Volpenhein 1964; Senior 1964]. Both these products are soluble in bile salt

Transcription

1 Q. Ji exp. Physiol. (1969) 54, THE MECHANISM OF FAT ABSORPTION IN THE BILE FISTULA RAT. By R. G. H. MORGAN' and B. BORGSTROM. From the Department of Physiological Chemistry, University of Lund, Lund, Sweden. (Received for publication 6th August 1968) Fat absorption was studied in normal and bile fistula rats using test meals of corn oil containing radioactively labelled triolein. Two types of test meal were used, which would produce differently labelled monoglyceride and fatty acid under the action of pancreatic lipase. Triolein containing 3H labelled oleic acid in the 1 and 3 positions and 14C labelled oleic acid in the 2 position was fed to normal and bile fistula rats with and without lymph fistulae. Short term pancreatic lipolysis of this triolein produced 3H labelled fatty acid and 14C labelled 2-monoolein. Fat balance studies, Cori type experiments and the recovery of label in the lymph showed that in bile fistula rats 3H was absorbed in excess of 14C during the first few hr after feeding. In contrast, in lymph fistula rats with a normal bile flow 14C was absorbed in excess of 3H except in the earliest sample. These results indicate that after digestion triglyceride is absorbed mainly as fatty acid in the bile fistula rat, but as a mixture of fatty acid and monoglyceride in the normal animal. Analysis of lymph lipids confirmed that the mechanism of absorption is different in the bile fistula and normal animals. A mixture of triolein labelled with 3H in the glycerol moiety and triolein labelled with 14C in the fatty acid moieties was also used as a marker in corn oil test meals fed to lymph fistula rats with and without bile diversion. In bile fistula rats the early lymph samples contained more 3H than 14C, but from 6 hr onward 14C predominated, and overall more 14C than 3H was recovered in the lymph. It seems probable that the early high recovery of 3H was due to the incorporation of free glycerol into lymph lipids in these animals. The overall recovery of 14C in excess of 3H is consistent with the concept that triglyceride is absorbed chiefly as fatty acid in the absence of bile. In animals with a normal bile flow 14C was absorbed in excess of 3H throughout the recovery period. It is concluded that the mechanism of fat absorption in the bile fistula rat is not the same as it is in the presence of bile. Possibly fatty acid may be absorbed as soaps in the absence of bile, but no mechanism for the absorption of intact monoglyceride by these animals can be suggested. IN the normal animal dietary triglyceride is hydrolysed to a mixture of fatty acid and monoglyceride in the upper small intestine [Mattson and Volpenhein 1964; Senior 1964]. Both these products are soluble in bile salt solutions under conditions found in the small intestine [Hofmann 1963], and in the intact animal bile salts act as a transport vehicle, carrying the products of fat digestion to the absorptive surface in the form of mixed micelles [Hofmann and Borgstrom 1964; Senior 1964]. As a result of recent studies it has become apparent that this micellar solubilization may be of fundamental importance. Thus Ugolev [1965] has calculated that the microvilli of the intestinal mucosa form a close packed array with spaces of less than 200 A between the microvilli. Since fat absorption almost certainly occurs either through the walls of the microvilli [Hogben 1960; C. J. Martin Travelling Fellow of the National Health and Medical Research Council of Australia. Present address: Department of Physiology University of Western Australia, Nedlands, Western Australia. 228

