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Effects of Controlled Interruption of the

Enterohepatic Circulation of Bile Salts by Biliary
Diversion and by Ileal Resection on Bile Salt Secretion,
Synthesis, and Pool Size in the Rhesus Monkey
technical assistance of JOHN Picorr
From the Departments of Medicine and Surgery, Boston University School of
Medicine, Boston, Massachusetts 02118

ABSTRA CT The effects of controlled interruption The effects of interruption of the EHC by the stream-
of the enterohepatic circulation (EHC) of bile salts splitter were compared with those produced by resection
by biliary diversion on bile volume, bile salt secretion of the distal one-third or two-thirds of small bowel.
and synthesis rates, bile salt pool size, and the rela- While ileal resection appreciably reduced bile salt se-
tionship to fecal fat excretion were studied in 16 rhesus cretion, the EHC was by no means abolished. Bile salt
monkeys. reabsorption from the residual intestine was greater
Bile from a chronic bile fistula was returned to the after one-third than after two-thirds small bowel re-
intestine through an electronic stream-splitter which, by section. These observations suggest that jejunal reab-
diverting different percentages of bile to a collecting sorption of bile salts occurs and may well contribute to
system, provided graded and controlled interruption of the normal EHC.
the EHC.
The increase in hepatic bile salt synthesis in response INTRODUCTION
to interruption of the EHC was limited and reached a
maximum rate at 20% interruption of the EHC. Up to Bile salts are synthesized in the liver from cholesterol
this level of biliary diversion, the increased hepatic syn- and after conjugation with glycine or taurine are se-
thesis compensated for bile salt loss so that bile salt se- creted in the bile and pass into the duodenum. Having
cretion and pool size were maintained at normal levels. contributed to fat absorption in the upper small bowel,
With diversion of 33% or more, there was no further in- they then pass along the intestine and are largely reab-
crease in hepatic bile salt synthesis to compensate for sorbed by an active transport mechanism in the ileum
external loss, and as a result there was diminished bile (1, 2) to return to the liver via the portal vein (3-5).
salt secretion, a reduction in bile salt pool size, and The efficiency of this enterohepatic circulation (EHC)
steatorrhea was observed. means that only a small percentage (less than 5% each
day) (6-8) of the circulating bile salts escape reabsorp-
This work was presented in part at the Annual Meeting tion to appear in the feces. To maintain a steady state,
of the American Society for Clinical Investigation, Atlantic therefore, the liver is normally required to synthesize
City, N. J. and reported in part in abstract form (Dowling, only enough bile salts to replace the fecal loss.
R. H., and D. M. Small. 1968. The effect of controlled in- It is now well-known that ileal disease or resection
terruptions of the enterohepatic circulation on the composi-
tion of bile in the Rhesus monkey. J. Clin. Invest. 47: 26a. may cause bile salt malabsorption (9-16), but it is not
[Abstr.] ). known to what extent the liver may compensate for
Dr. Dowling's present address is Department of Medicine, such interruption of the EHC by increasing its bile
Royal Postgraduate Medical School, London, W. 12, England. salt synthesis. Furthermore, the net effect of ileal dys-
Dr. Small is a Markle Scholar in Academic Medicine.
Received for publication 22 June 1969 and in revised form function on bile salt secretion clearly depends not only on
18 September 1969. the adaptive increase in hepatic synthesis, but also upon

232 The Journal of Clinical Investigation Volume 49 1970

converted diphosphopyridine nucleotide ing degrees of interruption of the EHC and to show (DPN) to its reduced form DPNH which was then mea- how this is related to the effective circulating bile salt sured in a Beckman DU2 spectrophotometer at 340 miu. bile samples were brought to room tem- bile salt secretion (total bile salt output by the liver. This enzyme. phospholipid. and cholesterol with feces. version was recorded at 12.) D. Ahrens. Large female rhesus monkeys were and Miettinen (8). salt loss (8). Small. six animals having small biliary diversions (5. pool size.24 Amole/0. bile salt synthesis was also obtained in eight control monkeys agement of the experimental animals. bile salt synthesis of interruption of the EHC on the composition of bile must equal loss.4 To determine bile salt synthesis. 33. and 0. only fecal and biliary bile salt loss were mea- turn to the duodenum. R. dium taurocholate in methanol were also measured enzy- matically to obtain a standard curve. diluted in spectranalyzed methanol (usually 1: 20 dilution). (TLC). Bile composition was then studied in of man (18. 66.. Boston. fold: (a) to measure the changes in bile volume and For analysis. The stools for bile acid determination were collected during the last 3 days of the steady-state period The general design of the experimental model used in these at each of the different levels of biliary diversion.200C until analyzed. perature. 0. a representative cooker. Since. 5% of the bile was collected (the mini- other areas of the intestine. the type of restraining by measuring fecal bile acid excretion before abdominal chair and electronic equipment used. therefore. and (d) containing 0. Experimental mnodel. Cholesterol and bile composition after interruption of the enterohepatic circulation of the Rhesus 3 Details of the method may be obtained on request from monkey. When this residue (containing the bile acids) was vestigation. and in turn bile salt loss is the sum of that removed by the stream-splitter plus that excreted in the in respect to bile salt. 1969. and the 3-OH bile acids were then trained to sit in restraining chairs before surgical im. The biliary scribed above for biliary bile acids. and the bile acid concentration measured. The vol- the enterohepatic circulation of bile. Fecal bile acids were extracted with (17) but may be summarized as follows: modifications of the method described by Grundy. 20. duced by the stream-splitter with that produced by ileal Bile acid synthesis. R. Laboratory procedures (collection of samples). described (8). the steady state for 1 wk intervals at 10. measured with essentially the same enzymatic method de- plantation of biliary and upper intestinal fistulae. by oxidizing the 3-OH group of the bile acid molecules. E. which includes both newly synthesized and recycled Biliary bile acids. Controlled Interruption of the Enterohepatic Circulation of Bile Salts 233 . we designed an ex. Boston University Medical School. (22). 17: 301. this level of inter- perimental model to study the rhesus monkey (17) in ruption of the EHC was taken as the "base line" for the which the composition of the bile closely resembles that experimental model. and turned to the intestine 2 through an electronic stream-splitter extraction of neutral sterols were followed as previously which diverted different percentages of the bile to a col. procedure followed. or in some cases to the distal stomach. was measured enzymatically with hydroxysteroid dehy- tion of the EHC. Rigorous saponification was carried out in lecting system. acid excretion was measured in 3-day stool collections from stone formation (21 ). M. steel bombs overnight at 110°C. synthesis. rather than in a pressure trolled degree of interruption of the EHC. of bile were then quickly frozen at . 