(i.e. specific activity) of C16: 0, C18: 0 and C18: 1 acids between the

Transcription

1 Quarterly Journal of Experimental Phy8iology (1974), 59, ORIGIN AND FORMATION OF LYMPH LIPIDS IN THE SHEEP. By W. M. F. LEAT and F. A. HARRISON. From the Agricultural Research Council Institute of Animal Physiology, Babraham, Cambridge. (Received for publication 5th September, 1973) 3H and 14C-labelled palmitic, stearic, oleic, linoleic and linolenic acids introduced into the duodenum of sheep were recovered in thoracic duct lymph, and the specific radioactivities of the fatty acids in the various lipid fractions were determined. Lymph fatty acids were transported mainly as triglycerides (77 %) and phospholipids ( 18%), probably mostly in the form of very low density lipoproteins rather than as chylomicrons. Lymph triglycerides transported most of the palmitic, stearic and oleic acids (74-79%) whereas phospholipids were important in the transport of the essential fatty acids, linoleic and linolenic acids (54% and 38% respectively). Specific radioactivity measurements indicated that part of the lymph cholesteryl ester and phospholipid fatty acids were of a non-radioactive origin. In lymph phospholipids 52% of the linoleic acid, 81% of the linolenic acid and more than 88% of the palmitic, stearic and oleic acids were derived from endogenous esterified sources. Evidence is presented to indicate that the major part of lymph phospholipids could be derived from biliary phospholipid. The possible roles of bile and pancreatic juice in fat absorption in the ruminant are discussed. The major fatty acids ingested by ruminant animals are linolenic acid (C18: 3) when the animal is grazing pasture, and linoleic acid (C18: 2) when concentrates are the main component of the diet. These fatty acids are present in the diet in esterified forms, but in the rumen extensive hydrolysis occurs followed by hydrogenation of the C18: 2 and C18: 3 acids to monoenoic acids and stearic acid [see Garton, 1967]. The small amounts of C18: 2 and C18: 3 acids that escape hydrogenation account for less than 1% of the total energy of the diet [Leat and Harrison, 1972]. As a result of this microbial action in the rumen, the major lipid in digesta reaching the small intestine is free fatty acid consisting mainly of palmitic (C 16: 0) stearic (C18: 0) and monoenoic (C18: 1) acids. The free fatty acids are rendered soluble by the action of bile and pancreatic juice and absorbed into the lymphatics where they appear as esterified lipids. Harrison and Leat [1972] showed that radioactively labelled C16: 0, C18: 0 and C18: 1 acids introduced into the duodenum of the sheep are absorbed rapidly into the lymphatics and most of the radioactivity (90%) was associated with the triglyceride fraction. Few quantitative data are available on the synthesis and formation of lymph lipids in the ruminant, and to obtain more information on the origin of lymph lipids the distribution by weight and by radioactivity (i.e. specific activity) of C16: 0, C18: 0 and C18: 1 acids between the various esterified lipids has been determined. Additional observations were made on the absorption from the intestine of the small amounts of essential fatty acids (C 18: 2 and C18: 3 acids) which have escaped hydrogenation in the rumen; and the specific activities of these acids in lymph lipids have been compared with the values obtained for C16: 0, C18: 0 and C18: 1 acids. 131

2 132 Leat and Harrison MATERIALS AND METHODS Surgical preparation A 2 year old Clun Forest ewe ('Jane') weighing kg was used. After the usual 3-4 week period of acclimatization in the animal house to a dry diet of 1000 g chaffed hay and 200 g crushed oats given once daily, the sheep was fitted with a rumen cannula and, at a subsequent operation, with a duodenal cannula placed opposite the orifice of the common bile and pancreatic duct. At this stage 100 g concentrates were added to the diet to increase the lipid intake to 20 g daily before the experiments on fat absorption. At a final operation the thoracic lymph duct was permanently catheterized as described previously [Harrison and Leat, 1970, 1972]. All surgery was performed aseptically under general anaesthesia induced with intravenous pentobarbitone sodium and, after endotracheal intubation with a cuffed Magill tube, maintained with a mixture of oxygen and halothane ('Fluothane', I.C.I. Ltd.) in a closed-circuit rebreathing system. For thoracic duct cannulation, positive pressure respiration was used whilst the thorax was open and the operation took approximately 11 hr. Experiments on lymph were not started until 4 