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During fat digestion, fatty acids and monoglycerides are liberated in the gastrointestinal tract. Generally, these lipids are potent inhibitors of gram-positive bacteria in vitro but have less effect on gram-negative microbes. Considering this, we hypothesized that increased intake of bovine milk fat would result in enhanced gastrointestinal killing of Listeria monocytogenes (gram-positive) but have little effect on infection with Salmonella enteritidis (gram-negative) in rats. To test this, rats were fed either low milk fat diets (10% of energy obtained from milk fat, corresponding to 4. 2 g fat/100 g diet) or high milk fat diets (40% of energy obtained from milk fat, corresponding to 19.6 g fat/100 g diet). After adaptation to these diets, rats were orally infected with Listeria or Salmonella. Greater milk fat consumption in Listeria-infected rats diminished intestinal colonization of Listeria (P < 0.05) and reduced diarrhea (P < 0.05). Analysis of gastrointestinal contents showed that killing of Listeria occurred predominantly in the stomach. High milk fat intake significantly augmented this gastric listericidal capacity (P < 0.05) and raised the concentration of medium-chain and saturated long-chain free fatty acids and of monoglycerides of C12:0, C14:0, C16:0, C18:0, and C18:1 in gastric chyme (P < 0.05). Considering the in vitro listericidal capacity of these agents, it was concluded that particularly the free fatty acids C10:0, C12:0 and C14:0 and the monoglycerides of C12:0, C14:0, and C16:0 seem to play a pivotal role in this enhanced Listeria killing. In contrast, Salmonella infection was not affected by milk fat consumption. In conclusion, high milk fat intake results in higher concentrations of gastric bactericidal lipids and thereby protects against Listeria infection but not against Salmonella. [ABSTRACT FROM AUTHOR]

We have shown recently that dietary calcium phosphate (CaPi) has a trophic effect on the intestinal microflora and strongly protects against salmonella infection. It was speculated that precipitation by CaPi of intestinal surfactants, such as bile acids and fatty acids, reduced the cytotoxicity of intestinal contents and favored growth of the microflora. Because lactobacilli may have antagonistic activity against pathogens, the main purpose of the present study was to examine whether this CaPi-induced protection coincides with a reinforcement of the endogenous lactobacilli. In vitro, Salmonella enteritidis appeared to be insensitive to bile acids and fatty acids, whereas Lactobacillus acidophilus was killed by physiologically relevant concentrations of these surfactants. Additionally, after adaptation to a purified diet differing only in CaPi concentration (20 and 180 mmol CaHPO4. 2H2O/kg), rats (n = 8) were orally infected with S. enteritidis. Besides reducing the cytotoxicity and the concentration of bile acids and fatty acids of ileal contents and fecal water, CaPi notably changed the composition of ileal bile acids in a less cell-damaging direction. Significantly greater numbers of ileal and fecal lactobacilli were detected in noninfected, CaPi-supplemented rats. As judged by the lower urinary NOx excretion, which is a biomarker of intestinal bacterial translocation, dietary CaPi reduced the invasion of salmonella. Additionally, the colonization resistance was improved considering the reduction of excreted fecal salmonella. In accordance, fewer viable salmonella were detected in ileal contents and on the ileal mucosa in the CaPi group. In conclusion, reducing the intestinal surfactant concentration by dietary CaPi strengthens the endogenous lactobacilli and increases the resistance to salmonella. [ABSTRACT FROM AUTHOR]

