Tests of Bile-acid and Vitamin B 12 Metabolism in Ileal Crohn's Disease

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1 Tests of Bile-acid and Vitamin B Metabolism in Ileal Crohn's Disease SIRUS FARIVAR, M.D., HANS FROMM, M.D., DETLEF SCHINDLER, M.D., BRITTAIN McJUNKIN, M.D., AND FRIEDRICH W. SCHMIDT Farivar, Sirus, Fromm, Hans, Schindler, Detlef, Mcjunkin, Brittain, and Schmidt, Friedrich W.: Tests of bile-acid and vitamin B metabolism in ileal Crohn's disease. Am J Clin Pathol 7: 9-7, 9. The bile-acid breath test, fecal analysis of labeled bile acids, and Schilling test were used to study bile-acid and vitamin B metabolism in patients with ileal Crohn's disease. Results of the bile-acid breath test were positive for % of the patients; Schilling test, %; fecal analysis of bile-acid labels, 9%. Combination of the tests increased the percentage of positive cases to. About % of the patients who had positive breath tests had evidence of normal bile-acid absorption, indicating increased bile-acid deconjugation by small-intestinal bacteria. The other % had evidence of various degrees of bile-acid malabsorption. Disease activity did not correlate with results of any test. Extent of ileal involvement correlated with results of the bile-acid tests, but not with those of the Schilling test. The study demonstrates that there is a wide spectrum of disturbances of bile-acid and vitamin B, metabolism in ileitis, and that the tests should be useful in the diagnostic evaluation of patients with proven or questionable Crohn's disease who have diarrhea and malabsorptive abnormalities that could be related to disturbances of bile-acid and vitamin B metabolism. (Key words: Bile-acid metabolism; Vitamin B, metabolism; Crohn's disease; Ileum). TWO MAJOR DISTURBANCES of bile-acid and vitamin B metabolism are known to occur in Crohn's disease with involvement of the terminal ileum. The firstbile-acid and vitamin B malabsorptionresults from ileal dysfunction due to ileal disease. 7 The other disturbance, increased deconjugation of bile acids in the small intestine and binding and/or consumption of vitamin B by intestinal bacteria, is caused by small-intestinal stasis and bacterial overgrowth due to stricture or stenosis., " IH The frequency of disturbances of bile-acid metabolism in patients who have Crohn's ileitis that has not been subjected to surgical treatment has not adequately been studied. The aims of the present study, therefore, were to investigate () Received November 7, 97; received revised manuscript and accepted for publication January, 979. Supported in part by research grant S- from the United Way Health Research and Services Foundation, and by research grant Fr-- from the Deutsche Forschungsgemeinschaft. Dr. Fromm is recipient of Research Career Development Award AM 9 from the National Institute of Arthritis, Metabolism, and Digestive Diseases. Address reprint requests to Dr. Fromm: Montefiore Hospital, 9 Fifth Avenue, Pittsburgh, Pennsylvania. Gastroenterology and Nutrition Unit, Montefiore Hospital, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, and Division of Gastroenterology and Hepatology, Department of Medicine, Hannover Medical School, Hannover, West Germany. how often disturbances of bile-acid metabolism occur in Crohn's ileitis in comparison with disturbances of vitamin B, metabolism, and () whether the results of tests of altered bile-acid and vitamin B metabolism correlate with the activity and extent of the disease. If a correlation could be shown between the test results and the activity and extent of the disease, then the tests could be used for the assessment of the efficacy of different modes of treatment for Crohn's disease, which are presently being studied by several groups of investigators. The tests could then also be useful in the diagnostic evaluation of patients with clinical symptoms of Crohn's disease, when radiological findings are questionable. Methods Experimental Design Both bile-acid and vitamin B, metabolism were studied in one -hour test, as previously described." Bile-acid absorption was studied by measuring the excretion of fecal H, after administration of cholyl-,- H taurine (ring-labeled taurocholate), and that of C, after administration of cholylglycine-l- C. Although the radioactive labels originate from different parts of the bile-acid molecule, H from the steroid ring and C from the glycine moiety, it has been shown that there is a close correlation between the fecal excretion of this C label and that of either total bile acids or H after administration of cholyl-,- H taurine." Vitamin B absorption was assessed by measuring "Co in urine, after administration of "Co-labeled vitamin B and intrinsic factor (Schilling test). Bile-acid deconjugation was determined by measuring C in breath after administration of cholyl-glycine-l- C (bile-acid breath test). Intestinal transit was determined by analyzing fecal Cr activity after administration of l CrCI ) a nonabsorbable marker. -97///9 $. American Society of Clinical Pathologists Downloaded from on February 9

