Role of Bile Acid Malabsorption in Pathogenesis of Diarrhea and Steatorrhea in Patients with Ileal Resection

Vol. 62, No. 5 Printed in U.S.A.

GASTROENTEROLOGY

Copyright © 1972 by The Williams & Wilkins Co.

ROLE OF BILE ACID MALABSORPTION IN PATHOGENESIS OF DIARRHEA AND STEATORRHEA IN PATIENTS WITH ILEAL RESECTION I. Response to cholestyramine or replacement of dietary long chain triglyceride by medium chain triglyceride ALAN F. HOFMANN, M.D., AND J. RAINER POLEY, M.D.

Gastroenterology Unit, Mayo Clinic and Mayo Foundation, Rochester, Minnesota

Balance studies, which focused on bile acid metabolism, were carried out on 9 patients with ileal resection in order to define the importance of bile acid malabsorption in the pathogenesis of the diarrhea and steatorrhea. The influence of cholestyramine (Q) or type of dietary fat-long chain triglyceride (LCT) or equicalorically substituted medium chain triglyceride (MCT)-on bile acid synthesis, diarrhea, steatorrhea, and fecal electrolyte excretion was measured during steady state intervals of four randomized periods (LCT, LCT + Q, MCT, and MCT + Q) . During control (LCT) periods, all patients had bile acid malabsorption, excreting > 75% of administered taurocholate-i'C within 24 hr, and this malabsorption was accompanied by a 10- to 20-fold increase in bile acid synthesis. Patients with ileal resection of less than 100 cm had greatly increased concentrations of bile acids in fecal water compared with normal control subjects. Administration of cholestyramine significantly decreased fecal weight, frequency, and sodium ion excretion as well as the concentration of bile acid in fecal water. These observations, together with our previous demonstration that bile acids induce water and electrolyte secretion by the human colon, provide evidence that a major cause of diarrhea in these patients was the increased passage of bile acids into the colon. Cholestyramine caused the already increased bile acid synthesis to increase still more in some of these patients; maximal synthesis was 0.1 to 0.3 mmole per kg of body weight per day, based on gas chromatographic measurements of fecal bile acids. All patients had mild steatorrhea « 20.g per day), Received April 29, 1971. Accepted December 7, 1971. This work was presented in part at the meetings of the American Society for Clinical Investigation, May, 1968, I and the American Gastroenterological Association, May, 1969. 2 A preliminary report describing certain aspects of the clinical response has been published. 3 Address requests for reprints to: Dr. Alan F. Hofmann, Gastroenterology Unit, Mayo Clinic, Rochester, Minnesota 55901. This investigation was supported in part by Research Grant AM-6908 from the National Institutes of Health, Public Health Service.

The authors acknowledge the technical assistance of Mary Kaye Merrick, Janet A. Carter, Mrs. Kathleen Rydell, Carol A. Gronseth, Charles B. Betts, and Alan Strange; the aid in composing diets from Corazon Guevara and Esperanza R. Briones; the excellent nursing care directed by Lee E . Fast and the personnel of the Clinical Study Unit; and, most important, the cooperation of the patients described in this study. The authors are also indebted to their colleagues Dr. William F. Taylor and Mrs. Mary M. Ho for planning and executing the statistical analyses. Supplies of cholestyramine and medium chain triglyceride were generously provided by Mead Johnson and Company. 918

May 1972

BILE ACID METABOLISM AFTER ILEAL RESECTION. I

919

and malabsorption of fat appeared to contribute little to diarrhea because diarrhea persisted during MCT periods when steatorrhea was not present. Cholestyramine did increase steatorrhea when LCT was fed, but this was of no caloric significance. Patients with ileal resection of more than 100 cm had greater steatorrhea (> 20 g per day); fat malabsorption appeared to be a major cause of diarrhea, since replacement of LCT by MCT caused a decrease in fecal weight, sodium, and potassium as well as a striking decrease in steatorrhea. Unabsorbed fatty acids were converted in part to hydroxy fatty acids by intestinal bacteria and it is proposed that unabsorbed fatty acid or its bacterial product, hydroxy fatty acid, or both, contributed to diarrhea. Bile acid malabsorption appeared to play no direct role in the diarrhea in 2 of 3 patients, since there was no response to cholestyramine and the concentration of bile acids in fecal water was low. Malabsorption of bile acids was nonetheless important in the syndrome, since jejunal bile acid concentrations were decreased (causing fat maldigestion) in 2 of 3 patients, and fat maldigestion, together with decreased mucosal surface, was the probable explanation for the severe steatorrhea. These studies define two syndromes of bile acid malabsorption, present evidence for different mechanisms of diarrhea in each, and describe a therapeutic program. It is now well established that most patients with ileal resection have bile acid malabsorption. 4-6 In animal studies,7-9 as well as in limited human studies,10-12 an increase in hepatic synthesis of bile acids from cholesterol has been observed when the enterohepatic circulation of bile acids is interrupted, indicating that patients with bile acid malabsorption should have an increased passage of bile acids into the colon. The demonstration that dihydroxy bile acids induce water secretion in the perfused human colon 13 led to the proposal 14 that the diarrhea associated with ileal resection could be caused, in part, by a direct action of bile acids on the colonic mucosal cell. If this were true, bile acidbinding agents such as cholestyramine should ameliorate the diarrhea, and such has been reported. 3 This paper is the first of three summarizing studies aimed at characterizing bile acid metabolism in patients with ileal resection and defining the role of disturbed bile acid metabolism in the diarrhea and steatorrhea frequently present in such patients. Ideally, such a study would be carried out in a homogeneous group of patients and would include not only balance measurements during prolonged steady

state intervals of randomized periods but also characterization of secretion, digestion, and absorption along the entire alimentary canal. Furthermore, the site and magnitude of bacterial biotransformations should be quantified. Our studies partially fulfill such criteria in that we have carried out balance studies, characterized digestion of a test meal by jejunal sampling, and determined, by gas chromatography, the extent to which bacterial degradation of bile acids and fatty acids occurred during intestinal passage. As will be shown in this paper, our patient group was not homogeneous but comprised at least two groups clearly distinguishable by clinical features, nature of disturbance in bile acid metabolism, and therapeutic response. Thus, presentation of data comparing responses during different periods among different patient groups in a lucid manner is difficult, and we find it necessary to divide our results for presentation according to three major topics. This paper defines bile acid kinetics and the influence of cholestyramine and type of dietary fat on fat, bile acid, water, and electrolyte excretion on the basis of balance techniques. Subsequent papers

920

HOFMANN AND POLEY

will characterize the role of bacteria as well as the effect of interrupted enterohepatic circulation of bile acids on fat digestion.

Patients, Design, and Methods Patients Relevant clinical and laboratory data on patients are given in table 1. All patients had diarrhea, but the 6 group C patients (referred to henceforth as "mild steatorrhea") had an ileal resection of less than 100 cm and steatorrhea of less than 20 g per day. Three of these 6 patients had normal results on Schilling tests. Group D patients (referred to henceforth as "severe steatorrhea") had larger resections, similar diarrhea, significantly greater steatorrhea, and little vitamin BI2 absorption.

