Concentrations Of Secondary Bile Acids Research Articles

Comparison of diets enriched in stearic, oleic, and palmitic acids on inflammation, immune response, cardiometabolic risk factors, and fecal bile acid concentrations in mildly hypercholesterolemic postmenopausal women—randomized crossover trial

Direct comparisons between SFAs varying in chain length, specifically palmitic acid (16:0) and stearic acid (18:0), relative to the latter's metabolic product, oleic acid (18:1), on cardiometabolic risk factors are limited. The aim of this study was to determine the relative comparability of diets enriched in palmitic acid, stearic acid, and oleic acid on inflammation and coagulation markers, T lymphocyte proliferation/ex-vivo cytokine secretion, plasma cardiometabolic risk factors, and fecal bile acid concentrations. Hypercholesterolemic postmenopausal women (n=20, mean±SD age 64±7 y, BMI 26.4±3.4 kg/m2, LDL cholesterol≥2.8 mmol/L) were provided with each of 3 diets [55% energy (%E) carbohydrate, 15%E protein, 30%E fat, with ∼50% fat contributed by palmitic acid, stearic acid, or oleic acid in each diet; 5 wk/diet phase] using a randomized crossover design with 2-wk washouts between phases. Outcome measures were assessed at the end of each phase. Fasting LDL-cholesterol and non-HDL-cholesterol concentrations were lower after the stearic acid and oleic acid diets than the palmitic acid diet (all P<0.01). Fasting HDL-cholesterol concentrations were lower after the stearic acid diet than the palmitic acid and oleic acid diets (P<0.01). The stearic acid diet resulted in lower lithocholic acid (P=0.01) and total secondary bile acid (SBA) concentrations (P=0.04) than the oleic acid diet. All other outcome measures were similar between diets. Lithocholic acid concentrations were positively correlated with fasting LDL-cholesterol concentrations (r=0.33; P=0.011). Total SBA, lithocholic acid, and deoxycholic acid concentrations were negatively correlated with fasting HDL cholesterol (r=-0.51 to -0.44; P<0.01) concentrations and positively correlated with LDL cholesterol:HDL cholesterol (r =0.37-0.54; P<0.01) ratios. Dietary stearic acid and oleic acid had similar effects on fasting LDL-cholesterol and non-HDL-cholesterol concentrations and more favorable ones than palmitic acid. Unlike oleic acid, the hypocholesterolemic effect of stearic acid may be mediated by inhibition of intestinal hydrophobic SBA synthesis. These findings add to the data suggesting there should be a reassessment of current SFA dietary guidance and Nutrient Facts panel labeling.This trial was registered at clinicaltrials.gov as NCT02145936.

Potential biomarkers to predict outcome of faecal microbiota transfer for recurrent Clostridioides difficile infection

Background & AimsFaecal microbiota transplantation (FMT) has proven high clinical efficacy in the management of recurrent Clostridioides difficile infection (rCDI) with cure rates of over 80% after a single treatment. Nevertheless, the reasons for failure in the remaining 20% remain elusive. The aim of the present study was to investigate different potential predictors of response to FMT. MethodsFaecal specimens of sixteen patients undergoing FMT for rCDI, as well as samples from the respective donors were collected and analyzed by 16S rRNA gene profiling, bile acid-inducible (baiCD) gene specific qPCR, and liquid chromatography tandem-mass spectrometry (LC–MS/MS) to quantify the concentrations of primary and secondary bile acids. ResultsUsing the faecal concentration of the secondary bile acid lithocholic acid (LCA)within the patient specimens, we were able to predict response to FMT (accuracy 95.2%, sensitivity 100%, specificity 90.9%). By combining the faecal LCA concentration with the urinary pCS concentration, an accuracy of 100% was achieved. ConclusionLCA appears to be a promising marker candidate for prediction of clinical response to FMT. Other makers, such as urinary concentration of pCS, but not 3-IS, might be used to improve accuracy of prediction. Further studies are warranted to validate these candidate markers.