2 7 FARIVAR 7-,Z.. Table. Data for Patients with Crohn's Disease* A.J.C.P. January 9 Age (Yr.), Sex Length of Ileal Involvement (cm) Additional Sites of Involvement Activity Index of Disease "C in Breath Inh x ) Inh x ) -h Stoc C :, H ' >D r "Cr "Co in -h Urine) -h Fecal wt (g) Fecal Fat (g per h) Patient Patient Patient Patient Patient Patient Patient 7 Patient Patient 9 Patient Patient Patient Patient Patient Patient Patient Patient 7 Patient Patient 9 Patient Patient Patient Patient Patient Patient Patient Patient 7 Patient Patient 9 Patient Patient, F, M, F, F, F, F, M 7, M, F, F, M, M 9, F, M, F, F, F, M 7, M 9, F 7, M, F, F, M 7, F, M, F, F, F, F, M 9 7 Entire Entire Cecum, descending, sigmoid Sigmoid, rectum Transverse, descending transverse Entire, transverse Esophagus, sigmoid Descending, sigmoid, transverse , 7, , 9 9,79, li 7 ' Abnormal results are underlined. = Not measured. Subjects The study was approved by the Protection of Human Subjects Committee of Montefiore Hospital. Informed consent was obtained from all subjects. The patients were initially referred for the study of diarrhea. Patients Patient data are summarized in Table. Thirty-one patients studied, 9 women and men, with a mean age of 9 years, had evidence of Crohn's disease of the distal ileum, without prior intestinal resection. The extent of ileal disease was estimated by measuring the length of the segment of ileum that, roentgenographically, showed signs of involvement. In of the patients there was also evidence of ic involvement, and, in a thirteenth, evidence of esophageal disease as well (Table ) All patients except three had evidence of active disease. The disease activity was expressed numerically, utilizing eight selected clinical variables, as proposed by Best and co-workers (Table ). This activity index has been adopted by the National Cooperative Crohn's Disease Study for the evaluation of the response of the disease to treatment. In this so-called activity index, numbers become higher with increasing disease activ- Downloaded from on February

3 Vol. 7 No. I BILE-ACID AND VITAMIN B, METABOLISM 7 ity. An index below is consistent with quiescent disease. The patients were not acutely ill, and had no evidence of intestinal obstruction at the time of the study. They had not received any medication for at least one week prior to the study, with the exception of three patients (Patients,, and ), who were being treated with corticosteroids. Experimental and Analytic Procedure Control Data The limits of normalcy used in this study are based on control data, which were obtained in healthy subjects, in ten subjects with non-gastroenterologic disorders, and in 7 patients with diarrhea not related to inflammatory disease or resection of the ileum. Materials Cholyl-glycine-l- C (specific radioactivity. /xci/ mmol) and cholyl-, - H taurine (specific radioactivity mci/jumol) were purchased from New England Nuclear Corporation and found to be 9% pure by zonal scanning on thin-layer chromatogram. CrCl was purchased from New England Nuclear Corporation. Trapping solutions for breath C (. mmol/ml) were prepared, as previously described, using equal amounts of M hyamine hydroxide (Packard Instrument Corporation, Downers Grove, Illinois) and absolute ethanol (containing thymolphthalein, mg/, as indicator). % administered C expired in breath x In this calculation, percentage of dose per mmol C is multiplied by body weight and the average C production per kilogram in healthy subjects (9 mmol/kg/h). The percentage of dose per mmol of C represents a normalized specific activity, which is calculated by dividing the specific activity of expired C by dose. The results of the calculation are multiplied by for convenience in order to avoid small numbers, which are frequently only a fraction of. This measurement unit converts a specific activity value obtained in a --min collection to the amount of radioactivity calculated to be present in a one-hour collection, assuming excretion of H C continued at the same rate. Any value above the highest value for control subjects was considered abnormal. The upper limits of normal used were at one hour, at two hours, at three hours, 9 at four hours, at six hours and at eight hours. The cumulative C output was calculated by assuming that the arithmetic mean of the C excretion rate, at the beginning and end of each time interval, represents the average C excretion rate. Details of the procedures have been described. '' " At : A.M., fasting subjects received a nutritionally balanced liquid meal containing calories ( ml of Metrecal), into which tracer doses of /JLC'\ of cholylglycine-l- l C, /LiCi of cholyl-, - H taurine, and /i.ci of CrCl, as nonabsorbable marker of intestinal transit, were mixed. In addition, a capsule of labeled cyanocobalamin (. yu,g containing. /uci of 7 Co) and intrinsic factor was administered to each subject. Two hours later, mg vitamin B was given by intramuscular injection. Duplicate breath samples were collected before the administration of the isotopes and one, two, three, four, six, and eight hours afterwards. Feces and urine were