Enterohepatic Circulation of C14-Labeled Bile Salts in Disorders of the Distal Small Bowel

Vol. 55, No. 1 Printed i n U.S.A.

GASTRO ENTEROLOGY

Copyright © 1968 by The Willia ms & Wilkins Co.

14

ENTEROHEPATIC CIRCULATION OF C -LABELED BILE SALTS IN DISORDERS OF THE DISTAL SMALL BOWEL K. W. HEATON, M .B., M .R .C.P ., W. I. AusTAD, M .B., M.R.C .P., L. LAcK, PH.D., AND M. P. TYOR, M .D .

Department of Medicine and Department of Physiology and Pharmaco logy , Duke University Medical Center, Durham , Nor th Carolina

a bsence of approximately the distal twothirds of the small intestine and 3 patients had less marked changes. Our interests were primarily 3-fold: (1) to measure the enterohepatic circulation of the major cholate conjugate in man, glycocholate, in these patients, since this bile salt may be more subject to absorption via passive diffusion processes; (2) to obtain additional and more precise information concerning the localization of the active bile salt transport system, throughout the ileal length ; and (3) to assess further the possible contribution of the colon to the enterohepatic circulation of bile salts in these types of patients.

Studies of intestinal bile salt transport have identified the ileum as the sole site of active transport in a variety of species of animals. 1 • 2 It has also been shown in dogs that an intact ileal transport system is necessary to maintain enterohepatic recirculation of taurocholate. 3 Our recent observations4 confirm those of Borgstrom et aJ.5 that taurocholate absorption in roan occurs at a more distal lo cation than fat absorption. In addition, we demonstrated a virtual absence of enterohepatic circulation of taurocholate in a patient with ileal disease and in a patient who had a jejunocolic fistula. 4 In the course of these investigations into the physiological significance of this ileal bile salt transport system, we have studied the enterohepatic recirculation of conjugates of cholic acid in 6 patients. Three had a fun ctional

Materials and Methods All patients were hospitalized on the Clinical Resea rch Unit at Duke University Medical Center. In 3, the jejunum 30 inches from the ligament of Treitz was anastomosed end to side to the transverse colon (approximately 73 inches of distal small intestine bypassed). The operations had been performed at another institution 4 to 36 months previously for the managemf'nt of intractible obesity. One patient, C. B., had a cholecystectomy in 1954. OtherwisE', there had been no significant past illness. All had rf'cf'ived injections of vitamin B, and supplf'mr n ts of potassium and magnesium since thf'ir surgery . They were all moderately obese despite an average postopera tive weight loss of 47 kg. During their 2 weeks of hospitalization, thf'y were asymptomatic save for two to six bowel movements per day. Mensuremf'nts of SE'rtim electrolytes wE're normal. The remaining pertinent clinical data are presented in table 1. Three additional patients had varying degrees of abnormalit ies of the distal small intf'st ine, as indicated in table 1. The clinical data in patient N . S. have been presented previously in detail.• Peroral biopsies were ob-

R eceived November 24, 1967. Accepted F ebruary 9, 1968. This paper was presented in part to t he Ameri can Gastroenterological Association, Colorado Springs, Colorado, M ay 27, 1967. Address requests for reprints to: Dr. M alcolm P. Tyo r, Duke University Medical Center, Durham, North Carolina 27706. This investigation was primarily supported by Research GrantR AM 11730 and AM 09582 from the National Institu te-s of H ealth , Uni tf'd StatPs Public Healt h Service, Bethesda, M aryland. The services of the Clinical RPsearch Unit werP supportPd by Grant M0-1FR30 from the National Insti tutes of H ealth. ThP work of Dr. Austad was supported by Training Grant 2-A 5093 from the National Institute of Arthritis and Metabolic Diseases. The authors express t heir gratitude to Dr. Irvin B. Moore, Cleveland, Ohio, for arranging the hospitalization and study of patients C. B., E. D., and J . H .

5

6

HEATON ET AL.