Perturbation of the intestinal microbiota by antibiotics predisposes the host to food-borne pathogens like Salmonella. The effects of antibiotic treatment on intestinal permeability during infection and the efficacy of dietary components to improve resistance to infection have not been studied. Therefore, we investigated the effect of clindamycin on intestinal barrier function in Salmonella-infected rats. We also studied the ability of dietary calcium and tannic acid to protect against infection and concomitant diarrhea and we assessed intestinal barrier function. Rats were fed a purified control diet including the permeability marker chromium EDTA (CrEDTA) (2 g/kg) or the same diet supplemented with calcium (4.8 g/kg) or tannic acid (3.75 g/kg). After adaptation, rats were orally treated with clindamycin for 4 d followed by oral infection with Salmonella enteritidis. Two additional control groups were not treated with antibiotics and received either saline or Salmonella. Urine and feces were collected to quantify intestinal permeability, diarrhea, cytotoxicity of fecal water, and Salmonella excretion. In addition, Salmonella translocation was determined. Diarrhea, CrEDTA excretion, and cytotoxicity of fecal water were higher in the clindamycin-treated infected rats than in the non-clindamycin-treated infected control group. Intestinal barrier function was less in the Salmonella-infected rats pretreated with antibiotics compared with the non-clindamycin- treated rats. Both calcium and tannic acid reduced infection-associated diarrhea and inhibited the adverse intestinal permeability changes but did not decrease Salmonella colonization and translocation. Our results indicate that calcium protects against intestinal changes due to Salmonella infection by reducing luminal cytotoxicity, whereas tannic acid offers protection by improving the mucosal resistance.

We have shown in several controlled rat and human infection studies that dietary calcium improves intestinal resistance and strengthens the mucosal barrier. Reinforcement of gut barrier function may alleviate inflammatory bowel disease (IBD). Therefore, we investigated the effect of supplemental calcium on spontaneous colitis development in an experimental rat model of IBD. HLA-B27 transgenic rats were fed a purified high-fat diet containing either a low or high calcium concentration (30 and 120 mmol CaHPO4/kg diet, respectively) for almost 7 wk. Inert chromium EDTA (CrEDTA) was added to the diets to quantify intestinal permeability by measuring urinary CrEDTA excretion. Relative fecal wet weight was determined to quantify diarrhea. Colonic inflammation was determined histologically and by measuring mucosal interleukin (IL)-1beta. In addition, colonic mucosal gene expression of individual rats was analyzed using whole-genome microarrays. The calcium diet significantly inhibited the increase in intestinal permeability and diarrhea with time in HLA-B27 rats developing colitis compared with the control transgenic rats. Mucosal IL-1beta levels were lower in calcium-fed rats and histological colitis scores tended to be lower (P = 0.08). Supplemental calcium prevented the colitis-induced increase in the expression of extracellular matrix remodeling genes (e.g. matrix metalloproteinases, procollagens, and fibronectin), which was confirmed by quantitative real-time PCR and gelatin zymography. In conclusion, dietary calcium ameliorates several important aspects of colitis severity in HLA-B27 transgenic rats. Reduction of mucosal irritation by luminal components might be part of the mechanism. These results show promise for supplemental calcium as effective adjunct therapy for IBD.

In contrast to most expectations, we showed previously that dietary fructooligosaccharides (FOS) stimulate intestinal colonization and translocation of invasive Salmonella enteritidis in rats. Even before infection, FOS increased the cytotoxicity of fecal water, mucin excretion, and intestinal permeability. In the present study, we tested whether FOS has these effects in humans. A double-blind, placebo-controlled, crossover study of 2 x 2 wk, with a washout period of 2 wk, was performed with 34 healthy men. Each day, subjects consumed lemonade containing either 20 g FOS or placebo and the intestinal permeability marker chromium EDTA (CrEDTA). On the last 2 d of each supplement period, subjects scored their gastrointestinal complaints on a visual analog scale and collected feces and urine for 24 h. Fecal lactic acid was measured using a colorimetric enzymatic kit. The cytotoxicity of fecal water was determined with an in vitro bioassay, fecal mucins were quantified fluorimetrically, and intestinal permeability was determined by measuring urinary CrEDTA excretion. In agreement with our animal studies, FOS fermentation increased fecal wet weight, bifidobacteria, lactobacilli, and lactic acid. Consumption of FOS increased flatulence and intestinal bloating. In addition, FOS consumption doubled fecal mucin excretion, indicating mucosal irritation. However, FOS did not affect the cytotoxicity of fecal water and intestinal permeability. The FOS-induced increase in mucin excretion in our human study suggests mucosal irritation in humans, but the overall effects are more moderate than those in rats.

