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The Effect of a Conjugated Bile Salt on Oleic Acid Absorption in the Rat
GASTROENTEROLOGY
Vol. 49, No.6 Printed in U.S.A.
Copyright © 1965 by The Williams & Wilkins Co
THE EFFECT OF A CONJUGATED BILE SALT ON OLEIC ACID ABSORPTION IN THE RAT .FRED KERN , JR .,
M.D .,
AND BENGT BORGSTROM ,
M.D.
Department of Physiological Chemistry, University of Lund , Lund, Sweden
Recent studies have elucidated some of the important physicochemicaF· 2 and biochemicaP· 4 functions of bile salts in the absorption of fatty acids. There continue to be, however, conflicting and contradictory reports about the role of bile salts in lipid absorption in intact animals. For example, in bile fistula dogs Pessoa et al. found that there was 54 to 86% absorption of oleic acid, 5 whereas Cohen reported finding much less absorption of a similar quantity of oleic acid. 6 Knoebel and Ryan noted that after 4 hr bile fistula dogs had absorbed almost as much oleic acid as normal dogs, but there was marked impairment in intracellular esterification of the fatty acid. 7 In the biledeficient rat numerous investigators have shown an impairment in fatty acid absorption,8· 9 but others have shown less impairment of fatty acid absorption in similar experiments.10 Conjugated bile salts participate in the intestinal absorption of fatty acid by several mechanisms: (a) within the lumen of the intestine they solubilize fatty acids and monoglycerides by the production of mixed micelles, and it is in this form that the lipids are normally absorbed; 11 • 12 (b) within the intestinal epithelial cell they facilitate the synthesis of triglyceride 3 and stimulate the Received June 14, 1965. Accepted August 5, 1965. Address requests for reprints to: Dr. Fred Kern, Jr., Department of Medicine, Division of ·Gastroenterology, University of Colorado Medical Center, Denver, Colorado 80220. This investigation was supported by Grant H-5302 from the United States Public Health :Service, and by the Swedish Medical Research Council. Dr. K ern was the recipient of a Commonwealth Fund (New York) Fellowship Award. The expert assistance of L. Krabisch and Mrs. G. Bjorklund is gratefully acknowledged.
incorporation of glucose and glycerol into lipid ;4 and (c) they exert a controversial and poorly understood effect upon gastrointestinal motility. In the study reported here the absorption of orally administered oleic acid has been compared in bile fistula and control-operated rats. The effects of administration of a conjugated bile salt to both groups of animals has also been measured. Since even small amounts of free bile salts damage the mucosa and probably impair some of the intracellular mechanisms for triglyceride synthesis, a pure conjugated bile salt was used. The purpose of this study was to evaluate separately each of the actions of conjugated bile salts which influences fatty acid absorption. Materials and Methods
White rats, 150 to 250 g, were fasted overnight and then operated upon under ether anesthesia. In the experimental group a polyethylene tube was placed in the distal end of the bile duct, and bile and pancreatic juice were diverted to the outside. In an equal number of control animals a similar polyethylene tube was inserted into the peritoneal cavity, sutured to the lateral abdominal wall and brought through the abdominal incision to the outside. Both groups of animals were placed in Bollman-type restraining cages. The biliary fistula rats were fed a solution containing 5% glucose, 0.6% NaCl and 0.04% KCl, and the control animals were fed 5% sucrose. Twenty-four hours after operation they were lightly anesthetized and a test meal (3 ml) was administered by stomach tube. The test meal given to 32 animals, 16 biliary fistula and 16 control, was essentially the same as that previously employed in studies of digestion and absorption. 13 Each milli-
623
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KERN AND BORGSTROM
liter contained 0.126 g of skim milk powder, 0.138 g of glucose and 0.067 g of oleic acid including approximately 1.0 p.c oleic acid9, 10-H3 (Amersham). Another 32 animals, one-half biliary fistula and one-half control, were given the same test meal except that it contained in addition 40 p.moles per ml of sodium taurodesoxycholate (NaTDC). At the time the test meal was administered two or three similar portions were placed in flasks for subsequent extraction and measurement of radioactivity. The test meal was prepared by adding the H 3 -oleic acid in heptane to the nonradioactive oleic acid, blowing off the heptane, then adding the other components and quickly homogenizing with an Ultra Turrax mixer. An homogeneous emulsion was produced that was stable for about 2 weeks in the refrigerator. It was always brought to room temperature prior to use. The NaTDC was prepared by the method of Norman, 14 modified by Hofmann.15 It was approximately 99% pure by thin layer chromatography. After administration of the test meal the rats were replaced in the restraining cages and sacrificed at intervals. After each test meal four experimental and four control animals were sacrificed at 2, 4, 6 and 9 hr. The gastrointestinal tract was immediately removed and divided into four parts: stomach, upper one-half of the small intestine, lower one-half of the small intestine and colon. The stomach, the lower one-half of the small intestine and the colon along with their contents were individually placed in 10 to 15 ml of ethanolic NaOH (100 g NaOH, 100 ml water, 400 ml ethanol). Feces passed after administration of the test meal were put in the digestion mixture with the colon. The material in the ethanolic NaOH was digested at 40 to 50 C for at least 24 hr. The mixture was then acidified with 6 N HCl; occasionally ethanol had to be added to produce one phase. An equal quantity (usually 20 ml) of the pre-equilibrated upper phase of a two-phase extraction mixture consisting of ether, petroleum ether, ethanol and water (1:1:1:1) was added and thoroughly mixed. Two milliliters of the upper phase were placed in a
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counting vial and taken to dryness. Ten milliliters of a scintillation mixture 16 were added and the radioactivity measured in a Packard Tri Carb liquid scintillation spectrometer. Because of color quenching (0 to 50%) an internal standard was always added to each vial which was then counted again and the activity calculated. The upper one-half of the small intestine with its contents was immediately placed in 20 volumes of chloroform: methanol (2: 1). The tissue was cut into small pieces with a scissors, homogenized with the Ultra Turrax mixer and allowed to stand in the chloroform methanol mixture at least 24 hr. It was then filtered and the filtrate washed according to Folch et al. 17 A portion of the chloroform fraction was placed in the counting vial, taken to dryness, scintillation mixture added and the radioactivity was assayed. A similar portion was evaporated to dryness under nitrogen, dissolved in ether and petroleum ether (1: 1), applied to silicic acid thin layer chromatographic plates and developed." The plates were sprayed with iodine and the spots scraped and counted as described above. Several samples of test meal were extracted by each extraction technique and their radioactivity was assayed in the same manner. Since there was a slight difference in the amount of radioactivity recovered from the two extraction mixtures, the activity of each was regarded as 100% in the calculation. Results
The total radioactivity recovered in the gastrointestinal tract (table 1) was subtracted from the amount given, and the per cent absorbed was calculated (fig. 1). The biliary fistula animals that were fed test meal without bile salt absorbed the oleic acid slowly. At 2 hr they had absorbed none, and at 9 hr they had absorbed 38%. The administration of NaTDC to the biliary fistula rats increased absorption especially during the first 4 hr. The addition of bile salt to the test meal of the control animals caused as great an increase in absorption as it did in the biliary fistula animals. In order to understand the mechanism
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CONJUGATED BILE SALT TABLE
1. Per cent of test meal activity in gastrointestinal tract Comparison with other groups (P )
Group and time of sacrifice (hours after feeding )
Mean(±
Per cent of activity
SE)
Biliary fistula-NaTDC
Control
Control-NaTDC
<0.001 <0.05 NSa NS
<0.01 NS NS <0.01
< 0 .001 <0 .01 <0 .01 <0.01
NS NS NS < 0.001
NS <0 .05 <0.05 <0.05
Biliary fistula 2 4 6 9 Biliary fistula-N aTDC 2 4 6 9 Control 2 4 6 9 Control-NaTDC 2 4 6 9 a
106, 96, 102 104, 97, 71 , 58 89, 46, 68 81, 47, 58
101.3 82.5 67.5 61.9
± ± ± ±
1.2 10.7 12. 5 19 .8
74, 63, 43, 52,
54, 27, 47, 47,
49, 72 49, 68 45 49
62.2 51.7 44.9 49.3
± ± ± ±
6.1 9.0 1.3 1.2
79, 71, 62, 20,
63, 81, 33, 28,
82 34, 63 40, 60 33, 34
72.0 62.3 48.8 28.9
± ± ± ±
5.8 10.0 3.8 3.1
53, 27, 11, 11,
39, 25, 14, 11,
57 35, 39 41 22, 27
49.7 31.7 22 .0 17 .8
± ± ± ±
5.3 3.4 9.3 3.9
<0.01 <0.001 <0.05 <0.05
NS = not significant.