tained from the distal jejunum and mid and distal ileum (identification of biopsy site was determined from radiographs following instillation of Gastrografin). From these observations, it appeared that there was marked mucosal alteration of the distal one-half of the jejunaileum. Surgery had been performed in patients E. R. and E. B. for intestinal obstruction due to volvulus. Estimates made at surgery indicated that the distal one-half (56 inches) and distal one-third (34 inches) of the small intestine had been resected, respectively. The anastomosis was end to end with the ascending colon. Other than having three to five watery bowel movements per day, these patients were essentially asymptomatic during their period of study. There had been no significant past illness. All appeared malnourished, but the physical examination was otherwise unremarkable. Measurements of serum electrolytes were normal. The remainder of the pertinent clinical data are shown in table 1. Measurements of intestinal absorption. Fecal lipid analysis was performed by the method of Van de Kamer et al." with minor modifications.' Urinary o-xylose was measured by the method of Roe and Rice7 in aliquots of urine collected for a 5-hour period following the oral administration after an overnight fast of 25 g in 600 ml of water. Vitamin B12 absorption was estimated by the method of Schilling.• The excretion of 5-hydroxy-3-indolacetic acid (5HIAA) in the urine was measu red by the method of Udenfriend et al." in a 24-hr urine collection. Measurements of sodium glycocholate-C" and sodium taurocholate-C" recirculation. These studies were performed in the 6 patients and in 8 male medical student volunteers, ages 22 to 25 years. Control subjects were ambulatory and their feeding schedule and daily habits were restricted only as indicated. The dietary intake of the patients was constant throughout the study in that they ate 70 to 80 g of fat dHily. Their total caloric intake varied between 1800 and 2500, consumed during three meals per day. Their feeding schedule was restricted only as indicated. In all instances, 5.0 to 7.0 JLC of sodium glycochola te-24-C" or ta urocholate-24-C" were administered intravenously. The bile salts were dissolved in 30 ml of 0.15 M NaCI. This material was given at approximately 8:30 AM after an overnight fast. All subjects remained fasting for a 3-hr period following the administration of the labeled bile salt. The method for collection of duodenal contents which included intubation and aspiration following the intravenous injection of chole-

Vol. 55. No. 1

cystokinin (Cecekin, Vitrum Chemical Company, Stockholm, Sweden) has been described previously.• Duodenal samples which were obtained within 2 to 3 hr after injection of a labeled bile salt were combined and kept on ice. Upon completion of the sampling, 2 to 6 ml of the pooled duodenal contents were saved for analysis. This never amounted to more than 5% of the total aspirate. The remainder of the sample was delivered into the duodenum via the indwelling tube which was flushed with water and then removed. The entire procedure of intubation, cholecystokinin infusion, and duodenal aspiration was repeated in control subjects after an overnight fast at 24, 72, and 120 hr after the injection of the labeled bile salt. In the patients, duodenal sampling was repeated at 24 and 48 hr after the injection of the labeled bile salt. Preparation and chromatographic analysis of duodenal samples. Duodenal samples were prepared for chromatographic analysis by protein precipitation and extraction with ethanol.• Conjugated bile salts were separated by thin layer chromatography using isoamyl acetatepropionic acid-n-propanol-water (30: 30:20: 15 vj v) (A. F. Hofmann, personal communication) . The methods of identification of taurocholic acid and subsequent extraction from the silica gel with ethanol have been described previously.' Similar methods were used for glycocholic acid, using an appropriate standard. In each instance, 0.1 ml of either the taurocholic acid ethanol extract or glycocholic acid · ethanol extract was transferred to a standard 20-ml counting vial. In addition, 0.5-ml aliquots were subjected to the modified Pettenkofer reaction of Irvin et al.' 0 which permitted the determination of taurocholic acid and glycocholic acid concentration. In this manner, the specific activity, counts per minute per milligram of the taurocholate or glycocholate present in the aspirated duodenal contents, could be determined (table 2). Total radioactivity per unit volume was ascertained in each duodrnal aspirate obtained from all subjects.' In selected experiments, the precipitated and ethanol extracted samples obtained 24 hr after injection of the labeled bile salt were scanned for radioactivity, using thin layer chromatography. The plates were sprayed with Neotan (Brinkm ann Instruments, Inc ., Great Neck, Long Island , N. Y.) and, after drying in air, they were divided into consecutive 5-mm strips, beginning at the point of origin and continuing to the solvent front. Each strip was identified by appropriate standards and individually transferred to a counting vial. The radioactivity

43

79

48

67

J. H.

N. S.b

E . R.

E . B.

I

25

E. D.

.Jcj uno -transverse colostomy, distal two-thi rds of small intestine bypassed Jejuno-transverse colostomy, distal two-thirds of small intestine bypassed J ejuno-transverse colostomy, distal two-thirds of small intestine bypassed Amyloid of d istal one-half of jej unoi leum (posti rradiation) J ej uno-ascending colostomy, distal one -half of small intestine resec ted Ileal-ascend ing colostomy, d istal one -third of small intest ine resec t ed

Diagnosis

• 5-Hydroxy-3 -indoleacetic acid. b P revi ously reported in detai l. 4

Normal value

47

- --

Age

C. B.

Patient

I ntestinal obstruc tion Intestinal obstr nction

Obesity

Obesity

Obesity

surgery

12/66

1/54

1/64

7/66

11 / 65

su 1gery

Date of

68

133

145

95

~ft~~ kg

Pre- ~

34 36

45

36

63

100

68

sent

Pre-

Weight

2/67 35 6/67 '

2/67

10/ 66

1/67

11/66

10/66

---

Date of study

1. Summary of clinical data

Indication for

TABLE

Serum

a lbumin

Serum

cholest.e10l

I

32 40

32

35

39

42

34

>3.5

4 .3 4.5

4 .1

3 .5

3 .7

3 .6

3 .7

g/ 100 ml

> 150

139 119

127

110

87

200

140

m g/100 ml

- - - - - - - -vol/ 100 ml

Bematocrit

3.9

2 .5

3.1

2 7

0.5

1.8

g/5 hr

---

Urinea

5-HIAA

>8 .5

2.5

0.5

2 .0

0.3

<8.4

3 .8

2.9

4.8

4. 1

3.1

--- --% dose/ m g/24hr 48 hr

o-Xy lose Vitamin Bu absorpabsorption tion

< 5.5 >4.2

14 .2

25.9

28.0

21. 1

48 . 1

16.7

g/24 hr

- --

Fecal fat

~

.......

