- Email: [email protected]
Role of Biliary Lecithin in Lymphatic Transport of Fat
73:1362-1367, 1977 Copyright © 1977 by the American Gastroenterological Association
Vol. 73, No. 6
GASTROENTEROLOGY
Printed in U.S A.
ROLE OF BILIARY LECITHIN IN LYMPHATIC TRANSPORT OF FAT PATRICK Tso, B.Sc (HoNs), JoHN A. BALINT, M.D., F.R.C.P., AND WILFRED J. SIMMONDS, M.B. B.S., D.PHIL., F.R.A.C.P. Department of Physiology , The University of Western Australia, Nedlands , Western Australia
This study was undertaken to assess the role of luminal lecithin in the lymphatic transportation of fat as chylomicrons. Two doses of fat, the low and high dose, were fed to two different groups of rats, control and bile fistula. At low dose, infusing at 35 p,moles of total fatty acid per hr of a mixture of oleic acid and monoolein, molar ratio 2:1, solubilized in 55 p,moles of sodium taurocholate, there was no difference in the lymphatic output of absorbed fat during steady state (7th and 8th hour) absorption. Infusing at a high dose, 173 p,moles of total fatty acid per hr of a mixture of oleic acid and monoolein, molar ratio 2:1, solubilized in 55 p,moles of sodium taurocholate, the bile fistula rats had lower triglyceride and phospholipid output, with a higher proportion of oleic acid in lymph lecithin than did control rats. These alterations in bile fistula rats returned to normal by adding 10 p,moles per hr of biliary lecithin to the infusate. We conclude that intraluminal biliary lecithin plays a significant role in the translocation of high doses of absorbed fat into lymph and in the amount and type of lecithin synthesized. Bile salts promote the uptake of fat from the intestinal lumen by solubilizing the products of digestion, fatty acid and monoglyceride. 1 Whether biliary lecithin plays a part in triglyceride absorption is uncertain. Lecithin has been shown to impede uptake of fatty acid by everted sacs in vitro, 2• 3 possibly by expanding micelles and slowing diffusion up to the cell membrane, but such an effect seems unlikely in vivo. However, an effect at a later stage of sbsorption is possible. Mucosal phospholipid (PL) and membrane turnover is increased during fat absorption and the output of PL in lymph, mainly as coating for chylomicrons, is enhanced. It is possible that the luminal supply of PL, much of it of biliary origin, 4 could modulate the formation and extrusion of chylomicrons, by providing lysolecithin or a component such as choline. O'Doherty et al.S found that mucosal content of absorbed radioactive fatty acid was decreased and liver content increased when egg lecithin was added to a gastric test meal of lipid and bile salts fed to bile fistula rats. Egg phosphatidylethanolamine or inositol had no effect. Rodgers, 6 on the other hand, found no difference in mucosal content or absorption of fatty acid Received February 16, 1977. Accepted June 18, 1977. Address requests for r eprints to: Professor W. J . Simmonds, Department of Physiology, The University of Western Australia, Nedlands 6009 , Western Australia. This study was supported by grants from the National Health and Medical Research Council of Australia and The University of Western Australia. Professor Balint's present address is: Department of Medicine, Division of Gastroenterology, Albany Medical College of Union University, Albany, New York 12208. The authors are grateful for the skilled research assistance of Mrs. Heather Kendrick.
during steady lipid in bile salt infusions when phosphatidylethanolamine replaced phosphatidylcholine as a supplement. Neither group monitored the transport of absorbed lipid in lymph. This paper describes experiments in which the transport of absorbed fat was measured directly by lymph fistula while the absorbing cells were placed under a steady load by duodenal infusion of micellar monoolein and oleic acid. Both small and large doses of micellar lipid were given, as it seemed likely that any effect of deficiency of biliary lecithin would be related to the rate of handling of fat by the absorptive cells. From 6th to 8th hr of infusion, output of triglyceride (TG) and PL in lymph was steady whereas luminal recoveries of infused fat were small, indicating that the rate of lipid uptake was about the same as the rate of lipid infusion. Thus the output and composition of PL in lymph could be related to concomitant TG output and to metabolic turnover in the absorptive cell more readily than during the rapid changes after a single dose.
Materials and Methods Animals . Male adult albino rats of the Wistar strain, weighing 200 to 250 g were used. The rats were fasted overnight before operation. The animals were anesthetized with ether before operation. Two groups of rats were used, the control and the test. In both groups, the thoracic duct was cannulated with a clear vinyl tubing (outer diameter 0.8 mm) according to the method of Bollman et al. 7 A silicone tubing (outer diameter 1.6 mm) tipped inside with clear vinyl tubing (outer diameter 1.0 mm) was introduced about 2 em down the duodenum through the fundus of the stomach. The tubing was secured in the duodenum through a transmural suture and the fundal incision was closed by a purse-string suture. In the test animals, the common bile duct was cannulated just below the two hepatic ducts with silicone tubing (outer
1362
December 1977
BILE LECITHIN AND FAT TRANSPORT
diameter 0.5 mm) tipped with polyethylene 10 tubing. Postoperatively, the animals were infused via the i!ltraduodenal tube at a rate of 2.9 ml per hr with saline (145 mM NaCl and 4 mM KCl). The operated animals were allowed to recover for at least 36 hr in restraint cages kept in a warm room (-30°C) before lipid infusions were given. Experimental plan. On the day of experiment, lipid was infused in the same volume of fluid, 2.9 ml per hr, as postoperatively. Control animals received a mixture of oleic acid (OA) and glyceryl-1-monooleate (MO) in molar ratio OA 2:MO 1. This was given either in low dose, 35 fLmoles of total fatty acid per hr, or in high dose, 173 fLmoles per hr. The test animals, all of which had bile fistulae, comprised two subgroups. One subgroup, hereafter designated BF, received either the low dose or high dose lipid mixture used for controls. The other subgroup received a high dose of infusate which was modified by substituting purified biliary lecithin 3.3 mM, for an equivalent amount of fatty acids 6.6 mM, in the OA:MO mixture. This subgroup is hereafter designated BF + PL. No subgroup BF + PL was included for the low dose of lipid because no difference was found between control and BF animals in the lymphatic output of absorbed fat. Composition and preparation of infusate . The lipid composition of infusate in micromoles per milliliter was low doseOA 8, MO 4; high dose-OA 40, MO 20; high dose for group BF + PL-OA 35.6, MO 17.8, biliary lecithin 3.3. In some experiments the lipid was labelled with [V 4 C]oleic acid -0.01 1-LCi per ml. All infusates contained sodium taurocholate 19 1-Lmoles per ml in phosphate-buffered saline (HP04= 6.75mM, H2P04- 16.50 mM, Na 30.0 mM), pH 6.4. On the day of experiment, the appropriate volumes of the stock lipid were taken and evaporated under a stream of nitrogen. Sodium taurocholate, dissolved in phosphate buffer was then added to make up to the lipid concentration and the mixture was sonically disrupted. The low dose infusate was a clear micellar solution, and the high dose infusate with or without phospholipid formed a stable, slightly milky mixture. Samples of infusate were analyzed for glyceride ester and radioactivity at the beginning and end of infusion. Analyses agreed within 5%. Experimental procedure. Lymph was collected into conical graduated centrifuge tubes (containing 2ml of methanol) for 2 hr before lipid infusion. The methanol was added to prevent any possible enzymatic degradation of lipids. This lymph sample was analyzed as the basal lymphatic output before lipid infusion. Lymph samples were collected as above between 0 and 2, 2 and 4 hr, and at the 5th, 6th, 7th, and 8th hr during the 8-hr infusion. At the end of the infusion, the animal was anesthetized and exsanguinated. In some animals, the stomach, the upper small intestine, the lower small intestine, and the colon were tied off separately and contents were eluted twice with 5-ml aliquots of sodium taurocholate solution (5 mM). Aliquots were taken from these washings and radioactivity was determined after mixing with a water-miscible scintillant Aquasolv. Mucosa from the upper and lower intestinal wall were scraped and lipid was extracted with toluene-ethanol 2:1 as described by Clark. 