DURING the absorption of a fatty meal the lipid content of intestinal and. from the intestine to the plasma in the chylomicra as triglycerides [Yoffey

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1 FATTY ACID TRANSPORT IN TORACIC DUCT, EPATIC AND INTESTINAL LYMP DURING FASTING AND AFTER FEEDING GLUCOSE. By R. V. COXON and D. S. ROBINSON.* From the University Laboratory of Physiology and the Sir William Dunn School of Pathology, Oford. (Received for publication 12th March 1962) Analyses of thoracic duct lymph in the rat during fasting and after glucose feeding have shown the presence of esterified fatty acids additional to those which are contributed to the lymph by plasma lipoprotein lipid. The ecess may amount to an output in thoracic duct lymph of 5 mg. esterified fatty acid a day. In the dog, analyses of hepatic and intestinal lymph during fasting have shown that ecess esterified fatty acid is present in intestinal, but not hepatic, lymph. Calculations based on these findings do not support the view that the lymnplhaties are a major route for the mobiliation of fatty acid during fasting. DURING the absorption of a fatty meal the lipid content of intestinal and thoracic duct lymph rises markedly as the dietary fatty acids are transported from the intestine to the plasma in the chylomicra as triglycerides [Yoffey and urtice, 1956]. The possibility of transport of triglyceride by the lymphatics under conditions other than during fat absorption has not hitherto received much attention though Rony, Mortimer and Ivy [1932] reported the presence of lipid particles in dog thoracic duct lymph during fasting and suggested that triglyceride mobilied from the fat depots might be conveyed in the lymph. In recent years the importance of the blood unesterified fatty acids in the transport of mobilied lipid has been recognied [Fredrickson and Gordon, 1958], and so it seemed desirable to assess the possible quantitative significance of the alternative route suggested by Rony et al. [1932]. In the present study esterified fatty acids have been estimated in thoracic duct lymph in the rat during fasting and after feeding glucose, and also in the hepatic and intestinal lymph of the dog during fasting. EXPERIMENTAL Wistar strain albino rats weighing between 16 and 2 g. were used. They had been fed the laboratory stock diet, containing 4 per cent of fat, until the beginning of the eperiment. Thoracic duct fistule were established by the technique of Bollman, Cain and Grindlay [1948] and at the time of operation gastrostomy tubes were inserted so that controlled amounts of fluid could be given. After the operation the rats were put into restraining cages and 6 hr. later collection of lymph was started. Samples of lymph were collected in 3-8 per cent (w/v) trisodium citrate solution using approimately 1 volume of citrate for 9 volumes of lymph. The samples were collected at room temperature and stored at C. until they were analyed. During the 6 hr. before the first lymph samples were collected a rate of lymph * Eternal Staff of the Medical Research uncil. 252

2 Fatty Acids in Lymph flow in ecess of 1 ml./hr. was established by giving 45 per cent (w/v) sodium chloride solution through the gastrostomy tube at a rate of 5 ml./hr. Subsequently the rats were given either '45 per cent (w/v) sodium chloride solution (fasting animals) or 1 per cent (w/v) glucose in -45 per cent sodium chloride solution (glucose-fed animals) to drink. If the rate of lymph flow fell below 1 ml./hr. at anv time during the eperiment, additional sodium chloride solution was given by gastrostomy tube so that a lymph flow of between 1 ml. and 2 ml./hr. was maintained throughout. The total amount of glucose taken during the periods of feeding varied between 7 g. and 9 g./24 hr., being considerably in ecess of that required for the caloric need, which for a rat weighing 2 g. at rest is about 2 kcal./day [Kleiber, 1947; Prosser, 1952]. For the eperiments on dogs, animals of either se and weighing between 8 and 16 kg. were used. They were fasted for 48 hr. before eperiment ecept for one instaince when the fast lasted only 24 hr.-but were allowed free access to water. Anaesthesia was induced by means of intraperitoneal pentabarbitone sodium (Nembutal, Abbott Laboratories) in a dosage of -6 mg./kg. The abdomen was opened by a midline incision from the iphisternum to below the umbilicus and this was later joined to a right subcostal incision. By the use of diathermy the latter incision could be rapidly etended through all layers of the abdominal wall with very little loss of blood and, by turning caudally a flap in the shape of an inverted "V", a good eposure of the posterior abdominal wall and root of the mesentery could be obtained, after retracting the duodenum to the left and packing off the intestinal loops. The general topography of the mesenteric lymphatics has been fully described by the workers at the Mayo Clinic [Cain, Grindlay, Bollman, Flock and Mann, 1947; Grindlay, Cain, Bollman and Mann, 195]. In carrying out cannulation, for which polythene tubing of 1-5 mm. eternal diameter was used, it was found that much time and difficulty were saved by a procedure which permitted the tubing to be correctly aligned with the lymphatic channel before incising the latter. This alignment was achieved by suturing to the peritoneum of the posterior abdominal wall a sleeve of tubing somewhat larger than the cannula. The sleeve was positioned along the line of the lymphatic to be entered and it terminated just short of the proposed site of cannulation. The narrower tubing of the cannula could then be slid through the sleeve and after the lymph-channel had been opened could be slipped smoothly into the lumen without its ehibiting any of the troublesome whip to which it was otherwise prone. A demonstration illustrating this technique has been given to the Physiological Society [sgrove, on and Spenser, 196]. After collecting specimens of lymph, the area drained by the cannula was determined by infiltrating the tissues with Evans blue dye which initially stained only the locally produced lymph. It was generally found that the hepatic and intestinal lymph channels lay cephalad and caudad respectively to the superior mesenteric artery; their dispositions, with cannulae inserted through the sleeves, are shown in fig. 2. The free ends of the cannulse were drawn through stab wounds in the right flank and drained into tubes containing a trace of heparin. The rate of lymph flow usually observed was about 1 ml./min. from both intestinal and hepatic vessels which is somewhat less than that recorded by Cain et al. [1947]. owever, in our eperiments no attempt was made to occlude other vessels draining the same territories. Analytical Determinations The method of Stern and Shapiro [1953] was used to measure total esterified fatty acids. Values are epressed as mg. of esterified fatty acid, 28 being used as the fatty acid equivalent weight. Protein nitrogen was determined by a micro- Kjeldahl procedure [Campbell and anna, 1937]. A suitable aliquot of the lymph or plasma sample was blown from a micro-pipette into 4 ml. of a 1 per cent (w/v) 2553

