FDA-approved drug screening in patient-derived organoids demonstrates potential of drug repurposing for rare cystic fibrosis genotypes.

BACKGROUND: Preclinical cell-based assays that recapitulate human disease play an important role in drug repurposing. We previously developed a functional forskolin induced swelling (FIS) assay using patient-derived intestinal organoids (PDIOs), allowing functional characterization of CFTR, the gene mutated in people with cystic fibrosis (pwCF). CFTR function-increasing pharmacotherapies have revolutionized treatment for approximately 85% of people with CF who carry the most prevalent F508del-CFTR mutation, but a large unmet need remains to identify new treatments for all pwCF. METHODS: We used 76 PDIOs not homozygous for F508del-CFTR to test the efficacy of 1400 FDA-approved drugs on improving CFTR function, as measured in FIS assays. The most promising hits were verified in a secondary FIS screen. Based on the results of this secondary screen, we further investigated CFTR elevating function of PDE4 inhibitors and currently existing CFTR modulators. RESULTS: In the primary screen, 30 hits were characterized that elevated CFTR function. In the secondary validation screen, 19 hits were confirmed and categorized in three main drug families: CFTR modulators, PDE4 inhibitors and tyrosine kinase inhibitors. We show that PDE4 inhibitors are potent CFTR function inducers in PDIOs where residual CFTR function is either present, or created by additional compound exposure. Additionally, upon CFTR modulator treatment we show rescue of CF genotypes that are currently not eligible for this therapy. CONCLUSION: This study exemplifies the feasibility of high-throughput compound screening using PDIOs. We show the potential of repurposing drugs for pwCF carrying non-F508del genotypes that are currently not eligible for therapies. ONE-SENTENCE SUMMARY: We screened 1400 FDA-approved drugs in CF patient-derived intestinal organoids using the previously established functional FIS assay, and show the potential of repurposing PDE4 inhibitors and CFTR modulators for rare CF genotypes.

Genetic and nongenetic variation in concentration of selenium, calcium, potassium, zinc, magnesium, and phosphorus in milk of Dutch Holstein-Friesian cows.

Minerals found in milk, such as Se, Ca, K, Zn, Mg, and P, contribute to several vital physiological processes. The aim of this study was to quantify the genetic variation in levels of Se, Ca, K, Zn, Mg, and P in milk and to quantify the between-herd variation in the levels of these minerals in milk. One morning milk sample from each of 1,860 Dutch Holstein-Friesian cows from 388 commercial herds in the Netherlands was used. Concentration of minerals was determined by inductively coupled plasma-atomic emission spectrometry. Variance components were estimated using an animal model with covariates for days in milk and age at first calving; fixed effects for season of calving and effect of test or proven bull; and random effects for animal, herd, and error. Heritability and proportion of phenotypic variation that can be explained by herd were estimated using univariate analysis. The intraherd heritability for Se was low (0.20) whereas herd explained 65% of the total variation in Se. Variation between herds most likely results from variation in Se content in the feed, which partly reflects variation in Se levels in the soil. Intraherd heritabilities for Ca, K, Zn, Mg, and P were moderate to high and were 0.57, 0.46, 0.41, 0.60, and 0.62, respectively. For Ca, K, Zn, Mg, and P, the proportions of phenotypic variation that could be explained by herd were low (0.13-0.24). This study shows that there are possibilities for altering the mineral composition of milk. For Ca, K, Zn, Mg, and P, there are good prospects for selective breeding whereas, for Se, measures at farm level may be more effective.

Red meat and colon cancer: dietary haem-induced colonic cytotoxicity and epithelial hyperproliferation are inhibited by calcium.

