In Vitro Phospholipid Synthesis in Normal and Atheromatous Rabbit Aortas

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1 In Vitro Phospholipid Synthesis in Normal and Atheromatous Rabbit Aortas By H. A. I. Newman, Ph.D., Allen J. Day, M.D., D.Phil., and D. B. Zilversmit, Ph.D. ABSTRACT The incorporation of P"-phosphate and C^-choline into normal and atherosclerotic rabbit aortas was studied extensively in vitro. The same results as observed in vivo were found, namely an enhanced synthesis of phospholipids by the atheromatous aorta, localized primarily in the intima. Aortic intimal phospholipids incorporated more choline than phosphate. Tests with cyanide and fluoride showed that the higher incorporation of choline could not be attributed to exchange with preformed aortic phospholipid but presumably was due to differences in intermediate metabolic pools. The difference in distribution of P" between the two most active phospholipid classes, phosphatidyl choline and phosphatidyl inositol, of normal and atherosclerotic aortas was similar in vivo and in vitro. In the normal aorta, phosphatidyl inositol had a higher percentage of P" than phosphatidyl choline; in the atheromatous aorta, these percentages were reversed. The studies in vitro were used as evidence to show that the enhanced synthesis observed in vivo was probably not mediated by neural factors or stretch of the aorta, and that synthesis in situ could account for the higher amounts of phospholipid present in the atheromatous aorta. ADDITIONAL KEY WORDS atherosclerosis phosphatidyl choline An exponential increase in both aortic cholesterol and aortic phospholipid of cholesterol-fed rabbits has been observed previously. 1 The increase in cholesterol and cholesterol ester appears to stem directly from an increased influx from plasma, 2 whereas the accumulation of phospholipid in the atherosclerotic aorta can be accounted for by synthesis in situ. 3 The mechanism whereby this elevated synthesis of phospholipids in atheromas is produced is unknown, but the increase does not appear to depend on ele- From the Department of Physiology and Biophysics, University of Tennessee Medical Units, Memphis, Tennessee. Supported by Grant HE-2181 from the U. S. Public Health Service. Dr. Day is on study leave from the University of Adelaide, supported by U. S. Public Health Service Fellowship Award FO5-TW Dr. Zilversmit is a Career Investigator of the American Heart Association. Dr. Newman's present address is Lipid Research Laboratory, Akron City Hospital, Akron, Ohio. Accepted for publication February 15, C^-choline P"-phosphate phosphatidyl inositol vated serum cholesterol levels.* It could, however, be mediated by neural or humoral factors or by altered pressure gradients in the aorta. To assess the importance of these factors the following studies in vitro on the incorporation of P"-orthophosphate and C^-choline into phospholipids of normal and atherosclerotic rabbit aortas were performed. Methods New Zealand albino rabbits were fed 100 g Purina rabbit chow per day. In experimental rabbits the chow diet was supplemented, with 1 g of cholesterol* and 2.8 g of cottonseed oil.t up to 124 days. For these studies rabbits were anesthetized with di-ethyl ether and killed by exsanguination from the abdominal aorta. In each of the incubations the thoracic portion Nutritional Biochemical Company, Cleveland, Ohio. fkindly supplied by Humko Products, Memphis, Tennessee, a division of National Dairy Products Corporation. 132 Grculatioa Raeuch. VoL XTX. July 1966

PHOSPHOLIPID SYNTHESIS IN RABBIT AORTA 133 of the aorta was removed and washed briefly with the incubation medium. While wet, the adventitia of the aorta was freed of superficial fat.

