R.W. van der Meer

Saturated fat stimulates obesity and hepatic steatosis and affects gut microbiota composition by an enhanced overflow of dietary fat to the distal intestine

We studied the effect of dietary fat type, varying in polyunsaturated-to-saturated fatty acid ratios (P/S), on development of metabolic syndrome. C57Bl/6J mice were fed purified high-fat diets (45E% fat) containing palm oil (HF-PO; P/S 0.4), olive oil (HF-OO; P/S 1.1), or safflower oil (HF-SO; P/S 7.8) for 8 wk. A low-fat palm oil diet (LF-PO; 10E% fat) was used as a reference. Additionally, we analyzed diet-induced changes in gut microbiota composition and mucosal gene expression. The HF-PO diet induced a higher body weight gain and liver triglyceride content compared with the HF-OO, HF-SO, or LF-PO diet. In the intestine, the HF-PO diet reduced microbial diversity and increased the Firmicutes-to-Bacteroidetes ratio. Although this fits a typical obesity profile, our data clearly indicate that an overflow of the HF-PO diet to the distal intestine, rather than obesity itself, is the main trigger for these gut microbiota changes. A HF-PO diet-induced elevation of lipid metabolism-related genes in the distal small intestine confirmed the overflow of palm oil to the distal intestine. Some of these lipid metabolism-related genes were previously already associated with the metabolic syndrome. In conclusion, our data indicate that saturated fat (HF-PO) has a more stimulatory effect on weight gain and hepatic lipid accumulation than unsaturated fat (HF-OO and HF-SO). The overflow of fat to the distal intestine on the HF-PO diet induced changes in gut microbiota composition and mucosal gene expression. We speculate that both are directly or indirectly contributive to the saturated fat-induced development of obesity and hepatic steatosis.

Increasing the intestinal resistance of rats to the invasive pathogen Salmonella enteritidis: additive effects of dietary lactulose and calcium.

BACKGROUND AND AIMS: Lactulose fermentation by the intestinal microflora acidifies the gut contents, resulting in an increased resistance to colonisation by acid sensitive pathogens. The extent of fermentation should be controlled to prevent acid induced epithelial cell damage. Considering the buffering capacity of calcium phosphate and its intestinal cytoprotective effects, whether supplemental calcium phosphate adds to the increased resistance to intestinal infections by lactulose fermentations was studied. METHODS: In a strictly controlled experiment, rats were fed a purified low calcium control diet, a low calcium/lactulose diet, or a high calcium/lactulose diet, and subsequently infected orally with Salmonella enteritidis. RESULTS: Lactulose fermentation lowered the pH and increased the lactic acid concentration of the intestinal contents, which significantly reduced excretion of this pathogen in faeces; thus it improved the resistance to colonisation. This agreed with the high sensitivity of S enteritidis to lactic acid (main metabolite of lactulose fermentation) in vitro. Calcium phosphate decreased translocation of S enteritidis to the systemic circulation, an effect independent of lactulose. The unfavourable increased cytotoxicity of faecal water caused by lactulose fermentation was more than counteracted by supplemental calcium phosphate. Moreover, calcium phosphate stimulated lactulose fermentation, as judged by the reduced lactulose excretion in faeces and increased lactic acid, ammonia, and faecal nitrogen excretion. CONCLUSION: Extra calcium phosphate added to a lactulose diet improves the resistance to colonisation and translocation of S enteritidis. This is probably mediated by a calcium induced stimulation of lactulose fermentation by the intestinal microflora and reversion of the lactulose mediated increased luminal cytotoxicity, which reduces damage inflicted on the intestinal mucosa.

Dietary fructo-oligosaccharides and lactulose inhibit intestinal colonisation but stimulate translocation of salmonella in rats

Background and aims: It is frequently assumed that dietary non-digestible carbohydrates improve host resistance to intestinal infections by stimulating the protective gut microflora. However, compelling scientific evidence from in vivo infection studies is lacking. Therefore, we studied the effect of several non-digestible carbohydrates on the resistance of rats to Salmonella enteritidis infection. Methods: Rats (n=8 per group) were fed “humanised” purified diets containing 4% lactulose, fructo-oligosaccharides (FOS), resistant starch, wheat fibre, or cellulose. After an adaptation period of 2 weeks the animals were orally infected with S enteritidis. Supplement induced changes in faecal biochemical and microbiological parameters were studied before infection. Colonisation of salmonella was determined by studying the faecal excretion of this pathogen and translocation by analysis of urinary nitric oxide metabolites over time and classical organ cultures. Intestinal mucosal myeloperoxidase activity was determined to quantify intestinal inflammation after infection. Results: Despite stimulation of intestinal lactobacilli and bifidobacteria and inhibition of salmonella colonisation, FOS and lactulose significantly enhanced translocation of this pathogen. These supplements also increased cytotoxicity of faecal water and faecal mucin excretion, which may reflect mucosal irritation. In addition, caecal and colonic, but not ileal, mucosal myeloperoxidase activity was increased in infected rats fed FOS and lactulose. In contrast, cellulose, wheat fibre, and resistant starch did not affect the resistance to salmonella. Conclusions: In contrast to most expectations, FOS and lactulose impair the resistance of rats to intestinal salmonella infection. Obviously, stimulation of the endogenous lactobacilli and bifidobacteria is no guarantee of improved host defence against intestinal infections.