8Aliquots were taken for both radioactivity determination and separation into lipid classes by thin layer chromatography (TLC) using the solvent system petroleum ether-diethyl etheracetic acid (75:15:0.6, v/v/v). Materials. [1-1 4 C]Oleic acid was purchased from the Radiochemical Centre, Amersham, England, and was found by TLC using the above described solvent system to be 97% pure. This was used without purification. Using the same solvent system, unlabeled oleic acid (May & Baker, Victoria,
1363
Australia) and glyceryl-1-monooleate (Calbiochem, San Diego, Calif.) each moved as one spot on TLC. The fatty acid composition of the oleic acid as determined by gas-liquid chromatography was: 14:0, 3%; 15:0, 4.6%; 16:1, 8.1%; 18:1, 76.2%; 18:2, 8.1 %. Monoolein was found to contain >86% oleic acid and the rest as palmitate and palmitoleate. Lipid from rat bile was extracted by the method of Bligh and Dyer, 9 and lecithin was purified by silicic acid column. 10 The purified bile lecithin was found to be chromatographically pure as one spot on TLC using a chloroform-methanol-water system (65:25:4, v/v/v). Sodium taurocholate was synthesized by the method of Lack et al., 11 and checked on TLC using a propionic acid-isoamyl acetate-water-n-propanol system (15:20:5:10, v/v/ v/v). All reagents used were of analytical grade. Solvents were of analytical grade and used without redistillation. Ethanol was redistilled before use. Analytical. Lymph lipid was extracted by the method of Bligh and Dyer 9 and dissolved in chloroform with butylated hydroxy toluene added to prevent oxidation. Aliquots were taken for esterified fatty acid determination 12 and PL determination. 13 Aliquots were also taken and separated into different classes of phospholipids by two dimensional TLC (Silica Gel H, 0.5 mm thickness), according to .the method of Turner and Rouser. 14 The different phospholipids were identified against standards (Serdary Research Laboratories, Ontario, Canada) after spraying with Dittmer-Lester spray for phospholipids. 1" After scraping off the TLC plate, each of the spots was analyzed for its phosphorus content. 13 Lymph lecithin was isolated by TLC using a chloroformmethanol-water system (65:25:4 , v/v/v) and methyl esters of fatty acid were prepared. 16 The fatty acid methyl esters were then analyzed in a 2-m by 4-mm inner diameter column of 5% EGSS-X (ethylene glycol succinate polyester combined with a methyl silicone) on 100 to 120 mesh Aeropak 30 (Varian Aerograph, Walnut Creek, Calif.) in a Varian Aerograph 1400 gas chromatograph. Peaks were identified by their retention time relative to known standards (Harmel Institute, University of Minnesota, Austin, Minn.) and quantified by peak height by width method. 18 Radioactivity determination. Radioactivity of extracted lymph and mucosal lipid was determined after the solvent was evaporated under nitrogen and 10 ml of scintillant containing 4 g of 2,5-diphenyloxazole, 0.05 g of 4-bis-2-(4-methyl5-phenyloxazolyl) benzene in 1 liter of toluene were added. Radioactivity of unextracted lymph and of intestinal contents was measured by adding an aliquot to a water-miscible scintillant, "Aquasolv". This was made by mixing 9 volumes of Readisolv (8.5 g per liter of Fluoralloy TLA as supplied by Beckman Instruments, Palo Alto, Calif.) in toluene with 1 volume of Bio-Solv Formula BBS-3 (supplied by Beckman). Counting was done in a Nuclear Chicago (Des Plaines, Ill.) Isocap 300 counter and the samples were counted for 10 min. The counting efficiency was 92%. Quenching was corrected by the channel ratio method. w Statistics . Where applicable, the results from different groups of experiments were tested by Student's t-test. 20 Differences were considered significant if P < 0.05.
Results Low Load of Infused Fat Lipid output. The fasting output of total esterified fatty acid was lower in the BF group (P < 0.01), as • · expected. Proportionately, TG output was depressed more than PL output, as shown by a significantly higher PL:TG ratio in the BF group (P < 0.05).
1364
TSO ET AL .
Steady infusion of the test meal increased lipid output to steady values within 5 to 6 hr. Total esterified fatty acid and TG output were significantly less in the BF group (P < 0.05 to P < 0.01). The PL output and PL:TG ratios were higher but not significantly so. These results are summarized in figure 1, left. Because the fasting lymph flow and lipid output were measured in each rat before giving the test meal, the effect of the lipid load may be expressed as the excess over fasting value in each rat. The results of this treatment of the data are summarized in figure 1, right. It can be seen that the excess output of total esterified fatty acid in the test group was virtually the same as in controls. There was, on average, more PL and slightly less TG and a higher PL:TG ratio in BF rats but these differences were not statistically significant. Thus, the BF group started with a lower basal
. ·-
2-
<: ~
~
g it
:1 ~
J-
2-8=
o ...... g :::afi!·a·o·o · "2·o
i5
J-5-
IP..,....
1-
1-
0/
·5-
g
'8
6 o- =~to'o
/-
1'0 .1.
:r
i
120-
60-
§
eo-
AD-
~
~
i
AD-
2-
.:
1-
0 ...... 0
T
o- 1--'
0
T
T
0
o- g-o·o·g.2 T
-o·2·(l.g
·5-
"'0 ........
15-
8~
20-
JO-
!2
1o-
- o-o·g·o·D ,o - o· ·o·o Q- , o- 0
T
.,. 'i(''To CJ'
0...
1-
JO-
o
o-o=8•oo
1-S-
o-
i
20-
g-g·g·o-o - · ·o·c
J-
~
§
o- :8:::
/~-?~·~·b 1 1
~,
5-
0
..,....o ,.o•~O·~
J·21-
TIME
IN
0......_.,.
T
T
T o ...o-2-o·O o T l. 1-u ... o·o· T o·oT 1 1
HOURS
FIG. 1. Effect of a low fat infusion on the flow and lipid composition of the lymph. A lipid mixture composed of 35 ~-tmoles per hr of monoolein and oleic acid in molar ratio 1:2 dissolved in 55 /Lmoles of sodium taurocholate was infused in 2.9 ml per hr of buffered saline; 4 BF ( 0 - 0 ) and 3 control (D-0) rats were used. Left, values during and before infusion; right, values with basal output subtracted. All values expressed as mean ± SEM. PL, phospholipid; TG , triglyceride; TEFA , total esterified fatty acid. For BF rats see experimental plan under Materials and Methods.
Vol . 73, No.6
output of endogenous lipid but the response to a small exogenous load was not materially affected. Phospholipid. In fasting lymph of BF rats, lecithin was 20 to 25% lower than in controls (P < 0.01), and other PL classes were correspondingly elevated. During fat infusion these differences progressively diminished until the steady state was reached, when the pattern in BF rats was almost the same as in controls (table 1). Even in fasting lymph the differences were not large and could possibly be explained by a larger proportion of soluble lipoprotein of nonintestinal origin in BF rats. Despite the similarity in the percentage of phospholipid as lecithin, the fatty acid composition of lecithin was strikingly different in the two groups during steady fat absorption (table 2). These differences will be considered later in conjunction with the effects of a higher lipid load.