whereby bile salt improved absorption, the effects on gastric emptying and on absorption rate after the test meal entered the small intestine were examined separately. The test meal bile salt seemed to accelerate gastric emptying in both the bile fistula and the control rats, particularly during the first few hours after its administration (fig. 2). The individual differences among animals in each group were great, however, so that this interpretation was statistically supported only when the control animals fed NaTDC were compared with the two biliary fi stula groups (table 2). Table 3 and figure 3 show the per cent absorption of the test meal after it had left the stomach. Once again the added bile salt had an effect in both groups. When bile salt was in the test meal of the biliary fistula rats, absorption from the small intestine varied from 73 to 86% ; this was in striking contrast with the slow absorption from the small intestine of the biliary fistula animals that were not fed NaTDC. After 9 hr, however, absorption in the absence of bile salt was nearly normal. Even though the major
3
ABSORPTION OF H 0LEIC ACID
75
50
25
Hours
FIG. 1. The total amount of radioactivity recovered from the gastrointestinal tract was subtracted from the amount administered in order to calculate the per cent absorbed. In figures 1 to 3, each point represents the mean of three or four animals.
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KERN AND BORGSTROM PER CENT OF RADIOACTIVITY IN STOMACH
75 c ~
...~
50
25
Hours
Fm. 2. The proportion of H 3-oleic acid remaining in the stomach reflects the rate of gastric emptying. The difference between the biliary fistula animals and the controls given NaTDC is statistically significant at 2 and 4 hr. The biliary fistula animals given NaTDC differ significantly from the controls given NaTDC at each time point. The standard errors and statistical significance are shown in table 2. TABLE
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change produced by NaTDC in the control animals was an increase in small intestinal absorption during the first 2 hr from 47 to· 76%, there was an increase in absorption at each time period when NaTDC was fed to· the controls. The total amount of activity extracted from the upper half of the small intestine,. including intestinal content, was largest in the control group (table 4). There were no differences in distribution of activity at the various time periods; therefore, the data for all the animals in each test group ( 14 to· 16 rats) were pooled. Since an insignificant proportion of the activity was in the lower glycerides this was omitted from consideration. In the biliary fistula animals that were fed test meal without bile salt, most of the activity was in the free fatty acid fraction and only a small proportion was in either triglyceride or phospholipid. On the other hand, in the other groups, even though there was free fatty acid activity present, probably from unabsorbed test meal in the lumen, a substantial portion was also incorporated into triglyceride and phospholipid
2. Per cent of test meal activity in stomach Comparison with other group( (P )
Group and time of sacrifice (hours after feeding)
Per cent of activity
Mean(±
SE )
Biliary fi stula-NaTDC
Control
Control-NaTDC
NS• NS NS NS
NS NS NS NS
<0 .05 <0.01 NS NS
NS NS NS <0.01
<0.05 <0 .05 <0 .05 <0.001
Biliary fistula 2 4 6 9 Biliary fistula-NaTDC 2 4 6 9 Control 2 4 6 9 Control-N aTDC 2 4 6 9 a
NS
=
not significant.
79, 38, 84, 56 21 , 63, 55, 53 30, 15, 29 2, 31 , 54
64.2 48.1 24.8 29 . 1
± ± ± ±
12.9 9.2 4.9 15 .2
64, 55 , 26, 34,
52 .3 34.5 33.5 38.3
± ± ± ±
6. 7 8.5 9.7 2.5
45, 39, 31 , 66 47, 50 , 19, 62 23 , 28 , 25, 11.1 3, 4, 13, 18
45 44.5 21.6 9. 3
± ± ± ±
7.4 9. 1 3.6 3.6
37, 16, 40 3, 6, 25, 20 2, 2, 24 1, 1, 11, 11
30.8 13.4 9. 3 6.0
± ± ± ±
7.7 6.5 7.2 2.8
42, 14, 29, 38,
39, 64 31 , 39 17, 62 43
NS <0.05 NS NS
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CONJUGATED BILE SALT TABLE
3. P er cent of test meal activity absorbed from the small intestine• Comparison with other groups (P )
Group and time of sacrifice (hours alter feeding )
Per cent of activity
Mean (±
SE)
Biliary listula-NaTDC
Control
Control-NaTDC
<0 .001 NSb <0.05 NS
<0.001 NS NS NS
<0.001 NS < 0.01 NS
<0. 001 NS NS NS
< 0.05 NS < 0.05 < 0.05
Biliary fistula 2 4 6 9 Biliary fistula-N aTDC 2 4 6 9 Control 2 4 6 9 Control-NaT DC 2 4 6 9
0, 0,0 0, 8, 66, 89 15, 63, 46 26 , 84, 92
0 40.6 ± 33. 5 41.3 ± 13 .8 67.4 ± 27 .7
73, 84, 78, 78,
80, 85, 74, 80,
83, 78 73 , 54 66 86
78 .5 73 .9 72.5 81.2
± ± ± ±
2.2 7.0 3. 1 2.1
34, 55, 78, 82,
53, 40, 56, 76,
52 81 , 97 49 , 76
46.5 68.1 64.7 77.9
± ± ± ±
6.3 12.7 7. 1 1.5
75, 76, 90, 90,
72, 72
72 .9 79.4 85 87.5
± ± ± ±
1.0 2. 6 3.8 1.6
77, 77
79, 87, 76 88, 77 90, 87, 83
< 0.01 NS <0.05 <0 .01
The data were calculated as fo llows: (100 - A) - (B + C + D) X 100 100- A A = per cent of administered radioactivity in the stomach; B, C, D = per cent of administered radioactivity in the upper small intestine (B), lower small intestine (C), and colon (D). b NS = not significant. a
3
indicating greater absorption and metabolism by the epit helial cell.