3 254 on and Robinson trichloroacetic acid solution. The resulting protein suspension was centrifuged and the precipitate was washed twice with 4 ml. of 5 per cent (w/v) trichloroacetic acid before being digested with acid. The protein nitrogen values have been multiplied by 6-25 and the results epressed as mg. of protein. All determinations were made in duplicate. RESULTS The rate of flow and the composition of thoracic duct lymph is influenced by several factors. When large volumes of water are given by mouth, lymph flow increases rapidly and the protein concentration in the lymph falls, though the total protein output may remain constant. If salt solutions, or glucose dissolved in salt solutions, are given, the lymph flow increases more slowly and, though the protein concentration of the lymph still falls, there is a rise in total protein output which is believed to be due to increased transfer of protein into the lymph from the plasma [Borgstrom and Laurell, 1953; Simmonds, 1954]. Any such increase in the transfer of plasma protein will also raise the amount of plasma lipoprotein and hence of esterified fatty acids in the lymph. Thus, in order to assess the possibility of an increase in the transport of triglyceride in the lymph under any specified conditions it is necessary to relate the esterified fatty acid concentration to the protein concentration. Only a sustained rise in the esterified fatty acid: protein ratio may be considered to indicate an increase in lymphatic transport of triglyceride. In the present eperiments such a rise did not occur in the rat with fasting for up to 3 hr. (fig. 1B). Nor was there any fall in the ratio when glucose was fed after an initial period of fasting (fig. Ic and D). Data on four rats are presented and similar findings have been obtained with four other animals. Thus, fasting does not appear to be associated with an increased transport of triglyceride in thoracic duct lymph in the rat. The ratio mg. esterified fatty acid: mg. protein in the thoracic duct lymph is, however, much higher in both fasting and glucose-fed animals (fig. IA and B) than the same ratio in the plasma. Thus, in the present study, it varied in the.lymph between.15 and.3 and in the plasma between.2 and.4 (there was no change in the ratio in the plasma during the period of lymph collection). Data cited by Yoffey and urtice [1956] also show that in the cat, dog, rabbit and rat in the post-absorptive state the ratio mg. esterified fatty acid: mg. protein in thoracic duct lymph is, in all cases, higher than that in the plasma of these animals. These findings suggest that, after either fasting or feeding glucose, there is more esterified fatty acid present in thoracic duct lymph than can be accounted for by the lipid of the lymph lipoproteins derived from the plasma. To investigate whether this ecess of esterified fatty acid in the thoracic duct lymph was also demonstrable in the hepatic or intestinal lymph, further eperiments were carried out on dogs which had been fasted for 48 hr. Table I shows the results. In three dogs, in which samples were obtained from both the hepatic and intestinal lymphatics, the fatty acid: protein ratio was higher in the intestinal lymph than in the