collected for hours. Feces were analyzed for l C, H and l Cr radioactivity, and urine was analyzed for 7 Co radioactivity. Fecal fat excretion was studied in patients, in of them by analysis of a 7-hour balance, and in the remaining, by analysis of the -hour stool specimen (, 9). Assessment of Measurements Isotope Studies. Measured radioactivity in breath, stool or urine was expressed as percentage of administered dose., C in Breath. The data of the bile-acid breath test were calculated for each collection time as the percentage of the administered radioactivity in breath per hour x by the following formula: C in breath x mmol COo x administered C x 9 mmol C x kg body wt x " Increases in the C excretion rate in breath (positive bile-acid breath test) indicate the presence of increased bile-acid deconjugation in the intestine (). Positive bile-acid breath tests are due to either bacterial overgrowth in the small intestine or bile-acid malabsorption. The two conditions can be distinguished from each other by additional fecal C analysis. Fecal l C is normal in bacterial overgrowth, but increased in bileacid malabsorption. Large increases in fecal l C indicate the presence of severe bile-acid malabsorption, whereas small increases may be due to either slight bile-acid malabsorption alone or a combination of bile-acid malabsorption and bacterial overgrowth., C and H in Stool. A -hour fecal C excretion of to 9% of the dose was considered to be borderline, and excretion of more than 9% was considered to represent definite bile-acid malabsorption. The corresponding limits for fecal H were to 9% for borderline and more than 9% for definite bile-acid malabsorption. - Borderline bile-acid malabsorption may be due either to Downloaded from on February

4 7 FARIVAR ETAL. A.J.C.P. January 9 9 (/) or w 7 < Z rr O z CD - - FIG.. Percentages of ileitis patients with abnormal results of tests of bile-acid and vitamin B,.. metabolism. UJ BILE ACID BREATH TEST FECAL H SCHILLING TEST ALL TESTS COMBINED rapid intestinal transit without ileal dysfunction or to ileal dysfunction with moderately increased bile-acid loss., CV in Stool and Ratio u C/', Cr and n HI^Cr in Stool. The presence of definite bile-acid malabsorption was further documented by determining the ratios of C/ l Cr and WCr. The ratios were above. for C/, Cr and above. for H/ Cr for all patients who had definite bile-acid malabsorption. In contrast, diarrhea-control subjects who had rapid intestinal transit without evidence of ileal disease had ratios less than these values." False-positive ratios appear to occur frequently when the -hour excretion of Cr is less than %. However, in patients with evidence of severe bileacid malabsorption, l Cr excretion always exceeded %. "Co in Urine. Excretion of less than % of the 7 Co dose in the -hour specimen was considered to be abnormal (positive Schilling test), indicating vitamin B malabsorption. Fecal Weight and Fat. Fecal weight of more than g/ h was considered to be abnormally high. Steatorrhea was considered to be present when fecal fat excretion exceeded 7 g/ h. Means and correlation coefficients were calculated by standard methods. Results The results of the tests in the group of patients with ileitis are shown in Figure and Table. Positive results were found with the bile-acid breath test in % (/) of the patients, with the Schilling test in % (/), and with the fecal :! H and C analyses in 9% (/). Patients with at least one positive test increased to % (/) when the bile-acid breath test and Schilling test were combined. Patients with positive tests did not increase further when fecal l C and H analysis were also done. Of the patients who had positive bile-acid breath tests, fecal C and/or H excretion were normal for seven, borderline for two, and definitely abnormal for the remaining four. Of the patients with increased fecal C or H or both, six had evidence of borderline bile-acid loss, while the other six had evidence of definite bile-acid malabsorption. There was no significant difference between the frequencies of positive tests in the patients who had only ileal involvement and those who had signs of ic disease also. The length of ileum involved by Crohn's disease, as documented by barium-contrast studies of the small bowel, correlated with total l C excretion in four hours (r =., P <.) and six hours (r =., P <.), and with fecal C excretion (r =.7, P <.), fecal H excretion (r =.9, P <.), and fecal fat (r =., P <.). However, there was no significant correlation between the length of ileum involved and urinary 7 Co excretion. The activity of the disease (activity index) did not correlate with length of ileum involved, C in breath, fecal C, fecal H, fecal fat, or urinary "Co. Discussion There is only limited information in the medical literature with regard to the frequency and types of disturbances of bile-acid and vitamin B metabolism in Downloaded from on February

