GASTROENTEROLOGY
1992;103:51-56
Effect of Butyrate Enemas on the Colonic Mucosa in Distal Ulcerative Colitis WOLFGANG SCHEPPACH, HARTMUT SOMMER, THOMAS KIRCHNER, GIAN-MARIA PAGANELLI, PETER BARTRAM, STEFAN CHRISTL, FRANK RICHTER, GERDA DUSEL, and HEINRICH KASPER Departments of Medicine and Pathology, University of Wiirzburg, Wiirzburg, Germany; Municipal Hospital of Heidenheim, Heidenheim, Germany; and Istituto di Clinica Medica e Gastroenterologia, University of Bologna, Bologna, Italy
Short-chain fatty acid irrigation has been shown to ameliorate inflammation in diversion colitis. In this study the effect of butyrate enemas was tested in 10 patients with distal ulcerative colitis who had been unresponsive to or intolerant of standard therapy for 8 weeks. They were treated for 2 weeks with sodium butyrate (100 mmol/L) and 2 weeks with placebo in random order (single-blind trial). Before and after treatment, clinical symptoms were noted and the degree of inflammation was graded endoscopically and histologically. Rectal proliferation was assessed by autoradiography. After butyrate irrigation, stool frequency (n/day) decreased from 4.7 + 0.5 to 2.1 k 0.4 (P < 0.01) and discharge of blood ceased in 9 of 10 patients. The endoscopic score fell from 6.5 + 0.4 to 3.8 f 0.8 (P < 0.01). The histological degree of inflammation decreased from 2.4 f 0.3 to 1.5f 0.3(P < 0.02). Overall crypt proliferation was unchanged, but the upper crypt-labeling index fell from 0.086 + 0.019 to 0.032 + 0.003 (P < 0.03). On placebo, all of these parameters were unchanged. These data support the view that butyrate deficiency may play a role in the pathogenesis of distal ulcerative colitis and that butyrate irrigation ameliorates this condition.
T
here is good evidence that short-chain fatty acids (SCFAs; mainly acetate, propionate, and n-butyrate) derived from bacterial breakdown of carbohydrate and protein in the large bowel are important regulators of the colonic mi1ieu.l Of these acids, butyrate is a major energy-yielding substrate to colonocytes2~3 and also affects mucosal cell proliferation in humans4 and rats.5 The hypothesis has been put forward that a lack of luminal SCFAs leads in the short term to mucosal atrophy and in the long term to “nutritional colitis.“6 This is especially evident in diversion colitis, which develops after complete diversion of the fecal stream and subsides after restoration of colorectal continuity.7*8
Harig et al.g report 4 patients with diversion colitis in whom SCFA (acetate, 60 mmol/L; propionate, 30 mmol/L; and butyrate, 40 mmol/L) irrigation for 2-3 weeks resulted in macroscopic and histological resolution of inflammation. They also discuss similarities between diversion colitis and ulcerative colitis (UC). Decreased fecal concentrations of SCFAs have indeed been shown to occur in patients with UC but not in those with Crohn’s colitis.” On the basis of these data, we used butyrate (100 mmol/L) enemas to treat 10 patients with distal UC in a placebocontrolled, single-blind, randomized trial. We preferred butyrate alone rather than the SCFA mixture because evidence was strongest for butyrate to affect the colonic mucosa. A preliminary report (uncontrolled study) has been published recently suggesting effective treatment of distal UC by an SCFA mixture.” Materials and Methods the effect of butyrate enemas on the patients (5 men, 5 women, aged 38.8 -t 3.9 years: personal data given in Table 1) with active UC were studied. Because rectal irrigation is effective in the treatment of the rectum, sigmoid, and descending colon,‘2 only patients with a maximum involvement from the rectum to the splenic flexure were included in the study. Patients had been unresponsive to or intolerant of standard treatment [oral sulfasalazine/5-acetylsalicyclic acid (5-ASA)/low-dose prednisone; rectal 5-ASA/corticoids] for at least 8 weeks. Oral medication was kept constant throughout the study, but no topical treatment other than butyrate/placebo irrigation was allowed. Antibiotics were not taken for 4 weeks before or during the study. The patients gave informed written consent to participate in the study, which was approved by the Ethical Committee of the Faculty of Medicine, University of Wiirzburg. In a randomized, single-blind study with cross-over design, the patients received sodium butyrate or sodium chloTo evaluate
inflamed mucosa,
10
0 1992 by the American
Gastroenterological 0016-5085/92/$3.00
Association
52
SCHEPPACH ET AL.
Table 1. Personal Patient 1
2 3 4 5 6 7 8 9 10
GASTROENTEROLOGY
Data of 10 Patients
Age (yrl/sex 28/M 55/M 42/M 33/F 27/M 42/F 65/M 36/F 24/F 36/F
NOTE. Oral medication
Vol. 103, No. 1
With Distal UC
Duration of UC (mo) 60 16 120
22 24 120
6 24 54 2
Colonic involvement Rectum Rectum Rectum Rectum Rectum Rectum Rectum Rectum Rectum Rectum
to splenic flexure to splenic flexure to sigmoid colon to sigmoid colon to sigmoid colon only to sigmoid colon to sigmoid colon to mid descending to splenic flexure
was kept constant for 4 weeks before and throughout
ride (placebo) enemas for 2-week periods with a washout phase of 2 weeks when butyrate was followed by NaCl (no washout phase when NaCl was given first). Butyrate enemas contained 100 mmol/L sodium butyrate (pH 7.0) made isotonic by addition of NaCl (40 mmol/L). Placebo enemas contained 140 mmol/L NaCl. Two enemas per day (volume, 100 mL) were instilled into the rectum, and the patient was to remain supine for 30 minutes thereafter. In 3 patients the butyrate course was extended to a total of 4 weeks. Before and after both treatment periods, sigmoidoscopic examinations were performed between 8 AM and 11 AM using an Olympus CF 20 M sigmoidoscope (Olympus Optical Europe, Hamburg, Germany) after bowel preparation with a tap-water enema. This mode of preparation has been shown not to interfere with rectal epithelial proliferation.13 At endoscopy (performed by an endoscopist blinded to the mode of treatment), the macroscopic appearance of the mucosa was evaluated by an index based on the presence or absence of erythema, edema, friability, granularity, and erosions (range of O-10).’ Biopsy specimens were taken with the regular forceps from the rectosigmoid junction and the rectal ampulla for evaluation of inflammation. They were fixed in formalin, embedded in paraffin, cut into 3ym sections, and stained with H&E. The changes evaluated as present or absent were accumulation of lymphocytes and plasma cells, infiltration of crypt epithelium by neutrophils, crypt abscesses, ulcerations, mucus depletion of epithelial cells, and distortion of crypts. Overall inflammatory change was graded 0 (absent), 1 (mild), 2 (moderate), or 3 (severe). All biopsy specimens were interpreted in a random and blinded manner by a single pathologist. In 6 of 10 patients with distal UC, biopsy specimens were also taken 10 cm from the anus for a study of rectal cell proliferation. Similarly, biopsy specimens were obtained from a group of 30 sex- and age-matched controls (15 men, 15 women, aged 39.8 + 2.2 years) with no pathological findings during routine colonoscopy. The specimens were immersed in prewarmed cell culture medium [basal medium with Earle’s salts (BME), fetal calf serum, and antibiotic-antimycotic solution; Gibco, Paisley, Scotland] and processed within 30 minutes. The specimens
Oral medication (mg/day)
colon
5-ASA, 3 X 500; prednisone, 5-ASA, 3 X 500; prednisone, 5-ASA, 3 X 500 Sulfasalazine, 3 X 1000 5-ASA, 3 x 500 Sulfasalazine, 3 X 1000 5-ASA, 2 x 500 None 5-ASA, 3 x 500 5-ASA, 3 x 500
2.5 7.5
the trial.