~

"-l

1:-<

0

0:;

1:-< 1:-<

~

~

Ct:l

1:-<

~

tn....,

t>:l b

::t:

....,

"'l

0

~ Ct:l

b

~

b

tn

~

._

~

8

HEATON ET AL.

p resent m each bile salt fraction was compared with the total recoverable radioactivity from the plate . The scintillation mixture and counting methods used including considerations of selfTABLE

2. Total C 14 -radioactivity in duodenal samples 24 hr after administration of cholate-C 14 conjugates Bile salt administered

Patient

Tau rocholate-C"

I

Glycocholate-C"

cpm fmg cholate (percentage of initial value)a

C. B. E. D. J. H. N. S. E. R . E. B.

12 5 2 1

7 0 4 1 1 31

17 ()b

Methods of calculation detailed in text. Value obtained from second study in patient E. B., during tetracycline administration . a

b

TAHLE

3.

M e a.~urements

Vol . 55 , No. 1

quenching, have been described in detail previously.'·" Chemicals. The sodium taurocholate-24-C" and sodium glycocholate-24-Cu were either synthesized from cholic acid-24-C14 (New England Nuclear Corporation, Boston, Mass.) and taurine or glycine (Sigma Chern. Company, St. Louis, Mo.) by the method of Norman 12 or obtained as sodium taurocholate-24-C 14 (Trace rlab Company, Waltham, Mass. ). When the latter material was used, between 1.0 and 1.5 mg were administered as shown in table 3. The studies utilizing bile salts synthesized in our laboratory required the administration of between 30 to 45 mg as indicated in table 3. I n all instances, the labeled material was diluted in 30 ml of sterile physiological saline subsequent to its injection through a M illipore filter. In each case, 1 ml was retained for analysis . Chromatographic analysis of these materials showed greater than 99% of the radioactivity recoverable in the appropriate bile salt spot. The standards used in these experiments were obtained from Maybridge Chemical Company, Ltd ., Cornwall, United Kingdom.

of sodium taurocholate-C 14 and sodium glycocholale-C 14 recirculation and metabolism Glycocholate-C"

Tau rocholate·C" Subject

Student volunteers A. B . ~ R . K.b

M. E.b D . V.b R . A.• i::L L.•

B. D. D . R. Mean ± IH .D. Patie nts E. D. • J. H .• N. S.• E. R.• E . B.•

I T urnove ra

Half-time Cty,)a

Exchan~eab le poo a

mg

mg / day

days

mg

2.20 2.25 1.85 1.75 1.45 1.70

355 272 633 300 367 337

112 84 237 11 9 175 137

1 .87 ± 0.32

377 ± 130

144 ± 55

Half-time

Exchangeable

(ty,)•

poola

days

64 41 40 59 56•

1.35 1.55 1. 25

926 1600 735 1400" 1. 38 ± 0 . 16 1165 ± 403 836 825 625 322 535

Method of calculation detailed in text. Sodium taurocholate-C 14, 30 to 45 mg. • Sodium taurocholate-C'', 1 to 1.5 mg . • Calculated from initial (3 hr) sample. • Values obtained in second study in patient E. B., during tetracycli ne admin istration. a

b

T urnoveru mg/ da y

476 715 407 533 ± 162

DISORDERS OF THE DISTAL SMALL BOWEL

July 1968

Calculation of results. In these calculations, specific activity, counts per minute per milligram of sodium glycocholate or sodium taurocholate obtained from duodenal aspirates 2 to 3 hr after injection of the labeled bile salt, has been assigned a value of 100% and the specific activity of duodenal samples obtained subsequently has been related to this initial value . The methods used to calculate the halftime (t112) of the rate of disappearance of injected sodium glycocholate-C14 or sodium taurocholate-C", as well as those used for estimating

Student Volunteers (Average Values}

"' .....c: -;;e. ~ >

u

<( (.)

·;:;

., 0.

en 0

en

4

~

ii5

2

Fw. 1. Rate of disappearance of sodium taurocholate-24-C 14 ( e ) and sodium glycocholate24-C" (Q) from the enterohepatic circulation in normal subj ects and patient J . H. who had a jejunocolic fistu la. In these calculations, the specific activity, counts per minute per milligram, of sodium taurocholate or sodium glycocholate obtained from the initial (2 to 3 hr) duodenal aspirate after injection of the labeled bile salt has been considered to be 100%, and the specific activities of duodenal samples ob- tained subsequently have been related to the initial value. In the student volunteers, the average values for sodium taurocholate-C" were obtained from studies in 6 subjects; the average values for sodium glycocholate-C14 were obtained from 3 subjects.