High Fat Load with and without Biliary Lecithin Lipid output. The fasting lymph flows and lipid outputs in control and bile fistula groups used in these experiments were the same as for the corresponding groups used in the low dose experiments. For conciseness, the results with high dose are therefore presented as a comparison of outputs in excess of fasting for high and low infused lipid loads, in control rats with bile ducts intact (fig. 2, left) and in bile fistula rats (fig. 2, right). In rats with intact bile supply, the TG output increased in proportion to the load and the PL output increased to a lesser extent (fig. 2, left). In bile fistula rats, group BF, TG output also increased but much less than in controls (fig. 2, right, middle curve), whereas the PL output was no greater than with low load. The differences in TG and PL output between control and BF groups were statistically significant (P < 0.01). When lecithin was replaced, group BF + PL, there was a large and significant increase in TG and PL outputs, P < 0.01 compared with group BF. The response in fact, was indistinguishable from controls with bile ducts intact (compare upper curves, fig. 2, left and right). Phospholipid. As for low infused loads, control and bile fistula rats did not differ significantly in the class distribution of lymph PL, when steady outputs had been established. Replacement of lecithin, group BF + PL, did not alter the distribution significantly (table 1). However, there were considerable differences between the groups in the fatty acid composition of lymph lecithin with both low and high loads (table 2). With the low fat load, there was less palmitic acid (C 16:0) and more oleic acid (C 18:1) in lymph lecithin of bile fistula rats than in controls. With the high fat load these differences were accentuated. It is also of interest to compare the effect of increasing the absorptive load in controls with that in the bile fistual groups (BF). In controls, the output of lecithin was considerably greater with the higher load, but changes in the fatty acid composition were small. On the other hand, in groups BF, the lecithin output was no greater with the high load than with the low load,
December 1977
1365
BILE LECITHIN AND FAT TRANSPORT TABLE
Treatment Preinfusion Control (3) BF (4) Low dose Control (3) BF (4) High dose Control (3) BF (4) BF + PL (3)
1. Lymph phospholipid classes (percentage of total phosphorus) a
PC
PE
SPH
PI
LPC
Other
80.2 ::!: 0.5 64.3 ::!: 1.8
2.4 ::!: 0.8 5.6::!: 0.7
3.1 ::!: 0.5 8.7::!:1.0
2.2::!: 0.1 4.4 ::!: 0.8
7.5 ::!: 1.8 15.6 ::!: 1.0
2.5::!: 0.5 1.4 ::!: 1.4
82.5 ::!: 0.8 78.5::!: 2.5
7.5::!: 1.3 4.3 ::!: 1.8
2.3 ::!: 0.4 3.0 ± 1.1
3.4 ::!: 0.2 3.9::!: 0.7
3.7::!: 1.2 6.3 ::!: 1.2
0.7::!: 0.7 4.0 ::!: 1.6
75.9 ::!: 5.1 76.1::!: 1.8 86.0 ::!: 4.0
6.0 ::!: 2.3 3.3::!: 0.8 2.3 ± 0.2
1.7::!: 0.5 3.9 ::!: 1.2 2.4 ::!: 0.4
6.1::!: 1.2 5.5::!: 0.2 2.0 ::!: 1.3
6.6 ::!: 0.4 9.2 ::!: 1.0 4.2 ::!: 1.1
3.7::!: 1.9 2.0::!: 2.0 3.1::!: 1.5
a Values are expressed as mean ::!: SEM. Numbers in parenthesis , number of animals studied. Results for low and high dose are steady values at 7 and 8 hr. Abbreviations: PC, phosphatidyl choline; PE , phosphatidyl ethanolamine; SPH, sphingomyelin; PI, phosphatidyl inositol; LPC, lysophosphatidyl choline. For BF rats and BF + PL rats , see experimental plan under Materials and Methods.
TABLE
Treatment Preinfusion Control BF Low dose Control BF Control vs. BF High dose Control BF Control vs. BF BF + PL a
Values are expressed as m ean ±
SEM .
2. Fatty acid composition of lymph lecithin (mass per cent) a 16:0
18:0
18:1
18:2
20:4
34.6 ::!: 1.6 32 .8 ::!: 2.9
18.7::!: 1.1 20.6::!: 2.7
6.9 ::!: 0.4 14.8 ::!: 1.6
24.9 ::!: 1.6 23.8::!: 3.5
14.9 ::!: 2.5 8.0::!: 1.6
31.3 ::!: 1.2 21.0 ::!: 1.3 p < 0.05
17.6::!: 1.1 22.5::!:0.7 p < 0.05
18.2 ::!: 1.6 31.8 ::!: 2.2 p < 0.02
23.8 ::!: 1. 7 21.7::!: 1.3 NS
8.9::!: 2.3 ~.0::!: 0.2 NS
27 .5::!: 1.0 15.0 ::!: 1.0 p < 0.001 25.4 ± 0.7
13.1 ::!: 0.3 11.4 ± 1.0 NS 13.8 ± 0.9
24.0 ::!: 0.9 48 .7 ± 1.9 p < 0.001 27.3 ± 2.2
27.0 ::!: 0.5 21.8 ± 0.9 p < 0.001 26.8 ± 1.1
8.4 ± 0.2 3.2 ± 0.5 p < 0.001 6.6 ± 1.5
Same animals used as in table 1. NS , not significant. For BF and BF
+ PL, see experimental
plan under Materials and Methods.
but there was a large increase inC 18:1with complemen- on total lymph lipid, mainly TG, was not attributable tary decreases in C 16:0 and C 18:0. These differences to increased synthesis from unlabeled endogenous fatty were statistically significant (P < 0.01). Against this acids. background the effect of biliary lecithin supplement, Discussion group BF + PL, was striking. Although the output of These experiments showed that absence of biliary PL, and of TG, rose to double that in group BF, the percentage of C 18:1 in lymph lecithin was almost lecithin from the intestinal lumen did not impede clearhalved with a commensurate increase in C 16:0. The ance of absorbed fat into the lymph when the absorptive composition as well as the output of lymph lecithin in load was small. However, when the load was increased 5-fold a deficiency in lymphatic transport of absorbed group BF + PL thus became the same as in controls. fat became apparent and this was remedied by addition Recoveries of Labeled Fatty Acid of biliary lecithin to the intestinal infusate. Before In an additional series of rats given the high load, considering the interpretation of these findings three the infusate was labeled with [14 C]oleic acid and recov- aspects will be briefly discussed. These concern the eries of labeled fat were measured in the lymph, the relevance of the experimental conditions to the physiomucosa of the small intestine, and the lumen of the logical situation, the use of chemical estimates of the whole intestinal canal. Recovery from the lumen was output of absorbed fat, and the evidence that differences small in all three groups, with means from 4 to 8.5% of in transport and not uptake were involved. the dose infused (table 3). Very little of the luminal The higher dose of lipid was not physiologically lipid had passed into the colon. These results show that excessive. It corresponded to an absorptive load of 0.4 g lecithin in the. lumen did not promote lipid output in of fat in 8 hr. The PL supplement required to abolish the lymph merely by enhancing uptake from the lumen. the absorptive defect, 10 fLmoles of lecithin per hr, was In group BF + PL (table 3), lymphatic output was also within the physiological range. Similar outputs in also estimated chemically, as output of esterified fatty bile have been recorded in rats shortly after establishacid during infusion in excess of fasting values per ing bile fistulae, before phospholipid output was rehour: Chemical and radioactive estimates agreed duced by reduction of bile acid pool and secretion closely (40.8 and 39.1% of the infused dose, respec- rate. 21• 22 The total amount of bile salts given in 8 hr, tively). This indicated that the effect ofluminallecithin 450 /Lmoles as taurocholate, was similar to the amount
1366
TSO ET AL.
·-
:1:
3-
"i 3:
g
. :1:
:t
2-
~-~
/o-o"~~·o •
1-
~
..~
!;'/
:1:
1120-
T
~ Sao-
..
0
_/
~AO-
~- 0- 0•0•0•0
0
l ~
5
3-
2-
/
0
0::
f/
T
t;•6 6·~'~ ...
~-x
.,~,
X..~
t;
1-
~-g-o·2·g-~
:1:
x-~
l~
...-/
~
::>
t:::.. ..,., ... •t; ~
6
~
al-
~
T/'-
)()-
;-o-D•O•D•D
6
0
·30 ;:
:
T t; ~~~.. 0-;:J:~:~ TTT
·2-
g 0::
·I-
TIME
IN
FIG. 2. Flow and lipid composition of lymph at low and high dose when basal output was subtracted. Left, values are compared for control rats at low ( 0 - 0 ) and high ( 6 - 6 ) fat infusion; right, values for BF rats at low ( 0 - 0 ) and high ( e - e ) dose and BF + PL (e· · · ·e) rats at high dose. PL, phospholipid; TG , triglyceride; TEFA, total esterified fatty acid. For BF rats andBF + PL rats, see experimental plan under Materials and Methods. TABLE
3. Labeled oleic acid recoveries from lymph, lumen, and mucosa expressed as per cent total infused•
Rats
Control (5) BF(3) BF + PL (3)
Lymph
Lumen
Mucosa
Total
40.7 ± 2.4 27 .5 ± 6.2 40.3 ± 4.3
4.0 ± 2.6 8.5 ± 4.2 7.1 ± 3.1
25.5 ± 3.1 15.8 ± 4.5 14.0 ± 3.0
70.1 ± 4.6 51.8 ± 6.7 61.4 ± 4.4
.ci All values expressed as mean ± SEM . Numbers in parenthesis, number of animals studied. For BF and BF + PL rats, see experimental plan under Materials and Methods.
