999

Fecal Bile Acid Excretion and Composition in Response to Changes in Dietary Wheat Bran, Fat and Calcium in the Rat Marie L. Borum a, Kathleen L. Shehan a, Hans Fromm a,*, Saleem Jahangeer b, Marianne K. Floor b and Oliver Alabaster ~ aDivision oI Gastroenterology and Nutrition and bInstitute for Disease Prevention, Department of Medicine, The George Washington University Medical Center, Washington, D.C. 20037 The effect and possible interactive influence of different dietary amounts of wheat bran, fat and calcium on the fecal excretion, concentration and composition of bile acids was studied in Fischer-344 rats. The fecal bile acids were analyzed using gas-liquid chromatography. Dietary wheat bran increased both total bile acid excretion and fecal weight without changes in fecal bile acid concentration. The proportion of fecal hyodeoxycholic acid decreased with increasing dietary fiber, whereas that of lithocholic and deoxycholic acids increased significantly with fiber intake. The percent content of fecal chenodeoxycholic acid did not change. Increasing dietary fat led to an increase in bile acid excretion without changes in either fecal weight or bile acid concentration. In contrast, the level of dietary calcium did not affect the total excretion of bile acids. However, since calcium increased the fecal weight, it consequently diluted bile acids and decreased their fecal concentration. Dietary fat and calcium had no influence on fecal bile acid composition. There were no interactive effects of wheat bran, fat and calcium on fecal bile acids. The finding in this study that dietary fiber, fat and calcium induce significant changes in fecal bile acids may be of relevance to the potential of bile acids to promote carcinogenesis. Lipids 27, 999-1004 (1992). Bile acids have been shown to express cancer~pmmoting pr~ perties which are modified by dietary factors (1-18). In particular, fiber, fat and calcium have received increased attention for their ability to induce changes in the metabolism of bile acids which affect their potential for promoting the development of experimental colon cancer (6-18). Whereas both dietary fiber and calcium can suppress the experimental induction of colon cancer by carcinogens (10,12,14-16,18), increased ingestion of fat has the opposite effect (7,13,19). The potential changes in bile acid metabolism, which are considered to be relevant to the promotion of colon cancer, consist of increases in the fecal excretion and concentration of bile acids as well as their modification by bacteria Most authors have reported that diets high in any type of fiber lead to both an increase in the total excretion and a suppression in the bacterial degradation of bile acids (6,9,11, 15,20-23), but the effect on fecal bile acid concentration appears to be less certain (11,23-26). Several studies in human subjects indicate that fecal concentrations of bile acids decrease after the ingestion of a diet high in either wheat bran or a mixtue of dietary fibers (24,25). In other human studies that involved diets high in oat bran and citrus pectin, respectively, no decreases in fecal bile acid concentrations were observed (20,22). On the other hand, a high fat intake has been reported *To whom correspondence should be addressed at Division of Gastroenterology and Nutrition, The George Washington University Medical Center, 2150 Pennsylvania Avenue NW, Suite 5-405. Washington, D.C. 20037. Abbreviation: GLM, group least square means.

to result in an increased fecal excretion and concentration of bile acids (7,14). Although it is known that calcium binds to bile acids (27,28), little information is available regarding the effect of dietary calcium on fecal bile acids. There also appear to be no systematic studies of the combined or interactive effects of fiber, fat and calcium on bile acids. The aim of the study reported in the present paper, therefore, was to determine the individual effects of different levels of wheat bran, fat and calciun% and the influence of their possible interactions on the fecal excretion, concentration and composition of bile acids.