4 May 1969. Studies in one 'Small quantities of bile salts are also lost each day in monkey showed no significant difference in the levels of bile the urine and through the skin. 2The bile was returned either to the proximal duodenum Mass. (b) to examine the adaptive increase drogenase using a modification (20) 8 of Talalay's method in de novo hepatic bile salt synthesis in relation to vary. at each level of interruption resection. and the experimental surgery.or 24-hr intervals and samples The purpose of the present communication is four. CGn. With each series of bile acid estimations. Small. After column chromatography with Florisil (in an sample of bile for analysis. M. and a measure of the volume of attempt to separate bile acids from fatty acids) and extrac- bile produced during any given period of time. steady-state conditions were reached for bile A subsequent publication' will deal with the effect salt secretion (and for bile volume). Total bile acid concentration in bile bile salts) in response to varying degrees of interrup. 02118. excretion are responsible for less than 2%o of the total bile first with bile returned to the stomach and later with re. (c) to show how bile salt secretion. Since bile salt secretion mum level of biliary diversion which consistently provided cannot be studied directly in man. The model thus provided a varied and con.the amount of bile salts which may be reabsorbed from In each monkey.1 of pure so- to compare the effects of interruption of the EHC pro. H. of the EHC. mild saponification. tion with ethyl acetate and water. 19). has already been published in detail Fecal bile acids. and 100%o The model has been used to study various aspects of biliary diversion. cannula drained the total bile output which was then re. Rcs. three solutions and pool size are related to fecal fat excretion. but since these routes of volume and bile salt secretion when these were measured. care and man. 10 and 20%o diversions) and six animals having large biliary diversions METHODS (33 and 66%o). Normal studies. sufficient volume for analysis) and since 95%o of the bile was still being returned to the intestine. (Dowling. and this paper is ume of bile produced at the various levels of biliary di- the first of a series of articles describing our findings.12. together with details of the surgery. The steps of homogenization. fecal bile emphasis on cholesterol solubility (20) in bile and gall. as long-chain fatty acids on thin-layer chromatography cal Research and the American Society for Clinical In. sured in this study. and dissolved in methanol and added to the mixture for enzy- D. Mack.06. the residue in the flask usually still contained an oily material which had the same 1 Presented in part to the American Federation for Clini.

Dowling. and Sjdvall (25) have shown by gas-liquid chroma- acids from the bile acid containing residue by TLC using tography (GLC) and mass spectrometry that. Bile salt secretion after the low point is due exclusively to bile salt synthesis by the liver. and Eneroth. Lindstedt. in addition to Florisil column chromatog. and Sjdvall (23). Bile salt pool size. in man. Mack. before the addition of enzyme. 1). Mass. An estimate of basal synthesis (the solid bar on the left. the bile acids remained at or near the of the fecal bile acids contain the 3-OH group. and sulphuric acid identification. bile salt secretion is similar to that seen at 5% interruption. is shown by the stippled bar.01 mmoles. '34 R. phos. 29). For the first 1-3 hr after the acute recovered. which rises gradually over the next 24 hr to reach a new steady state. The 24 hr secretion rate over the subsequent 7 days and the mean value in the new steady state are shown on the right.0 BILE 16. The bile salts secreted from the time of interruption until the low point is reached give a measure of the circulating bile salt pool size (hatched area) which here was 1. H.01 mmoles/24 hr). quate for the present investigation. By plotting the changes in bile salt The bile acids were eluted from the silica with methanol secretion at short time intervals after an "intact" EHC and then estimated enzymatically. M." the secre- tion rate falls and a low point is reached in 4i hr.or 18. E. Eneroth.0 _ 6. the enzy- point of application and were clearly separated from the matic bile acid determination used here was considered ade- contaminating fatty acids. Deoxycholic acid-24-14C (5% biliary diversion) is interrupted with a complete bile (Tracerlab. The increase in hepatic synthesis at 100% interruption of the EHC. during the next 3 hr. PER CENT INTERRUPTION a 5. the a hexane: ether: acetic acid (60: 40: 1) solvent system. a "washout curve" is ob- homogenate as an internal standard to check the recovery tained (26-28) from which several measurements may be of bile acids. OF EHC *01 ACUTE CHANGE 20. Small .58 mmole/24 hr) may be obtained by extrapolating (the lower broken hori- zontal line) from the low point (26. was often bile acids which might be present in feces.0 24 hr t ~ I 10. Usually > 90%o of the internal standard was made (refer to Fig. as the bile salts which remain in the EHC are "washed out. Radioactivity was counted in Bray's solution change from 5 to 100%o biliary diversion. ~~~~INCREASED ~~~~~~~~ ~~~~SYNTHESIS ~~~~~~~~ LOW 4. For this reason. Hellstr6m. since so high as to make bile acid determination impossible. over the basal synthesis. bile salt secretion is using a Packard Tri-Carb liquid scintillation counter (model maintained at the same level as that observed during the 314E). predominant fecal bile acids are nonketonic and that most With this system.0 BASAL SYNTHESIS 0 0 1 2 3 4 5 0 3 6 9 12 1S 18 21 1 2 3 4 S 6 7 DAYS HOURS DAYS FIGURE 1 Changes in bile salt secretion rate in monkey 18 seen immediately after breaking the enterohepatic circulation (EHC). Danielsson. Eneroth. and D.0 - POINT 2. Then. Ryhage and Sj6vall (24). Then by adding standards of "4C in toluene.0 _ 8. This estimate of synthesis is similar to that cal- culated by the fecal excretion method (0. It should be noted that this method measured only 3-OH veloped so that the initial background reading with the bile acids and does not measure keto forms of the different spectrophotometer. raphy. we found it necessary to separate further the fatty Gordon. and the samples were then corrected for quenching preceding 24-hr estimations in the base line state. For the first 90 min.*.0 SALT SECRETION RATE mmoles I 12.0 . the upper broken horizontal line being the mean value over 6 days (14. an emulsion frequently ae. Gordon. phomolybdic. the changes in bile salt secretion rate are followed at 90-min intervals for a total of 21 hr after changing abruptly from 5 to 100% interruption. The daily base line secretion rate (5% interruption cf the EHC) is plotted on the left. 28. 0.) was added to each fecal fistula (100% biliary diversion).70 mmole/24 hr).matic determination of bile acids. However. as demonstrated by iodine. In the center portion of the figure. secretion falls rapidly to a low point. Waltham.

