Association between low colonic short-chain fatty acids and high bile acids in high colon cancer risk populations.

We propose that the influence of diet on colon cancer risk is mediated by the microbiota. To investigate how dietary fat influences risk, we compared the colonic contents of 12 adult high-risk African Americans (AAs) and 10 Caucasian Americans (CAs) who consumed a high-fat diet (123 ± 11 g/d and 129 ± 17 g/d, respectively) to 13 native Africans (NAs) who subsisted on a low-fat (38 ± 3.0 g/d) diet, all aged 50-60 yr. The colonic bile acids were measured by LC-MS and the short-chain fatty acids (SCFAs) by GC. The chief secondary colonic bile acids, deoxycholic acid and lithocholic acid, were correlated with fat intake and similar between AAs and CAs, but 3-4 times higher than in AAs (p < 0.05). The major SCFAs were lower in AAs (p < 0.001) and CAs (p < 0.001) compared to AAs, but conversely, the branched chain fatty acids (BFCA) were higher. Our results suggest that the higher risk of colon cancer in Americans may be partly explained by their high-fat and high-protein, low complex carbohydrate diet, which produces colonic residues that promote microbes to produce potentially carcinogenic secondary bile acids and less antineoplastic SCFAs. The role of BCFA in colonic carcinogenesis deserves further study.

Products of the colonic microbiota mediate the effects of diet on colon cancer risk.

It is estimated that most colon cancers can be attributed to dietary causes. We have hypothesized that diet influences the health of the colonic mucosa through interaction with the microbiota and that it is the milieu interior that regulates mucosal proliferation and therefore cancer risk. To validate this further, we compared colonic contents from healthy 50- to 65-y-old people from populations with high and low risk, specifically low risk Native Africans (cancer incidence <1:100,000; n = 17), high risk African Americans (risk 65:100,000; n = 17), and Caucasian Americans (risk 50:100,000; n = 18). Americans typically consume a high-animal protein and -fat diet, whereas Africans consume a staple diet of maize meal, rich in resistant starch and low in animal products. Following overnight fasting, rapid colonic evacuation was performed with 2 L polyethylene glycol. Total colonic evacuants were analyzed for SCFA, vitamins, nitrogen, and minerals. Total SCFA and butyrate were significantly higher in Native Africans than in both American groups. Colonic folate and biotin content, measured by Lactobacillus rhamnoses and Lactobacillus plantarum ATCC 8014 bioassay, respectively, exceeded normal daily dietary intakes. Compared with Africans, calcium and iron contents were significantly higher in Caucasian Americans and zinc content was significantly higher in African Americans, but nitrogen content did not differ among the 3 groups. In conclusion, the results support our hypothesis that the microbiota mediates the effect diet has on colon cancer risk by their generation of butyrate, folate, and biotin, molecules known to play a key role in the regulation of epithelial proliferation.

Identification of enzymes involved in the metabolism of 17alpha-hydroxyprogesterone caproate: an effective agent for prevention of preterm birth.

Preterm delivery, that is delivery before 37 completed weeks of gestation, is the major determinant of neonatal morbidity and mortality. Until recently, no effective therapies for prevention of preterm birth existed. In a recent multicentered trial, 17alpha-hydroxyprogesterone caproate (17-OHPC) reduced the rate of preterm birth by 33% in a group of high-risk women. Limited pharmacologic data exist for this drug. The recommended dose is empiric; the metabolic pathways are not well defined especially in pregnant women; and the fetal exposure has not been quantified. To define the metabolic pathways of 17-OHPC we used human liver microsomes (HLMs), fresh human hepatocytes (FHHs), and expressed enzymes. HLMs in the presence of NADPH generated three metabolites, whereas two major metabolites were observed with FHHs. Metabolism of 17-OHPC was significantly inhibited by the CYP3A4 inhibitors ketoconazole and troleandomycin in HLM and FHH. Metabolism of 17-OHPC was significantly greater in FHH treated with the CYP3A inducers, rifampin and phenobarbital. Furthermore, studies with expressed enzymes showed that 17-OHPC is metabolized exclusively by CYP3A4 and CYP3A5. The caproic acid ester was intact in the major metabolites generated, indicating that 17-OHPC is not converted to the primary progesterone metabolite, 17alpha-hydroxyprogesterone. In summary, this study shows that 17-OHPC is metabolized by CYP3A. Because CYP3A is involved in the oxidative metabolism of numerous commonly used drugs, 17-OHPC may be involved in clinically relevant metabolic drug interactions with coadministered CYP3A inhibitors or inducers.