normally recycled in 24 hr, 23 · 24 thus providing an adequate replacement in bile fistula rats. To standardize the physical state of the infused lipid, the same dose of bile salts was also given to rats without bile fistulae. These rats therefore received an excess, but there was no indication that expansion of their bile acid pool affected absorption or other gastrointestinal functions during the 8-hr infusion. The clearance of absorbed fat into the lymph was estimated chemically by subtracting the hourly fasting output of total esterified fatty acid during the 2 hr
Vol . 73, No.6
before fat loading from the successive hourly outputs during fat infusion. This corrected for the differences in fasting (endogenous) output between rats with and without bile fistulae. 21 · 22 There might have been an increase in endogenous fatty acid esterification during fat absorption, but comparison of radioactive (exogenous) and chemical estimates suggested that this was small. At high absorptive rates most of the lymph lipid mass is TG in which the proportion of endogenous fatty acids is low. 25· 26 The small recovery of isotopic lipid from the lumen even in the absence of lecithin showed that the effect of lecithin could not be attributed merely to increased uptake from the lumen. However, absorbed lipid did not accumulate in the mucosa in the absence of luminal phospholipid as it did in the experiments of O'Doherty et al. 5 Control tests gave no evidence of technical deficiencies in recovery or extraction of lipid in the present experiments. Whether the discrepancy is attributable to differences in experimental design can only be resolved by further work. Interrelations of TG and PL output. Normally, PL output in lymph seems to be determined by the output ofTG-rich particles, chylomicrons, and very low density lipoproteins. Exogenous PL added to the normal biliary supply does not increase PL output.27· 28 The low output of PL in fasting lymph of bile fistula rats is associated with a low TG synthesis in the absence of endogenous biliary lipid from the lumen. 21 · 22 Moreover, with a low load of absorbed fat both TG and PL output increased as much in rats with bile fistulae as in those with bile ducts intact; that is, a moderate increase in mucosal PL synthesis was not dependent on the biliary luminal supply. However, a luminal supply of PL appeared to be necessary for transport of a high fat load. Several features of the results indicated that the defect in bile fistula rats was primarily a limited ability to increase mucosal PL turnover. The TG output in lymph, although considerably less than in rats with bile duct intact, was still double that with the low load, whereas the PL output was no higher. Furthermore, substitution of PL, 10 J.Lmoles per hr for an equivalent amount of infused MO and OA, 20 J.Lmoles per hr increased the lymphatic output of TG fatty acid by 50 to 60 J.Lmoles per hr. The concurrent doubling of PL output represented an increase in esterified fatty acid of only 3 J.Lmoles per hr . Lecithin is hydrolyzed to lysolecithin before absorption.29 The results so far have not shown whether it is the supply of lysolecithin or of its component choline which facilitates a high rate of mucosal PL turnover. Analysis of biliary lecithin indicated that it was rich in a-palmityllecithin. When added to the infusate in bile fistula animals there was an increase not only in output of lymph lecithin but also in its palmitic acid content to equal that in bile duct-intact animals. It may be that lecithin synthesis from absorbed lysolecithin is faster than from the Kennedy pathway. 30 On the other hand, O'Doherty et al. 5 reduced mucosal accumulation of isotopic fat in bile fistula rats by adding choline to a gastric test meal.
BILE LECITHIN AND FAT TRANSPORT
December 1977
The mechanism by which increased mucosal turnover of PL facilitated the lymphatic output of triglyceride is also not clear . The assembly and coating of chylomicrons5 or the turnover of all membranes involved in exocytosis are obvious possibilities. 17' 31 Whatever the precise site of lecithin action will prove to be, these studies provide evidence for a role of luminal biliary lecithin in the exit of chylomicrons, synthesized from absorbed fatty acid, from the enterocytes to the lymph. REFERENCES 1. Hoffman AF: Functions of bile in the alimentary canal. In Handbook of Physiology, sect 6: Alimentary Canal vol V. Edited by CF Code . Washington, DC, American Physiological Society, 1968,p.2507-2533 2. Rampone AJ: The effects of bile salt and raw bile on the intestinal absorption of micellar fatty acid in the rat in vitro. J Physiol (Land) 222:679-690, 1972 3. Rodger JB , O'Connor PJ: Effect of phosphatidylcholine on fatty acid and cholesterol absorption from mixed micellar solutions. Biochim Biophys Acta 409:192-200, 1975 4. Borgstrom B: Phospholipid absorption. In Lipid Absorption: Biochemical and Clinical Aspects. Edited by K Rommel , H Goebel!, co-edited by R Bohmer. Lancaster, England, MTP Press Ltd, 1976, p 65-72 5. O'Doherty PJA, Kakis G, Kuksis A: Role of luminal lecithin in intestinal fat absorption. Lipids 8:249-255, 1973 6. Rodgers JB: Lipid absorption in bile fistula rats-lack of a requirement for biliary lecithin. Biochim Biophys Acta 398:92100, 1975 7. Bollman JL, Cain JC, Grindlay, JH: Techniques for the collection of lymph from the liver, small intestine or thoracic duct of the rat. J Lab Clin Med 33:1349-1352, 1949 8. Clark SB: The uptake of oleic acid by rat small intestine. J Lipid Res 12:43-55, 1971 9. Bligh EG, Dyer WJ: A rapid method of total lipid extraction and purifica tion. Can J Biochem Physiol37:911-917, 1959 10. Hanahan DJ, Dittmer JC, Warashina E: A column chromatographic separation of classes of phospholipides. J Bioi Chern 228:685-700, 1957 11. Lack L, Dorrity FO Jr, Walker T, et al: Synthesis of conjugated bile acids by means of a peptide coupling agent. J Lipid Res 14:367-370, 1973 12. Stern I , Shapiro B: A rapid and simple method for the determination of esterified fatty acids and for total fatty acids in blood. J Clin Pathol 6:158-160, 1953 13. Parker F , Peterson NF: Quantitative analysis of phospholipids and phospholipid fatty acids from silica gel thin-layer chromatograms. J Lipid Res 6:455-460, 1965 14. Turner JD, Rouser G: Precise quantitative determination of
1367
human blood lipids by thin-layer and triethylaminoethylcellulose column chromatography. II. Plasma lipids. Anal Biochem 38:437-445, 1970 15. Vaskovsky VE, Kostetsky EY: Modified spray for the detection of phospholipids on thin layer chromatograms. J Lipid Res 9:396, 1968 16. Balint JA, Nyhan WL, Lietman P, et a!: Lipid patterns in Niemann-Pick's disease. J Lab Clin Med 58:548-558, 1961 17. Sabesin SM: Ultrastructural aspects of the intracellar assembly, transport and exocytosis of chylomicrons by rat intestinal absorptive cells. In Lipid Absorption: Biochemical and Clinical Aspects. Edited by K Rommel, H Goebell, co-edited by R Bohmer, Lancaster, England, MTP Press Ltd, 1976, p 113-148 18. Kates M: Techniques of Lipidology-Isolation, Analysis and Identification of Lipids. Amsterdam, North-Holland Publishing Co, 1972, p 466-467 19. Hendler RW: Procedures for simultaneous assay of two ,8-emitting isotopes with the liquid scintillation counting technique. Anal Biochem 7:110-120, 1964 20. Snedecor GW, Cochran WG: Statistical Methods. Sixth edition. Ames, Iowa , Iowa State University Press, 1967 21. Baxter JH: Origin and characteristics of endogenous lipid in thoracic duct lymph in rat. J Lipid Res 7:158-166, 1966 22. Shrivastava BK, Redgrave TG, Simmonds, WJ: The source of endogenous lipid in the thoracic duct lymph of fasting rats. Q J Exp Physiol 52:305-312, 1967 23. Bergstrom S: Metabolism of bile acids. Fed Proc 21 (suppl11):2832, 1962 24. Mok HYI, Perry PM, Dowling RH: The control of bile acid pool size: effect of jejunal resection and phenobarbitone on bile acid metabolism in the rat. Gut 15:247-253, 1974 25. Karmen A, Whyte M, Goodma n DS: Fatty acid esterification and chylomicron formation during fat absorption. 1. Triglycerides and cholesterol esters. J Lipid Res 3:312-321, 1963 26. Simmonds WJ: Fat absorption and chylomicron formation. In Blood Lipids and Lipoproteins: Quantitation, Composition and Metabolism. Edited by GJ Nelson , New York, Wiley - lnterscience, 1962, p 705-743 27. Byers SO, Friedman M: Effect of ingestion of various lipids upon intestinal absorption of phospholipid. Am J Physiol191:8789, 1957 28. Scow RO , Stein Y, Stein 0 : Incorporation of dietary lecithin lysolecithin into lymph chylomicrons in the rat. J Bioi Chern 242:4919-4924, 1967 29. Parthasarathy S, Subbaiah PV, Ganguly J: The mechanism of intestinal absorption of phosphatidylcholine in rats. Biochem J 140:503-508, 1974 30. Kennedy EP: Biosynthesis of complex lipids. Fed Proc 20:934940, 1961 31. Redgrave TG: The role in chylomicron formation of phospholipase activity of intestinal Golgi membranes. Aust J Exp Biol Med Sci 51:427-434, 1973
Vol. 73, No. 6
GASTROENTEROLOGY
Printed in U.S A.
