Medicine, Memorial University of Newfoundland, St. John's, Newfoundland, Canada, AIB 3V6.

Transcription

1 Quarterly Journal ofexperimentalphysiology (1978) 63, ENHANCED INTESTINAL LYMPH FORMATION DURING FAT ABSORPTION: THE IMPORTANCE OF TRIGLYCERIDE HYDRO- LYSIS. By S. G. TURNER* and J. A. BARROWMAN. From the Faculty of Medicine, Memorial University of Newfoundland, St. John's, Newfoundland, Canada, AIB 3V6. (Receivedforpublication 22ndJune 1977) The effect of intraduodenal administration of fats was studied in the rat to define the mechanisms responsible for the substantial increase in intestinal lymph flow and protein transport which follows fat ingestion. Triglyceride in the intestinal lumen, protected from hydrolysis, does not appear to enhance intestinal lymph production. Giving both long- and medium-chain fatty acids, however, causes intestinal lymph flow and protein transport to increase in a manner similar to that found after giving triglyceride which is allowed to undergo hydrolysis. Bile by itself does not seem to be responsible for the phenomenon. When experimental animals are fed fat, flow and protein transport increase in the intestinal lymphatics; this does not happen when protein or carbohydrates are given [Borgstrom and Laurell, 1953; Simmonds, 1954, 1955]. We have previously shown that this effect on intestinal lymph occurs whether absorbed fat leaving the intestinal cell passes into the lacteal or into the portal venous system [Turner and Barrowman, 1977a]. The present experiments were carried out to determine whether changes in lymph flow and protein transport occur in response to one of the products of triglyceride hydrolysis, namely fatty acids, or whether the presence of unhydrolysed triglyceride in the small intestinal lumen is an adequate stimulus. METHODS Animal Preparation Wistar rats weighing between g were anaesthetized with ether, followed by sodium pentobarbitone (Nembutal, Abbott Laboratories Ltd., 30 mg/kg, I.P.). The main small intestinal lymphatic vessel and the duodenum were cannulated as described by Turner and Barrowman [1977a]. Additionally, in one group of rats, the pancreatic ducts were ligated so that pancreatic juice but not bile was prevented from entering the duodenum (Fig. 1). A PP 10 cannula (Portex Ltd., U.K.) was used for re-routing bile flow. The preparation has the advantage that continuous saline and bile flow into the duodenum can be checked at a glance. Experimental Procedure Post-operatively, the animals were kept in restraining cages [Bollman, 1948], and left in a quiet warm room. Experiments were performed on the conscious animals on the following day, approximately 18 h after surgery. A continuous infusion of physiologically normal saline (0 9 g% NaCl) into the duodenum at 2 5 ml. h1 was maintained from the end of surgery onwards using a 'Blue Line Infant Feeding Tube' (Portex Ltd., U.K.). Three 40 min control collections of intestinal lymph were made, and the test oil was then washed into the *Present address-faculty of Science, The Open University, Milton Keynes, MK7 6AA, U.K. 255

2 256 Turner and Barrowman duodenum over a period of 1 min with a volume of saline sufficient to make the total volume given equal to 1 ml. A further eight 40 min collections of lymph were made after which the animal was killed. Test Meals Three different test meals were given: a long-chain triglyceride, L.C.T. (olive oil, which is chiefly triolein and has the following fatty acid composition-85% oleic, 10% palmitic and small amounts of linoleic and stearic acids); a long-chain fatty acid, L.C.F.A. (oleic acid, approximately 99 % purity, Sigma); and a medium-chain fatty acid, M.C.F.A. (octanoic acid, % purity, Sigma). COM MON \fi PANCREATIC FiG. 1. Diagram illustrating pancreatic exclusion S: stomach; D: duodenum; L: liver I and E: Internal and External to the animal. Saline (2.5 ml per h) is infused intraduodenally through the feeding tube which passes into the animal in the subcostal area. Five groups of experiments were carried out on rats with an intestinal lymphatic cannula and a duodenal cannula. (a) L.C.T. (0 5 ml) + saline (0 5 ml) -5 animals (b) L.C.T. (0 5 ml) + saline (0 5 ml) + pancreatic duct ligation 5 animals (c) L.C.F.A. (0A42 ml, 1V42 mmol) + saline (0 58 ml)+ pancreatic duct ligation -3 animals (d) L.C.F.A. (0A42 ml, 1 24 mmol) + saline (0-58 ml) 5 animals (e) M.C.F.A. (0-20 ml, 1-24 mmol) + saline (0-80 ml) 5 animals On the basis that lipolysis in the small intestine results in the release of roughly two moles of fatty acid per mole of triglyceride, the amounts of fatty acid given to groups (c), (d) and (e) were calculated to be approximately equivalent to two-thirds digestion of the amount of triglyceride given to groups (a) and (b).