Dietary fructo-oligosaccharides and inulin decrease resistance of rats to salmonella: protective role of calcium

Background: We have shown recently that rapid fermentable fructo-oligosaccharides (FOS) decreased resistance of rats towards salmonella. It is not known whether inulin (which is fermented more gradually) has similar effects or whether buffering nutrients can counteract the adverse effects of rapid fermentation. Aims: To compare the effects of dietary inulin and FOS on resistance of rats to Salmonella enterica serovar Enteritidis and to determine whether calcium phosphate counteracts the effects of fermentation. Methods: Male Wistar rats (nu200a=u200a8 per group) were fed a human “Western style diet”. Diets with 60 g/kg cellulose (control), FOS, or inulin had either a low (30 mmol/kg) or high (100 mmol/kg) calcium concentration. After an adaptation period of two weeks, animals were orally infected with 2×109 colony forming units of Salmonella enterica serovar Enteritidis. Colonisation of salmonella was determined by quantification of salmonella in caecal contents. Translocation of salmonella was quantified by analysis of urinary nitric oxide metabolites in time. Results: Inulin and FOS decreased intestinal pH and increased faecal lactobacilli and enterobacteria. Moreover, both prebiotics increased the cytotoxicity of faecal water and faecal mucin excretion. Both prebiotics increased colonisation of salmonella in caecal contents and enhanced translocation of salmonella. Dietary calcium phosphate counteracted most of the adverse effects of inulin and FOS. Conclusions: Both inulin and FOS impair resistance to intestinal infections in rats. This impairment is partially prevented by dietary calcium phosphate. The results of the present study await verification in other controlled animal and human studies.

Non-Invasive Detection of Low-Intestinal Lactase Activity in Children by Use of a Combined 13CO2/H2 Breath Test

BACKGROUNDnThe aim of the study was to diagnose hypolactasia with a higher accuracy than with the traditional H2 breath test.nnnMETHODSnWe used a combined 13C-lactose 13CO2/H2 breath test, which was performed in 33 patients in whom lactase activity was measured.nnnRESULTSnLactase activity was reduced in 13 cases. The sensitivity and specificity of the H2 test were 54% and 90%; those of the 13CO2 test 69% and 70%. False-negative results did not always occur in the same patients. In five of six patients with both test results abnormal, lactase activity was low. In 13 of 15 patients with both test results normal, lactase activity was normal. In 6 of 12 cases with only 1 test abnormal, lactase activity was low.nnnCONCLUSIONnThe combined H2/13CO2 breath test (sensitivity, 85%; specificity, 65%) is more adequate for diagnosis of hypolactasia than the H2 breath test alone.

Calcium in milk and fermentation by yoghurt bacteria increase the resistance of rats to Salmonella infection.

Calcium in milk products stimulates gastric acid secretion and inhibits the cytolytic activity of intestinal contents. Based on these effects, it was hypothesised that calcium might lessen the severity of food borne intestinal infections. The possible differential effects of a low calcium milk and normal milk products (milk, acidified milk, and pasteurised yoghurt) on the resistance of rats to a salmonella infection was therefore studied. Rats were infected orally with Salmonella enteritidis just after food consumption. The first day after infection, faecal salmonella counts of the yoghurt fed rats were significantly lower than those of the other groups. Thereafter, faecal salmonella excretion declined rapidly in all high calcium groups, whereas rats fed the low calcium milk continued to excrete high numbers of salmonella. The reduced colonisation resistance to salmonella of rats fed low calcium milk might be caused by the high cytolytic activity of faecal water or a high iron concentration in faecal water, already present before infection, or both. The reduced resistance of these rats corresponded with a large infection induced increase in the cytolytic activity of faecal water, an appreciable reduction in apparent iron absorption, and a large increase in faecal mucin and alkaline phosphatase excretion. In yoghurt fed rats, only minor infection induced changes in luminal parameters were noticed. The rats fed milk and acidified milk always showed intermediate reactions. In conclusion, in addition to fermentation by yoghurt bacteria, calcium in milk products strongly enhanced the resistance to salmonella infection by lowering luminal cytolytic activity or diminishing the availability of iron for pathogen growth, or both.