MATERIALS AND METHODS

Study design and animal groups. Male albino Fisher-344 rats (F344/NHsdBR, Hilltop Lab Animals, Inc., Scottsdale, PA), weighing approximately 190 to 225 g, were used to measure the effects of 36 different experimental diets. The latter were characterized, in a factorial s t u d y (4 • 3 • 3), by the combinations of different concentrations, by weight, of wheat bran (0, 2.5, 10 and 20%), fat (1, 5, 10%), and calcium (0.18, 0.52 and 1.04%). The dietary fat, 1/3 of it saturated, 1/3 monounsaturated and 1/3 polyunsaturated, consisted of 44.2% lard, 46.2% corn oil and 9.6% hydrogenated coconut oil. Calcium carbonate served as the source of calcium in the diets. Two standard additional diets, Purina Rat Chow 5001 and AIN-76A (Purina, St. Louis, MO) served as controls. The calcium content of the Purina Rat Chow is 1%, and t h a t of the AIN-76A diet is 0.52%. The rats were kept for 28 d on these diets. Each of the 38 study groups consisted of 10 animals. Preparation of diets and collection of fecal samples. The above described combinations of wheat bran, fat and calcium were mixed into a basal diet. There were no other sources of fiber, fat and calcium. The only cholesterol within the diet was derived from the fat mixture. The basal diet had the following composition (g/kg): casein, 198; DL-methionine, 2; choline bitartrate, 2; sucrose, 369 to 269; dextrose, 369 to 269; a vitamin mixture, 10; and a salt mixture, 35. The salt mixture provided the phosphorus to the diets and did not vary. The diets followed the standard guidelines established for experimental animals (29). The diets were produced and pelleted by Dyets Inc. (Bethlehem, PA). The designated composition of the experimental diets was independently verified by analyses which were performed by two other laboratories. Following a one-week quarantine, the rats were housed in groups of two, under standard conditions of 23~ with a 12-h dark-light cycle, in the core animal facility of The George Washington University Medical Center, Washington, D.C. The animal care was provided according to HHS/N IH guidelines. The respective diets were fed along with water ad libitum for 28 d. To ensure an accurate assessment of animal intake, any food t h a t was spilled or uneaten was measured and the weight was subtracted from the total weight of food ration. Three days before the LIPID& Vol. 27, no. 12 (1992)

1000 M.L. BORUM

E T AL.

completion of the diet treatment, the animals were transferred, in groups of two, into metabolic cages which contained a funneled tray for accurate collection of feces, without contamination by urine or food. All feces were collected for 72 h, weighed and then stored in polypropylene test tubes at - 8 0 ~ overnight. Following lyophilization and mechanical homogenization, the fecal material was reweighed in order to determine the wet/dry ratio. The feces from each paired group were pooled and immediately stored at - 2 0 ~ until analyzed for fat and bile acid content. Fecal bile acid analysis9 The fecal bile acids were qualitatively and quantitatively analyzed using gas-liquid chromatography, essentially as previously described by this laboratory and others {30-33}. In brief, aliquots of fecal material, with nordeoxycholic acid added as an internal standard, were refluxed with absolute ethanol for 30 min, cooled and then decanted through filter paper and collected. This process was repeated using chloroform/ methanol (1:1, vol]vol). In order to separate the bile acids from the neutral sterols, the filtrate with the extracted bile acids was evaporated, then redissolved in 15 mL of 0.01 N HC1, and percolated through a prewashed Lipidex column (Lipidex 1000, Packard Instrument Corporation, Downers Grove, ILL The column was then washed with 20 mL of distilled water. The bile acids were eluted with 20 mL of 68% methanol. After elution from the column, the bile acids were subjected to alkaline hydrolysis. The deconjugated bile acids were extracted with dichloromethane, and the bile acid methyl ester acetates were formed as previously described (31-33). The recovery of bile acids in the extraction and deconjugation procedures was evaluated with the use of ~C-labelled bile acids. The recovery was 64 +_ 2.66 (M +_ SEM) % for lithocholic acid, 79 _+ 0.44% for cholic acid and 62 __ 3.77% for chenodeoxycholic acid. The derivatized bile acids were analyzed on a Packard Gas Chromatograph {Packard Instrument Corporation} using a 6-ft coiled column with a cyanosilicone stationary phase (3% AN-600 on Gas Chrom Q; Analabs, Camden, CT). Fecal fat analysis9 The fecal fat content was determined by the method of van de Kamer et al. {34). Fecal calcium analysis9 Fecal calcium was assayed by atomic absorption spectroscopy (Varian 2~chtron AA-5, Palo Alto, CA) using commercially prepared standards {Fisher Scientific, Silver Spring, MD) {35,36}. A 0.5% LaC13 standard was included in all calcium analyses to correct for phosphate interference. Statistical analysis. The effects of fiber, fat and calcium and their interactions were evaluated by three-way analysis of variance, using the group least square means (GLM) procedure in the SAS statistical computer package {37}. GLM were then compared using the conservative Bonferroni adjustment for multiple comparisons {38}9 Pearson correlations were also computed between selected pairs of variables. The criterion for statistical significance was set at a = 0.01.

