Volpenhein [1964] found fat equivalent to approximately 150 mg. oleic acid

Quart. J. exp. Physiol. (1967) 52, 305-312 THE SOURCE OF ENDOGENOUS LIPID IN THE THORACIC DUCT LYMPH OF FASTING RATS. By B. K. SHRIVASTAVA,* T. G. REDGRAVE t and W. J. SIMMONDS. From the Department of Physiology, The University of Western Australia, Nedlands. (Received for publication 12th December 1966) The output of esterified fatty acid in the thoracic duct lymph of fasting, unanaesthetized rats decreased by 80 per cent when a bile fistula was produced. Steady infusion of sodium taurocholate, 5 or 10 mg./hr. in saline, did not reverse this fall. This suggested that it was the loss of biliary lipids and not primarily that of bile salts which was responsible for the decreased lymph lipid. The output of esterified fatty acid in bile of rats with chronic bile fistulae was small. However, experiments in which bile was returned by re-entrant cannula and sampled only for short periods showed that operation depressed biliary lipid output for about 24 hr. and drainage of bile clearly decreased biliary lipids within 2-4 hr. In each of a group of rats with re-entrant biliary cannulae the output of biliary lipids in the first 2 hr. of drainage was more than enough to account for the decrease in output of esterified fatty acid in fasting lymph in each rat after continuous biliary drainage was established. Gas liquid chromatography showed that before biliary drainage was established, fasting lymph lipids were rich in fatty acids characteristic of bile but after biliary drainage the fatty acid pattern closely resembled that of plasma. It is suggested that fatty acid liberated from phospholipids of bile in the intestinal lumen contribute a major portion of the esterified fatty acid of fasting lymph and that lipoproteins passing from plasma into lymph provide much of the remainder. The prompt effect of biliary drainage on phospholipid output in bile is briefly discussed. IN fasting rats or on a fat free diet the lipid present in thoracic duct lymph is clearly endogenous - that is, not derived directly from the diet. Mattson and Volpenhein [1964] found fat equivalent to approximately 150 mg. oleic acid per day in thoracic duct lymph of rats fed a fat free diet. The non-dietary contribution may be increased by fat feeding. For example, Karmen et al. [1963] and Whyte et al. [1963], fed labelled fatty acids and found that unlabelled, endogenous lipid subsequently constituted up to 40 per cent of the esterified fat in chylomicrons. The sources of endogenous lipid are not clearly known. A contribution by lipoproteins which leak through the capillaries into tissue fluids, particularly in the liver, was demonstrated by Morris [1956]. A metabolic origin from newly synthesized fatty acids in the mucosa was supported by Karmen et al. [1963]. Various methods have also been demonstrated by which non-dietary fat can reach the intestinal lumen and then be reabsorbed. Weld [1961] gave evidence of epithelial secretion into the lumen of lipomicron particles containing about 50 per cent fat. Burr et al. [1960] demonstratede xudation of lipoprotein and even chylomicrons from plasma into the lumen. Pessoa et al. [1953] suggested that desquamated epithelial cells were a major source of endogenous lipid. Balint et al. [1963] observed that bile 'may be a source of endogenous * Colombo Plan Fellow. t Australian and New Zealand Life Insurance Medical Research Fellow. 305

306 Shrivastava, Redgrave and Simmonds fatty acids' and Baxter [1966] reported that the endogenous lipid was 'derived in part from bile - possibly to the extent of roughly 50 per cent'. The aim of the present investigation was to evaluate the contribution to endogenous lymph lipid of fatty acid reabsorbed from the bile in the intestinal lumen. The output and composition of endogenous fatty acid in bile and lymph were compared in animals either with bile fistulae or with unimpaired entry of bile into the intestine. METHODS Male albino rats of Wistar strain, interbred locally for 10 years, and weighing 200-220 gm. were used throughout the experiment. All operative procedures were carried out under ether anaesthesia