2 Bile and Absorption of Fat 229 Sj6strand 1963] or from between their bases [Palay and Karlin 1959], this imposes a maximum size of ca. 200 A on the liquid droplet if absorption is to occur. Furthermore, it has been shown that in most mammals the intestinal mucosa possesses a tough integral glycocalyx [Ito 1965, Hladik 1966] which 'acts as a mechanical barrier to large particles, but colloidal material, large molecules and nutrients in solution are probably not restricted'-[ito 1965]. In view of this apparently vital role of bile in solubilizing fatty acids and monoglycerides, it is surprising to find that in the absence of bile about 60 per cent of a moderate dose of fat is still absorbed [Borgstrom 1953; Gallagher, Webb and Dawson 1965]. Presumably there is some other mechanism for solubilizing the products of fat digestion in these animals. One possibility is that fatty acids are absorbed slowly as water soluble soaps but the insoluble intact monoglycerides are not absorbed. A study was therefore undertaken to investigate whether in bile fistula rats fat was absorbed as a mixture of fatty acids and monoglyceride as in the normal animal, or as fatty acid alone. This was done by feeding test meals which on digestion produced monoglyceride and fatty acid fractions labelled with different radioactive isotopes. Absorption was studied by a balance technique and by measuring the recovery of radioactive label in the lymph. METHODS Two differing radioactive test meals were used. 1. Doubly labelled triolein (1, 3-oleyl-9, 10-3H-2-oleyl-1-'4C glycerol). - Triolein containing 3H labelled oleic acid in the 1 and 3 positions and 14C labelled oleic acid in the 2 position was prepared by Dr. L. Krabisch of this laboratory. The starting material was 99 + per cent pure 1, 3 dipalmitin isolated from commercial dipalmitin by repeated crystallization from hexane at room temperature. This pure diglyceride was reacted with dihydropyrane under acid conditions to produce the 2-tetrahydropyranyl ether of 1, 3 dipalmitin. The ester bonds were then hydrolysed by alkaline saponification to produce 2-0-tetrahydropyranyl glycerol. This material was reacted with 9, 10-3H oleyl chloride in pyridine and the resultant 1, 3-di (oleyl-9, 10-3H) 2-0-tetrahydropyranyl glycerol isolated. After removal of the protecting tetrahydropyranyl radical by acid hydrolysis, the 1, 3 diolein was further esterified with 1-14C oleyl chloride to produce the final doubly labelled product. The labelled triolein was purified by silicic acid column chromatography and preparative thin layer chromatography on silica gel G (Merck). The final product had a ratio of 3H to 14C activity of 4-2 when counted with the instrumental settings used throughout these studies, and showed better than 98 per cent radiopurity for both isotopes by thin layer chromatography. Lipolysis of the material by rat pancreatic lipase in vitro produced a fatty acid fraction labelled almost exclusively with 3H and a monoglyceride fraction labelled only with 14C (Table VII). The doubly labelled triolein was diluted with commercial triolein to give an activity of approximately 6 x 105 c/min of 3H activity in each 0 4 ml. test meal. 2. Glycerol-fatty acid labelled triolein. - Triolein labelled with 3H in the glycerol moiety and triolein labelled with 14C in the fatty acid moiety were prepared by standard techniques and purified to 99 + per cent radiopurity. These radioactive products were mixed and diluted with commercial triolein to produce a test meal containing approximately 4 x 106 cpm 3H activity per 0 4 ml. test meal. The ratio of 3H to 14C activity in this test meal was 10:1. VOL. LIV, NO

3 230 Morgan and Borgstr6m Absorption of these labelled test meals was studied in six groups of male albino rats weighing approximately 250 gm. Groups 1, 2, 3 and 4 received doubly labelled triolein while Groups 5 and 6 were fed the glycerol-fatty acid labelled meal. The groups used and the test meals fed them are summarized in Table I. TABLE I. Method for studying Group No. Operation Test meal absorption 1. 4 Bile cannulation Emulsified doubly Faecal balance labelled triolein 2. 2 Bile cannulation Emulsified doubly Cori labellad triolein 1 Normal 3. 4 Bile and lymph Unemulsified doubly Lymphatic recovery cannulae labelled triolein Gastric fistula 4. 2 Lymph cannula Unemulsified doubly Lymphatic recovery Gastric fistula labelled triolein 5. 3 Bile and lymph Unemulsified glycerol- Lymphatic recovery cannuloe fatty acid labelled Gastric fistula triolein 6. 3 Lymph cannula Unemulsified glycerol- Lymphatic recovery Gastric fistula fatty acid labelled triolein Group 1. - In four rats bile was diverted by standard techniques and the animals placed in restraint cages. Ninety six hr later 2 ml. of an emulsified triolein test meal containing doubly labelled triolein was fed by gavage. This emulsion was prepared by insonation in 0-15M NaCl solution, and contained 100 jumoles triolein and 5 mg egg phosphatide per ml. The faeces were collected for 72 hr after feeding in 3 x 24 hr fractions. Group 2. - Two bile fistula rats were fed 2 ml. of the emulsified test meal used in Group 1, and killed 3 and 6 hr after feeding. One normal animal was also fed the test meal and killed 6 hr after feeding. The stomach, small intestine and large intestine were removed. The small intestine was washed with 10 ml. saline and the intestinal contents collected. Group 3. - Four bile and lymph fistula rats were prepared as previously described [Morgan, 1964]. At the time of operation a gastric cannula of silicone rubber or flexible PVC was also inserted. The animals were placed in restraint cages and allowed a mixture of 0-64 per cent NaCl and 0-04 per cent KC1 in water to drink. Twenty four hr after operation two rats (rats 21 and 23) were fed, via the gastric cannula, 0-4 ml. unemulsified doubly labelled triolein, followed by 2-0 ml. of 0-9 per cent NaCl solution. Forty eight hr after surgery all animals received a similar test meal. After feeding, the lymph was collected in graduated heparinized tubes hourly for eight hr, then as a four hr fraction followed by a 12-hr sample. In the case of rats 21 and 23, a 0-5 hr fraction collected before feeding the second test meal showed no residual activity from the first meal. Group 4. - In two rats lymph and gastric cannule were inserted, but the bile flow was left intact. These animals were fed 0-4 ml. of unemulsified doubly labelled triolein plus 2 ml. saline 48 hr after operation and lymph was collected as for Group 3.

