Gut 1993; 34:365-370

Effects of dietary calcium and phosphate on theintestinal interactions between calcium, phosphate,fatty acids, and bile acids

M J A P Govers, R Van der Meer

AbstractLuminal free fatty acids and bile acids maydamage the colonic epithelium and stimulateproliferation, which may increase the risk ofcolon cancer. It has been suggested that onlysoluble calcium ions (Ca'+) precipitate fattyacids and bile acids, thus reducing their lyticactivity. Consequently, precipitation ofluminal Ca2' by dietary phosphate shouldinhibit these effects. To evaluate the proposedantagonistic effects of dietary calcium andphosphate, we studied the intestinal inter-actions between calcium, phosphate, fattyacids, and bile acids in rats fed purified dietsthat differed only in the concentrations ofcalcium and phosphate. Increased dietarycalcium drastically decreased the solubility offatty acids in the ileum, colon, and faeces, aswell as the solubility of bile acids in the colonand faeces. Although dietary calcium stronglyincreased the total faecal fatty acid concentra-tion and hardly affected the total faecal bileacid concentration, the fatty acid and bile acidconcentrations in faecal water were drasticallydecreased by dietary calcium. Consequently,the lytic activity offaecal water was decreased.Dietary phosphate did not interfere with theseintestinal effects of calcium. These resultsindicate that dietary phosphate does not inhibitthe protective effects of dietary calcium onluminal solubility and the lytic activity of fattyand bile acids.(Gut 1993; 34: 365-370)

Department of Nutrition,Netherlands Institute forDairy Research, Ede,The NetherlandsM J A P GoversR Van der MeerCorrespondence to:Dr R Van der Meer,Department of Nutrition,NIZO, PO Box 20, 6710 BAEde, The Netherlands.Accepted for publication31 August 1992

Epidemiological studies indicate that the incid-ence of colon cancer is positively correlated withthe dietary intake of fat,' and negatively corre-

lated with the intake of calcium.4 To explainthese associations it has been hypothesised" thatdietary fat increases the colonic concentrations ofsoluble free fatty acids and secondary bile acids.These hydrophobic surfactants may damagecolonic epithelial cells and consequently stimu-late the proliferation of colonic crypt cells,67which may increase the risk of colon cancer.'With regard to the protective effects of dietarycalcium, Newmark et all hypothesised thatsoluble calcium (Ca2) in the intestinal lumenprecipitates fatty acids and bile acids and thusprevents their lytic effects on colonic epithelium.They suggested that dietary phosphate inhibitsthis protective effect of calcium, because phos-phate will also bind luminal calcium ions andthus reduce the amount of calcium available forfatty acid and bile acid precipitation.The protective effects of dietary calcium on

the lytic activity of faecal water and on colonic

proliferation have been ascertained in manyrodent and human studies."'"'2 However, themechanism of these protective effects as well asthe proposed inhibitory effect of phosphate havehardly been studied in vivo. Only Hu et alI3reported recently that phosphate inhibits theprotective effects of calcium on epithelial pro-liferation caused by intrarectally instilled deoxy-cholate. Their study design, however, precludedthe normal physiological interactions betweendietary calcium, phosphate, fatty acids, and bileacids in the small and large intestine. In vitroexperiments performed in our laboratory haveshown that the lytic activity of bile acidsincreases in the presence of Ca2+ and decreaseswhen phosphate is added. This inhibition iscaused by the formation of insoluble precipitatesof calcium, phosphate, and bile acid micelles. 14 Itshould be noted that these in vitro results arecontrast with the hypothesis mentioned above.Because phosphate is approximately equimolarto calcium in animal diets and is in molar excessof calcium in human diets, quantification of theintestinal interactions between calcium, phos-phate, fatty acids, and bile acids in vivo isrelevant for a proper evaluation of the effects ofdietary calcium and phosphate. Therefore, in thepresent study these interactions were studied in astrictly controlled experiment with rats, usingdietary calcium and phosphate concentrationswhich mimic their concentrations in animal andhuman diets. In addition, to determine thedietary effects on the cytolytic potency of theintestinal contents, luminal lytic activity wasmeasured as lysis oferythrocytes by faecal water.