after starving overnight. The thoracic duct was cannulated according to the method of Bollman et al. [1948] using a polyethylene cannula having an internal diameter of 0-5 mm. The common bile duct was cannulated by inserting a polyethylene tube (P.E. 10), internal diameter 0 375 mm., proximally, leaving the pancreatic duct intact as suggested by Colwell [1950]. An elastic silicone tube was passed through a small incision in the stomach into the duodenum and its tip was secured near the entrance of the bile duct. The internal diameter of this duodenal cannula was such that it gave a snug fit for the distal end of the biliary cannula. This allowed bile to be returned to the intestine by making the connection between the tube or drained by breaking it. In some rats another tube was passed into the stomach and secured there. After operation the rats were transferred to Bollman restraining cages. Thereafter, a solution of NaCl 0 9 per cent, KCI 0-03 per cent (W./V.) was infused continuously through the intestinal or stomach tube at 1-5 ml./hr. until the end of the experiment. In some cases sodium taurocholate, 3-3 or 66 mg./ml., was added to the infusate so that 5 or 10 mg. bile salt entered the intestine per hour. The sodium tatrocholate (Koch Light Laboratories, or synthesized by the method of Hofmann [1963], contained only a trace impurity of cholic acid when examined by thin layer chromatography [Usui, 1963]. Rats were allowed to drink a solution of NaCl, 0-64 per cent, KCI 0 04 per cent (W./V.) ad lib except during experiments in which uniform hydration and steady lymph flow were important. Determination of Lipid. - The total esterified fatty acid (e.f.a.) content of the lymph and bile was determined according to the method of Stern and Shapiro [1953]. The lipid phosphorus content of bile was determined using the method of Zilversmit [1958]. The total cholesterol in lymph was determined according to the method of Abell et al. [1958]. The total lipid of lymph and bile were extracted either with chloroform: methanol (2: 1) Folch et al. [1957] or with water: heptane: alcohol: ether (1:1: 1:1) as described by Blankenhorn and Ahrens [1955]. The different lipid components of the total bile and lymph lipid were separated and qualitatively examined by thin layer chromatography using silica gel G and a solvent system of hexane : ether acetic acid (80 20 : 2) or by using silica gel and a two step development system as described by Skipski et al. [1965]. Standards were used to assist in the location of different lipid components of unknown samples. The spots were identified by spraying them either with 50 per cent sulphuric acid or with 1 per cent iodine in methanol. The fatty acid pattern of total lipids of bile, lymph and plasma and of phospholipids of bile was studied by gas chromatography. Methyl esters from phospholipids were prepared according to the method of Morrison and Smith [1964]. For preparation of methyl esters from total lipids, the lipids were saponified, the fatty acids extracted with petroleum ether, the ether evaporated and the fatty acids converted into methyl esters according to the method of Rogozinski [1964]. Gas

Lipid in Thoracic Duct Lymph 307 chromatographic analyses were performed with a Pye-Argon Gas Chromatograph using 4 ft. columns containing diethylene glycol succinate (10 per cent) on 100-120 mesh silanized Chromosorb G (British Drug Houses) at 175 C. NIH and Hormel standards were used and the results calculated by multiplying peak height with retention time [Bartlet and Iverson, 1966]. RESULTS Morgan [1964] had shown that the output of lipid in the lymph of fasting rats dropped by 64 per cent on production of bile fistula. To determine if this drop was due principally to malabsorption of endogenous lymph lipids from sources other than bile, bile salts alone were returned to the intestine. Bile fistula-lymph fistula rats were prepared and 48 hr. after operation collection of lymph and bile was started. Drinking water was removed and the rats were kept only on saline infusion. Lymph and bile were collected hourly for 4 hr. After this bile salts, 5 or 10 mg./hr., were added to the saline