days after surgery when the animal was eating the full daily ration of food. Experimental Single injection of isotope Fatty acids labelled with H (C16: 0) and 14C (U-14C 18: 0, U-14C 18: 1, 1_14C 18: 2 and 1-14C 18: 3) (The Radiochemical Centre, Amersham, Bucks, U.K.) were dissolved as their potassium soaps and injected as a single dose into the duodenum [see Harrison and Leat, 1972]. Two acids were studied simultaneously, 3H palmitic acid (100,uCi) with each of the 14C fatty acids (10 uci) in turn. In this way the extent of absorption of the various fatty acids could be compared by reference to the common acid. Lymph was collected in min samples, centrifuged to remove blood cells and stored at -20 C if not analysed immediately. Constant infusion of isotope 300,uCi 3H-palmitic acid was dissolved in 2 ml. water containing 50 mg potassium soaps, diluted to 300 ml. with warm water and infused into the duodenum at 0 5 ml./ min using a Technicon Autoanalyzer pump connected to the reservoir of isotope solution which was immersed in a heated water bath (3900). On mixing with the acid contents of the duodenum (ph ) the free fatty acids would be released from their potassium soaps and would then mix with the free fatty acids of digesta. Analytical Lymph lipids were extracted by the method of Folch, Lees and Sloane Stanley [1957] and suitable aliquots were chromatographed on thin layer plates of silica gel H (Merck) using the solvent systems light petroleum/diethyl ether/acetic acid (90: 10: 1 and 60: 40: 1) [Bowyer, Leat, Howard and Gresham, 1963]. The lipids were located with iodine vapour and, after allowing the iodine to sublime, the neutral lipids were extracted with chloroform/methanol (1: 1) and the phospholipids by three extractions with chloroform/methanol/water (45: 45: 10). Portions of these extracts were taken for assay of radioactivity [Cunningham and Leat, 1969] and for chemical estimations. Phosphorus was determined by a modification [Bottcher, van Gent and Pries, 1961] of the method of Bartlett [1959] and glyceride/glycerol according to Carlson and Wadstrom [1959]. Free cholesterol and cholesteryl esters were determined by the method of Zlatkis, Zak and Boyle [1953], modified by Klungs6yr, Haukenes and Closs [1958]. The methyl esters of the lymph and plasma lipids separated by thin layer chromatography were prepared as described by Bowyer et al. [1964]. The fatty acid composition was determined by gas-liquid chromatography [see Leat, 1963] using a

3 Origin of Ovine Lymph Lipids 133 Pye Unicam Chromatograph (Series 104) coupled to a digital integrator (Kent Chromatog 2). From these values the specific radioactivities of the fatty acids in each lymph lipid were calculated and expressed as m,uc/mg fatty acid. Separation of the fatty acids into saturated and mono-unsaturated fractions was achieved by the method of Morris [1964]. RESULTS Table I shows that the major lipids of thoracic duct lymph were triglycerides (67%) and phospholipids (22%). Minor components included free cholesterol (2%), cholesteryl esters (6%), free fatty acids (1%), diglycerides (1 %) and monoglycerides ( < 1%). This composition is in reasonable agreement with values previously recorded for ovine thoracic duct and intestinal-duct lymph lipids [Felinski, Garton, Lough and Phillipson, 1964]. The high content of phospholipids in sheep thoracic duct lymph suggests that fat is transported in the form of very low density lipoproteins rather than as chylomicrons [see Levy, Bilheimer and Eisenberg, 1971]. Over 90% of the fatty acids transported in lymph were located in the triglycerides and phospholipids, but the distribution of the individual fatty acids both by weight and by radioactivity was not random between these lipids (Table II). Triglycerides contained the major part of the TALE I. Lipid Composition Composition of thoracic duct lymph lipids and plasma lipids of the sheep Lymph % of total Mean±S.E.M.(n) % of total lymph fatty Plasma (mg/100 ml.) lymph lipid acid (mg/100 ml.) Cholesterol (20) 2*2 n.d. Cholesteryl esters 55-7±2.2 (20) Triglycerides 658.7±37.9 (20) Phospholipids 219.0±11.1 (20) Free fatty acid 8*5±0.8 (10) 0*9 1.0 n.d. Diglycerides 12.9±0*8 (20) 1i3 1.4 n.d. Monoglycerides 2.2±0t2 (20) n.d. Major fatty acid composition (% of total fatty acids) Fatty acid 16:0 16:1 18:0 18:1 18:2 18:3 20:4 other acids* Cholesteryl lymph 14X esters tplasma * Triglycerides flymph trt 12.2 Tplasma X7 0X6 trt lymph Phospholipids Phshd ~plasma Free fatty acid Flymph ~plasma trt 9-6 Total lipids diet *9 * containing minor even, odd and branched chain fatty acids. n.d. = not determined. t actre.

