON, R. F., AND BARCLAY, M.: Quantitative analysis of phospholipis by thin-layer chromatography. Biochem. J. 90: 374, NEWMAN, H. A. L, DAY, A. J., AND ZILVER- SMIT, D. B.: In vitro phospholipi synthesis in normal an atheromatous rabbit aortas. Circulation Res. 19: 132, SNYDER, F.: Raioassay of thin-layer chroma tograms: A high-resolution zonal scraper for quantitative C 14 an H 3 scanning of thin-layer chroma tograms. Anal. Biochem. 9: 183, GLOMSET, J. A.: The plasma lecithin: cholesterol acyl transferase reaction. J. Lipi Res. 9: 155, STEIN, Y., STEIN, O., AND SHAPIRO, B.: Enzymic pathways of glycerie an phospholipi synthesis in aortic homogenates. Biochim. Biophys. Acta 70: 33, EISENBERG, S., STEIN, Y., AND STEIN, O.: Role of lysolecithin in phospholipi metabolism of human umbilical an og caroti arteries. Biochim. Biophys. Acta 137: 221, PARKER, F., ODLAND, G. F., ORMSBY, J. W., AND WILLIAMS, R. H.: Some ultra structural observations on the eveloping experimental atherosclerotic plaque in rabbit coronary artery an aorta. In Evolution of the Atherosclerotic Plaque, eite by R. J. Jones. Chicago, The University of Chicago Press, 1963, p WISSLER, R. W.: The arterial meial cell, smooth muscle, or multifunctional mesenchyme? Circulation 36: 1, CHOBANIAN, A. V., AND HOLLANDER, W.: Stuies on fatty aci metabolism in human bloo vessels. Clin. Res. 11: 216, Circulation Research, Volume XXIII, December 1968