Diets high in red meat and low in green vegetables are associated with an increased risk of colon cancer. In rats, dietary heme, mimicking red meat, increases colonic cytotoxicity and proliferation of the colonocytes, whereas addition of chlorophyll from green vegetables inhibits these heme-induced effects. Chlorophyllin is a water-soluble hydrolysis product of chlorophyll that inhibits the toxicity of many planar aromatic compounds. The present study investigated whether chlorophyllins could inhibit the heme-induced luminal cytotoxicity and colonic hyperproliferation as natural chlorophyll does. Rats were fed a purified control diet, the control diet supplemented with heme, or a heme diet with 1.2 mmol/kg diet of chlorophyllin, copper chlorophyllin, or natural chlorophyll for 14 d (n = 8/group). The cytotoxicity of fecal water was determined with an erythrocyte bioassay and colonic epithelial cell proliferation was quantified in vivo by [methyl-(3)H]thymidine incorporation into newly synthesized DNA. Exfoliation of colonocytes was measured as the amount of rat DNA in feces using quantitative PCR analysis. Heme caused a >50-fold increase in the cytotoxicity of the fecal water, a nearly 100% increase in proliferation, and almost total inhibition of exfoliation of the colonocytes. Furthermore, the addition of heme increased TBARS in fecal water. Chlorophyll, but not the chlorophyllins, completely prevented these heme-induced effects. In conclusion, inhibition of the heme-induced colonic cytotoxicity and epithelial cell turnover is specific for natural chlorophyll and cannot be mimicked by water-soluble chlorophyllins.

We showed previously that fructooligosaccharides (FOS) decrease the resistance to salmonella infection in rats. However, the mechanism responsible for this effect is unclear. Therefore, we examined whether dietary FOS affects intestinal permeability before and after infection with Salmonella enterica serovar Enteritidis. Male Wistar rats were fed restricted quantities of a purified diet that mimicked the composition of a Western human diet. The diet was supplemented with 60 g/kg cellulose (control) or 60 g/kg FOS and with 4 mmol/kg of the intestinal permeability marker chromium EDTA (CrEDTA) (n = 8 or 10). After an adaptation period of 2 wk, rats were orally infected with 10(8) colony-forming units (cfu) of S. enteritidis. Mucin concentrations in intestinal contents and mucosa were measured fluorimetrically, as markers of mucosal irritation. Intestinal permeability was determined by measuring urinary CrEDTA excretion. Translocation of salmonella was quantified by analysis of urinary nitric oxide metabolites with time. Before infection, FOS increased mucosal lactobacilli and enterobacteria in cecum and colon, but not in the ileum. However, FOS increased cytotoxicity of fecal water and intestinal permeability. Moreover, FOS increased fecal mucin excretion and mucin concentrations in cecal and colonic contents, and in cecal mucosa before infection. After infection, mucin excretion and intestinal permeability in the FOS groups increased even further in contrast to the control group. In addition, FOS increased translocation of salmonella to extraintestinal sites. Thus, FOS impairs the intestinal barrier in rats, as indicated by higher intestinal permeability. Whether these results can be extrapolated to humans requires further investigation.

Red meat intake is associated with colon cancer risk. Puzzlingly, meat does not promote carcinogenesis in rat studies. However, we demonstrated previously that dietary heme promotes aberrant crypt foci (ACF) formation in rats given a low-calcium diet. Here, we tested the hypothesis that heme-rich meats promote colon carcinogenesis in rats treated with azoxymethane and fed low-calcium diets (0.8 g/kg). Three meat-based diets were formulated to contain varying concentrations of heme by the addition of raw chicken (low heme), beef (medium heme), or black pudding (blood sausage; high heme). The no-heme control diet was supplemented with ferric citrate and the heme control diet with hemoglobin to match iron and heme concentrations in the beef diet, respectively. After 100 d, colons were scored for ACF and mucin-depleted foci (MDF). Fecal water was assayed for lipoperoxides and cytotoxicity. Only diets with heme promoted the formation of MDF, but all meat diets promoted ACF formation. The number of MDF/colon was 0.55 +/- 0.68 in controls, but 1.2 +/- 0.6 (P = 0.13), 1.9 +/- 1.4 (P < 0.01), and 3.0 +/- 1.2 (P < 0.001) in chicken-, beef-, and black pudding-fed rats. MDF promotion by the high-heme black pudding diet was greater than that by the medium-heme beef diet. The number of ACF/colon was 72 +/- 16 in controls, but 91 +/- 18, 100 +/- 13, and 103 +/- 14 in chicken-, beef-, and black pudding-fed rats (all P < 0.001). ACF and MDF did not differ between rats fed the beef diet and those fed the heme control diet. MDF promotion was correlated with high fecal water lipoperoxides and cytotoxicity (r = 0.65, P < 0.01). This is the first study to show the promotion of experimental carcinogenesis by dietary meat and the association with heme intake.