Effects of long-term oral L-arginine on esophageal motility and gallbladder dynamics in healthy humans.

Inhibitory nitrergic neurons are known to play a role in the regulation of motility patterns of the distal esophagus, the lower esophageal sphincter (LES), and the gallbladder. Our study aim was to investigate the effects of long-term (i.e., prolonged) oral intake of L-arginine (L-Arg), the endogenous source for nitric oxide (NO) synthesis, on postprandial LES pressure (LESP), esophageal motility, gastroesophageal reflux, and gallbladder motility. L-Arg (30 g/day) or glycine (placebo; 13 g/day; isosmolar) was given orally to 10 healthy male volunteers for 8 days, according to a randomized, crossover design. Twenty-four-hour urinary nitrite/nitrate excretion was measured to indicate NO synthesis. Basal early postprandial LESP was lower after L-Arg ingestion (2.2 kPa) than after glycine ingestion (2.7 kPa) (P < 0.05). L-Arg abolished the physiological late postprandial rise in LESP. Transient LES relaxations were longer lasting after L-Arg ingestion (P < 0.02). Esophageal motility and reflux were not affected (not significant). Fasting and residual gallbladder volumes were greater after L-Arg ingestion (P < 0.05). Urinary nitrite/nitrate excretion was higher after L-Arg intake (P < 0.05). In conclusion, long-term oral L-Arg suppresses late postprandial LESP increase, prolongs transient LES relaxations, and increases fasting and residual gallbladder volumes. These effects may be mediated by increased NO synthesis.Inhibitory nitrergic neurons are known to play a role in the regulation of motility patterns of the distal esophagus, the lower esophageal sphincter (LES), and the gallbladder. Our study aim was to investigate the effects of long-term (i.e., prolonged) oral intake ofl-arginine (l-Arg), the endogenous source for nitric oxide (NO) synthesis, on postprandial LES pressure (LESP), esophageal motility, gastroesophageal reflux, and gallbladder motility. l-Arg (30 g/day) or glycine (placebo; 13 g/day; isosmolar) was given orally to 10 healthy male volunteers for 8 days, according to a randomized, crossover design. Twenty-four-hour urinary nitrite/nitrate excretion was measured to indicate NO synthesis. Basal early postprandial LESP was lower after l-Arg ingestion (2.2 kPa) than after glycine ingestion (2.7 kPa) ( P < 0.05).l-Arg abolished the physiological late postprandial rise in LESP. Transient LES relaxations were longer lasting after l-Arg ingestion ( P < 0.02). Esophageal motility and reflux were not affected (not significant). Fasting and residual gallbladder volumes were greater afterl-Arg ingestion ( P < 0.05). Urinary nitrite/nitrate excretion was higher after l-Arg intake ( P < 0.05). In conclusion, long-term oral l-Arg suppresses late postprandial LESP increase, prolongs transient LES relaxations, and increases fasting and residual gallbladder volumes. These effects may be mediated by increased NO synthesis.

Dietary haem stimulates epithelial cell turnover by downregulating feedback inhibitors of proliferation in murine colon

Objective Colon cancer is a leading cause of cancer deaths in Western countries and is associated with diets high in red meat. Haem, the iron-porphyrin pigment of red meat, induces cytotoxicity of gut contents and damages the colon surface epithelium. Compensatory hyperproliferation leads to epithelial hyperplasia which increases the risk of colon cancer. The aim of this study was to identify molecules signalling from the surface epithelium to the crypt to initiate hyperproliferation upon stress induced by haem. Methods C57Bl6/J mice (n=9/group) received a ‘westernised’ control diet (40 en% fat) with or without 0.5u2005μmol/g haem for 14u2005days. Colon mucosa was used to quantify cell proliferation and for microarray transcriptome analysis. Gene expression profiles of surface and crypt cells were compared using laser capture microdissection. Protein levels of potential signalling molecules were quantified. Results Haem-fed mice showed epithelial hyperproliferation and decreased apoptosis, resulting in hyperplasia. Microarray analysis of the colon mucosa showed 3710 differentially expressed genes (false discovery rate (q) <0.01), with many involved in the cell cycle. Expression levels of haem- and stress-related genes showed that haem affected surface cells but did not directly affect crypt cells. Injured surface cells should therefore signal to crypt cells to induce compensatory hyperproliferation. Haem downregulated the inhibitors of proliferation, Wnt inhibitory factor 1, Indian Hedgehog and bone morphogenetic protein 2. Interleukin-15 was also downregulated. Haem upregulated amphiregulin, epiregulin and cyclo-oxygenase-2 mRNA in surface cells. Their protein/metabolite levels were, however, not increased as haem induced surface-specific inhibition of translation by increasing 4E-BP1. Conclusions Haem induces colonic hyperproliferation and hyperplasia by inhibiting the surface to crypt signalling of feedback inhibitors of proliferation.