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RESULTS

The overall results of the measurements of body weight and fecal weights and bile acids, as well as fecal fat and calcium content derived from rats in the combined dietary groups are presented in Table 1. The correlations between L I P I D S , Vol. 27, no. 12 (1992)

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1001

FIBER, FAT AND CALCIUM EFFECTS ON BILE ACIDS TABLE 2 Correlations Between Fecal Bile Acid Excretion and Fecal Weight, Fat and Calcium in the Different Dietary Groups a

Dietary groups 0 Fecal wt, wet (g/24 h) n.s. Fecal wt, dry (g/24 h) n.s. Fecal fat (mg fat/24 h) n.s. Fecal calcium (mEq/24 h) -0.549 b aCorrelation coefficients which

Fiber (%) 2.5 10

20

0.18

Calcium (%) 0.52

1.04

1

Fat (%) 5

10

All groups combined

n.s.

0.413 a

n.s.

0.407 a

0.635 c

0.563 c

n.s.

0.452 b

0.656 c

0.454 c

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

0.710 c

n.s.

n.s.

0.539 c

0.439 c

0.400 b

0.403 b

0.382 b

0.356 c

n.s. 0.448 b n.s. n.s. n.s. n.s. n.s. n.s. n.s. are statistically not significant are not presented (n.s.). ap < 0.01, bp < 0.005, cp < 0.001.

n.s.

TABLE 3 Correlations Between Fecal Bile Acid Concentration a and Fecal Weight, Fat and Calcium in the Different Dietary Groups a

Dietary groups Fiber (%) 2.5 10

20

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0 Fecal wt, wet (g/24 h) Fecal wt, dry (g/24 h) Fecal fat (g/24 h) Fecal calcium (mEq/24 h)

Calcium (%) 0.52 1.04

1

Fat (%) 5

10

All groups combined

n.s.

n.s.

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aFecal bile acid concentration per fecal dry weight. bSee Table 2, footnote a.

t h e fecal bile acids, a n d fecal w e i g h t , fat, a n d c a l c i u m , r e s p e c t i v e l y , are p r e s e n t e d in Table 2 a n d Table 3. T h e effect of v a r y i n g c o n c e n t r a t i o n of d i e t a r y fiber, f a t a n d c a l c i u m u p o n r a t b o d y w e i g h t , fecal weight, bile a c i d conc e n t r a t i o n s , p e r c e n t c o m p o s i t i o n of t h e i n d i v i d u a l bile acids, a s well a s t o t a l a n d i n d i v i d u a l fecal bile a c i d excretion was analyzed. Body weight. T h e a v e r a g e w e i g h t gain, w h i c h w a s v e r y s i m i l a r in t h e d i f f e r e n t g r o u p s , w a s a p p r o x i m a t e l y 65 g. T h e r e w a s n o s i g n i f i c a n t difference in t h e a m o u n t of d i e t consumed between the different experimental groups. N e i t h e r t h e fiber n o r t h e c a l c i u m c o n t e n t of t h e d i e t h a d a n y a p p r e c i a b l e i n f l u e n c e on w e i g h t gain. However, t h e r e w a s a small, b u t s t a t i s t i c a l l y s i g n i f i c a n t i n c r e a s e in b o d y w e i g h t w i t h i n c r e a s i n g d i e t a r y fat. T h e r e w a s a signific a n t i n c r e a s e (P < 0.001) in a n i m a l w e i g h t in t h e 10% d i e t a r y f a t g r o u p (Table 1). Dietary fiber. F e c a l w e t w e i g h t (Fig. 1) a s well a s fecal dry weight increased significantly with increasing dietary fiber (P < 0.001). T h e t o t a l fecal e x c r e t i o n of bile a c i d s inc r e a s e d s i g n i f i c a n t l y w i t h i n c r e a s i n g d i e t a r y f i b e r (Fig. 2). I t i n c r e a s e d f r o m a m e a n v a l u e of 5.48 in t h e 0% fiber g r o u p t o t h a t of 23.66 m m o l / 2 4 h in t h e a n i m a l s w h i c h r e c e i v e d 20% fiber (P < 0.001) (Table 1). T h i s s i g n i f i c a n t increase of fecal bile acid excretion w i t h i n c r e a s i n g d i e t a r y fiber c o n t e n t w a s seen w i t h lithocholic, c h e n o d e o x y c h o l i r d e o x y c h o l i c a n d h y o d e o x y c h o l i c acids, w h i c h c o n s t i t u t e d t h e m a j o r fecal bile a c i d s (Fig. 3, Table 4). Total b i l e a c i d excretion showed significant positive correlations with

