THE RABBIT maintained on a highcholesterol

Transcription

1 Fatty Acid Cmpsitin f Serum and Artic Intimal Lipids in Rabbits Fed Lw- and High-Chlesterl Diets By DONALD B. ZILVEKSMIT, PH.D., CHARLES C. SWEELEY, PH.D., AND HOWARD A. I. NEWMAN, PH.D. THE RABBIT maintained n a highchlesterl diet rapidly accumulates lipids in the artic intima. The rigin f these lipids has been much debated. On the ne hand, evidence has been presented that artic lipid plaques are residues frm lipids riginally present in serum, whereas ther experiments have demnstrated the ability f the arta f the rabbit t synthesize varius lipid fractins. Istpic studies in particular have cntributed t ur knwledge n the develpment f athermatus lesins. Thus it has been shwn that varius phsphlipids f the athermatus arta turn ver mre rapidly than thse f the nrmal artery and that, f the phsphlipids present at any ne time, few if any f their cnstituents are derived frm plasma. 1 In the case f chlesterl, hwever, the situatin seems t be different. Althugh a measurable turnver f chlesterl in the athermatus lesin fakes place, a cnsiderable prtin f the ttal chlesterl present in the lesin was nce a plasma cnstituent. 2 In rder t thrw further light n the rigin f athermatus lipids, we have cmpared the cmpsitin f different lipid fractins in the plasma t their cunterparts in the atliermatus lesin. If lipids were depsited frm plasma at randm and subsequently did nt underg metablic alteratins in the arta, then the cmpsitin f artic lipid fractins shuld be identical t that f plasma. Differences in cmpsitin culd, therefre, be interpreted as evidence against the depsitin r filtra- Frm the Department f Physilgy, University f Tennessee, Memphis, Tennessee. Supprted by a grant frm the Natinal Heart Institute (H-2181). Dr. Zilversmit is a Career Investigatr f the American Heart Assciatin. Received fr publicatin August 19, Circulatin Research, Vlume IX, March tin thery r as evidence that lipids, nce depsited, underg metablic alteratins in the arterial wall. Methds Albin New Zealand rabbits were maintained n Purina chw fr abut eight weeks and then divided int tw grups. Half f them were cntinued fr 16 additinal weeks n the chw diet whereas the ther half received daily 1 Gm. f chlesterl disslved in 2.6 Gm. f Humk hydrgenated cttnseed il* added t the diet. At the end f the perid, all animals were bled by heart puncture int syringes wetted by 2 per cent Mepesulfatet in saline and the entire thracic arta and prtins f adipse tissue remved fr analysis. The intima and adventitia were separated and grund up separately befre extractin. Lipids frm all tissues were extracted with chlrfrm-methanl and purified by an adaptatin f the Flch prcedure. 3 Neutral lipids were separated frm phsphlipids n small activated silicic acid clumns. 4 The neutral lipids were further fractinated by the methd f Hrning et al. 5 whereas the phsphlipids were separated accrding t Newman et al. 6 as fllws: 4 Gm. f Mallinekrdt 100 mesh silicic acid (suitable fr ehrmatgraphic analysis), freshly activated by heating at 120 t 130 C. fr 48 hurs, was slurried with chlrfrm and then transferred t a clumn f the dimensins 10 X 100 mm. having a tapered tip and a glass-sealed 250-ml. reservir. The silicic acid was packed with sufficient pressure frm dried air (apprximately 1 lb. per square inch) t maintain a flw rate f 0.5 ml. per minute. The phsphlipid sample in chlrfrm was added, and the pressure increased gradually t 4 t 5 lbs. t maintain the 0.5-ml.-per-minute flw rate. Lads frm t 1.23 nig. phsphlipid phsphrus were used. The eluates frm the clumn were autmatically cllected thrugh a vlumetric siphn in 5-ml. fractins. When the sample had cmpletely entered the clumn, it was washed with 30 ml. mre chlrfrm, which eluted mst f the neutral lipids. Apprximately 40 t Chlesterl TJSP grade was graciusly dnated by Merck C., Eahway, New Jersey, and hydrgenated cttnseed il by Humk C., Memphis, Tennessee. t Anticagulant, Hffmann La Bche.