621. Treatment of Recurrent Clostridium difficile Infection with SER-109 Increases the Concentration of Secondary Bile Acids in a Dose-Dependent Manner

Background C. difficile recurs when dormant spores germinate in the dysbiotic gut, facilitated by an increase of 1° vs. 2° bile acids. SER-109, an ecology of bacterial spores purified from stool of healthy donors, is an investigational first-in-class microbiome therapeutic intended to facilitate microbiome restoration and reduce risk of recurrent C. difficile (rCDI). Rapid engraftment of spore-forming species is associated with (i) higher doses of SER-109 in our dose-ranging Phase 1b study (Ph1b) and (ii) reduced rCDI in our Phase 2 trial (Ph2). We explored whether higher doses of SER-109 were associated with an increase in 2° bile acids.MethodsWhole metagenomic shotgun (WMS) data were generated from stool, and species were identified using a proprietary build of MetaPhlAn. Evaluation of spore-forming species richness and bile acid concentrations identified effects of SER-109 treatment. A triple stage bioreactor model of the human gut and rCDI was used to evaluate the impact of microbial therapeutics.ResultsPh1b subjects who received a higher dose (>1.5 × 108 SporQ) had significantly higher spore-forming species richness than subjects who received a low dose (<1.5 × 108 SporQ) at Week 1 post-treatment (P = 0.017, Figure 1). Spore-forming species richness in patients receiving a low dose in Ph1b was comparable to that observed in non-recurrent patients in Ph2, who received the same mean dose (Figure 1). Ph1b subjects in the high dose group had a significantly higher concentration of 2° bile acids as compared with Ph1b low dose subjects and non-recurrent Ph2 subjects (P = 0.036, P < 0.001, respectively, Figure 2). A higher dose (3 × 108 SporQ × 3 days) suppressed recurrence in a gut model of rCDI; a single dose did not.ConclusionHigher doses of SER-109 are significantly associated with (i) higher spore-forming species richness, (ii) concentrations of secondary bile acids, and (iii) prevention of recurrence in an gut model of CDI. These results suggest that SER-109 in the Phase 2 trial was biologically active and catalyzed a functional change in the microbiome of a subset of subjects; a dose increase may optimize efficacy across a broad population. Seres has initiated a Phase 3 study of SER-109 to reduce rCDI, which includes an increase in dose titer and frequency. Figure 1:Figure 2:Disclosures M. Henn, Seres Therapeutics, Inc.: Employee and Shareholder, Salary. C. Ford, Seres Therapeutics, Inc.: Employee and Shareholder, Salary. E. O’Brien, Seres: Employee and Shareholder, Salary. J. Wortman, Seres Therapeutics, Inc.: Employee and Shareholder, Salary. L. Diao, Seres Therapeutics, Inc.: Employee and Shareholder, Salary. C. Desjardins, Seres Therapeutics, Inc.: Employee and Shareholder, Salary. A. Tomlinson, Seres Therapeutics, Inc.: Employee and Shareholder, Salary. K. Litcofsky, Seres Therapeutics, Inc.: Employee and Shareholder, Salary. M. Wilcox, Seres Therapeutics, Inc.: Consultant, Research Contractor, Scientific Advisor and Shareholder, Research support. A. Buckley, Seres Therapeutics, Inc.: Research Contractor, Research support. P. Bernardo, Seres Therapeutics, Inc.: Employee and Shareholder, Salary. B. McGovern, Seres Therapeutics, Inc.: Employee and Shareholder, Salary. J. G. Aunins, Seres Therapeutics, Inc.: Employee and Shareholder, Salary. D. N. Cook, Seres Therapeutics, Inc.: Employee and Shareholder, Salary. M. Trucksis, Seres Therapeutics, Inc.: Employee and Shareholder, Salary.

The effects of amoxicillin and vancomycin on parameters reflecting cholesterol metabolism

BackgroundChanges in the microbiota composition have been implicated in the development of obesity and type 2 diabetes. However, not much is known on the involvement of gut microbiota in lipid and cholesterol metabolism. In addition, the gut microbiota might also be a potential source of plasma oxyphytosterol and oxycholesterol concentrations (oxidation products of plant sterols and cholesterol). Therefore, the aim of this study was to modulate the gut microbiota by antibiotic therapy to investigate effects on parameters reflecting cholesterol metabolism and oxyphytosterol concentrations. DesignA randomized, double blind, placebo-controlled trial was performed in which 55 obese, pre-diabetic men received oral amoxicillin (broad-spectrum antibiotic), vancomycin (antibiotic directed against Gram-positive bacteria) or placebo (microcrystalline cellulose) capsules for 7days (1500mg/day). Plasma lipid and lipoprotein, non-cholesterol sterol, bile acid and oxy(phyto)sterol concentrations were determined at baseline and after 1-week intervention. ResultsPlasma secondary bile acids correlated negatively with cholestanol (marker for cholesterol absorption, r=−0.367; P<0.05) and positively with lathosterol concentrations (marker for cholesterol synthesis, r=0.430; P<0.05). Fasting plasma secondary bile acid concentrations were reduced after vancomycin treatment as compared to placebo treatment (-0.24±0.22μmol/L vs. −0.08±0.29μmol/L; P<0.01). Vancomycin and amoxicillin treatment did not affect markers for cholesterol metabolism, plasma TAG, total cholesterol, LDL-C or HDL-C concentrations as compared to placebo. In addition, both antibiotic treatments did not affect individual isoforms or total plasma oxyphytosterol or oxycholesterol concentrations. ConclusionDespite strong correlations between plasma bile acid concentrations and cholesterol metabolism (synthesis and absorption), amoxicillin and vancomycin treatment for 7days did not affect plasma lipid and lipoprotein, plasma non-cholesterol sterol and oxy(phyto)sterol concentrations in obese, pre-diabetic men.