9

exchangeable bile salt pool, and turnover rate constant in the student volunteers have been described previously.' In the patients, the available bile salts (body pool) accumulated in the gall bladder prior t o discharge represented newly synthesized material (see "Discussion"). This pool has been estimated by the isotope dilution formula using the specific activity of the initial duodenal sample and the specific activity of the known amount of injected glycocholate-24-C14 or taurocholate-24-C14. In these calculations, we have assumed complete hepatic clearance of the injected bile salt, as well as complete mixing with endogenous bile salts. Studies in animals indicate the likelihood of the former assumption.13 The latter assumption may not be entirely valid, since endogenous bile salts may have remained sequestered in other regions of the enterohepatic space during the 3-hr period in which the labeled material was inj ected and collected. Additionally, incomplete evacuation of the gall bladder14 and inadequate mixing of the contents of the biliary tree might also lead to an error in sampling. There are no means of critically evaluating these potential sources of error when the 3-hr isotope dilution method is used. Nevertheless, a comparison of such results in normal volunteers with values obtained from the conventional method (employing a 5-day exponential decay of specific activity) shows minimal differences. The pool size in student volunteers calculated by the isotope dilution method was slightly higher in 6 of 8 studies with taurocholate-C14 and in all 3 studies with glycocholate-C14 when compared with calculations using the conventional method. Thus, the taurocholate pool averaged 9% and the glycocholate pool 13% greater when the specific activity was calculated from the initial 2- to 3-hr duodenal samples. In 2 volunteers the taurocholate pool was slightly lower (9% ) with the 3-hr isotope dilution method.

Results

Measurements of intestinal absorption. The results of these measurements are presented in table 1. In general, patients with jejunocolic fistula and those with lesser abnormalities of the distal small intestine exhibited similar values for several measurements of intestinal absorption. All had significant steatorrhea. n-Xylose absorption was uniformly decreased. The urinary excretion of 5-HIAA was

10

Vol. 55, No. 1

HEATON ET AL . ..,100

GLYCOCHOLIC ACID

TAUROCHOLIC ACID

:> 0

> 0 ~ 80 1-

c:

~60 -

•

-

,

•

•

'

.::: :;£ 40 -~ u

"' r.n

0.

-

0

20

-

r.n 0

0

Student Volunteers

Jejuno- Co he Fistula

Other D1stal Small Bowel Disorders

Student Volunteers

Je)uno-Cohc Fistula

Other D1stal Small Bowel Disorders

Fw. 2. Disappearance of C"-labeled bile salts from the enterohepatic circulation 24 hr after intravenous inj ection in student volunteers, patients with jejunocolic fistul a, and patients with lesser disorders of the distal small bowel. In t hese calculations, the specific activity, counts per minute per milligram, of sodium taurocho late or sodium glycocholate obtained from the initial duodenal aspirate after inj ection of the labeled bile salt has been considered to be 100%, and t he specific activities of duodenal samples obtained at 24 hr have been related to the initial value.

normal. Values for the Schilling test were decreased in all patients studied. M easurements of sodium glycocholate C 14 and sodium taurocholate-C 14 recirculation and metabolism. The average time course curves of disappearance of sodium glycocholate-C 14 and sodium taurocholat e-C14 from the enterohepatic circulation in student volunteers are shown in figure 1. These studies agree with the exponential rate of disappearance of labeled taurocholate reported previously and originally described by Lindstedtl 5 using C 14 -cholic acid. Not previously reported is the expon enti al rate of disappearance of glycocholat e-C14, which behaves similarly. In contrast, a n excessively rapid fall in specific activity of these bile salts is evident in patient J. H., who had a jejunocolic fistula , figure 1. It is apparent from figure 2 that the data obtained in patient J. H. are representative, not only of patients with jejunocolic fistula but a lso those obtained from the other patients studied, i.e., those retaining larger amounts of functioning small bowel. At 24 hr, the specific activity in all patients had fallen to virtually 0 ; whereas, in

the student volunteers 24-hr values were 62 to 84% of the 2- to 3-hr specific activity when t aurocholate-C 14 was inj ected and 57 to 64% after glycocholate-C 14 injection. Values for total C 14 -radioactivity, normalized for volume changes, in duodenal samples obtained from all patients 24 hr after the labeled bile salts were administered are presented in table 2. These results represent the sum of t he remaining unaltered bile salt and its microbial metabolites. It is apparent from these observations that most patients exhibited not only a virtual disappearance of enterohepatically circulating sodium taurocholate-C14 a nd sodium glycocholate-C 14 at 24 hr but also a negligible recirculation of total C 14 -radioactivity. The exceptions appeared to be patient E. B . and , to a lesser extent, patient C. B. However, it is to be emphasized that there was no detectable radioactivity in bile samples obtained at 48 hr in all patients. The relative chemical compositions of this residual total C 14 -radioactivity obtained 24 hr after administration of labeled bile salts are shown in figure 3. It is apparent that the