2 236 ZILVEESMIT, SWEELEY, NEWMAN Table 1 Per Cent Fatty Acids f Diet, Adipse Tissue, Plasma, and Intimal Triglycerides* Nrmal chw High chlesterl -f- fat chw- Adipse cntrlf Adipse chlesterl-fed t Plasma cntrl t Plasma chlesterl-fed f Intima cntrl i Intima chlesterl-fed *Little r n CM acid was present in any f these samples. t All values are means f three animals. {Pled intima frm six animals. Mean value f tw intimas. One value was 11.4 and the ther Table 2 Per Cent Fatty Acids f Plasma and Intimal Chlesterl Esters in Chlesterl-Fed Rabbits 14-0 * * *Less thiin 0.5 per cent PI Int nil. f 20 per cent methanl in chlrfrm (v/v) was then added t the clumn t elute the nnchline-entaining phsphlipids. The chline-cntaining phsphlipids, which were left n the clumn, were separated by adding a series f slvents as fllws: 125 t 140 ml. f 40 per cent methanl in chlrfrm (v/v) eluted lecithin; 50 t 80 nil. f 60 per cent methanl in chlrfrm (v/v) eluted sphingmyelin; finally, 50 ml. f methanl eluted lyslecithin. Fatty acids were determined by gas-liquid chrmatgraphy. Methyl esters frm each lipid fractin were btained by transmethylatin in methanl-cneentrated sulfuric acid (95/5, v/v) at 75 C. fr 12 hurs in sealed tubes. The esters were extracted frm the reactin mixtures with redistilled hesane. Analyses were made n a 6 ft. by 4 mm. glass tube packed with ethylene glycl-suecinic acid plyester n Chrmsrb W- S0 t 100 mesh, 16 per cent (w/w) f plyester. The clumn was maintained at 1S5 C with an inlet pressure f 22 t 25 punds per square inch f argn. The retentin time fr methyl stearate was 10 minutes, and the separatin factr fr methyl leate and methyl stearate was 1.1S. The clumn lad was usually 25 t 50 ftg. f mixed methyl esters. The gas-liquid partitin chrmatgraphy instrument was equipped with an argn 18-1 PI Int PI. -P Int. 3.0 inizatin detectr. 7 Area calculatins were based n measurements f peak height and width at half height. The results, which are reprted in the tables, are in weight per cent, btained by a nrmalizatin calculatin frm the cmpnent areas. 8 Results In rder t btain sufficient lipid fr the varius fractinatins, intimas frm six nrmal rabbits were pled befre extractin, but intimas frm chlesterl-fed animals were analyzed separately. All plasma and adipse tissue samples were als analyzed separately. Althugh the fatty acid cmpsitin f the diet f the chlesterl-fed animals differed frm that f the cntrls by the additin f 2.6 per cent hydrgenated cttnseed il, n remarkable differences appeared in the fatty acid cmpsitin f plasma r adipse tissue triglycerides (table 1). The fatty acid cmpsitin f adipse tissue was very similar t the fatty acid cmpsitin f the chlesterl- and hydrgenated-fat-supplemented diet. The high percentage f linleie (18-2) acid f the unsupplemented chw was Circulatin Research. Vlume IX. March 1B61

3 AORTIC IXTIMA LIPIDS l40n 60 ; 100% CHjOH TUBE NUMBER Figure 1 Chrmatgraphic separatin f plasma phsphlipids in nrmal (left) a<nd chlesterlfed (right) rabbit; diagnal lines = lecithin analyzed by gas-liquid chrviatgraphy (GLC); vertical lines = sphingmyelin analyzed by GLC ; hrizntal lines = lyslecithin analyzed by GLC. nt reflected in a crrespnding enrichment f that acid in adipse tissue. It shuld be nted, hwever, that althugh the ttal fat intake f the tw grups f animals differed, the ttal intake f liuleie acid was apprximately the same. A cmparisn f fatty acids in plasma and arterial triglycerides in the nrmal r chlesterl-fed animals als failed t shw any cnsistent differences. Nr did the medial and adventitial triglyceride fattj r acid pattern differ significantly frm that in the intima r plasma. The chlesterl esters f plasma and intima shwed mre striking differences. Althugh analyses f the pled nrmal intima were made, t little chlesterl ester was present t give reliable results. Hwever, it is clear frm table 2 that the chlesterl esters accumulating in the athermatus intima cntained primarily leie (18-1), palmitic (16-0), and linleie (18-2) acid. A cmparisn f chlesterl esters in the plasma and intima 50 f chlesterl-fed animals reveals a remarkable similarity in cmpsitin, except fr the presence f abut 3 per cent plyunsatu rated 18-carbn acids in the athermatus intima, whereas nne were fund in the plasma esters. Smaller but cnsistent differences were nted in the leic (18-1) and linleie (18-2) acid fractins. A typical distributin f phsphlipids in plasma and arta nrmal and chlesterl-fed rabbits is shwn in figures 1 and 2. In cnfirmatin f previus results," the ehline-cntaining phsphlipids in the athermatus intimas increased much mre than the nnchline types. In tw f the three artas frm chlesterl-fed animals the separatin f lecithin and sphingmyelin was made difficult by the appearance f a cnsiderable fractin which cnsisted f a mixture f these tw lipids (seefig.2, right). Gas chrmatgraphic analyses are reprted n material btained frm the lecithin, sphingmyelin, and lyslecithin fractins as indicated Circulatin Research, Vlume IX. March 1961