A Bayes Study of Bile Acid Constituents on Cholelithiasis and Carcinoma of the Gallbladder

Background: Significantly high concentration of secondary bile acids and low concentration of primary acids are reported by the surgeons in patients with gallbladder carcinoma (GBC) compared to control and cholelithiasis groups. Aim: To examine the effect of primary and secondary bile acids on the development of cholelithiasis and GBC and to investigate the association, if any, among the two groups of bile acids. Methods: The study uses two groups of patients at a time selected in accordance with some pre-fixed inclusion and exclusion criteria. Informed consent was obtained from all patients. The demographic characteristics such as mean age, sex ratio and body mass index, etc. are obtained for the selected groups of patients. The study defines dichotomous responses and the four bile acid constituents, namely cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA) and lithocholic acid (LCA), as the predictors. It then assumes logistic regression model to associate the binary responses with the predictors by using probability scores. Bayes analysis is developed using Markov chain Monte Carlo (MCMC) pack in R software for the posterior simulation. Results: Twenty one cholelithiasis patients and twenty patients in each of control and GBC groups are studied. It is seen that a unit decrement in the level of CA (CDCA) increases the log (odds ratio) for cholelithiasis by an amount of 0.49 (0.14) and odds ratio by almost 1.5 (1.12). Similarly, a unit increment in the level of DCA (LCA) provides the log (odds ratio) for cholelithiasis as 0.18 (1.3) and odds ratio as 1.16 (2.95). Comparing GBC with control population, it is noted that a unit decrease in the level of CA (CDCA) in the control population increases the log (odds ratio) for GBC by an amount of 1.16(0.26) and odds ratio by almost 2.63 (1.24) times. Similarly, the log(odds ratio) for GBC increases by 0.77(1.94) and the odds ratio increases by 1.9 (5.0) for the unit increment in the level of DCA(LCA). Conclusions: The study observes relatively high variations in the primary and secondary bile acids in the cholelithiasis and GBC groups as compared with the control group. It, in turn, reflects strong association among the two categories of bile acids in gallbladder diseases.

Effects of legume kernel fibres and citrus fibre on putative risk factors for colorectal cancer: a randomised, double-blind, crossover human intervention trial

BackgroundIn some studies, high intake of dietary fibre has been associated with a lower risk of colorectal cancer. The present study aimed to compare physiological effects of three legume kernel fibres and citrus fibre on blood lipids (primary outcome: LDL cholesterol) and colonic health.MethodsNinety-two subjects were recruited for the double-blind, controlled crossover trial. Seventy-eight participants were randomly divided into three groups. Following run-in, half the volunteers from each group consumed 25 g/d of a legume fibre, comprising blue lupin fibre, white lupin fibre, and soya fibre for two weeks. The other half received the same amount of citrus fibre (active comparator). The intervention was crossed within each group after two weeks wash-out. At the end of run-in and intervention, a quantitative faeces collection took place and fasting blood samples were drawn. Repeated measures ANOVA with the general linear model were applied to evaluate changes following interventions.ResultsSeventy-six subjects completed the study. Dietary fibre intake during all interventions was approximately twice the fibre intake at run-in. The lupin fibre supplementations increased daily faecal dry matter and faecal weight compared to run-in, representing an increase of 1.76 g faeces/g additional dietary fibre contributed by blue lupin and of 1.64 g faeces/g by white lupin, respectively. Both lupin interventions led to a significantly enhanced formation of short-chain fatty acids, and blue lupin fibre to a decrease in faecal pH compared to run-in (0.27 units, P < 0.01). Further, blue lupin increased primary bile acids-excretion (P = 0.02). All legume fibres reduced faecal concentrations of total and secondary bile acids (blue lupin: 16%; white lupin: 24%; soya: 16%). Blood lipids were not influenced by any intervention. No serious adverse effects were observed.ConclusionsThe tested fibre preparations do not affect lipid metabolism through bile acid-binding in normocholesterolaemic subjects. However, particularly blue lupin kernel fibre improve colonic function and have beneficial effects on putative risk factors for colorectal cancer such as faecal mass, transit time, SCFA, faecal pH, and secondary bile acid concentration. Therefore, enhancing dietary fibre intake through blue lupin up to about 50 g/d can be recommended.Trial registrationNCT01036308