ROLE OF BILIARY LECITHIN IN LYMPHATIC TRANSPORT OF FAT PATRICK Tso, B.Sc (HoNs), JoHN A. BALINT, M.D., F.R.C.P., AND WILFRED J. SIMMONDS, M.B. B.S., D.PHIL., F.R.A.C.P. Department of Physiology , The University of Western Australia, Nedlands , Western Australia
This study was undertaken to assess the role of luminal lecithin in the lymphatic transportation of fat as chylomicrons. Two doses of fat, the low and high dose, were fed to two different groups of rats, control and bile fistula. At low dose, infusing at 35 p,moles of total fatty acid per hr of a mixture of oleic acid and monoolein, molar ratio 2:1, solubilized in 55 p,moles of sodium taurocholate, there was no difference in the lymphatic output of absorbed fat during steady state (7th and 8th hour) absorption. Infusing at a high dose, 173 p,moles of total fatty acid per hr of a mixture of oleic acid and monoolein, molar ratio 2:1, solubilized in 55 p,moles of sodium taurocholate, the bile fistula rats had lower triglyceride and phospholipid output, with a higher proportion of oleic acid in lymph lecithin than did control rats. These alterations in bile fistula rats returned to normal by adding 10 p,moles per hr of biliary lecithin to the infusate. We conclude that intraluminal biliary lecithin plays a significant role in the translocation of high doses of absorbed fat into lymph and in the amount and type of lecithin synthesized. Bile salts promote the uptake of fat from the intestinal lumen by solubilizing the products of digestion, fatty acid and monoglyceride. 1 Whether biliary lecithin plays a part in triglyceride absorption is uncertain. Lecithin has been shown to impede uptake of fatty acid by everted sacs in vitro, 2• 3 possibly by expanding micelles and slowing diffusion up to the cell membrane, but such an effect seems unlikely in vivo. However, an effect at a later stage of sbsorption is possible. Mucosal phospholipid (PL) and membrane turnover is increased during fat absorption and the output of PL in lymph, mainly as coating for chylomicrons, is enhanced. It is possible that the luminal supply of PL, much of it of biliary origin, 4 could modulate the formation and extrusion of chylomicrons, by providing lysolecithin or a component such as choline. O'Doherty et al.S found that mucosal content of absorbed radioactive fatty acid was decreased and liver content increased when egg lecithin was added to a gastric test meal of lipid and bile salts fed to bile fistula rats. Egg phosphatidylethanolamine or inositol had no effect. Rodgers, 6 on the other hand, found no difference in mucosal content or absorption of fatty acid Received February 16, 1977. Accepted June 18, 1977. Address requests for r eprints to: Professor W. J . Simmonds, Department of Physiology, The University of Western Australia, Nedlands 6009 , Western Australia. This study was supported by grants from the National Health and Medical Research Council of Australia and The University of Western Australia. Professor Balint's present address is: Department of Medicine, Division of Gastroenterology, Albany Medical College of Union University, Albany, New York 12208. The authors are grateful for the skilled research assistance of Mrs. Heather Kendrick.
during steady lipid in bile salt infusions when phosphatidylethanolamine replaced phosphatidylcholine as a supplement. Neither group monitored the transport of absorbed lipid in lymph. This paper describes experiments in which the transport of absorbed fat was measured directly by lymph fistula while the absorbing cells were placed under a steady load by duodenal infusion of micellar monoolein and oleic acid. Both small and large doses of micellar lipid were given, as it seemed likely that any effect of deficiency of biliary lecithin would be related to the rate of handling of fat by the absorptive cells. From 6th to 8th hr of infusion, output of triglyceride (TG) and PL in lymph was steady whereas luminal recoveries of infused fat were small, indicating that the rate of lipid uptake was about the same as the rate of lipid infusion. Thus the output and composition of PL in lymph could be related to concomitant TG output and to metabolic turnover in the absorptive cell more readily than during the rapid changes after a single dose.
Materials and Methods Animals . Male adult albino rats of the Wistar strain, weighing 200 to 250 g were used. The rats were fasted overnight before operation. The animals were anesthetized with ether before operation. Two groups of rats were used, the control and the test. In both groups, the thoracic duct was cannulated with a clear vinyl tubing (outer diameter 0.8 mm) according to the method of Bollman et al. 7 A silicone tubing (outer diameter 1.6 mm) tipped inside with clear vinyl tubing (outer diameter 1.0 mm) was introduced about 2 em down the duodenum through the fundus of the stomach. The tubing was secured in the duodenum through a transmural suture and the fundal incision was closed by a purse-string suture. In the test animals, the common bile duct was cannulated just below the two hepatic ducts with silicone tubing (outer
1362
December 1977
BILE LECITHIN AND FAT TRANSPORT
diameter 0.5 mm) tipped with polyethylene 10 tubing. Postoperatively, the animals were infused via the i!ltraduodenal tube at a rate of 2.9 ml per hr with saline (145 mM NaCl and 4 mM KCl). The operated animals were allowed to recover for at least 36 hr in restraint cages kept in a warm room (-30°C) before lipid infusions were given. Experimental plan. On the day of experiment, lipid was infused in the same volume of fluid, 2.9 ml per hr, as postoperatively. Control animals received a mixture of oleic acid (OA) and glyceryl-1-monooleate (MO) in molar ratio OA 2:MO 1. This was given either in low dose, 35 fLmoles of total fatty acid per hr, or in high dose, 173 fLmoles per hr. The test animals, all of which had bile fistulae, comprised two subgroups. One subgroup, hereafter designated BF, received either the low dose or high dose lipid mixture used for controls. The other subgroup received a high dose of infusate which was modified by substituting purified biliary lecithin 3.3 mM, for an equivalent amount of fatty acids 6.6 mM, in the OA:MO mixture. This subgroup is hereafter designated BF + PL. No subgroup BF + PL was included for the low dose of lipid because no difference was found between control and BF animals in the lymphatic output of absorbed fat. Composition and preparation of infusate . The lipid composition of infusate in micromoles per milliliter was low doseOA 8, MO 4; high dose-OA 40, MO 20; high dose for group BF + PL-OA 35.6, MO 17.8, biliary lecithin 3.3. In some experiments the lipid was labelled with [V 4 C]oleic acid -0.01 1-LCi per ml. All infusates contained sodium taurocholate 19 1-Lmoles per ml in phosphate-buffered saline (HP04= 6.75mM, H2P04- 16.50 mM, Na 30.0 mM), pH 6.4. On the day of experiment, the appropriate volumes of the stock lipid were taken and evaporated under a stream of nitrogen. Sodium taurocholate, dissolved in phosphate buffer was then added to make up to the lipid concentration and the mixture was sonically disrupted. The low dose infusate was a clear micellar solution, and the high dose infusate with or without phospholipid formed a stable, slightly milky mixture. Samples of infusate were analyzed for glyceride ester and radioactivity at the beginning and end of infusion. Analyses agreed within 5%. Experimental procedure. Lymph was collected into conical graduated centrifuge tubes (containing 2ml of methanol) for 2 hr before lipid infusion. The methanol was added to prevent any possible enzymatic degradation of lipids. This lymph sample was analyzed as the basal lymphatic output before lipid infusion. Lymph samples were collected as above between 0 and 2, 2 and 4 hr, and at the 5th, 6th, 7th, and 8th hr during the 8-hr infusion. At the end of the infusion, the animal was anesthetized and exsanguinated. In some animals, the stomach, the upper small intestine, the lower small intestine, and the colon were tied off separately and contents were eluted twice with 5-ml aliquots of sodium taurocholate solution (5 mM). Aliquots were taken from these washings and radioactivity was determined after mixing with a water-miscible scintillant Aquasolv. Mucosa from the upper and lower intestinal wall were scraped and lipid was extracted with toluene-ethanol 2:1 as described by Clark. 