were then oriented mucosal-side-up on a membrane filter in a Petri dish containing 3 mL BME medium and tritiumlabeled thymidine (3H-Thy, 5 pCi/mL; DuPont Nemours Germany, Bad Homburg, Germany). The Petri dish was placed in an airtight chamber (Modular Incubator Chamber; Billups-Rothenberg Inc., Del Mar, CA) on a rocking platform (10 cycles/min). During the l-hour incubation period, carbogen gas (95% oxygen, 5% carbon dioxide) was administered at 1 atm additional pressure and the assembly was kept at 37°C. The biopsy specimens were then washed three times with saline and fixed in formalin (3.5% vol/vol; Merck, Darmstadt, Germany) contained in phosphate-buffered saline (PBS; Gibco) solution. For autoradiography, biopsy specimens were embedded in paraplast, cut into s-pm sections mounted on slides, and stained with acid-Schiff’s reagent (Feulgen reaction). Slides were dipped in Ilford K2 emulsion (Ilford, Essex, England) and exposed for 15 days in the dark following standard techniques described elsewhere.14 Rectal epithelial cell proliferation was assessed by recording the frequency of 3H-Thy-labeled cells in longitudinally sectioned crypts under the light microscope. At least 15 crypts (control group, at least 12 crypts) were studied per experiment. The numbers of labeled (a) and unlabeled (b) cells per crypt were determined and the labeling index (LI) computed (a/a + b).” Crypts were divided into five equal longitudinal compartments (compartment 1, crypt base; compartment 5, crypt surface), and in addition to the whole-crypt LI, an LI for each compartment was calculated. In addition, the $h value was assessed as the number of labeled cells in compartments 4-5 divided by the total number of labeled cells per crypt.16 Cell kinetics counting was performed in a blinded manner. Statistical
Analysis
All values are expressed as mean + SEM. Comparisons of quantitative data were made by the Wilcoxon rank sum test (paired data) or the Mann-Whitney U test (unpaired data). Qualitative data were compared by the x2 test. The Spearman rank coefficient was used for correlations. Differences were considered significant at P values of 10.05.
BUTYRATE AND ULCERATIVE COLITIS 53
July 1992
Results Clinical Symptoms At entry in the study, all 10 patients with distal UC had been suffering constantly from diarrhea for at least 3 weeks. During the control period, no change in stool frequency was seen (before NaCl, 3.9 * 0.7;after NaCl, 3.9 f 0.7;NS). However, after treatment with butyrate enemas the number of defecations per day decreased significantly (P < 0.01) from 4.5 -t 0.5 to 2.1 * 0.4. Fecal blood was observed in 9 of 10 patients before NaCl; this figure remained unchanged during the control phase. When butyrate enemas were administered, the number of patients with fecal blood decreased from 9 of 10 to 1 of 10 (P < 0.05, x2 test). The response of patient 8 (no additional medication) to butyrate enemas did not differ from that seen in the other patients who received concomitant standard treatment (Table 1). Butyrate and control enemas were retained for at least 30 minutes in every case; in many instances, they were kept for a longer period (up to several hours). They were tolerated without side effects. Endoscopic
Appearance
Before treatment, the endoscopic appearance score was 6.0+ 0.9(before NaCl) and 6.5f 0.4(before butyrate; NS) (Figure 1). After a 2-week course of NaCl, no change was seen (5.9 f 0.9). After 2 weeks of butyrate irrigation, the endoscopic appearance improved in 9 of 10 patients (3.8f 0.8;P < 0.01; n = 10). In 3 patients in whom only slight improvement was noted after 2 weeks, butyrate enemas were continued for another 2 weeks. In these patients, a further regression of inflammation was noted (2-week scores, 7, 4, and 5; 4-week scores, 2, 2, and 2). Histological
Data
Overall inflammatory change was graded (H&E stain) 0 (absent), 1 (mild), 2 (moderate), or 3
Figure 1. Endoscopic score (n = lo), histological grading (n = lo), and upper-crypt labeling frequency (n = 6) in patients with distal UC before and after treatment with sodium butyrate (0) or sodium chloride (0) (control) enemas. Vertical bars indicate 1 SEM; *significant differences (endoscopic score, P < 0.01; histological grading, P < 0.02; upper-crypt labeling, P < 0.03; Wilcoxon test).
6.
6.
4-
%
*
2.
o* before
afta
Edoscopic
Score
(severe). Before treatment, the histological score was 2.1 + 0.3 (NaCl) and 2.4 + 0.3 (butyrate; NS) (Figure 1). In the control run (NaCl enemas), no change was After 2 weeks of butyrate irrigaobserved (2.4k 0.3). tion, a decrease of mucosal inflammation was noted in 7 of 10 patients (score, 1.5f 0.3;P < 0.02;n = 10). In the 3 patients who showed minor endoscopic improvement after 2 weeks (histology unchanged, n = 2;slight histological improvement, n = l), treatment was continued for another 2 weeks. In these 3 patients, a further decrease in histological score was observed (2-week scores, 3,1,and 2; 4-week scores, 2,0,and 1). Proliferation
Pattern
In 6 of 10 patients, rectal cell-proliferation data were available and compared with data obtained from 30 healthy control persons (Figure 1; Table 2). Overall proliferation (whole-crypt LI) was not different between groups. However, cell labeling in compartments 4 and 5 was significantly higher in patients with UC before treatment and after NaCl treatment than in healthy controls. Butyrate irrigation resulted in a significant reduction of 3H-Thy incorporation in compartments 4 and 5. The LI in compartment 4 was reduced to values found in healthy controls. Similarly, the $h value indicated expansion of the proliferative zone in patients with untreated UC (before/after NaCl, before butyrate) but not in patients after butyrate irrigation or healthy controls. There was no correlation between the degree of inflammation (endoscopic score, histological grading) and parameters of an expanded proliferative zone (upper-crypt LI, 4h value). Discussion A connection between SCFAs and colitis was first established by Harig et al.’ who successfully treated four patients with diversion colitis using acetate (60mmol/L), propionate (30 mmol/L), and buty-
54
SCHEPPACH ET AL.
