MINIMAL BILE ACID MALABSORPTION AND NORMAL BILE ACID BREATH TESTS IN CYSTIC FIBROSIS AND ACQUIRED PANCREATIC INSUFFICIENCY

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1 GASTROENTEROLOGY 72:661--&i5, 1977 Copyright 1977 by The American Gastroenterological Association Vol. 72, No. 4, Part 1 Printed in U.s.A. MINIMAL BILE ACID MALABSORPTION AND NORMAL BILE ACID BREATH TESTS IN CYSTIC FIBROSIS AND ACQUIRED PANCREATIC INSUFFICIENCY RICHARD J. ROLLER, M.D., AND FRED KERN, JR., M.D. Division ofgastroenterowgy, University o f C olorado School of Medicine, Denver, Colorado This study was undertaken because of reports of a marked incrase in fecal bile acid excretion by childre with cystic fibrosis. We attempted to confirm this finding by performing [V 4 Clcholylglycine breath tests and by measuring fecal bile acid and fat excretion in patients with cystic fibrosis and acquired pancreatic insufficiency. Studies were done when patients were taking pancreatic enzymes (Cotazym) and also without medication. 14C0 2 excretion in breath was normal in patients with acquired pancreatic insufficiency and even lower in cystic fibrosis, both with and without Cotazym therapy. Fecal bile acid excretion was slightly elevated in both groups without Cotazym and became normal with Cotazym in patients with acquired pancreatic insufficiency. Steatorrhea was present in both patient groups and improved during Cotazym therapy. Bile acid malabsorption in cystic fibrosis and acquired pancreatic insufficiency is minimal and probably not clinically important. The enterohepatic circulation of bile acids in normal subjects is an efficient process. During each day the bile acid pool circulates 6 to 10 times, yet only 2 to 5% of the total pool is lost in the feces with each cycle. 2 These losses are greatly magnified when the enterohepatic circulation is broken, as it is in patients with ileal disease or resection. 3 The bile acid pool shrinks, often resulting in steatorrhea and lithogenic bile, and the excess bile acids entering the colon cause diarrhea. 3, 4 Weber et aj.5 found that the enterohepatic circulation is also broken in children with cystic fibrosis, who lose almost 7 times more fecal bile acids than do control subjects. The magnitude of the loss is comparable to that which occurs after ileal resection. Watkins et al. 6 reported a decrease in bile acid pool size and an increase in fractional turnover rate of cholic acid in a group of 6 children with untreated cystic fibrosis. Evidence of intact mucosal bile acid absorption and normal hepatic regulation of synthesis led them to implicate an intraluminal factor causing bile acid malabsorption. In a few adults with acquired pancreatic insufficiency, bile acid Received July 12, Accepted September 28, An abstract of this work has been published. 1 Address requests for reprints to: Dr. Richard J. Roller, University of Colorado Medical Center, Division of Gastroenterology, 4200 East 9th Avenue, Denver, Colorado This study was supported by Grant AM12626 from the United States Public Health Service, National Institutes of Health and by the Clinical Research Center RR-51, of the University of Colorado Medical Center. Dr. Roller's work was supported by Veterans Administration Gastroenterology Training Grant 110. The authors are grateful to Dr. Ernest Cotton, Department of Pediatrics, Dr. William R. Waddell, Department of Surgery, for referring patients, and to Patricia Coan for expert technical assistance. absorption was normal. 