Prebiotics, such as fructo-oligosaccharides (FOS), stimulate the protective gut microflora, resulting in an increased production of organic acids. This may result in increased luminal killing of acid-sensitive pathogens. However, host defense against invasive pathogens, like salmonella, also depends on the barrier function of the intestinal mucosa. Rapid fermentation of prebiotics leading to high concentrations of organic acids may impair the barrier function. Therefore, we determined the dose-dependent effect of dietary FOS on the resistance of rats to Salmonella enteritidis. Male Wistar rats were fed restricted quantities of a "humanized" purified diet supplemented with 0, 3 or 6 g/100 g of FOS (n = 7 in the 6% FOS group and n = 8 in the other diet groups). After an adaptation period of 2 wk, rats were orally infected with 1.7 x 10(10) colony-forming units of S. enteritidis. Supplement-induced changes in the intestinal microflora and fecal cation excretion were determined before and after infection. Cytotoxicity of fecal water was determined with an in vitro bioassay, and fecal mucins were quantified fluorimetrically. Colonization of S. enteritidis was determined by quantification of salmonella in cecal contents and mucosa. Translocation of S. enteritidis was quantified by analysis of urinary nitric oxide metabolites in time. Before infection, FOS decreased cecal and fecal pH, increased fecal lactic acid concentration and increased bifidobacteria and enterobacteria. FOS also increased cytotoxicity of fecal water and fecal mucin excretion, indicating mucosal irritation. Remarkably, FOS dose-dependently increased salmonella numbers in cecal contents and mucosa and caused a major increase in infection-induced diarrhea. In addition, FOS enhanced translocation of salmonella. Thus, in contrast to most expectations, FOS dose-dependently impairs the resistance to salmonella infection in rats. These results await verification by other controlled animal and human studies.

Most Gram-positive bacteria are susceptible to the bactericidal action of fatty acids and bile acids. Because dietary calcium phosphate (CaP(i)) lowers the intestinal concentration of these antimicrobial agents, high CaP(i) intake may enhance intestinal colonization of Gram-positive pathogens and the subsequent pathogenesis. In this study, we tested this hypothesis in a rat model using Listeria monocytogenes. Rats were fed diets containing low (20 micromol/g diet) or high (160 micromol/g diet) amounts of CaP(i). Dietary fat was provided as corn oil or milk fat. Rats were orally inoculated with L. monocytogenes. When rats consumed diets containing corn oil, high CaP(i) intake indeed stimulated colonization of L. monocytogenes and increased L. monocytogenes translocation and diarrhea. In addition, supplemental CaP(i) enhanced ex vivo growth of L. monocytogenes in fecal extracts of rats fed corn oil diets, suggesting that high CaP(i) intake decreased a luminal inhibitory factor. The concentrations of bile salts and fatty acids, which were highly listericidal in vitro, were indeed considerably decreased in fecal water of rats in the high calcium corn oil group. Surprisingly, dietary CaP(i) did not affect colonization and translocation of L. monocytogenes in rats fed the milk fat diet, nor did CaP(i) enhance ex vivo growth in fecal extracts. This absence of Listeria stimulation was associated with a lack of effect of dietary CaP(i) on fecal soluble fatty acids. In addition, residual soluble bile salts were higher in rats fed the high CaP(i) milk fat diet compared with the high CaP(i) corn oil diet. These results suggest that the stimulating effect of CaP(i) on L. monocytogenes infection depends on the type of dietary fat consumed.