GASTROENTEROLOGY
Vol. 103,No. 1
Table 2. Rectal Cell-Proliferation Data of Patients With UC and Healthv Controls Patients with UC Na-Butyrate Before Group A No. of subjects No. of crypts assayed No. of labeled cells No. of all crypt cells Whole-crypt LI LI by crypt compartment Compartment 1 Compartment 2 Compartment 3 Compartment 4 Compartment 5 bh value
NaCl (control) After Group B
Before Group C
After Group D
Controls Group E
6 152 2085 14,330 0.144f 0.015
6 176 1860 16,995 0.115f 0.013
6 115 1669 11,825 0.150f 0.020
6 120 1827 11,975 0.155* 0.017
30 438 2478 20,124 0.125+ 0.006
0.158+ 0.208 f 0.179 f 0.112f 0.060 + 0.223 f
0.187f 0.025 0.203f 0.022 0.127+ 0.014* 0.047rt0.006A.D.E 0.011+ o.o04*J-~E 0.103+_0.012A*D.E
0.177f 0.196f 0.189f 0.128+ 0.062+ 0.249k
0.196f 0.027 0.219+ 0.027 0.181?I0.020' 0.122i-0.024BsC 0.055+ 0.009B~C 0.227t 0.020B,C
0.190f 0.013 0.254+ 0.011 0.132+ 0.013 0.057+ 0.018A.D,E 0.008+ 0.003A.BsD.E 0.070I!T 0.017A.D.E
0.009 0.018 0.028 0.026Bsc 0.014B*c 0.031B.c
0.044 0.032 0.031 0.026B,c 0.018Bsc o.034B.c
NOTE. Data represent means f SEM. bh value used according to Lipkin et al.‘” Patients with UC, n = 6; healthy control persons, n = 30. Superscripts A-E indicate significant differences (P < 0.05) vs. other groups. LI, labeling index.
rate (40 mmol/L). After 2 weeks of SCFA irrigation, they observed endoscopic and histological amelioration of inflammation. They concluded that a prolonged deprivation of preferred nutrients (SCFAs) frequently leads to colitis in the rectal stump. The resupply of nutrients, either by colonic reanastomosis or by SCFA irrigation, induces remission of colitis by endoscopic and histological criteria. Because there are striking similarities between diversion colitis and UC, a controlled trial was undertaken in patients with distal UC (maximum involvement, rectum to splenic flexure). Rectal enemas have been shown to provide homogeneous delivery of the drug to the inflamed lower colon reaching the splenic flexure.” In this study, butyrate was tested alone and not in a mixture of SCFAs because evidence from the literature was strongest in favor of butyrate to affect the mucosa.4’” After 2 weeks of butyrate irrigation, 9 of 10 patients responded endoscopically and 7 histologically. On placebo, no change in inflammation was observed. These data from a controlled study are supported by an uncontrolled preliminary report on the effect of an SCFA mixture (acetate, 80 mmol/L; propionate, 30 mmol/L; and butyrate, 40 mmol/L) as a topical treatment of distal UC. Breuer et a1.l’ defined a disease activity index based on stool frequency, rectal bleeding, endoscopic appearance, and impact of symptoms on lifestyle (range, O-12). This index and histological parameters improved partially by week 2 but markedly by week 6. This finding is in agreement with our data showing further improvement after 4 weeks in patients with a partial response after 2 weeks. How do these data compare with standard topical treatment of distal UC? For 2-week trials testing cor-
ticosteroid enemas, clinical response rates of 38%78% have been reported.“‘l* The clinical response to 5-ASA enemas for 2 weeks (93%) has been found superior to response to hydrocortisone (57%); in the same study, the histological improvement has been less pronounced (77% for 5-ASA vs. 33% for hydrocortisone).‘g Other authors have argued that prolonged therapy (>4 weeks) is required to achieve remission in a majority of patients.20~2* To evaluate the role of butyrate/SCFA in topical treatment of distal UC, direct comparisons with standard drugs remain to be made. An advantage of butyrate is the entire lack of side effects because this compound is present in the colon under physiological conditions. By which mechanism may butyrate affect the coionic mucosa in UC? On the basis of in vitro experiments using isolated colonocytes from patients with UC, Roediger”,22*23 has described pathobiochemical changes in acute and quiescent colitis. He found as an important defect in diseased colonocytes a reduction (but not complete failure) of mitochondrial fatty acid (SCFA) oxidation. In acute UC, the rate of oxygen consumption by colonocytes from the descending colon was reduced by 46% compared with cells from healthy controls when butyrate (10 mmol/L) was present as a fuel.” Instead, compensatory pathways of fuel utilization were used (glutamine and glucose oxidation enhanced). Considering data from experimental colitis, impaired butyrate oxidation is thought to be because of a diminution of free coenzyme A (CoA), which is essential for SCFA activation and subsequent oxidation to CO, and ketone bodies.23 Levels of free CoA have in fact been found low in human UC.24 Soergel” has speculated that treatment of distal UC by SCFA enemas might overcome
BUTYRATE
July 1992
this partial metabolic block by “mass action”; this means that higher than normal SCFA concentrations in the colonic lumen may improve energy supply to colonocytes despite an abnormality of mitochondrial SCFA oxidation. This concept differs from other, mainly anti-inflammatory approaches (sulfasalazine, 5-ASA, corticosteroids). To date, there is no information on a potential interference of 5-ASA with SCFA metabolism in the colonocyte. Clinical results concerning the treatment of distal UC with SCFAs” or butyrate (present work) are encouraging; however, more work is needed to clarify the role of SCFAs in the pathogenesis of UC. Butyrate has different effects on proliferation of normal colonocytes and colon cancer cell lines. While the physiological pattern of proliferation (confined to the basal crypt) is stimulated in the normal human mucosa,4 growth rates are inhibited in various human colon cancer cell lines.26-31 Therefore, it was of interest whether butyrate could influence rectal cell proliferation in UC, known as a principally preneoplastic condition. The upper-crypt labeling frequency (labeling in crypt compartments 4 and 5) in patients with UC decreased significantly after butyrate treatment and was no longer different from values obtained in 30 healthy control subjects. Expansion of the proliferative zone to the crypt surface has been considered by Lipkin a “biomarker of increased susceptibility to cancer formation.“32 This phenomenon is observed in patients at risk of malignancy (familial polyposis coli, familial nonpolyposis colon cancer, sporadic colonic adenoms, UC.16,33-3* Biasco et a1.3ghave shown that expansion of the proliferative zone in UC is independent of the degree of inflammation and the duration of disease; this finding is in agreement with our data. The effect of butyrate on upper-crypt labeling may be due to its potent differentiating property demonstrated in colon cancer cell lines in permanent culture.26-31 In conclusion, butyrate as an end product of bacterial fermentation in the large bowel profoundly affects the colonic epithelium (inflammation and proliferation) in UC. It is suggested that the effect of an SCFA mixture” on the inflamed mucosa in UC is largely, if not entirely, attributable to its butyrate moiety. References Cummings JH. Fermentation in the human large intestine: evidence and implications for health. Lancet 1983;1:12061209. Roediger WEW. Role of anaerobic bacteria in the metabolic welfare of the colonic mucosa in man. Gut 1980;21:793-798. Roediger WEW. Utilization of nutrients by isolated epithelial cells of the rat colon. Gastroenterology 1982;83:424-429. Scheppach W, Bartram P, Richter A, Richter F, Liepold H, Dusel G, Hofstetter G, Rtithlein J, Kasper H. The effect of
short-chain J Parenter
AND
IJLCERATIVE
fatty acids on the human Enter
Nutr
colonic
COLITIS
mucosa
55
in vitro.
1992:16:43-48.
JL. Stim5. Kripke SA, Fox AD, Berman jM, Settle RG. Rombeau ulation of intestinal mucosal growth with intracolonic infusion of short-chain fatty acids. J Parenter Enter Nutr 1989;13:109-116. mucosal nu6. Roediger WEW. The starved colon-diminished trition, diminished absorption, and colitis. Dis Colon Rectum 1990;33:858-862. H. Proctitis and colitis follow7. Glotzer DJ. Glick ME, Goldman ing diversion of the fecal stream. Gastroenterology 1981;80: 438-441. VP, Schimmel EM. Diversion colitis: 8. Agarwal deficiency syndrome? Nutr Rev 1989;47:257-261.
a nutritional
RA, Wood CM. Treatment 9. Harig JM. Soergel KH, Komorowski of diversion colitis with short-chain fatty acid irrigation. N Engl J Med 1989;320:23-28. A. Hauck W. Breuer RI. Organic anions 10. Vernia P, Gnaedinger and the diarrhea of inflammatory bowel disease. Dig Dis Sci 1988;33:1353-1358. 11. Breuer RI, Buto SK, Christ ML, Bean J. Vernia P. Paoluzi P, DiPaolo MC, Caprilli R. Rectal irrigation with short-chain fatty acids for distal ulcerative colitis. Preliminary report. Dig Dis Sci 1991;36:185-187. 12. Kruis LV. Bull U, Eisenburg Ionic spread of sulfasalazine 1982:17:933-938.