7,8 To elucidate these puzzling findings, we measured fecal bile acid and fecal fat excretion in patients with cystic fibrosis and acquired pancreatic insufficiency, before and during pancreatic enzyme replacement. We also performed the [1-14Clcholylglycine breath test,8-10 a sensitive test of bile acid malabsorption, and compared the results to those in control subjects and in patients with ileal resection. Patients and Methods Patients. Investigations were performed in the Clinical Research Unit and in the Gastrointestinal Laboratory under a research protocol approved by the Radiation Safety Committee and the Human Research Committee, University of Colorado Medical Center. Informed consent was obtained from adult patients or the parents of minors. Only males with cystic fibrosis were studied, because they are sterile and would be endangered less by irradiation. Normal control subjects were 7 men and 4 women, ages 19 to 68 (mean 51), with no evidence of pancreatic, hepatobiliary, or gastrointestinal disease. Diarrhea controls were 6 men and 4 women, ages 24 to 72 (mean 45), with diarrhea of various causes (ulcerative colitis, postgastrectomy, functional) but without roentgenographic or clinical evidence of ileal disease or bacterial overgrowth. There were 5 men and 5 women, ages 23 to 79 (mean 54), who had ileal resections of 28 to 300 cm, measured from the ileocecal valve, for Crohn's disease (5), mesenteric vascular insufficiency (3), actinomycosis (1), and carcinoid tumor (1). Nine patients, 7 men and 2 women, ages 32 to 73 (mean 53) had acquired pancreatic insufficiency as a result of chronic alcoholic pancreatitis (3), Whipple's operation for carcinoma (5), or traumatic pancreatitis (1). The 8 cystic fibrosis patients were males, ages 11 to 24 (mean 18), with pancreatic and pulmonary involvement. One additional patient with cystic fibrosis had had the distal 50% of his small bowel resected for meconium ileus at birth and was considered separately. All had stopped taking antibiotics at least 1 week before the study. 661

2 662 ROLLER AND KERN Vol. 72, N o.4, Part 1 Methods. The [V 4 Clcholylglycine breath test was performed in all subjects. Patients with cystic fibrosis or acquired pancreatic insufficiency had the test while they were receiving Cotazym (Organon, Inc., Toronto, Canada) and again at least 4 days after Cotazym had been discontinued. [1-14C1Cholylglycine, 5 to 10 JLC, specific activity 52 JLC per mmole (Amersham/Searle, Arlington Heights, Ill.) dissolved in 1 ml of ethanol, was given orally, followed immediately by 150 ml of water, to fasting, sedentary patients. s A standard hospital meal was given 2 hr later. Exhaled breath was collected hourly for 6 hr by passage over anhydrous CaS04, and 1 mmole of COz was trapped in a scintillation vial containing 1 ml of 1 M Hyamine hydroxide in methanol (Packard Instrument Co., Inc., LaGrange, Ill.), and 2 ml of methanol. Phenolphthalein was used to indicate neutralization. The exact quantity of COz collected was calculated from the concentration of Hyamine, which was determined by titration of the stock solution with hydrochloric acid. Radioactivity of 14COz in the vial was measured in a liquid scintillation counter (2425, Packard Instrument Company, Inc.). Results were expressed as total 14CO Z in expired breath after 6 hr, as a percentage of administered dose, with the assumption that the average CO 2 in expired breath is 9 mmoles per kg of body weight per hr." For fecal fat determinations, patients with cystic fibrosis or acquired pancreatic insufficiency were fed a measured 100-g fat diet and stools were collected for 72 hr. Fecal fat and fecal bile acid excretion were measured while the patients were taking Cotazym and again at least 4 days after it had been discontinued. The dose of Cotazym varied from 12 to 51 capsules per day. In every patient it was the same as or more than the patient's customary dose, depending on symptoms and gross appearance of stools. Fecal fat was determined by the method of Van de Kamer et al., 12 as modified by Anderson et al.,13 and expressed as grams of fat per 100 g of dietary fat per 24 hr (normal <5). Total fecal bile acids were measured by the hydroxy steriod dehydrogenase (Sigma Chemical Co., St. Louis, Mo.) assay, using a previously described modification. 