Experimental Design The experimental design used for patients C I through C 5 and all D patients featured four 6- to lO-day periods: long chain triglyceride (LCT), medium chain triglyceride (MCT), LCT + cholestyramine (Q), and MCT + Q. Their order was randomized (table 2) to give an incomplete randomized Latin square design and to permit statistical treatment by conventional analysis of variance techniques. Patient C. had an a-b-a-b- design (LCT, LCT + Q) with MCT and MCT + Q periods omitted. Patients were hospitalized in a metabolic study unit. All patients were on constant fluid and sodium intake, a 2-day repeating diet, generally at 40 kcal per kg of body weight, and all except C. received chromium sesquioxide, 0.5 g before each meal, as a steady state nonabsorbable indicator. For LCT periods, 40% of calories was as conventional dietary fat; for MCT periods, 90% of LCT calories was replaced by MCT calories by using MCT oil (table 2). Cholestyramine (Questran), 4 g, was ingested 15 min before each meal and on retiring.

Analytical Methods Processing of stools. Feces were collected in preweighed paint cans and kept refrigerated during the 24-hr collection periods. After weighing, a known volume of saline, 0.5-inch Burundum cylinders, and 5-mm glass beads (A. H. Thomas, Philadelphia) were added and the sample was shaken for 3 min in a commercial paint shaker. The pH of the homogenate was then determined using a combination electrode. A 100-ml aliquot was frozen and stored

Vol. 62, No.5

at -20 C. For subsequent analyses, samples were thawed for V2 hr in a warm bath or overnight at 4 C and rehomogenized by shaking as described, and weighed aliquots were taken. Fat. Fecal fat was determined by saponification, acidification, and extraction of liberated fatty acids into toluene, as described by Jover and Gordon. 15 One aliquot of the toluene phase was titrated with 0.08 N tetrabutyl ammonium hydroxide in methanol, using bromothymol blue as indicator I. ; obtained values were converted to mass by using the molecular weight of oleic acid. No correction was made for the small amount of bile acids in the toluene extract. More than 97% of added synthetic 9(10)-hydroxystearic acid was recovered by this procedure. The mean deviation of analyses on duplicate aliquots of fecal homogenates was 3.6%. A second aliquot of the toluene phase was stored at -20 C for subsequent gas chromatographic analysis. Bile acids. Bile acid malabsorption was assessed by measuring the fraction of taurocholate-24- 14 C (Tracerlabs, Waltham, Mass.; purity-checked by thin layer chromatography I 7) excreted in stool 24 and 48 hr after intravenous administration of it before breakfast. The radioactivity present in stool was isolated by alkaline saponification, acidification, extraction into ethyl acetate, bleaching with hydrogen peroxide, and liquid scintillation spectroscopy.18 Samples were analyzed in duplicate with a mean deviation of analyses of duplicate aliquots of 2.5%. Total fecal bile acid excretion-that is, daily synthesis-was determined by gas chromatography. A 5.0-g aliquot of stool was gently saponified, essentially as described by Grundy et al.,19 and extracted with petroleum hydrocarbon to remove sterols. To an aliquot of the aqueous phase an internal standard, 1 mg of synthetically prepared, chromatographically pure nordeoxycholic acid, 20 was added; ethanol was removed by an air stream at 37 C. Sodium hydroxide was added to make the sample 2 N in NaOH, and vigorous saponification (4 hr, 115 C) was carried out in a nickel bomb (Parr Instrument Company, Moline, I1l.). After acidification, free bile acids were extracted three times with 3 volumes of diethyl ether, esterified with freshly prepared diazomethane, and oxidized with chromic oxide as described by Evrard and Janssen. 2I The keto bile acid methyl esters were dissolved in diglyme (2methoxyethyl ether) and 2 to 8 tLg were analyzed on 1% or 3% OV -17 columns isothermally at 270 to 280 C. The amount of bile acid in the sample was calculated from area of

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Fat

Measured by pathologist at time of operation. II, ileum; Ce, cecum ; As, ascending colon; Tr, transverse colon. ' Steady state interval (see text) for all patients (mean ± SE) . d The following were within normal limits in all patients except as noted: hemoglobin; hematocrit value ; erythrocyte, leukocyte, and reticulocyte counts; serum electrolytes, Fe, Ca, P , alkaline phosphatase, serum glutamic oxaloacetic transaminase, bilirubin; prothrombin time ; sulfobromophthalein (BSP) retention; albumin: globulins (lgG, IgM, IgA) ; blood urea; blood glucose; and urinalysis.

D: patients with large resection and severe steatorrhea

C: patients with small resection and mild steatorrhea

Group

Pa-

TABLE

BSP, 14%; Mg,1.58

IgA, 1.9

Albumin, 2.8; IgA, 1.6 Mg,1.80

Ca,8.8; albumin, 2.6 IgA, 1.5

serum values d

Abnormal

HOFMANN AND POLEY

922

TABLE

2. Experimental design a

Order of experimenta l periods!> Caloric intake

Patient

LeT

LeT + Q

2 3 1 2, 5 4 1,3

1 2 4 1 3 2, 4

MeT

Vol . 62, No . 5

LeT period, per· centage of calories

MeT + Q

MeT period. percent· age of calories as LeT

MeT

keal/kg

C1 C, C3 C. C, C.

3 4 2 3 1

4 1 3 4 2

38 .7 39.9 38.6 51 - 53' 37.8 26.4

37 .3 36.9 37.3 36.1 37.4 37 .2

4.6 4.9 5.9 5.6 3.4

33.9 32.6 31.1 30 .5 33.6

01 0, 03

4.9 1 2 3, 5 4 35.2 35.2 29.9 4.5 31.8 4 2 1 43.2 36.9 3 4.9 32.0 42.7 37.2 4 3 1 2 a Patients received a 2-day repeating diet and constant water and electrolyte intake throughout the study. Periods, randomized as shown, were 6 to 12 days long and the last 4 to 5 days were considered to be a steady state interval. Laboratory tests and assessment of bile acid and vitamin B 12 absorption were carried out prior to the study. Jejunal sampling during digestion of two sequential test meals was carried out prior to the study to assess bile acid concentration during digestion; the procedure was repeated at the end of the LCT + Q period, but 4 g of cholestyramine were given to the patient just prior to the ingestion of each test meal. b LCT, long chain triglyceride; Q, cholestyramine ; MCT, medium chain triglyceride. , Adolescent boy.