4 134 Leat and Harrison mass (74-79%) and radioactivity (85-90%) of the C16: 0, C18: 0 and C18: 1 acids in lymph whereas phospholipids were more important in the transport of C18:2 and C18:3acids. TABLE II. Percentage distribution by weight and by radioactivity of the major fatty acids in lymph after injecting radioactively labelled fatty acids in the duodenum (see text for details). Fatty acid 16:0 18:0 18:1 18:2 18:3 Triglycerides By By weight radioactivity Phospholipids By By weight radioactivity Two fatty acids were studied simultaneously, 3H palmitic acid (16: 0) with each, in turn, of the other fatty acids labelled with 14C. Values are calculated from the weight of lipids and total radioactivity in lymph collected during the experimental period (4-6 hr.) A 0 E 50 CL0~~~~~~ Time (h) FIG. 1. Specific activities (m,uc/mg) of 3H-palmitic acid in triglycerides (0) diglycerides (A) and monoglycerides (0) in ovine lymph lipids. Values are expressed as a percentage of the maximum specific activity in triglycerides (100 = 949*1 m,uc/mg palmitic acid). Time of single injection of isotope into duodenum = 0. (see text for details). The specific activity of palmitic acid in diglycerides and monoglycerides was similar to that in triglycerides (Fig. 1). The specific activities of the various fatty acids in phospholipids and cholesteryl esters differed from each other, but were always less than those found in triglycerides, indicating a dilution from non-radioactive sources (Figs 2 and 3). In phospholipids the maximum specific activities of C16: 0, C18: 0 and C18: 1 acids were 4, 12 and 6% of the corresponding values in triglycerides (Fig. 2). Values were higher for the essential fatty acids where the maximum specific activities of C18: 2 and C18: 3 acids in phospholipids were 48 and 19% of the values found in triglycerides (Fig. 3).

5 Origin of Ovine Lymph Lipids 135 A B C Fio Time (h) Specific activities (mpc/mg) of palmitic acid (A), stearic acid (B) and octadecenoic acid (C) in ovine lymph lipids. Values are expressed as a percentage of the maximum specific activity in triglycerides (for A, m,uc/mg palmitic acid; for B, 100 _ 63-3 m,&c/mg stearic acid; for C, 100 _ 76*5 ml&c/mg octadecenoic acid). Time of single injection of isotope into duodenum = 0 (see text for experimental details). *-*, triglycerides; A-A, cholesteryl esters; 0-0, phospholipids. A B 0,50- E V CL U)0 T Time (h) FIG. 3. Specific activities (myic/mg) of linoleic acid (A) and linolenic acid (B) in ovine lymph lipids. Values are expressed as a percentage of the maximum specific radioactivity in triglycerides (for A, m,uc/mg linoleic acid; for B, *9 m,c/mg linolenic acid). Time of single injection of isotope into duodenum = 0 (see text for experimental details). 0-0, triglycerides; *-A, cholesteryl esters; 0-0, phospholipids.