infusion. Lymph and bile were collected in hourly samples for 4 hr. and then pooled overnight for 16 hr. Next day 4 hourly collections were made while the rats were still on bile salt-saline infusion. After this bile salt-saline infusion was replaced by infusion of saline alone and lymph and bile were collected hourly for 4 hr. and then overnight for 16 hr. Exactly the same procedure was also followed in rats with ly;mph fistula but with intact bile ducts. The hourly collections showed that any changes persisting from the previous regime had passed off within 1 hr. of changing the infused solution. The results from the overnight samples (Table I) agreed closely with the hourly samples collected on the same regime during the first 4 hr. of the following day's experiment. In rats without bile fistula (right hand side, Table I) the infusion of bile salts produced a small but statistically significant increase in output of esterified fat in fasting lymph, from 15-3 to 20-8,uEq/hr. (P < 0 01). In rats with bile fistulae (left hand side, Table I) the fasting output of esterified fat was only about 20 per cent of that when bile ducts were intact and the slight increase, from 2-8 to 4-8,uEq/hr., following bile salt infusion was not statistically significant. It seemed clear then that the low output in bile fistula rats could not be ascribed to the absence of bile salts and loss of biliary lipids from the small intestine seemed a more likely cause. When bile salts were given to lymph fistula rats in which bile ducts were intact the increase in lipid output could have been due to stimulation of lipid output in the bile with subsequent reabsorption. In Table I, middle column, there was a significant increase in lipid concentration and lipid output in fistula bile following bile salt infusion. However, the output of esterified fat even when stimulated by bile salts was only 5-4,uEq/hr. This would not account for the difference in esterified fatty acid output in lymph, 12-5 RuEq/hr., between rats with bile fistulae and those with bile ducts intact. It seemed important then to determine the output of lipid in bile under conditions in which acute effects of operation and anaesthesia and chronic effects of interrupted entero-bepatic recirculation could be excluded.

308 Shrivastava, Redgrave and Simmonds A group of rats was prepared with re-entrant cannulae in the bile ducts, that is the cannula draining the bile could be fitted snugly into the cannula entering the small intestine, returning bile to the lumen of the intestine until the time of sampling. Only one sampling period was used in a given rat. In different groups of rats biliary samples were collected in tubes immersed in ice for two successive 2-hourly periods immediately after operation and 24, 48, 72 and 96 hr. thereafter. Considering the first 2-hr. sample (0-2, Table II) the flow of bile, concentration and output of esterified fatty acid were considerably lower immediately after operation than on subsequent days TABLE I. THE EFFECT OF BILE SALTS ON ESTERIFIED FATTY ACIDS (E.F.A.) OF BILE AND LYMPH. Samples were collected for 16 hr. (from 5th to 20th hr.) during steady intraduodenal infusion at 1-5 ml./hr. of 09 per cent NaCl and 0 03 per cent KCl solution or bile salts, 5 or 10 mg./hr., in the above mentioned solution. Average values (±s.e.m.) are given for four rats with bile and lymph fistulae and for six rats with lymph fistulae only. Each rat received both saline and bile salt-saline infusion, on successive days. Lymph fistula-bile fistula rats Lymph fistula only Lymph Bile Lymph SALINE INFUSION Flow in ml./hr. 1-09±0-18 0.30±0.10 1*28+0-07 Concentration of e.f.a. in ;LEq/ml. 2-60±0-31 7-60±1-60 1175±124 Output of e.f.a. injcleq/hr. 2-76±031 2*54±0-62 15-26±1-59 BILE SALT-SALINE INFUSION Flow in ml./hr. 1 10±0 19 0-36±0 15 1-53±006 Concentration of e.f.a. in,eq/ml. 410±1-18 14-98±1*18 13-22±0-59 Output of e.f.a. in,ueq/hr. 4-82±1-76 5-41±0±67 20*84±089 (P < 0-01). There were no significant differences in output of esterified fatty acid from 24 hr. onwards, showing that recovery from effects of operation was fairly rapid. In these animals in which the effects