Methods

ANIMALS AND DIETSEight week old female outbred Wistar rats(Laboratory Animal Center of the WageningenAgricultural University) were housed individu-ally. For 2 weeks, eight groups of sit rats eachwere fed purified diets, differing only in theconcentrations of calcium and phosphate. Thebasal diet contained (per kg) 200 g casein (acidcasein, DMV Veghel, The Netherlands), 471 gdextrose, 20 g cellulose, 35 g mineral mix, 10 gvitamin mix, 180 g palm oil, 20 g corn oil, and10 g cholesterol. Cholesterol was added toincrease the intestinal concentration of bileacids, because dietary cholesterol stimulates bileacid synthesis in rats.' Because of the presenceof casein, a phosphorylated protein, this basaldiet contained 50 [imol/g phosphate. Varyingamounts of CaCO3 and NaH2PO4/KH2PO4 (25,75, and 225 Ftmol/g) were added to the basal diet

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in exchange for washed sand. These concentra-tions were chosen to cover a wide range aroundthe recommended dietary concentration of 130[imol/g CaHPO4 in the standard AIN-76 diet.'6The diets were balanced for sodium and potas-sium (final concentrations 125 and 100 [tmol/g)with Na2CO3 and K2CO3 respectively. Dietaryconcentrations of other minerals and of vitaminswere according to the AIN-76 recommenda-tions. 16

Diets and water were supplied ad libitum.Food consumption was recorded every 3 daysand the animals were weighed weekly. Faeceswere collected quantitatively from days 11 to 14.The interactions between calcium, phosphate,

fatty acids, and bile acids in the distal smallintestine as well as in the colon were studied indetail in the groups with the lowest and thehighest concentrations of dietary calcium andphosphate. Rats fed the low calcium (25 ,tmol/g)and phosphate (75 ,umol/g) control diet or thisdiet supplemented with either 200 ,umol/gcalcium or 200 [imol/g calcium and phosphatewere killed by decapitation on day 14 between10.00 and 12.00 am. The distal third part of thesmall intestine (ileum) and the colon were

excised and their contents collected.

TREATMENT OF ILEAL, COLONIC, AND FAECALSAMPLESIleal contents were incubated in a shaking water-bath (5 minutes at 37°C) and subsequentlycentrifuged for 10 minutes at 15 000 g(Eppendorf5415). Supernatant was stored at -20°C untilfurther use and the pellet was freeze dried.Colonic contents and faeces were freeze dried.Colonic and faecal water were prepared byreconstituting freeze dried samples with doubledistilled water to respectively 20% and 35% dryweight, to mimic the conditions in the proximaland distal colon. After homogenisation, thesamples were incubated in a shaking waterbath(1 hour at 37°C), followed by centrifugation for10 minutes at 15000 g. The supernatant was

stored at -20°C until further use. Using thisprocedure, faecal water from freeze dried faecesin control experiments did not differ from faecalwater prepared from fresh faeces with regard toconcentrations of calcium, phosphate, fattyacids, and bile acids'0 and with regard to lyticactivity. II

ANALYSES

After dry ashing ofthe freeze dried samples, totalcalcium was measured using an atomic absorp-tion spectophotometer (Model 1100, Perkin-Elmer Corp, Norwalk, CT, USA) and totalphosphate was measured as described by Fiskeand Subbarow. 7 Supernatants were acidifiedwith trichloroacetic acid (final concentration 5%w/v) and analysed for calcium and phosphate as

described above. Total and soluble fatty acidswere determined as follows. Freeze driedsamples and supernatants were acidified withHCI (final concentration 4 M) and fatty acidswere subsequently extracted three times with 5volumes of diethyl ether. After evaporation ofdiethyl ether, fatty acids were resolubilised in

ethanol and quantified using a colorimetricenzymatic assay (NEFA-C, Wako Chemicals,Neuss, Germany). In control experiments thisextraction procedure resulted in >95% recoveryof added fatty acids. Total bile acids wereextracted from freeze dried samples with at-butanol/water mixture (1:1, v/v), as describedpreviously. 18 Extracts and supernatants wereassayed for bile acids using a fluorimetric enzy-matic assay (Sterognost 3ca-flu, Nycomed AS,Oslo, Norway).