6 136 Leat and Harrison The peak in the specific activity of absorbed fatty acids in lymph phospholipids was 05-1 hr later than that found in triglycerides, and in the later stages of absorption the specific activities of the fatty acids in phospholipids exceeded those in triglycerides; this was particularly marked for C18: 2 and 018: 3 acids. These observations suggest that there is a delay in the synthesis or secretion of the phospholipid component of lymph lipids, and might indicate that the lower incorporation of radioactively labelled fatty acids into lymph phospholipids compared to triglycerides is due to the synthesis of phospholipids occurring in a larger substrate pool of slower turnover rate compared to the triglyceride pool. However specific activity data obtained when radioactively labelled palmitic acid was infused continuously into the duodenum for 4 hr showed an apparent plateau in the phospholipid fraction at 3-4 h when the specific activity had reached only 12% of that in the lymph triglycerides (Fig. 4) E 0 Q60/ 4_0 FIG. 4. 0~~~~~~ Time (h) Specific activities (m,uc/mg) of 3H-palmitic acid in triglycerides (@) and phospholipids (0) of ovine lymph. Time for commencement of continuous infusion of isotope (0.5,uc/min) = 0 (see text for details). Bickerstaffe and Annison [1969a] showed that subcellular preparations of sheep intestinal mucosa could desaturate stearic acid in vitro. In the present experiments, in vivo, when U-14C stearic acid was injected into the duodenum, 97.5% of the radioactivity in lymph was located in the saturated fatty acid fraction and 1.9% in the monoenoic acids. The corresponding values when U-14C oleic acid was injected into the duodenum were 1-4% and 92.6%, respectively. These data indicate that the interconversion of saturated and unsaturated fatty acids was negligible in the whole animal preparation. DISCUSSION Incorporation of absorbed fatty acids into lymph lipids The fatty acid compositions of the lipids of thoracic duct lymph (Table I) are in good agreement with values previously recorded [Felinski et al., 1964; Heath,

7 Origin of Ovine Lymph Lipids Adams and Morris, 1964]. Lymph cholesteryl esters and triglycerides were more saturated than the corresponding plasma lipids, whereas lymph phospholipids contained more C18: 2 and C18: 3 acids than plasma phospholipids. The high content of stearic acid in lymph triglycerides is a reflection of hydrogenation of dietary fatty acids in the rumen, and there is no evidence from the present experiments to indicate that this process is reversed to any significant extent during the process of absorption. In the monogastric animal dietary triglycerides are hydrolysed by pancreatic juice to monoglyceride and free fatty acids which form a micelle in the presence of bile salts. After absorption into the intestinal mucosa resynthesis to triglyceride occurs by the monoglyceride and oc-glycerophosphate pathways [see Hiibscher, 1970]. In the ruminant, however, dietary lipids are hydrolysed to free fatty acids in the rumen and, since little monoglyceride is absorbed from the small intestine [Leat and Harrison, 1969], the major pathway of glyceride synthesis must be the a-glycerophosphate pathway [Cunningham and Leat, 1969; Bickerstaffe and Annison, 1969b]. The radioactively labelled free fatty acids (FFA) introduced into the duodenum should mix with the pool of FFA in the intestinal digesta and reflect their fate. If all esterified lipids were synthesized from this pool of free fatty acids, the specific activities of the fatty acids in the various esterified lipids should be similar. This appears to be true for triglycerides, diglycerides and monoglycerides, but not for cholesteryl esters and phospholipids, indicating that part of these lipids must have been derived from unlabelled, esterified sources. The low specific activity of 018: 2 acid in lymph cholesteryl esters is further evidence that the intestine plays a negligible role in the synthesis of plasma cholesteryl linoleate, and that its source is elsewhere [Leat and Baker, 1969]. 