2 PHOSPHOLIPID SYNTHESIS IN RABBIT AORTA 133 of the aorta was removed and washed briefly with the incubation medium. While wet, the adventitia of the aorta was freed of superficial fat. The aorta was cut longitudinally into two approximately equal parts and incubated at 37 C in an atmosphere of 95% O 2 and 5% CO 2. The lipids from these strips or from the whole aorta were extracted with chloroform-methanol (2:1, v/v) as described by Folch et al. 5 Aliquots from the lipid extracts were assayed for P" and C 1 * in a liquid scintillation counter by the procedure of Gordon and Wolfe. 9 Lipid phosphorus was determined in the lipid extracts by the method of Bartlett. 7 Inorganic phosphate in the serum used for the incubation medium was determined by the same procedure after precipitation of serum protein with 10% trichloroacetic acid. The amount of phosphate (m^moles/hour) incorporated into phospholipids in the aortas in these experiments was calculated from the measured specific activity of inorganic phosphate in the medium. The amount of choline incorporated into phospholipid was calculated from the specific activity of the choline salts (Cl~ and Br~) added to the medium. This presupposes that the amount of free choline originally present in serum was negligible comparedto that added. To test this hypothesis, to one normal aortic half was added 3.90 ^moles of choline and to the other 18.2 ^imoles. The incorporation of chorine thus calculated was essentially the same, 25 and 23 ^unoles respectively, in four hours incubation. The distribution of phospholipids in aortic extracts was determined by thin-layer chromatography by the method of Skipski et al. 8 After development the chromatogram was sprayed with iodine vapor to visualize the lipid spots, which were marked, scraped and added to vials containing the dioxane-napthalene-water-scintillator mixture of Snyder. 9 No significant loss of phospholipid radioactivity occurred in the presence of silica gel with this procedure. In one experiment spots separated by thin-layer chromatography were identified as follows: All phospholopids gave a positive reaction with molybdate applied by the method of Dittmer and Lester. 10 Cholinecontaining phospholipids were identified by the Dragendorf reagent as prepared by Skidmore and Entenman. 11 Amino-containing phospholipids were detected by the ninhydrin spray as used by Skipski et al. 8 The standard phospholipids, phosphatidy] ethanolamine and phosphatidyl serine were obtained from Applied Science Laboratories, State College, Pennsylvania. Phosphatidyl choline containing lysophosphatidyl choline was obtained from Mann Research Laboratories, New York, New York. Phosphatidyl inositol was kindly supplied by Dr. M. Faure, Pasteur Institute, Paris, France. As a secondary standard rat plasma Grcuktion Research. Vol. XIX, July 1966 phospholipids identified previously 12 were also employed. P 3f -labeled phospholipid was identified by comparing R F values of radioactive spots located by autoradiography with standards and by eochromatography as described elsewhere. 18 Cochromatography was performed on intimal lipid from one atheromatous aorta incubated for six hours with 10 ml of hypercholesterolemic serum containing 500 fie P M. In this experiment the antioxidant. butylated hydroxytoluene (1%), was added to the solvents employed in the extraction and chromatography. Results In preliminary experiments whole blood was employed as an incubation medium, but incorporation of P 1 into aortic phospholipids was variable and appeared to be less with increased degree of hemolysis. To test this, in one experiment 10 mg hemoglobin added to serum was shown to inhibit the incorporation of P" into phospholipids. In additional, preliminary incubations plasma was employed as the incubation medium. However, the EDTA added to the blood for obtaining plasma also inhibited by about hah 0 P 3I -phosphate uptake into aortic phospholipids. Thus, serum was the medium of choice for the metabolic studies carried out on the thoracic portion of the aorta. This serum was obtained free of visible hemoglobin by collecting and centrifuging the blood at 4 C for 10 min at 13,000 X g, removing the plasma and allowing it to clot at room temperature. To test the viability of aortic strips in serum prepared in the manner just described one-half of a normal aorta was incubated with P" for two hours and the other half similarly after four hours of prior incubation. For three aortas treated in this way, prior incubation resulted in a 35% decrease in incorporation of P". Preincubation of aortas for longer periods sharply decreased incorporation of P" into phospholipid. In the early studies 10 ml of hypercholesterolemic serum were used with atheromatous aortas and 10 ml normal serum with normal aortas. Prior to the two-hour incubation period, 1 ml of Krebs Ringer phosphate containing 7 to 40 /*c P"-phosphate (Abbott

3 z m N m 5 TABLE 1 Comparison of Atheromatous and Normal Aortic Phospholipid-P s * after Incubation in Hypercholesteroleinic and Normal Serum* for Two Hours Expt no. 1 a" b 2 a** b Dan on diet* Incorp. m^moloa/2 hr Normal aortmf AtheromatouB aortaf Specific Dayi on Lipid P activity! Expt. diet* Incorp. Lipid P mg no iott m/unolo/2 hr Specific activity! 26.1 ± ± ± ± ± ± Normal aortas were incubated with normal serum; atheromatous aortas, with hypercholesteroleinic serum. tlipid P and incorporation are expressed per half thoracic portion of the aorta. $Days on high and low cholesterol diets respectively. 5! added P 3I /mg lipid P. The specific activities are about one-tenth of those in table 2. This is due to differences in incubation time, volume of medium, and the presence of some added Krebs-Ringer phosphate in the first set of experiments. *a and b are two halves of the same aorta. ttno. 10 was not considered atheromatous and was omitted from means, calculated from variance between different aortas

