Hans-Peter Bartram

histological changes in the colonic mucosa following irrigation with short-chain fatty acids

Objectives: Short‐chain fatty acids (SCFAs) derived from bacterial fermentation of complex carbohydrates are preferred luminal nutrients of the colonic mucosa. Starvation of colonocytes through lack or impaired metabolism of luminal SCFAs may be a cofactor in the pathogenesis of ulcerative colitis. Design: A detailed histological evaluation of colonic biopsy specimens was performed in patients with active distal ulcerative colitis who were treated with rectal enemas containing a mixture of SCFAs, n‐butyrate alone or saline placebo. Together with light microscopic parameters of mucosal inflammation, the pattern of crypt cell proliferation (proliferating cell nuclear antigen) and the mucosal activity of factor XIII were assessed. Results: Butyrate reduced the density of polymorphonuclear leucocytes in the lamina propria (4 weeks: P = 0.063; 8 weeks: P = 0.091); other inflammatory parameters remained unchanged. Both butyrate and the SCFA mixture reduced significantly the number of proliferating cells in the upper 40% of crypts. Tissue factor XIII activity in active ulcerative colitis was significantly lower than in mucosa from normal colons; however, it was not affected by SCFA or butyrate irrigation. Conclusion: SCFAs and butyrate have a more marked effect on crypt cell proliferation than on parameters of inflammation in patients with active ulcerative colitis.

Bile acid metabolism by colonic bacteria in continuous culture: effects of starch and pH.

Secondary bile acids (BA) have been shown to be involved as a promoting agent in the adenoma-carcinoma sequence of colorectal cancer. In previous studies, fermentation of starch has been shown to inhibit the degradation of primary to secondary BA by the colonic microflora. This study was designed to investigate BA metabolism in continuous cultures of mixed fecal bacteria to get further insights into the mechanisms of this inhibition. Fermentation vessels were fed with media containing cholic (0.6 g/l) and chenodeoxycholic acid (0.4 g/l). Cultures were either starch-free or enriched with starch (10 g/l). pH was controlled and adjusted to 7.0 or 6.0. Total culture duration was 28 days and concentrations of BA, short-chain fatty acids (SCFA), and starch were measured periodically. At pH 6, significantly more primary BA remained in the media and less secondary BA were produced. Total BA concentrations were lower at pH7. SCFA concentrations were higher in the vessels supplemented with starch. Starch was completely fermented and not present in significant amounts in any fermentation vial after the first week. These data indicate that bacterial breakdown of primary to secondary BA is inhibited when starch is simultaneously fermented. This effect can be explained by the reduction of pH resulting from SCFA production. Considering these findings, resistant starch which escapes assimilation in the small bowel may be a protective factor against colorectal cancer.

Effects of Deoxycholic Acid and Butyrate on Mucosal Prostaglandin E2 Release and Cell Proliferation in the Human Sigmoid Colon

BACKGROUND A high-fat, low-fiber diet resulting in increased excretion of fecal secondary bile acids is regarded as a major risk factor for colon cancer. Incubation of human colonic biopsies with the secondary bile acid deoxycholic acid (DCA) leads to hyperproliferation with expansion of the proliferative zone, ie, a biomarker of increased cancer risk. Antiproliferative effects on various colon cancer cell lines, however, were reported for butyrate (BUT), a fermentation product of dietary fiber. METHODS In the following in vitro study we incubated biopsies from the normal sigmoid colon of 12 patients (age 55.8 +/- 3.6 years) with 5 microM DCA or a combination of 5 microM DCA plus 10 mM BUT (DCA/BUT) and determined epithelial proliferation by bromodeoxyuridine immunohistochemistry. As a possible mediator for the DCA effects on colonic cell proliferation, mucosal prostaglandin E2 (PGE2) release into the incubation medium was measured by 125I-PGE2 radioimmunoassay. RESULTS Incubation with DCA alone revealed a significantly higher labeling index for the whole crypt (.17 vs .11, p < .01) and for the upper 40% of the crypt (.05 vs .01, p < .01) compared with DCA/BUT. Mucosal PGE2 release during DCA/BUT incubation showed a trend toward lower values compared with DCA incubation (357.07 vs 434.29 pg/mg per hour; p = .07). CONCLUSION The results indicate a normalization of DCA-induced hyperproliferation of colonic epithelium by butyrate that is not clearly mediated by PGE2. Considering that nutrition affects the luminal concentrations of DCA and butyrate, our findings may have implications for colonic carcinogenesis.