of chronic bile fistulae were avoided the output of esterified fatty acid in bile was much higher than in the ffist series at a comparable time after operation. The effect of interrupting entero-hepatic recirculation of bile on the output of bile lipid was surprisingly rapid, as can be seen from the results for the second 2-hourly samples (hour 3-4, Table II). The rapid decline was due to decrease in concentration of esterified fatty acid in bile as well as to decrease in flow (both being statistically significant P < 0-001). Immediately after operation, however, the output of esterified fatty acid in bile was greater in the second of the 2-hourly periods due to a significant improvement of flow (P < 0-01) without change in concentration. It seemed clear that when the effects of operation and chronic biliary drainage were avoided the output of esterified fatty acid in bile, 20--30,uEq/hr., was more than adequate to explain the difference in output in esterified fatty acid in fasting lymph between bile fistula and control rats in the first series. To provide a closer comparison, a group of twelve rats were prepared with lymph

Lipid in Thoracic Duct Lymph 30'3 fistulae and re-entrant bile cannulae. After operation a steady infusion of saline was given and bile was returned to the intestine through the re-entrant cannula. A steady flow of lymph was established. Forty-eight hours after TABLE II. THE EFFECT OF POST-OPERATIVE RECOVERY AND DURATION OF BILIARY DRAINAGE ON ESTERIFIED FATTY ACIDS (E.F.A.) IN BILE. N o. of hrs. after Operation 0 24 48 72 96 Rats had re-entrant biliary cannulae so that bile was returnedl to the duodenum until the sample was taken. Each rat provided two successive 2-hourly samples (0-2 3-4) and was used only once. No. of rats 6 6 6 76 Flow in ml./hlr. 0-2 3-4 0-37 054 0-61 0-42 0*66 0*41 0-76 0-46 0*87 0-56 Concentration e.f.a.,ueq/ml. 0-2 3-4 16-38 18-91 32-66 16-67 31-09 19-14 26*33 16*65 32-87 21-72 Output of e.f.a. yieq/hr. r-- ---.' 0-2 3-4 6*20 10-33 19-84 7(08 20-99 7-57 19-90 6-47 28-61 12-24 operation a 1-hr. sample of lymph was collected and then the biliary cannula was disconnected and the bile was collected for 1 hr. The output of esterified fatty acid in lymph immediately before collecting the bile and the output in bile immediately after disconnecting the biliary from the intestinal end of TABLE III. THE OUTPUT OF ESTERIFIED FATTY ACID (E.F.A.) IN /IEQ/HR. IN LYMPH AND BILE. Output of e.f.a. in lymph in,eq/hr. Rat No. (collected for 0-1 hr.) 1 7-44 2 18-58 3 10-70 4 11-64 5 16-91 6 23-36 7 16-46 8 4*10 9 17-91 10 8-96 11 9-54 12 13-47 Output of e.f.a. in bile in REq/hr. (collected for 1-2 lhr.) 7-35 15-81 9 98 10-57 15-32 21-24 14-40 4-10 17-92 10-52 10-76 12-89 the re-entrant cannula are shown in Table III for each of twelve rats. It can be seen that the output of esterified fatty acid in lymph was very close to the output of esterified fatty acid in bile. In those rats in which the output of esterified fatty acid in bile was low, it was also low in lymph and where it was high in bile it was high in lymph. If biliary lipids were the chief source of endogenous esterified fatty acid in the lymph as the above results suggested then the fatty acid pattern of esterified

310 Shrivastava, Redgrave and Simmonds biliary lipids should resemble that of lymph lipid of rats with intact bile ducts. The fatty acid pattern as determined by gas chromatography of total bile lipids and of total lymph lipids of rats with bile entering into the intestine are given in Table IV. It will be seen that the fatty acid patterns of the two are very similar. Most of the biliary lipid was phospholipid as seen by thin layer chromatography. On biochemical estimation of lipid phosphorus of bile it was found that 80 per cent or more of the esterified fatty acid content of bile was contributed by phospholipid. By gas chromatography the fatty acid pattern of bile phospholipid and total bile lipid was found to be similar. The fatty acid pattern of lymph lipid of bile fistula rats (L, Table IV) was quite different from that of bile lipid as well as from that of lymph lipids of TABLE IV. The fatty acid pattern of