14 The recovery of added deoxycholate, lithocholate, and conjugates of cholate averaged 88%, and measured values were appropriately corrected. The statistical significance of the differences between groups was evaluated by Student's t-test, and paired data were analyzed by the paired t-test. Results 14C0 2 excretion in breath after administration of [1-14Clcholylglycine did not differ significantly between normal and diarrhea control groups (table 1). They were therefore considered together as a combined control group. The range of normal cumulative 6-hr 14C0 2 breath excretion for our laboratory was defined as the combined control group mean ± 2 SD (0 to 4.97%). Patients with ileal resection, except 1 with only a 28-cm resection, had abnormal breath tests, implying bile acid malabsorption (control 2.7 ± 0.44%, patients ± 1.41 %; P < 0.001). 14C0 2 breath excretion in the acquired pancreatic insufficiency group, despite Cotazym therapy, was the same as in the control group, implying normal bile acid absorption (table 1, fig. 1). Two patients in this group did have slightly abnormal breath tests. Both had had operations for pancreatic carcinoma, and neither had clinical evidence of small intestinal bacterial overgrowth or ileal disease. No patient with cystic fibrosis alone had an abnormal breath test either with or without Cotazym. In fact, 14C0 2 breath excretion in this group was significantly lower than that of the controls (P < 0.005). The 1 patient with cystic fibrosis and ileal resection had abnormal breath tests, not affected by Cotazym therapy, indicating bile acid malabsorption. All patients with cystic fibrosis or acquired pancreatic insufficiency had steatorrhea (table 2). During pancreatic enzyme replacement therapy fecal fat decreased in both groups (P < 0.05), but many individuals had a poor response. Therapy was more effective in the ac- TABLE 1. Results of [l -'"'C]cholylglycine breath tests for each patient and control subject Acquired pancreatic insufficiency Cystic fibrosis Normal Diarrhea Ileal controls controls resection With Without With Without Cotazyrn Cotazyrn Cotazyrn Cotazyrn '"'C0 2 breath excretion/6 hr as % of administered dose b ' Mean ± SEM ±0.44 ±0.34 ±1.41 ±0.62 ±0.66 ±0.09 pc < NS NS <0.005 a Results for the patient with cystic fibrosis and ileal resection: with Cotazym, 8.63%; without Cotazym, 9.82.%. b Normal range, defined as mean of normal and diarrhea controls ± 2 so: (0 to 4.97%). c Significance of difference between each group mean and combined control group mean, 2.38 ± 0.25, analyzed by Student's t-test; NS, not significant ±0.21 <0.005

3 April BILE ACID ABSORPTION IN PANCREATIC INSUFFICIENCY 663 N'lO N l ~ ~ 5 0 N 9 ILEAL RE5(CTION N l FIG CO. breath excretion in 6 hr as percentage of administered dose for each group. breath at the rate of9 mmoles per kg of body weight per hr in the basal state. If this assumption were not valid for adolescents with cystic fibrosis, our conclusions could be erroneous. If one assumes that cystic fibrosis patients, because of age and disease, excrete CO 2 at double the normal rate, an unlikely but theoretically possible rate, the results of the breath test would be doubled. This is, the mean 14C0 2 excretion for the untreated patients would be 1.16% in 6 hr, which is still less than one-half of the control value. There are no data about endogenous CO2 production in such patients, but the basal oxygen consumption of adults with pulmonary emphysema is about 10% less than that of controls. 16 Watkins et al. 6 speculated that bile acids are malabsorbed because they are bound intraluminally to some product of digestion. If such binding prevented exposure quired pancreatic insufficiency group than in the cystic fibrosis group, as shown by lower fecal fat during Cotazym therapy (P < 0.05). This occurred despite the higher average dose of Cotazym in the cystic fibrosis group (38 capsules per day) than in the acquired pancreatic insufficiency group (20 capsules per day). The 1 patient with cystic fibrosis and ileal resection had steatorrhea which was only slightly improved by therapy. There was a minimal increase in fecal bile acid excretion in untreated patients with cystic fibrosis and acquired pancreatic insufficiency (table 3). Treatment with Cotazym caused a significant decrease in fecal bile acids in the acquired pancreatic insufficiency, but not in the cystic fibrosis group. Discussion These studies failed to confirm the marked increase in fecal bile acid excretion found by Weber et al. 