3 Enhanced intestinal lymph formation during fat absorption 257 Analysis oflymph Lymph flow. The lymph weight per h from the fistula was determined by weighing lymph collected over 40 min periods. For protein and triglyceride concentrations lymph weights have been converted to volumes assuming a specific gravity of I 0 for lymph. Protein assay. To avoid turbidity interfering with the chemical assay of protein, lymph lipid was removed by diluting samples with saline and centrifuging at 120,000 g for 1 h. Duplicate samples from the clear infranatant were assayed for total protein using the Biuret reaction. Triglyceride assay. Triglyceride was measured in lymph from animals in groups (a) and (b). Aliquots of freshly collected lymph were stored at - 20 C until assayed. Initial assays were based on the enzymic method of Eggstein [1966], but in later experiments a partition method advocated by DADE was used, (DADE Division, American Hospital Supply Corporation, Miami, Florida. TRI-25 Triglyceride Method) [Soloni, 1971; Giegel, Soloni, Trinidad, Cohen and Clema 1972]. This method depends upon initial triglyceride extraction with nonane and isopropanol. Triglycerides in the non-polar phase are then converted to glycerol and through a number of intermediate steps to 3, 5-diacetyl-1,4 dihydrolutidine which has a maximum absorbance at 415 nm. A linear calibration curve allowed direct calculation of triglyceride concentrations. RESULTS (a) L.CT. Group Initial flow of intestinal lymph was 1.8 ± 0-2 g. h'- (mean ± s.e. of the mean). After giving the triglyceride, lymph flow increased and was maximal min later when it reached 4 5 ± 0 4 g. h- 1. Initial lymph protein concentration was mg.ml-'. At the peak flow rate this increased slightly to mg.ml1' and continued to increase until the second hour after the meal, reaching 18&7 ± 2-3 mg.ml-1, whereupon a decline in concentration commenced. Total protein in lymph (lymph flow x lymph protein concentration) remained steady prior to fat administration with a value of 21*7 ± 19 mg. h - 1. Giving the triglyceride increased the total protein carried by the lymph and this reached a peak of 79 1 ± 10-6 mg. h1 in the second 40 min collecting period after the meal, coincidental with the peak rate of flow. Triglyceride, measured in 3 of the 5 animals, was found to be present in pre-meal intestinal lymph at a concentration between mg. 100 ml-'. The rats had been without food since the start of surgery the previous day. No change in lymph triglyceride concentration occurred in the first 40 min after feeding L.C.T., but it then rose and reached a plateau of about 2000 mg. 100 ml 1, min after the meal, which was maintained until the experiment was concluded, 320 min from the time of feeding. Initial triglyceride transport in lymph was between 6A4-7 1 mg. h- 1. A rise was detectable in the first 40 min after L.C.T. and this reached a peak of 72-2 ( ) mg.h-1 in the third 40 min, after the peak in flow and protein transport (Figs. 2 and 3). During the 320 min after L.C.T., 5-4 ± 1.31 g of lymph and 126&5 ± 13-7 mg of protein were lost over and above control amounts. (b) L. C. T.-Group with Pancreatic Duct Ligation No significant change in lymph flow rate from the control value of 1*7 ± 0.2 g. h -I occurred after giving L.C.T. This is in marked contrast to the substantial