4 PHOSPHOLIPID SYNTHESIS IN RABBIT AORTA 135 Laboratories, North Chicago, Illinois) was added to the medium. The incorporation of P"-phosphate into phospholipids by atherosclerotic and normal aortas is shown in table 1. To ascertain the reproducibility of incubation of aortic halves under these conditions, we measured P" incorporation into phospholipids from each half of two normal rabbit aortas. Incubation gave specific activities for halves of the first aorta of and and for the second aorta and In the atheromatous aortas the amount of lipid phosphorus was six times that of the normal aorta. Incorporation of P"-phosphate into phospholipids of the atheromatous aortas was eight times that of the normal, a highly significant difference (P<0.01). That this difference was not the result of a difference in dilution of added P" with serum and readily exchangeable tissue phosphate was demonstrated by the lack of difference in the specific activities of acid soluble phosphate in the incubation medium af the end of the experiment. The mean specific activity of phospholipid in the atheromatous aortas was about twice that of the normal arteries but this difference lacked statistical significance. In the following studies the same medium was employed for normal and atherosclerotic rabbit aortas and the incorporation of two different precursors into aortic phospholipids was determined. To 5 ml of normal rabbit serum was added 19.5 /imoles nonradioactive choline chloride (0.1 ml) containing approximately 3 /AC of choline-1, 2-C J *-bromide (Nuclear Research Chemicals, Inc., Orlando, Florida) along with 5 to 10 /AC P sf -labeled phosphate. After incubation for four hours the aortic halves were removed, washed with 0.9$ sodium chloride solution and then with a solution of nonradioactive choline chloride (5 mg/ml) in 0.1 M phosphate buffer, ph 7.4. In all but one of the experiments 0.5 ml of a solution of KCN and of NaF were added to one of the halves to make final concentrations of 5 X 10~ 2 M, and the incubation was carried out in an atmosphere of 95% N2, 5% CO 2. An equivalent amount of water was Circulation Roe«rch, VoL XIX, July 1966 added to the other half which was incubated in 95$ O 2, 5% CO 2. The CN~ and F~ concentrations employed were similar to those used by Shapira et al. u to stop metabolic activity in normal rabbit aortas. This concentration of CN~, however, was found to raise the ph of the medium to 11. Following incubation and washing, each half aorta was split into two portions, one consisting of intima (containing some media) and one of residual aorta. The data, from experiments in which the relative incorporation of choline and P" into phospholipid were examined, are given in table 2. In the first experiment the two halves of the normal aorta were treated similarly. The incorporation of both P"-phosphate and C^-choline into phospholipid was similar and the specific activity was almost identical in the two halves. In this experiment the normal artery was extracted in toto. In the other experiments, and for all the atherosclerotic arteries, the intima was stripped from the residua] aorta. These two portions were extracted separately and data are presented in table 2 for both portions. It is apparent that the incorporation of both phosphate and choline into phospholipid is increased many fold in the atherosclerotic intimas as compared with the normal intimas. The increase in phospholipid synthesis in the atherosclerotic aorta is due almost entirely to the intimal portion, which of course shows the greatest increase in phospholipid content. The normal residual aorta incorporates as much P Sf and choline-c" into phospholipid as the corresponding intimal-medial portion. The amount of incorporation in the residual aorta of the atherosclerotic artery is of the same order as that in the normal. The effect of 5 X 10-2 M CN" and F~ on the incorporation of P"-phosphate and C H - choline into phospholipid was tested in two normal and three atherosclerotic aortas. One half of the aorta served as a control for the other half. Where CN~ and F~ were present, incorporation of P" into phospholipids in both normal and atherosclerotic intimas and residual aortas was completely inhibited. In-