High intake of red meat is associated with increased colon cancer risk. We have shown earlier that this may be due to the high haem content of red meat, because dietary haem increased cytolytic activity of faecal water and colonic epithelial proliferation. Dietary calcium inhibits diet-induced epithelial hyperproliferation. Furthermore, it has been shown that supplemental calcium inhibited the recurrence of colorectal adenomas. Therefore, we studied whether dietary calcium phosphate can exert its protective effects by inhibiting the deleterious effects of haem. In vitro, calcium phosphate precipitated haem and inhibited the haem-induced cytotoxicity. Subsequently, rats were fed diets, differing in haem (0 or 1.3 micromol/g) and calcium phosphate content only (20 or 180 micromol/g). Faeces were collected for biochemical analyses. Cytolytic activity of faecal water was determined from the degree of lysis of erythrocytes by faecal water. Colonic epithelial proliferation was measured in vivo using [(3)H]thymidine incorporation. In rats fed low calcium diets, dietary haem increased cytolytic activity of faecal water (98 +/- 1 versus 1 +/- 1%, P < 0.001) and the concentration of cations in faeces (964 +/- 31 versus 254 +/- 20 micromol/g), when compared with controls. This indicates that dietary haem increased colonic mucosal exposure to luminal irritants. Colonic epithelial proliferation was increased compared with controls (70 +/- 4 versus 48 +/- 8 d.p.m./microg DNA, P < 0.001). This was accompanied by metabolism of the ingested haem and solubilization of haem compounds in the faecal water. A high calcium diet largely prevented this metabolism and solubilization. It also inhibited the haem-induced cytolytic activity of faecal water and increase in faecal cation concentration. In accordance, the haem-induced colonic epithelial hyperproliferation was prevented. We therefore suggest that dietary calcium phosphate acts as a chemopreventive agent in colon carcinogenesis by inhibiting the cytolytic and hyperproliferative effects of dietary haem.

Microencapsulation: its application in nutrition.

The development of new functional foods requires technologies for incorporating health-promoting ingredients into food without reducing their bioavailability or functionality. In many cases, microencapsulation can provide the necessary protection for these compounds, but in all cases bioavailability should be carefully studied. The present paper gives an overview of the application of various microencapsulation technologies to nutritionally-important compounds, i.e. vitamins, n-3 polyunsaturated fatty acids, Ca, Fe and antioxidants. It also gives a view on future technologies and trends in microencapsulation technology for nutritional applications.

Red meat and colon cancer: the cytotoxic and hyperproliferative effects of dietary heme.

The intake of a Western diet with a high amount of red meat is associated with a high risk for colon cancer. We hypothesize that heme, the iron carrier of red meat, is involved in diet-induced colonic epithelial damage, resulting in increased epithelial proliferation. Rats were fed purified control diets, or purified diets supplemented with 1.3 micromol/g of hemin (ferriheme), protoporphyrin IX, ferric citrate, or bilirubin (n = 8/group) for 14 days. Feces were collected for biochemical analyses. Fecal cytotoxicity was determined from the degree of lysis of erythrocytes by fecal water. Colonic epithelial proliferation was measured in vivo using [3H]thymidine incorporation into colonic mucosa. The colonic epithelial proliferation in heme-fed rats was significantly increased compared to control rats [55.2 +/- 5.8 versus 32.6 +/- 6.3 dpm/microg DNA (mean +/- SE); P < 0.05]. The fecal water of the heme group was highly cytotoxic compared to the controls (90 +/- 2% versus 2 +/- 1%; P < 0.001), although the concentrations of cytotoxic bile acids and fatty acids were significantly lower. Organic iron was significantly increased compared to the controls (257 +/- 26 versus 80 +/- 21, microM; P < 0.001). Spectrophotometric analyses suggest that this organic iron is heme-associated. Thiobarbituric acid-reactive substances were greatly increased in the fecal water of heme-fed rats compared to the controls (177 +/- 12 versus 59 +/- 7 microM; P < 0.05). Heme itself could not account for the increased cytotoxicity because the addition of heme to the fecal water of the control group, which was equimolar to the organic iron content of the fecal water of the heme group, did not influence the cytotoxicity. Hence, an additional heme-induced cytotoxic factor is involved, which may be modulated by the generation of luminal-reactive oxygen species. Protoporphyrin IX, ferric citrate, and bilirubin did not increase proliferation and cytotoxicity. In conclusion, dietary heme leads to the formation of an unknown, highly cytotoxic factor in the colonic lumen. This suggests that, in heme-fed rats, colonic mucosa is damaged by the intestinal contents. This results in a compensatory hyperproliferation of the epithelium, which supposedly increases the risk for colon cancer.