Effects of fish oil on fecal bacterial enzymes and steroid excretion in healthy volunteers: implications for colon cancer prevention.

Diet-induced changes in fecal excretion of secondary bile acids, certain neutral sterols, and bacterial enzyme activities are known to play a role in colon cancer development. Dietary fish oil (FO) has been implicated as a protective agent in colon carcinogenesis. In the present study, the effects of FO and corn oil (CO) on these fecal parameters were investigated in 24 healthy volunteers consuming a low- or a high-fat diet (30% or 50% of energy derived from fat). After four weeks of FO or CO supplementation (4.4 g of n-3 fatty acids/day), no significant differences were noted for fecal activities of beta-glucuronidase, beta-glucosidase, and sulfatase, nor was fecal bile acid excretion significantly affected by FO or CO consumption. However, daily excretion of the putative colon carcinogen 4-cholesten-3-one was significantly lower in the FO than in the CO period during low- and high-fat experiments. This may be another biochemical mechanism by which FO exerts its protective effect on colon cancer development.

Comparisons of the anti-proliferative effects of butyrate and aspirin on human colonic mucosa in vitro.

The short-chain fatty acid butyrate is regarded as a regulative agent in haemostasis of mucosal cell turnover. Inhibition of prostaglandin E2 synthesis is particularly involved in this regulation process. In the present study, proliferation was stimulated in colonic biopsies of 12 healthy subjects (age 51.3 years, range 25-81) by incubation with deoxycholic acid (5 micromol/l DCA). The anti-proliferative and cyclo-oxygenase-inhibiting properties of butyrate (10 mmol/l BUT) and of aspirin (555 micromol/l ASA) were investigated. Colonic cell proliferation was determined by bromodeoxyuridine immunohistochemistry. PGE2 release into the incubation medium was measured by radioimmunoassay. Incubation with DCA/ASA, DCA/BUT and DCA/ASA/BUT revealed a significant reduction in crypt cell proliferation as measured by the labelling index of the whole crypt in comparison to incubation with DCA alone (DCA/ASA: 0.14, P < 0.01; DCA/BUT: 0.15, P < 0.05; DCA/ASA/BUT: 0.15, P < 0.05, versus DCA: 0.18). The labelling index for the upper 40% of the crypt was only lower after incubation with DCA/ASA (0.023) compared to DCA (0.028) (P < 0.05). PGE2 release from biopsy specimens was only significantly decreased in the incubation media where ASA was added (DCA/ASA: 29.0 pg/mg mucosa/h, P < 0.005; DCA/ASA/BUT: 31.4 pg/mg mucosa/h, P < 0.01 versus DCA: 56.9 pg/mg mucosa/h). Butyrate and aspirin showed no synergistic effects. The results indicate a normalization of DCA-induced hyperproliferation of colonic mucosa by butyrate, and, even more efficiently, by aspirin. The data support the hypothesis that butyrate and aspirin can act as chemopreventive agents in colon carcinogenesis.