4 Fatty Acids in Lymph plasma, but, in the hepatic lymph, the ratio was the same as in the plasma. In the remaining three dogs, in each of which only one region was sampled, the same type of relationship held. The difference between the ratios in intestinal lymph and plasma was less than that between thoracic duct lymph and plasma in the rat. owever, dogs have a much higher plasma lipid concentration than rats and hence a greater proportion of the total esterified fatty acids of the dog's lymph is derived from plasma lipoprotein lipid. 255 a.4- ct.-.4 Vn E - D UJ Zj D U B 14 2 CL > E - Li I D U /ax I/ (TIME hours) FIG. 1.-The cumulative output of protein ( ) and esterified fatty acid (@) in rat thoracic duct lymph in animals fed glucose (A), in fasting animals (B) and in animals fed glucose after a period of fasting (C and D). DIscuSSION The foregoing findings suggest that, in the rat, ecess esterified fatty acids are present in thoracic duct lymph during fasting and after feeding glucose and that, in the dog, the ecess during fasting is characteristic of the intestinal, but not the hepatic, lymph. The source of this ecess fat in the thoracic duct and intestinal lymph is not clear. It may represent fatty acids which have been held up in the intestinal cells from the time of ingestion

5 256 on and Robinson - F L =E F > Cn < ca - - CD S '.: " ONDO N Pq It:OC: ece * I~ S 3) E-4. C q i 1 5.E t Ce CS t C - a m It IC = * oci X:

6 A i ma 1 ::'1 &-fr ]FIG. 2-. I)riwing to illustrate the teehniqute us(1d to iiis-eit caindie inito the lelat'lti( aind intestinoal lynilohatie duets in the (lug. A =lhe)patite(-ldut eannula. B =intestinil (dlult oannula. [To face page 236

7 Fatty Acids in Lymph 257 of the last meal containing fat-though, if this were so, a gradual fall with time in the esterified fatty acid content of the thoracic duct lymph might have been epected in the eperiments in rats in which lymph samples were collected for up to 3 hr. (fig. ib). Alternatively, in the glucose-fed animal. fatty acids synthesied in the tissues may be released into the intestinal lymph or, in the fasting animal, esterified fatty acids may be mobilied into the lymph from the fat depots. In either eventuality the amount of fat involved is small. In the rat, the esterified fatty acid output, in ecess of that present in the lymph lipoproteins derived from the plasma, would be of the order of 5 mg. a day. This is only a small proportion of the total fatty acids which would need to be mobilied during fasting to provide for all the caloric needs-namely, at least 2 g. of fat daily for a rat weighing 2 g. Assuming that the lymphatics in the mesentery and in the subcutaneous regions of the abdomen and hind limb, which eventually enter the thoracic duct, drain 5 per cent of the total fat depots of the body, such a mobiliation of fat occurring by way of the lymphatics would lead to an increase in the fatty acid content of the thoracic duct lymph from between 2 and 4 mg./hr. to between 4 and 5 mg./hr. Such a rise was not observed in our study. The conclusion which was drawn by Rony et al. [1932] from their eperiments in the dog that about 2 per cent of the fatty acids mobilied during fasting were transported in the lymph was based on the total fatty acid content of the lymph. It did not allow for the esterified fatty acids contributed by those lymph lipoproteins derived directly from the plasma. REFERENCES BOLLMAN, J. L., CAIN, J. C. and GRINDLAY, J.. (1948). J. Lab. clin. Med. 33, BORGSTR6M, B. and LAUTRELL, C-B. (1953). Acta physiol. scand. 29, 264. CAIN, J. C., GRINDLAY, J.., BOLLMAN, J. L., FLOCK, E. V. and MANN, F. C. (1947). Surg. Gyn. Obst. 85, 559. CAMPBELL, W. R. and ANNA, M. I. (1937). J. biol. Chem. 189, 35. sgrove, P. J., on, R. V. and SPENSER, A. A. (196). J. Physiol. 153, 12P. FREDRICKSON, D. and GORDON, R. S. (1958). Physiol. Rev. 38, 585. GRINDLAY, J.., CAIN, J. C., BOLLMAN, J. L. and MANN, F. C. (195). Surgery, 27, 152. KLEIBER, M. (1947). Physiol. Rev. 27, 511. PROSSER, C. L. (1952). mparative Animal Physiology. Philadelphia and London: W. B. Saunders. RONY,. R., MORTIMER, B. and Ivy, A. C. (1932). J. biot. Chem. 96, 737. SIMMONDS, W. J. (1954). Aust. J. ep. Biol. med. Sci. 32, 285. STERN, I. and SAPIRO, B. (1953). J. clin. Path. 6, 158. YOFFEY, J. M. and COURTICE, F. C. (1956). Lymphatics, Lymph and Lymphoid Tissue. London: Edward Arnold Ltd.