8Aliquots were taken for both radioactivity determination and separation into lipid classes by thin layer chromatography (TLC) using the solvent system petroleum ether-diethyl etheracetic acid (75:15:0.6, v/v/v). Materials. [1-1 4 C]Oleic acid was purchased from the Radiochemical Centre, Amersham, England, and was found by TLC using the above described solvent system to be 97% pure. This was used without purification. Using the same solvent system, unlabeled oleic acid (May & Baker, Victoria,
1363
Australia) and glyceryl-1-monooleate (Calbiochem, San Diego, Calif.) each moved as one spot on TLC. The fatty acid composition of the oleic acid as determined by gas-liquid chromatography was: 14:0, 3%; 15:0, 4.6%; 16:1, 8.1%; 18:1, 76.2%; 18:2, 8.1 %. Monoolein was found to contain >86% oleic acid and the rest as palmitate and palmitoleate. Lipid from rat bile was extracted by the method of Bligh and Dyer, 9 and lecithin was purified by silicic acid column. 10 The purified bile lecithin was found to be chromatographically pure as one spot on TLC using a chloroform-methanol-water system (65:25:4, v/v/v). Sodium taurocholate was synthesized by the method of Lack et al., 11 and checked on TLC using a propionic acid-isoamyl acetate-water-n-propanol system (15:20:5:10, v/v/ v/v). All reagents used were of analytical grade. Solvents were of analytical grade and used without redistillation. Ethanol was redistilled before use. Analytical. Lymph lipid was extracted by the method of Bligh and Dyer 9 and dissolved in chloroform with butylated hydroxy toluene added to prevent oxidation. Aliquots were taken for esterified fatty acid determination 12 and PL determination. 13 Aliquots were also taken and separated into different classes of phospholipids by two dimensional TLC (Silica Gel H, 0.5 mm thickness), according to .the method of Turner and Rouser. 14 The different phospholipids were identified against standards (Serdary Research Laboratories, Ontario, Canada) after spraying with Dittmer-Lester spray for phospholipids. 1" After scraping off the TLC plate, each of the spots was analyzed for its phosphorus content. 13 Lymph lecithin was isolated by TLC using a chloroformmethanol-water system (65:25:4 , v/v/v) and methyl esters of fatty acid were prepared. 16 The fatty acid methyl esters were then analyzed in a 2-m by 4-mm inner diameter column of 5% EGSS-X (ethylene glycol succinate polyester combined with a methyl silicone) on 100 to 120 mesh Aeropak 30 (Varian Aerograph, Walnut Creek, Calif.) in a Varian Aerograph 1400 gas chromatograph. Peaks were identified by their retention time relative to known standards (Harmel Institute, University of Minnesota, Austin, Minn.) and quantified by peak height by width method. 18 Radioactivity determination. Radioactivity of extracted lymph and mucosal lipid was determined after the solvent was evaporated under nitrogen and 10 ml of scintillant containing 4 g of 2,5-diphenyloxazole, 0.05 g of 4-bis-2-(4-methyl5-phenyloxazolyl) benzene in 1 liter of toluene were added. Radioactivity of unextracted lymph and of intestinal contents was measured by adding an aliquot to a water-miscible scintillant, "Aquasolv". This was made by mixing 9 volumes of Readisolv (8.5 g per liter of Fluoralloy TLA as supplied by Beckman Instruments, Palo Alto, Calif.) in toluene with 1 volume of Bio-Solv Formula BBS-3 (supplied by Beckman). Counting was done in a Nuclear Chicago (Des Plaines, Ill.) Isocap 300 counter and the samples were counted for 10 min. The counting efficiency was 92%. Quenching was corrected by the channel ratio method. w Statistics . Where applicable, the results from different groups of experiments were tested by Student's t-test. 20 Differences were considered significant if P < 0.05.
Results Low Load of Infused Fat Lipid output. The fasting output of total esterified fatty acid was lower in the BF group (P < 0.01), as • · expected. Proportionately, TG output was depressed more than PL output, as shown by a significantly higher PL:TG ratio in the BF group (P < 0.05).
1364
TSO ET AL .
Steady infusion of the test meal increased lipid output to steady values within 5 to 6 hr. Total esterified fatty acid and TG output were significantly less in the BF group (P < 0.05 to P < 0.01). The PL output and PL:TG ratios were higher but not significantly so. These results are summarized in figure 1, left. Because the fasting lymph flow and lipid output were measured in each rat before giving the test meal, the effect of the lipid load may be expressed as the excess over fasting value in each rat. The results of this treatment of the data are summarized in figure 1, right. It can be seen that the excess output of total esterified fatty acid in the test group was virtually the same as in controls. There was, on average, more PL and slightly less TG and a higher PL:TG ratio in BF rats but these differences were not statistically significant. Thus, the BF group started with a lower basal
. ·-
2-
<: ~
~
g it
:1 ~
J-
2-8=
o ...... g :::afi!·a·o·o · "2·o
i5
J-5-
IP..,....
1-
1-
0/
·5-
g
'8
6 o- =~to'o
/-
1'0 .1.
:r
i
120-
60-
§
eo-
AD-
~
~
i
AD-
2-
.:
1-
0 ...... 0
T
o- 1--'
0
T
T
0
o- g-o·o·g.2 T
-o·2·(l.g
·5-
"'0 ........
15-
8~
20-
JO-
!2
1o-
- o-o·g·o·D ,o - o· ·o·o Q- , o- 0
T
.,. 'i(''To CJ'
0...
1-
JO-
o
o-o=8•oo
1-S-
o-
i
20-
g-g·g·o-o - · ·o·c
J-
~
§
o- :8:::
/~-?~·~·b 1 1
~,
5-
0
..,....o ,.o•~O·~
J·21-
TIME
IN
0......_.,.
T
T
T o ...o-2-o·O o T l. 1-u ... o·o· T o·oT 1 1
HOURS
FIG. 1. Effect of a low fat infusion on the flow and lipid composition of the lymph. A lipid mixture composed of 35 ~-tmoles per hr of monoolein and oleic acid in molar ratio 1:2 dissolved in 55 /Lmoles of sodium taurocholate was infused in 2.9 ml per hr of buffered saline; 4 BF ( 0 - 0 ) and 3 control (D-0) rats were used. Left, values during and before infusion; right, values with basal output subtracted. All values expressed as mean ± SEM. PL, phospholipid; TG , triglyceride; TEFA , total esterified fatty acid. For BF rats see experimental plan under Materials and Methods.
Vol . 73, No.6
output of endogenous lipid but the response to a small exogenous load was not materially affected. Phospholipid. In fasting lymph of BF rats, lecithin was 20 to 25% lower than in controls (P < 0.01), and other PL classes were correspondingly elevated. During fat infusion these differences progressively diminished until the steady state was reached, when the pattern in BF rats was almost the same as in controls (table 1). Even in fasting lymph the differences were not large and could possibly be explained by a larger proportion of soluble lipoprotein of nonintestinal origin in BF rats. Despite the similarity in the percentage of phospholipid as lecithin, the fatty acid composition of lecithin was strikingly different in the two groups during steady fat absorption (table 2). These differences will be considered later in conjunction with the effects of a higher lipid load.