DETERMINATION OF LYTIC ACTIVITYHuman erythrocytes were isolated and washed asdescribed by Coleman et al.'9 Increasing volumesof faecal water (0-160 1d) were mixed with 154mM NaCl to a total volume of 160 1d. Afterpreincubation for 5 minutes at 37°C in a shakingwaterbath, 40 p1 erythrocytes (final haematocrit5%) were added. Simultaneously, erythrocyteswere incubated in 154 mM NaCl (0% lysis) andin double distilled water (100% lysis). Afterincubation for 30 minutes at 37°C, samples werecentrifuged for a minute at 10 000 g. Haemolysiswas determined by measuring the Fe content ofthe supernatant using an atomic absorptionspectrophotometer (Perkin-Elmer 1100). Thelytic activity was quantified as the area under thelytic curve and expressed as a percentage of themaximal area, which implies 100% lysis oferythrocytes at each dilution of faecal water. Werecently found a high correlation betweenluminal lytic activity and in vivo colonic pro-liferation as determined by lysis of erythrocytesand 3H-thymidine incorporation, respectively. '.

STATISTICSResults are given as means of six rats per groupwith standard errors. After analysis of variance,differences between means were subjected to theleast significant difference (LSD) test. Differ-ences were regarded as significant ifp<0 05.

ResultsNo differences were observed between groups infood intake (mean 13-0 g/day) or in final bodyweight (mean 175 g). The daily output of faeceswas slightly enhanced by dietary calcium anddecreased by dietary phosphate (Table I).

First, we studied the intestinal interactions ofcalcium, phosphate, fatty acids, and bile acids inanimals fed the low calcium and phosphatecontrol diet or this diet supplemented with eithercalcium or calcium and phosphate. Figure 1shows the effects of these diets on the concentra-

TABLE I Effects of diets onfaecal output (giday) (values,mean (SEM), n=6)

Dietary Dietary calcium ([tmol/g)phosphate(pmol/g) 25 75 225

75 1-19 (0 09)A 1-16 (0-05)" 1-60 (0-07)"'125 1-02 (0 10)A 160 (0 09)"'275 0-80 (0-03)A 102 (0 09)" 1-21 (009)""'

Values in the same row that do not share the same capital letter,and those in the same column that do not share the same letter aresignificantly different (p<005).

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Figure 1: Effects of the lowcalcium (25 smollg) andphosphate (75 [tmol/g)control diet (open bars) andofthe control dietsupplemented with either200imollg calcium (hatchedbars) or 200 ptmollg calciumand phosphate (filled bars)on the intestinalconcentrations ofsoluble andinsoluble calcium andphosphate. Whole barsreflect the totalconcentrations (mean(SEM), n=6). *Significantv the control diet (p<0 05).**Significant v the highcalcium diet (p<0 05).

Figure 2: Effects ofthe lowcalcium (25 [tmollg) andphosphate (75 [tmollg)control diet (open bars) andofthe control dietsupplemented with either 200[tmollg calcium (hatchedbars) or 200 [smollg calciumand phosphate (filled bars)on the intestinal solubility offatty acids and bile acids(mean (SEM), n=6).*Significant v the control diet(p<0 05). No significanteffects ofdietary phosphatewere observed.

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tions of soluble and insoluble calcium and phos-phate in the ileum, colon, and faeces. Highdietary calcium, as well as additional supple-mentation with phosphate, hardly affected thesoluble concentrations of calcium and phosphatein the ileum, colon, and faeces. On the otherhand, these treatments drastically increased theconcentrations of insoluble calcium. Calciumsupplementation increased the intestinal concen-trations of insoluble phosphate, which indicatesformation of insoluble calcium phosphate. Thiswas stimulated further by additional supple-

TABLE ii Effects ofdiets on the total concentrations ([mol/g)offatty acids and bile acids in the ileum, colon, andfaeces(values, mean (SEM), n=6)

Dietary calcium ([tmollg) 25 225 225Dietary phosphate (molfg) 75 75 275

Ileum:Fatty acids 35 (4)a 150 (14)h 162 (19)hBile acids 80 (10) 56 (7) 68 (11)

Colon:Fatty acids 39 (3)a 495 (14)b 297 (35)'Bile acids 19 (4) 16 (2) 18 (2)

Faeces:Fatty acids 97 (10) 951 (29)h 704 (68)'Bile acids 38 (5) 25 (2) 27 (3)

Values in the same row that do not share the same letter aresignificantly different (p<O0OS).