total bile lipid (B), of total lymph lipid of rats with bile entering into the intestine (LB), of total lymph lipid of bile fistula rats (L) and of total plasma lipids of bile fistula rats (P). No. of rats C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:4 B 4 40 1 4 13 29 Nil 13 LB 4 33 1 7 12 28 3 16 L 4 21 1 8 25 19 1 25 P 3 23 1 9 12 17 Tr. 38 (pooled) rats in which bile was entering the intestine. In bile fistula animals, the pattern of lymph lipid closely resembled that in the plasma of these rats (P, Table IV). In both, C16: 0 and C18: 2 were relatively low and C20: 4 was high Ṫhe above results suggest that biliary lipid is the chief source of endogenous lipid present in the thoracic duct lymph of normal rats. The small amount of esterified fatty acid (about 3 jteq/hr.) which is present in the lymph of rats with bile fistula probably comes from plasma as suggested by the similarity between the fatty acid pattern of plasma lipids and lymph lipid of bile fistula rats. DIsCUSSION The decreased output of esterified fatty acid in the fasting lymph following bile fistula was clearly not due primarily to malabsorption of non-dietary fat from other sources. Morgan [1964] has shown in bile fistula rats that taurocholate, 15 mg./hr., completely reversed malabsorption of triglyceride given at a steady rate of 136*5 [ueq/hr. into the intestine. In the present experiments, taurocholate, 5 or 10 mg./hr., had no effect of physiological i;mportance on the output of fasting lymph lipid in bile fistula rats. These experiments also excluded the possibility that a metabolic contribution of esterified fatty acid by the intestinal epithelium was diminished by the absence of bile salts in bile fistula rats. Moreover, evidence of an intracellular role of bile salts in esterification in the

Lipid in Thoracic Duct Lymph 311 intestinal epithelium is conflicting [Johnston and Borgstrom, 1964; Morgan, 1964; Saunders and Dawson, 1963]. If fatty acids from the phospholipid in bile are absorbed to provide the main part of the esterified fatty acid in fasting lymph then the output of esterified fatty acid in the bile should be at least equal to the deficiency in output of esterified fatty acid in lymph following bile fistula. The present investigations showed that this was so, provided that bile was analyzed from animals which had recovered from the effects of operation and anaesthesia and in which there was a minimal period of interruption of the entero-hepatic recirculation of biliary constituents. Further evidence for the important contribution from reabsorbed biliary lipids to the esterified fatty acid outpuit in fasting lymph was given by the close resemblance of the fatty acid spectruim in fasting lymph with bile circulation intact to the rather characteristic spectrum in bile. The resemblance between the plasma and lymph totals of fatty acid in bile fistula animals is consistent with the view that the other main source of endogenous fatty acids other than bile is transudation of lipoprotein from circulating plasma into tissue fluid and lymph. The effects of operation and anaesthesia and of biliary loss itself on the concentration and output of esterified fatty acid in bile warrant further study. Baxter [1966] found in rats a total esterified fatty acid output of 10,tEq/hr. in bile collected during the first 6 hr. after operation, which is the same as the value for the 3rd and 4th hour after operation in the present experiments. He also showed that the output fell to 3-5 RuEq/hr. in later samples, which is similar to 2-5,uEq/hr. found in chronic bile fistula rats in the present series. An effect of biliary depletion is clearly apparent in Baxter's results but his experimental design did not show up the full extent of the operative depression nor the rapid fall in the output of esterified fatty acid in bile when biliary drainage was commenced after post-operative recovery. Gas liquid chromatography showed in the present series that the fatty acid distribution was very similar in chronic fistula bile to that in bile collected immediately after opening the re-entrant cannula. The decrease in lipid output was thus not due to