ten Bokkel Huininck. Thus.8 8. Relationship to fecal fat excretion. while bile salt secretion proximately equal to 4% of the monkeys' body weights.45 ±0. The interruption of the EHC was a progressive increase in fecal fat to reciprocate by ileectomy was also compared with that produced by a com.6 ml/5 kg per 24 hr (+SEM 9. 5-16 monkeys is shown graphically in Fig. mon. 10 6 5 185 ± 7.001). At 5% diversion. 3). fe. as indicated by the standard error of the In three monkeys. 29). The relationship the cecum was also resected and the proximal intestinal between fecal fat excretion and bile salt secretion in remnant anastomosed end-to-end with the ascending colon.33 was used in four monkeys to measure the bile salt pool size. 'an aliquot of homogenized stool is However. TABLE I creases to levels severalfold greater than the low point Effect of Graded Interruption of the Enterohepatic Circulation (Fig.001.7 3. 5 22 13 178 i 9. of No. Lund.5 9. in individual monkeys. there was returned to the intestine. ever. as the percentage of bile diverted increased acidified to pH 4. After studying bile salt secretion at the different levels of biliary diversion in five weight. and the subsequent rise in the bile salt secretion represented the compensatory %0 ml/24 hr mmoles/24 hr increase in hepatic bile salt synthesis in response to the in.13 The same technique was also applied to measure pool size at 5.5 9.and finally over the ensuing 12-24 hr. washout includes not only the circulating pool. approximately two.6 g/day. ence between this normal level of fat excretion and that Controlled Interruption of the Enterohepatic Circulation of Bile Salts 235- . bile salt all results in all the monkeys studied show that with secretion was measured when 95%o of the biliary output diversion of 33% or more of bile to the exterior. The mean control fecal fat excretion in seven monkeys trolled interruption of the EHC.001. when measurements of bile volume monkeys. In each case. distal bowel resections were performed.98 40. the distal one-third of the combined lengths of jejunum and ileum was resected (26-34 inches or mean in these corrected measurements was the same as 66-86 cm). bile satt secretion in. ruption of the EHC when compared with the base line cal fat excretion was measured in pooled 3-day collections of feces using the method of Bowers. This technique gives very com- parable results with the more widely used method of Van de these values were less than base line values by statistically Kamer.74 ±1. How- plete bile fistula when no bile was returned to the shortened intestine. The amount of bile salts secreted from the time of changeover until the low point is reached. and 66%o interruption of the EHC in one * Significantly less than "base line" value t5% interruption) P < 0. (30). thus giving a die. No. The scatter of re- bowel resection in the same animals.71t 40. estimations with the two methods on 14 stool samples gave The values for bile volume and bile salt secretion in a correlation coefficient (r) of 0. individual monkeys did not appear to be related to body Distal small bowel resection. 100 It 9 94* 7. Bile volume.35) which was 19-28% of the dietary were reached at the d fferent levels of interruption of the fat intake.8 5. it was suggested that tion of obser. 28. I Significantly less than base line value (5% interruption) P < 0. but a con. in two other animals.51 33 8 8 134* i 9. the in 13 monkeys at 5% interruption were normalized per effects of interruption of the EHC produced by the stream. of Bile salt interrupted by a complete bile fistula. There was no statist cally significant differ- EHC are given in Table I. Per cent In previous studies in which an intact EHC was abruptly interrup.62 terrupted EHC (26. 20.4-12. with the decreasing levels of bile salt secretion.2 l. this relationship between bile salt secretion and the degree of steatorrhea did not RESULTS always correlate well.211 40.88 The technique of changing abruptly from steady-state 20 5 5 176 i 7.62) (Fig. was 178 ml/24 hr (±SEM 9. and Weyers (31) as paired significant amounts. 2. 33.35 g/day per ume and bile salt secretion when steady-state conditions monkey (+SEM 0. from 33% to 66% to 100%. (Table I). The results of bile vol. represents (EHC) of Bile on Bile Voluem and Bile Salt the effective bile salt pool which is "washed out" during one Secretion circulation.005- monkey. 5 kg body weight. averaged 9.98 mmoles/24 hr (+SEM 0.975 (P < 0.5). sults. there was a progressive fall and the lipid residue from the chloroform phase is then both in bile volume and in bile salt secretion and all measured gravimetrically. the mean 24 hr bile volume was splitter could be compared with that produced by small 170.34) by weight. the mean bile volume for 13 monkeys Fecal fat determination. Periodical analyses of the chow showed that its mean lipid There was no significant difference in the daily levels of content was 7% (+SEM 0. and Mathies (5%) values. secretion the low point in the washout curve represented the basal rate the EHC vations keys Mean ±SEM Mean 4SEM of bile salt synthesis in the normal state. The total quantity of bile salts obtained in thz 0. therefore. tribution from endogenous synthesis occurring during col- lection. The dietary intake of Purina monkey chow in healthy animals (120-180 g/day) was ap. 1). For example. In this method. bile volume and bile salt secretion at 10 and 20% inter- tary fat intake of 8. with an intact EHC before surgery was 2. that observed in the raw data.91 i0. The over- When the animals recovered from this second operation and were again eating normally (some 2 wk later). extracted with chloroform: methanol (2: 1). Bile volume and bile salt secretion in response to con. thirds of the distal small bowel was removed.53 conditions with an intact EHC to a complete bile fistula 66 7 7 105* ±10.6). On this dietary regime.