4 258 Turner and Barrowman rise seen in rats with unligated pancreatic ducts given a similar meal (Fig. 2a). Protein concentrations in the lymph remained steady and hence total protein carried per unit time did not alter. Lymph protein concentration before giving the L.C.T. was 7*3 ± 12mg.ml-1 and protein transport was X6 mg.h-'. Neither triglyceride concentrations nor total triglyceride carried in lymph increased to any extent (Fig. 3) showing that exclusion of pancreatic :E 5-4- (a) :W- z z -80 m 0 WY _ I C J = E (b) I _J o 40 - ;- z.-i Ln z FIG. 2a, 2b TIME ( min Intestinal lymph flow (a) and intestinal lymph transport of protein (b) in response to 05 ml olive oil given to rats with (open circles) and without (closed circles) pancreatic juice exclusion. Vertical lines represent the s.e. of mean, and the arrow indicates when the intraduodenal dose was given. juice largely prevented triglyceride absorption and its subsequent appearance in lymph. Triglyceride transport rate in intestinal lymph before L.C.T. was 3-4 ± 0O8 mg.h-'. At the time when the transport rate was maximal (72-2 mg triglyceride.h-') in rats with uninterrupted pancreatic ducts given L.C.T., the rate of triglyceride transport in this group was only 4-6 ± 1-2 mg.h- 1. It was noted that lymph protein concentration in these animals was less than half that in animals with intact pancreatic ducts.

5 Enhanced intestinal lymph formation during fat absorption 259 (c) L.C.F.A.-Group with Pancreatic Duct Ligation This group of animals was studied to ascertain whether the process of fatty acid absorption was normal in the absence of pancreatic secretions. Only three experiments were carried out in this group and these showed that intestinal lymph flow and protein transport increased after oleic acid in a manner similar _ 60 LLI 0 I _~40 4 0~ 20 FIG. 3. A #. w TIME (min) Total triglyceride in intestinal lymph in 8 animals before and after a test meal of 0.5 ml olive oil, followed by 0 5 ml of 09 % NaCl. Circles represent the means from 5 animals which had had their pancreatic ducts tied the previous day. Triangles represent the means from 3 animals with intact pancreatic ducts. Vertical lines in the former represent the s.e. of mean and in the latter the highest and lowest values from the three experiments. The arrow indicates the time at which the meal was given. to that in animals with uninterrupted pancreatic ducts (d). Peak lymph flow after oleic acid in the latter group was g.h-'; in animals with duct ligation it was 4'7 ( ) g. h-. Peak protein transport after oleic acid in rats with intact pancreatic ducts was 505 ± 6-8 mg.h-' and with pancreatic duct ligation it was 57-5 ( ) mg. h'-. Mean increases in peak flow rate and protein transport after the fatty acid expressed as a percentage of control values were 80% and 83 % respectively for animals without (d), and 106% and 990 for those with (c) pancreatic exclusion. D

6 260 Turner and Barrowman (d) L.C.F.A.-Group Control lymph flow was 1P8 ± 0-1 g.h-. On giving oleic acid the flow rate rose slowly, reaching a peak of 3-2 ± 0-6 g. h -' three hours after the test dose. Flow then began to fall but had not reached control levels by the end of the experiment, just over five hours after the fatty acid had been given. Lymph protein concentration before the fatty acid was 15-4 ± 0-7 mg.ml-'. This rose slightly in the first 40 min after the fat reaching a maximum of mg. ml' after min and then began to decrease. Before the end of the experiment the concentration had returned to control levels, and even decreased below them. Total protein carried in the lymph was initially mg. h-. This increased on giving L.C.F.A. to reach a peak of 50-5 ± 16-8 mg.h 1, min after the meal, then started to decrease but had not returned to control values by the end of the experiment, despite the decrease in protein concentration (Fig. 4). During the time of the experiment, g of lymph and 60-0 ± 17-8 mg of protein were lost through the fistula over and above the basal rates as a result of giving oleic acid. (e) M.C.F.A.-Group Basal lymph flow rate was 1-5 ± 0-1 g. h '. Intraduodenal M.C.F.A. caused the rate of flow to increase quickly, achieving a maximal rate of 2-8 ± 0-2 g. h 1 in the first 40 min, returning to control levels 120 min after the fatty acid was given. In the 320 min following the meal the rise in lymph flow caused 2-9 ± 0-8 g to be lost through the cannula over and above basal levels. Lymph protein concentration was mg.ml-' in the control period. After giving octanoic acid the concentration of protein in intestinal lymph fell, reaching 16-0 ± 2-9 mg.ml-' after min. In spite of this, total protein carried in the lymph rose from 30-1 ± 2-2mg.h ' to 46-5 ± 2.5 mg.h'- in the first 40 min. Two hours after the fatty acid was given, total protein carried in lymph had returned to control levels and did not fall below them. An extra 16-1 ± 10-6 mg of protein were lost through the cannula as a result of giving M.C.F.A. DIsCUSSION Throughout the experiments the animals seemed in good health. The volumes and amounts of fluid and fat given were considered to be not unphysiological being roughly equivalent to a human test meal of 250 ml containing about 100 g oflipid. The volume delivered to the duodenum was always 1 ml and was given over a period of approximately 1 min so that the purely mechanical effects of administering the test meal would be reasonably uniform. Figure 2 shows that an increase in intestinal lymph flow and protein output occurs after L.C.T., confirming previous work [Borgstrom and Laurell, 1953; Simmonds, 1954, 1955; Wollin and Jaques, 1973]. In 1954, Simmonds found that the extra amount of thoracic duct lymph produced in the whole experimental period after 0-5 ml of olive oil intragastrically was 4-6 ml. In the present