fecal w e t w e i g h t (P < 0.01), fecal f a t c o n t e n t (P < 0.001) a n d fecal c a l c i u m e x c r e t i o n (P < 0.005), in t h e 10% fiber group, a s well a s a n e g a t i v e c o r r e l a t i o n (P < 0.005} w i t h fecal c a l c i u m c o n t e n t in t h e 0% f i b e r g r o u p (Table 2 a n d Table 3). T h e fecal bile a c i d c o n c e n t r a t i o n s , b a s e d on b o t h w e t a n d d r y fecal w e i g h t , were n o t a f f e c t e d b y i n c r e a s i n g d i e t a r y fiber. W i t h t h e e x c e p t i o n of t h e 0% f i b e r group, t h e r e w a s no c o r r e l a t i o n b e t w e e n fecal bile a c i d concent r a t i o n p e r fecal d r y w e i g h t or fecal w e t w e i g h t , a n d fecal w e t w e i g h t , d r y w e i g h t , f a t a n d calcium. I n t h e 0% fiber group, t h e bile a c i d c o n c e n t r a t i o n s h o w e d a s i g n i f i c a n t n e g a t i v e c o r r e l a t i o n (P < 0.001) w i t h fecal d r y w e i g h t a n d c a l c i u m (P < 0.001). T h e d i e t a r y w h e a t b r a n - a s s o c i a t e d c h a n g e s in fecal acid c o m p o s i t i o n are s h o w n in Table 4. T h e p r o p o r t i o n of h y o d e o x y c h o l i c a c i d d e c r e a s e d s i g n i f i c a n t l y (r -- - 0 . 7 9 9 9 , P < 0.001) f r o m a m e a n v a l u e of 50.34% in t h e 0% fiber g r o u p t o t h a t of 39.09% on 20% fiber. I n c o n t r a s t , lithocholic and deoxycholic acids increased significantly (r -- 0.7871, P < .001 a n d r = 0.7580, P < .001, respectively) w i t h f i b e r intake. T h e r e s p e c t i v e v a l u e s in t h e 0% a n d 20% fiber g r o u p s were 1.91 a n d 15.34% for lithocholic acid a n d 15.74 a n d 28.59% for d e o x y c h o l i c acid. T h e p e r c e n t c o n t e n t of fecal c h e n o d e o x y c h o l i c a c i d d i d n o t change. Cholic, o~muricholic and/3-muricholic acids, w h i c h were n o t p r e s e n t in all a n i m a l s , d i d n o t s h o w s i g n i f i c a n t c h a n g e s in r e s p o n s e t o t h e d i f f e r e n t d i e t s (Table 4). Dietary fat. Total bile a c i d e x c r e t i o n i n c r e a s e d signifiLIPIDS, Vol. 27, no. 12 (1992)

1002

M.L. BORUM ET AL.

30

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Dietlry C l l d u . 1 104%

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10

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2.5

10

20

Dietary Fiber ( % of Total Diet)

20%

Oiitlr,# Flbir

FIG. 2. The effect of increasing dietary fiber content on the total

Diellry Calcium 1.04

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FIG. 1. Effects of dietary fiber, fat and calcium on fecal wet weight (g/24 h). The bars represent the effects of increasing dietary fiber and calcium on fecal wet weight in relation to dietary fat content. Panel A, 1% fat diet; panel B, 5% fat diet; panel C, 10% fat diet.

cantly from 12.37 umol/24 h in the 1% fat group to 15.19 i~mol/24 h in the animals which received a 10% fat diet (P < 0.05) (Table 1). Fecal fat showed significant increases with increasing dietary fat content (Table 1). The fecal wet and dry weights as well as fecal bile acid concentrations and composition were not affected by dietary fat. Fecal bile acid excretion showed a significant positive correlation with fecal wet weight (r = 0.452, P < .005 for the 5% fat group and r = 0.656, P < .001 for the 10% fat group) (Table 2). Significant positive correlations existed also between bile acid excretion and fecal fat in all three dietary fat groups (Table 2). There was a significant negative correlation (r -- -0.357, P < .005) between the fecal bile acid concentration per fecal dry weight and fecal calcium excretion in the 5% dietary fat group (Table 3). Dietary calcium. Fecal weight (wet) increased signifiLIPIDS, VoI. 27, no. 12 (1992)

FIG. 3. Effect of dietary fiber on the excretion of the major fecal bile adds. LC, llthocholic; CDC, chenodeoxycholic; DC, deoxycholic; HDC, byodeoxycholic.