4 Bile and Absorption of Fat 231 A third rat in this group was fed doubly labelled triolein 24 hr after operation but died during the second test meal. Group 5. - Three animals were prepared as for Group 3 and fed 0 4 ml. glycerolfatty acid labelled triolein 48 hr after operation. Lymph was collected by the same time schedule as for Group 3. Group 6. - Three rats were prepared as for Group 4 but were fed the glycerolfatty acid labelled triolein test meal, 48 hr after surgery. Lymph was collected as for the other groups. Analytical procedures. Faeces. - Each 24 hr collection of faeces was homogenized in 100 ml. methanol/chloroform 1:1 using an MSE homogenizer. After filtration the solvent was evaporated and the lipids saponified by warming for 1 hr at 600 with 50 ml. of 10 per cent potassium hydroxide in 50 per cent aqueous methanol. The mixture was acidified and 25 ml. each of heptane and diethyl ether added to extract the fatty acids. The upper phase was removed from the resultant two-phase system and taken to dryness. The residue was dissolved in 15 ml. of a toluene based liquid scintillation mixture containing 5 g PPO and 0 3 g dimethyl POPOP per litre, and counted in a Packard Tricarb Model Intestinal tissues. - The stomach, small intestine wall and large intestine were added to 10 ml. of a potassium hydroxide solution (50 ml. KOH pellets, 100 ml. distilled water and 300 ml. methanol) and 15 ml. 75 per cent aqueous methanol. The organs were digested for 3 hr in a boiling water bath. After acidification, the fatty acids were extracted into the upper phase by adding water/methanol/ethyl ether/heptane to give final proportions of 1: 1: 1: 1. The upper phase was removed and treated as for the faeces extract. Lymph. - The volume of each lymph fraction was recorded, and the samples were then centrifuged at 2,000 rpm for 10 min. The cell-free lymph was decanted from the pellet of cells. 0 5 ml. of each fraction was pipetted into a liquid scintillation counting vial and freeze dried overnight. Fifteen ml. of a dioxane based scintillation mixture [Brown and Johnston, 1962] was added and the vial was insonated with an ultrasonic generator (Branson, setting 4) for 20 sec to disperse the lyophilized lymph. The vial was then counted for 3H and 14C activity in a Packard Tricarb liquid scintillation spectrometer Model Instrumental settings which gave no tritium counts in the 14C channel and 22-3 per cent 14C counts in the tritium channel were used. A pure 14C sample therefore gave a 3H:14C ratio of C efficiency was 42 per cent and 3H efficiency was 12-5 per cent. Lymph samples prepared by this technique showed no greater quenching than the scintillation mixture alone, as measured by the external standard incorporated in this spectrometer. In Groups 3, 4 and 5 an aliquot of lymph ( ml.) was also extracted with 20 volumes of chloroform/methanol 2:1, according to Folch et al., [1957]. The chloroform extract was taken to dryness, then dissolved in a small volume of petroleum ether and applied to 0 5 ml. thick silica gel G thin layer plates. The plates were developed in hexane/ether/methanol/acetic acid, 90:20:3:2 [Brown and Johnston, 1962]. After evaporating the solvent the lipid fractions were detected with iodine vapor and outlined. The iodine was removed by gentle heating and the spots scraped off into small chromatography columns. Lipids other than phospholipid were eluted from the silicic acid into liquid scintillation counting vials with 6 ml. of ether or ether/chloroform 2: 1. Phospholipids were eluted with 4 ml. of a mixture of chloroform/methanol/formic acid/water (9:9:1:1 by volume) followed by 4 ml. of a mixture of chloroform/methanol/formic acid/water (4: 4: 1: 1 by volume) [Nilsson and Borgstrom 1967]. The solvents were evaporated from each fraction and the lipids dissolved in 15 ml. of the dioxane scintillant and counted, as for the lyophilized lymph samples. The total recovery after extraction and separation on thin layer plates was between 80 and 90 per cent of the expected recovery, based upon activity in lyophilized lymph samples. In all cases where sufficient counts were present to allow accurate counting statistics, the ratio of the total 3H activity