Fatty acids

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mentation with phosphate, especially in thecolon and faeces.The total fatty acid and bile acid concentra-

tions along the intestine are shown in Table II.Because of these diet induced differences in totalfatty acid concentrations, the solubility of fattyacids and bile acids is expressed relative to theirtotal concentrations (Fig 2). Dietary calciumsupplementation drastically reduced thesolubility of fatty acids in the ileum, and thiseffect was maintained in the colon and faeces.Calcium supplementation did not influence thesolubility of bile acids in the ileum, but itreduced their solubility to about 20% in the colonand faeces. Additional phosphate supplementa-tion did not affect the calcium induced reductionin solubility of fatty acids and bile acids.

Because of the similarity of the effectsobserved in the colon and faeces, we subse-quently studied the concentration dependence ofthe effects of dietary calcium and phosphate inthe faeces. Figures 3 and 4 show the effects of thedifferent diets on the total faecal concentrationsof calcium, phosphate, fatty acids, and bileacids. As expected, increased dietary calciumincreased the total calcium in faeces and this wasonly slightly stimulated by phosphate supple-mentation of the high calcium diet (Fig 3).Increased dietary calcium increased faecal phos-phate because the formation of insoluble calciumphosphate inhibits the absorption of phosphate.This effect was more pronounced in rats takingthe high phosphate diets. Dietary calciumsupplementation drastically increased the totalfaecal fatty acid concentration (Fig 4) and thisreflects the inhibition of the intestinal absorptionoffatty acids. This inhibition was partly counter-acted by increased dietary phosphate. In contrastto fatty acids, the total bile acid concentrationwas hardly influenced by the different diets. Theobserved slight decrease in faecal bile acid con-centration with increasing dietary calciumconcentrations reflects the concomitant increasein faecal mass (Table I). Therefore no significant

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Dietary calcium (pmol/g)Figure 3: Effects ofdietary calcium and phosphate (O 75;t 125; 0 275 Itmol/g) on the total faecal concentrations ofcalcium and phosphate (mean (SEM), n =6). Different curvesindicate a significant effect ofdietary phosphate. SEMs areeither smaller than symbols or indicated by bars.

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Figure 4: Effects ofdietarycalcium and phosphate (O75; 4 125; 0 275 [omollg)on the total faecalconcentrations offatty acidsand bile acids (mean(SEM), n=6). Differentcurves indicate a significanteffect ofdietary phosphate.SEMs are either smallerthan symbols or indicated bybars.

1000 FFatty acids

750 -

500 -

250 F

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Bile acids

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25 75-v

Dietary calcium (pmc

effect of the different diets on theoutput of bile acids was observed.We subsequently determined the s

concentrations of calcium, phospacids, and bile acids. Supplemercalcium did not increase the conceisoluble calcium (Fig 5), notwithstlarge increase in total faecal calcium.low calcium diet was soluble calciiantly decreased, as soluble phozincreased by phosphate supplementsdiet. Phosphate supplementation omediate and high calcium diets insoluble phosphate value, but decreasefaecal water. The net result of these;effects was that no significant changcalcium was observed. The logariconcentration product of calcium an(was negatively correlated with the Fwater (r=-0-75, n=48; p<O-(indicates that the soluble faecal concecalcium and phosphate are controlle

8-0

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6 5

Figure 5: Effects ofdietarycalcium and phosphate (O75; C 125; 0 275 pmollg)on thepH offaecal water onthe solublefaecalconcentrations ofcalciumand phosphate (mean(SEM), n=6). Differentcurves indicate a significanteffect ofdietary phosphate.SEMs are either smallerthan symbols or indicated bybars.