7 Enhanced intestinal lymph formation during fat absorption 261 experiments, the extra amount of intestinal lymph after the same quantity of olive oil intraduodenally was 5-4 ± 1P3 g. As suggested by Simmonds, the change he found in thoracic duct lymph flow must have been almost entirely due to an increase in intestinal lymph flow. 41 (a) 3-c I (b) EZ z 1-J o' I- ' FIG. 4a, 4b TIME (min.) Intestinal lymph flow (a) and intestinal lymph transport of protein (b) in response to 1-24 mmol oleic acid (closed circles) and 1 24 mmol octanoic acid (open circles). Vertical lines represent the s.e. of the mean, and the arrow indicates the time at which the test meal was given. No such change in lymph flow and protein transport were seen in animals in which pancreatic juice was excluded from the intestinal lumen. This is not because of any impairment in the ability of the animal to respond to a fat meal by increased formation of lymph, since similar changes in lymph flow and protein transport were seen to occur in rats with and without ligated pancreatic ducts in response to intraduodenal oleic acid.

8 262 Turner and Barrowman To determine whether pancreatitis had developed in animals with pancreatic exclusion, sections of pancreas from one animal 26 h after ligating the duct were examined. Low power microscopic examination revealed no obvious abnormalities of the tissue, but under higher power, slight, sparse inflammation and oedema were seen, and there were signs of incipient fat necroses. However, the bulk of the parenchyma seemed normal which agrees with observations made under similar conditions by others [for instance: Short, 1961, and Edstrom and Falkmer, 1968]. Thus during the experimental period our animals were not in a severely abnormal physiological state. It is possible that lack of access to food until the fat test load was given kept pancreatic secretion at basal levels and, therefore, back pressure might develop only slowly. However, damaged tissue may cause the release of vasoactive agents which might interfere with the effect induced by fat. Since basal lymph flow in duct-ligated rats did not differ from that of control rats such substances were apparently not present in amounts sufficient to affect the splanchnic circulation. Since there was no apparent increase in either triglyceride concentration or total triglyceride in the intestinal lymph of the duct-ligated rats after giving fat (Figure 3), and the lymph remained clear and fat-free, pancreatic juice was judged to have been effectively excluded. It appears from our results that triglyceride in the duodenum, protected from pancreatic juice, does not provoke change in lymph flow or transport of protein. Fatty acids exert an effect on lymph flow and protein transport in that lymph similar to that produced by triglycerides (Figure 4) but different in certain aspects. The magnitude of the effect due to fatty acids appears to be roughly similar to that caused by an approximately equivalent dose of their respective triglycerides. However, a distinct difference is found in the pattern of lymph response. Following L.C.F.A. the rise in lymph flow and protein transport is both slower and longer-lasting than after L.C.T. This has also been observed by Simmonds [1954, 1955]. After M.C.F.A., the increase in lymph flow and protein content of the lymph is rapid, reaching a peak earlier, and having a shorter duration of effect than after the corresponding triglyceride [Turner and Barrowman, 1977a). These differences may be related to dissimilarities in the handling of the fatty acid and the triglyceride. Because of the complex nature of lipolysis of triglyceride in the intestinal lumen, it is difficult to make further close comparison of the effects of triglyceride versus fatty acids on intestinal lymph flow and composition. It is possible that the ability of fatty acids to inhibit ileal water absorption [Ammon and Phillips, 1974] might have an influence on lymph flow in our study. Although the mechanism underlying the variation in response seen after triglyceride and fatty acid is not clear, the overall effect is similar, demonstrating that the presence of fatty acids in the intestine is capable of providing a stimulus for increased lymph flow and protein transport. It is possible that the response following intraluminal fat is brought about by fatty acids releasing humoral agents such as cholecystokinin which is known to have vasodilator