5 232 Morgan and Borgstrom to the total 14C activity was within 0 5 of the 3H/14C ratio in lyophilized lymph samples. Test meals. - The emulsified test meal fed to Group 1 was carried through the same analytical procedure and analysed in conjunction with the faeces samples. The test meals for Group 2 were analysed as for intestinal tissues. Unemulsified triolein test meals fed to Group 3 to 6 were analysed by adding a known weight (20 to 50 mg) of the labelled oil to a counting vial and adding 15 ml. of dioxane scintillant, then counting directly. RESULTS Doubly labelled test meal. - In animals fed doubly labelled triolein, 3H activity should indicate the presence of fatty acid from the 1 and 3 position of the original triglyceride (1, 3-fatty acids) while 14C activity should be a measure of fatty acid from the 2 position (2-fatty acid), either free or as monoglyceride. In the presentation of the results which follow, this equivalence will be assumed. TABLE II. FAECAL RECOVERY OF 3H AND 14C AFTER FEEDING 200,LMOLES EMULSIFIED TRIOLEIN CONTAINING 3H OLEIC ACID IN THE 1, 3 POSITION AND 14C OLEIC ACID IN THE 2 POSITION. Faecal recovery (per cent dose fed) Recovery 3H Rat Day 3H 14C Recovery 14C * Total P Total X X2 61X X Total Total Group 1. - In all four bile fistula rats fed emulsified doubly labelled triolein, more of the fed dose of 14C than of 3H was excreted in the first 24 hr (Table II), i.e. the faeces contained a relative excess of 2-fatty acid. However, in the second and third 24 hr collections more of the fed 3H than 14C was recovered, indicating a relative excess of 1, 3-fatty acid. Over the 72 hr collection period there was approximately equal recovery of both isotopes. These results are shown in Table II in which the faecal recovery of each isotope is presented as a percentage of the dose fed.

6 Bile and Absorption of Fat 233 Group 2. - In the bile fistula rat fed emulsified doubly labelled triolein and killed 3 hr later, the stomach contained a small amount of the test meal showing an isotopic ratio the same as that fed. Most of the test meal was in the small intestine, where the isotope ratio of the contents indicated a relative excess of 2-fatty acid (14C labelled), while the ratio in the intestinal wall indicated a relative excess of 1, 3-fatty acid (3H labelled). Very little TABLE III. RECOVERY OF 3H AND 14C IN STOMACH, SMALL INTESTINE AND LARGE INTESTINE AFTER FEEDING EMULSIFIED TRIOLEIN CONTAINING 3H OLEIc ACID IN 1 AND 3 POSITION AND 14C OLEIc ACID IN THE 2 POSITION. Recovery Time (per cent after dose fed). Recovery 8H Rat Feeding Recovery in 3H 14C R Recovery 14C Bile Diverted.5. 3 Hrs Stomach Small int F content Small int wall Large int Total Hrs Stomach *02 Small int *89 content Small int wall Large int Total O92 Normal 7. 6 Hrs Stomach Small int content Small int wall Large int Total *03 activity was recovered in the large intestine. In the animal killed 6 hr after feeding, the stomach again contained unchanged test meal, but less activity was recovered in the intestine, and the isotopic ratio of the contents indicated only a small excess of 2-fatty acid. The ratio in the wall in this animal also showed a slight excess of 2-fatty acid. This is shown in Table III. In the normal animal very little activity was recovered in any organ. Group 3. - When unemulsified doubly labelled triolein was fed to bile and lymph fistula rats 48 hr after operation very little radioactivity was recovered in the lymph until the second hr after feeding. At this time the ratio of 3H/14C in the lymph was higher than the ratio in the test meal, indicating that a relative excess of 1, 3-fatty acid, compared with 2-fatty acid, had been

7 234 Morgan and Borgstrom ' Q) Hours after feeding Fig. 1. The recovery of 3H and 14C in the lymph of rats after feeding triolein containing 3H labelled oleic acid in the 1 and 3 positions and 14C labelled oleic acid in the 2 position. Lymph activity is expressed as recovery fed 3H recovery fed 14C A value greater than 1 therefore indicates absorption of 1, 3 fatty acid in excess of 2-fatty acid, while a value of less than 1 indicates absorption of 2-fatty acid in excess of 1, 3 fatty acid. * 0 bile fistula rats (Group 3). X-- -X rats with normal bile flow (Group 4).