Influence of starch fermentation on bile acid metabolism by colonic bacteria

Secondary bile acids (BA) may be involved in the pathogenesis of colorectal cancer. In vivo, starch malabsorption has been shown to reduce fecal excretion of secondary BA. The present in vitro study was performed to investigate the effect of starch fermentation on BA metabolism by colonic bacteria. Fecal samples of healthy volunteers were incubated in anaerobic batch cultures for 48 hours with the primary bile acids cholic (0.6 g/l) and chenodeoxycholic acid (0.4 g/l). Media were starch free or enriched with starch (10 g/l). The pH was controlled and held at 6 or 7. In the starch-free incubations, secondary BA were rapidly formed, and degradation of primary to secondary BA was complete within 24 hours. The formation of secondary BA was partially inhibited by the addition of starch to the media. This effect was stronger at pH 6 than at pH 7. Starch was rapidly and completely fermented. In conclusion, this study showed that formation of secondary BA by fecal bacteria is inhibited when starch is simultaneously fermented, an effect that is mainly, but not completely, explained by reduction of pH.

Effects of calcium and deoxycholic acid on human colonic cell proliferation in vitro.

A high-fat and low-fiber diet is regarded as a major risk factor for colon cancer by increasing luminal contents of secondary bile acids. Calcium, on the other hand, has been implicated as a possible preventive agent in colon tumor development. In in vitro studies with human colonic epithelium, incubation with the secondary bile acid deoxycholic acid (DCA) induced hyperproliferation of colonic crypt cells which is regarded as a sign of preneoplastic transformation. In the present study the effects of calcium chloride (CaCl2) on DCA-induced hyperproliferation were tested at different stages of DCA-induced cell injury. Colonic biopsies from 36 patients (no tumors, polyps or IBD) were incubated with CaCl2 (1 and 10 mM) and 5 microM DCA which was added to the incubation medium either together with (experiment A), after (experiment B), or before CaCl2 (experiment C). Coincubation of the biopsies with DCA and 10 mM CaCl2 at the same time (experiment A) resulted in a significant reduction of whole crypt labeling index by 12% (p < 0.05), whereas in the other incubation experiments no significant growth-inhibitory effects could be demonstrated for CaCl2. These findings may best be explained by the formation of calcium-bound bile acid salts which lost most of their toxicity for the colonic cells.

Antiproliferative effect of 1,4-phenylenebis(methylene)selenocyanate (p-XSC) on colonic epithelium of patients with adenomatous polyps in vitro.

We have consistently shown that the organoselenium compound 1,4–phenylenebis(methylene)selenocyanate (p-XSC) is a superior cancer chemopreventive agent and less toxic than selenite or certain naturally-occurring selenoamino acids. To elucidate the effects of p-XSC on human colonic mucosa, biopsies from endoscopically normal sigmoid colon of 30 patients with adenomatous polyps were incubated with p-XSC at concentrations of 1, 2 and 5 μmol/l dissolved in dimethylsulphoxide (DMSO). Biopsies incubated with DMSO or pure culture medium served as a control. Proliferating cells were labelled by bromodeoxyuridine immunohistochemistry and the labelling index (LI) was computed. Upper crypt labelling index (LI of crypt compartments 4+5) and Φh value, which are both discriminators of the expansion of the proliferative zone, were significantly lower after incubation with 1 and 5 μmol/l p-XSC, respectively (LI 4+5: 0.8 and 1.0; Φh value: 2.1 and 2.4), as compared with DMSO (LI 4+5: 3.6 and 4.5; Φh value: 7.0 and 8.3) or culture medium (LI 4+5: 3.3 and 4.5; Φh value: 7.2 and 8.1) (P<0.005 and P<0.05 by Friedmans block test). A trend towards lower levels of LI 4+5 (P=0.059) and Φh value (P=0.075) were seen after 2 μmol/l p-XSC incubation compared with DMSO. Since hyperproliferation of colonic crypt cells with expansion of the proliferative zone is regarded as a biomarker of increased cancer risk, the antiproliferative effects of p-XSC especially on upper crypt LI and Φh value may indicate a possible protective effect of this organoselenium compound in the prevention of human colon cancer development.