20

15

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5

0

50

40

30

20

10

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pH

. /~~~~_ -

Calcium

Phosphate

25 75

Dietary calcium ([mo

dependent solubility product of calcium phos-phate.'0

Increased dietary calcium decreased the fattyacid concentration in faecal water (Fig 6).Supplementary dietary phosphate decreased thefatty acid concentration only on the low calciumdiet. The bile acid concentration in faecal wateralso decreased with increasing dietary calcium,and this was not influenced by dietary phos-phate. The logarithms of soluble fatty acidconcentrations in the faeces were highly corre-lated with the logarithms of precipitated faecalcalcium and phosphate concentrations (respect-ively -0 85 and -0-76, n=48; p<0-001).Similar negative correlations were found for thelogarithms of soluble bile acids and of precipi-tated calcium and phosphate (respectively -0-80and -0-60, n=48; p<0-001).

Finally, using lysis of erythrocytes, we ascer-225 tained whether the decreased luminal solubility

Al/g) of fatty acids and bile acids affected the cytolyticpotency of the intestinal contents. The lyticactivity of faecal water was high on the lowcalcium diets (Fig 6). Corresponding with the

total daily decreased solubility of fatty acids and bile acids,the lytic activity was completely inhibited by the

oluble faecal highest level ofdietary calcium and no significanthate, fatty effect of dietary phosphate was observed.ital dietaryntrations oftanding the DiscussionOnly on the These results show for the first time to ouram signific- knowledge, the interactions between calcium,sphate was phosphate, fatty acids, and bile acids along theation of this intestine. Extrapolating these results to humanbf the inter- diets, the lowest calcium concentration used inicreased the the present study (25 ,tmol/g) reflects a dailyed the pH of intake of calcium of about 12-5 mmol (500 mg)antagonistic which is about 60% of the recommended dietaryre in soluble allowance for calcium. Hence, increasing thethm of the calcium concentration to 75 and 225 ,tmol/gd phosphate mimics a supplementing the human diet with 25)H of faecal and 100 mmol/day, respectively. The phosphate)01). This concentrations were changed accordingly tontrations of incorporate the molar calcium to phosphate ratiod by the pH of about 0 5, which is typical for human diets.20

It was shown that dietary phosphate does notinterfere with the protective effects of dietarycalcium on luminal solubility and lytic activity of

5 fatty acids and bile acids. This is supported byour study with human volunteers,20 in which anexcess of dietary phosphate did not prevent theprotective effects of supplemental dietarycalcium on solubility and lytic activity of luminalsurfactants. Our study does not, therefore, offeran explanation for the recent observations of Huet al,3 who found that phosphate inhibits the

I protective effect of dietary calcium on colonicproliferation in rats. These authors, however,

e used intrarectal instillation of deoxycholate com-bined with short term (about 24 hours) oralintubations of calcium and phosphate. Thisdesign precludes, in our opinion, the normalphysiological interaction between calcium, phos-phate, and bile acids in the small and largeintestine. Our results are in accordance withother studies in rodents, which showed thatdietary supplementation with calcium phosphate

225 inhibits lytic activity of faecal water and colonicI/g) proliferation."""'" The extent of the reported

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Effects ofdietary calcium andphosphate on the intestinal interactions between calcium, phosphate, fatty acids, and bile acids3

Figure 6: Effects ofdietarycalcium and phosphate (O75; C 125; * 275 [tmollg)on the solublefaecalconcentrations offatty acidsand bile acids and on the lyticactivity offaecal water(mean (SEM), n=6).Different curves indicate asignificant effect ofdietaryphosphate. SEMs are eithersmaller than symbols orindicated by bars.

40

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Dietary calcium (,umol/g)

inhibition is comparable with that found in otherstudies using similar amounts of calcium carbon-ate or calcium lactate.72"2" In our opinion, theseresults already suggest that the type of calciumsalt does not interfere with the protective effectsof dietary calcium on lytic activity of faecal wateran on proliferation. In none of these studies,however, were the intestinal interactionsbetween calcium, phosphate, fatty acids, andbile acids determined. Quantification of theseinteractions is essential for a proper discussion ofthe intestinal effects of dietary calcium andphosphate.