8 Bile and Absorption of Fat 235 absorbed and transported to the lymph. This ratio rose further, to reach a peak in the third hr, and then fell slowly, reaching the test meal ratio (4.2) between the eighth and twelfth hr. This is shown graphically in fig. 1. In this figure the ratio (recovery 3H/recovery 14C) has been plotted for each lymph fraction collected. A value greater than 1.0 thus indicated a relative excess of 1, 3-fatty acid in the lymph, while a value less than 1-0 indicates a relative excess of 2-fatty acid. Over the 24 hr collection period 38 per cent of the fed 3H activity and 36 per cent of the fed 14C activity was recovered in the lymph (Table IV). Most of this activity appeared between 8 and 24 hr after feeding as is shown in fig. 3, in which the cumulative recovery of the activity as a percentage of total fed is plotted against time. TABLE IV. LYMPHATIC OUTPUT OF RADIOACTIVITY AFTER FEEDING 0 4 ml TRIOLEIN CONTAINING BOTH 3H AND 14C. Percentage recovery of dose fed Group Isotope 0-8 hr 8-12 hr hr Total 3. 3H Bile diverted 14C H 57 3 nil 60 Bile flow normal 14C 63 2 nil H Bile diverted 14C H 80 3 nil 83 Bile flow normal 14C 90 4 nil 94 Groups 3 and 4 received doubly labelled triolein, Groups 5 and 6 glycerol-fatty acid labelled triolein (see text). When the doubly labelled test meal was fed 24 hr after surgery, only one rat produced sufficient activity in the lymph to allow accurate estimation of the 3H/14C ratio. In this animal (rat 21) 22 per cent of the fed 3H and 23 per cent of the fed 14C was recovered, and the 3H/14C ratio in the lymph fluctuated widely. Nevertheless, the same general trend as in the others was apparent. Group 4. - When rats with normal bile flow were fed doubly labelled triolein, a slight excess of 1, 3-fatty acid was recovered in the first hour after feeding. From the second hr onward, however, 2-fatty acid was recovered in excess (fig. 1) and the overall recovery of 3H was less than the overall recovery of 14C. Thus in one rat 54 per cent of fed 3H and 59 per cent of fed 4C was recovered in 8 hr (the 8-12 hr sample was lost) while in the other rat 66 per cent of 3H and 71 per cent of 14C was recovered in 24 hr (Table IV). Absorption of both isotopes was almost complete by 8 hr (fig. 3). In the rat fed the doubly labelled test meal 24 hr after operation, the 3H/14C ratios in the lymph were very similar to those for others in Group 4. In this animal 60 per cent of the fed 3H and 70 per cent of the fed 14C was recovered.

9 236 Morgan and Borgstrom Glycerol-fatty acid labelled test meal. - With this test meal 14C should indicate the presence of fatty acid from any position on the triglyceride, while 3H should be a measure of the glycerol backbone (free or as monoglyceride) SI) 2.0 Q3 Q l Hours after feeding Fig. 2. The recovery of 3H and 14C in the lymph of rats fed a mixture of triolein labelled with 3H in the glycerol moiety and triolein labelled with 14C in the oleic acid moiety. The lymph activity is expressed as in fig. 1. * * bile fistula rats (Group 5). X- - -X rats with normal bile flow (Group 6). Group 5. - When the glycerol-fatty acid labelled triolein test meal was given to three bile and lymph fistula rats, very little activity was recovered in the lymph until the third hr after feeding, at which time the ratio of 3H/14C in the lymph was much higher than the ratio in the test meal. This indicated an initial absorption of glycerol in excess of the fatty acid moiety. As more