Dietary calcium phosphate stimulates intestinal lactobacilli and decreases the severity of a salmonella infection in rats.

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.

Dietary calcium inhibits the intestinal colonization and translocation of Salmonella in rats.

BACKGROUND & AIMS: Dietary calcium decreases the cytotoxicity of intestinal contents and intestinal epitheliolysis by precipitating cytotoxic surfactants such as bile acids. A decreased luminal cytotoxicity might not only strengthen the barrier function of the gut mucosa but also reinforce the protective, endogenous microflora. We hypothesized, therefore, that dietary calcium increases the resistance to intestinal infections. METHODS: Rats on a low-, medium-, or high-calcium purified diet were orally infected with a single dose of Salmonella enteritidis. The kinetics of fecal Salmonella excretion was studied to determine the colonization resistance. Intestinal bacterial translocation was quantitated by measuring urinary oxidation products of nitric oxide (NOx) excretion and culturing bacteria from tissues. RESULTS: Compared with the low-calcium group, the medium- and high-calcium diet-fed rats had a substantially improved colonization resistance. Calcium supplementation also reduced translocation of Salmonella, considering the diminished urinary NOx excretion and viable Salmonella counts in the Ileal Peyer's patches and spleen. Dietary calcium decreased the bile acid concentration and cytotoxicity of fecal water. Several indicators of fecal bacterial mass were significantly increased by supplemental calcium. CONCLUSIONS: Dietary calcium improves the colonization resistance and reduces the severity of gut-derived systemic infections, which is probably attributable to its luminal cytoprotective effects.

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.

Oral calcium phosphate: a new therapy for Crigler-Najjar disease?

BACKGROUND & AIMS: Calcium phosphate binds unconjugated bilirubin in vitro, and dietary calcium phosphate supplementation reduces the serum bilirubin level in rats with hereditary unconjugated hyperbilirubinemia (Gunn rats). The aim of this study was to evaluate the effect of oral calcium phosphate supplementation on plasma bilirubin levels in patients with Crigler-Najjar disease. METHODS: A placebo-controlled, double-blind, crossover design was used. Eleven patients, 2-42 years of age, participated. The group included 5 patients with type I disease who were all treated with phototherapy and 6 patients with type II disease who were primarily treated with phenobarbital. In addition to plasma bilirubin levels, dietary intake and urinary and fecal excretion of calcium and phosphate were evaluated. RESULTS: A modest but significant decrease in serum bilirubin was observed in patients with type I disease (18% +/- 6%, P = 0.03) but not in patients with type II disease during treatment with calcium phosphate. Urinary output of calcium and phosphate did not change during the treatment period. CONCLUSIONS: Oral calcium phosphate may be a useful adjuvant to photo-therapy in Crigler-Najjar type I disease.

Dietary ascorbic acid raises iron absorption in anaemic rats through enhancing mucosal iron uptake independent of iron solubility in the digesta.

We studied Fe absorption from FeSO4 in rats with Fe deficiency-induced anaemia that were given an Fe-sufficient purified diet without or with ascorbic acid (10.4 g/kg diet). Attention was focused on mucosal Fe uptake as measured in vivo by a double-isotope technique. Haemoglobin repletion and liver Fe levels were not affected when the ascorbic acid-supplemented diet was given, but apparent Fe absorption and retention of orally administered 59Fe were significantly enhanced. The distribution of Fe between liquid and solid phases of contents of both the stomach and the proximal intestine was not affected by the feeding of the ascorbic acid, but ascorbic acid significantly enhanced mucosal Fe uptake. It is concluded that ascorbic acid in the diet raises mucosal Fe uptake through a mechanism independent of the intestinal Fe solubility.

Protective effects of dietary lactulose and calcium phosphate against Salmonella infection.