137 Origin and formation of lymph phospholipids The most probable cause of the low incorporation of radioactivity into lymph phospholipids is dilution in the substrate pool with non-radioactive phospholipids of endogenous origin. Some endogenous phospholipid could have been derived from capillary filtrate, but this source is unlikelyto exceed 10-15% of the phospholipids in lymph. The remaining endogenous phospholipid probably originates from unlabelled phospholipid absorbed from the gut, e.g. microbial, dietary and biliary phospholipid. The minimal flow of biliary phospholipid into the lumen of the sheep intestine, based on the collection of bile or mixed secretions from other fistulated animals with non-return to the intestine, is 0x160 g/hr (range of 3 experiments of 3-4 hr drainage = g/hr) [F. A. Harrison and W. M. F. Leat, unpublished observations]. This flow of biliary phospholipid could account for a major part of the lymph phospholipids secreted in sheep Jane (range of 3 experiments of 4 hr duration = g/hr; mean = g/hr) [W. M. F. Leat and F. A. Harrison, unpublished observations]. Evidence in the rat suggests that lecithin is hydrolyzed to lysolecithin before absorption [Scow, Stein and Stein, 1967; Nilsson, 1968]; and that after

8 138 Leat and Harrison absorption the lysolecithin is reacylated to lecithin by enzymes in the intestinal mucosa [Subbaiah, Sastry and Ganguly, 1969]. In the sheep the major phospholipid of bile is lecithin which is rapidly hydrolysed by pancreatic juice to 1- and 2-lysolecithins [Leat, 1965; Leat and Harrison, 1969]. After absorption into the mucosa 1-lysolecithin would require unsaturated fatty acids for reesterification whereas the 2-lysolecithin would be esterified preferentially with saturated fatty acids. The greater incorporation into lymph phospholipids of radioactively labelled C18: 2 and C18: 3 acids compared to C16: 0 and C18: 0 acids suggests that esterification of I-lysolecithin is the predominant pathway. The relatively low specific activity of absorbed fatty acids in phospholipids would indicate that de novo synthesis of phospholipids via oc-glycerophosphate is of minor importance, although for triglycerides it is the major synthetic pathway. Phospholipids could also be formed by the transesterification of 1- and 2-lysolecithin which would give one molecule of unlabelled lecithin, but the significance of this pathway is difficult to assess. Possible mode of action of bile and pancreatic juice Malabsorption of fat in the absence of bile is usually attributed to a deficiency of bile salts adversely affecting micellar solubility in the gut lumen. However, if phospholipids are an essential component of the lipoprotein absorbed into lymph, and the lymph phospholipids originate mainly from bile phospholipids, a deficiency of bile could also affect the formation and secretion of lipid particles into lymph. This may be particularly so for the sheep where phospholipids account for more than 20% of lymph lipids. Bile may therefore have a dual action, with bile salts acting in the lumen and the phospholipids acting in the mucosal cell. Further, since the lecithin must first be hydrolysed to lysolecithin before absorption, a deficiency of pancreatic juice could result in biliary lecithin being unabsorbed [Leat and Harrison, 1969] and again adversely affect lipoprotein formation in the mucosa. Because dietary lipids are hydrolysed in the rumen, the role of pancreatic juice in lipid absorption in the ruminant must be other than the hydrolysis of triglycerides. It has been suggested that pancreatic juice functions by converting biliary lecithin to lysolecithin which may be necessary for optimum solubility of fatty acids in the intestinal lumen [Leat and Harrison, 1969; Harrison and Leat, 1972]. However, it is possible that lysolecithin may function through its ability to enter the mucosal cell (in contrast to lecithin) where it would be esterified to form the lecithin necessary for synthesis of the chylomicron-very low density lipoprotein particle of sheep lymph. ACKNOWLEDGMENTS We thank Miss G. Needham, Mrs G. A. Griggs and Mr F. 0. T. Kubasek for valuable technical assistance. REFERENCES BARTLETT, G. R. (1959). Phosphorus assay in column chromatography. Journal of Biological Chemistry, 234,