July 1968

r

11

DISORDERS OF THE DISTAL SMALL ROWEL

residua l radioactivity after taurocholate injection is chiefly present as reconjugated microbial metabolites. The residual radioactivity 24 hr after glycocholate administration to patient E. B. is also present chiefly as such metabolites. The similarity in distribution of radioactivity following administration of either bile salt to patients E. B. and J. H. is striking (see "Discussion"). The entire study with sodium taurocholate-C 14 was repeated in E. B. following the administration of tetracycline 0.25 g every 6 hr. The drug was initiated 48 hr prior to administration of the bile salt and continued until the aspiration of the fin al sample. During tetracycline administration, C 14 -radioactivity of the untreated duodenal sample was 0 at 24 hr (table 2). The exponential declin e in specific activity of sodium taurocholate and sodium glycocholate in the control subj ects (fig. 1) permitted .estimation of the t112 for the rate of disappearance of these bile salts, the exchangeable sodium tauro cholate and sodium glycocholate pool, and their respective turnover rates (table 3) . The data ob tained after labeled taurocholate inj ection confirm our previous observations in student volunteers. 4 They are presented in detail to facilitate comparison with those values derived from experiments in normal subj ects who were given taurocholate-C14 and glycocholate-C 14 . The combined studies in subj ects R. A. and S. L. suggest that the pool size of th e two cholate conjugates derived directly may vary between 1293 and 1937 mg. Furthermore, between 18 and 28% consists of cho li c acid conjugated wit h taurine. I n the patients, the sodium taurocholate and sodmm glycocholate pools were each estimated from the specific activity of the ini tial 2- to 3-hr sample (table 3) . It is apparent that the size of the taurocholate-C14 pool in all patients was considerably sma ller than values obtained from student volunteers. With glycocholate-C14, estimations of pool size were lower on the average in patients with jejunocolic fis tula than in normal subjects. Overlap was evident with one control subject. However, values were uniformly

BILE SALT ADMINISTERED ci4_TAUROCHOLIC C 14-GLYCOACID CHOLIC ACID

-0 100 ~

,QQf

~

>

00

80 1-

0

:Oo ~0 _1-60 1-

GDC

GDC

go

~~ ., a: ~40

-

GC

GC

GC

GC

-" 0~

5v "'0c.

1--

:i: N20 1E 0

(.)

0

1--

GC

1--TC C.B.

Tc ~ J.H.

GC

1-TC

E.B. N.S. Pa t ients

1--

TC t;=Dc J.H. E. B.

FIG. 3. Composition of residual radioactivity in duodenal aspirates obtained 24 hr after the intravenous injection of either C 14-taurocho lic acid or C"-glycocholic acid in patients with disorders of th e distal small bowel. Data are expressed in terms of percentage of radioactivity contributed by each bile salt fraction to the total recoverable radioactivity. GDC, glycodeoxycholate; GC, glycocho late; TDC, taurodeoxycholate ; TC, taurocholate .

reduced in the 3 other patients who had lesser abnormalities of the distal small intestine. In addition, it would appear that there may be a relative increase in the ratio of the glycocholate pool to the taurocholate pool of patients, as compared with values from the 2 student volunteers studi ed with both bile salts : range 9.5 to 20.1 as compared wi th 2.5 and 4.7, respectively. Discu ssion

These studies demonstrate a virtual absence from the enterohepatic circulation of sodium glycocholate-C 14 and sodium taurocholate-C 14 24 hr a fter their administration to 3 patients who had a jejunatransverse colostomy. Such findings were not unexpected in view of our previous observations with labeled taurocholate in a similar patient, 4 and the reported results of taurocholate recirculation in dogs following resection of the distal one-half of the small intestine. 3 Additionally, t he

12

HEATON ET AL.

results were essentially identical in patients with abnormalities involving lesser amounts of distal small intestine. The problems of precise localization of the extent of involvement of disease, as well as the extent of surgical excision of the small intestinal length, are well recognized. 16 Nevertheless, the present data suggest that ~he chief site of absorption of bile salts may be localized to the distal one-third of the small intestine. Resection of the distal one-third of the small intestine, as in E. B., or extensive inflammatory disease of the distal one-half of the jejunoileum, as in N. S., resulted in a virtual loss of enterohepatic recirculation of bile salts when studied in this manner. The results of these studies are in accordance with the previous identification of the ileum as the only intestinal site of active transport of bile salts both unconjugated.l· 2 • 17 • 18 conjugated and Absorption of unconjugated bile acids due to nonionic passive diffusion has been demonstrated in the rat.l 8 • 19 Indeed, it has been suggested recently that absorption via diffusion may be quantitatively important in the over-all recirculation of bile salts. 18 Previous observations in this laboratory 4 in two patients with abnormalities of the distal small intestine have indicated that such absorption, i.e., diffusion through proximal small intestine, may not contribute significantly to the enterohepatic recirculation of sodium taurocholate-C14. The present studies extend these observations with taurocholate and provide similar information with the major primary cholic acid conjugate, glycocholate. However, it should be reemphasized that the present experimental design only permitted measurement of the over-all recirculation of injected bile salts after 24 hr. It has been estimated that bile salts recirculate at least twice during each meal. 20 Therefore, it may be assumed that these materials recirculated at least 6 times during this 24-hr period of study. A partial loss of absorptive capacity compounded over six enterohepatic cycles would deplete these patients of the injected isotope. This sequence