J. Paumgartner G. Retrograde coenemas. Stand J Gastroenterol
Z. Rozen P, Fine N, Chetrit A. Reproducibility 13. Fireman ies and effects of bowel preparations on measurements tal epithelial proliferation, Cancer Lett 1989;45:59-64.
studof rec-
M. Fujita M, Lipkin M, Palmer R, Friedman E, Au14. Ilsugane genlicht L. Cell proliferation in explant cultures of the human colon. Digestion 1982;24:225-233. SJ. Tritiated-thymidine label15. Lipkin M. Enker WE. Winawer ing of rectal epithelial cells in “non-prep” biopsies if individuals at increased risk for colonic neoplasia. Cancer Lett 1987;37:153-161, 16. Lipkin M, Blattner WE, Fraumeni JF. Lynch HT, Deschner E, Winawer S. Tritiated thymidine ($p, $h) labeling distribution as a marker for hereditary predisposition to colon cancer. Cancer Res 1983;43:1899-1904. 17
Hamilton I, Pinder IF, Dickinson RJ, Ruddell WS, Dixon MF, Axon ATR. A comparison of prednisolone enemas with lowdose oral prednisolone in the treatment of acute distal ulcerative colitis. Dis Colon Rectum 1984;27:701-702.
18
McIntyre PB. Macrae FA, Berghouse 1,. English J. LennardJones JE. Therapeutic benefits from a poorly absorbed prednisolone enema in distal ulcerative colitis. Gut 1985;26:822824.
19
Campieri M, Lanfranchi GA, Bazzocchi G, Brignola C, Sarti F, Franzin G. Battocchia A, Labo G, Dal Monte PR. Treatment of ulcerative colitis with high-dose 5-aminosalicylic acid enemas. Lancet 1981;2:270-271.
20. Sutherland LR, Martin F. Greer S, Robinson M, Creenberger N, Saibil F, Martin T, Sparr J, Prokipchuk E, Borgen L. 5-Aminosalicylic acid enemas in the treatment of distal ulcerative colitis. proctosigmoiditis and proctitis. Gastroenterology 1987;92:1894-1898. 21. Danish 5-ASA Group. Topical 5-aminosalicylic acid versus prednisolone in ulcerative proctosigmoiditis. Dig Dis Sci 1987:32:598-602. 22. Roediger WEW. The colonic epithelium in ulcerative colitis: an energy-deficiency disease? Lancet 1980:2:712-715. 23. Roediger LVEW. The role of colonic mucosal metabolism in the pathogenesis of ulcerative colitis. In: Goebell H, Peskar BM. Malchow H, eds. Inflammatory bowel diseases-basic re-
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search and clinical implications. Lancaster, England: MTP, 1988:69-78. 24. Ellestad-Sayad JJ, Nelson RA, Adson MA, Palmer WM, Soule EH. Pantothenic acid, coenzyme A and human chronic ulcerative and granulomatous colitis. Am J Clin Nutr 1976;29: 1333-1338. 25. Soergel KH. Colitis and short-chain fatty acids. IBD Forum Symposium, Digestive Disease Week, San Antonio, TX, 1990. 26. Dexter DL, Lev R, McKendall GR, Mitchell P, Calabres P. Sodium butyrate-induced alteration of growth properties and glycogen levels in cultured human colon carcinoma cells. Histochem J 1984;16:137-149, 27. Augeron C, Laboisse CL. Emergence of permanently differentiated cell clones in a human colonic cancer cell line in culture after treatment with sodium butyrate. Cancer Res 1984;44:3961-3969. 28. Gum JR, Kam WK, Byrd JC, Hicks JW, Sleisenger MH, Kim YS. Effects of sodium butyrate on human colonic adenocarcinoma cells. J Biol Chem 1987;262:1092-1097. 29. Siddiqui B, Kim JS. Effects of sodium butyrate, dimethyl sulfoxide, and retinoic acid on glycolipids of human rectal adenocarcinoma cells. Cancer Res 1984;44:1648-1652. 30. Chung YS, Song IS, Erickson RH, Sleisenger MH, Kim YS. Effect of growth and sodium butyrate on brush border membrane-associated hydrolases in human colorectal cancer cell lines, Cancer Res 1985;45:2976-2982. 31. Whitehead RH, Young GP, Bhatal PS. Effects of short chain fatty acids on a new human colon carcinoma cell line (LIM 1215).Gut 1986;27:1457-1463. 32. Lipkin M. Biomarkers of increased susceptibility to gastrointestinal cancer: new application to studies of cancer prevention in human subjects. Cancer Res 1988;48:235-245. 33. Lipkin M, Blattner WA, Gardner EJ, Burt RW, Lynch H, Deschner E, Winawer S, Fraumeni JF. Classification and risk assessment of individuals with familial polyposis, Gardner’s syndrome, and familial non-polyposis colon cancer from
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(3H)thymidine labeling patterns in colonic epithelial cells. Cancer Res 1984;44:4201-4207. 34. Terpstra OT, van Blankenstein M, Dees J, Eilers GA. Abnormal pattern of cell proliferation in the entire colonic mucosa of patients with colon adenoma or cancer. Gastroenterology 1987;92:704-708. 35. Risio M, Lipkin M, Candelaresi GL, Bertone A, Coverlizza S, Rossini FP. Correlations between rectal mucosa cell proliferation and the clinical and pathological features of nonfamilial neoplasia of the large intestine. Cancer Res 1991;51:19171921. 36. Biasco G, Lipkin M, Minarini A, Higgins P, Miglioli M, Barbara L. Proliferative and antigenic properties of rectal cells in patients with chronic ulcerative colitis. Cancer Res 1984;44: 5450-5454. 37. Serafini EP, Kirk AP, Chambers
TJ. Rate and pattern of epithelial cell proliferation in ulcerative colitis. Gut 1981;22:648652. 38. Kanemitsu T, Koike A, Yamamoto S. Study of the cell proliferation kinetics in ulcerative colitis, adenomatous polyps, and cancer. Cancer 1985;56:1094-1098. 39. Biasco G, Paganelli GM, Miglioli M, Brillanti S, Di Febo G, Gizzi G, Ponz de Leon M, Campieri M, Barbara L. Rectal cell proliferation and colon cancer risk in ulcerative colitis. Cancer Res 1990;50:1156-1159.
Received August 19, 1991. Accepted December 11,1991. Address requests for reprints to: Wolfgang Scheppach, M.D., Department of Medicine, University of Wiirzburg, Josef-SchneiderStraBe 2, D-8700 Wtirzburg, Germany. The cooperation of the patients in this study is gratefully acknowledged. The authors thank Dr. Englert [Pharmacy of the Wtirzburg University Hospital) for providing sodium butyrate in sterile solution. The help of the Endoscopy Unit’s staff is appreciated.