01-0. 0) E en 0 2. gressively and these values were significantly greater The normal bile salt synthesis in animals before sur- than the normal level of fat excretion (t 3. There was a rapid qb. I 6- 5- FECAL FAT 4- g/day 3- 2- 0' .03. and 100% of the various levels of interruption of the EHC are shown the EHC. in Fig. Mack.21 mmoles/day. associated with diversion of 5. synthesis when steady-state conditions were reached at However. 3.001 ).5- E E 2. or 20% of the bile Bile salt snitliesis. The bars represent mean values and the vertical lines. PER CENT INTERRUPTION OF EHC 0 (PRE-OP) 5 lo 20 33 66 100 12- 10- BLE SALT 8 SECRETION 6- mmoles /24 hr 4- 2- 7. 3. The results of de novo bile salt to the exterior. with interruption of 33. H.0- Un Un w I 1.0- n. Dowling.95-6. 10. 236 R. 66. the SEM. Small . excretion) was 0. and D. gery (which of course is simply the normal fecal bile salt P < 0. I'm''1 FIGURE 2 The results of bile salt secretion in 5-16 monkeys at various levels of interruption of the enterohepatic circulation and their relationship to fecal fat excretion.5- z CA 1. the average fecal fat excretion increased pro.0- t]4 U) 0. E. M.5- -I 6 1b io 3o 40 5o 6 70 80 90 100 % INTERRUPTION OF THE EHC FIGURE 3 Bile salt synthesis in 5-16 monkeys at different levels of interrup- tion of the enterohepatic circulation (mean values ±sEM).

and 66% interruption of and progressive increase in bile salt synthesis as the per the EHC. The normal bile salt pool size in value for bile salt secretion was more than double that four monkeys. and 2. bile secretion.73 21 33 4. while this represents considerable reduction in bile salt terruptions (5. this postresection Bile salt pool size.01 1. as measured by the on bile salt diversion with the stream-splitter.57 57. TABLE I I biliary diversion (see Fig.60 10.25 mmoles/24 hr (+SEM 0.1 synthesis was almost able to maintain the bile salt pool 21 20 6.06.98 37.96 1. and 20% diversion).10 0. there was no subsequent the actual values being 0.21 1. The normal fecal levels to the duodenum. ranged from 0. 1).8 studies of bile salt secretion and synthesis. therefore. 10.5 size at the base line level with 20% bile diversion (0. 0.3-13.23 0. 1) was reached.61 0.01 13.09). pool interrup.61). reabsorption % Reab- Monkey No.01). and there was little further increase in bile Ileal resection. once the low point of the washout curve (re- cent of bile diverted increased from 5% to 10% to 20%.59 1.53 mmoles at 33% and 0. 1.46 1.02 62.89 2. These calculations No. of synthesis.01 hr (+SEM 0. The effects of ileal resection on bile salt synthesis with greater degrees of biliary diversion. 33.03). the increased 21 5 7. 18 5 14.60. and the over-all results in approximately a 10-fold increase above the normal rate five monkeys are given in Table III.29) and of bile salt excretion.65 1. In fact.27 mmoles). 100% col.86 1. fer to Fig. However. bowel resected lection lection after resection sorbed mmoles/24 hr mmoles/24 hr % 9 Distal one-third 4.88 8. 4). the EHC secretion pool size day show that.21 mmole/25 hr (+SEM 0. did not differ significantly from those either with small in. was 4. one may calculate the number of times Per cent No.60 Bile Salt Pool Size and Circulation Frequency at Different Levels mmoles (mean = 1. 0.88-1. or with large interruptions (33 and mmoles/day +SEM 0.84 2.53 52. the bile salt pool must cir- % mmoles/ mmoles culate 9. Since the corresponding of Interruption of the Enterohepatic Circulation values of bile salt secretion at 5% interruption of the EHC are known. At 20. but also stream-splitter when all but 5% of the bile was returned on fecal bile salt excretion levels. circula- the pool must circulate each day by dividing the pool Monkey tion of Bile Bile salt tions per size into the 24 hr bile secretion.7 18 Distal one-third 4.8 (range 6.22 mmole at 66%.09 1. rise in bile salt secretion. represents animal (monkey 18) in Fig.27 0.27 mmoles/day. 4.1 19 Distal two-thirds 2. confirming that synthesis was Synthesis was almost maximal at 20% interruption of already maximal.7 mmole).26 mmole/24 secretion as compared with the base line state (14.22 10. on the average. although the increased synthesis was 21 66 2. After resection of the distal one-third of the small The values for bile salt synthesis were based not only bowel (Fig.2 21 Distal one-third 4.8 intact monkeys Controlled Interruption of the Enterohepatic Circulation of Bile Salts 237 .6 20 Distal two-thirds 2.9 Mean results from all normal None 97.8) times each day (Table II). as measured from washout curves at 5% found with a complete interruption of the EHC pro- TABLE I I I Effect of One-Third or Two-Thirds Distal Small Bowel Resection on Bile Salt Secretion and Reabsorption Bile salt secretion after resection Bile salt Length of small 5% col.53 7.84 3. salt secretion are illustrated by the results from one The maximal rate of synthesis.3 sured at different levels of interruption of the EHC.59 6.0 maximal at 33 and 66%.56 58.73 9.90 0.30 2.88 mmole. it was no longer able to main- tain the pool size which fell to 0. 24 hr In monkey 21 in which bile salt pool size was mea- 15 5 9. 0.8 the base line pool was 0. was by no means abolished.84 mmoles/day (+SEM 0. the enterohepatic circulation 66%). the EHC. As suggested by the 19 5 17.