Diet, microbiota, and microbial metabolites in colon cancer risk in rural Africans and African Americans

Epidemiologic studies have suggested that most cases of sporadic colon cancer can be attributed to diet. The recognition that colonic microbiota have a major influence on colonic health suggests that they might mediate colonic carcinogenesis. To examine the hypothesis that the influence of diet on colon cancer risk is mediated by the microbiota through their metabolites, we measured differences in colonic microbes and their metabolites in African Americans with a high risk and in rural native Africans with a low risk of colon cancer. Fresh fecal samples were collected from 12 healthy African Americans aged 50-65 y and from 12 age- and sex-matched native Africans. Microbiomes were analyzed with 16S ribosomal RNA gene pyrosequencing together with quantitative polymerase chain reaction of the major fermentative, butyrate-producing, and bile acid-deconjugating bacteria. Fecal short-chain fatty acids were measured by gas chromatography and bile acids by liquid chromatography-mass spectrometry. Microbial composition was fundamentally different, with a predominance of Prevotella in native Africans (enterotype 2) and of Bacteroides in African Americans (enterotype 1). Total bacteria and major butyrate-producing groups were significantly more abundant in fecal samples from native Africans. Microbial genes encoding for secondary bile acid production were more abundant in African Americans, whereas those encoding for methanogenesis and hydrogen sulfide production were higher in native Africans. Fecal secondary bile acid concentrations were higher in African Americans, whereas short-chain fatty acids were higher in native Africans. Our results support the hypothesis that colon cancer risk is influenced by the balance between microbial production of health-promoting metabolites such as butyrate and potentially carcinogenic metabolites such as secondary bile acids.

Comparative Effects of Cellulose and Soluble Fibers (Pectin, Konjac Glucomannan, Inulin) on Fecal Water Toxicity toward Caco-2 Cells, Fecal Bacteria Enzymes, Bile Acid, and Short-Chain Fatty Acids

The aim of this study was to compare the effects of cellulose and three soluble dietary fibers, pectin, konjac glucomannan (KGM), and inulin, on the cytotoxicity and DNA damage of fecal water-treated Caco-2 cells, a human colon adenocarcinoma cell line, and to investigate the fecal components that potentially modulate the fecal toxicity, that is, bacterial enzymes, bile acids, and short-chain fatty acids. Six-week-old BALB/cJ mice were randomly allocated to consume an AIN-93 diet that contained no dietary fiber (fiber-free) or 5% (w/w) cellulose, pectin, KGM, and inulin for 3 weeks. Feces were collected during days 18-21. Fecal waters were co-incubated with Caco-2 cells to determine the cytotoxicity and DNA damage. In addition, the fecal bacterial enzymes, bile acids, and short-chain fatty acids were determined. Results indicated that all fiber diets similarly increased the survival rate (%) of fecal water-treated Caco-2 cells as compared with the fiber-free diet. The inhibition of fecal water-induced DNA damage in Caco-2 cells was greater for the pectin and inulin diets than for the cellulose and KGM diets. In contrast, cellulose exerted the greatest inhibitory effect on the fecal β-glucuronidase activity. Cellulose and all soluble dietary fibers reduced the secondary bile acid concentrations in the fecal water, but only soluble fibers increased the fecal concentrations of short-chain fatty acids, as compared with no fiber. Therefore, this study suggests that all dietary fibers substantially reduced the fecal water toxicity, which is associated with decreased secondary bile acid levels by all fibers, reduced fecal β-glucuronidase activity by cellulose, and increased short-chain fatty acid levels by soluble dietary fibers.

Physiological Effects of Extraction Juices from Apple, Grape, and Red Beet Pomaces in Rats

In comparison to classical fruit juice processing, polyphenols and dietary fiber can be extracted from pomace by means of pectinases and cellulases. In the present study, rats were fed with such produced extraction juices from apples, grapes, and red beets as drinking fluids instead of water for 4 weeks to evaluate their physiological effects. In all test groups, the intake of extraction juices was greater as compared to control (water intake), resulting in a higher urine excretion. In the apple and grape group, pH values in feces was lower than control. Administration of extraction juices from apples increased fecal counts of Lactobacillus and Bifidobacterium. More acetate and total short-chain fatty acids appeared in intestinal contents of the apple and red beet group. Furthermore, the intestinal contents of test groups contained higher concentrations of primary bile acids, cholesterol, and cholesterol metabolites but lower concentrations of secondary bile acids. The total amount of steroids excreted by these groups was also greater than control. Quercetin and isorhamnetin appeared in urine of rats fed extraction juices from apples and grapes; in urine of the former group, phloretin was found also. Administration of the extraction juices, enriched in secondary plant metabolites and dietary fiber, resulted in beneficial nutritional effects in rats.