the peaks determined by electronic integrat.ion by using factors for varying flame ionization detector response and standard formulas. 22 Mean detector response correction factors were 0.93 for monoketo bile acids, 0.96 for diketo bile acids, and 1.15 for triketo bile acids. The coefficient of variation of replicate analyses (N = 49), performed over a 4 -month interval, of a standard mixture (sample size, 1 to 14 j.Lg) was: monoketo acids, 4.5%; diketo acids, 3.0%; and triketo acids, 7.5%. The method of saponification and extraction has been shown 1 9 to give complete recovery of added radioactive bile acids, and we confirmed this by recovery after vigorous alkaline saponification of greater than 95% of added cholate- 1 ·C. We chose to analyze keto derivatives in preference to trimethyl silyl ethers or acetates because of the possibility of nonbile acid components having retention times nearly identical to those of the bile acids which have been demonstrated to occur in healthy subjects 23 and which might be increased in disease; after conversion of bile acids to keto derivatives, it was unnecessary to remove fatty acids prior to gas chromatography. The chromatograms obtained gave two or three well resolved peaks of keto acids. We used OV-17 rather than JXR since this phase (obtained from Applied Science Laboratories, State College, Pa.) afforded a better separation of the 23-keto bile acid internal standard from the mono- and diketo bile acids. Reten-

tion times on 1% OV-17 (relative to methyl 3,12-diketonorcholanoate) were: monoketo, 0.69; diketo, 1.31; triketo, 2.08. The absolute retention time of the internal standard was 8 to 9 min. The precision of the method was only fair, with a mean deviation on duplicate aliquots of 12 fecal homogenates of 15.6%. The validity of the method was confirmed by replicate analyses of a standard mixture of taurocholate, glycocholate , taurodeoxycholate, glycodeoxycholate, chenodeoxycholate, and lithocholate, which showed good agreement between expected and observed results. An aliquot of the ether phase obtained after alkaline saponification was methylated, and the acetate methyl esters"' of the bile acids were then prepared and chromatographed on 1 % QF-l (Applied Science Laboratories) and 1% AN-600 (Analabs, Hamden, Conn.) columns to identify individual fecal bile acids. From peak areas and published 25 or experimentally determined retention times of known bile acids, it was possible to calculate cholic and chenodeoxycholic acid synthesis, assuming that peaks with retention times corresponding to known bile acids represented only those bile acids and that the detector response was similar to that of the nearest neighbor bile acid of a standard mixture of lithocholic, deoxycholic, chenodeoxycholic, and cholic acids. Typical correction factors (relative to nordeoxycholic acid) for nonlinear detector response were: lithocholic, 0.91; deoxycholic,

May 1972

BILE ACID METABOLISM AFTER ILEAL RESECTION. I

1.00; chenodeoxycholic, 1.28; cholic, 1.37; and 7-ketocholic, 1.14. The coefficient of variation for 14 replicate analyses of a standard mixture carried out over a I-week interval was: lithocholic, 3.7%; deoxycholic, 3.5%; chenodeoxycholic, 4.7%; and cholic, 6.1%. Bile acids in aqueous phase of stool. Homogenates (stools + water) were centrifuged at 100,000 x g for 2 to 4 hr at 25 to 40 C in a Spinco model L. The side of the tube was pierced with a needle and all of the clear aqueous supernate was aspirated. Bile acids were quantified by gas chromatography of the methyl ester acetates on AN-600, using nordeoxycholic acid as an internal standard, and also by the steroid dehydrogenase method 2. after deproteination with methanol-acetone. 27 The concentration of bile acids in fecal water was calculated by assuming that all stools were 85% water and correcting for water added during homogenization. Values for healthy persons were obtained on two 24-hr stool collections from 5 normal subjects. Total hydroxy fatty acid excretion (hydroxy fatty acid present in both aqueous and particulate phases of stool) was determined by gas chromatography. Fatty acids were esterified by using boron trifluoride-methanol (Applied Science Laboratories, State College, Pa.). The methyl esters were than acetylated 2. and chromatographed at 181 C on 3% OV-17 (Applied Science Laboratories) using a 6-foot column. Detector response, based on a standard reference mixture containing added synthetic 9(10)-hydroxystearic acid, was directly proportional to mass injected. The percentage of hydroxy fatty acid was calculated and multiplied by fecal excretion to give hydroxy fatty acid excretion in grams per day. Marker. Chromium sesquioxide (Cr 2 0 3) was determined colorimetrically after nitric acidsodium molybdate-perchloric acid oxidation 2S or by atomic absorption spectroscopy (Perkin Elmer Model 303) after nitric acid-sulfuric acid oxidation (R. S. Goldsmith, personal communication). Duplicate analyses showed a mean deviation of 3.4%. Electrolytes. Fecal Na+ and K+ and urinary Na+ were determined by flame emission spectroscopy. Fecal Ca++ and Mg++ were determined by atomic absorption spectroscopy after nitric acid oxidation.

Calculation of Results The last 4 days of each study period (days 6 to 10) were considered to be a steady state interval and values from them were used for the determinations reported. Although much

923

longer study periods would have been preferable, we were unable to find patients who would consent to longer hospitalization. Fecal outputs were corrected by multiplying obtained values by the reciprocal of the fraction of Cr 2 0 S recovered; such data are termed "corrected. " Fecal frequency was based on records kept by the patient. Fecal weight was determined directly, and the quotient of fecal mass divided by fecal frequency is defined as "mean bowel movement mass." To determine the distribution of sodium loss between urine and stool, the quotient [fecal Na+/(fecal Na + + urine Na+») x 100 was calculated.

Statistical Treatment Data for steady state intervals were analyzed by a conventional analysis of variance technique 29 which tested drug effect, diet effect, patient variability, and all possible interactions. The compensated patients were analyzed as a group, with the assumption that they would be similar in response to drug or diet or both. The decompensated patients were analyzed similarly.

Results Bile Acid Kinetics Radioactive taurocholate excretion rate was increased in all patients (table 3), indicating bile acid malabsorption. Bile acid synthesis rates were markedly increased in all patients and the increase in synthesis involved both cholic acid and chenodeoxycholic acid. The degree to which increased bile acid synthesis compensated for the interrupted enterohepatic circulation of bile acids was inferred by measurements of bile acid concentration in jejunal contents obtained during digestion of two sequential test meals. Analyses of jejunal aspirates, to be detailed in a subsequent paper, showed that bile acid concentrations of the aqueous (micellar) phase were within normal limits in 4 of 6 of the patients with mild steatorrhea (group C), whereas they were decreased in 2 of 3 of the patients with severe steatorrhea (group D).

Marker Recovery Total chromium sesquioxide recovery for steady state intervals was satisfactory

924

Vol. 62, No.5

HOFMANN AND POLEY TABLE

3. Assessment of bile acid absorption, bile acid synthesis, and postprandial jejunal bile acid concentration Taurocholate-I·e excretion

Patient

24·hr

Bile acid

synthesis, mean ± SE

48·hr % of dose

gldoy

C, C. C3 C, C, C.