1992;103:51-56
Effect of Butyrate Enemas on the Colonic Mucosa in Distal Ulcerative Colitis WOLFGANG SCHEPPACH, HARTMUT SOMMER, THOMAS KIRCHNER, GIAN-MARIA PAGANELLI, PETER BARTRAM, STEFAN CHRISTL, FRANK RICHTER, GERDA DUSEL, and HEINRICH KASPER Departments of Medicine and Pathology, University of Wiirzburg, Wiirzburg, Germany; Municipal Hospital of Heidenheim, Heidenheim, Germany; and Istituto di Clinica Medica e Gastroenterologia, University of Bologna, Bologna, Italy
Short-chain fatty acid irrigation has been shown to ameliorate inflammation in diversion colitis. In this study the effect of butyrate enemas was tested in 10 patients with distal ulcerative colitis who had been unresponsive to or intolerant of standard therapy for 8 weeks. They were treated for 2 weeks with sodium butyrate (100 mmol/L) and 2 weeks with placebo in random order (single-blind trial). Before and after treatment, clinical symptoms were noted and the degree of inflammation was graded endoscopically and histologically. Rectal proliferation was assessed by autoradiography. After butyrate irrigation, stool frequency (n/day) decreased from 4.7 + 0.5 to 2.1 k 0.4 (P < 0.01) and discharge of blood ceased in 9 of 10 patients. The endoscopic score fell from 6.5 + 0.4 to 3.8 f 0.8 (P < 0.01). The histological degree of inflammation decreased from 2.4 f 0.3 to 1.5f 0.3(P < 0.02). Overall crypt proliferation was unchanged, but the upper crypt-labeling index fell from 0.086 + 0.019 to 0.032 + 0.003 (P < 0.03). On placebo, all of these parameters were unchanged. These data support the view that butyrate deficiency may play a role in the pathogenesis of distal ulcerative colitis and that butyrate irrigation ameliorates this condition.
T
here is good evidence that short-chain fatty acids (SCFAs; mainly acetate, propionate, and n-butyrate) derived from bacterial breakdown of carbohydrate and protein in the large bowel are important regulators of the colonic mi1ieu.l Of these acids, butyrate is a major energy-yielding substrate to colonocytes2~3 and also affects mucosal cell proliferation in humans4 and rats.5 The hypothesis has been put forward that a lack of luminal SCFAs leads in the short term to mucosal atrophy and in the long term to “nutritional colitis.“6 This is especially evident in diversion colitis, which develops after complete diversion of the fecal stream and subsides after restoration of colorectal continuity.7*8
Harig et al.g report 4 patients with diversion colitis in whom SCFA (acetate, 60 mmol/L; propionate, 30 mmol/L; and butyrate, 40 mmol/L) irrigation for 2-3 weeks resulted in macroscopic and histological resolution of inflammation. They also discuss similarities between diversion colitis and ulcerative colitis (UC). Decreased fecal concentrations of SCFAs have indeed been shown to occur in patients with UC but not in those with Crohn’s colitis.” On the basis of these data, we used butyrate (100 mmol/L) enemas to treat 10 patients with distal UC in a placebocontrolled, single-blind, randomized trial. We preferred butyrate alone rather than the SCFA mixture because evidence was strongest for butyrate to affect the colonic mucosa. A preliminary report (uncontrolled study) has been published recently suggesting effective treatment of distal UC by an SCFA mixture.” Materials and Methods the effect of butyrate enemas on the patients (5 men, 5 women, aged 38.8 -t 3.9 years: personal data given in Table 1) with active UC were studied. Because rectal irrigation is effective in the treatment of the rectum, sigmoid, and descending colon,‘2 only patients with a maximum involvement from the rectum to the splenic flexure were included in the study. Patients had been unresponsive to or intolerant of standard treatment [oral sulfasalazine/5-acetylsalicyclic acid (5-ASA)/low-dose prednisone; rectal 5-ASA/corticoids] for at least 8 weeks. Oral medication was kept constant throughout the study, but no topical treatment other than butyrate/placebo irrigation was allowed. Antibiotics were not taken for 4 weeks before or during the study. The patients gave informed written consent to participate in the study, which was approved by the Ethical Committee of the Faculty of Medicine, University of Wiirzburg. In a randomized, single-blind study with cross-over design, the patients received sodium butyrate or sodium chloTo evaluate
inflamed mucosa,
10
0 1992 by the American
Gastroenterological 0016-5085/92/$3.00
Association
52
SCHEPPACH ET AL.
Table 1. Personal Patient 1
2 3 4 5 6 7 8 9 10
GASTROENTEROLOGY
Data of 10 Patients
Age (yrl/sex 28/M 55/M 42/M 33/F 27/M 42/F 65/M 36/F 24/F 36/F
NOTE. Oral medication
Vol. 103, No. 1
With Distal UC
Duration of UC (mo) 60 16 120
22 24 120
6 24 54 2
Colonic involvement Rectum Rectum Rectum Rectum Rectum Rectum Rectum Rectum Rectum Rectum
to splenic flexure to splenic flexure to sigmoid colon to sigmoid colon to sigmoid colon only to sigmoid colon to sigmoid colon to mid descending to splenic flexure
was kept constant for 4 weeks before and throughout
ride (placebo) enemas for 2-week periods with a washout phase of 2 weeks when butyrate was followed by NaCl (no washout phase when NaCl was given first). Butyrate enemas contained 100 mmol/L sodium butyrate (pH 7.0) made isotonic by addition of NaCl (40 mmol/L). Placebo enemas contained 140 mmol/L NaCl. Two enemas per day (volume, 100 mL) were instilled into the rectum, and the patient was to remain supine for 30 minutes thereafter. In 3 patients the butyrate course was extended to a total of 4 weeks. Before and after both treatment periods, sigmoidoscopic examinations were performed between 8 AM and 11 AM using an Olympus CF 20 M sigmoidoscope (Olympus Optical Europe, Hamburg, Germany) after bowel preparation with a tap-water enema. This mode of preparation has been shown not to interfere with rectal epithelial proliferation.13 At endoscopy (performed by an endoscopist blinded to the mode of treatment), the macroscopic appearance of the mucosa was evaluated by an index based on the presence or absence of erythema, edema, friability, granularity, and erosions (range of O-10).’ Biopsy specimens were taken with the regular forceps from the rectosigmoid junction and the rectal ampulla for evaluation of inflammation. They were fixed in formalin, embedded in paraffin, cut into 3ym sections, and stained with H&E. The changes evaluated as present or absent were accumulation of lymphocytes and plasma cells, infiltration of crypt epithelium by neutrophils, crypt abscesses, ulcerations, mucus depletion of epithelial cells, and distortion of crypts. Overall inflammatory change was graded 0 (absent), 1 (mild), 2 (moderate), or 3 (severe). All biopsy specimens were interpreted in a random and blinded manner by a single pathologist. In 6 of 10 patients with distal UC, biopsy specimens were also taken 10 cm from the anus for a study of rectal cell proliferation. Similarly, biopsy specimens were obtained from a group of 30 sex- and age-matched controls (15 men, 15 women, aged 39.8 + 2.2 years) with no pathological findings during routine colonoscopy. The specimens were immersed in prewarmed cell culture medium [basal medium with Earle’s salts (BME), fetal calf serum, and antibiotic-antimycotic solution; Gibco, Paisley, Scotland] and processed within 30 minutes. The specimens
Oral medication (mg/day)
colon
5-ASA, 3 X 500; prednisone, 5-ASA, 3 X 500; prednisone, 5-ASA, 3 X 500 Sulfasalazine, 3 X 1000 5-ASA, 3 x 500 Sulfasalazine, 3 X 1000 5-ASA, 2 x 500 None 5-ASA, 3 x 500 5-ASA, 3 x 500
2.5 7.5
the trial.