5 [1-14C]Cholylglycine breath tests did not detect any bile acid malabsorption in our cystic fibrosis patients, and fecal bile acids were only minimally elevated. The mean fecal bile acid excretion of normal infants and children is 6.1 mg per kg per day, with a range of 1.9 to 11.6 mg per kg per day. 14 Normal adults excrete 200 to 650 mg per day, or for a 70-kg man, 3 to 9 mg per kg per day,2.15 and patients with ileal resection excrete a mean of 2553 mg per day, or 36.5 mg per kg per day. 7 Fecal bile acid excretion in our cystic fibrosis patients was only about twice normal, and not nearly as much as in patients with ileal resection. Almost all patients with ileal resection have increased breath 14C0 2 excretion after oral administration of [l- 14 C]cholylglycine If patients with cystic fibrosis have bile acid malabsorption of comparable magnitude, this breath test should detect it. 14C0 2 excretion was increased in 9 of 10 patients with ileal resection and in the 1 with cystic fibrosis and ileal resection, but in none with cystic fibrosis alone. 14C0 2 excretion was actually lower in the cystic fibrosis group than in controls. The calculation of the percentage of the administered [1-14C]cholylglycine excreted is based on the assumption that the average individual excretes CO 2 in expired TABLE 2. Fecal {at values {or patients with and without Cotazym Mean ± SEM pd Cystic fibrosis" Acquired insufficiency With Without With Without Cotazym Cotazym Cotazym Cotazym g/24 hr/loo g dietary {atb ND c ND ND ND < 0.05 < 0.05 a Results for the patient with cystic fibrosis and ileal resection: with Cotazym 24.2; without Cotazym b Normal less than 5. C Not done. d Significance of differences analyzed by paired t-test. TABLE 3. Fecal bile acid values {or patients with and with Cotazym Cystic fibrosis Acquired insufficiency With Without With Without Cotazym Cotazym Cotazym Cotazym mg/kg body wt/24 hr NDb ND ND ND Mean ± SEM ±2.02 ±1.96 ± 1.98 ±3.95 p c NS <0.05 a Results for the patient with cystic fibrosis and ileal resection: with Cotazym 27.3; without Cotazym b Not done. (. Significance of differences analyzed by paired t-test; NS, not significant.

4 664 ROLLER AND KERN Vol. 72, No.4, Part 1 of the bile acid-amide bond from colonic bacteria, deconjugation might be prevented, causing false negative breath tests. Yet binding of bile acids by cholestyramine does not prevent d ~ c o n j u 8 galthough a t i o n colonic. bacteria in cystic fibrosis patients have not been studied, it seems unlikely that their microflora would lack the deconjugating enzymes, which are present in so many different species offecal microorganismsy We are unable to explain why 14C02 excretion in the cystic fibrosis patients was even lower than in control subjects. In the two published studies, pancreatic replacement therapy in patients with cystic fibrosis decreased fecal bile acid loss,5 increased bile acid pool, and decreased bile acid fractional turnover rate. 6 Similar studies have not been reported in patients with acquired pancreatic insufficiency. In pancreatic exocrine insufficiency of any cause, such therapy decreases steatorrhea. 5,18-20 Although Cotazym significantly improved steatorrhea in both cystic fibrosis and acquired pancreatic insufficiency patients, the response was variable, and mean fecal fat excretion remained above normal in both groups. Despite a smaller dose of Cotazym, acquired pancreatic insufficiency patients had a better response than did those with cystic fibrosis, possibly explaining why fecal bile acid excretion returned to normal during therapy in the acquired pancreatic insufficiency patients but not in the cystic fibrosis patients. There is little published information on bile acid malabsorption in acquired pancreatic insufficiency. Fecal bile acid excretion was normal in 1 patient, 7 but in unpublished studies by Mietinnen et ai., 21 "slight bile acid malabsorption" was found. Bile acid breath tests have also been normal in several such patients. 