ABSORPTION OF H 0LEIC
ACID
FROM THE SMALL INTESTINE
Discussion
These results show that H 3 -oleic acid is very slowly absorbed in the rat with a biliary fi stula and that the simultaneous feeding of a pure conjugated bile salt increases the rate of absorption of oleic acid, probably by several mechanisms. The data suggest, but do not establish conclusively, that the rate of gastric emptying is increased by the bile salt thereby making more fatty acid available to the small intestinal mucosa. The bile salt clearly increases the rate of absorption from the small intestine itself. These effects are present in biliary fistula animals and in control-operated animals without biliary fistula . Bile salts also increase the percentage of labeled oleic acid in the triglyceride fraction extracted from the small intestinal mucosa of biliary fi stula
75
50
25
H ours
Fro. 3. The per cent absorbed from the small intestine after the t est meal left the stomach was calculated as shown in tab le 3.
628
KERN AND BORGSTROM TABLE
Vol. 49, No. 6
4. Radioactivity in lumen and wall of the upper half of the small -intestine• Per cent administered (mean ±so)
Group Total
1. Biliary fistula (n = 14) 2. Biliary fistulaNaTDC (n = 15) 3. Control (n = 16) 4. Control-N aTDC (n = 16)
Free fatty acid
Triglyceride
Phospholipid
6.1 ± 6 .0
4.1
± 0.9
0.64 ± 0 .38
0.91 ± 0.63
5 .9 ± 3.7
2.9
± 0.88
1.3
± 0.62
1.28 ± 0 .57
4 .2 ± 1.62 2. 26 ± 0 .55
4.0 1.7
± 2.6 ± 0.7
1.69 ± 0 .8 1.59 ± 1.84
10 .8 ± 5.3 6 . 2 ± 4.3
Significance of comparison between groups (P valueb) Total
Free fa tty acid
Triglyceride
Phospholipid
Group 2
3
4
2
-- -- ---- -
1 2 3
NS• <0.01 <0.01
3
4
-
- -
2
-- -
3
4
-
- -
2
3
4
-- ----
NS <0.01 NS <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 NS <0.01 <0.01 <0.01 NS <0.01 ,<0 .01 <0.01 <0 .01 <0 .01 NS
• The data for all animals in each test group were pooled because there were no differences in distribution of activity at the various time periods. b Student's t-test. c NS = not significant.
rats, probably reflecting an increase in rate of triglyceride synthesis. This effect of bile salts was not present in the control-operated rats. Indeed, the percentage of activity in triglyceride in the mucosa of the control animals given NaTDC was less than in the control animals not given N aTDC (table 4).
One effect of bile salts on gastric emptying in these experiments was clear: when control-operated rats were fed NaTDC the stomach emptied faster than in any other experimental group. There seemed to be a slight delay in gastric emptying in the biliary fistula rats compared with the controloperated rats, but this was not definitely established. The reported effects of bile salts or of biliary diversion on gastric emptying are contradictory. In 1941, Ackerman and colleagues studied the effect of bile salts on gastric emptying in dogs with an internal biliary fistula (gall bladder-renal pelvis with the common bile duct ligated) .18 They used X-ray examination to estimate the disappearance of barium sulfate from the stomach. They found that when the dogs
were fed a meal of barium sulfate, oil and water, gastric emptying was decreased. This delayed gastric emptying was restored to normal by the addition of bile salts to the oil and water meal. In 1953, Borgstrom, in an experiment similar to the one reported here, found slow gastric emptying in biliary fistula rats given palmitic acid-1-C 14 dissolved in corn oil. 8 The rats were sacrificed at intervals and for about 6 hr after the fat administration there was more radioactivity in the stomach of the biliary fistula group than of the rats with a normal flow of bile into the intestinal tract. In experiments of an entirely different design, Menguy reached the conclusion that the inhibition of gastric motility and secretion secondary to the presence of fat in the duodenum was really caused by bile salts in the duodenum. 19 He reported that olive oil in the duodenum of intact rats caused decreased gastric secretion and motility (measured by a balloon). When bile was diverted to the rectum, leaving the flow of pancreatic juice undisturbed, fat in the duodenum had no inhibitory action on the
December 1965
CONJUGATED BILE SALT
629
stomach. In such animals oil and ox bile tine, only about one-fourth of absorbed salts placed in the duodenum also caused palmitic acid-1-C 14 was transported by the diminished gastric secretion and motility. lymphatics and suggested the existence of Menguy also found that the intravenous in- a different pathway of fat absorption. 8 This jection of ox bile salts inhibited gastric se- observation has recently been confirmed cretion and motility. Since ox bile salts con- and extended by Saunders and Dawson, who tain a mixture of conjugated and free bile fed C 14 -oleic acid to biliary fistula and salts, it is difficult to interpret clearly the thoracic duct fistula rats and found but little radioactive lipid in the thoracic duct.9 effects attributed to them. In yet a different kind of experiment Further, most of the tagged oleic acid in the Morgan and Simmonds concluded the gas- thoracic duct was not esterified and there tric emptying and the movement of a non- was a considerable increase in portal vein absorbable marker (polyethylene glycol) radioactive oleic acid in the form of free through the small intestine were normal in fatty acid. The details of this portal vein rats when bile was diverted into the ileum. 20 pathway for the absorption of long chain On the other hand, when they diverted bile fatty acids have not been studied. In a reto the urinary bladder, the rate of gastric cent study Morgan questioned the existence emptying was increased and passage of the of this pathway, as he found that emulsified marker through the small intestine was de- fatty acid was absorbed, re-esterified and layed. Knoebel and Ryan found that bile transported in the lymph in the absence of fistula dogs had a decreased gastric evacua- bile as well as in its presence. 10 This investion of both cottonseed oil and oleic acid. 7 tigator employed emulsifying agents of unIt is difficult to reconcile these strikingly certain physiological effect. He also used different results. It should be noted, how- commercial sodium taurocholate which, ever, that the experimental designs were when analyzed by thin layer chromatogdifferent; the operative procedures and raphy, contained a number of components, postoperative care of the animals were dif- including conjugated and unconjugated bile ferent; the test meals were different and the acids. Although the amount of free bile acid experimental procedures and analytical present in these experiments may have no techniques were different. The experiments physiological importance, it has been demreported here confirm earlier studies show- onstrated, both in vitro 3 and in vivo, 23 that ing considerable absorption of fatty acid free bile acids damage the intestinal epithein the absence of bile. They also demon- lial cells. strate the marked delay in absorption withConjugated bile salts facilitate the about bile salts and the return to normal when sorption of fatty acid by an intraluminal bile salts are replaced. and an intracellular mechanism. Within There were small, but generally signifi- the lumen they form mixed micelles with cant differences in the rate of absorption of the fatty acid and it is in this physical state the fatty acid from the small intestine in the that the fatty acid is absorbed. Within the three groups of animals in which bile salts cell they stimulate synthesis of triglyceride, were available, suggesting that the rate of as shown in these studies. Very little radiointestinal absorption is directly related to active oleic acid in the upper small intestine the amount of bile salts present. There was was incorporated into triglyceride in the considerable delay in absorption in the bili- absence of bile salts and it was increased ary fistula animal without bile salts in the two- to sixfold in the presence of bile salt. test meal. Poor fatty acid absorption in the Since there was more radioactivity in the absence of bile salts has been known since upper one-half of the small intestine of con21 the report of Munk in 1890 and confirmed than in the biliary fistula anitrol animals 22 by many investigators. Numerous studies , it is likely that the greater mals (table 4), in different animals have also established proportion of triglyceride in the controls is the fact that some fat is absorbed in the absence of bile. Borgstrom noted in 1953 not an artifact produced by slow absorption that in the absence of bile from the intes- of H 3 -oleic acid in the biliary fistula rats.