97.0 79 .0 96 . 1 60.2 40.0 90.6

98.0 93 . 2 97.0 97 .5 69 .0 102.0

3.0 3.9 6.5 2.0 1.4 2.0

0,

97 .8 90 .6 72 .9

98.3 98.0 91.8

2.0 2.8 2.2

O. 0

3

In health

12

±

5"

23

±

8"

± ± ± ± ± ± ± ± ±

Cholic acid synthesis

Bile acid concentration in aqueous phase of jejunal aspirate, mean

a

Meal I

gldoy

%

Meal 2 mM

mM

0.4 0.5 2.1 0.3 0.3 0 .9

1.6 3.2 3.5 1.6 1.0 1.2

57.7 82 .2 59.7 77.4 66.0 61.0

5.7 8 .2 10 .8 11.1 3.3 4.3

4.9 7 .0 5.4 8 .8 1.9 5.0

0 .2 0.3 0.3

1.0 2.2 1.6

53.3 79.5 67 .0

2.6 5.4 0.7

1.8 4.0 0 .1

0.1-0.4<

69

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Means of dally chohc aCid productIOn (durmg steady state mterval of LCT penod) were obtamed by summing all gas chromatographic peaks with retention times of bile acids derived from cholic acid. This mean was divided by the sum of all peaks to give percentage of cholic acid in fecal bile acids. The percentage value was multiplied by total bile acids which had been determined independently by gas chromatography of ketoderivatives to give grams per day. " Based on references 4 and 5. Mean ±SE.
for all patients (means ± SE: 101 ± 8, 109 ± 9,88 ± 7,103 ± 6, 91 ± 14, 104 ± 7, 101 ± 11, and 95 ± 9% for patients Dl through D 5 and C 1 through C a, respectively). By analysis of variance, marker recovery did not differ significantly between individual study periods. However, when recovery data for the days at the beginning of each study period were compared with those of the days comprising the subsequent steady state intervals, marker recovery was significantly decreased (P < 0.02) at the beginning of those study periods when cholestyramine was given. Thus, intestinal retention of marker occurred when diarrhea abated, and corrections of fecal data based on chromium sesquioxide output were invalid until a new steady state had obtained-when output equalled intake. Diarrhea Electrolytes and water. Cholestyramine caused a consistent decrease in fecal Na + in patients with mild steatorrhea (group C) (fig. 1), which was statistically signi-

ficant (P <0.001). These patients continued to have diarrhea when MCT was substituted for LCT, and cholestyramine also was effective on this dietary regimen. The change in dietary fat had no influence on fecal Na +. The decreased fecal Na + excretion caused by cholestyramine was associated with a parallel decrease in fecal water, manifested by significantly (P <0.001) decreased fecal weight (fig. 2). Fecal K + excretion behaved similarly to fecal Na + excretion (P = 0.06) (fig. 3) . Urine Na + increased when fecal Na + decreased (P <0.02), but the effect was marked in only 4 patients (C 1 , C a, C 4 , and C s) in whom daily excretion doubled. In patients with severe steatorrhea (group D), replacement of LCT by MCT (low fat diet) caused a significant (P < 0.04) decrease in fecal Na + in all 3 patients (fig. 4), although the magnitude of the diet effect in these patients was less than that of the drug effect in the compensated patients. Cholestyramine decreased fecal Na + in only 1 of these 3 patients; when data for all patients were pooled, the effect was

May 1972

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FIG. 1. Influence of cholestyramine or medium chain triglyceride-long chain triglyceride (MCT-LCT) exchange on daily fecal sodium (corrected by chromium sesquioxide data) during steady state intervals in patients with mild steatorrhea (group C). In this and subsequent figures, horizontal bar indicates mean; LCT and MeT periods are indicated by solid circles; LCT + Q and MCT + Q periods are indicated by open circles; and the direction of cholestyramine effect is indicated by a vertical arrow .

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925

BILE ACID METABOLISM AFTER ILEAL RESECTION. I

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not significant. Fecal water, manifested by fecal weight, was significantly (P < 0.001) decreased by the low fat diet, whereas the effect of drug was not significant (fig. 5). Fecal K + was decreased by the low fat diet (P = 0.06) and, again, drug had no consistent effect (fig. 6). Urinary Na + was not consistently altered by diet or drug in group D patients.

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Fecal and urinary Na + values from each patient were added to give total Na + excretion. In patients with mild steatorrhea (group e) the fraction ofthis sum excreted in stool decreased from 49 to 15% when cholestyramine was given (P < 0.03), whereas diet had no effect. In the patients with severe steatorrhea (group D), the change from LeT to MeT decreased the fraction of Na + in stool from 19 to 8% (P < 0.02) and cholestyramine had no effect. Fecal frequency. Fecal frequency was significantly (P = 0.03) decreased by drug but not by diet in patients with mild stea-

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Vol . 62, No.5

HOFMANN AND POLEY

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patients (P < 0.005) , but the magnitude of the response was small. Thus, frequency was decreased only by drug in the patients with mild steatorrhea but much more by diet than by drug in patients with severe steatorrhea . Since fecal mass and fecal frequency were correlated, mean bowel movement mass changed little during periods; differences between patients were highly significant (P < 0.001 for subject variation in each group), but differences caused by

e2

C1

LeT MeT

FIG . 5. Effect of drug or diet on daily corrected fecal weight during steady state intervals in patients with severe steatorrhea (group D) .

14

12 10

O2

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~

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~

~

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·

.

.

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LeT

MeT

LeT MCT

LCT MeT

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-·i ·· LeT

0

LeT MeT

MeT

FIG. 6. Effect of drug or diet on daily corrected fecal potassium during steady state intervals in patients with severe steatorrhea (group D) .

torrhea (group C) (fig. 7). In the patients with severe steatorrhea (group D), however, the change from LCT to MCT rather than drug caused a consistent and marked decrease in fecal frequency (P 0.025) (fig. 8) . Cholestyramine also decreased fecal frequency in all group D

D3

D2

14

S-

~...

12

'b

-Q

E:

'"

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00

FIG. 7. Effect of drug or diet on daily fecal frequency during steady state intervals in patients with mild steatorrhea (group C) .

0 0

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• •

... ...

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•

•

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:

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FIG. 8. Effect of drug or diet on daily fecal frequency during steady state intervals in patients with severe steatorrhea (group D).

May 1972

~ILE

927

ACID METABOLISM AFTER ILEAL RESECTION. I

drug or diet were not. Bowel movement mass averaged about 125 g, but was as low as 90 g in some patients and as high as 240 g in others.

Bile Acid Synthesis Bile acid synthesis, already greatly increased during control (LeT) periods, increased still further when cholestyramine was given in 5 of9 patients (table 4). In patients with mild steatorrhea (group e), the 2 patients with the highest synthesis rate during LeT (e. and e a) showed the least increase in bile acid synthesis during cholestyramine treatment (table 5) . This lack of further increase suggests that these patients were already synthesizing bile acids at the maximal rate. Fecal Fat Figures 9 and 10 show fecal fat excretion; uncorrected values have been used because the corrected numbers were impossible on several occasions, indicating separation of marker and fat during intestinal transit. Fecal fat in patients with mild steatorrhea (group e) was tripled by cholestyramine administration (P < 0.001). Fecal fat was significantly (P < 0.01) lower when MeT was substituted for LeT, and steatorrhea increased little when cholestyramine was given. In the patients with severe steatorrhea (group D), cholestyra-

mine also tripled the fecal fat in 2 of 3 patients (P < 0.001). MeT substitution decreased fecal fat significantly (P = 0.03), and only a small increase in fecal fat was observed when cholestyramine was added.