decrease in a particular class of fatty acid. It is not clear by what mechanism esterified fatty acid concentration in bile is reduced by 40-50 per cent and output reduced by about 70 per cent within 3-4 hr. of starting biliary drainage. The bile acid output was not measured but must have been very considerably reduced. With bile ducts intact 4-2 of the total bile acid pool is cycled each hour by the enterohepatic recirculation. Possibly the output of phospholipid - the main source of esterified fatty acid in bile - depends on the rate of excretion of bile salts by the liver cells. However, infusion of bile salts in animals with chronic biliary fistulae had a negligible effect on output of esterified fatty acid in bile. Perhaps a prolonged depletion of bile salts affected phospholipid metabolism. The decrease in esterified fatty acid output cannot readily be explained by loss of the biliary lipid since the esterified fatty acid absorbed from bile before commencing drainage should have formed only a minor component of total fatty acid turnover, even in the case of special constituents such as arachidonic acid. It may be relevant that VOL. LII, NO. 3.- 1967 21

312 Shrivastava, Redgrave and Simmonds Zilversmit and van Handel [1958] and Balint et al. [1965, 1966] have suggested the possibility of a phospholipid compartment in the liver specifically concerned with production of biliary phospholipid and distinguishable from the phospholipid compartment which exchanges with plasma. It would be interesting if bile acid metabolism could be shown to influence a particular compartment of hepatic phospholipid. Further experiments will be necessary, including measurement of bile acid concentration and output in animals with re-entrant cannulae in which the returm of bile can be interrupted and replaced by equivalent amounts of purified bile salts. ACKNOWLEDGMENTS This work was carried out with the aid of grants from National Health and Medical Research Council of Australia, the Medical Research Grants Committee, The University of Western Australia and Colombo Plan. REFERENCES ABELL, L. L., LEVY, B. B., BRODIE, B. B. and KENDAL, F. E. (1958). In Standard Methods in Clinical Chemistry, Vol. II, p. 26. New York: Academic Press. BALINT, J. A., BEELER, D. A. and SPITZER, H. L. (1966). Fed. Proc. 25, 210. BALINT, J. A., KYRIAKIDES, E. C., SPITZER, H. L. and MORRISON, E. S. (1965). J. Lipid Res. 6, 96. BALINT, J. A., SPITZER, H. L. and KYRIARIDES, E. C. (1963). Clin. Res. 11, 32. BARTLET, J. C. and IVERSON, J. L. (1966). J. Assoc. Official Anal. Chem. 49, 21. BAXTER, J. H. (1966). J. Lipid Res. 7, 158. BLANKENHORN, D. H. and AHRENS, E. H. Jr. (1955). J. Biol. Ohem. 212, 69. BOLLMAN, J. L., CAIN, J. C. and GRINDLAY, J. H. (1948). J. Lab. & Clin. Med. 33, 1349. BURR, W. W., MCPHERSON, J. C. and TIDWELL, H. C. (1960). J. Nutr. 70, 171. COLWELL, A. R. (1950). Am. J. Dig. Dis. 17, 270. FOLCH, J., LEES, M., STANLEY, G. H. S. (1957). J. Biol. Chem. 226, 497. HOFMANN, A. F. (1963). Acta Chem. Scand. 17, 173. JOHNSTON, J. M. and BORGSTROM, B. (1964). Biochem. Biophys. Acta 84, 412. KARMEN, A., WHYTE, M. and GOODMAN, D. S. (1963). J. Lipid Res. 4, 322. MATTSON, F. H. and VOLPENHEIN, R. A. (1964). J. Biol. Chem. 239, 2772. MORGAN, R. G. H. (1964). Quart J. exp. Physiol. 49, 458. MORRIS, B. (1956). Quart. J. exp. Physiol. 41, 318. MORRISON, W. R. and SMITH, L. M. (1964). J. Lipid Res. 5, 600. PESSOA, V. C., KIM, K. S. and IVY, A. C. (1953). Am. J. Physiol. 174, 209. ROGOZINSxI, M. (1964). J. of Gas Chromatog., p. 136. SAUNDERS, D. R. and DAWSON, A. H. (1963). In Biochemical Problems of Lipids, p. 55. Amsterdam: Elsevier Publishing Company. SKIPSKI, V. P., SMOLOWE, A. F., SULLIVAN, R. C. and- BARCHY, M. (1965). Biochem. Biophys. Acta 106, 386. STERN, I. and SHAPIRO, B. (1953). J. Clin. Path. 6, 158. UsuI, T. (1963). J. Biochem. 54, 283. WELD, C. B. (1961). Canad. J. Biochem. Physiol. 39, 15. WHYTE, M., KARMEN, A. and GOODMAN, D. S. (1963). J. Lipid Res. 4, 312. ZILVERSMIT, D. B. (1958). In Standard Methods of Clinical Chemistry, Vol. II, p. 132. New York: Academic Press. ZILVERSMIT, D. B. and VAN HANDEL, E. (1958). Arch. Biochem. Biophys. 73, 224.