630
KERN AND BORGSTROM
Bile salt in excess of the amount usually present did not increase the percentage of labeled oleic acid in triglyceride in the mucosa suggesting that excess bile salt increased absorption by an intraluminal effect, perhaps by increasing the micellar solubilization of the fatty acid. A number of important questions remain. By what mechanism do conjugated bile salts accelerate gastric emptying? In what physical state is oleic acid absorbed in the absence of bile salts? How do excess bile salts augment fatty acid absorption from the small intestine in the intact animal?
erties of dilute micellar solutions of conjugated bile salts. Biochem. J. 89: 57-68. 3. Dawson, A. M., and K. J. Isselbacher. 1964. Studies on lipid metabolism in the small intestine with observations on the role of bile salts. J. Clin. Invest. 39: 730-740. 4. Holt, P. R., H. A. Haessler, and K. J. Isselbacher. 1963. Effects of bile salts on glucose metabolism by slices of hamster small intestine. J. Clin. Invest. 42 : 777-786. 5. Pessoa, V. C., K. S. Kim, and A. C. Ivy. 1953. Fat absorption in the absence of bile and pancreatic juice. Amer. J . Physiol. 174: 209218. 6. Cohen, B. J. 1961. Fat excretion in dogs lack-
Summary
The effect of bile salts on the absorption of H 3 -oleic acid has been studied in biliary fistula rats and in rats subjected to a control operation. A total bile fistula was performed in 32 rats and the control operation was performed in the same number of rats. Half of each group was fed by stomach tube a test meal containing emulsified oleic acid tagged with H 3 . The other half of each group was fed a similar test meal except that it contained sodium taurodesoxycholate (120 !-'moles). The results indicate that: (a) H 3 -oleic acid absorption was slow in the biliary fistula animals and it was restored to normal by NaTDC in the test meal. (b) NaTDC accelerated gastric emptying in the control animals. (c) Oleic acid absorption from the small intestine (after it had left the stomach) was markedly delayed in the biliary fistula rat and was very rapid in all groups in which either endogenous or exogenous bile salt was present. The rate of absorption seemed to be dependent upon the amount of bile salt present. (d) There was very little triglyceride containing radioactive oleic acid in the mucosa of the upper small intestine in the absence of bile salts. The inclusion of bile salts in the test meal restored this toward normal. REFERENCES 1. Hofmann, A. F., and B. Borgstrom. 1962.
Physico-chemical state of lipids in intestinal content in digestion and absorption. Fed. Proc. 21: 43-50. 2. Hofmann, A. F. 1963. The function of bile salts in fat absorption. The solvent prop-
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7.
8.
9.
10.
11.
12.
13.
14.
15.
ing both bile and pancreatic juice. Proc. Soc. Exp. Biol. Med. 107: 40-42. Knoebel, L. K., and J. M. Ryan. 1963. Digestion and mucosal absorption of fat in normal and bile-deficient dogs. Amer. J. Physiol. 204: 509-514. Borgstrom, B. 1953. The mechanism of intestinal fat absorption. V. The effect of bile diversion on fat absorption in the rat. Acta Physiol. Scan d. 28: 279-286. Saunders, D. R., and A. H. Dawson. 1963. The absorption of oleic acid in the bile fistula rat. Gut 4: 254-260. Morgan, R. G. H . 1964. The effect of bile salts on the lymphatic absorption by the unanesthetized rat of intraduodenally infused lipids. Quart. J. Exp. Physiol. 49: 457-465. Hofmann, A. F., and B. Borgstrom. 1964. The intraluminal phase of fat digestion in man: the lipid content of the micellar and oil phases of intestinal content obtained during fat digestion and absorption. J. Clin. Invest. 43: 247-257. Johnston, J. M., and B. Borgstrom. 1964. The intestinal absorption and metabolism of micellar solutions of lipids. Biochim. Biophys. Acta. 84: 412-423. Borgstrom, B., A. Dahlqvist, G. Lundh, and J. Sjovall. 1957. Studies of intestinal digestion and absorption in the human. J. Clin. Invest. 36: 1521-1536. Norman, A. 1955-56. Preparation of conjugated bile acids using mixed carboxylic acid anhydrides. Bile acids and steroids. Arkiv Kemi 8: 331-342. Hofmann, A. F. 1963. The preparation of chenodeoxycholic acid and its glycine and taurine conjugates. Acta Chern. Scand. 17:
173-186. 16. Brown, J. L., and J. M. Johnston. 1962. Radio-
assay of lipid components separated by thin-layer chromatography. J. Lipid Res. 3: 480-481.
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17. Folch, J., M. Lees, and G. H. Sloane-Stanley. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chern. 226 : 497-509. 18. Ackerman, R. F., H . Curl, and A. A. Crandall, Jr. 1941. Gastrointestinal tract motility in the absence of bile . Amer. J. Physiol. 134: 32-36. 19. Menguy, R. 1960. Role of biliary and pan-
creatic secretions in the inhibition of gastric motility by fat in the intestine. An experimental study in the rat. Amer. J. Dig. Dis. 5: 792-800. 20. Morgan, R. G. H., and W. J. Simmonds. 1962. The relative effects of diversion of bile to
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the ileum or to the urinary bladder on fat absorption and gastrointestinal motility in the rat. Quart. J. Exp. Physiol. 47: 352-359. 21. Munk, I. 1890. Ueber die Resorption von Fetten und festen Fettsauren nach Ausschluss der Galle vom Darmkanal. Arch. Path. Anat.122: 302-325. 22. Wilson, T. H . 1962. Intestinal absorption, p. 158-189. W. B. Saunders Company, Philadelphia. 23. Fry, R. J. M., and E . Staffeldt. 1964. Effect of a diet containing sodium deoxycholate on the intestinal mucosa of the mouse. Nature (London) 203 : 1396-1398.