Functional evolution of the vitamin D and pregnane X receptors.

BACKGROUND: The vitamin D receptor (VDR) and pregnane X receptor (PXR) are nuclear hormone receptors of the NR1I subfamily that show contrasting patterns of cross-species variation. VDR and PXR are thought to have arisen from duplication of an ancestral gene, evident now as a single gene in the genome of the chordate invertebrate Ciona intestinalis (sea squirt). VDR genes have been detected in a wide range of vertebrates including jawless fish. To date, PXR genes have not been found in cartilaginous fish. In this study, the ligand selectivities of VDRs were compared in detail across a range of vertebrate species and compared with those of the Ciona VDR/PXR. In addition, several assays were used to search for evidence of PXR-mediated hepatic effects in three model non-mammalian species: sea lamprey (Petromyzon marinus), zebrafish (Danio rerio), and African clawed frog (Xenopus laevis). RESULTS: Human, mouse, frog, zebrafish, and lamprey VDRs were found to have similar ligand selectivities for vitamin D derivatives. In contrast, using cultured primary hepatocytes, only zebrafish showed evidence of PXR-mediated induction of enzyme expression, with increases in testosterone 6beta-hydroxylation activity (a measure of cytochrome P450 3A activity in other species) and flurbiprofen 4-hydroxylation activity (measure of cytochrome P450 2C activity) following exposure to known PXR activators. A separate assay in vivo using zebrafish demonstrated increased hepatic transcription of another PXR target, multidrug resistance gene (ABCB5), following injection of the major zebrafish bile salt, 5alpha-cyprinol 27-sulfate. The PXR target function, testosterone hydroxylation, was detected in frog and sea lamprey primary hepatocytes, but was not inducible in these two species by a wide range of PXR activators in other animals. Analysis of the sea lamprey draft genome also did not show evidence of a PXR gene. CONCLUSION: Our results show tight conservation of ligand selectivity of VDRs across vertebrate species from Agnatha to mammals. Using a functional approach, we demonstrate classic PXR-mediated effects in zebrafish, but not in sea lamprey or African clawed frog liver cells. Using a genomic approach, we failed to find evidence of a PXR gene in lamprey, suggesting that VDR may be the original NR1I gene.

Pancreatic and Intestinal Function Post Roux-en-Y Gastric Bypass Surgery for Obesity.

OBJECTIVES: Despite the fact that the most effective treatment for morbid obesity today is gastric bypass surgery, some patients develop life-threatening nutritional complications associated with their weight loss. METHODS: Here we examine the influence of the altered anatomy and digestive physiology on pancreatic secretion and fat absorption. Thirteen post Roux-en-Y gastric bypass (RYGB) patients who had lost >100 lbs in the first year following surgery and who gave variable histories of gastrointestinal (GI) dysfunction, were selected for study. Food-stimulated pancreatic enzyme secretion and GI hormone responses were measured during 2 h perfusions of the Roux limb with a standard polymeric liquid formula diet and polyethylene glycol marker, with collections of secretions from the common channel distal to the anastomosis and blood testing. Fat absorption was then measured during a 72 h balance study when a normal diet was given containing ~100 g fat/d. RESULTS: Result showed that all patients had some fat malabsorption, but eight had coefficients of fat absorption <80%, indicative of steatorrhea. This was associated with significantly lower feed-stimulated secretion rates of trypsin, amylase, and lipase, and higher plasma peptide-YY concentrations compared with healthy controls. Five steatorrhea patients were subsequently treated with low quantities of pancreatic enzyme supplements for 3 months, and then retested. The supplements were well tolerated, and fat absorption improved in four of five patients accompanied by an increase in lipase secretion, but body weight increased in only three. Postprandial breath hydrogen concentrations were elevated with some improvement following enzyme supplementation suggesting persistent bacterial overgrowth and decreased colonic fermentation. CONCLUSIONS: Our investigations revealed a wide spectrum of gastrointestinal abnormalities, including fat malabsorption, impaired food stimulated pancreatic secretion, ileal brake stimulation, and bacterial overgrowth, in patients following RYGB which could be attributed to the breakdown of the normally highly orchestrated digestive anatomy and physiology.

Fat, fibre and cancer risk in African Americans and rural Africans.