Fecal Divalent Cations Fecal ea ++ appeared to be influenced little by drug or diet in patients e., e 2, and e a and all D patients, indicating that marked changes in fecal fat and Na + exTABLE 5. Influence of cholestyramine (Q) and type of dietary fat on bile acid synthesis· LeT

MeT

Patient

NoQ

+ Q

%

change

No Q

+ Q

C, C, C2 C, C,

0.022 0.037 0.049 0 .075 0 . 110

D, D2 D,

0.030 0.039 +30 0 .013 0 .022 0 .050 0.139 +159 0.030 0.054 0.051 0.053 + 4 0.032 0.048 0 .003-0 .010

In health b

0 .055 + 150 0.010 0.051 + 38 0.020 0.066 + 35 0 .028 0.080 +7 0.053 0.100 - 9 0.051

% change

0 .055 + 450 0.041 + 105 0.051 +82 0.063 +19 0.055 +8 + 69 +80 + 50

• Synthesis shown as grams per kilogram per day. LCT, long chain triglyceride; MCT, medium chain triglyceride. b Based on references 30 to 32. No reliable data have been published for bile acid synthesis in normal individuals on MCT diet or given cholestyramine.

TABLE 4. Total fecal bile acids· Mean ( ± SE) Patient

LCT

LeT + Q

MeT

MeT + Q

g/ciay

C, C2 C, C, C, C.

3 .0 3.9 6.5 2.0 1.4 2.0

± 0.2 ± 0.5 ± 2.7 ± 0.3 ± 0.3 ± 0 .9

D, D2 D,

2.0 ± 0.2 2.8 ± 0.3 2. 2 ± 0 . 3

3.2 5 .3 5 .9 2.8 2.7 2.6

± 0.3 ± 0.5 b ± 0.7 ± 0 . 4b ± LOb ± 0.2

2 .6 ± 0.3 7.8 ± 0 . 9 b 2.3 ± 0.4 b

2.1 2.2 3.0 1.1 0.5

± 0.3 ± 0.2 ± 0.6 ± 0.1 ± 1.0

0.9 ± 0 . 1 1.7 ± 0.3 1.4 ± 0 . 1

2.5 4.1 2. 9 2. 2 1. 2

± 0.4 ± 0 . 3b ± 0 .6 ± 0.5 b ± 0.5 b

1.5 ± 0.4 4 .0 ± 0 . 5b 2 . 1 ± O. 2b

• Corrected for Cr 20, recovery. b P < 0.05 for increased bile acid synthesis during cholestyramine periods by Wilcoxon rank sum test for paired replicates (LCT versus LCT + Q; MCT versus MCT + Q); for calculations, data from both dietary periods were pooled. LCT, long chain triglyceride; Q, cholestyramine; MCT, medium chain triglyceride.

928

~

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0 0

40 30

~.

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0

0

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0 0

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t

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LeT "leT

FIG . 9. Effect of drug or diet on daily uncorrected fecal fat during steady state intervals in patients with mild steatorrhea (group C).

80

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90

~ ~

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58, 980 ± 30, and 326 ± 29 mg per day, respectively. Fecal Mg++ appeared to vary less than fecal Ca + +, and there also was no effect of drug or diet despite the marked changes in steatorrhea and fecal electrolyte secretion already discussed. Mean (±SE) fecal Mg++ excretion in these patients was 112 ± 4, 240 ± 10, 176 ± 16, 148 ± 9, 96 ± 2, 68 ± 3, and 61 ± 3 mg per day, respectively . Fecal pH

The mean fecal pH for all study periods varied from 6.4 to 7.3 (data not given), which is within normal limits.34 No consistent change in pH was induced by diet or drug in patients with mild steatorrhea. In the patients with severe steatorrhea (group D) the addition of cholestyramine to an MCT diet increased fecal pH in all patients. Thus, in these patients, fecal pH was highest when diarrhea was least. Concentration of Bile Acids in Aqueous Phase of Centrifuged Stool

0

-,

60 50

:,

I

0 0

a c::

'-

03 0

~

"t;,'

Vol . 62, No.5

HOFMANN AND POLEY

0

o·

I•

•

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FIG. 10. Effect of drug or diet on daily uncorrected fecal fat excretion during steady state intervals in patients with severe steatorrhea (group D).

cretion were not associated with changes in fecal Ca++ excretion. Patient C 4, a growing boy, showed a consistent decrease in fecal Ca ++ and Mg+ + when M CT replaced LCT. Ca ++ excretion varied widely from patient to patient but was constant for each patient during the study. Mean ( ± SE) fecal Ca + +excretion in patients C 1 through C 4 and Dl through D3 was 164 ± 14, 341 ± 11, 1,148 ± 92, 429 ± 34, 914 ±

Table 6 shows that bile acid concentrations were strikingly increased in patients with mild steatorrhea (group C), when compared with healthy controls; bile acid concentrations were consistently and markedly decreased by cholestyramine (P < 0.001) with either diet. In 2 of 3 patients with severe steatorrhea (group D), the concentration of bile acids was not different from that observed by us in the aqueous phase of stool from healthy individuals. These low concentrations are thought to be caused by (1) the lower concentration of bile acids in small intestinal content entering the colon and (2) bacterial 7a-dehydroxylation, causing bile acid precipitation. Patient D 2 , who had an increased bile acid concentration in fecal water, differed from the other 2 patients in having a greater increase in bile acid synthesis, higher jejunal bile acid concentrations, and little bacterial 7a-dehydroxylation. The chemical composition of fecal bile acids in relation to physical state in these patients will be detailed in a subsequent paper in this series.