were then oriented mucosal-side-up on a membrane filter in a Petri dish containing 3 mL BME medium and tritiumlabeled thymidine (3H-Thy, 5 pCi/mL; DuPont Nemours Germany, Bad Homburg, Germany). The Petri dish was placed in an airtight chamber (Modular Incubator Chamber; Billups-Rothenberg Inc., Del Mar, CA) on a rocking platform (10 cycles/min). During the l-hour incubation period, carbogen gas (95% oxygen, 5% carbon dioxide) was administered at 1 atm additional pressure and the assembly was kept at 37°C. The biopsy specimens were then washed three times with saline and fixed in formalin (3.5% vol/vol; Merck, Darmstadt, Germany) contained in phosphate-buffered saline (PBS; Gibco) solution. For autoradiography, biopsy specimens were embedded in paraplast, cut into s-pm sections mounted on slides, and stained with acid-Schiff’s reagent (Feulgen reaction). Slides were dipped in Ilford K2 emulsion (Ilford, Essex, England) and exposed for 15 days in the dark following standard techniques described elsewhere.14 Rectal epithelial cell proliferation was assessed by recording the frequency of 3H-Thy-labeled cells in longitudinally sectioned crypts under the light microscope. At least 15 crypts (control group, at least 12 crypts) were studied per experiment. The numbers of labeled (a) and unlabeled (b) cells per crypt were determined and the labeling index (LI) computed (a/a + b).” Crypts were divided into five equal longitudinal compartments (compartment 1, crypt base; compartment 5, crypt surface), and in addition to the whole-crypt LI, an LI for each compartment was calculated. In addition, the $h value was assessed as the number of labeled cells in compartments 4-5 divided by the total number of labeled cells per crypt.16 Cell kinetics counting was performed in a blinded manner. Statistical
Analysis
All values are expressed as mean + SEM. Comparisons of quantitative data were made by the Wilcoxon rank sum test (paired data) or the Mann-Whitney U test (unpaired data). Qualitative data were compared by the x2 test. The Spearman rank coefficient was used for correlations. Differences were considered significant at P values of 10.05.
BUTYRATE AND ULCERATIVE COLITIS 53
July 1992
Results Clinical Symptoms At entry in the study, all 10 patients with distal UC had been suffering constantly from diarrhea for at least 3 weeks. During the control period, no change in stool frequency was seen (before NaCl, 3.9 * 0.7;after NaCl, 3.9 f 0.7;NS). However, after treatment with butyrate enemas the number of defecations per day decreased significantly (P < 0.01) from 4.5 -t 0.5 to 2.1 * 0.4. Fecal blood was observed in 9 of 10 patients before NaCl; this figure remained unchanged during the control phase. When butyrate enemas were administered, the number of patients with fecal blood decreased from 9 of 10 to 1 of 10 (P < 0.05, x2 test). The response of patient 8 (no additional medication) to butyrate enemas did not differ from that seen in the other patients who received concomitant standard treatment (Table 1). Butyrate and control enemas were retained for at least 30 minutes in every case; in many instances, they were kept for a longer period (up to several hours). They were tolerated without side effects. Endoscopic
Appearance
Before treatment, the endoscopic appearance score was 6.0+ 0.9(before NaCl) and 6.5f 0.4(before butyrate; NS) (Figure 1). After a 2-week course of NaCl, no change was seen (5.9 f 0.9). After 2 weeks of butyrate irrigation, the endoscopic appearance improved in 9 of 10 patients (3.8f 0.8;P < 0.01; n = 10). In 3 patients in whom only slight improvement was noted after 2 weeks, butyrate enemas were continued for another 2 weeks. In these patients, a further regression of inflammation was noted (2-week scores, 7, 4, and 5; 4-week scores, 2, 2, and 2). Histological
Data
Overall inflammatory change was graded (H&E stain) 0 (absent), 1 (mild), 2 (moderate), or 3
Figure 1. Endoscopic score (n = lo), histological grading (n = lo), and upper-crypt labeling frequency (n = 6) in patients with distal UC before and after treatment with sodium butyrate (0) or sodium chloride (0) (control) enemas. Vertical bars indicate 1 SEM; *significant differences (endoscopic score, P < 0.01; histological grading, P < 0.02; upper-crypt labeling, P < 0.03; Wilcoxon test).
6.
6.
4-
%
*
2.
o* before
afta
Edoscopic
Score
(severe). Before treatment, the histological score was 2.1 + 0.3 (NaCl) and 2.4 + 0.3 (butyrate; NS) (Figure 1). In the control run (NaCl enemas), no change was After 2 weeks of butyrate irrigaobserved (2.4k 0.3). tion, a decrease of mucosal inflammation was noted in 7 of 10 patients (score, 1.5f 0.3;P < 0.02;n = 10). In the 3 patients who showed minor endoscopic improvement after 2 weeks (histology unchanged, n = 2;slight histological improvement, n = l), treatment was continued for another 2 weeks. In these 3 patients, a further decrease in histological score was observed (2-week scores, 3,1,and 2; 4-week scores, 2,0,and 1). Proliferation
Pattern
In 6 of 10 patients, rectal cell-proliferation data were available and compared with data obtained from 30 healthy control persons (Figure 1; Table 2). Overall proliferation (whole-crypt LI) was not different between groups. However, cell labeling in compartments 4 and 5 was significantly higher in patients with UC before treatment and after NaCl treatment than in healthy controls. Butyrate irrigation resulted in a significant reduction of 3H-Thy incorporation in compartments 4 and 5. The LI in compartment 4 was reduced to values found in healthy controls. Similarly, the $h value indicated expansion of the proliferative zone in patients with untreated UC (before/after NaCl, before butyrate) but not in patients after butyrate irrigation or healthy controls. There was no correlation between the degree of inflammation (endoscopic score, histological grading) and parameters of an expanded proliferative zone (upper-crypt LI, 4h value). Discussion A connection between SCFAs and colitis was first established by Harig et al.’ who successfully treated four patients with diversion colitis using acetate (60mmol/L), propionate (30 mmol/L), and buty-
54
SCHEPPACH ET AL.