8 The slight bile acid malabsorption in our acquired pancreatic insufficiency patients is of the same magnitude as in cystic fibrosis patients and may have a common cause. The cystic fibrosis patients we studied were older than those studied by Weber et aj.5 (mean age 4.9 years) and Watkins et al. 6 (range 3 months to 4 1 /2 years). These older patients represent a selected group-the survivors from the population of younger patients with cystic fibrosis - but severity of pulmonary disease, not pancreatic disease, is the major influence on survival. 22 Pancreatic insufficiency was as severe in our group as in those previously studied. In patients with cystic fibrosis and acquired pancreatic insufficiency, bile acid malabsorption is minimal and probably not clinically important. We were unable to confirm by either of two techniques the marked bile acid malabsorption found in other studies of cystic fibrosis. Note Added in Proof In an important paper which we previously overlooked, Goodchild et al. 23 published data that resolves, to a large extent, the discrepancy between our findings and those of Weber et al. 5 and Watkins et al. 6 Goodchild and her associates studied fecal bile acid excretion in 29 patients with cyctic fibrosis, varying in age from a few months to 20 years. Their data show that bile acid excretion was clearly greater in young patients «12 years, N = 21) than in the older group (N = 8): 24.2 mg per kg per 24 hr in the younger group versus 5.1 in the older group. In both groups, patients with liver disease, who presumably had a decreased bile acid pool size, had a smaller fecal bile acid excretion. For example, in the 4 older patients with liver disease the mean daily fecal bile acid excretion was 3.4 mg and in those without liver disease it was 7.2 mg per kg per 24 hr. The latter figure is identical to the fecal bile acid excretion in their 11 control subjects and slightly less than we found in our older cystic fibrosis patients, none of whom had liver disease. REFERENCES 1. Roller J, Kern F Jr: Minimal bile acid (BA) malabsorption and normal BA breath tests in cystic fibrosis (CF) and acquired pancreatic insufficiency (API) (abstr). Gastroenterology 70:930, Small DM, Dowling RH, Redinger RN: The enterohepatic circulation of bile acids. Arch Intern Med 130: , Hofmann AF: The syndrome of ileal disease and the broken enterohepatic circulation: cholerheic enteropathy. Gastroenterology 52: , Kelly TR, Klein RL, Woodford JW: Alterations in gallstone solubility following distal ileal resection. Arch Surg 105: , Weber AM, Roy CC, Merin CL, et al: Malabsorption of bile acids in children with cystic fibrosis. 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J BioI Chern 177: , Anderson CM, Frazer AC, et al: Coeliac disease: gastrointestinal studies and the effect of dietary wheat flour. Lancet 1: , Weber AM, Chartrand L, Deyen G, et al: The quantitative determination of fecal bile acids in children by the enzymatic method. Clin Chim Acta 39: , Ali SS, Kuksis A, Beveridge JM: Excretion of bile acids by three men on a fat-free diet. Can J Biochem 44: , McGregor M, Becklake M: The relationship of oxygen cost of breathing to respiratory mechanical work and respiratory force. J Clin Invest 40: , Lewis R, Gorbach S: Modification of bile acids by intestinal bactera. Arch Intern Med 130: , Ross CA: Fat absorption studies in the diagnosis and treatment of pancreatic fibrosis. Arch Dis Child 30: , KaIser MH, Leite CA, Warren WD: Fat assimilation after mas-

5 April 1977 BILE ACID ABSORPTION IN PANCREATIC INSUFFICIENCY 665 sive distal pancreatectomy. N Engl J Med 279: , Jordan P, Grossman M: Effect of dosage schedule on the efficacy of substitution therapy in pancreatic insufficiency. Gastroenterology 36: , Miettinen TA, Ahrens EH, Grundy 8M: Quantitative isolation and gas-liquid chromatographic analysis of total dietary and fecal neutral steroids. J Lipid Res 6: , Guide to Diagnosis and Management of Cystic Fibrosis. Prepared by Professional Education Committee, Chairman CF Doershuk, National Cystic Fibrosis Research Foundation, 1971, Atlanta 23. Goodchild MC, Murphy GM, Howell AM, et al: Aspects of bile acid metabolism in cystic fibrosis. Arch Dis Child 50: , 1975