reticulo-endothelial cells in rat lymph nodes has been further investigated

FATTY ACID PATTERNS OF CHOLESTEROL ESTERS SYNTHE- SIZED BY RETICULO-ENDOTHELIAL CELLS.* By A. J. DAY, N. H. FIDGE, P. R. S. GoULD-HURST and D. J. RISELY. From the Department of Human Physiology and Pharmacology, University of Adelaide. (Received for publication 5th March 1963) The fatty acid pattern of cholesterol esters synthesized by RE cells in rat lymph nodes was determined by gas phase chromatography. Following the uptake of cholesterol by these cells esterification with predominantly saturated fatty acids occurred. Where corn oil or coconut oil was administered together with the cholesterol the fatty acid pattern of the cholesterol esters synthesized became more unsaturated or more saturated respectively. The significance of these findings in terms of the metabolism of cholesterol by RE cells and its relevance to atherosclerosis is discussed. CHOLESTEROL suspensions taken up by reticulo-endothelial cells in rat lymph nodes or by rabbit macrophages in vitro, are partially converted to cholesterol ester [Day and French, 1959; Day and Gould-Hurst, 1961]. The fatty acid pattern of cholesterol esters may also influence their subsequent metabolism [Klein and Martin, 1959; Lewis et at., 1961] and much interest has arisen concerning fatty acid patterns of cholesterol esters in various situations, particularly in the arterial wall in atherosclerosis [Sinclair, 1956; Bottcher et al., 1960]. Some workers have suggested that cholesterol may be esterified in situ in the arterial wall in atherosclerosis [Zilversmit et al., 1961] and Day [1962] has shown that macrophages may play a role in lipid metabolism in this situation. In the present paper the esterification of cholesterol by reticulo-endothelial cells in rat lymph nodes has been further investigated using gas phase chromatography. The fatty acid pattern of the cholesterol esters synthesized following the uptake of cholesterol has been determined and the effect of administering either corn oil or coconut oil together with the cholesterol on the fatty acid pattern investigated. MATERIALS AND METHODS Cholesterol suspensions, corn oil and coconut oil emulsions were prepared as previously described [Day, 1960]. Experimental Procedure.-Male Norwegian hooded rats 180-220 g. were used. Batches of 18-25 rats were injected intraperitoneally with 2 ml. of a preparation containing 8 mg. cholesterol and 50 mg. bovine albumin (Armour). In experiments where corn oil or coconut oil was added the rats received in addition to the cholesterol suspension and albumin, 1 ml. of 10 per cent corn oil emulsion or coconut oil emulsion, respectively. Extraction of Lipids.-24 hr. after injection of the cholesterol the sternal lymph * This work was aided by a grant from the National Heart Foundation of Australia. 298

Cholesterol Ester Fatty Acids nodes were dissected out and a combined extract of nodes from all rats in one batch was prepared using 2: 1 chloroform: methanol as described by Folch et al. [1957]. An aliquot of the combined extract was taken for chromotographic separation of the free and ester cholesterol by the method of Kerr and Bauld [1953] and its subsequent determination by the method of Zlatkis et al. [1953]. The remainder of the extract was separated into its lipid components on silicic acid columns as described by Hirsch and Ahrens [1958]. Gas Phase Chromatography.-The cholesterol ester fraction was transesterified with HCI in methanol by the method of Stoffel, et al. [1959] in order to prepare the methyl esters of the fatty acids. Gas phase chromatography was then carried out using a Pye Argon Chromatograph with polyethylene glycol adipate (10 per cent on Celite 545, 100-120 mesh) at 1800 C. as stationary phase. Identification of individual fatty acids was made by comparison with standards or by reference to published retention data. Peak areas were calculated by triangulation as described by James [1959]. RESULTS Table I gives the amount of ester synthesized by the nodes in all the experiments. No ester cholesterol was present in the nodes prior to their uptake of free cholesterol, but the percentage ester present rose following cholesterol uptake to a mean of 16 per cent in the group receiving cholesterol alone. Where either corn oil or coconut oil was administered together with TABLE I.-ESTERIFICATION OF CHOLESTEROL BY RE CELLS IN RAT LYMPH NODES EXPt NO Of Rats Amount of ester Ester E N (mg.) (percentage) Cholesterol alone B6 28 1-04 12 B7 29 2*28 18 B17 24 2-77 16 B21 21 2*04 16 B22 19 1-84 20 Cholesterol with corn oil B9 25 2.01 24 B1O 25 1.90 18 Bl 20 1.01 25 B14 18 1.43 46 Cholesterol with coconut oil B13 22 1-88 25 B15 25 1-89 25 B16 24 2-25 29 B19 24 2-65 13 299 the cholesterol the percentage esterification was higher than was the case with cholesterol administration alone. Table II gives the percentage distribution of fatty acids from cholesterol esters synthesized by the nodes under the three experimental conditions. The fatty acid composition of the corn oil and of the coconut oil used is shown in Table III. In the group receiving cholesterol alone the principal