May 1972

929

BILE ACID METABOLISM AFTER ILEAL RESECTION. I

Discussion

of bile acids in fecal water and jejunal contents, mechanism of diarrhea, and response to treatment. Bile acid diarrhea . In patients with mild steatorrhea and smaller ileal resection the major pathophysiologic disturbance is the increased passage of bile acids into the colon, producing diarrhea. The effective compensatory hepatic bile acid synthesis in most of these patients is evidenced by their normal jejunal bile

These studies show that two separate and well defined clinical entities may be found among patients with ileal resection and bile acid malabsorption (table 7). Common to both groups is bile acid malabsorption followed by a compensatory increase in hepatic bile acid synthesis. Yet the groups differ in degree of steatorrhea, length of resection, concentration TABLE

6. Bile acids in aqueous plw.se of stool a Mean ( ±

SE)

concentration

(mM)

Patient LeT + Q

Le T

C1 C. C3 C. Dl D. D3 In health (N = 5) a

7.5 13 .4 12.0 1l.2

± 0.8 ± 2.0 ± 0 .5 ± 0.6

0.8 0.9 0.6 0 .9

1.7 ± 0.3 12.0 ± 2.8 1.1±0. 2 1.9 ± 0 . 5

± 0.1 ± 0.1 ± 0 .1 ± 0.2

2.4 ± 1.4 2 . 2 ± 0.6 0 .5 ± 0.2

MeT

2.9 5.1 5.3 6.2

± 0.2 ± 1.9 ± 0.1 ± 0.8

1.4±0.4 11.4 ± 1.8 1.1±0.4

1.1 0.5 1.1 1.1

± ± ± ±

0.2 0 .2 0.1 0. 3

0.3 ± 0 5.1 ± 0.5 0.5 ± 0 . 1

LCT, long chain triglyceride; Q, cholestyramine; MCT, medium chain triglyceride. TABLE

7. Two syndromes of interrupted enterohepatic circulation in patients with ileal resection Bile acid diarrhea

Clinical features Ileal resection (cm) Fecal weight (g/day) Steatorrhea (g/day) Bile acid metabolism Bile acid malabsorption Bile acid synthesis Jejunal bile acid concentration Fecal water bile acid concentration Bacterial biotransformations Hydroxy fatty acid production Deoxycholic acid production Response to therapy Cholestyramine

LCT/MCTa exchange

30-100 250- 1000 8- 20

" Fatty acid" diarrhea

~100

250-1000 > 20

Present Increased Normal or decreased Increased

Present Increased Usually decreased Normal

Normal

Increased

Decreased

Normal

Decreased diarrhea, of sympto· matic benefit Increased steatorrhea, of no caloric significance No decrease in diarrhea

Small decrease in diarrhea, of no symptomatic benefit Increased steatorrhea, causing sig· nificant caloric loss Decrease in diarrhea, of symptomatic benefit Decrease in steatorrhea, of probable caloric benefit

Decrease in steatorrhea a

MeT + Q

LCT, long chain triglyceride; MCT, medium chain triglyceride.

930

HOFMANN AND POLEY

acid concentration, which permits relatively unimpaired fat digestion and absorption. The 2 patients with the smallest resections had somewhat decreased jejunal bile acid concentrations; yet little steatorrhea was present, and it is hypothesized that the remaining intestinal surface area was adequate to compensate for the slight decrease in jejunal bile acid concentration. The diarrhea of these patients is believed to be caused in large part by bile acid stimulation of colonic secretion of water and sodium. The concentration of bile acids in solution in the stools of the patients with bile acid diarrhea was similar to that which we have shown to cause secretion of water and electrolytes during colonic perfusion in man. 13 Cholestyramine is considered to have abolished diarrhea in these patients by binding bile acids and thus preventing them from having this effect. Although cholestyramine was beneficial in the treatment of diarrhea in these patients, it also bound bile acids in the jejunum and induced fat mal digestion (to be presented in detail subsequently). It thus antagonized the beneficial effect (in regard to fat digestion) of the compensatory increase in hepatic synthesis of bile acids. The cholestyramine-augmented steatorrhea in the patients with small resection and bile acid diarrhea is of no caloric or symptomatic consequence, based on our clinical experience with patients receiving cholestyramine for up to 3 years. Thus, diarrhea and steatorrhea are dissociated in the patient with bile acid diarrhea, both in the treated and untreated states. "Fatty acid" diarrhea. In the 3 patients with severe steatorrhe~ and larger resection (group D), fat absorption was markedly impaired because of deficient intraluminal micellar dispersion of digested lipids and because of a greater decrease of mucosal surface area. In these patients there was an increased passage of fatty acids into the colon, which seemed causally related to the diarrhea because fecal weight diminished when steatorrhea was ameliorated by replacing LCT with

Vol. 62, No.5

MCT. To define the chemical form of fecal fatty acid, we carried out gas chromatographic analyses which showed that, in these patients, hydroxy fatty acid excretion was greatly increased (D I, 2.9 g per day; D 2 , 2.6 g per day; D 3 , 12.6 g per day), whereas all patients with mild steatorrhea (group C) excreted less than 0.4 g per day. Hydroxy fatty acids were produced by colonic bacteria in vitro 35 and are present among the fecal fatty acids in patients with diarrhea and steatorrhea. 36, 37 Hydroxy fatty acids resemble ricinoleic acid in structure, which is the hydroxy acid responsible for the cathartic action of castor oil, 3 8 but their role in the diarrhea of these patients cannot be defined by this type of study since they will invariably be associated with an increase in nonhydroxy fatty acids. Therefore, we suggest that the term "fatty acid" diarrhea be used until the role of the hydroxy fatty acids can be assessed. Furthermore, the possibility that fatty acids cause diarrhea by inducing fecal flora to produce other cathartic substances cannot be excluded. In principle, the 3 patients with "fatty acid" diarrhea should also have had bile acid diarrhea. Yet, during MCT periods, diarrhea was not present in these patients although it persisted unchanged in the patients with mild steatorrhea (group C). This paradox-the absence of diarrhea in group D patients during MCT periodswas finally explained by the finding that bile acid concentration in fecal water was low during LCT and MCT periods and this, in turn, was explained by chromatographic analyses (to be presented) showing extensive bacterial 7a-dehydroxy lation of bile acids with consequent bile acid precipitation. 39 Such 7a-dehydroxylation did not occur in most of our group C patients. To summarize, diarrhea in patients with severe steatorrhea appeared to be in part a consequence of increased passage of fatty acid into the colon. Concomitantly, bacterial degradation of bile acids caused bile acid precipitation, precluding bile acid diarrhea (fig. 11). Patient D2 did not form deoxycholic acid, and fecal bile acid concentrations were high in this patient. Diarrhea was de-

May 1972

BILE ACID METABOLISM AFTER ILEAL RESECTION. I 101

" FATTY ACIDS "

I

FiG. 11. Mechanism of diarrhea in patients with small resection and mild steatorrhea (left) is contrasted to that in patients with large resection and severe steatorrhea (right). The unabsorbed fatty acid is converted in part to hydroxy fatty acid, but whether hydroxy fatty acid plays an important role in the pathogenesis of diarrhea has not been defined, nor can balance experiments exclude the possibility that unabsorbed fatty acid alters bacterial flora, with this change in tum associated with the production of new substances with cathartic activity.