5 Vol. 7 No. I Crohn's disease and their relation to the activity and extent of the disease. Data published by other investigators are based on studies in small groups of patients without direct measurements of bile-acid losses. The present study shows that tests of bile-acid and vitamin B l metabolism are not very sensitive indicators of ileal disease. The bile-acid breath test and the Schilling test were of comparable sensitivities. Each of the two tests was positive for only about % of the patients. However, a combination of the bile-acid breath test and Schilling test increased the percentage of patients with positive tests to. The additional measurement of fecal excretion of the bile-acid labels, which was positive for only 9%, did not increase the overall number of patients with positive tests. These findings indicate, as a consequence of either ileal dysfunction or bacterial overgrowth in the small intestine, the presence of a wide spectrum of disturbances of bile-acid and vitamin B, metabolism. Any of these disturbances, i.e., increased bile-acid deconjugation due to bile-acid malabsorption or bacterial overgrowth, or both, in the small intestine, and vitamin B malabsorption, may occur separately or together. Indeed, it was observed in the present study that about half of the patients with positive bile-acid breath tests had evidence of normal bile-acid absorption.the finding of positive breath tests for patients with evidence of normal bile-acid absorption with or without steatorrhea indicated the presence of increased deconjugation of bile acids due to bacterial overgrowth in the small intestine.'' " Once bile acids are deconjugated by bacteria in the small intestine, they are rapidly absorbed by passive non-ionic diffusion, and do not depend any more on active transport in the ileum. Small-intestinal bacterial overgrowth can, therefore, mask ileal dysfunction by preventing bile-acid malabsorption. If, however, definite bile-acid malabsorption is demonstrated, this unequivocally indicates the presence of ileal dysfunction. Indeed, bile-acid malabsorption is the only reliable indicator of ileal dysfunction, since both vitamin B, malabsorption and bile-acid deconjugation can also be caused by bacterial overgrowth in the small intestine. The results of the tests were not significantly influenced by the involvement of the, in addition to that of the ileum. When two subgroups of patients with comparable extents of ileal disease, one with and the other without evidence of ic involvement, were compared, the numbers of abnormal test results were very similar. No study in which the effects of ic disease on tests of bile-acid and vitamin B metabolism in ileitis have been examined could be found in the medical literature. There are only data from studies in ileal resection, which indicate that bile-acid malabsorption is more pronounced when the is removed in addition to the ileum. However, our study shows that ileal BILE-ACID AND VITAMIN B,., METABOLISM 7 disease causes bile-acid malabsorption considerably less frequently than has been reported for ileal resection, suggesting that ileal function is less compromised in disease than in resection of the ileum. This apparent difference between the effect of inflammatory disease and that of resection on intestinal function may be similar in the and in the ileum, explaining the lack of a detectable influence of ic disease on the development of bile-acid malabsorption in ileal disease. An attempt was made in this study to relate the results of the tests to the extent of ileal involvement and disease activity. However, in the interpretation of these correlations, one has to consider that the reliability of the presently available clinical methods for determining extent and activity of Crohn's disease is limited. Radiologic measurement of the length of ileum involved in Crohn's disease is probably not very accurate, although there is a lack of good studies in which radiologic and histologic evidence of disease are compared. By.the same token, one must consider the limitations inherent in the so-called disease activity index, which is based mainly on subjective criteria such as the patient's symptoms, and the physician's physical findings. A correlation was found between the length of ileum involved and the results of tests of bile-acid absorption and deconjugation. The length of ileum involved correlated also with fecal fat excretion. However, there was no correlation between ileal involvement and the Schilling test or between the index of disease activity and results of any of the tests. The data in the literature that can be compared with those of the present study are conflicting. Scarpello and Sladen, who studied the bile-acid breath test and Schilling test in patients with ileal disease not subjected to operation, found no correlation between results of the tests and the extent or severity of ileal or ic disease. However, Lenz, who conducted a similar study in a small group of patients, found evidence that the results of the bile-acid breath test correlated with the activity of the disease. The main reason for the disagreement between the results of the different studies is probably the lack of a uniform expression of disease activity. Unlike studies by other investigators, in the present study disease activity was expressed numerically, as proposed by Best and colleagues. This so-called activity index should be more accurate for statistical analysis than other means of grading disease activity. That bile-acid excretion and deconjugation correlate only with the length of ileum involved, but not with disease activity, may be related to the finding that there is no correlation between the length of ileum involved and the activity or prognosis 7 of Crohn's disease. The effect of the length of ileum involved on bile-acid excretion and deconjugation observed in this study appears to be similar to that of the extent of ileal resection reported by Downloaded from on February