Vol. 49, No.6 Printed in U.S.A.
Copyright © 1965 by The Williams & Wilkins Co
THE EFFECT OF A CONJUGATED BILE SALT ON OLEIC ACID ABSORPTION IN THE RAT .FRED KERN , JR .,
M.D .,
AND BENGT BORGSTROM ,
M.D.
Department of Physiological Chemistry, University of Lund , Lund, Sweden
Recent studies have elucidated some of the important physicochemicaF· 2 and biochemicaP· 4 functions of bile salts in the absorption of fatty acids. There continue to be, however, conflicting and contradictory reports about the role of bile salts in lipid absorption in intact animals. For example, in bile fistula dogs Pessoa et al. found that there was 54 to 86% absorption of oleic acid, 5 whereas Cohen reported finding much less absorption of a similar quantity of oleic acid. 6 Knoebel and Ryan noted that after 4 hr bile fistula dogs had absorbed almost as much oleic acid as normal dogs, but there was marked impairment in intracellular esterification of the fatty acid. 7 In the biledeficient rat numerous investigators have shown an impairment in fatty acid absorption,8· 9 but others have shown less impairment of fatty acid absorption in similar experiments.10 Conjugated bile salts participate in the intestinal absorption of fatty acid by several mechanisms: (a) within the lumen of the intestine they solubilize fatty acids and monoglycerides by the production of mixed micelles, and it is in this form that the lipids are normally absorbed; 11 • 12 (b) within the intestinal epithelial cell they facilitate the synthesis of triglyceride 3 and stimulate the Received June 14, 1965. Accepted August 5, 1965. Address requests for reprints to: Dr. Fred Kern, Jr., Department of Medicine, Division of ·Gastroenterology, University of Colorado Medical Center, Denver, Colorado 80220. This investigation was supported by Grant H-5302 from the United States Public Health :Service, and by the Swedish Medical Research Council. Dr. K ern was the recipient of a Commonwealth Fund (New York) Fellowship Award. The expert assistance of L. Krabisch and Mrs. G. Bjorklund is gratefully acknowledged.
incorporation of glucose and glycerol into lipid ;4 and (c) they exert a controversial and poorly understood effect upon gastrointestinal motility. In the study reported here the absorption of orally administered oleic acid has been compared in bile fistula and control-operated rats. The effects of administration of a conjugated bile salt to both groups of animals has also been measured. Since even small amounts of free bile salts damage the mucosa and probably impair some of the intracellular mechanisms for triglyceride synthesis, a pure conjugated bile salt was used. The purpose of this study was to evaluate separately each of the actions of conjugated bile salts which influences fatty acid absorption. Materials and Methods
White rats, 150 to 250 g, were fasted overnight and then operated upon under ether anesthesia. In the experimental group a polyethylene tube was placed in the distal end of the bile duct, and bile and pancreatic juice were diverted to the outside. In an equal number of control animals a similar polyethylene tube was inserted into the peritoneal cavity, sutured to the lateral abdominal wall and brought through the abdominal incision to the outside. Both groups of animals were placed in Bollman-type restraining cages. The biliary fistula rats were fed a solution containing 5% glucose, 0.6% NaCl and 0.04% KCl, and the control animals were fed 5% sucrose. Twenty-four hours after operation they were lightly anesthetized and a test meal (3 ml) was administered by stomach tube. The test meal given to 32 animals, 16 biliary fistula and 16 control, was essentially the same as that previously employed in studies of digestion and absorption. 13 Each milli-
623
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KERN AND BORGSTROM
liter contained 0.126 g of skim milk powder, 0.138 g of glucose and 0.067 g of oleic acid including approximately 1.0 p.c oleic acid9, 10-H3 (Amersham). Another 32 animals, one-half biliary fistula and one-half control, were given the same test meal except that it contained in addition 40 p.moles per ml of sodium taurodesoxycholate (NaTDC). At the time the test meal was administered two or three similar portions were placed in flasks for subsequent extraction and measurement of radioactivity. The test meal was prepared by adding the H 3 -oleic acid in heptane to the nonradioactive oleic acid, blowing off the heptane, then adding the other components and quickly homogenizing with an Ultra Turrax mixer. An homogeneous emulsion was produced that was stable for about 2 weeks in the refrigerator. It was always brought to room temperature prior to use. The NaTDC was prepared by the method of Norman, 14 modified by Hofmann.15 It was approximately 99% pure by thin layer chromatography. After administration of the test meal the rats were replaced in the restraining cages and sacrificed at intervals. After each test meal four experimental and four control animals were sacrificed at 2, 4, 6 and 9 hr. The gastrointestinal tract was immediately removed and divided into four parts: stomach, upper one-half of the small intestine, lower one-half of the small intestine and colon. The stomach, the lower one-half of the small intestine and the colon along with their contents were individually placed in 10 to 15 ml of ethanolic NaOH (100 g NaOH, 100 ml water, 400 ml ethanol). Feces passed after administration of the test meal were put in the digestion mixture with the colon. The material in the ethanolic NaOH was digested at 40 to 50 C for at least 24 hr. The mixture was then acidified with 6 N HCl; occasionally ethanol had to be added to produce one phase. An equal quantity (usually 20 ml) of the pre-equilibrated upper phase of a two-phase extraction mixture consisting of ether, petroleum ether, ethanol and water (1:1:1:1) was added and thoroughly mixed. Two milliliters of the upper phase were placed in a
Vol. 49, No. 6
counting vial and taken to dryness. Ten milliliters of a scintillation mixture 16 were added and the radioactivity measured in a Packard Tri Carb liquid scintillation spectrometer. Because of color quenching (0 to 50%) an internal standard was always added to each vial which was then counted again and the activity calculated. The upper one-half of the small intestine with its contents was immediately placed in 20 volumes of chloroform: methanol (2: 1). The tissue was cut into small pieces with a scissors, homogenized with the Ultra Turrax mixer and allowed to stand in the chloroform methanol mixture at least 24 hr. It was then filtered and the filtrate washed according to Folch et al. 17 A portion of the chloroform fraction was placed in the counting vial, taken to dryness, scintillation mixture added and the radioactivity was assayed. A similar portion was evaporated to dryness under nitrogen, dissolved in ether and petroleum ether (1: 1), applied to silicic acid thin layer chromatographic plates and developed." The plates were sprayed with iodine and the spots scraped and counted as described above. Several samples of test meal were extracted by each extraction technique and their radioactivity was assayed in the same manner. Since there was a slight difference in the amount of radioactivity recovered from the two extraction mixtures, the activity of each was regarded as 100% in the calculation. Results
The total radioactivity recovered in the gastrointestinal tract (table 1) was subtracted from the amount given, and the per cent absorbed was calculated (fig. 1). The biliary fistula animals that were fed test meal without bile salt absorbed the oleic acid slowly. At 2 hr they had absorbed none, and at 9 hr they had absorbed 38%. The administration of NaTDC to the biliary fistula rats increased absorption especially during the first 4 hr. The addition of bile salt to the test meal of the control animals caused as great an increase in absorption as it did in the biliary fistula animals. In order to understand the mechanism
December 1965
625
CONJUGATED BILE SALT TABLE
1. Per cent of test meal activity in gastrointestinal tract Comparison with other groups (P )
Group and time of sacrifice (hours after feeding )
Mean(±
Per cent of activity
SE)
Biliary fistula-NaTDC
Control
Control-NaTDC
<0.001 <0.05 NSa NS
<0.01 NS NS <0.01
< 0 .001 <0 .01 <0 .01 <0.01
NS NS NS < 0.001
NS <0 .05 <0.05 <0.05
Biliary fistula 2 4 6 9 Biliary fistula-N aTDC 2 4 6 9 Control 2 4 6 9 Control-NaTDC 2 4 6 9 a
106, 96, 102 104, 97, 71 , 58 89, 46, 68 81, 47, 58
101.3 82.5 67.5 61.9
± ± ± ±
1.2 10.7 12. 5 19 .8
74, 63, 43, 52,
54, 27, 47, 47,
49, 72 49, 68 45 49
62.2 51.7 44.9 49.3
± ± ± ±
6.1 9.0 1.3 1.2
79, 71, 62, 20,
63, 81, 33, 28,
82 34, 63 40, 60 33, 34
72.0 62.3 48.8 28.9
± ± ± ±
5.8 10.0 3.8 3.1
53, 27, 11, 11,
39, 25, 14, 11,
57 35, 39 41 22, 27
49.7 31.7 22 .0 17 .8
± ± ± ±
5.3 3.4 9.3 3.9
<0.01 <0.001 <0.05 <0.05
NS = not significant.