5 136 NEWMAN, DAY, ZILVERSMIT corporation of choline into phospholipid was inhibited 15 to 2035 of that in control intimas in both the normal and atherosclerotic aortas, but in the residual aorta the incorporation in the presence of the inhibitors was reduced only about The reason for this difference is not clear. The in vitro uptake of P" into phospholipid classes of normal and atheromatous aortas was determined by incubation with 5 to 10 /xc of P' in 5 ml of normal serum for four hours. In addition, normal and hypercholesterolemic rabbits were injected with 700 to 800 \uc P"-phosphate. Six hours later they were killed by intracardiac air, the thoracic portions of aortas were removed, separated into intima and residual aorta and lipids extracted as previously described. Table 3 shows that both in vitro and in vivo in the normal aorta about two-thirds of the label is found in the phosphatidyl inositol and an appreciable amount in phosphatidyl choline. In the atherosclerotic aorta in the two experimental conditions about two-thirds of the label was incorporated into phosphatidyl choline and a lesser amount in phosphatidyl inositol. Other phospholipid fractions including phosphatidyl ethanolamine, sphingomyelin and lysophosphatidyl choline contained the rest of the P 5>. These data are not pre- TABLE 2 Incorporation of Serum CH-choline and P 3 '-Phosphate into Normal and Atheromatous Aortic Phospholipids Dayi on lab. diet Specimens tested 75 Intact aorta* Intact aorta* Intimat Residual aorta 102 Intimat Residual aorta 125 Intimat Residual aorta Normal aorta Incorp. C'< P" Lipld P m/unolei/4 hr mg Specific activity^ C» P» Halves of the same aorta. faortic halves. Intimal portion of the aorta contains some media. Per cent added radioactivity/mg lipld P Incorp. C" P" m/imolw/4 hr AtheromatoTis aorta Llpid P TABLE 3 Phospholipid P" of Rabbit Atheromatous Aortic Intima in vivo Six Hours after Injecting P" and in vitro, Four Hours after Addition of pit* Specific activity} C'< P*» Atheromatous Normal in vivo(2)t in vitro(3) in vlvo(3) in vitro(2) Phosphatidyl choline^ 65.5 ± ± ± ± 1.6 Phosphatidyl inositol** 20.2 ± ± ± ± 2.56 *Thin-layer chromatography on alkaline plates by the method of Skipski et al. 8 fnumerals in parentheses indicate numbers of aortas. $Rechromatography on silica gel G with CHCl 3 -MeOH-H 2 O showed two spots by radioautography with 99* of the radioactivity in the lecithin. Mean ± SE. "Rechromatography of phosphatidyl inositol on alkaline plates with the solvent system di-isobutyl ketone-acetic acid-water (60:25:5, v/v/v) 16 gave three spots with over 802 of the radioactivity in phosphatidyl inositol and 101 in phosphatidyl serine. Grculitioo Research. Vol. XIX, July

$PHOSPHOLIPID SYNTHESIS IN RABBIT AORTA 137 sented in detail because of low counting rates as well as because of the presence of some radio-impurities in these fractions.$

6 PHOSPHOLIPID SYNTHESIS IN RABBIT AORTA 137 sented in detail because of low counting rates as well as because of the presence of some radio-impurities in these fractions. Discussion The amount of phospholipid synthesized in the atherosclerotic aorta in vitro was much greater than that in the normal aorta. This finding agrees closely with previous observations in vivo. 3 The incorporation of serum P'-phosphate and C^-choline into the residual aorta was not altered by the presence of atheromas indicating that the increased synthesis of phospholipid was localized in the thickened intima. This observation is in accord with the studies in vivo of Zilversmit and McCandless. 4 Choline-C J * was originally employed as a precursor for aortic phospholipids as it would be incorporated more specifically into phospholipid than P"-phosphate and thus provide a suitable precursor for autoradiographic studies concerning localization of" phospholipid synthesis in the aortic wall. These investigations so far, however, have yielded equivocal results. As background information for these studies it was necessary to incubate C 14 -choline and P'-phosphate with atheromatous aortas. Although conditions of incubation were not identical to those in which a single label was employed, the conclusions derived from both experiments are similar. Serum P 3f -phosphate enters many intermediate compounds before becoming incorporated into phospholipids. Therefore, calculations of synthesis based on the specific activity of phosphate in the incubation medium may give low results. With choline-c 1 * as a precursor, a more direct pathway is involved. The higher incorporations of serum choline than phosphate into both normal and atherosclerotic aortic phospholipids could be due to differences in intermediate metabolic pools. An alternate explanation based on nonmetabolic exchange between free choline and preformed phospholipid choline, as has been observed in liver microsomes, 16 was excluded by the study with metabolic inhibitors. The addition of cyanide and fluoride to aortic Grculition Re«e»rch. VoL XDC, Joly 1966 intimas reduced the incorporation of choline by four-fifths, a finding not compatible with exchange. Previous studies on intact rabbits have shown a shift in the pattern of P" incorporation from the noncholine phospholipid in the normal aorta to choline containing phospholipid in atheromatous artery. This is confirmed by our findings in vitro in which phosphatidyl inositol was the most active fraction in the normal aorta and phosphaudyl choline in the atheromatous aorta. In previous studies on intact animals, the role of hypercholesterolemia on the synthesis of aortic phospholipids was examined by comparing the incorporation of P" into aortic lipids of animals on a high cholesterol intake (hypercholesterolemic) and animals that had been changed from a high to a low cholesterol intake (normocholesterolemic).* No differences were observed, but the serum cholesterol concentration in the normocholesterolemic animals was always slightly" elevated. Moreover, some other humoral factors responsible for the high rate of aortic phospholipid synthesis might have been present in the blood of normocholesterolemic animals. The in vitro studies reported in table 2, however, give some evidence on this point. Both normal and atheromatous aortas were incubated in a medium containing P" and serum from normal rabbits. Under these conditions the intimas from the atheromatous aortas still incorporated seven times as much P" into phospholipid as did the normal intimas. The agreement between in vivo and in vitro phospholipid synthesis means that differences in aortic stretch or neural factors could not account for the accelerated phospholipid synthesis in the atherosclerotic aorta of intact animals. The possibility of synthesis of phospholipids elsewhere and deposition in the aorta is also eliminated in the present studies since the only source of labeled phospholipids in the aorta in vitro is through synthesis in situ. Acknowledgment We thank Mrs. P. Thompson and Miss R. Ward for their excellent technical assistance.