cantly (P < 0.001) with increasing dietary intake of calcium, La from 23.70 tJmol/g in the 0.18% calcium group to 11.67 iJmol]g in the rats which received a 1.04% calcium diet (Table 1, Fig 4). The total excretion (Table 1) and percent composition of fecal bile acids did not change with dietary calcium. Correlations in combined dietary groups. Total fecal bile acid excretion (Table 2 and Table 3) correlated significantly with fecal wet weight (r = 0.4536, P < 0.001) and with fecal fat (r = 0.3555, P < 0.001). Total fecal bile acid concentrations showed a significant negative correlation with fecal calcium when the data from all dietary groups were combined (r = 0.2835, P < .001). Cholic~ a-muricholic and/3-muricholic acids were not present in all animals and did not show significant changes in response to the different diets {Table 4). Additive effects of diets. The only additive effect of any combination of the diets was observed with fiber and calcium in relation to increasing fecal wet and dry weight. DISCUSSION

In agreement with work b y others, this s t u d y shows t h a t a diet high in wheat bran is associated with significant increases in fecal bile acid excretion (6,9,11,15,20-23}. The

1003

FIBER, FAT AND CALCIUM EFFECTS ON BILE ACIDS TABLE 4 Percent Composition of Fecal Bile Acids in Relation to Fiber Content of Diet (M -----SEM)

Bile acid Lithocholic Deoxycholic Chenodeoxycholic Hyodeoxycholic Cholic a-Muricholic /]-Muricholic Other

0 1.91 +_ 0.33 15.74 + 1.37 7.71 _+ 0.80 50.34 +_ 3.34 2.94 + 0.89 17.44 + 9.25 12.61 _ 11.09 2.04 + 0.77

Dietary fiber content (%) 2.5 10 4.38 + 1.55 7.77 + 1.23 20.64 +_ 1.69 26.69 +_ 1.17 8.66 +- 0.54 7.83 + 0.33 53.80 + 2.85 47.15 + 1.66 2.07 _+ 0.32 3.33 + 0.82 9.34 +_ 2.08 7.35 + 1.56 2.27 +_ 0.75 2.74 + 0.72 1.33 +- 0.72 1.11 + 0.34

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FIG. 4. Fecal bile acid concentration per dry fecal weight in relation to increasing dietary intake of calcium.

main m e c h a n i s m of the increased bile acid excretion appears to be bile acid binding to fiber (39-42). In comparison to dietary fiber, dietary fat exerted a smaller, b u t significant influence on fecal bile acid excretion. The effect of fat on fecal bile acids appears to depend upon the t y p e of fat in the diet (43,44). For example, the feeding of a high fat corn oil-based diet has been reported to be associated with an increased bile acid excretion, whereas t r a n s fat in the diet had no significant effect (44). I n cont r a s t to dietary fiber and fat, calcium exerted no significant effect on bile acid excretion. In c o n t r a s t to our study, v a n de Meer e t al. 145) found t h a t supplemental dietary calcium increased the fecal bile acid excretion in healthy men. The authors speculated this to be due to the binding of calcium to intraluminal bile acids. The difference between our d a t a and those by v a n der Meer e t al. (45) seems to indicate t h a t the rat model m a y not completely correlate with h u m a n s as far as calcium-bile acid interactions in the intestine are concerned. Although fecal bile acid concentrations were not affected by either the fiber or fat content of the diet, they decreased significantly with increasing dietary calcium. The absence of an effect of w h e a t b r a n on fecal bile acid concentration appears to be in disagreement with reports by other investigators (45-47). However, although all these authors observed decreased bile acid concentrations, there were considerable variations in the total fecal bile acid excretion measurements. The increases in fecal bile acid concentration were associated with either a rise (46) or no change {45,47) in bile acid excretion. I t appears, therefore.

20 15.34 + 1.91 28.59 + 1.76 8.08 + 1.39 39.09 + 2.92 2.10 + 0.56 5.09 _ 2.66 1.98 +_ 0.55 1.10 + 0.56

t h a t the differences a m o n g the studies are mainly attributable to the v a r y i n g degrees of dilution induced by dietary fiber. I n the present study, b o t h dietary fiber and calcium exerted a dilutional effect, as indicated b y their close correlation with fecal wet as well as d r y weight. The absence, in this study, of a correlation between fecal bile acid concentrations and the a m o u n t of fat in the diet is in c o n t r a s t to the results of m o s t other authors who found t h a t fecal bile acid concentrations increased with increasing dietary fat (7,19). The lack of a correlation in this s t u d y appears to be due to the finding t h a t dietary fat had only a moderate effect on total fecal bile acid excretion and t h a t there was no correlation between dietary fat and fecal weight. In addition, these findings m a y have been affected by the fact t h a t in some of the other studies the dietary fat was considerably higher t h a n in the present experiments (>~20% vs.