High Fat Load with and without Biliary Lecithin Lipid output. The fasting lymph flows and lipid outputs in control and bile fistula groups used in these experiments were the same as for the corresponding groups used in the low dose experiments. For conciseness, the results with high dose are therefore presented as a comparison of outputs in excess of fasting for high and low infused lipid loads, in control rats with bile ducts intact (fig. 2, left) and in bile fistula rats (fig. 2, right). In rats with intact bile supply, the TG output increased in proportion to the load and the PL output increased to a lesser extent (fig. 2, left). In bile fistula rats, group BF, TG output also increased but much less than in controls (fig. 2, right, middle curve), whereas the PL output was no greater than with low load. The differences in TG and PL output between control and BF groups were statistically significant (P < 0.01). When lecithin was replaced, group BF + PL, there was a large and significant increase in TG and PL outputs, P < 0.01 compared with group BF. The response in fact, was indistinguishable from controls with bile ducts intact (compare upper curves, fig. 2, left and right). Phospholipid. As for low infused loads, control and bile fistula rats did not differ significantly in the class distribution of lymph PL, when steady outputs had been established. Replacement of lecithin, group BF + PL, did not alter the distribution significantly (table 1). However, there were considerable differences between the groups in the fatty acid composition of lymph lecithin with both low and high loads (table 2). With the low fat load, there was less palmitic acid (C 16:0) and more oleic acid (C 18:1) in lymph lecithin of bile fistula rats than in controls. With the high fat load these differences were accentuated. It is also of interest to compare the effect of increasing the absorptive load in controls with that in the bile fistual groups (BF). In controls, the output of lecithin was considerably greater with the higher load, but changes in the fatty acid composition were small. On the other hand, in groups BF, the lecithin output was no greater with the high load than with the low load,
December 1977
1365
BILE LECITHIN AND FAT TRANSPORT TABLE
Treatment Preinfusion Control (3) BF (4) Low dose Control (3) BF (4) High dose Control (3) BF (4) BF + PL (3)
1. Lymph phospholipid classes (percentage of total phosphorus) a
PC
PE
SPH
PI
LPC
Other
80.2 ::!: 0.5 64.3 ::!: 1.8
2.4 ::!: 0.8 5.6::!: 0.7
3.1 ::!: 0.5 8.7::!:1.0
2.2::!: 0.1 4.4 ::!: 0.8
7.5 ::!: 1.8 15.6 ::!: 1.0
2.5::!: 0.5 1.4 ::!: 1.4
82.5 ::!: 0.8 78.5::!: 2.5
7.5::!: 1.3 4.3 ::!: 1.8
2.3 ::!: 0.4 3.0 ± 1.1
3.4 ::!: 0.2 3.9::!: 0.7
3.7::!: 1.2 6.3 ::!: 1.2
0.7::!: 0.7 4.0 ::!: 1.6
75.9 ::!: 5.1 76.1::!: 1.8 86.0 ::!: 4.0
6.0 ::!: 2.3 3.3::!: 0.8 2.3 ± 0.2
1.7::!: 0.5 3.9 ::!: 1.2 2.4 ::!: 0.4
6.1::!: 1.2 5.5::!: 0.2 2.0 ::!: 1.3
6.6 ::!: 0.4 9.2 ::!: 1.0 4.2 ::!: 1.1
3.7::!: 1.9 2.0::!: 2.0 3.1::!: 1.5
a Values are expressed as mean ::!: SEM. Numbers in parenthesis , number of animals studied. Results for low and high dose are steady values at 7 and 8 hr. Abbreviations: PC, phosphatidyl choline; PE , phosphatidyl ethanolamine; SPH, sphingomyelin; PI, phosphatidyl inositol; LPC, lysophosphatidyl choline. For BF rats and BF + PL rats , see experimental plan under Materials and Methods.
TABLE
Treatment Preinfusion Control BF Low dose Control BF Control vs. BF High dose Control BF Control vs. BF BF + PL a
Values are expressed as m ean ±
SEM .
2. Fatty acid composition of lymph lecithin (mass per cent) a 16:0
18:0
18:1
18:2
20:4
34.6 ::!: 1.6 32 .8 ::!: 2.9
18.7::!: 1.1 20.6::!: 2.7
6.9 ::!: 0.4 14.8 ::!: 1.6
24.9 ::!: 1.6 23.8::!: 3.5
14.9 ::!: 2.5 8.0::!: 1.6
31.3 ::!: 1.2 21.0 ::!: 1.3 p < 0.05
17.6::!: 1.1 22.5::!:0.7 p < 0.05
18.2 ::!: 1.6 31.8 ::!: 2.2 p < 0.02
23.8 ::!: 1. 7 21.7::!: 1.3 NS
8.9::!: 2.3 ~.0::!: 0.2 NS
27 .5::!: 1.0 15.0 ::!: 1.0 p < 0.001 25.4 ± 0.7
13.1 ::!: 0.3 11.4 ± 1.0 NS 13.8 ± 0.9
24.0 ::!: 0.9 48 .7 ± 1.9 p < 0.001 27.3 ± 2.2
27.0 ::!: 0.5 21.8 ± 0.9 p < 0.001 26.8 ± 1.1
8.4 ± 0.2 3.2 ± 0.5 p < 0.001 6.6 ± 1.5
Same animals used as in table 1. NS , not significant. For BF and BF
+ PL, see experimental
plan under Materials and Methods.
but there was a large increase inC 18:1with complemen- on total lymph lipid, mainly TG, was not attributable tary decreases in C 16:0 and C 18:0. These differences to increased synthesis from unlabeled endogenous fatty were statistically significant (P < 0.01). Against this acids. background the effect of biliary lecithin supplement, Discussion group BF + PL, was striking. Although the output of These experiments showed that absence of biliary PL, and of TG, rose to double that in group BF, the percentage of C 18:1 in lymph lecithin was almost lecithin from the intestinal lumen did not impede clearhalved with a commensurate increase in C 16:0. The ance of absorbed fat into the lymph when the absorptive composition as well as the output of lymph lecithin in load was small. However, when the load was increased 5-fold a deficiency in lymphatic transport of absorbed group BF + PL thus became the same as in controls. fat became apparent and this was remedied by addition Recoveries of Labeled Fatty Acid of biliary lecithin to the intestinal infusate. Before In an additional series of rats given the high load, considering the interpretation of these findings three the infusate was labeled with [14 C]oleic acid and recov- aspects will be briefly discussed. These concern the eries of labeled fat were measured in the lymph, the relevance of the experimental conditions to the physiomucosa of the small intestine, and the lumen of the logical situation, the use of chemical estimates of the whole intestinal canal. Recovery from the lumen was output of absorbed fat, and the evidence that differences small in all three groups, with means from 4 to 8.5% of in transport and not uptake were involved. the dose infused (table 3). Very little of the luminal The higher dose of lipid was not physiologically lipid had passed into the colon. These results show that excessive. It corresponded to an absorptive load of 0.4 g lecithin in the. lumen did not promote lipid output in of fat in 8 hr. The PL supplement required to abolish the lymph merely by enhancing uptake from the lumen. the absorptive defect, 10 fLmoles of lecithin per hr, was In group BF + PL (table 3), lymphatic output was also within the physiological range. Similar outputs in also estimated chemically, as output of esterified fatty bile have been recorded in rats shortly after establishacid during infusion in excess of fasting values per ing bile fistulae, before phospholipid output was rehour: Chemical and radioactive estimates agreed duced by reduction of bile acid pool and secretion closely (40.8 and 39.1% of the infused dose, respec- rate. 21• 22 The total amount of bile salts given in 8 hr, tively). This indicated that the effect ofluminallecithin 450 /Lmoles as taurocholate, was similar to the amount
1366
TSO ET AL.
·-
:1:
3-
"i 3:
g
. :1:
:t
2-
~-~
/o-o"~~·o •
1-
~
..~
!;'/
:1:
1120-
T
~ Sao-
..
0
_/
~AO-
~- 0- 0•0•0•0
0
l ~
5
3-
2-
/
0
0::
f/
T
t;•6 6·~'~ ...
~-x
.,~,
X..~
t;
1-
~-g-o·2·g-~
:1:
x-~
l~
...-/
~
::>
t:::.. ..,., ... •t; ~
6
~
al-
~
T/'-
)()-
;-o-D•O•D•D
6
0
·30 ;:
:
T t; ~~~.. 0-;:J:~:~ TTT
·2-
g 0::
·I-
TIME
IN
FIG. 2. Flow and lipid composition of lymph at low and high dose when basal output was subtracted. Left, values are compared for control rats at low ( 0 - 0 ) and high ( 6 - 6 ) fat infusion; right, values for BF rats at low ( 0 - 0 ) and high ( e - e ) dose and BF + PL (e· · · ·e) rats at high dose. PL, phospholipid; TG , triglyceride; TEFA, total esterified fatty acid. For BF rats andBF + PL rats, see experimental plan under Materials and Methods. TABLE
3. Labeled oleic acid recoveries from lymph, lumen, and mucosa expressed as per cent total infused•
Rats
Control (5) BF(3) BF + PL (3)
Lymph
Lumen
Mucosa
Total
40.7 ± 2.4 27 .5 ± 6.2 40.3 ± 4.3
4.0 ± 2.6 8.5 ± 4.2 7.1 ± 3.1
25.5 ± 3.1 15.8 ± 4.5 14.0 ± 3.0
70.1 ± 4.6 51.8 ± 6.7 61.4 ± 4.4
.ci All values expressed as mean ± SEM . Numbers in parenthesis, number of animals studied. For BF and BF + PL rats, see experimental plan under Materials and Methods.