whereby bile salt improved absorption, the effects on gastric emptying and on absorption rate after the test meal entered the small intestine were examined separately. The test meal bile salt seemed to accelerate gastric emptying in both the bile fistula and the control rats, particularly during the first few hours after its administration (fig. 2). The individual differences among animals in each group were great, however, so that this interpretation was statistically supported only when the control animals fed NaTDC were compared with the two biliary fi stula groups (table 2). Table 3 and figure 3 show the per cent absorption of the test meal after it had left the stomach. Once again the added bile salt had an effect in both groups. When bile salt was in the test meal of the biliary fistula rats, absorption from the small intestine varied from 73 to 86% ; this was in striking contrast with the slow absorption from the small intestine of the biliary fistula animals that were not fed NaTDC. After 9 hr, however, absorption in the absence of bile salt was nearly normal. Even though the major
3
ABSORPTION OF H 0LEIC ACID
75
50
25
Hours
FIG. 1. The total amount of radioactivity recovered from the gastrointestinal tract was subtracted from the amount administered in order to calculate the per cent absorbed. In figures 1 to 3, each point represents the mean of three or four animals.
626
KERN AND BORGSTROM PER CENT OF RADIOACTIVITY IN STOMACH
75 c ~
...~
50
25
Hours
Fm. 2. The proportion of H 3-oleic acid remaining in the stomach reflects the rate of gastric emptying. The difference between the biliary fistula animals and the controls given NaTDC is statistically significant at 2 and 4 hr. The biliary fistula animals given NaTDC differ significantly from the controls given NaTDC at each time point. The standard errors and statistical significance are shown in table 2. TABLE
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change produced by NaTDC in the control animals was an increase in small intestinal absorption during the first 2 hr from 47 to· 76%, there was an increase in absorption at each time period when NaTDC was fed to· the controls. The total amount of activity extracted from the upper half of the small intestine,. including intestinal content, was largest in the control group (table 4). There were no differences in distribution of activity at the various time periods; therefore, the data for all the animals in each test group ( 14 to· 16 rats) were pooled. Since an insignificant proportion of the activity was in the lower glycerides this was omitted from consideration. In the biliary fistula animals that were fed test meal without bile salt, most of the activity was in the free fatty acid fraction and only a small proportion was in either triglyceride or phospholipid. On the other hand, in the other groups, even though there was free fatty acid activity present, probably from unabsorbed test meal in the lumen, a substantial portion was also incorporated into triglyceride and phospholipid
2. Per cent of test meal activity in stomach Comparison with other group( (P )
Group and time of sacrifice (hours after feeding)
Per cent of activity
Mean(±
SE )
Biliary fi stula-NaTDC
Control
Control-NaTDC
NS• NS NS NS
NS NS NS NS
<0 .05 <0.01 NS NS
NS NS NS <0.01
<0.05 <0 .05 <0 .05 <0.001
Biliary fistula 2 4 6 9 Biliary fistula-NaTDC 2 4 6 9 Control 2 4 6 9 Control-N aTDC 2 4 6 9 a
NS
=
not significant.
79, 38, 84, 56 21 , 63, 55, 53 30, 15, 29 2, 31 , 54
64.2 48.1 24.8 29 . 1
± ± ± ±
12.9 9.2 4.9 15 .2
64, 55 , 26, 34,
52 .3 34.5 33.5 38.3
± ± ± ±
6. 7 8.5 9.7 2.5
45, 39, 31 , 66 47, 50 , 19, 62 23 , 28 , 25, 11.1 3, 4, 13, 18
45 44.5 21.6 9. 3
± ± ± ±
7.4 9. 1 3.6 3.6
37, 16, 40 3, 6, 25, 20 2, 2, 24 1, 1, 11, 11
30.8 13.4 9. 3 6.0
± ± ± ±
7.7 6.5 7.2 2.8
42, 14, 29, 38,
39, 64 31 , 39 17, 62 43
NS <0.05 NS NS
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627
CONJUGATED BILE SALT TABLE
3. P er cent of test meal activity absorbed from the small intestine• Comparison with other groups (P )
Group and time of sacrifice (hours alter feeding )
Per cent of activity
Mean (±
SE)
Biliary listula-NaTDC
Control
Control-NaTDC
<0 .001 NSb <0.05 NS
<0.001 NS NS NS
<0.001 NS < 0.01 NS
<0. 001 NS NS NS
< 0.05 NS < 0.05 < 0.05
Biliary fistula 2 4 6 9 Biliary fistula-N aTDC 2 4 6 9 Control 2 4 6 9 Control-NaT DC 2 4 6 9
0, 0,0 0, 8, 66, 89 15, 63, 46 26 , 84, 92
0 40.6 ± 33. 5 41.3 ± 13 .8 67.4 ± 27 .7
73, 84, 78, 78,
80, 85, 74, 80,
83, 78 73 , 54 66 86
78 .5 73 .9 72.5 81.2
± ± ± ±
2.2 7.0 3. 1 2.1
34, 55, 78, 82,
53, 40, 56, 76,
52 81 , 97 49 , 76
46.5 68.1 64.7 77.9
± ± ± ±
6.3 12.7 7. 1 1.5
75, 76, 90, 90,
72, 72
72 .9 79.4 85 87.5
± ± ± ±
1.0 2. 6 3.8 1.6
77, 77
79, 87, 76 88, 77 90, 87, 83
< 0.01 NS <0.05 <0 .01
The data were calculated as fo llows: (100 - A) - (B + C + D) X 100 100- A A = per cent of administered radioactivity in the stomach; B, C, D = per cent of administered radioactivity in the upper small intestine (B), lower small intestine (C), and colon (D). b NS = not significant. a
3
indicating greater absorption and metabolism by the epit helial cell.