7 138 NEWMAN, DAY, ZILVERSMIT References 1. NEWMAN, H. A. I., AND ZTLVERSMIT, D. B.: Accumulation of lipid and nonlipid constituents in rabbit atheroma. J. Atherosclerosis Res. 4: 261, NEWMAN, H. A. I., AND ZTLVERSMTT, D. B.: Quantitative aspects of cholesterol flux in rabbit atheromatosis. J. Biol. Chem. 237: 2078, ZILVEHSMIT, D. B., SHORE, M. L., AND ACKEH- MAN, R. F.: The origin of aortic phospholipid in rabbit atheromatosis. Circulation 9: 581, ZtLVEBSMTT, D. B., AND McCANDLESS, E. L.: Independence of arterial phospholipid synthesis from alterations in blood lipids. J. Lipid Res. 1: 118, FOLCH, J., LEES, M., AND SLOANE-STANLEY, G. H.: A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226: 497, GORDON, C. F., AND WOLFE, A. L.: Liquid scintillation counting of aqueous samples. Anal. Chem. 32: 574, BARTLETT, G. R.: Phosphorus assay in column chromatography. J. Biol. Chem. 234: 466, SKTPSKL, V. P., PETERSON, R. F., AND BARTLEY, M.: Quantitative analysis of phospholipids by thin-layer chromatography. Biochem. J. 90: 374, SNYDER, F.: Radio-assay of thin-layer chromatograms: A high-resolution zonal scraper for quantitative C'4 and H 3 scanning of thinlayer chromatograms. Anal. Biochem. 9: 183, DITTMER, J. C, AND LESTER, R. L.: A simple, specific spray for the detection of phospholipids on thin-layer chromatograms. J. Lipid Res. 5: 126, SKTDMORE, W. D., AND ENTENMAN, C: Two-dimensional thin-layer chromatography of rat liver phosphatides. J. Lipid Res. 3: 471, NEWMAN, H. A. I., Lro, C.-T., AND ZTLVERSMTT, D. B.: Evidence for the physiological occurrence of lysolecithin in rat plasma. J. Lipid Res. 2: 403, DAY, A. J., NEWMAN, H. A. I., AND ZILVER- SMIT, D. B.: Synthesis of phospholipid by foam cells isolated from rabbit atherosclerotic lesions. Circulation Res. 19: 122, SHAPTRA, J., GLASGOW, J. L., PERKINS, W. H., AND HARVEY, C: Effect of cyanide and fluoride on in vitro incorporation of H s -cholesterol into rabbit artery. Proc. Soc. Exptl. Biol. Med. 109: 675, MARINETTI, G. V.: Chromatographic separation, identification and analysis of phosphatides. J. Lipid Res. 3: 1, DTLS, R. R., AND HUBSCHER, G.: Metabolism of phospholipids. III. The effect of calcium ions on the incorporation of labelled choline into rat liver microsomes. Biochim. Biophys. Acta 46: 505, Grcuktion Roearch, VoL XTX, July 1966

Incorporation of Oleic Acid into Lipid by Foam Cells in Human Atherosclerotic Lesions

Incorporation of Oleic Acid into Lipid by Foam Cells in Human Atherosclerotic Lesions By Mark L. Wohlqvist, B.Med.Sc, M.B., B.S., Allan J. Day, M.D., D.Phil., and Ronald K. Tume, B.Sc. ABSTRACT The incorporation