GASTROENTEROLOGY
Vol. 103,No. 1
Table 2. Rectal Cell-Proliferation Data of Patients With UC and Healthv Controls Patients with UC Na-Butyrate Before Group A No. of subjects No. of crypts assayed No. of labeled cells No. of all crypt cells Whole-crypt LI LI by crypt compartment Compartment 1 Compartment 2 Compartment 3 Compartment 4 Compartment 5 bh value
NaCl (control) After Group B
Before Group C
After Group D
Controls Group E
6 152 2085 14,330 0.144f 0.015
6 176 1860 16,995 0.115f 0.013
6 115 1669 11,825 0.150f 0.020
6 120 1827 11,975 0.155* 0.017
30 438 2478 20,124 0.125+ 0.006
0.158+ 0.208 f 0.179 f 0.112f 0.060 + 0.223 f
0.187f 0.025 0.203f 0.022 0.127+ 0.014* 0.047rt0.006A.D.E 0.011+ o.o04*J-~E 0.103+_0.012A*D.E
0.177f 0.196f 0.189f 0.128+ 0.062+ 0.249k
0.196f 0.027 0.219+ 0.027 0.181?I0.020' 0.122i-0.024BsC 0.055+ 0.009B~C 0.227t 0.020B,C
0.190f 0.013 0.254+ 0.011 0.132+ 0.013 0.057+ 0.018A.D,E 0.008+ 0.003A.BsD.E 0.070I!T 0.017A.D.E
0.009 0.018 0.028 0.026Bsc 0.014B*c 0.031B.c
0.044 0.032 0.031 0.026B,c 0.018Bsc o.034B.c
NOTE. Data represent means f SEM. bh value used according to Lipkin et al.‘” Patients with UC, n = 6; healthy control persons, n = 30. Superscripts A-E indicate significant differences (P < 0.05) vs. other groups. LI, labeling index.
rate (40 mmol/L). After 2 weeks of SCFA irrigation, they observed endoscopic and histological amelioration of inflammation. They concluded that a prolonged deprivation of preferred nutrients (SCFAs) frequently leads to colitis in the rectal stump. The resupply of nutrients, either by colonic reanastomosis or by SCFA irrigation, induces remission of colitis by endoscopic and histological criteria. Because there are striking similarities between diversion colitis and UC, a controlled trial was undertaken in patients with distal UC (maximum involvement, rectum to splenic flexure). Rectal enemas have been shown to provide homogeneous delivery of the drug to the inflamed lower colon reaching the splenic flexure.” In this study, butyrate was tested alone and not in a mixture of SCFAs because evidence from the literature was strongest in favor of butyrate to affect the mucosa.4’” After 2 weeks of butyrate irrigation, 9 of 10 patients responded endoscopically and 7 histologically. On placebo, no change in inflammation was observed. These data from a controlled study are supported by an uncontrolled preliminary report on the effect of an SCFA mixture (acetate, 80 mmol/L; propionate, 30 mmol/L; and butyrate, 40 mmol/L) as a topical treatment of distal UC. Breuer et a1.l’ defined a disease activity index based on stool frequency, rectal bleeding, endoscopic appearance, and impact of symptoms on lifestyle (range, O-12). This index and histological parameters improved partially by week 2 but markedly by week 6. This finding is in agreement with our data showing further improvement after 4 weeks in patients with a partial response after 2 weeks. How do these data compare with standard topical treatment of distal UC? For 2-week trials testing cor-
ticosteroid enemas, clinical response rates of 38%78% have been reported.“‘l* The clinical response to 5-ASA enemas for 2 weeks (93%) has been found superior to response to hydrocortisone (57%); in the same study, the histological improvement has been less pronounced (77% for 5-ASA vs. 33% for hydrocortisone).‘g Other authors have argued that prolonged therapy (>4 weeks) is required to achieve remission in a majority of patients.20~2* To evaluate the role of butyrate/SCFA in topical treatment of distal UC, direct comparisons with standard drugs remain to be made. An advantage of butyrate is the entire lack of side effects because this compound is present in the colon under physiological conditions. By which mechanism may butyrate affect the coionic mucosa in UC? On the basis of in vitro experiments using isolated colonocytes from patients with UC, Roediger”,22*23 has described pathobiochemical changes in acute and quiescent colitis. He found as an important defect in diseased colonocytes a reduction (but not complete failure) of mitochondrial fatty acid (SCFA) oxidation. In acute UC, the rate of oxygen consumption by colonocytes from the descending colon was reduced by 46% compared with cells from healthy controls when butyrate (10 mmol/L) was present as a fuel.” Instead, compensatory pathways of fuel utilization were used (glutamine and glucose oxidation enhanced). Considering data from experimental colitis, impaired butyrate oxidation is thought to be because of a diminution of free coenzyme A (CoA), which is essential for SCFA activation and subsequent oxidation to CO, and ketone bodies.23 Levels of free CoA have in fact been found low in human UC.24 Soergel” has speculated that treatment of distal UC by SCFA enemas might overcome
BUTYRATE
July 1992
this partial metabolic block by “mass action”; this means that higher than normal SCFA concentrations in the colonic lumen may improve energy supply to colonocytes despite an abnormality of mitochondrial SCFA oxidation. This concept differs from other, mainly anti-inflammatory approaches (sulfasalazine, 5-ASA, corticosteroids). To date, there is no information on a potential interference of 5-ASA with SCFA metabolism in the colonocyte. Clinical results concerning the treatment of distal UC with SCFAs” or butyrate (present work) are encouraging; however, more work is needed to clarify the role of SCFAs in the pathogenesis of UC. Butyrate has different effects on proliferation of normal colonocytes and colon cancer cell lines. While the physiological pattern of proliferation (confined to the basal crypt) is stimulated in the normal human mucosa,4 growth rates are inhibited in various human colon cancer cell lines.26-31 Therefore, it was of interest whether butyrate could influence rectal cell proliferation in UC, known as a principally preneoplastic condition. The upper-crypt labeling frequency (labeling in crypt compartments 4 and 5) in patients with UC decreased significantly after butyrate treatment and was no longer different from values obtained in 30 healthy control subjects. Expansion of the proliferative zone to the crypt surface has been considered by Lipkin a “biomarker of increased susceptibility to cancer formation.“32 This phenomenon is observed in patients at risk of malignancy (familial polyposis coli, familial nonpolyposis colon cancer, sporadic colonic adenoms, UC.16,33-3* Biasco et a1.3ghave shown that expansion of the proliferative zone in UC is independent of the degree of inflammation and the duration of disease; this finding is in agreement with our data. The effect of butyrate on upper-crypt labeling may be due to its potent differentiating property demonstrated in colon cancer cell lines in permanent culture.26-31 In conclusion, butyrate as an end product of bacterial fermentation in the large bowel profoundly affects the colonic epithelium (inflammation and proliferation) in UC. It is suggested that the effect of an SCFA mixture” on the inflamed mucosa in UC is largely, if not entirely, attributable to its butyrate moiety. References Cummings JH. Fermentation in the human large intestine: evidence and implications for health. Lancet 1983;1:12061209. Roediger WEW. Role of anaerobic bacteria in the metabolic welfare of the colonic mucosa in man. Gut 1980;21:793-798. Roediger WEW. Utilization of nutrients by isolated epithelial cells of the rat colon. Gastroenterology 1982;83:424-429. Scheppach W, Bartram P, Richter A, Richter F, Liepold H, Dusel G, Hofstetter G, Rtithlein J, Kasper H. The effect of
short-chain J Parenter
AND
IJLCERATIVE
fatty acids on the human Enter
Nutr
colonic
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mucosa
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JL. Stim5. Kripke SA, Fox AD, Berman jM, Settle RG. Rombeau ulation of intestinal mucosal growth with intracolonic infusion of short-chain fatty acids. J Parenter Enter Nutr 1989;13:109-116. mucosal nu6. Roediger WEW. The starved colon-diminished trition, diminished absorption, and colitis. Dis Colon Rectum 1990;33:858-862. H. Proctitis and colitis follow7. Glotzer DJ. Glick ME, Goldman ing diversion of the fecal stream. Gastroenterology 1981;80: 438-441. VP, Schimmel EM. Diversion colitis: 8. Agarwal deficiency syndrome? Nutr Rev 1989;47:257-261.
a nutritional
RA, Wood CM. Treatment 9. Harig JM. Soergel KH, Komorowski of diversion colitis with short-chain fatty acid irrigation. N Engl J Med 1989;320:23-28. A. Hauck W. Breuer RI. Organic anions 10. Vernia P, Gnaedinger and the diarrhea of inflammatory bowel disease. Dig Dis Sci 1988;33:1353-1358. 11. Breuer RI, Buto SK, Christ ML, Bean J. Vernia P. Paoluzi P, DiPaolo MC, Caprilli R. Rectal irrigation with short-chain fatty acids for distal ulcerative colitis. Preliminary report. Dig Dis Sci 1991;36:185-187. 12. Kruis LV. Bull U, Eisenburg Ionic spread of sulfasalazine 1982:17:933-938.