Recently we showed that soybean protein compared with casein stimulates the fecal excretion of endogenous magnesium. In the present study we investigated whether this differential effect of soybean protein is due to intestinal epithelial cell damage and thus results in a compensating increase in proliferation of epithelial cells. Two groups of six rats were fed purified high fat, low calcium diets, differing only in the type of protein. When compared with casein feeding, soybean protein feeding stimulated the fecal excretion of magnesium, fat and fatty acids, but had no effect on the excretion of bile acids. In fecal water, the concentration of bile acids was lower when soybean protein was fed. In contrast, free fatty acid concentration, as well as luminal cytolytic activity, was higher in fecal water of rats fed soybean protein. Furthermore, epithelial cell damage and proliferation of colonic epithelium (measured as in vivo incorporation of tritiated thymidine into DNA) were greater in rats fed soybean protein. The stimulation of colonic proliferation by soybean protein is consistent with the observed increase in luminal cytolytic activity and epithelial cell damage. We conclude that the stimulatory effect of soybean protein on endogenous magnesium excretion is due to a soybean protein-specific damage of colonic epithelial cells, which results in a compensatory epithelial cell hyperproliferation.

The effects of the type of dietary fat (180 g/kg diet) and of calcium phosphate (CaHPO4) supplementation (25 vs. 225 mmol/kg diet) on luminal solubility of fatty acids and bile acids, cytotoxicity of fecal water and intestinal epitheliolysis were studied in rats. In rats fed the low and high calcium phosphate diets, fecal excretion of fatty acids diminished in the order palm oil > milk fat > corn oil. Palm oil also caused the highest concentration of fatty acids measured in fecal water followed by milk fat and corn oil when fed at both calcium phosphate levels. The differences in concentrations of luminal surfactants in fecal water of rats fed the three fat diets resulted in a fat type-dependent cytotoxicity of fecal water, with that of palm oil-fed rats the most cytotoxic. The concentrations of fatty acids as well as bile acids in fecal water were, however, significantly lowered by calcium phosphate supplementation in rats fed all types of dietary fat. This reduction in concentration of fecal water surfactants resulted in a lower cytotoxicity of fecal water. The concentration of surfactants in fecal water and cytotoxicity were correlated by multiple regression analysis (R = 0.89). Intestinal epitheliolysis measured as alkaline phosphatase activity in fecal water was lowered comparably to the reduction in cytotoxicity by supplemental calcium phosphate. Intestinal epitheliolysis and cytotoxicity of fecal water were correlated (r = 0.92, P < 0.001). The type of dietary fat and the amount of dietary calcium phosphate influence the concentrations of surfactants in fecal water and consequently affect cytotoxicity of fecal water and intestinal epitheliolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Apparent magnesium absorption is depressed in rats fed diets containing soybean protein or enriched with sodium phytate or phosphate in comparison with casein, whereas it is raised in rats fed lactose. However, the possibility that changes in apparent absorption are caused by changes in fecal excretion of endogenous magnesium cannot be excluded. We studied the effects of casein, soybean protein, sodium phytate, lactose and phosphate on apparent and true absorption of magnesium. True magnesium absorption was measured with the use of oral and intraperitoneal administration of tracer doses of 28Mg. Fecal excretion of endogenous magnesium was calculated from apparent and true absorption. True magnesium absorption was not affected by either substitution of soybean protein for casein or by the addition of sodium phytate to a diet containing casein. Endogenous magnesium excretion in feces was significantly increased by soybean protein and sodium phytate. Thus the observed impairment of apparent magnesium absorption in rats fed soybean protein or sodium phytate is due to enhanced fecal excretion of endogenous magnesium. With the other dietary treatments, enhanced fecal excretion of endogenous magnesium was not associated with a discrepancy in the effects on apparent and true magnesium absorption. Dietary lactose vs. dextrose and supplemental phosphate both stimulated fecal excretion of endogenous magnesium, but lactose raised both true and apparent magnesium absorption, and phosphate depressed both true and apparent magnesium absorption.