M. in the two animals in which two-thirds of the clearly illustrated in Fig. the postresection value for bile salt secretion was approximately equivalent to 50% interruption of the EHC as measured by the stream-splitter. the greater loss of absorptive surface salt reabsorption by the residual intestine. After distal INTERRUPTION OF THE ENTEROHEPATIC CIRCULATION PRODUCED BY STREAM SPLITTER ILEAL RESECTION CMDRESECTION DISTAL * INTACT SMALL INTESTINE 1/3 SMALL OFINTESTINE 10 E 14t 2 0 (204 - FIGURE 4 Example of bile salt secretion values in one monkey. The postresection bile salt secretion was measured by sampling 57o of the biliary output with the stream- splitter and compared with bile salt secretion with a complete bile fistula (100O7o interruption of the enterohepatic circulation) .59 and 2. H. 4).65 mmoles/24 with a complete bile fistula and after ileal resection. 2. and while these values are still more difference between bile salt secretion after resction and than those with complete bile fistula (1. when the enterohepatic. before resection. tion in bile acid absorption from the ileum. As is shown by the broken horizontal line. Small . 4. It is worth emphasizing.09 that found with 100% bile diversion must represent bile mmoles/24 hr). small bowel was removed. and D.30 mmoles/day ±SEM after two-thirds distal resection was associated with a 0. Similar centage interruption of biliary flow by the stream-splitter.the electronic stream-splitter compared with that produced by distal one-third small bowel resection. perhaps. Dowling. findings were true for the two other monkeys with a for example 33%. does not imply a comparable interrup- distal one-third small bowel resection (Table III).61 and 1. This is However. this level was approximately equiva. 238 R. the hr respectively. Mack.14) and comparing bile salt secretion after ileectomy more marked effect on the enterohepatic circulation of with that found with controlled interruption of the EHC bile salts (Table III).duced by 100% bile diversion (2. circulation was interrupted mechanically by . there was a more drastic Assuming that bile salt synthesis is maximal both reduction in bile salt secretion. that a certain per- lent to 50% interruption of the EHC (Fig. E.

19). effects of anesthesia and surgery (17). The model also pro. they reabsorb about even this level of biliary diversion does not represent a 98% of all secreted bile salts. acids comparable with those found in man (18. the extent of the resultant in.53. the levels of bile salt secre- (34-36). by a bile fistula. various ingenious methods have been used or more of the bile. still required to synthesize the amount of bile salts be- crete only 0. monkey 21) at differ. Since tau- marked reduction in bile salt pool size occurred at 33 rine-conjugated bile acids are strong acids with low and 66% interruption of the EHC.21 mmole bile salts/day and secrete a total ing diverted by 5% interruption of the EHC so that of 9.56 mmoles (Table III). the obvious problems created secretion which follows the low point of the washout Controlled Interruption of the Enterohepatic Circulation of Bile Salts 239 . time of the operation to insert the bile cannula. they largely remain in the ionized Although in the past few years it has often been shown form in the intestinal lumen. This being the case. studies of ileal resection in that ileectomy virtually abolished the EHC of '4C-labeled the rat might be expected to yield very different re- taurocholate. These Thus. although the liver is capable of increasing its bile methods were reviewed by Thureborn (38) whose bile salt synthesis. sults from studies in the rhesus monky or in man where Subject studies in man confirmed these results (10. taurine-conjugated b le acids predominate. even the 10-fold increase in hepatic bile salt the present model. In ent degrees of interruption of the EHC also confirmed this respect. and Weiner (9) showed tion.02 mmoles. As a result. thought that the gradual increase in bile salt secretion splitter provides sampling access to bile for direct mea. 14. transport system of an intact ileum for their reabsorp- Using the dog.98 mmoles into their intestine. because the normal level of synthesis is sampling technique was modified in the development of so small. 32. while the two monkeys were largely avoided. animal than the more widely used rat in which the tion in the pool size occurred at 20% interruption. As long ago as 1928. there is a surgery (38-40). reabsorbed from the jejunum by passive nonionic dif- sorption (9-16. the adaptive increase in hepatic ternal drainage (7) or to maintain the EHC until the bile salt synthesis is limited and is almost maximal at time of the experiment (37). 12. only 5% of the bile produced and returning the remain. Since the rates of absorption and excre. 16. In small laboratory DISCUSSION animals. With diversion of 33% In man. resection. is inadequate to compensate for bile salt loss mon bile ducts of patients who had undergone biliary produced by the stream-splitter. As such. fusion (1) and are largely dependent on the active terruption of the EHC has not been directly quantitated. Eriksson (26) first suggested that the rise in bile salt ing 95% to the intestine. Whipple and Smith tion of the different bile acids may vary considerably (45) noted that with time. Several previous investigators have also attempted to sorb 38-58% of total bile salt entering the intestine. similar to those described in this study. By collecting have also been noted in rabbits (47) and in man (48). Lack. the liver was 1. and 3. 20% interruption of the EHC. The animals with small truly "normal" or "intact" EHC.98 and bile was being returned to the intestine. avoid the artifacts produced by surgical operation or by simple bile fistula preparations. by using both intestinal and biliary cannulae. the glycine-conjugated bile acids predominate. These results have shown by direct measurement that. In this monkey. Since the normal monkeys ex. 33).57. tions were made by Thompson and Vars (46) who In the present investigation. the estimated bile salt reabsorption by interruption of the EHC with simple bile fistulae was 2. they cannot be that ileal disease or resection may cause bile salt malab. the use of the stream. although 95% of the with more extensive resections reabsorbed only 0. it is difficult to extrapolate the findings with a tion tended to rise in bile fistula dogs. bowel resections secrete less total bile salt but only reab. vided a means of varying and controlling the degree of Washout curves for bile salt secretion after acute interruption of the EHC in the steady state long after the bile fistulation. the T-tube or by transnasal duodenal intubation. the liver after ileal resection was suggested by similar isotope was followed either by duodenal drainage or by adaptive responses to complete interruption of the EHC fecal analysis. pKa values (41-44). 