Changes in Bile Acid Composition and Effect on Cytolytic Activity of Fecal Water by Ursodeoxycholic Acid Administration: a Placebo-Controlled Cross-over Intervention Trial in Healthy Volunteers

Background: Ursodeoxycholic acid (UDCA) has been shown to affect membrane-damaging effects of bile acids in vitro and fecal bile acid composition in rats. This study evaluates the effect of UDCA on fecal bile acid composition and on cytolytic activity of fecal water in man to clarify the potential chemopreventive role of UDCA for colorectal cancer. Methods: In this placebo-controlled crossover intervention trial, the effect of 900 mg/day UDCA orally in 15 healthy volunteers was studied. At the end of each 4-week period, 72 h feces were collected. Total and individual bile acids in feces were determined by gas chromatography and soluble bile acids were analyzed by high-performance liquid chromatography. Cytolytic activity of fecal water was measured using an erythrocyte lysis assay. Results: In feces, the percentages of primary bile acids--cholic acid (CA) and chenodeoxycholic acid (CDCA)--and of secondary bile acid--deoxycholic acid (DCA)--decreased after supplementation with UDCA, whereas those of UDCA and LCA increased from 2.7 ± 0.4% to 23.7 ± 2.6%, P < 0.0001 and from 26.2 ± 1.2% to 49.4 ± 1.8%, P < 0.0001 respectively. The concentrations of these two bile acids in fecal water also increased after UDCA administration from 7.8 ± 1.9 μmol/l to 47.0 ± 6.7 μmol/l (UDCA), P < 0.0001 and from 2.5 ± 0.6 μmol/l to 18.3 ± 4.1 μmol/l (LCA), P < 0.002, respectively. Cytolytic activity of fecal water was not affected by UDCA. Conclusion: These results do not support a protective effect of UDCA supplementation against colorectal cancer in man.

Real‐time PCR of host DNA in feces to study differential exfoliation of colonocytes between rats and humans

Background: Colonic mucosa has a high turnover rate. At the end of their lifespan, colonocytes become senescent and die. Histological studies indicate that senescent colonocytes are shed (exfoliated) into the fecal stream in rats, but phagocytosed by mucosal macrophages in humans. We study whether quantification of host DNA in feces can be used as a non‐invasive marker for this differential disposal of colonocytes. Methods: Selective primers and probes for the rat and human β‐globin genes were designed and used in real‐time PCR reactions. Results: Host DNA was quantitatively extracted and detected in fecal samples of both species. Feces of rats fed a humanized diet contained approximately 100u2005μg rat DNA peru2005g freeze‐dried feces. In human feces, however, only 5 out of 12 samples contained detectable, though very low (less than 0.35u2005μg/g), levels of host DNA. This about 300‐fold difference could not be attributed to differences in DNase activities in the fecal stream. Conclusion: Our results indicate that there is considerable luminal shedding of senescent colonocytes in rats, whereas mucosal phagocytosis is the main route of colonocyte disposal in humans. Thus, real‐time PCR of host DNA in feces can be applied as a non‐invasive method for studying the differential exfoliation of colonocytes.Background: Colonic mucosa has a high turnover rate. At the end of their lifespan, colonocytes become senescent and die. Histological studies indicate that senescent colonocytes are shed (exfoliated) into the fecal stream in rats, but phagocytosed by mucosal macrophages in humans. We study whether quantification of host DNA in feces can be used as a non‐invasive marker for this differential disposal of colonocytes. Methods: Selective primers and probes for the rat and human β‐globin genes were designed and used in real‐time PCR reactions. Results: Host DNA was quantitatively extracted and detected in fecal samples of both species. Feces of rats fed a humanized diet contained approximately 100u2005μg rat DNA peru2005g freeze‐dried feces. In human feces, however, only 5 out of 12 samples contained detectable, though very low (less than 0.35u2005μg/g), levels of host DNA. This about 300‐fold difference could not be attributed to differences in DNase activities in the fecal stream. Conclusion: Our results indicate that t...