T04-P-044 The impact of plasma homocysteine level on neointimal proliferation in patients after PCI with reduced barotrauma

Changes in the microbiota composition have been implicated in the development of obesity and type 2 diabetes. However, not much is known on the involvement of gut microbiota in lipid and cholesterol metabolism. In addition, the gut microbiota might also be a potential source of plasma oxyphytosterol and oxycholesterol concentrations (oxidation products of plant sterols and cholesterol). Therefore, the aim of this study was to modulate the gut microbiota by antibiotic therapy to investigate effects on parameters reflecting cholesterol metabolism and oxyphytosterol concentrations.A randomized, double blind, placebo-controlled trial was performed in which 55 obese, pre-diabetic men received oral amoxicillin (broad-spectrum antibiotic), vancomycin (antibiotic directed against Gram-positive bacteria) or placebo (microcrystalline cellulose) capsules for 7 days (1500 mg/day). Plasma lipid and lipoprotein, non-cholesterol sterol, bile acid and oxy(phyto)sterol concentrations were determined at baseline and after 1-week intervention.Plasma secondary bile acids correlated negatively with cholestanol (marker for cholesterol absorption, r = −0.367; P < 0.05) and positively with lathosterol concentrations (marker for cholesterol synthesis, r = 0.430; P < 0.05). Fasting plasma secondary bile acid concentrations were reduced after vancomycin treatment as compared to placebo treatment (-0.24 ± 0.22 μmol/L vs. −0.08 ± 0.29 μmol/L; P < 0.01). Vancomycin and amoxicillin treatment did not affect markers for cholesterol metabolism, plasma TAG, total cholesterol, LDL-C or HDL-C concentrations as compared to placebo. In addition, both antibiotic treatments did not affect individual isoforms or total plasma oxyphytosterol or oxycholesterol concentrations.Despite strong correlations between plasma bile acid concentrations and cholesterol metabolism (synthesis and absorption), amoxicillin and vancomycin treatment for 7 days did not affect plasma lipid and lipoprotein, plasma non-cholesterol sterol and oxy(phyto)sterol concentrations in obese, pre-diabetic men.

Regulation of CYP3A4 by the bile acid receptor FXR: evidence for functional binding sites in the CYP3A4 gene.

CYP3A4, the most abundant cytochrome P450 in human liver, is responsible for the metabolism of numerous xenobiotics and endobiotics. CYP3A4 expression is highly variable and is induced by numerous compounds of exogenous and endogenous origin, including elevated concentrations of secondary bile acids via the pregnane X receptor (PXR). We show that physiological concentrations of the primary bile acid chenodeoxycholic acid regulate the expression of CYP3A4 via the bile acid receptor FXR. Experiments performed in vitro in different cell culture systems, gel-mobility shift assays and experiments performed in vivo in transgenic mice lacking FXR or PXR and treated with the synthetic FXR agonist GW4064 were undertaken to study the implication of FXR in the regulation of CYP3A. Our data provide evidence for the presence of two functional FXR recognition sites located in a 345-bp element within the 5'-flanking region of CYP3A4. Mutational analysis of these sites and experiments in transgenic mice lacking FXR or PXR support the relevance of FXR activation for CYP3A regulation. Thus, whereas elevated concentrations of precursors of bile acids and secondary bile acids induce CYP3A via PXR, primary bile acids can modulate the expression of CYP3A via FXR. These findings may explain elevated CYP3A expression in cholestasis and part of the variability of drug responsiveness and toxicity between individuals.

Increased GADD gene expression in human colon epithelial cells exposed to deoxycholate.