9 Enhanced intestinal lymph formation during fat absorption action on the splanchnic circulation [Fara, Rubinstein and Sonnenschein, 1969; Fara and Madden, 1975]. Increased capillary flow in the intestine could then result in enhanced lymph flow and protein transport from plasma to lymph [Turner and Barrowman 1977b]. The presence of fat in the intestine causes release of bile by neurohormonal mechanisms [Meyer and Grossman, 1972] and the question arises as to whether an increased flow of bile rather than intraluminal fat is responsible for the changes in the intestinal lymph after a meal of fat. However, in the group of rats with pancreatic exclusion increase in bile flow was not prevented, but no increase in lymph flow or lymph protein transport followed introduction of L.C.T. The explanation for the lower protein concentration in lymph of rats with pancreatic exclusion is not immediately obvious but deserves further study. ACKNOWLEDGMENTS We thank Jon Hyde for help in preparation of pancreas sections for microscopy and Dr. K. B. Roberts for constructive criticism of the manuscript. REFERENCES 263 AMMON, H. V. and PHILLIPS, S. F. (1974). Inhibition of ileal water absorption by intraluminal fatty acids. Journal ofclinical Investigation, 53, BOLLMAN, J. L. (1948). A cage which limits the activity of rats. Journal of Laboratory and Clinical Medicine, 33, BORGSTROM, B. and LAURELL, C-B. (1953). Studies on lymph and lymph-proteins during absorption of fat and saline by rats. Acta Physiologica Scandinavica, 29, EDSTROM, C. and FALKMER, S. (1968). Pancreatic morphology and blood glucose levels in rats at various intervals after duct ligation. Virchows Archiv Abteilung A. Pathologische Anatomie, 345, EGGSTEIN, M. (1966). Eine neue Bestimmung der Neutralfette in Blutserum und Gewebe. II. Zuverlassigkeit der Methode, andere Neutralfettbestimmungen, Normalwerte fur Triglyceride und Glycerin in menschlichen Blut. Klinische Wochenschrift, 44, FARA, J. W. and MADDEN, K. S. (1975). Effect of secretin and cholecystokinin on small intestinal blood flow distribution. American Journal ofphysiology, 229, FARA, J. W., RUBINSTEIN, E. H. and SONNENSCHEIN, R. R. (1969). Visceral and behavioural responses to intraduodenal fat. Science (N. Y.) 166, GIEGEL, J., SOLONI, F., TRINIDAD, E., COHEN, B. and CLEMA, W. (1972). Manual and semiautomated procedures for the measurement of triglycerides. Clinical Chemistry 18, 693 (Abstract). MEYER, J. H. and GROSSMAN, M. I. (1972). Release of secretin and cholecystokinin. In Gastrointestinal Hormones, L. Demling, Editor. Thieme Verlag, Stuttgart, SHORT, D. W. (1961). The effect of drugs upon experimental pancreatitis in the rat, with special reference to blood amylase level. British Journal ofsurgery, 48, SIMMONDS, W. J. (1954). The effect of fluid, electrolyte and food intake on thoracic duct lymph flow in unanaesthetized rats. Australian Journal of Experimental Biology and Medical Science, 32, SIMMONDS. W. J. (1955). Some observations on the increase in thoracic duct lymph flow during intestinal absorption of fat in unanaesthetized rats. Australian Journal of Experimental Biology and Medical Science, 33,

10 264 Turner and Barrowman SOLONI, F. G. (1971). Simplified manual micromethod for determination of serum triglycerides. Clinical Chemistry 17, TURNER, S. G. and BARROWMAN, J. A. (1977a). Intestinal lymph flow and lymphatic transport of protein during fat absorption. Quarterly Journal of Experimental Physiology, 62, TURNER, S. G. and BARROWMAN, J. A. (1977b). The effect of cholecystokinin and cholecystokinin-octapeptide on intestinal lymph flow in the rat. Canadian Journal ofphysiology andpharmacology, 55, WOLLIN, A. and JAQuEs, L. B. (1973). Plasma protein escape from the intestinal circulation to the lymphatics during fat absorption. Proceedings of the Society for Experimental Biology and Medicine, 142,