Vol. 55, No. 1

could occur in the patients presently described if proximal absorption of bile salts was significant. A study of the decline of specific activity of injected labeled bile salt during the initial 24-hr period would be required to assess the relative contribution of the proximal region of the small intestine to the enterohepatic circulation of bile salts. Such data are presently not available in these patients. However, previous studies in the dog, with surgical resections of the small bowel, suggest that such proximal contribution is minimal for taurocholate. 3 • 21 Bacterial modification of primary bile salts enables such materials to traverse the intestinal mucosa via nonionic diffusion processes. A small intestinal bacterial flora, developed in these patients following surgery, could provide bacterial modification of the injected primary bile salt with resultant deconjugation and passive absorption. One could reason that this mechanism of absorption might also be enhanced in situations of loss of ileal transport, since bile salts in greater abundance would enter the large bowel and be subject to bacterial modification and passive diffusion. In these cases, considerable total C 14 -radioactivity would then persist in sequential duodenal aspirates. This metabolic sequence was most apparent from measurements of total C 14 -radioactivity at 24 hr in patient E. B., who had a resection of the distal one-third of the small intestine and the cecum, and to a lesser extent in all other patients studied (table 2). It is apparent that the residual radioactivity at 24 hr was composed of bile salts (fig. 3) derived from well recognized metabolic pathways. 22 These observations suggest that there is a tendency in some patients for microbial metabolites of taurocholate and glycocholate to persist in the enterohepatic circulation longer than the parent compound. The extent of metabolic alteration of primary cholate conjugates was variable in the present study. Deconjugation was operative in all instances and was most apparent in studies which followed the administration of taurocho-

July 1968

DISORDERS OF THE DISTAL SMALL BOWEL

late-C 14 . Here, it can be seen readily that the glycocholate-Cl 4 obtained from duodenal aspirates at 24 hr may be considered a product of bacterial metabolism, since it could only arise as a result of bacterial hydrolysis followed by reabsorption of the C 14 -cholate and subsequent hepatic conjugation with glycine. Thus it is not representative of newly synthesized glycocholate or glycocholate which recirculated intact enterohepatically. It is also apparent that dehydroxylation was chiefly operative in patient E. B. with subsequent reabsorption of the resultant deoxycholic acid and hepatic reconjugation with glycine and taurine. Although firm statements cannot be made regarding the origin of the residual glycocholate-C 14 obtained 24 hr after the intravenous administration of glycocholate-Cl 4 to patients J. H. and E. B., the patterns of distribution of radioactivity are strikingly similar whether taurocholate-C 14 or glycocholate-C14 was administered (fig. 3). This similarity is in accord with the concept that the radioactivity present in the 24-hr sample was derived from a common precursor; in this case, C 14 -cholate was formed from bacterial hydrolysis. It should be emphasized that no measurable radioactivity was present in duodenal samples obtained from any patient at 48 hr. When taurocholate-C 14 was readministered to patient E. B. during tetracycline administration, no detectable radioactivity was present in the 24-hr sample. Although the intestinal flora was not studied bacteriologically, this observation with tetracycline lends additional support to the role of passive diffusion as a mechanism of absorption of deconjugated bile acids in some patients with ileal disorders. These considerations of bacterial metabolism of primary bile salts are not quantitatively important to the enterohepatic circulation of the present patients, suggesting that the colon need not assume a quantitatively important role. However, they do serve to point up the complexity of the qualitative contribution to the bile salt pool in certain pathophysiological states, which result in a loss of the ileal transport system. Ad-

13

ditionally, studies in patients utilizing unconjugated labeled bile acids may not detect these phenomena. In such experiments, the injected C 14 -cholate would be partitioned by the liver between the taurocholate and glycocholate pools, and bacterially dependent interconversions of taurocholate and glycocholate (or other primary bile salts) could not be ascertained. Consequently, one would not be able to delineate whether residual radioactivity in cholate bile salts resulted from an enhanced bacterial action or from a lesser degree of ileal pathology. The present studies which include the kinetics of the enterohepatic recirculation of taurocholate in normal man (control values ) confirm our previous observations in 2 normal medical students. 4 The kinetics of the enterohepatic recircula tion of glycocholate have not been previously reported in man. It is apparent that the t 112 of the disappearance of labeled glycocholate is comparable to that of taurocholate-C 14 . Also, the average exchangeable glycocholate-C14 pool is approximately 3 times greater than the average taurocholate-C 14 pool (table 3). Thus, the cholate pool in normal man , derived from the summation of taurocholate and glycocholate pools separately determined, approximates 1500 mg, which is consistent with previous observations in control subjects which followed the oral administration of cholic acid-24-C14.15, 23, 24 We have previously reported estimates of taurocholate pool in patients who exhibit a marked decrease in enterohepatic bile salt recirculation. 4 The available bile salts (body pool) accumulated in the gall bladder prior to discharge may be assumed to represent newly synthesized material. In the present study, estimates of taurocholate pool were also uniformly reduced in all patients studied when compared with the normal values. Similarly, estimates of glycocholate pool were reduced in 3 of 5 patients studied when compared with normal subjects. It is apparent that a reduction in combined conjugated cholate pool was exhibited by all patients studied (table 3). Compared with nor-

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HEATON ET AL.