ABSORPTION OF H 0LEIC
ACID
FROM THE SMALL INTESTINE
Discussion
These results show that H 3 -oleic acid is very slowly absorbed in the rat with a biliary fi stula and that the simultaneous feeding of a pure conjugated bile salt increases the rate of absorption of oleic acid, probably by several mechanisms. The data suggest, but do not establish conclusively, that the rate of gastric emptying is increased by the bile salt thereby making more fatty acid available to the small intestinal mucosa. The bile salt clearly increases the rate of absorption from the small intestine itself. These effects are present in biliary fistula animals and in control-operated animals without biliary fistula . Bile salts also increase the percentage of labeled oleic acid in the triglyceride fraction extracted from the small intestinal mucosa of biliary fi stula
75
50
25
H ours
Fro. 3. The per cent absorbed from the small intestine after the t est meal left the stomach was calculated as shown in tab le 3.
628
KERN AND BORGSTROM TABLE
Vol. 49, No. 6
4. Radioactivity in lumen and wall of the upper half of the small -intestine• Per cent administered (mean ±so)
Group Total
1. Biliary fistula (n = 14) 2. Biliary fistulaNaTDC (n = 15) 3. Control (n = 16) 4. Control-N aTDC (n = 16)
Free fatty acid
Triglyceride
Phospholipid
6.1 ± 6 .0
4.1
± 0.9
0.64 ± 0 .38
0.91 ± 0.63
5 .9 ± 3.7
2.9
± 0.88
1.3
± 0.62
1.28 ± 0 .57
4 .2 ± 1.62 2. 26 ± 0 .55
4.0 1.7
± 2.6 ± 0.7
1.69 ± 0 .8 1.59 ± 1.84
10 .8 ± 5.3 6 . 2 ± 4.3
Significance of comparison between groups (P valueb) Total
Free fa tty acid
Triglyceride
Phospholipid
Group 2
3
4
2
-- -- ---- -
1 2 3
NS• <0.01 <0.01
3
4
-
- -
2
-- -
3
4
-
- -
2
3
4
-- ----
NS <0.01 NS <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 NS <0.01 <0.01 <0.01 NS <0.01 ,<0 .01 <0.01 <0 .01 <0 .01 NS
• The data for all animals in each test group were pooled because there were no differences in distribution of activity at the various time periods. b Student's t-test. c NS = not significant.
rats, probably reflecting an increase in rate of triglyceride synthesis. This effect of bile salts was not present in the control-operated rats. Indeed, the percentage of activity in triglyceride in the mucosa of the control animals given NaTDC was less than in the control animals not given N aTDC (table 4).
One effect of bile salts on gastric emptying in these experiments was clear: when control-operated rats were fed NaTDC the stomach emptied faster than in any other experimental group. There seemed to be a slight delay in gastric emptying in the biliary fistula rats compared with the controloperated rats, but this was not definitely established. The reported effects of bile salts or of biliary diversion on gastric emptying are contradictory. In 1941, Ackerman and colleagues studied the effect of bile salts on gastric emptying in dogs with an internal biliary fistula (gall bladder-renal pelvis with the common bile duct ligated) .18 They used X-ray examination to estimate the disappearance of barium sulfate from the stomach. They found that when the dogs
were fed a meal of barium sulfate, oil and water, gastric emptying was decreased. This delayed gastric emptying was restored to normal by the addition of bile salts to the oil and water meal. In 1953, Borgstrom, in an experiment similar to the one reported here, found slow gastric emptying in biliary fistula rats given palmitic acid-1-C 14 dissolved in corn oil. 8 The rats were sacrificed at intervals and for about 6 hr after the fat administration there was more radioactivity in the stomach of the biliary fistula group than of the rats with a normal flow of bile into the intestinal tract. In experiments of an entirely different design, Menguy reached the conclusion that the inhibition of gastric motility and secretion secondary to the presence of fat in the duodenum was really caused by bile salts in the duodenum. 19 He reported that olive oil in the duodenum of intact rats caused decreased gastric secretion and motility (measured by a balloon). When bile was diverted to the rectum, leaving the flow of pancreatic juice undisturbed, fat in the duodenum had no inhibitory action on the
December 1965
CONJUGATED BILE SALT
629
stomach. In such animals oil and ox bile tine, only about one-fourth of absorbed salts placed in the duodenum also caused palmitic acid-1-C 14 was transported by the diminished gastric secretion and motility. lymphatics and suggested the existence of Menguy also found that the intravenous in- a different pathway of fat absorption. 8 This jection of ox bile salts inhibited gastric se- observation has recently been confirmed cretion and motility. Since ox bile salts con- and extended by Saunders and Dawson, who tain a mixture of conjugated and free bile fed C 14 -oleic acid to biliary fistula and salts, it is difficult to interpret clearly the thoracic duct fistula rats and found but little radioactive lipid in the thoracic duct.9 effects attributed to them. In yet a different kind of experiment Further, most of the tagged oleic acid in the Morgan and Simmonds concluded the gas- thoracic duct was not esterified and there tric emptying and the movement of a non- was a considerable increase in portal vein absorbable marker (polyethylene glycol) radioactive oleic acid in the form of free through the small intestine were normal in fatty acid. The details of this portal vein rats when bile was diverted into the ileum. 20 pathway for the absorption of long chain On the other hand, when they diverted bile fatty acids have not been studied. In a reto the urinary bladder, the rate of gastric cent study Morgan questioned the existence emptying was increased and passage of the of this pathway, as he found that emulsified marker through the small intestine was de- fatty acid was absorbed, re-esterified and layed. Knoebel and Ryan found that bile transported in the lymph in the absence of fistula dogs had a decreased gastric evacua- bile as well as in its presence. 10 This investion of both cottonseed oil and oleic acid. 7 tigator employed emulsifying agents of unIt is difficult to reconcile these strikingly certain physiological effect. He also used different results. It should be noted, how- commercial sodium taurocholate which, ever, that the experimental designs were when analyzed by thin layer chromatogdifferent; the operative procedures and raphy, contained a number of components, postoperative care of the animals were dif- including conjugated and unconjugated bile ferent; the test meals were different and the acids. Although the amount of free bile acid experimental procedures and analytical present in these experiments may have no techniques were different. The experiments physiological importance, it has been demreported here confirm earlier studies show- onstrated, both in vitro 3 and in vivo, 23 that ing considerable absorption of fatty acid free bile acids damage the intestinal epithein the absence of bile. They also demon- lial cells. strate the marked delay in absorption withConjugated bile salts facilitate the about bile salts and the return to normal when sorption of fatty acid by an intraluminal bile salts are replaced. and an intracellular mechanism. Within There were small, but generally signifi- the lumen they form mixed micelles with cant differences in the rate of absorption of the fatty acid and it is in this physical state the fatty acid from the small intestine in the that the fatty acid is absorbed. Within the three groups of animals in which bile salts cell they stimulate synthesis of triglyceride, were available, suggesting that the rate of as shown in these studies. Very little radiointestinal absorption is directly related to active oleic acid in the upper small intestine the amount of bile salts present. There was was incorporated into triglyceride in the considerable delay in absorption in the bili- absence of bile salts and it was increased ary fistula animal without bile salts in the two- to sixfold in the presence of bile salt. test meal. Poor fatty acid absorption in the Since there was more radioactivity in the absence of bile salts has been known since upper one-half of the small intestine of con21 the report of Munk in 1890 and confirmed than in the biliary fistula anitrol animals 22 by many investigators. Numerous studies , it is likely that the greater mals (table 4), in different animals have also established proportion of triglyceride in the controls is the fact that some fat is absorbed in the absence of bile. Borgstrom noted in 1953 not an artifact produced by slow absorption that in the absence of bile from the intes- of H 3 -oleic acid in the biliary fistula rats.