J. Paumgartner G. Retrograde coenemas. Stand J Gastroenterol
Z. Rozen P, Fine N, Chetrit A. Reproducibility 13. Fireman ies and effects of bowel preparations on measurements tal epithelial proliferation, Cancer Lett 1989;45:59-64.
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M. Fujita M, Lipkin M, Palmer R, Friedman E, Au14. Ilsugane genlicht L. Cell proliferation in explant cultures of the human colon. Digestion 1982;24:225-233. SJ. Tritiated-thymidine label15. Lipkin M. Enker WE. Winawer ing of rectal epithelial cells in “non-prep” biopsies if individuals at increased risk for colonic neoplasia. Cancer Lett 1987;37:153-161, 16. Lipkin M, Blattner WE, Fraumeni JF. Lynch HT, Deschner E, Winawer S. Tritiated thymidine ($p, $h) labeling distribution as a marker for hereditary predisposition to colon cancer. Cancer Res 1983;43:1899-1904. 17
Hamilton I, Pinder IF, Dickinson RJ, Ruddell WS, Dixon MF, Axon ATR. A comparison of prednisolone enemas with lowdose oral prednisolone in the treatment of acute distal ulcerative colitis. Dis Colon Rectum 1984;27:701-702.
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McIntyre PB. Macrae FA, Berghouse 1,. English J. LennardJones JE. Therapeutic benefits from a poorly absorbed prednisolone enema in distal ulcerative colitis. Gut 1985;26:822824.
19
Campieri M, Lanfranchi GA, Bazzocchi G, Brignola C, Sarti F, Franzin G. Battocchia A, Labo G, Dal Monte PR. Treatment of ulcerative colitis with high-dose 5-aminosalicylic acid enemas. Lancet 1981;2:270-271.
20. Sutherland LR, Martin F. Greer S, Robinson M, Creenberger N, Saibil F, Martin T, Sparr J, Prokipchuk E, Borgen L. 5-Aminosalicylic acid enemas in the treatment of distal ulcerative colitis. proctosigmoiditis and proctitis. Gastroenterology 1987;92:1894-1898. 21. Danish 5-ASA Group. Topical 5-aminosalicylic acid versus prednisolone in ulcerative proctosigmoiditis. Dig Dis Sci 1987:32:598-602. 22. Roediger WEW. The colonic epithelium in ulcerative colitis: an energy-deficiency disease? Lancet 1980:2:712-715. 23. Roediger LVEW. The role of colonic mucosal metabolism in the pathogenesis of ulcerative colitis. In: Goebell H, Peskar BM. Malchow H, eds. Inflammatory bowel diseases-basic re-
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search and clinical implications. Lancaster, England: MTP, 1988:69-78. 24. Ellestad-Sayad JJ, Nelson RA, Adson MA, Palmer WM, Soule EH. Pantothenic acid, coenzyme A and human chronic ulcerative and granulomatous colitis. Am J Clin Nutr 1976;29: 1333-1338. 25. Soergel KH. Colitis and short-chain fatty acids. IBD Forum Symposium, Digestive Disease Week, San Antonio, TX, 1990. 26. Dexter DL, Lev R, McKendall GR, Mitchell P, Calabres P. Sodium butyrate-induced alteration of growth properties and glycogen levels in cultured human colon carcinoma cells. Histochem J 1984;16:137-149, 27. Augeron C, Laboisse CL. Emergence of permanently differentiated cell clones in a human colonic cancer cell line in culture after treatment with sodium butyrate. Cancer Res 1984;44:3961-3969. 28. Gum JR, Kam WK, Byrd JC, Hicks JW, Sleisenger MH, Kim YS. Effects of sodium butyrate on human colonic adenocarcinoma cells. J Biol Chem 1987;262:1092-1097. 29. Siddiqui B, Kim JS. Effects of sodium butyrate, dimethyl sulfoxide, and retinoic acid on glycolipids of human rectal adenocarcinoma cells. Cancer Res 1984;44:1648-1652. 30. Chung YS, Song IS, Erickson RH, Sleisenger MH, Kim YS. Effect of growth and sodium butyrate on brush border membrane-associated hydrolases in human colorectal cancer cell lines, Cancer Res 1985;45:2976-2982. 31. Whitehead RH, Young GP, Bhatal PS. Effects of short chain fatty acids on a new human colon carcinoma cell line (LIM 1215).Gut 1986;27:1457-1463. 32. Lipkin M. Biomarkers of increased susceptibility to gastrointestinal cancer: new application to studies of cancer prevention in human subjects. Cancer Res 1988;48:235-245. 33. Lipkin M, Blattner WA, Gardner EJ, Burt RW, Lynch H, Deschner E, Winawer S, Fraumeni JF. Classification and risk assessment of individuals with familial polyposis, Gardner’s syndrome, and familial non-polyposis colon cancer from
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(3H)thymidine labeling patterns in colonic epithelial cells. Cancer Res 1984;44:4201-4207. 34. Terpstra OT, van Blankenstein M, Dees J, Eilers GA. Abnormal pattern of cell proliferation in the entire colonic mucosa of patients with colon adenoma or cancer. Gastroenterology 1987;92:704-708. 35. Risio M, Lipkin M, Candelaresi GL, Bertone A, Coverlizza S, Rossini FP. Correlations between rectal mucosa cell proliferation and the clinical and pathological features of nonfamilial neoplasia of the large intestine. Cancer Res 1991;51:19171921. 36. Biasco G, Lipkin M, Minarini A, Higgins P, Miglioli M, Barbara L. Proliferative and antigenic properties of rectal cells in patients with chronic ulcerative colitis. Cancer Res 1984;44: 5450-5454. 37. Serafini EP, Kirk AP, Chambers
TJ. Rate and pattern of epithelial cell proliferation in ulcerative colitis. Gut 1981;22:648652. 38. Kanemitsu T, Koike A, Yamamoto S. Study of the cell proliferation kinetics in ulcerative colitis, adenomatous polyps, and cancer. Cancer 1985;56:1094-1098. 39. Biasco G, Paganelli GM, Miglioli M, Brillanti S, Di Febo G, Gizzi G, Ponz de Leon M, Campieri M, Barbara L. Rectal cell proliferation and colon cancer risk in ulcerative colitis. Cancer Res 1990;50:1156-1159.
Received August 19, 1991. Accepted December 11,1991. Address requests for reprints to: Wolfgang Scheppach, M.D., Department of Medicine, University of Wiirzburg, Josef-SchneiderStraBe 2, D-8700 Wtirzburg, Germany. The cooperation of the patients in this study is gratefully acknowledged. The authors thank Dr. Englert [Pharmacy of the Wtirzburg University Hospital) for providing sodium butyrate in sterile solution. The help of the Endoscopy Unit’s staff is appreciated.