10 Bile and Absorption of Fat 237 activity was recovered the lymph ratio gradually fell, to equal the ratio in the test meal at about the seventh hr, and fall below it from this time onwards (fig. 2). Over 24 hr 19 per cent of the fed 3H activity and 27 per cent of the fed 14C activity was recovered (Table IV.) About 75 per cent of the recovered activity for both isotopes was collected between 8 and 24 hr (fig. 3). ln - Ivv A* Q) '3 lb Uj) 0\1 Q), 75F ' 401-! I F ' I 0--- I I *0 Bila fistula rats fed doubly labelled test meal. (Group3) Rats with normal bile flow fed doub I y labe Iled te st mea 1. (Group 4) Bile fistula rats fed glycerol fatty acid labelled test maeal. Rats with normal bile flow fed glycerol fatty acid labelaldtst meal. (Group 6) I n Hours after feeding Fig. 3. Cumulative recovery of 14C expressed as a percentage of dose fed. * * bile fistula rats fed doubly labelled test meal (Group 3). A A rats with normal bile flow fed doubly labelled test meal (Group 4). O O bile fistula rats fed glycerol fatty acid labelled test meal (Group 5). A A rats with normal bile flow fed glycerol fatty acid labelled test meal (Group 6). Group 6. - In this group, the ratio of 3H to 14C activity in the lymph was always less than that in the test meal. The ratio fell progressively up to 7'hr after feeding, but then rose slightly in the last two samples (fig. 2). Over 24 hr 83 per cent of the fed 3H and 94 per cent of the fed 14C was recovered in the lymph, almost all of this in the first 8 hr (Table IV and fig. 3). Lymph composition. - In most samples activity was counted in fractions corresponding to phospholipid plus monoglyceride, diglyceride, fatty acid and triglyceride plus cholesterol ester. With bile fistula rats (rats in Groups 3 and 5) sufficient activity for accurate analysis was not present earlier than 2 hr after feeding, and in rats with normal bile flow the same applied to lymph

11 238 Morgan and Borgstr6m samples later than 12 hr after feeding. The composition of the lymph was fairly constant between 4 and 12 hr after feeding in all groups, but earlier and later samples showed variation. TABLE V. RADIOACTIVITY IN LYMPH LIPID FRACTIONS AFTER FEEDING TRIOLEIN LABELLED WITH 3H OLEIC ACID IN THE 1 AND 3 POSITION AND 14C OLEIC ACID IN THE 2 POSITION. PL AND MG: PHOSPHOLIPID AND MONOGLYCERIDE; DG: DIGLYCERIDE; FA: FATTY ACID; TG AND CE: TRIGLYCERIDE AND CHOLESTEROL ESTERS. Group 3 4 TABLE VI. Time after feeding hours * Time after feeding hours Percentage of activity in Isotope PL & MG DG FA 3H 14C 3H 14C 3H 14C 3H 14C 3H 14C TG & CE RADIOACTIVITY IN LYMPH LIPID FRACTIONS AFTER FEEDING A MIXTUTRE OF TRIOLEIN LABELLED WITH 3H IN THE GLYCEROL MOIETY AND TRIOLEIN LABELLED WITH 14C IN THE FATTY ACID MOIETY (GROUP 5). PL AND MG: PHOSPHOLIPID AND MONOGLYCERIDE; DG: DIGLYCERIDE; FA: FATY ACID; TG: TRIGLYCERIDE. Percentage of activity in Isotope PL & MG DG FA Unknown TG 3H 14C 3H 14C 3H 14C nil nil * 1 rat Group 3. - When the test meal was fed 48 hr after surgery the distribution of radioactivity between the various lymph fractions was almost the same for both isotopes (Table V.) Early and late the lymph contained considerable amounts of phospholipid, diglyceride and free fatty acid, but between 4 and

12 Bile and Absorption of Fat hr these fractions were minor, and almost 90 per cent of both isotopes was in the form of triglyceride. In some samples iodine vapor detected an unidentified fraction between the fatty acid and triglyceride spots. This was always diffuse and difficult to outline, but as it contained small amounts of radioactivity which showed a 3H/14C ratio similar to that in the triglyceride fraction, it has been included in the triglyceride fraction in Table V. TABLE VII. 3H/14C RATIo IN PRODUCTS OF SHORT TERM LIPOLYSIS OF DOUBLY LABELLED TEST MEAL AND LYMPH TRIGLYCERIDE. Ratio 3H/14C in Total Fatty acid Monoglyceride Triglyceride released by formed by fraction Lipolysis Lipolysis Doubly labelled test meal Rat hr bile diverted 8-12 hr hr *3 Rat hr bile flow 6-8 hr normal 8-12 hr hr Group 4. - The composition of the first and second hourly fractions and of the remaining lymph up to 12 hr is shown in Table V. In all fractions a greater percentage of 3H than 14C was incorporated in the diglyceride and fatty acid fractions, while 14C was preferentially incorporated into triglyceride. Triglyceride predominated in all fractions except the first. Group 5. - In Table VI the composition of the lymph collected 4 to 12 hr after feeding Group 5 is shown. In these analyses the silicic acid between the fatty acid and triglyceride spots was also scraped off and eluted to give a fraction of unknown composition which contained radioactivity. Attempts to isolate this material by repeated preparative thin layer chromatography failed to give a pure product after three separations. This isolated material tailed extensively on thin layer chromatography, extending from the diglyceride region to immediately below a triglyceride standard when visualized with iodine vapor. However, a diffuse darker area with an Rf between those of fatty acid and triglyceride could be distinguished. DISCUSSION With both the doubly labelled and the glycerol-fatty acid labelled test meals, bile fistula animals and animals with a normal bile flow showed different patterns of absorption (figs. 1 and 2). Thus it appears that bile fistula animals do not absorb a mixture of fatty acid and monoglyceride of the same composition as normal animals, and it seems likely that fatty acid is absorbed in excess of monoglyceride in the absence of bile. However, a precise estimate