Vol. 5li, No. 1

mal subjects this amounted to an average rocholate administration to patients with 43% decrease in patients with jejunocolic distal small bowel resection. fistula and 67% decrease in those patients Although the data are limited, the with lesser abnormalities of the distal present observations on the ratio of glycosmall intestine. cholate pool to taurocholate pool suggest It has been estimated that the bile salt a relative increase in glycine conjugation pool in normal man recirculates six to in patients with an absent enterohepatic eight times daily. 20 In the steady state circulation of bile salts. These patients situation, bile salt synthesis is equal to may have enhanced requirements for daily fecal loss. 22 In this way, the size taurine and its precursors. Similar elevaof the normal bile salt pool is maintained tions in the ratio of glycine to taurine at sufficient levels to allow for adequate conjugates of bile acids have been reported concentrations in the intestinal lumen. in patients with prolonged external biliary Thus, for a normal cholate pool size of drainage 26 and in patients with hypo1500 mg, the intestine is perfused with 9 thyroidism.27 to 12 g of cholate conjugates daily and, Summary therefore, around 18 to 24 g of total bile salts daily. On the other hand, in patients 1. The enterohepatic recirculation of with interruption of the enterohepatic sodium glycocholate-24-C 14 and sodium circulation, it may be assumed that the taurocholate-24-C 14 was studied in 8 volunbulk of bile salts present in the biliary teer medical studients, 3 patients with tree at any one time represents material jejunocolic fistula, and 3 patients with which has been synthesized de novo and lesser degrees of abnormality of the distal may only be available to the intestine small intestine. once. If this thesis maintains, then the 2. In the normal subjects, the halfquantity present in the gall bladder at the time of the disappearance of labeled glycoend of an overnight fast approximates the cholate was comparable to that of tauromaximal synthesizing capacity of the cholate-C14. The average glycocholate pool liver over a 12-hr period. This quantity, as was approximately 3 times greater than estimated by isotope dilution techniques, the average taurocholate pool. Thus, the was approximately 900 mg in 2 patients cholate pool in normal man derived from with jejunocolic fistula and approximately the summation of taurocholate and 550 mg in 2 patients with lesser abnormali- glycocholate pools, separately determined, ties of the distal small intestine (table 3). approximates 1500 mg. Therefore, in 24 hr perhaps no more than 3. A marked decrease in enterohepatic twice this amount, 1800 mg and 1100 mg recirculation of sodium glycocholate-C 14 of cholate bile salts, respectively, perfused and sodium taurocholate-C 14 was demonthe intestine of these patients. The present strated in all patients with jejunocolic study does not permit precise identifica- fistula. Additionally, resection of the distal tion of the mechanism of the steatorrhea one-third of the small intestine or extensive exhibited by these patients. However, it inflammatory disease of the distal one-half seems likely that hepatic bile salt syn- of the jejunoileum also resulted in a thesis, although enhanced when entero- similar loss of enterohepatic recirculation hepatic recirculation is compromised, is of these bile salts. not sufficient to meet the daily require4. Measurements of total C 14 -radioacments of the intraluminal bile salt concen- tivity in all patients suggest that there tration for digestive processes. This is a tendency in some instances for microsuggestion is further supported by the bial metabolites of taurocholate and glycorecent report of Hardison and Rosen- cholate to persist in the enterohepatic berg,25 demonstrating improvement of fat circulation longer than the parent comabsorption and micellarization of intra- pound. These microbial metabolites induodenal lipid following oral sodium tau- cluded two classes of substances: (a)