32. The measurements of taurine conjugates and of trihydroxy: dihydroxy bile bile salt pool size (in one animal. was due to recovery from liver damage inflicted at the surement of bile salt concentrations. 2. synthesis found in this study could not compensate for The bile of the rhesus monkey has ratios of glycine: more than 20% biliary diversion. although a modest reduc. 33). while jejunal resection had no such effect. the increased synthesis. Similar observa- single isotope to the total bile salt pool. the bile was returned progressive fall in bile salt secretion as the per cent of to the intestine either by bypassing the occluded limb of bile diverted increases from 33% to 66% to 100%. but most of these investigations suffer The compensatory increase in bile salt synthesis by the disadvantage that the fate of a single bile salt radio. However. After sampling. the monkey is a more suitable experimental this pattern. although to occlude the distal limb of T-tubes draining the com- maximal. in it has been possible to replace bile salts lost through ex- in the rhesus monkey.

man and Sj6vall (54) showed that in the rat more than The fecal fat measurements in control animals showed 50% of cholic acid-14C was recovered from the bile that a surprisingly high percentage of the dietary fat in. D. 240 R. Sj6vall. tion at least. 1955. Siperstein. ileal resection. glycine-conjugated bile salts in fact predominate in man Cholestyramine is an anionic binding resin which com. The increased EHC.. and A. such as the glycine-conjugated adaptive increase in bile salt synthesis comes from acids. 1936. Dietschy. were also absorbed in the jejunum. when the radioisotope was placed in the cecum. J. Fed. Acta Physiol. in a ratio of about 3:1 (52). since the ratio of glycine-: taurine-con- terrupted EHC functionally similar to that caused by a jugated bile acids is greatly increased after ileectomy bile fistula. previous investigations where it was suggested that disease or resection of the distal small intestine virtually REFERENCES abolished the EHC (9. by intestinal perfusion in man that the weaker bile acids Furthermore evidence that the liver is capable of an with higher pKa values. S. Mack. Small . This becomes of even greater significance after fecal bile acid excretion which results produces an in. although it has been suggested that endogenous fat excre.. Small and Dietschy gested that the liver was capable of a 10 to 20-fold in. Mosbach. remains to be evaluated.. these animals (32. From acute studies in the bile fistula rat. H. H. Nor- salt synthesis (49). E. the large bowel. this may be related to the 1966. examination by hot-stage polarizing micros. Grants 4-K6-AM-05589. 2. I. This high melting point sorption from different sites along the intestinal tract suggests that much of the dietary lipid would be un. 51). Studies on the mechanisms use of a nonrepresentative isotope such as the sodium of intestinal transport. perature. Hofmann. 5-fold increase). M. Weiner. Meilman. Clin. J. Proc. 30: 2224. Biochem. J. Scand. he sug. This work was carried out during the tenure of a Sir- tion may be increased in animals with biliary diversion Henry Wellcome Travelling Fellowship from the Medical Research Council (RHD) and was supported in part by- (50. salt of taurocholic acid-j4C which. Intestinal absorption of taurocholic acid in the rat. J. absorption by diffusion bines with bile acids in the small bowel lumen to form may well be an important component of the normal a complex which cannot be absorbed. larly in patients undergoing biliary surgery. Since the cholestyramine feeding experiments in mice (49). Dowling. Lack. plete interruption of the EHC. size. Samuel.. Salomon. 4. and I. 12). and as in the bile fistula rat. and M. at intestinal pH. it is known 273. in T-tube bile when cholic acid-14C was injected into. Egdahl. copy showed that much of the lipid remained in a The quantitative significance of the bile salt reab- crystalline state until 570-580C. and although the fecal fat excretion in the com- plete absence of bile was less than three times the ACKNOWLEDGMENTS normal fecal fat. segments of the intestinal tract (1). Using this isotope to study the effects of the bile acids from the intestines. AM-12890. 45: 832. but the limited data from the available for intestinal absorption at body temperature. However.9% of administered radioactivity while some of this lipid melted at or below body tem. the fecal fat excretion showed the ex. Akesson. Bile acid metabolism. lated to the crude nature of the dietary fat in the com- mercial monkey chow. Kay and Entenman (29) jejunum of the rat and showed that glycine-conjugated found similar results in the rat. As indicated previously. and Schoenfield (35) showed synthesis was more limited (4. typed the manuscript. L. Josephson. The resorption of reabsorption. The origin We wish to acknowledge the helpful criticism and support- of Doctors F. 34:. (36) defined the kinetics of bile salt diffusion from the crease in bile salt synthesis. but Myant and Eder bile salts were readily absorbed from the upper small bowel (28) thought that the adaptive increase in bile salt while Hislop. and AM-11453 from the- The findings of an incomplete interruption of the EHC U. This may be re. 53). absorption from the residual small bowel pected inverse relationship to bile salt secretion and pool may be an important factor in maintaining the EHC. in ileal resec- In spite of this. Simi- take was excreted each day in the stools. M. Invest. 1. Ingelfinger and R. J. it is well-recognized that the steator- rhea of biliary insufficiency is usually modest. would remain in the ionized form and would therefore 22: 1334. Rydin. present experiments would suggest that. and Chafizedeh (55) re-- chow and feces were solid at room temperature and covered an average 58. B. also develop a compensatory increase in hepatic bile Bile salts may also be reabsorbed from the colon. Intestinal absorption- of bile salts and some biological implications. and D. and by Grant AM-08726 from by ileal resection in the present study conflicts with the National Institutes of Health. We would of the fecal fat and the relative concentrations from also like to thank Miss Lucy Lee and Miss Ruth Sutton who dietary and endogenous sources were not studied here. Public Health Service. and I. M. Lipid extracts of both monkey Saypol. require the active transport system of the ileum for its 3.curve was due to increased bile salt synthesis by the that bile salts may be absorbed by diffusion from many liver. H. S. ileal (lisease might suggest that there was almost com.