Rates of colon cancer are much higher in African Americans (65:100,000) than in rural South Africans (<5:100,000). The higher rates are associated with higher animal protein and fat, and lower fibre consumption, higher colonic secondary bile acids, lower colonic short-chain fatty acid quantities and higher mucosal proliferative biomarkers of cancer risk in otherwise healthy middle-aged volunteers. Here we investigate further the role of fat and fibre in this association. We performed 2-week food exchanges in subjects from the same populations, where African Americans were fed a high-fibre, low-fat African-style diet and rural Africans a high-fat, low-fibre western-style diet, under close supervision. In comparison with their usual diets, the food changes resulted in remarkable reciprocal changes in mucosal biomarkers of cancer risk and in aspects of the microbiota and metabolome known to affect cancer risk, best illustrated by increased saccharolytic fermentation and butyrogenesis, and suppressed secondary bile acid synthesis in the African Americans.

Morphology and function of canine small intestinal autografts: with particular interest in the influence of ex vivo graft irradiation.

The intestinal transplantation procedure obligates an early functional deficit in intestinal grafts. Graft irradiation has been used to modulate post-transplant immune reactions; however, irradiation may cause further deterioration of the function of the transplanted intestine. Using the canine model, we investigated the influence of the transplant procedures, with and without ex vivo graft irradiation, on early intestinal graft function and histopathology. Outbred hound dogs underwent autointestinal transplantation with (n=8) or without (n=5) 7.5 Gy ex vivo graft irradiation. Mucosal cytochrome P450 and P-glycoprotein, routine immunohistopathology, and intestinal absorptive function were studied. Weight gain was slow after surgery, but was comparable in the irradiated and nonirradiated groups. During the early post-transplant period, both groups showed defects in intestinal absorption, associated with decreased cytochrome P450 3A4 activity and reduced P-glycoprotein expression, regardless of graft irradiation. These changes returned to normal in both groups by day 28. Histopathologically, epithelial apoptosis showed a slight increase 1 hour after transplantation; however, there was no evidence of histopathologic abnormalities including arterial changes associated with irradiation. In addition, the frequency of T and B lymphocytes in the lamina propria were not significantly influenced by the transplant surgery or ex vivo irradiation. Thus, early after transplantation, intestinal function was impaired and effectiveness of orally administered immunosuppressive drugs was significantly altered. Graft irradiation did not induce further defects in intestinal function or cause histopathologic abnormalities.

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

BACKGROUND: 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. OBJECTIVE: 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. DESIGN: 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. RESULTS: 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. CONCLUSION: 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.

Hydrogenotrophic microbiota distinguish native Africans from African and European Americans.

Reduced susceptibility to sporadic colorectal cancer in native Africans (NA) is correlated with low consumption of animal products and greater microbial production of colonic methane. In this context, two hydrogenotrophic microbial groups are of interest, methanogenic Archaea (MA) utilizing H2 to produce methane and sulfate-reducing bacteria (SRB) generating hydrogen sulfide, which has been linked with chronic inflammatory disorders of the colon. In the present study, stool samples from NA, consuming a diet high in resistant starch and low in animal products, and from African Americans (AA) and European Americans (EA), both consuming a typical Western diet, were examined for genetic diversity and structure of Archaea, MA and SRB communities. In general, a greater proportion of NA than AA and EA harboured the full range of targeted hydrogenotrophic groups. Terminal restriction fragment length polymorphism analysis of 16S rRNA genes and specific functional genes, combined with multivariate statistical analyses, revealed that NA harboured more diverse and different Archaea and MA populations than AA and EA. Also, NA harboured significantly distinct SRB populations compared with AA and EA. Taken together, these data are consistent with diet selecting for distinct hydrogenotrophic microbiota.

Effect of fiber supplementation on the microbiota in critically ill patients.