300 Day, Fidge, Gould-Hurst and Risely fatty acids esterified with the cholesterol were palmitic, oleic and linoleic, but predominantly palmitic. Where corn oil was given with the cholesterol, the same three fatty acids predominate but considerably more oleic acid and linoleic acid were present. Although the predominant fatty acid in the corn oil itself was linoleic acid, in the cholesterol ester oleic acid was predominant. TABLE II.-PERCENTAGE DISTRIBUTION OF FATTY ACIDS IN CHOLESTEROL ESTERS SYNTHESIZED BY RE CELLS (Mean of four or five experiments together with the estimated standard error is shown) Cholesterol with Cholesterol with Cholesterol alone Fatty acid (mean corn oil coconut oil oof ive) (mean of four) (mean of four) 12:0 (Lauric).. 0 7A0.2 0 7+001 1F6+0 3 14:0 (Myristic).. 4.2±1.0 1 1:0-2 14-4±3.3 16: 0 (Palmitic).. 39.1±4.4 21.0±009 28.9±1*3 16: 1 (Palmitoleic). 8-1±1-7 5.6±0*6 5-6+0A4 18: 0 (Stearic).. 5-2±0 5 4-6i12 6.6± 0 7 18: 1 (Oleic).. 28-7±5-2 42-6+1F3 31.3±1.5 18:2 (Linoleic).. 10.7±15 20.4±0-7 7.3±141 18: 3 (Linolenic).... trace 20:4 (Arachidonic). 3.1±0.8 3*9±1*4 4*4±1@3 Where coconut oil (containing a high proportion of lauric acid) was administered the fatty acids of the cholesterol esters became more saturated and of shorter chain length. Less linoleic acid was present than in the other two groups and a considerable amount of myristic acid appeared in the cholesterol ester fatty acid. Little increase in the lauric acid was apparent. TABLE III.-PERCENTAGE DISTRIBUTION OF FATTY ACIDS IN CORN OIL AND COCONUT OIL USED FOR EXPERIMENTS Fatty acid Corn oil Coconut oil 12: 0 (Lauric).. 0-6 47.0 14: 0 (Myristic). 06 26-7 16: 0 (Palmitic). 20.6 12-4 16: 1 (Palnitoleic). 18 : 0 (Stearic).. 13 3-4 18: 1 (Oleic).. 301 8.4 18 : 2 (Linoleic). 46.7 1.9 18: 3 (Linolenic). trace 20: 4 (Arachidonic). Fig. 1 shows the proportion of saturated, monounsaturated and polyunsaturated fatty acids under the three experimental conditions. In the nodes in which cholesterol alone was taken up 49.3 per cent of the cholesterol ester fatty acid was saturated and only 13.9 per cent polyunsaturated. There was little difference in this pattern when coconut oil was also administered, although a higher proportion of the saturated fatty acids were shorter chain acids. Where corn oil was administered only 27.4 per cent was saturated and 24.3 per cent polyunsaturated although even in this case the pattern contains less polyunsaturated fatty acids than might be expected.

Cholesterol Ester Fatty Acids 301 DISCUSSION The cholesterol ester fatty acid pattern of serum in those species where it has been examined contains a relatively large proportion of polyunsaturated fatty acids. The information regarding cholesterol ester fatty acids of rat serum is somewhat conflicting [Mukherjee et al., 1957; Klein and Jannsen, I S.. W Rw ċ J t ; ; ; ;J Saturated % Distribution 60 =....-.-.-.-.-.- -*:-:-:-:-:-: Mono Poly Unsaturated Unsaturated 50 40 30 20 10 Cholesterol Cholesterol Cholesterol alone with corn oil with coconut oil FIG. 1.-Percentage distribution of saturated, monounsaturated and polyunsaturated fatty acids in cholesterol esters formed by RE cells following their uptake of cholesterol either alone or together with corn oil or coconut oil. 1959; Swell et al., 1960]. However, using gas phase chromatography and determination of a wide range of fatty acids, Swell et al. [1960], found that up to 70 per cent of the cholesterol ester fatty acids of serum of normal rats were polyunsaturated. This is in marked contrast to the 13.9 per cent polyunsaturated fatty acid in the cholesterol ester synthesized by RE cells. This may reflect the type of fatty acid available for synthesis, but even where large amounts of polyunsaturated fatty acid were present the amount of polyunsaturated fatty acid in the cholesterol ester was only elevated to 24.3 per cent of the total. This implies that cholesterol may be selectively