4 238 ZILVERSMIT, SWBELEY, NEWMAN l % CHjOH 12- i 0_ TUBE NUMBER Figure 2 ChrmaitgrapMc separatin f intimal phsphlipids in nrmal (left) and chlesterlfed (right) rabbitj diagnal lines lecithin analyzed by GLC; vertical lines = sphingmyelin analyzed by GLCj hrizntal lines = lyslecithin analyzed by GLC. in figures 1 and 2. In ther animals the lecithin and lysleeithin were fund t cntain less than 1 per cent impurity by paper chrmatgraphy 10 whereas the sphingmyelin fractin cntained up t 10 per cent lecithin. Patty acid pattern f the nnchline fractins are nt reprted, since this fractin was relatively small and appeared t be cntaminated with sme glyceride. Chlesterl feeding appeared t have little r n effect n the lecithin fatty acids f plasma r intima (table 3). In bth nrmal and chlesterl-fed animals, hwever, the relative cntent f linleic (18-2) acid in plasma far exceeded that in the intima, whereas the palmitleic (16-1) was cnsistently less in the frmer. In the athermatus intima the elutin pattern shwed a prnunced tailing f the lecithin fractin. Fatty acids frm this prtin f the chrinatgram are nt included in table 3. Althugh the plasma samples f the chlesterl-fed animals shwed appreciable amunts f arachidic (20-0), arachidnic (20-4), behenic (22-0), tricsanic (23-0), lignceric (24-0), and particularly nervnic (24-1) acids in this prtin f the eluate, the intimal lesins shwed practically n such acids. This prtin f the eluate als shwed a relatively high palmitleic (16-1) acid cntent f artery cmpared t that f plasma. The sphingmyelin fractin f bth plasma and artic intima shwed an increase in cncentratin similar t r greater than that bserved fr lecithin. The fatty acids f plasma sphingmyelin f chlesterl-fed animals did nt differ t much frm thse f cntrl animals (table 4) except that there seemed t be a significantly higher percentage f palmitleie (16-1) acid in the cntrl animals. The intima f the chlesterl-fed animals appeared t have an elevated percentage f palmitleic acid and particularly f nervnic (24-1) acid which was nt fund in nrmal intima. Cnversely, the percentage f arachidic (20-0) and lignceric (24-0) acids decreased belw that f the nrmal iutima. A cmparisn f intimal and plasma sphingmyelin in the nrmal animals revealed a Circulatin Research, Vlume IX, March 1961

5 AORTIC INTIMA LIPIDS 239 t Ci t c TJ5 c cxi r-i CM lo «N Tji ra» : i Oi 115 r! ^ w ci ^ O i t * *' i * r-j OO cc ct^ ii N ^ I-! OO t Cj r-^ t>- ci i-* O Ci r-i Ci * in" (M r-i Ci j O r-i r-i O i i i 8. * *» t Ci OS Ci IO t- t- (M* IO (M* Ci C: t r^ 00 t- O I- ".i ei c i ei ci -* ci c - t-. 1O 2 a, 0 Circulatin Retearch, Vlume IX, March _ " "

6 240 ZILVERSMIT, SWEELEY, NEWMAN IO 00 t C-l 1.6 s 2 ^ c! r ~l t i,: 10 >; -^ IO Cl in cj ci ci <M r-. Cl <M O 06 c^i t N Ol Oi r-i ci ^ -^ t ci c> rh IO t d Cl r-i Cl r S 4" ci ci cc t i d * higher cntent f lignceric (24-0) acid in the intima, whereas in the chlesterl-fed animal the intima cntained much less palrnitie (18-0) acid and mre laurie (12-0), myristic (14-0), palmitleic (16-0), leic (18-1), and lignceric (24-0) acid than the crrespnding plasma. The amunt f lysleeithin phsphrus fund in plasma and arta f nrmal r chlesterl-fed animals varied frm 10 t 20 per cent f the ttal lipid phsphrus. This relatively high lyslecithin cntent f plasma parallels that bserved in the plasma f the rat 6 and is smewhat higher than that in the plasma f man. 11 The analysis f lyslecithin fatty acids (table 5) shwed that the nrmal intima cntained a smaller percentage f linleic (18-2) acid and a higher percentage f linlenie (18-3) acid than nrmal plasma, whereas the intima f the chlesterl-fed animal cntained relatively mre laurie (12-0), myristic (14-0), palmitleic (16-1), and linlenic (18-3) acid and less linleic (18-2) acid than the crrespnding plasmas. Discussin Previus studies in ur labratry have demnstrated that the phsphlipids f athermatus lesins in the arta f the chlesterl-fed rabbit turn ver rapidly. 1 Studies with P 32 and C 14 -acetate have indicated that the radiactive phsphlipids present in the arterial lesins f these rabbits are predminantly synthesized in situ. 12 ' ls The prnunced differences in fatty acid cmpsitin f arterial intima and plasma reprted in this paper als supprt the cncept that plaque phsphlipids are nt merely randm depsits f plasma phsphlipids. The case fr chlesterl appears t be smewhat different: in the intact chlesterl-fed rabbit the chlesterl synthesis by the artery makes little r n cntributin t the chlesterl f the lesin. 2 This bservatin is nt necessarily in cnflict with the finding that fragments f rabbit artery in vitr are capable f cnverting C 14 -acetate t sterl. 14 ' 1S If such cnversin ccurs in the intact chlesterl-fed rabbit it merely represents an insignificant prprtin f the ttal chlesterl flux int, Circulatin Research, Vlume IX, March 1S61