Dietary supplementation with calciumcarbonate did not increase the soluble calciumconcentrations along the intestine and in thefaeces (Figs 1 and 5), notwithstanding the largeincrease in the total faecal calcium concentra-tions (Figs 1 and 3). The present results indicatethat a substantial amount of this total calcium isassociated with insoluble phosphate, which is inaccordance with results obtained in otheranimal29 and human20 studies. As shownearlier,2930 calcium phosphate starts precipitat-ing at a pH of about 6. Therefore, high con-

centrations of soluble calcium can only exist atthe relatively low pH of the proximal smallintestine,3' or when no phosphate is present, as

can be derived from the observed negativecorrelation between the logarithm of thesolubility product of calcium phosphate and thepH of faecal water.

Increased dietary calcium drastically increasedthe faecal excretion of fatty acids (Fig 4), whichhas also been found in other studies.2032 Thisreflects inhibition of the absorption of dietaryfat. On the low phosphate diets this inhibition isprobably caused by precipitation of fatty acids byhigh concentrations of soluble calcium in theproximal small intestine. As shown in Figure 2,this effect of calcium on the solubility of fatty

acids persisted in the ileum and colon. Thestimulation of fatty acid excretion was slightlycounteracted by dietary phosphate (Fig 4),which indicates that under these conditions,formation of insoluble calcium phosphate hadalready occurred in the proximal small intestine.Our results indicate that this effect of phosphateis confined to the proximal small intestinebecause increased dietary phosphate did notsignificantly increase the solubility of fatty acidsin the ileum, colon, and faeces (Fig 2). We donot, at present, have an adequate explanation forthe apparently anomalous effect of phosphate onthe fatty acid concentration in faecal water in ratsfed the low calcium diet, and this requiresfurther investigation.The present results show that dietary calcium

has a site specific effect on the solubility of bileacids. It can be speculated that the calciumdependent precipitation of bile acids in the colonis a result of their microbial deconjugation anddehydroxylation in this compartment. Bile acidsin the rat are predominantly conjugated withtaurine.33 As shown earlier,'430 taurine con-jugated bile acids are not precipitated by calciumor calcium phosphate. In contrast, the uncon-jugated, carboxylic, bile acids are easily precipi-tated by calcium phosphate.3"The observation that bile acids are not precipi-

tated by calcium in the ileum, where reabsorp-tion of bile acids takes place, explains why totalfaecal bile acid output is hardly affected bydietary calcium (Fig 4). In contrast, fatty acidexcretion is strongly increased by dietarycalcium, suggesting that fatty acids are precipi-tated by calcium before the absorption site, theproximal small intestine, is reached. These find-ings indicate that the total faecal concentrationsof fatty acids and bile acids are not determined bythe amount of intraluminally formed complexes,as was recently suggested by Appleton et al,34 butare determined by the site at which this occurs.We found significant negative correlations in

faeces between soluble fatty acids and bile acidsand insoluble calcium and phosphate. Althoughcorrelations never prove a causal relationship,they may indicate that the solubilities of fattyacids and bile acids are determined by insolublecalcium phosphate. This is in accordance withour in vitro studies,'43 showing that calciumphosphate decreases the solubility of bile acids.

Finally, a pertinent finding of the presentstudy is that the protective effect of dietarycalcium on the lytic activity of faecal watercorrelated with its effect on the concentrations ofsoluble fatty acids and bile acids (Fig 6), but notwith their total concentrations (Fig 4). In accord-ance with the results of several other studies,"' '315this indicates that these soluble surfactants arethe main determinants of luminal lytic activity.We recently found that this luminal lytic activityis highly correlated with in vivo proliferation ofcolonic epithelium,"'` which indicates that thesesoluble surfactants may affect the risk of coloncancer.8 36

In conclusion these results show that dietarycalcium stimulates the formation of insolublecalcium phosphate and decreases the luminalsolubility of fatty acids and bile acids in rats.Luminal lytic activity is consequently inhibited.

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These protective effects of calcium are notinhibited by a fourfold increase in dietaryphosphate.

The authors thank Maria A W Peters and Gerrit Van Tintelen fortaking care of the animals and John A Lapre for helpfuldiscussions.

This study was supported by Grant 900-562-078 of the DutchOrganization for Scientific Research (NWO). Part of this work hasbeen presented at the Digestive Disease Week, New Orleans, LA,May 19-22, 1991.

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