1968. 23. S. J. 279: 41. Proc. Weiner. B. malabsorption syndromes. F. Lab. Lipids. Van Deest. F.) in disorders of the distal small bowel. Bile salt malab. Lab. On the mechanism of the intestinal 27. enterol.. Chem.. L. absorption of bile acids. E. 32. Scand. Dietschy. and J. Biochem. changes due to altered enterohepatic circulation. R. J. 46: 1070. J. Gallstones. F. Hofmann. 8. Dowling. M. Enzymic analysis of steroid hor. 263: 428. J. Vars. J. Berger. 1960. The conjugated bile salts of certain pri. Ekwall. S. Bloch. Determinants of the rate and site of bile acid ab- 1968. 26. Engl. H. fat in feces.. 42. 44. G. A. Sj6vall. Chem. Med. 197: 1330. E. tabolism. Hellstr6m. W. W. Fecal C14-bile tients. and D. 35.. Hislop. Biochem. Rosenberg. Van de Kamer. Invest. Med. Invest. 52: 638. Wilson. A. 1943. 95 344. and H. Lancet.. 9: 297. the lipids excreted by human subjects in normal and section steatorrhea responding to oral pure conjugated pathological states. Small.) induced diarrhea. and F. Whipple. 1949. Studies in Chir. B. and M. 1967. Experimental model for the study of rat bile. Eriksson. i: 873. 1968. 1953. Scand. sive diffusion process of bile acids across the small intes- sorption in regional ileitis. Gastro- chemical differences in bile salts..and dihydroxy bile acids in pH on the solubility of varying mixtures of free and human feces by gas-liquid chromatography and mass conjugated bile salts in solution. Lipid Res. Methods Biochem.. 7: 511. Schoenfield. M. Bile salts portance of bile acids and of an intact distal small in. and C. Enterohepatic circulation of C1'-labelled bile salts sorption in man.43: 1. G. J. Biliary excretion of bile acids and nism of bile acid absorption into the portal circulation.. and J. Clin. Isaksson. Riol. M. A of intestinal resection on bile salt absorption in dogs. E. [nter- 17. Hardison. D. 1966. Austad. Bile 33. 1933.) 31. feces. Sjbvall. D. and I. continuous collection of hepatic bile at "intact" entero- 19. Proc. N. K. J. W. Small. J. Human hepatic bile. and J. P. Austad. 20. Clin. C. 8: 119. Controlled Interruption of the Enterohepatic Circulation of Bile Salts 241 . I. Witmer. 177: 347. T. R. 7: 524. 15: 242. Mechanisms for the intestinal steroid-wasting syndromes secondary to ileal dysfunc. Fordtran. 54: 1272. drainage upon the synthesis of cholesterol in the liver.. Suppl. 1968. Physiol.. Picott. M. R. 277: 337.. D. J. Lipid Res. Bergstrom. 1968.. Gordon. In Lipide Me- absorption in the rat. Acta 18. E. Clin. A. 13. 1965. Biol. Grundy.. and salt deficiency in the steatorrhea following resection of J. B. and J. in small intestinal contents after ileal resection and other testine for fat absorption. 9. 24. and ruption of the enterohepatic circulation and its effect on D. John Wiley & Sons.. Gordon.. P. E. Thureborn. P. M1ed. Wooton. N. H. Small. 1961. and H.. M. Hofmann. K. bile salt treatment. J. How much bile salt circulates in the body? J. R. Borgstrbm. Probl. 303: 1. 36. J. E. 40. Miettinen. ileal resection. P. A. L. WNT. J. 209: 158. 72: 169. Jr. cholesterol in bile fistula rats. and D. M. 45. C.. Gastroenterology. III: 3. Im. A. H. A.. 1967. 1965. Wiggins. The effect of biliary Scand. The effect of Identification of mono. The biological significance of hepatic circulation in man. S. M. 1967. the bile acids. 1961. 1958.. D. P.. 12. Myant. F. Clin. 22. Rev. D. H. Biol. H. 200: 855. 1967. Engl. 39: 537. Jr. S. Meihoff. Detergent products of cholic and chenodeoxycholic acid in man. and H. (Abstr. H. Clin. B. A. 1967. Bergstrom. Lipid Res. 1957. Lack. Exp. Reinke. V. J. M. B. 1953. 238: 2299. G. J. and 11. Ternberg. 28: 279.) 25. Composition J. W. M. The solubilization of lipophilic sub- mones. 1928.. Morawski. L. Bile salt absorption in the irradi. Mathies. A modified T tube for quantitative collection of bile. Samuelsson. J. Quantitative isolation and gas-liquid chromato. 37. G. Acta Physiol. A. and D. and B. 1960. Physiol. tion of the monomer and micelle components of the pas- 16. D. W. S. 2: 363. 