BACKGROUND: Protection against intestinal infections by lactulose fermentation might be enhanced by calcium phosphate, due to the increased buffering capacity and/or inhibition of the cytotoxicity of luminal contents. METHODS: SPF rats were fed a low-calcium control diet, a low-calcium/lactulose diet or a high-calcium/lactulose diet and orally infected with Salmonella enteritidis. Faeces and urine were sampled for microbiological and chemical analyses. RESULTS: The lactulose-fed rats had a better colonization resistance. Translocation was reduced by dietary calcium, whereas lactulose was ineffective. In addition, calcium supplementation stimulated lactulose fermentation, reversed the lactulose-induced increase in cytotoxicity of faecal water and normalized growth and faecal consistency. CONCLUSIONS: The combination of dietary lactulose and calcium phosphate is protective against Salmonella infection.

Influence of dietary calcium phosphate on the disposition of bilirubin in rats with unconjugated hyperbilirubinemia.

The aim of this study was to test a possible form of therapy that could be used in the management of unconjugated hyperbilirubinemia. We hypothesized that unconjugated bilirubin (UCB) can permeate the intestinal wall and can thus be secreted with the feces. We have previously observed that UCB binds to amorphous calcium phosphate in vitro. Orally ingested amorphous calcium phosphate may act as a trapping agent for bilirubin in the intestine, thereby preventing back-diffusion across the intestinal wall. In this study, we tested whether feeding calcium phosphate leads to enhanced excretion of unconjugated bilirubin in Gunn rats. When a purified control diet was substituted by a high calcium phosphate diet, a decrease in bilirubin levels of 30% to 50% in male Gunn rats and of 23% in female rats was observed. The fecal output of bilirubin was more than doubled in Gunn rats in the first 3 days after the normal diet had been replaced by the high calcium-phosphate diet. The biological half-life of 3 H-labeled bilirubin in blood was 89.8 +/- 17.2 hours in rats fed the purified control diet and 50.9 +/- 1.4 hours in rats fed the high calcium phosphate diet (P = .004). After 30 weeks, plasma bilirubin levels were still significantly lower in Gunn rats fed a high calcium phosphate diet. No differences were found in plasma concentrations of calcium, magnesium, phosphate, urea, and creatinine in both Gunn rats and Wistar rats on control or high calcium phosphate diets. This therapy might be useful in the management of Crigler-Najjar patients, for example, as an adjunct to phototherapy.

Calcium and vitamin D: possible protective agents against colorectal cancer?

Nutritional factors are important determinants of colorectal cancer risk. Diets high in fat and/or low in fibre are especially recognised to increase risk. Dietary calcium and vitamin D have been suggested to be protective against colorectal cancer. With respect to calcium, its possible effect is thought to be mediated at least in part through intraluminal precipitation of hydrophobic, cytotoxic substances, in particular fatty and bile acids, which can promote colorectal cancer development. Data from studies in vitro and in animals support a protective effect of calcium, but studies in humans, both epidemiological and interventional, have given inconclusive results. With respect to vitamin D, data from only a small number of studies are available. Results suggest a protective effect by inhibition of cell proliferation, mediated through specific receptors. It is concluded that there are currently insufficient reasons to supplement subjects at increased colon cancer risk with calcium or vitamin D, especially when dietary intake of these substances is in agreement with general guidelines.

Randomized, double-blinded, placebo-controlled intervention study with supplemental calcium in families with hereditary nonpolyposis colorectal cancer.