We tested the hypothesis that increased intakes of calcium and phosphate lower magnesium solubility in the intestinal lumen, causing a decreased magnesium absorption. In in vitro experiments at a constant magnesium concentration, increasing calcium concentrations reduced magnesium solubility. This effect did not occur in the absence of phosphate. Increasing phosphate concentrations decreased the solubility of magnesium in the presence, but not in the absence, of calcium. These results suggest that the formation of an insoluble calcium-magnesium-phosphate complex determines magnesium solubility. To extend this concept to in vivo conditions, rats were fed purified diets containing a constant concentration of magnesium (16.4 mumol/g) but different concentrations of calcium (25, 100 or 175 mumol/g) and phosphate (58, 103 or 161 mumol/g). Increased intakes of calcium decreased magnesium solubility in the ileal lumen and lowered magnesium absorption. The latter result occurred only if the dietary phosphate concentration was at least 103 mumol/g. Increasing dietary phosphate concentrations reduced both magnesium solubility in the ileum and magnesium absorption, but only if the dietary calcium concentration was at least 100 mumol/g. These results support those obtained in vitro. We conclude that increased intakes of calcium and phosphate decrease magnesium absorption by the formation of an insoluble calcium-magnesium-phosphate complex in the intestinal lumen.

Treatment of casein with formaldehyde changes its tertiary structure and decreases its hypercholesterolemic properties in rabbits. To investigate whether formaldehyde-treated casein exerts this hypocholesterolemic effect in the same manner as soybean protein, rabbits were fed high or low cholesterol diets containing soybean protein, casein, formaldehyde-treated casein or a mixture of casein and formaldehyde-treated casein. Formaldehyde-treated casein was hypocholesterolemic when fed in a low, but not in a high, cholesterol diet. The hypocholesterolemic effect of soybean protein was independent of the amount of cholesterol included in the diet. In contrast to rabbits fed soybean protein, steroid absorption in those fed formaldehyde-treated casein did not differ from that in rabbits fed native casein. Furthermore, the absorption of phosphorus and nitrogen was lower in rabbits fed formaldehyde-treated casein than in those fed native casein, whereas the absorption found in rabbits fed soybean protein resembled that of their casein-fed counterparts. The diets containing soybean protein and formaldehyde-treated casein produced a comparable ratio of lysine to arginine in serum. The results presented in this paper indicate that the hypocholesterolemic action of dietary formaldehyde-treated casein does not resemble that of soybean protein.

Two groups of nine rabbits fed a purified diet containing soy protein were injected intravenously with [4-14C]cholesterol. One group was fed the same diet for 78 d while the other group was fed a diet containing casein. The feces of the rabbits were collected and analyzed for radioactivity present in the neutral steroid and bile acid fractions. The excretion of neutral steroids and bile acids was lowered within 3 d after soy protein was replaced by casein and before the serum cholesterol concentration was increased. However, in the casein-fed rabbits the specific radioactivity of the bile acids was greater, whereas the amount of excreted bile acids was lower than in the soy protein-fed rabbits. After the serum cholesterol level in the casein-fed animals became constant (d 35), a further injection of [4-14C]cholesterol was given to all animals. Kinetic parameters of cholesterol metabolism according to a two-pool model were estimated from the die-away curve of [4-14C]cholesterol in serum. The size of the rapidly exchangeable pool (pool A) was greater in the casein-fed rabbits than in the soy protein-fed rabbits, whereas the size of pool B was similar in the two groups. The partial turnover rate of pool A was lower, as was the partial turnover of the slowly exchangeable pool (pool B), and the production rate of cholesterol was significantly lower on the casein-fed rabbits than in the soy protein-fed rabbits. This study suggests that the reduced excretion of steroids in casein-fed rabbits could be the cause of the hypercholesterolemia and is not the result of it.