The colonic epithelium is often exposed to high concentrations of secondary bile acids, which stresses the epithelial cells, leading potentially to activation of stress-response genes. To examine this possibility in vitro, the purpose of this study was to determine if expression of certain growth arrest and DNA damage-inducible genes (GADD) is upregulated in human colonic epithelial cells exposed to deoxycholate (DOC). DNA macroarray screening of a small cluster of stress/apoptosis-related genes in DOC-treated HCT-116 colonocytes revealed clearly higher expression of only GADD45, which was confirmed by gene-specific relative RT-PCR analysis. Subsequently, it was found that DOC also increased GADD34 mRNA expression. However, mRNA expression of GADD153 was increased most markedly in DOC-treated HCT-116 colonocytes, which express wild-type p53. However, the upregulation of GADD34, GADD45, and GADD153 mRNA expression apparently did not require p53, based on the finding that DOC increased expression of all three GADD genes in HCT-15 colonocytes, which express mutant p53. In further studying GADD153 in particular, the effect of DOC on GADD153 mRNA was prevented by actinomycin-D (Act-D), but not by antioxidants or MAPK inhibitors. DOC also caused GADD153 protein to be expressed in close parallel with increased GADD153 mRNA expression. Induction of GADD153 protein by DOC was prevented by either anisomycin or cycloheximide. These findings suggest that DOC-induced upregulation of GADD153 mRNA expression occurred at the level of transcription without involving reactive oxygen species and MAPK signaling, and that the expression of GADD153 protein was due also to translation of pre-existing, and not just newly synthesized, mRNA.

Dietary fiber–rich colloids from apple pomace extraction juices do not affect food intake and blood serum lipid levels, but enhance fecal excretion of steroids in rats

The aim of this study was to investigate the effects of colloids isolated from apple pomace extraction juices (so-called B-juices) produced by enzymatic liquefaction on food intake, levels of blood serum lipids, and fecal excretion of bile acids (BA) and neutral sterols (NS) in vivo. Ten male Wistar rats per group were fed diets containing either no apple dietary fiber (DF) (control), a 5% supplementation with juice colloids, or an alcohol-insoluble substance (AIS) from apples for 6 weeks. Apple DF in diets led to lower weight gain in rats fed with B-juice colloids ( P< 0.05). For these rats, food intake was not affected but was highest with feeding AIS (10% more than control) to cover energy requirements. The supplementation of diet with apple DF from extraction juices or AIS had minor effects on blood serum lipids. In rats fed either juice colloids or AIS, up to 30% (5.31 μmol/g dry weight) and 88% (7.69 μmol/g dry weight) more primary BA were excreted in feces, respectively, as compared to that in the control group (4.10 μmol/g dry weight) ( P < 0.05). In cecal contents, a 15% (juice colloids) to 37% (AIS) increase in primary BA was found. In contrast, concentrations of secondary BA were lower in feces of test groups ( P < 0.05). Excretion of total BA and NS was higher in rats fed apple DF ( P < 0.05). Our study is the first to prove that there are beneficial physiologic effects of apple DF isolated from pomace extraction juices produced by enzymatic liquefaction. These results may help to develop such innovative juice products that are rich in DF of fruit origin for diminishing the lack of DF intake.

Deoxycholate induces DNA damage and apoptosis in human colon epithelial cells expressing either mutant or wild-type p53

Diets rich in fat result in higher concentrations of secondary bile acids or their salts in the colon, which may adversely affect cells of the colonic epithelium. Because secondary bile acids are thought to be genotoxic, exposing colon epithelial cells to secondary bile acids may induce DNA damage that might lead to apoptosis. The requirement for the p53 tumor suppressor gene in such events is unknown. In particular, the effects of secondary bile acids on colon epithelial cells having different p53 tumor suppressor gene status have not been examined. Therefore, HCT-116 and HCT-15 human colon adenocarcinoma cells, which express the wild-type and mutant p53 genes, respectively, were exposed to physiological concentrations of deoxycholate. The cells were then analyzed for evidence of DNA damage and apoptosis. After 2 h of incubation with 300 μM deoxycholate, both cell lines had greater levels of single-strand breaks in DNA as assessed by the comet assay. After 6 h of exposure to deoxycholate, HCT-116 and HCT-15 cells showed morphological signs of apoptosis, i.e., membrane blebbing and the presence of apoptotic bodies. Chromatin condensation and fragmentation were also seen after staining DNA with 4′,6-diamidino-2-phenylindole. Other apoptotic assays revealed greater binding of annexin V-fluorescein isothiocyanate, as well as greater post-enzymatic labeling with dUTP-fluorescein isothiocyanate, by both cell lines exposed to deoxycholate. These data suggest that deoxycholate caused DNA damage in colon epithelial cells that was sufficient to trigger apoptosis in a p53-independent manner.