BACKGROUND: A high-fat diet has been recognized for some time as a major risk factor for colorectal cancer. It is thought that fat promotes this disease by increasing the levels of fatty and bile acids within the colon. These acids irritate and damage the epithelial cells of the colon. As a result of this cellular destruction, an increase in the rate of cellular proliferation occurs. Oral calcium supplementation has been proposed as a dietary intervention for individuals at high risk of colorectal cancer because of its ability to reduce rectal epithelial cell proliferation through the binding of fatty and bile acids. Placebo-controlled studies, however, have yielded varying results. PURPOSE: We conducted a randomized, double-blinded, placebo-controlled trial to test oral calcium supplementation in patients at high risk of developing hereditary nonpolyposis colorectal cancer. METHODS: Thirty subjects at risk for this cancer, with an increased epithelial cell proliferation along the colon and rectum, were randomly assigned to either a placebo group (n = 15) or a treatment group (n = 15). They received either oral calcium carbonate (CaCO3) supplements (1.5 g) or placebo (cellulose and starch) three times a day during a 12-week period. Colonic biopsy specimens (rectal, sigmoidal, and descending) were obtained prior to and after the intervention trial, during endoscopy, for determination of labeling index (LI) of whole crypts and crypt compartments by 5-bromo-2'-deoxyuridine incorporation and immunohistochemistry. Proportional bile acid compositions in duodenal bile and cytolytic activity of fecal water were also determined. All P values represent two-tailed tests of statistical significance. RESULTS: Statistically significant reductions, comparing before with after intervention, in rectal whole-crypt LI after receiving either calcium supplements (from 10.9% +/- 5.2% [mean +/- SD] to 6.2% +/- 1.5%; P < .02) or placebo (from 11.7% +/- 4.7% to 8.2% +/- 3.1%; P < .05) were observed. In the three bowel segments, no statistically significant differences were observed between the supplemental calcium and placebo groups. A statistically significant reduction in cytolytic activity was determined during calcium supplementation (from 57% +/- 41% to 32% +/- 30%; P < .05), whereas in the placebo group, it did not change (from 42% +/- 41% to 36% +/- 27%; P > .10). CONCLUSIONS: Oral calcium supplementation was shown to cause only a minor nonstatistically significant reduction of epithelial cell proliferation in the rectum, compared with placebo, and to have no effect on the same parameter in the sigmoid and descending colon in first-degree relatives of hereditary nonpolyposis colorectal cancer patients. IMPLICATION: These results cast doubt on the value of calcium supplementation in the prevention of colorectal cancer, especially in individuals already consuming an adequate amount of dietary calcium.

Characterization of the adsorption of conjugated and unconjugated bile acids to insoluble, amorphous calcium phosphate.

Recently we showed that supplemental dietary calcium stimulates the intestinal formation of insoluble calcium phosphate and decreases the ratio of dihydroxy to trihydroxy bile acids in human duodenal bile. Because previous in vitro studies indicated that these effects could be due to differential adsorption of bile acids to amorphous calcium phosphate, we characterized the binding of bile acids to calcium phosphate. Freshly formed, amorphous, calcium phosphate bound and thus precipitated glycine-conjugated and unconjugated bile acids, whereas taurine-conjugated bile acids showed little binding. Glycochenodeoxycholic acid hardly adsorbed to other insoluble calcium phosphates, including hydroxyapatite. Adsorption studies using increasing amounts of glycine-conjugated and unconjugated bile acids showed that binding occurred above a bile acid-specific critical minimum concentration, dependent on bile acid hydrophobicity. The simultaneous use of a fluorescent hydrophobic probe indicated that this binding was due to ionic adsorption of monomers of bile acids, followed by their hydrophobic aggregation on the calcium phosphate surface, probably in the form of a bilayer. Finally, using human duodenal bile we found that amorphous calcium phosphate, but not Ca2+, preferentially bound and thus precipitated dihydroxy bile acids. We conclude that freshly formed, amorphous, calcium phosphate is a prerequisite for adsorption of bile acids and that monomers of glycine-conjugated and unconjugated dihydroxy bile acids have a high binding affinity for amorphous calcium phosphate.

Calcium supplementation as prophylaxis against colon cancer?

Dietary factors are major determinants of colorectal cancer risk. Especially a diet high in fat and low in fiber is recognized to be a risk factor. Dietary calcium has been suggested to be protective against colorectal cancer through the binding of intraluminal fatty acids and bile acids. Because of their cell-damaging properties these substances may stimulate colorectal epithelial cell proliferation and so promote colorectal cancer development. In this article data from in vitro, animal and human studies on the intraluminal effects of calcium, on its effects on colorectal epithelium and on the association between calcium intake and colorectal cancer are reviewed. It is concluded that at present it should be advised to bring dietary calcium intake into agreement with general dietary guidelines, but that high expectations of extra calcium as an effective mode of colon cancer prevention should not be encouraged.

Effects of supplemental dietary calcium on quantitative and qualitative fecal fat excretion in man.