13 240 Morgan and Borgstrom of the relative contribution of fatty acid and monoglyceride to fat absorption in these animals is difficult to obtain. The test meals used in these studies were chosen since short term lipolysis of both should produce fatty acid and monoglyceride fractions labelled with different isotopes. In the case of the doubly labelled triolein this was shown by incubating 10,umoles of the lipid with 10 jul. of rat pancreatic juice for 15 min. The resultant lipid mixture contained fatty acid labelled almost exclusively with 3H and monoglyceride labelled only with 14C (Table VII). However, longer hydrolysis would release 14C labelled fatty acid, either by direct breakdown of 2-monoglyceride [Borgstrom, 1964] or by hydrolysis after isomerization of the 2-monoglyceride to 1-monoglyceride [Mattson and Volpenhein, 1962]. In view of this, probably only the results obtained in the first few hr after feeding this give accurate indication of whether absorption occured as fatty acid or as monoglyceride. The rats in Group 1 absorbed more 1, 3-fatty acid (3H) than 2-fatty acid (14C) on the first day after feeding doubly labelled triolein (Table II). This probably indicates that fatty acid was absorbed in excess of monoglyceride in these animals, and the results in Group 2 are in agreement with these longer term studies. Three hr after feeding (Rat 5, Table III) there was less 3H than 14C in the small intestinal lumen, and more 3H in the wall, indicating preferential absorption of fatty acid at this time. By 6 hr, however, (Rat 6, Table III), the difference in the lumen was much less, and there was a slight excess of 14C over 3H in the wall. This probably resulted from the slow hydrolysis of 14C labelled monoglyceride which had accumulated in the lumen. Such an effect would produce a luminal fatty acid pool with increasing 14C label, and absorbed lipids would therefore show an increasing 14C content with the passage of time. This effect of an increasing contribution of 2-fatty acid in absorbed lipid with the passage of time is also apparent in Group 3 where absorption was monitored by lymph analysis. In these animals early fractions showed a high 3H/14C ratio, indicating absorption of 1, 3-fatty acids in excess of 2-fatty acids. However, the ratio fell slowly, and from about 12 hr onwards the ratio was below that in the test meal. Again this probably indicates that monoglyceride was slowly hydrolysed and absorbed as 14C labelled fatty acid. There is some evidence to support this theory in the analysis of the lymph lipids. In one bile fistula rat fed the doubly labelled test meal the triglyceride fraction was isolated from lymph collected from 6 hr onwards and purified by silicic acid column chromatography. This triglyceride was then subjected to short term hydrolysis by rat pancreatic juice (10 pul of juice for 15 min at room temperature) and the released fatty acid and monoglyceride was isolated and the 3H and I4C activity measured. If 14C was absorbed as monoglyceride and if the monoglyceride was metabolized in the same way in the absence of bile as in its presence. the absorbed 14C would be incorporated in lymph triglyceride almost exclusively in the 2 position. As a result, analysis of the triglyceride as described above should produce a fatty acid fraction with a higher 3H/14C ratio and a monoglyceride fraction with a lower