creased by both diet and drug, and we believe that she had both bile acid and "fatty acid" diarrhea. A combination of the two syndromes should exist in patients who have steatorrhea despite normal bile acid concentrations and atypical flora which do not biotransform bile acids in such a way as to cause precipitation from solution. Several investigators 40. 4' have noted a decrease in fecal fat when MCT was used in managing patients with diarrhea and steatorrhea. In these studies, as in ours, it is difficult to distinguish a beneficial effect of MCT from a deleterious effect of LCT.42 A major constituent of LCT is unsaturated fatty acid, the precursor of hydroxy fatty acid, whereas octanoic acid, the major constituent of MCT, cannot be hydroxylated by bacteria. Indeed, hydroxy fatty acid excretion decreased markedly in patients D" D2 and D3 during MCT periods (to be published). Chromium sesquioxide as reference marker. Major advantages derived from use of the chromium sesquioxide marker in our studies were: (1) documentation of complete fecal collections; (2) evidence for retention of intestinal contents during transition from diarrheal to nondiarrheal states; and (3) proof that a steady state

931

interval was attained. In addition, correction of outputs on the basis of marker excretion decreased variability of electrolyte excretion during nondiarrheal periods, which increased the sensitivity with which a therapeutic response could be detected. 43 The considerable coefficient of variation still present after correction (20 to 35%) probably represents the sum of true biologic variation in daily outputs as well as demixing of marker and intestinal contents. Such separation was observed with fat, corrected values being 149 and 195 g per day on two occasions; in agreement, the variation in daily output of fat was not decreased by the marker correction. Demixing of chromium sesquioxide from the fecal stream also has been observed in sheep.44. 45 Clinical features and parameters of diarrhea. Our patients with ileal resection differed from many patients with large intestinal resection in having fecal weights of less than 1 kg per day42 and having no problem with water, electrolytes, or acidbase balance. Their major clinical problem was the inconvenience of increased fecal frequency. Influence of cholestyramine on steatorrhea. Cholestyramine increased steatorrhea during "",LCT periods in all patients, indicating the importance of bile acids for fat absorption in our patients. Cholestyramine did not induce diarrhea even when it caused a marked increase in steatorrhea, presumably because of binding of fatty acids by the resin in the colon. 46 Lipolysis in jejunal aspirates was not disturbed by cholestyramine administration, although cholestyramine removed all bile acids from solution and greatly diminished the concentration of lipids in the micellar phase (to be published). Cholestyramine has been shown to decrease the absorption of dietary 47-49 and endogenous lipid 48 in experimental animals and to induce steatorrhea in healthy man. 49 However, cholestyramine does not interfere with the absorption of MfT in experimental animals 47 or man 49 ; in agreement with these observations, little increase in fecal fat occurred when cholestyramine was added to the MCT diet. 50 In contrast to long chain

932

HOFMANN AND POLEY

fatty acids, medium chain fatty acids are soluble in water and may be absorbed without requiring solubilization by bile acid micelles. 51. 52 Control of bile acid synthesis. Our data showing greatly increased bile acid synthesis in patients with bile acid malabsorption are consistent with recent studies indicating that bile acid synthesis is controlled by a negative feedback mechanism. 8 Although it has been proposed 53 that the amount of chylomicron cholesterol rather than the amount of bile acid returned to the liver regulates cholesterol synthesis, the greatly increased bile acid synthesis (and accordingly greatly increased cholesterol synthesis) observed in our patients with normal fat digestion, negligible steatorrhea, and probably good cholesterol absorption seems to us to be evidence in favor of the latter hypothesis. Our studies suggest that the maximal rate of bile acid synthesis, at least in ileal resection patients, is in the range of 0.1 to 0.3 mmole per kg per day. This would seem to be in fair agreement with the limited data published on normal human subjects 1o. 11. 54 and monkeys 9 and rats. 8 Thus, when derepressed, the liver can increase its synthesis rate 5 to 15 times. Iatrogenic bile acid and fatty acid diarrhea. The ileal bypass procedure which has been recommended for patients with hypercholesteremia should cause bile acid diarrhea, in principle. Published reports,55 however, claim that diarrhea is infrequent postoperatively. Patients with familial hypercholesteremia appear to have decreased bile acid synthesis compared with normal subjects under metabolic unit study conditions,56 and the few published data for bile acid synthesis after ileal bypass 12. 57 are significantly below those observed by us. Such could explain the reported low incidence of diarrhea after surgery; a second possibility is that patients with hypercholesteremia have intestinal flora differir:g from patients reported here. We selected ileal resection patients who had symptomatic diarrhea, and our data provide no information on the incidence of diarrhea in such

Vol. 62, No.5

patients. In patients undergoing large intestinal bypass surgery for life-threatening obesity, severe diarrhea and steatorrhea have been frequent. 58 On the basis of our data, such patients should have a "fatty acid" diarrhea and, in some instances, a coexisting bile acid diarrhea. Alternate approaches to therapy. The ideal therapy for these syndromes of bile acid malabsorption would be an ileal transplant. It seems unlikely that an inhibitor of bile acid synthesis would be efficacious in the compensated patients, since steatorrhea would occur and might well give rise to a fatty acid diarrhea. In decompensated patients, replacement therapy with a noncathartic bile acid would correct the digestive deficit but decreased mucosal surface would persist. Our present approach is to treat compensated patients with cholestyramine in doses of 4 to 16 g per day; although clinical experience has been good, the long term consequences are unknown. When cholestyramine is stopped, diarrhea returns. Decompensated patients are placed on a very low fat diet with MeT supplementation to ensure caloric balance; cholestyramine, if beneficial, is added to this dietary regimen. The major defect with this therapeutic program is its cost and inconvenience. REFERENCES 1. Poley JR, Hofmann AF: Diarrhea following ileal

2.

3. 4. 5.

6.

resection: pathogenesis and treatment (abstr) . J Clin Invest 47:79a-80a, 1968 Hofmann AF, Poley JR: Cholestyramine treatment of diarrhea associated with ileal resection: factors influencing response (abstr) . Gastroenterology 56:1168, 1969 Hofmann AF, Poley JR: Cholestyramine treatment of diarrhea associated with ileal resection. N Engl J Med 281:397-402, 1969 Meihoff WE, Kern F Jr: Bile salt malabsorption in regional ileitis, ileal resection, and mannitolinduced diarrhea. J Clin Invest 47:261-267, 1968 Stanley MM, Nemchausky B: Fecal C " ·bile acid excretion in normal subjects and patients with steroid-wasting syndromes secondary to ileal dysfunction. J Lab Clin Med 70:627-639, 1967 Heaton KW, Austad WI, Lack L, et al: Entero-