Oral calcium supplementation is thought to be a useful interventional agent to decrease colon cancer risk. This is supposedly due, at least in part, to the binding of bile acids and fatty acids by calcium in the colon, thus prohibiting the damaging effects of these substances to the epithelium. To determine the effects of calcium supplementation on fecal fat excretion, 24 subjects kept a fat and calcium constant diet for one week and were supplemented with either 0, 2 or 4 g elemental calcium as calcium carbonate in a double-blind fashion. At the end of the week 72-hour feces was collected, and total fat, neutral fat, fatty acids and the ratio of polyunsaturated and saturated fatty acids (P/S ratio) were measured. Calcium dose-dependently increased the percentual excretion of total fat as related to fat intake: 6.8 +/- 0.9% during 0 g, 7.4 +/- 1.0% during 2 g and 10.2 +/- 1.4% during 4 g, r = 0.44, p = 0.03. This was due to increased fatty acid excretion, excretion of neutral fat was not affected, nor was the P/S ratio. It is concluded that calcium supplementation modestly increases fecal fatty acid excretion. No adverse metabolic effects are to be expected from this in case of long-term calcium supplementation in subjects at increased risk for colon cancer.

Mechanism of the antiproliferative effect of milk mineral and other calcium supplements on colonic epithelium.

Recently we have shown that supplemental dietary calcium precipitates luminal cytolytic surfactants and thus inhibits colonic epithelial proliferation, which may decrease the risk of colon cancer. In Western diets, milk products are quantitatively the most important source of dietary calcium. However, they also contain large amounts of phosphate, which has been hypothesized to inhibit the antiproliferative effect of calcium. Therefore, we studied in rats the possible differential antiproliferative effects of dairy calcium, calcium carbonate, and calcium phosphate, supplemented to a Western high-risk control diet. We observed that fecal bile acid excretion was similar in the various diet groups, whereas fatty acid excretion was stimulated by the calcium supplements in the order calcium carbonate > calcium phosphate > milk mineral. In fecal water, concentrations of bile acids and fatty acids were drastically decreased in the supplemented groups, resulting in decreased cytolytic activity of fecal water. In vitro incubation of fecal water from the control group with insoluble calcium phosphate also decreased the high concentrations of surfactants and their cytolytic activity. The response of the colonic epithelium to these primary luminal effects of calcium was a decrease in cell damage and cell proliferation. Only minor differences between the supplements were observed. The concentration of serum gastrin, the possible trophic effect of which could counteract the antiproliferative effect of calcium, was increased by the supplements, but no significant correlation was observed between serum gastrin concentration and epithelial proliferation. We conclude that dietary calcium precipitates luminal surfactants and thus inhibits cytolytic activity, epithelial cell damage, and colonic proliferation. The similar efficacy of calcium carbonate, calcium phosphate, and milk mineral indicates that the antiproliferative effect of milk mineral is mediated by its calcium content and is not inhibited by phosphate.

Epithelial cell proliferation in the sigmoid colon of patients with adenomatous polyps increases during oral calcium supplementation.

To study the effect of oral supplemental calcium on colonic epithelial proliferation, 17 adenomatous polyp patients received 1.5 g Ca2+ as calcium carbonate daily during 12 weeks, while on a calcium constant diet, based on the patients' habitual diet. Seven subsequently continued calcium supplementation for 9 months without dietary restrictions. Epithelial proliferation rate in colonic biopsies, expressed as labelling index (%), was determined with 5-bromodeoxyuridine and immunohistochemistry. Biopsies were taken from the midsigmoid at time of polyp excision and at the end of the intervention period. Median labelling index increased from 6.1% before to 8.7% after 12 weeks calcium (n = 17, P < 0.02). This was due to increased labelling in the basal third of the crypts (11.9 vs 16%), whereas labelling in mid and luminal compartments was not affected. Labelling index remained increased after 1 year calcium supplementation at 8.8%. Crypt length was not affected by calcium. These results are in contrast to those of others, who have shown a decrease of rectal epithelial proliferation during similar doses of calcium. Therefore, the effect of nutritional intervention on colonic epithelial proliferation should be studied in biopsies taken not only from the rectum, but also from more proximal parts of the colon. Caution with respect to large scale intervention studies with calcium in high risk groups is mandatory.

Cytotoxicity of fecal water is dependent on the type of dietary fat and is reduced by supplemental calcium phosphate in rats.

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)