302 Day, Fidge, Gould-Hurst and Risely esterified with more saturated fatty acids by reticulo-endothelial cells in these situations. Fatty acid can be synthesized by R.E. cells in other situations [Day and Fidge, 1963], and preliminary work has shown that the more saturated and shorter chain fatty acids are turned over at a higher rate by rabbit macrophages. Such an increased turnover of these fatty acids probably influences the fatty acid pattern of cholesterol ester being synthesized. Generally tissue cholesterol ester fatty acids are more saturated than those of the serum. Rat liver cholesterol ester has been shown to have a pattern similar to that shown here for reticulo-endothelial cells [Swell et al., 1960]. In atherosclerosis in both the human and the rabbit the plasma cholesterol ester fatty acid differs from that of the plaque in that the latter is more saturated [Zilversmit et at., 1961; Tuna et al., 1958; Bottcher et al., 1960; Lewis, 1959; Swell et al., 1960]. The issue is confused, however, by the observation that increasing severity of lesions is associated with an increasing polyunsaturation of the cholesterol ester fatty acid present [Bottcher et at., 1960]. The differing composition of plaque and plasma fatty acid has led some workers [Zilversmit et al., 1961; Swell et al., 1960] to consider that esterification of cholesterol takes place in the arterial wall and it is relevant to suggest that since early lesions at least are characterized by cholesterol contained in reticulo-endothelial cells, that such esterification may be brought about within these cells. If this is so and RE cells in the arterial wall preferentially esterify the cholesterol with saturated fatty acids as RE cells in rat lymph nodes have been shown to do in the present experiments, this fact may in part account for the high percentage of saturated fatty acids in the cholesterol ester of the early atheromatous plaque. ACKNOWLEDGMENTS We are indebted to Misses M. Kleeman and D. Knapman-Jones for technical assistance. REFERENCES BOTTCHER, C. J. F., WOODFORD, F. P. and TER HAAR ROMENY-WACHTER, C. CH., BOELSMA-VAN HOUTE, E. and VAN GENT, C. M. (1960). Lancet 1, 1378. DAY, A. J. (1960). Aut. J. exp. Biol. Med. Sci. 38, 461. DAY, A. J. (1962). J. Athero8cler. Res. 2, 350. DAY, A. J. and FIDGE, N. H. (1963). [To be published.] DAY, A. J. and FRENCH, J. E. (1959). Quart. J. exp. Physiol. 44, 239. DAY, A. J. and GOULD-HURST, P. R. S. (1961). Quart. J. exp. Physiol. 46, 376. FOLCH, J., LEES, M. and SLOANE STANLEY, G. H. (1957). J. biol. Chem. 226, 497. HIRSCH, J. and AHRENS, E. H., Jr. (1958). J. biol. Chem. 233, 311. JAMEs, A. T. (1959). Methods of Biochemical Analysis. Ed. D. Glick. 8, 1. KERR, L. M. H. and BAULD, W. S. (1953). Biochem. J. 55, 872. KLEIN, P. D. and JANSSEN, E. T. (1959). J. biol. Chem. 234, 1417. KLEIN, P. D. and MARTIN, R. A. (1959). J. biol. Chem. 234, 1685.

Cholesterol Ester Fatty Acids 303 LEWIS, B. (1958). Lancet, 2, 71. LEWIS, B., PILKINGTON, T. R. E. and HODD, K. A. (1961). Clin. Sci. 20, 249. MU:KHERJEE, S., ACHAYA, K. T., DEUEL, H. J., Jr. and ALFIN-SLATER, R. B. (1957). J. biol. Chem. 226, 845. SINCLAIR, H. M. (1956). Lancet, 1, 381. STOFFEL, W., CHU, F. and AERENS, E. H., Jr. (1959). Anal. Chem. 31, 307. SWELL, L., LAW, M. D., FIELD, H., Jr. and TREADWELL, C. R. (1960). J. biol. Chem. 235, 1960. TUNA, N., RECKERS, L. and FRANTZ, I. D., Jr. (1958). J. clin. Invest. 37, 1153. ZILVERSMIT, D. B., SWEELEY, C. C. and NEW1AN, H. A. I. (1961). Circulation Res. 9, 235. ZLATKIS, A., ZAK, B. and BOYLE, A. J. (1953). J. Lab. clin. Med. 41, 486.