6 7 FARIVAR ET AL. A.J.C.P. January 9 some investigators. Scarpello and Sladen found a positive correlation between the length of ileum resected and l C excretion in the bile-acid breath test, whereas Lenz observed that the length of ileum resected correlated only with fecal C, but not with expired C. Other investigators found no correlation between the length of ileum resected and the result of any test of bile-acid or vitamin B metabolism. The lack of a correlation between the extent of ileal involvement and the results of the Schilling test may be explained by the finding, in another study, that vitamin B absorption is less affected than bile-acid absorption by ileal resection. The most important results of this study, clinically, can be summarized as follows: () Tests of bile-acid or vitamin B metabolism, carried out alone, are positive for only about % of patients with ileal disease. However, when the tests are used in combination, the percentage of positive results increases to approximately %. () Results of the tests of bile-acid absorption and deconjugation correlate with the length of ileum involved, but not with the so-called activity index of the disease. () The tests are clinically useful in the evaluation of selected patients with proven or questionable Crohn's disease who have diarrhea and malabsorptive abnormalities that could be related to disturbances of bile-acid and vitamin B metabolism. Acknowledgments. Janet Carter, Heidi Humke, Prafulla Amin, and Susan Ceryak provided technical assistance. References. Best Wr, Becktel JM, Singleton JW, et al: Development of a Crohn's disease activity index. National Cooperative Crohn's Disease Study. Gastroenterology 7:9-, 97. Dietschy JM, Salomon HS, Siperstein MD: Bile acid metabolism.. Studies on the mechanisms of intestinal transport. J Clin Invest :-, 9. Donaldson RM Jr: Studies on the pathogenesis of steatorrhea in the blind loop syndrome. J Clin Invest :-, 9. Donaldson RM Jr: Malabsorption of Co "-labeled cyanocobalamin in rats with intestinal diverticula.. Evaluation of possible mechanisms. Gastroenterology :7-, 9. Fromm H, Hofmann AF: Breath test for altered bile-acid metabolism. Lancet :-, 97. Fromm H, Thomas PJ, Hofmann AF: Sensitivity and specificity in tests of distal ileal function: Prospective comparison of bile acid and vitamin B, absorption in ileal resection patients. Gastroenterolgy :77-9, Fromm H, Wilson FA, Rodgers JB, et al: Granulomatous bowel (Crohn's) disease. A retrospective study of the course and treatment. Arch Intern Med :79-7, 97. Hardison WGM, Rosenberg IH: Bile-salt deficiency in the steatorrhea following resection of the ileum and proximal. New Engl J Med 77:7-, Hofmann AF: The syndrome of ileal disease and the broken enterohepatic circulation: Cholerheic enteropathy. Gastroenterology :7-77, 97. Hofmann AF, Poley JR: Role of bile acid malabsorption in the 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. Gastroenterology :9-9, 97. Kim YS, Spritz N, Blum M, et al: The role of altered bile acid metabolism in the steatorrhea of experimental blind loop. J Clin Invest :9-9, 9. Lenz K: The effect of the site of lesion and extent of resection on duodenal bile acid concentration and vitamin B absorption in Crohn's disease. Scand J Gastroenterol :-, 97. Lenz K: An evaluation of the "breath test'' in Crohn's disease. Scand J Gastroenterol :-7, 97. MeihoffWE, Kern FJr: Bile salt malabsorption in regional ileitis, ileal resection and mannitol-induced diarrhea. J Clin Invest 7:-7, 9. Roda A, Roda E, AldiniR.etal: Determination of H C in breath and H C in stool after oral administration of cholyl-l- H C- glycine: Clinical application. Clin Chem :7-, 977. Scarpello JHB, Sladen GE: H C-glycocholate test in Crohn's disease its value in assessment and treatment. Gut :7-7, Stanley MM, Nemchausky B: Fecal C H -bile acid excretion in normal subjects and patients with steroid-wasting syndromes secondary to ileal dysfunction. J Lab Clin Med 7:7-9, 97. Tabaqchali S, Hatzioannou J, Booth CC: Bile salt deconjugation and steatorrhea in patients with the stagnant loop syndrome. Lancet :-, 9 9. Van de Kamer JH, ten Bokkel Huinink H, Weyers HA: Rapid method for the determination of fat in feces. J Biol Chem 77:7-, 99. Winchell HS, Stahelin H, Kusubov N, et al: Kinetics of C -HC - in normal adult males. J Nucl Med :7-7, 97 Downloaded from on February