9 Origin of Ovine Lymph Lipids BICKERSTAFFE, R. and ANNISON, E. F. (1969a). Glycerokinase and desaturase activity in pig, chicken and sheep intestinal epithelium. Comparative Biochemistry and Physiology, 31, BICKERSTAFFE, R. and ANNIsoN, E. F. (1969b). Triglyceride synthesis by small intestinal epithelium of the pig, sheep and chicken. Biochemical Journal, 111, BOTTCHER, C. J. F., van GENT, C. M. and PRIEs, C. (1961). A rapid and sensitive submicro phosphorus determination. Analytica chimica acta, 24, BOWYER, D. E., LEAT, W. M. F., HOWARD, A. N. and GRESHAM, G. A. (1963). The determination of the fatty acid composition of serum lipids separated by thin layer chromatography; and a comparison with column chromatography. Biochimica et biophysica acta, 70, CARLSON, L. A. and WADSTROM, L. B. (1959). Determination of glycerides in blood serum. Clinica chimica acta, 4, CUNNINGHAM, H. M. and LEAT, W. M. F. (1969). Lipid synthesis by the monoglyceride and a-glycerophosphate pathways in sheep intestine. Canadian Journal of Biochemistry 47, FELINSKI, L., GARTON, G. A., LOUGH, A. K. and PHILLIPSON, A. T. (1964). Lipids of sheep lymph. Biochemical Journal, 90, FOLCH, J., LEES, M. and SLOANE STANLEY, G. H. (1957). A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry, 226, GARTON, G. A. (1967). The digestion and absorption of lipids in ruminant animals. World Review of Nutrition and Dietetics, 7, HARRISON, F. A. and LEAT, W. M. F. (1970). Studies of fat absorption in sheep with chronic fistulation of the thoracic duct. Journal of Physiology, 210, P. HARRISON, F. A. and LEAT, W. M. F. (1972). Absorption of palmitic, stearic and oleic acids in the sheep in the presence or absence of bile and/or pancreatic juice. Journal of Physiology, 225, HEATH, T. J., ADAMs, E. P. and MORRIS, B. (1964). The fatty acid composition of intestinallymph lipids in sheep and lambs. Biochemical Journal, 92, HtBSCHER, G. (1970). In Lipid Metabolism. Ed. Wakil, S. J., pp Academic Press, New York and London. KLUNGSOYR, L., HAUKENES, E. and CLOSS, K. (1958). A method for the determination of cholesterol in blood serum. Clinica chimica acta, 3, LEAT, W. M. F. (1963). Fatty acid composition of the serum lipids of pigs given different amounts of linoleic acid. Biochemical Journal, 89, LEAT, W. M. F. (1965). Possible function of bile and pancreatic juice in fat absorption in the ruminant. Biochemical Journal, 94, 21-22P. LEAT, W. M. F. and BAKER, J. (1970). Distribution of fatty acids in the plasma lipids of herbivores grazing pasture: a species comparison. Comparative Biochemistry and Physiology, 36, LEAT, W. M. F. and HARRISON, F. A. (1969). Lipid digestion in the sheep: effect of bile and pancreatic juice on the lipids of intestinal contents. Quarterly Journal of Experimental Physiology, 54, LEAT, W. M. F. and HARRISON, F. A. (1972). Intake and absorption of essential fatty acids by the sheep. Proceedings of the Nutrition Society, 31, 70-71A. LEVY, R. I., BILHEIMER, D. W. and EISENBERG, S. (1971). In Plasma Lipoproteins. Biochemical Society Symposium No. 33. Ed. Smellie, R. M. S., pp Academic Press, London and New York. MORRIS, L. J. (1964). In Metabolism and Physiological Significance of Lipids. Ed. Dawson, R. M. C. and Rhodes, D. N., pp London, John Wiley and Sons. NILSSON, A. (1968). Intestinal absorption of lecithin and lysolecithin by lymph fistula rats. Biochimica et biophysica acta, 152, Scow, R. O., STEIN, Y. and STEIN, 0. (1967). Incorporation of dietary lecithin and lysolecithin into lymph chylomicrons in the rat. Journal of Biological Chemistry, 242, SUBBAIAH, P. V., SASTRY, P. S. and GANGULY, J. (1969). Acylation of lysolecithin to lecithin by a brush-border-free particulate preparation from rat intestinal mucosa. Biochemical Journal, 113, ZLATKIS, A., ZAK, B. and BOYLE, A. J. (1953). A new method for the direct determination of serum cholesterol. Journal of Laboratory and Clinical Medicine, 41,