397. L. 1967. Lack. Hofmann. Eder. Gastroenterology. the enterohepatic circulation of bile in Rhesus monkeys. Jr. 70: 349.. A method for mates. Ryhage. Lack. McLeod.. 15. Light. Physiol. Eneroth. The effect of bile diversion on fat Formation and metabolismn of bile acids.. Playoust. Dietschy. 1958. S. S. and I. Stimulation of graphic analysis of total fecal bile acids. 47: 261. chemical basis of cholesterol gallstone formation in man. J. Sullivan. New 7. Bowers. Weyers. 47: 1314. 1968. Chim. Clin. Thureborn. tion in proximal small bowel contents of ileectomy pa- 14. Ahrens. 1959. S. Z. 39. Talalay. K. editor. 1968. Bile salt metab- terization of trisubstituted cholanoic acids in human olism IV. P. 29. 1968. Res. The physico. Conf. tion. Lipid Res. and M. and J. Entenman. H. Clin. H. A. Charac. function. Med.. 1962. J. Danielsson. Rapid method for determination of ated rat. Int. 80: 697. Clin. acid excretion in normal subjects and patients with 34. Tyor. Small. 1966. Med. Eneroth. P. Post re. Eneroth. R.. R. K. Aier. and H. Lund. Proc. On the turnover and excretory 43. R. Sj6vall. W. I. Bile salt and micellar fat concentra- the ileum and proximal colon. M. Lipid Res. 38.. 1964. M Small. 208: 363. Med. 47: 1043. Wiggins. P. 6. 291. J. (Abstr. Danielsson. Soc. Chem. Dowling. fat absorption. 21.. 1965. Invest. Stanley. and B. Effect 30. 1963. Grundy. 72: 517. Rev. Clin. J. and S. M. Peterson. A. Nemchausky. J. and T.. and J. P. Physiol. rapid reliable procedure for the determination of total Amer.. and J. tine of the rat. 70: 627. Mack.) 94: 578. Amer. (Abstr. The mecha. 18: 333. Danielsson. York. Kay. P. 2nd World Congr. Acta. Amer. 54: spectrometry. 6: taurocholic acid synthesis and biliary excretion of lipids. J. M. 1968. Tyor. B. ten Bokkel Huininck. H. Characteriza- 55: 5. Die Dissoziationskonstanten der 588. 1964. J. G. M. J. L. G.. fecal lipids with observations on the composition of 10. M. and mannitol. Invest. H. Admirand. 1968. N. Gallensauren. properties of bile salts: correlation with physiological Biol. Gastroenterology. (Abstr. M. 1291. H. 1965. Kern. H. Haslewood. tion of bile acid formation in the rat liver.. Anal. M. Biol. J. B. Med. On the regula. and E. Lindstedt. 1968. J. Small. M. Aach. and L. Clin. and H. Biochevi. 5.. 13: 254. J. G. N. Josephson. Res. Lab. Gastroenterology. C. stances by bile acid salts. Annu. M. Wilson. and I. S. Acta Physiol. (Abstr. 28. J. Heaton. I. Smith.

and J.. Meilman. cholestyramine.. Versuche an Ratten mit Gallengangverschluss. Physiol. 48. M. duodenal contents of infants and children. E. and J.) 54. K. Exp.46. J. 11: 421. Samuel. Biol. Absorption of bile acids from Sevler's Z. Garbutt. A. 1968. Heaton. the large bowel in man. H.. Lack. H. On the conjugation Acta. Sj6vall. W. (Abstr. J. 4: 793. E. Acta. and J.. Mack. T. and H. and B. Acta Chir. tfber den Ursprung des Kotfettes II. Chafizadeh. 47. 16: 284. and M. and M. S. 114: 439. Med. P. Beher. 1954. Chim. On the bile acids in Scand. 51. Glycine: taurine ratios of bile salts in ileal disease. and J. Ritzel.. Holasek. Clin. Ekdahl. wesenheit der Galle. Lindstedt. Sjovall. 1957. Soc. J-C. and E. Sjbvall. Hug. and formation of bile acids in the human liver. Scand. 49. M. Beher. Encrantz. A. Clin. On the transformation and cholesterol metabolism in the mouse as affected by and enterohepatic circulation of cholic acid in the rat. 1952. Physiol. On the formation of de. K. 50. 298: 219. Helv. tber cretion of cholic acid and cholesterol in hyper-. Small . 1958. 1959. hypo. Thompson. Exp.. 53. J. M. 52. Rao. Invest. Pharmacol. 1957. L. Proc. E. 55.. Saypol. Hoppe. Biol. 1966. oxycholic acid from cholic acid in the rabbit. G. P-H. Norman.. Bernhard. Soc.. 10: 68. Proc. 122: 881. and D. Dowling. Acta Chem. J. Bile acid Clin. E. 1968. M. Biol. 233: 872. 1953. die Sezernierung von Lipiden in das DarmIumen bei Ab- and euthyroid rats. Chem. Vars. Tyor. 242 R. Biliary ex. 47: 2070. C. Sj6vall. Mosbach. G. Chem. 83: 246. Res.