Rye Bread Improves Bowel Function and Decreases the Concentrations of Some Compounds That Are Putative Colon Cancer Risk Markers in Middle-Aged Women and Men

Cereal fiber may reduce the risk of colorectal cancer by diluting colonic contents due to increased fecal output, by accelerating intestinal transit, by increasing fecal frequency and by altering bacterial metabolism. The effects of whole-meal rye bread on some putative colon cancer risk markers were investigated in 17 healthy Finnish subjects using a randomized crossover trial with two 4-wk bread consumption periods and a 4-wk washout period between the bread periods. White wheat bread was used as a control. Test breads covered a minimum of 20% of the daily energy intake (range, 4330-14, 033 kJ/d). Intestinal transit time, stool weight, fecal bacterial enzyme activities and short-chain fatty acid, ammonia, diacylglycerol (DAG) and bile acid concentrations in feces (expressed per gram wet feces) were measured. Whole-meal rye bread significantly increased fecal output and fecal frequency and shortened mean intestinal transit time compared with wheat bread in both women and men. Activities of beta-glucuronidase and beta-glucosidase (expressed per gram wet feces) were significantly lower in men and urease activity significantly higher in women during the rye bread period (RBP). Fecal butyrate concentration was higher during the RBP in men. Fecal ammonia and DAG concentrations did not differ between bread periods. Fecal total and secondary bile acid concentrations were significantly lower during RBP in both women and men. This study shows that whole-meal rye bread significantly improves bowel function in healthy adults and may decrease the concentration of some compounds that are putative colon cancer risk markers.

Safety Evaluation of Phytosterol Esters. Part 4. Faecal Concentrations of Bile Acids and Neutral Sterols in Healthy Normolipidaemic Volunteers Consuming a Controlled Diet either with or without a Phytosterol Ester-enriched Margarine

A study was conducted in 12 healthy males and 12 females (mean age 36 years) to assess the impact of a margarine enriched with phytosterol esters on faecal concentrations of bile acids and sterols. During the run-in period, volunteers consumed 40 g of a control margarine for 21 consecutive days if male, and for 28 days if female. Half of the volunteers were then randomly allocated to consume the control margarine for another 21 or 28 days, respectively. The remaining subjects consumed 40 g of a margarine containing 8.6 g vegetable oil phytosterol (46% (w/w) β-sitosterol, 26% campesterol, 20% stigmasterol). Throughout the total study subjects consumed the same diet adjusted for individual energy requirements. The phytosterol ester-enriched spread significantly enhanced faecal neutral sterol concentrations from about 40 mg/g to 190 mg/g dry weight faeces. Faecal neutral sterol metabolites increased from about 30 mg/g to about 50 mg/g. The major parent sterols excreted were cholesterol, sitosterol, campesterol and stigmasterol. Sitosterol, campesterol and stigmasterol comprised 28%, 15% and 12% of the total faecal neutral sterols, reflecting the composition of the sterol enriched margarine. The major sterol metabolites excreted were metabolites formed by, predominantly, oxidation at the 3-position and metabolites saturated at the 5,6 position in a β-configuration. Faecal secondary bile acid concentration was reduced by vegetable oil sterols from 7.6 mg/g dry faeces to 6.0 mg/g. Consumption of vegetable oil phytosterols slightly but significantly increased the faecal concentration of 4-cholesten-3-one. However, 4-cholesten-3-one concentration remained very low (<2 mg/g) and in line with values reported in the literature for subjects fed high or low fat diets. No sterol oxides could be detected in the faeces. We conclude that in healthy adult males and females a high intake of vegetable oil phytosterol esters does increase the amount of neutral sterols in the faeces, as expected, but does not result in the increased formation of bile acids or sterol metabolites.

A Diet Containing Chickpeas and Wheat Offers Less Protection against Colon Tumors than a Casein and Wheat Diet in Dimethylhydrazine-Treated Rats

We examined the influence of extruded chickpeas and wheat relative to casein and wheat in a dimethylhydrazine (DMH)-induced colon tumor study in male Sprague-Dawley rats. The three diets, based on a modified AIN76 rodent diet with fat present at 10 g/100 g dry matter (DM), were as follows: casein with wheat starch (Cas/S) as control, casein with wheat (Cas/W) and chickpeas with wheat (CP/W). All diets were fed from 5 wk of age throughout the 28-wk study. At 28 wk, there was a significantly lower incidence of large intestinal tumors in rats fed Cas/W relative to those fed CP/W ( 11 vs. 56%, chi-square test, P = 0.018). The colonic tumor burden (tumors/tumor-bearing animal) was not different in Cas/W-fed and CP/W-fed rats (1 vs. 1.7), but the tumor mass index was significantly lower in the former group (0.22 vs. 1.21, P = 0.026). Rats fed the CP/W diet had significantly lower plasma cholesterol concentration (P< 0.01) than rats fed the other two diets. The cecal contents of rats fed the CP/W diet had significantly greater relative weights (46%, P < 0.05) than those of the Cas/W-fed rats; this was associated with higher concentrations of all short-chain fatty acids. Fecal analyses showed significantly (P < 0.05) higher concentrations of total fat (54%), total steroids (83%) and secondary bile acids (179%) in the CP/W-fed rats relative those fed Cas/W. There were higher concentrations of nitrogen in the feces of CP/W rats relative to the Cas/W-fed rats (84%, P < 0.05), associated with greater fecal weights (67%, P < 0.05). Although wheat and its fibers have been shown to be protective against DMH-induced cancers in rats, this was not the case in this study in which chickpeas (45 g/100 g diet) provided the protein and were an important source of soluble fiber. Elevated fat, secondary bile acid concentrations and/or nitrogenous compounds could be responsible for the increased colon tumorigenesis seen and may reflect a legume effect.