AIM: To determine tolerance to fiber supplementation of semi-elemental tube feeds in critically ill patients and measure its effect on colonic microbiota and fermentation. METHODS: Thirteen intensive care unit patients receiving jejunal feeding with a semi-elemental diet for predominantly necrotizing pancreatitis were studied. The study was divided into 2 parts: first, short-term (3-9 d) clinical tolerance and colonic fermentation as assessed by fecal short chain fatty acid (SCFA) concentrations and breath hydrogen and methane was measured in response to progressive fiber supplementation increasing from 4 g tid up to normal requirement levels of 8 g tid; second, 4 patients with diarrhea were studied for 2-5 wk with maximal supplementation to additionally assess its influence on fecal microbiota quantitated by quantitative polymerase chain reaction (qPCR) of microbial 16S rRNA genes and Human Intestinal Tract Chip (HITChip) microarray analysis. Nearly all patients were receiving antibiotics (10/13) and acid suppressants (11/13) at some stage during the studies. RESULTS: In group 1, tolerance to progressive fiber supplementation was good with breath hydrogen and methane evidence (P = 0.008 and P < 0.0001, respectively) of increased fermentation with no exacerbation of abdominal symptoms and resolution of diarrhea in 2 of 4 patients. In group 2 before supplementation, fecal microbiota mass and their metabolites, SCFA, were dramatically lower in patients compared to healthy volunteers. From qPCR and HITChip analyses we calculated that there was a 97% reduction in the predominant potential butyrate producers and starch degraders. Following 2-5 wk of fiber supplementation there was a significant increase in fecal SCFA (acetate 28.4 ± 4.1 µmol/g to 42.5 ± 3.1 µmol/g dry weight, P = 0.01; propionate 1.6 ± 0.5 vs 6.22 ± 1.1, P = 0.006 and butyrate 2.5 ± 0.6 vs 5.9 ± 1.1, P = 0.04) and microbial counts of specific butyrate producers, with resolution of diarrhea in 3 of 4 patients. CONCLUSION: Conventional management of critically ill patients, which includes the use of elemental diets and broad-spectrum antibiotics, was associated with gross suppression of the colonic microbiota and their production of essential colonic fuels, i.e., SCFA. Our investigations show that fiber supplementation of the feeds has the potential to improve microbiota mass and function, thereby reducing the risks of diarrhea due to dysbiosis.

Activity and expression of various isoforms of uridine diphosphate glucuronosyltransferase are differentially regulated during hepatic regeneration in rats.

PURPOSE: Glucuronidation pathway is very important in the detoxification of endogenous and exogenous compounds. The objective of this study was to evaluate the activity and expression of various hepatic uridine diphosphate glucuronosyltransferases (UGTs) in rats at various time points after initiation of hepatic regeneration by partial hepatectomy (PHx). METHODS: The mRNA expression of various UGTs was evaluated using real-time polymerase chain reaction (real-time PCR) with specific primers. The in vitro activity of UGTs was evaluated using different substrates such as estradiol (UGT1A1), acetaminophen (UGT1A6/7), morphine (UGT2B1), testosterone (UGT2B1/3/6), androsterone (UGT2B2), and (-)-borneol (UGT2B12). RESULTS: Whereas the activity and mRNA expression of UGT1A1, UGT2B1, UGT2B1/3/6, UGT2B2, and UGT2B12 were lower, the activity and mRNA expression of UGT1A6/7 were preserved during hepatic regeneration. The mRNA expression of UGT2B8 was down-regulated, whereas the mRNA expression of UGT1A5 and UGT1A8 was not altered by PHx. The mRNA expression of UGT1A2 and UGT1A3 was increased during hepatic regeneration. CONCLUSION: UGT-mediated drug-metabolizing ability of the liver was altered differentially in the regenerating rat liver. Individualized dosing regimen for different UGT substrates may be needed when using such substrates of these enzymes in patients with a regenerating liver, especially during the early postoperative period. However, the glucuronide conjugating capacity of the liver in the donor of a living donor liver transplantation is expected to completely return to normal with time after surgery.

Pharmacokinetics of tacrolimus and mycophenolic acid are altered, but recover at different times during hepatic regeneration in rats.

Hepatic regeneration is very critical to the success of living donor liver transplantation, which allows a reduced size liver to grow in size to accommodate the requirements of both the donor and the recipient. The objectives of this study were to evaluate 1) the hepatic metabolism of the two immunosuppressive drugs, tacrolimus and mycophenolic acid (MPA), and 2) the pharmacokinetics of tacrolimus and mycophenolic acid at various time points after initiation of hepatic regeneration by partial hepatectomy in rats. The hepatic intrinsic clearance of tacrolimus was decreased to 70% and 51% of the control level at the 24th h and the 6th day, respectively, but returned to normal level by day 14. The total body clearance of tacrolimus was reduced transiently but recovered completely by day 18. The hepatic intrinsic clearance of MPA was decreased to 52% and 51% of that in control rats at the 24th h and the 6th day, respectively, but recovered to normal level by day 14. The total body clearance of MPA was reduced at the 24th h but recovered by day 6. The magnitude of reduction in the clearance of tacrolimus and MPA was much smaller than what was predicted from in vitro data. The elimination clearance of MPA glucuronide was also impaired during hepatic regeneration but recovered to normal level with time. In conclusion, the pharmacokinetics of tacrolimus and mycophenolic acid were altered during hepatic regeneration but recovered completely at different rates over time. Caution must be exercised in extrapolating in vitro data to in vivo conditions during hepatic regeneration.