July 1968

DISORDERS OF THE DISTAL SMA LL BOWEL

bile salts (cholate ) which resulted from deconjugation of the administered cholate conjugates and (b) bile salts (deoxycholate) which resulted from deconjugation and reduction of the administered cholate conjugates. Both classes of substances were reconjugated by the liver. These metabolites had minimal quantitative importance in the present patients, since they were absent from the enterohepatic circulation by 48 hr. 5. These experiments demonstrate the importance of the distal small intestine in maintaining a normal enterohepatic recirculation of sodium glycocholate and sodium taurocholate. The mechanism of the steatorrhea, which was exhibited by all patients, has been discussed in relation to ileal disorders and the resultant decrease in bile salt enterohepatic recirculation. REFERENCES 1. Lack, L., and I. M. Weiner. 1961. In vitro absorption of bile salts by small intestine of rats and guinea pigs. Amer. J. Physiol. 200: 313-317. 2. Glasser, J., I. M . Weiner, and L. Lack. 1965. Comparative physiology of intestinal taurochola te transport. Amer. J. Physiol. 208: 359-362. 3. Playoust, M. R., L . Lack, and I. M. Weiner. 1965. Effect of intestinal resection on bile salt absorption in dogs. Amer. J. Physiol. 208 : 363-369. 4. Austad, W. I., L . Lack, and M. P. Tyor. 1967. Importance of bile acids and of an intact distal small intestine for fat absorption . Gastroenterology 52: 638-646. 5. Borgstrom, B., G. Lundh, and A. Hofmann. 1963. The site of absorption of conjugated bile salts in man. Gastroenterology 1,5: 229-238. 6. Van de Kamer, J. H., H. B . Huinink, and H. A. Weye rs. 1949. Rapid method for the determination of fat in feces. J . Bioi. Chern. 177: 347-355. 7. Ro e, J . H ., and E . W. Rice. 1949. Photometric method for t he determination of fre e pentoses in animal tissue. J. Bioi. Chern. 173 : 507-512. 8. Schilling, R. F. 1953. Intrinsic factor studies. II . The effect of gastric juice on the urinary excretion of radioactivity after the oral administration of radioactive vitamin B". J. Lab. Clio. Med . 1,2: 860-866.

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9. Udenfriend, S., E. Titus, and J. H. Wiessbach . 1955. The identification of 5-hydroxy-3-indoleacetic acid in normal urine and method for its assay. J. Bioi. Chern. 216: 499-505. 10. Irvin, J . L ., C. G. Johnston, and J . Kopala. 1944. A photometric method for the determination of cholates in bile and blood. J. Bioi. Chem.153 : 439-457. 11. M cCarthy, C. F., J. L. Borland, Jr., H. J. Lynch, Jr., E . E. Owen, and M. P . Tyor. 1964. Defective uptake of basic amino acids and L-cystine by intestinal mucosa of patients with cystinuria. J. Clio. Invest. 1,3: 1518-1524. 12. Norman, A. 1956. Preparation of conjugated bile acids using mixed carboxylic acid anhydrides. Bile acids and steroids. Arkiv. K emi. 8: 331-342. 13. Lack, L., and I. M . Weiner. 1963. The intestinal action of benzmalecene : the relationship of its hypocholesterolemic effect to active transport of bile salts and other substances. J . Pharmacal. Exp . Ther. 139: 248-258. 14. Englert, E., Jr., and U. S. W . Chin. 1966. Quantitative analysis of human biliary evacuation with a radioisotopic technique. Gastroenterology 50: 506-518. 15. Lindstedt, S. 1957. The turnover of cholic acid in man. Acta Physiol. Scand . 1,0 : 1-9. 16. Hirsch, J., E . H. Ahrens, Jr., and D. H . Blankenhorn. 1956. Measurement of the human intestinal length in vivo and some causes of variation. Gastroenterology 31: 274-284. 17. Holt, P. R. 1966. Competitive inhibi tion of in testinal bile salt absorption in the rat. Amer. J . Physiol. 210: 635--639. 18. Dietschy, J . M ., H. S. Salomon, and M . D. Siperstein. 1966. Bile acid metabolism. I. Studies on the mechanisms of intestinal transport. J. Clin. Invest. 1,5: 832- 846 . 19. Tidball, C. S. 1962. Intestinal transport of cholate and organic dyes. Amer. J. Physiol. 206: 239- 242. 20. Borgstrom, B., A. Dahlquist, G. Lundh, and J. Sjovall. 1957. Studies of intestinal digestion and absorption in the human. J. Clin. Invest. 36 : 1521-1536. 21. Lack, L ., and I. M . Weiner. 1963. Intestinal abso rption of bile salts and some biological implications. Fed. Pro c. 22: 1334-1338. 22. Bergstrom, S. 1962. Metabolism of bile acids. Fed. Proc. 21: 28-32. 23. Lindstedt, S., J. Avigan, D. S. Goodman,

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J. Sjovall, and D. Steinberg. 1965. The effect of dietary fat on the turnover of cholic acid and on the composition of the biliary bile acids in man. J. Clin. Invest.

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24. Danielsson, H ., P. Eneroth, K. H ellstrom, S. Lindstedt, and J. Sjovall. 1963. On the turnover and excretory products of cholic acid and chenodeoxycholic acid in man. J. Bioi. Chern. 238: 2299-2304. 25. Hardison, W. G. M., and I. H. Rosenberg. 1967. Bile salt deficiency in the steatorrhea

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following resection of the ileum and proximal colon. New Eng. J . Med. 277: 337-342. 26. Ekdahl, P. H., and J. Sjovall. 1958. On the conjugation and formation of bile acids in the human liver. I. On the excretion of bile acids by patients with postoperative choledochostomy drainage. Acta Chir. Scand. 114: 439-452. 27. Hellstrom, K., and J. Sjovall. 1961. Conjugation of bile acids in patients with hypothyroidism. J. Atheroscler. Res. 1: 20.5-210.