Effect of enteral feeding on intestinal epithelial proliferation and fecal bile acid profiles in the rat.

The preservation of colonic mucosal mass requires the presence of feces; thus, colonic hypoplasia develops with enteral feeding. Fermentable soy fiber may maintain fecal bulk and prevent mucosal atrophy. We therefore compared the effects of Enrich (containing soy fiber) with Ensure (fiber-free) on mucosal mass, fecal bile acids, and cell proliferation in the rat. Sprague-Dawley rats (n = 20) were randomized into groups receiving either standard rat food, Enrich, or Ensure. After 4 weeks, rats were weighed and killed at intervals after administration of vincristine sulfate (1 mg/kg intraperitoneally), which was given to induce metaphase arrest within the intestinal crypts and to allow calculation of crypt cell production rate. Fecal free bile acids and neutral steroids were analyzed using gas-liquid chromatography and gas-liquid chromatography mass spectrometry. Nucleic acid concentrations in the small and large intestine and crypt cell production rate in the small bowel were unaffected by diet. In the proximal large bowel, both enteral diets produced a marked reduction in crypt cell production rate to values only 5% to 7.5% of control values (p < .001). In the midcolon of rats fed Ensure, there was a similar reduction, but this hypoplastic effect was completely prevented by Enrich. Without an elevation in the lithocolic acid to deoxycholic acid ratio, secondary bile acid concentrations were increased in rats receiving Enrich (p < .05 to .01) but not in those receiving Ensure. Thus, fermentable soy fiber diets may prevent the midcolonic mucosal hypoplasia induced by enteral feeding.

ARE SECONDARY BILE ACIDS IN CHOLEDOCHAL CYSTS IMPORTANT AS A RISK FACTOR IN BlLlARY TRACT CARCINOMA?

Since secondary bile acids have mutagenic potency and choledochal cyst patients with or without cyst-enterostomy have a high risk of developing bile duct carcinoma, we examined the hypothesis that secondary bile acids are elevated in the choledochal cyst and possibly cause biliary tract carcinoma. Eleven choledochal cyst patients with or without previous cyst-enterostomy or biliary tract carcinoma, seven patients with biliary tract carcinoma not associated with choledochal cyst and five patients with cholecystolithiasis were examined. Samples were directly needle aspirated from the cyst or by cannulating a tube into the common bile duct through the cystic duct. The concentrations of each bile acid in these samples were quantified by gas-liquid chromatography and compared. Neither the relative composition nor the absolute concentration of secondary bile acids (deoxycholic acid, lithocholic acid) elevated in patients with choledochal cyst compared with the values in the non-choledochal cyst patients with or without biliary tract carcinoma. The presence of biliary tract carcinoma or previous cyst-enterostomy did not affect the concentrations of secondary bile acids. The results suggest that the factor other than secondary bile acids can be primarily responsible for the high risk of bile duct carcinoma in patients with choledochal cyst.

Effect of diets low and high in refined sugars on gut transit, bile acid metabolism, and bacterial fermentation.

Increasing consumption of refined sugar has been implicated in many gastrointestinal disorders on epidemiological grounds. Nine volunteers agreed to participate in a study comparing the effects of a diet containing 165 g refined sugar/day with a diet of only 60 g/day on gut transit, bile acid metabolism, and fermentative activity of the intestinal flora. The wet and dry weight, pH, and water content of the stools were similar on the two diets. On the high sugar diet mouth-to-anus transit time was significantly prolonged, despite a shortened mouth-to-caecum transit time. The faecal concentration of total bile acids and the faecal concentration of secondary bile acids increased significantly. Diet affected neither the serum bile acid pattern nor the concentration. Breath hydrogen tests showed significantly enhanced H2 production on the high sugar diet. We conclude that the quantity of refined sugar in the diet can significantly influence gut function and the composition of bowel contents.