630
KERN AND BORGSTROM
Bile salt in excess of the amount usually present did not increase the percentage of labeled oleic acid in triglyceride in the mucosa suggesting that excess bile salt increased absorption by an intraluminal effect, perhaps by increasing the micellar solubilization of the fatty acid. A number of important questions remain. By what mechanism do conjugated bile salts accelerate gastric emptying? In what physical state is oleic acid absorbed in the absence of bile salts? How do excess bile salts augment fatty acid absorption from the small intestine in the intact animal?
erties of dilute micellar solutions of conjugated bile salts. Biochem. J. 89: 57-68. 3. Dawson, A. M., and K. J. Isselbacher. 1964. Studies on lipid metabolism in the small intestine with observations on the role of bile salts. J. Clin. Invest. 39: 730-740. 4. Holt, P. R., H. A. Haessler, and K. J. Isselbacher. 1963. Effects of bile salts on glucose metabolism by slices of hamster small intestine. J. Clin. Invest. 42 : 777-786. 5. Pessoa, V. C., K. S. Kim, and A. C. Ivy. 1953. Fat absorption in the absence of bile and pancreatic juice. Amer. J . Physiol. 174: 209218. 6. Cohen, B. J. 1961. Fat excretion in dogs lack-
Summary
The effect of bile salts on the absorption of H 3 -oleic acid has been studied in biliary fistula rats and in rats subjected to a control operation. A total bile fistula was performed in 32 rats and the control operation was performed in the same number of rats. Half of each group was fed by stomach tube a test meal containing emulsified oleic acid tagged with H 3 . The other half of each group was fed a similar test meal except that it contained sodium taurodesoxycholate (120 !-'moles). The results indicate that: (a) H 3 -oleic acid absorption was slow in the biliary fistula animals and it was restored to normal by NaTDC in the test meal. (b) NaTDC accelerated gastric emptying in the control animals. (c) Oleic acid absorption from the small intestine (after it had left the stomach) was markedly delayed in the biliary fistula rat and was very rapid in all groups in which either endogenous or exogenous bile salt was present. The rate of absorption seemed to be dependent upon the amount of bile salt present. (d) There was very little triglyceride containing radioactive oleic acid in the mucosa of the upper small intestine in the absence of bile salts. The inclusion of bile salts in the test meal restored this toward normal. REFERENCES 1. Hofmann, A. F., and B. Borgstrom. 1962.
Physico-chemical state of lipids in intestinal content in digestion and absorption. Fed. Proc. 21: 43-50. 2. Hofmann, A. F. 1963. The function of bile salts in fat absorption. The solvent prop-
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7.
8.
9.
10.
11.
12.
13.
14.
15.
ing both bile and pancreatic juice. Proc. Soc. Exp. Biol. Med. 107: 40-42. Knoebel, L. K., and J. M. Ryan. 1963. Digestion and mucosal absorption of fat in normal and bile-deficient dogs. Amer. J. Physiol. 204: 509-514. Borgstrom, B. 1953. The mechanism of intestinal fat absorption. V. The effect of bile diversion on fat absorption in the rat. Acta Physiol. Scan d. 28: 279-286. Saunders, D. R., and A. H. Dawson. 1963. The absorption of oleic acid in the bile fistula rat. Gut 4: 254-260. Morgan, R. G. H . 1964. The effect of bile salts on the lymphatic absorption by the unanesthetized rat of intraduodenally infused lipids. Quart. J. Exp. Physiol. 49: 457-465. Hofmann, A. F., and B. Borgstrom. 1964. The intraluminal phase of fat digestion in man: the lipid content of the micellar and oil phases of intestinal content obtained during fat digestion and absorption. J. Clin. Invest. 43: 247-257. Johnston, J. M., and B. Borgstrom. 1964. The intestinal absorption and metabolism of micellar solutions of lipids. Biochim. Biophys. Acta. 84: 412-423. Borgstrom, B., A. Dahlqvist, G. Lundh, and J. Sjovall. 1957. Studies of intestinal digestion and absorption in the human. J. Clin. Invest. 36: 1521-1536. Norman, A. 1955-56. Preparation of conjugated bile acids using mixed carboxylic acid anhydrides. Bile acids and steroids. Arkiv Kemi 8: 331-342. Hofmann, A. F. 1963. The preparation of chenodeoxycholic acid and its glycine and taurine conjugates. Acta Chern. Scand. 17:
173-186. 16. Brown, J. L., and J. M. Johnston. 1962. Radio-
assay of lipid components separated by thin-layer chromatography. J. Lipid Res. 3: 480-481.
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17. Folch, J., M. Lees, and G. H. Sloane-Stanley. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chern. 226 : 497-509. 18. Ackerman, R. F., H . Curl, and A. A. Crandall, Jr. 1941. Gastrointestinal tract motility in the absence of bile . Amer. J. Physiol. 134: 32-36. 19. Menguy, R. 1960. Role of biliary and pan-
creatic secretions in the inhibition of gastric motility by fat in the intestine. An experimental study in the rat. Amer. J. Dig. Dis. 5: 792-800. 20. Morgan, R. G. H., and W. J. Simmonds. 1962. The relative effects of diversion of bile to
631
the ileum or to the urinary bladder on fat absorption and gastrointestinal motility in the rat. Quart. J. Exp. Physiol. 47: 352-359. 21. Munk, I. 1890. Ueber die Resorption von Fetten und festen Fettsauren nach Ausschluss der Galle vom Darmkanal. Arch. Path. Anat.122: 302-325. 22. Wilson, T. H . 1962. Intestinal absorption, p. 158-189. W. B. Saunders Company, Philadelphia. 23. Fry, R. J. M., and E . Staffeldt. 1964. Effect of a diet containing sodium deoxycholate on the intestinal mucosa of the mouse. Nature (London) 203 : 1396-1398.