normally recycled in 24 hr, 23 · 24 thus providing an adequate replacement in bile fistula rats. To standardize the physical state of the infused lipid, the same dose of bile salts was also given to rats without bile fistulae. These rats therefore received an excess, but there was no indication that expansion of their bile acid pool affected absorption or other gastrointestinal functions during the 8-hr infusion. The clearance of absorbed fat into the lymph was estimated chemically by subtracting the hourly fasting output of total esterified fatty acid during the 2 hr
Vol . 73, No.6
before fat loading from the successive hourly outputs during fat infusion. This corrected for the differences in fasting (endogenous) output between rats with and without bile fistulae. 21 · 22 There might have been an increase in endogenous fatty acid esterification during fat absorption, but comparison of radioactive (exogenous) and chemical estimates suggested that this was small. At high absorptive rates most of the lymph lipid mass is TG in which the proportion of endogenous fatty acids is low. 25· 26 The small recovery of isotopic lipid from the lumen even in the absence of lecithin showed that the effect of lecithin could not be attributed merely to increased uptake from the lumen. However, absorbed lipid did not accumulate in the mucosa in the absence of luminal phospholipid as it did in the experiments of O'Doherty et al. 5 Control tests gave no evidence of technical deficiencies in recovery or extraction of lipid in the present experiments. Whether the discrepancy is attributable to differences in experimental design can only be resolved by further work. Interrelations of TG and PL output. Normally, PL output in lymph seems to be determined by the output ofTG-rich particles, chylomicrons, and very low density lipoproteins. Exogenous PL added to the normal biliary supply does not increase PL output.27· 28 The low output of PL in fasting lymph of bile fistula rats is associated with a low TG synthesis in the absence of endogenous biliary lipid from the lumen. 21 · 22 Moreover, with a low load of absorbed fat both TG and PL output increased as much in rats with bile fistulae as in those with bile ducts intact; that is, a moderate increase in mucosal PL synthesis was not dependent on the biliary luminal supply. However, a luminal supply of PL appeared to be necessary for transport of a high fat load. Several features of the results indicated that the defect in bile fistula rats was primarily a limited ability to increase mucosal PL turnover. The TG output in lymph, although considerably less than in rats with bile duct intact, was still double that with the low load, whereas the PL output was no higher. Furthermore, substitution of PL, 10 J.Lmoles per hr for an equivalent amount of infused MO and OA, 20 J.Lmoles per hr increased the lymphatic output of TG fatty acid by 50 to 60 J.Lmoles per hr. The concurrent doubling of PL output represented an increase in esterified fatty acid of only 3 J.Lmoles per hr . Lecithin is hydrolyzed to lysolecithin before absorption.29 The results so far have not shown whether it is the supply of lysolecithin or of its component choline which facilitates a high rate of mucosal PL turnover. Analysis of biliary lecithin indicated that it was rich in a-palmityllecithin. When added to the infusate in bile fistula animals there was an increase not only in output of lymph lecithin but also in its palmitic acid content to equal that in bile duct-intact animals. It may be that lecithin synthesis from absorbed lysolecithin is faster than from the Kennedy pathway. 30 On the other hand, O'Doherty et al. 5 reduced mucosal accumulation of isotopic fat in bile fistula rats by adding choline to a gastric test meal.
BILE LECITHIN AND FAT TRANSPORT
December 1977
The mechanism by which increased mucosal turnover of PL facilitated the lymphatic output of triglyceride is also not clear . The assembly and coating of chylomicrons5 or the turnover of all membranes involved in exocytosis are obvious possibilities. 17' 31 Whatever the precise site of lecithin action will prove to be, these studies provide evidence for a role of luminal biliary lecithin in the exit of chylomicrons, synthesized from absorbed fatty acid, from the enterocytes to the lymph. REFERENCES 1. Hoffman AF: Functions of bile in the alimentary canal. In Handbook of Physiology, sect 6: Alimentary Canal vol V. Edited by CF Code . Washington, DC, American Physiological Society, 1968,p.2507-2533 2. Rampone AJ: The effects of bile salt and raw bile on the intestinal absorption of micellar fatty acid in the rat in vitro. J Physiol (Land) 222:679-690, 1972 3. Rodger JB , O'Connor PJ: Effect of phosphatidylcholine on fatty acid and cholesterol absorption from mixed micellar solutions. Biochim Biophys Acta 409:192-200, 1975 4. Borgstrom B: Phospholipid absorption. In Lipid Absorption: Biochemical and Clinical Aspects. Edited by K Rommel , H Goebel!, co-edited by R Bohmer. Lancaster, England, MTP Press Ltd, 1976, p 65-72 5. O'Doherty PJA, Kakis G, Kuksis A: Role of luminal lecithin in intestinal fat absorption. Lipids 8:249-255, 1973 6. Rodgers JB: Lipid absorption in bile fistula rats-lack of a requirement for biliary lecithin. Biochim Biophys Acta 398:92100, 1975 7. Bollman JL, Cain JC, Grindlay, JH: Techniques for the collection of lymph from the liver, small intestine or thoracic duct of the rat. J Lab Clin Med 33:1349-1352, 1949 8. Clark SB: The uptake of oleic acid by rat small intestine. J Lipid Res 12:43-55, 1971 9. Bligh EG, Dyer WJ: A rapid method of total lipid extraction and purifica tion. Can J Biochem Physiol37:911-917, 1959 10. Hanahan DJ, Dittmer JC, Warashina E: A column chromatographic separation of classes of phospholipides. J Bioi Chern 228:685-700, 1957 11. Lack L, Dorrity FO Jr, Walker T, et al: Synthesis of conjugated bile acids by means of a peptide coupling agent. J Lipid Res 14:367-370, 1973 12. Stern I , Shapiro B: A rapid and simple method for the determination of esterified fatty acids and for total fatty acids in blood. J Clin Pathol 6:158-160, 1953 13. Parker F , Peterson NF: Quantitative analysis of phospholipids and phospholipid fatty acids from silica gel thin-layer chromatograms. J Lipid Res 6:455-460, 1965 14. Turner JD, Rouser G: Precise quantitative determination of
1367
human blood lipids by thin-layer and triethylaminoethylcellulose column chromatography. II. Plasma lipids. Anal Biochem 38:437-445, 1970 15. Vaskovsky VE, Kostetsky EY: Modified spray for the detection of phospholipids on thin layer chromatograms. J Lipid Res 9:396, 1968 16. Balint JA, Nyhan WL, Lietman P, et a!: Lipid patterns in Niemann-Pick's disease. J Lab Clin Med 58:548-558, 1961 17. Sabesin SM: Ultrastructural aspects of the intracellar assembly, transport and exocytosis of chylomicrons by rat intestinal absorptive cells. In Lipid Absorption: Biochemical and Clinical Aspects. Edited by K Rommel, H Goebell, co-edited by R Bohmer, Lancaster, England, MTP Press Ltd, 1976, p 113-148 18. Kates M: Techniques of Lipidology-Isolation, Analysis and Identification of Lipids. Amsterdam, North-Holland Publishing Co, 1972, p 466-467 19. Hendler RW: Procedures for simultaneous assay of two ,8-emitting isotopes with the liquid scintillation counting technique. Anal Biochem 7:110-120, 1964 20. Snedecor GW, Cochran WG: Statistical Methods. Sixth edition. Ames, Iowa , Iowa State University Press, 1967 21. Baxter JH: Origin and characteristics of endogenous lipid in thoracic duct lymph in rat. J Lipid Res 7:158-166, 1966 22. Shrivastava BK, Redgrave TG, Simmonds, WJ: The source of endogenous lipid in the thoracic duct lymph of fasting rats. Q J Exp Physiol 52:305-312, 1967 23. Bergstrom S: Metabolism of bile acids. Fed Proc 21 (suppl11):2832, 1962 24. Mok HYI, Perry PM, Dowling RH: The control of bile acid pool size: effect of jejunal resection and phenobarbitone on bile acid metabolism in the rat. Gut 15:247-253, 1974 25. Karmen A, Whyte M, Goodma n DS: Fatty acid esterification and chylomicron formation during fat absorption. 1. Triglycerides and cholesterol esters. J Lipid Res 3:312-321, 1963 26. Simmonds WJ: Fat absorption and chylomicron formation. In Blood Lipids and Lipoproteins: Quantitation, Composition and Metabolism. Edited by GJ Nelson , New York, Wiley - lnterscience, 1962, p 705-743 27. Byers SO, Friedman M: Effect of ingestion of various lipids upon intestinal absorption of phospholipid. Am J Physiol191:8789, 1957 28. Scow RO , Stein Y, Stein 0 : Incorporation of dietary lecithin lysolecithin into lymph chylomicrons in the rat. J Bioi Chern 242:4919-4924, 1967 29. Parthasarathy S, Subbaiah PV, Ganguly J: The mechanism of intestinal absorption of phosphatidylcholine in rats. Biochem J 140:503-508, 1974 30. Kennedy EP: Biosynthesis of complex lipids. Fed Proc 20:934940, 1961 31. Redgrave TG: The role in chylomicron formation of phospholipase activity of intestinal Golgi membranes. Aust J Exp Biol Med Sci 51:427-434, 1973