14 Bile and Absorption of Fat 241 ratio than the triglyceride as a whole. However, it was found that the 3H/14C ratio was almost the same in the fatty acid as in the total triglyceride (Rat 23, Table VII), indicating that in these fractions extensive randomization of the two labels occurred at some stage during absorption and resynthesis of triglyceride. It seems most likely that this occurred in the gut lumen. In contrast to the bile fistula animal, triglyceride from the lymph of an animal with a normal bile flow showed a higher 3H/P4C in the 1 and 3 position than in the total triglyceride, the result which would be expected if absorption occurred as 14C labelled monoglyceride and 3H labelled fatty acid with direct synthesis of triglyceride from these compounds (Rat 12, Table VII). The composition of the lymph from the rats in Group 3 also suggests that absorption occurred as a randomized mixture of 3H and 14C labelled fatty acid. In these samples the distribution of radioactivity between the various lymph fractions was the same for both isotopes even in the earliest sample analysed (Table V). But in rats with a normal bile flow (Group 4), in which absorption probably occurred as two differently labelled molecular species, there was always a greater per cent of 3H than 13C in the diglyceride and fatty acid fractions. In rats with a normal bile flow the doubly labelled triolein test meal resulted in a ratio of 3H/'4C in the lymph below that in the test meal in all lymph samples except the first. This early high ratio is to be expected, as 3H labelled fatty acid will be produced before any significant amount of 14C labelled monoglyceride is formed. The later ratios below that in the test meal may be due to preferential absorption of monoglyceride in the presence of bile, which in turn may reflect the higher solubility of monoglyceride in bile salt solutions compared with fatty acid. Alternatively the low ratio may result from the failure of esterification of a fraction of absorbed fatty acid, with consequent transport of this free fatty acid in the portal blood [Hyun, Vahouny and Treadwell, 1967]. Although Group 4 contained only two animals, a third animal fed 24 hr after surgery showed exactly similar results. It is realized that it would be desirable to include more animals in this and some other groups but unfortunately no more of the doubly labelled triolein was available and the synthesis of more is a formidable task. When the glycerol-fatty acid labelled test meal was fed to bile fistula rats (Group 5) the 3H/14C ratio in the early fractions was higher than that in the test meal. This might indicate that monoglyceride is absorbed in excess of fatty acid soon after feeding, but such a conclusion is inconsistent with the results found in Groups 1, 2 and 3. It seems more likely that the early high ratio results from the rapid absorption of free glycerol and the incorporation of this labelled glycerol into lymph lipids. There is considerable evidence that such incorporation does indeed occur, especially soon after feeding, at least in animals with a normal bile flow [Holt 1964]. If this is the explanation then the fraction of triglyceride undergoing complete hydrolysis must be small, in view of the early excess of 1, 3 fatty acid over 2 fatty acid found in Group 2. However, even a small amount of complete hydrolysis

15 242 2Morgan and Borgstrom would produce an early high ratio, since glycerol is water soluble and would be completely absorbed as soon as it was released even in the absence of bile, whereas fatty acid absorption would be incomplete and delayed. In Group 5 the 3H/14C ratio fell rapidly and very little of either isotope appeared in the lymph before the ratio fell below that in the test meal. About 75 per cent of the total recovery of both isotopes occurred after the ratio had fallen below the ratio found in rats with normal bile flow, and over 24 hr a considerable excess of 14C compared with 3H was recovered (Table IV). These results are consistent with the theory that in the absence of bile the fatty acid produced by lipolysis is more readily absorbed than the monoglyceride. If fatty acid is absorbed in the absence of bile, some mechanism for its fine dispersion must be present. This may occur by solubilization as the sodium soaps. In the bile fistula animals absorption probably occurs from the distal ileum [Morgan 1964] where the ph may be as high as 8 [Robinson 1935]. Under these conditions oleic acid has a small but definite solubility in buffer alone [Hofmann 1968], and transport of oleic acid to the absorptive site could therefore occur by diffusion through the aqueous phase. Such a process would probably be slow, due to the low solubility of fatty acid, which might explain the prolonged fat absorption seen in the bile fistula rats. It is unlikely that significant absorption of intact monoglyceride occurs in the bile fistula animal. The concentration of fatty acid in solution would be below the CMC for these substances, so micellar solubilization of monoglyceride should not occur. However, it is possible that trace amounts of intact monoglyceride could be transported in solution, since, although the solubility of monoglycerides in buffer is very small, there is still slight molecular solubility. In view of the above discussion it might be expected that the aqueous phase from the intestinal lumen of bile fistula animals would contain small amounts of fatty acid and no other lipid. It has been shown that this is indeed the case, at least in humans [Hofmann 1968]. However, more studies of this type are needed. ACKNOWLEDGEMENTS We wish to thank Mr. L. Krabisch for synthesis of the labelled triglyceride and for technical assistance and advice throughout these studies. This investigation was supported in part by grant HE from the National Institute of Health, U.S. Public Health Service; by grant 13X-71 from the Swedish Medical Research Council; and by grants from the Scientific Council of the Swedish Magazine Industry and Albert Pahlssons Foundation, Malmo, Sweden.

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