May 1972

BILE ACID METABOLISM AFTER ILEAL RESECTION. I

hepatic circulation of C"-Iabeled bile salts in disorders of the distal small bowel. Gastroenterology 55:5-16, 1968 7_ Eriksson S: Biliary excretion of bile acids and cholesterol in bile fistula rats. Proc Soc Exp Bioi .Med 94:578-582, 1957 8. Shefer S, Hauser S, Bekersky I, et al: Feedback regulation of bile acid biosynthesis in the rat. J Lipid Res 10:646-655, 1969 9. Dowling RH, Mack E, Small DM: 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. J Clin Invest 49:232-242, 1970 10. Samuel P, Saypol GM, Meilman E, et al: Absorption of bile acids from the large bowel in man. J Clin Invest 47:2070-2078, 1968 11. Moutafis CO, Myant NB, Tabaqchali S: The metabolism of cholesterol after resection or by pass of the lower small intestine. Clin Sci 35: 537-545, 1968 12. Moore RB, Frantz ID Jr, Buchwald H: Changes in cholesterol pool size, turnover rate, and fecal bile acid and sterol excretion after partial ileal bypass in hypercholesteremic patients. Surgery 65:98-108, 1969 13. Mekhjian HS, Phillips SF, Hofmann AF: Colonic secretion of water and electrolytes induced by bile acids: perfusion studies in man. J Clin Invest 50:1569-1577, 1971 14. Hofmann AF: The syndrome of ileal disease and the broken enterohepatic circulation: cholerheic enteropathy. Gastroenterology 52:752-757, 1967 15. Jover A, Gordon RS Jr: Procedure for quantitative analysis of feces with special reference to fecal fatty acids. J Lab Clin Med 59:878-884, 1962 16. Cohen M, Morgan RGH, Hofmann AF: One-step quantitative extraction of medium-chain and long-chain fatty acids from aqueous samples. J Lipid Res 10:614-616, 1969 17. Hofmann AF: Thin-layer adsorption chromatography of free and conjugated bile acids on silicic acid. J Lipid Res 3:127-128, 1962 18. Hofmann AF, Schoenfield LJ, Kottke BA, et al: Methods for the description of bile acid kinetics in man. Methods Med Res 12:149-180,1970 19. Grundy SM, Ahrens EH Jr, Miettinen TA: Quantitative isolation and gas-liquid chromatographic analysis of total fecal bile acids. J Lipid Res 6:397- 410, 1965 20. Riegel B, Moffett RB, McIntosh AV: nor-Desoxycholic acid, Organic Syntheses, collective vol 3. Edited by EC Horning. New York, John Wiley and Sons Inc, 1955, p 234 21. Evrard E, Janssen G: Gas-liquid chromato-

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933

graphic determination of human fecal bile acids. J Lipid Res 9:226-236, 1968 Kuksis A: Newer developments in determination of bile acids and steroids by gas chromatography, Methods of Biochemical Analysis, vol 14. Edited by D Glick. New York, Interscience Publishers, Inc, 1966, p 325-454 Eneroth P, Hellstrom K, Sjovall J: A method for quantitative determination of bile acids in human feces. Acta Chem Scand 22: 1729-1744, 1968 Roovers J, Evrard E, Vanderhaeghe H: An improved method for measuring human blood bile acids. Clin Chim Acta 19:449-457, 1968 Eneroth P: Thin-layer chromatography of bile alcohols and bile acids, Lipid Chromatographic Analysis, vol 2. Edited by GV Marinetti. New York, Marcel Dekker Inc, 1969, p 149-183 Iwata T, Yamasaki K: Enzymatic determination and thin-layer chromatography of bile acids in blood. J Biochem (Tokyo) 56:424-431, 1964 Kottke BA, Wollenweber J, Owen CA Jr: Quantitative thin-layer chromatography of free and conjugated cholic acid in human bile and duodenal contents. J Chromatogr 21:439-447, 1966 Bolin DW, King RP, Klosterman EW: A simplified method for the determination of chromic oxide (Cr,O,) when used as an index substance. Science 116:634-635, 1952 Brownlee KA: Statistical Theory and Methodology in Science and Engineering. New· York, John Wiley and Sons Inc, 1965, p 504-526 Lindstedt S: The turnover of cholic acid in man. Acta Physiol Scand 40:1-9, 1957 Ali SS, Kuksis A, Beveridge JMR: Excretion of bile acids by three men on corn oil and butterfat diets. Can J Biochem 44:1377-1388, 1966 Vlahcevic ZR, Bell CC Jr, Buhac I, et al: Diminished bile acid pool size in patients with gallstones. Gastroenterology 59: 165-173, 1970 Go VLW, Poley JR, Hofmann AF, et al : Disturbances in fat digestion induced by acidic jejunal pH due to gastric hypersecretion in man. Gastroenterology 58:638-646, 1970 Wrong 0, Metcalfe-Gibson A, Morrison RBI, et al: In vivo dialysis of faeces as a method of stool analysis. I. Technique and results in normal subjects. Clin Sci 28:357-375, 1965 Thomas PJ: In vitro conversion of oleic acid to hydroxy stearic acid by intestinal bacteria (abstr). Clin Res 18:609, 1970 Kim YS, Spritz N: Hydroxy acid excretion in steatorrhea of pancreatic and non pancreatic origin. N Engl J Med 279:1424-1426,1968 James AT, Webb JPW, Kellock TD: The occurrence of unusual fatty acids in faecal lipids

934

38.

39.

40.

41. 42.

43.

44.

45.

46.

47.

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HOFMANN AND POLEY

from human beings with normal and abnormal fat absorption. Biochem J 78:333-339, 1961 Masri MS, Goldblatt LA, DeEds F, et al: Relation of cathartic activity to structural modifications of ricinoleic acid of castor oil. J Pharm Sci 51:999-1002, 1962 Gustafsson BE, Norman A: Physical state of bile acids in intestinal contents of germfree and conventional rats. Scand J Gastroenterol 3:625-631, 1968 Holt PR: Studies of medium chain triglycerides in patients with differing mechanisms for fat malabsorption, Medium Chain Triglycerides. Edited by JR Senior. Philadelphia, University of Pennsylvania Press, 1968, p 97-107 Greenberger NJ, Ruppert RD, Tzagournis M : Use of medium chain triglycerides in malabsorption. Ann Intern Med 66:727-734,1967 Bochenek W, Rodgers JB Jr, Balint JA: Effects of changes in dietary lipids on intestinal fluid loss in the short-bowel syndrome. Ann Intern Med 72:205-213, 1970 Grundy SM, Ahrens EH Jr, Salen G: Dietary ,B-sitosterol as an internal standard to correct for cholesterol losses in sterol balance studies. J Lipid Res 9:374-387,1968 Drennan MJ, Holmes JHG, Garrett WN: A comparison of markers for estimating magnitude of rumen digestion. Br J Nutr 24:961-970, 1970 MacRae JC, Armstrong DG: Studies on intestinal digestion in the sheep. I. The use of chromic oxide as an indigestible marker. Br J Nutr 23: 15- 23, 1969 Johns WH, Bates TR: Quantification of the binding tendencies of cholestyramine II: mechanism of interaction with bile salt and fatty acid salt anions. J Pharm Sci 59:329-333, 1970 Harkins RW, Hagerman LM, Sarett HP: Absorption of dietary fats by the rat in cholestyr· amine-induced steatorrhea. J Nutr 87:85-92, 1965 Morgan RGH, Hofmann AF: Use of 3H-labeled

49.

50.

51.

52.

53.

54. 55. 56.

57.

58.

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