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Effects of resistant potato starch on cholesterol and bile acid metabolism in the rat
Nutrition Research,Fbl. 17. Nos. 11/12, pp. 1671-1682.1997 Copyright0 1997 ElsevimScienceInc. Printedin theUSA. AU rightsresewed 0271~5317/97$17.00 + .oo EL.SEVIER
PII s0271-5317(97)00174-7
EFFECTS OF RESISTANT POTATO STARCH ON CHOLESTEROL AND BILE ACID METABOLISM IN THE RAT JoCarol Chezem, Ph.D., R.D.', Emily Furumoto, Ph.D., and Jon Story, Ph.D. Department of Foods and Nutrition, Purdue University, West Lafayette, IN
ABSTRACT This study examines the effects of two types of resistant starch (RS), raw starch from uncooked potatoes and retrograded starch in the form of cooked and cooled potatoes, on cholesterol and bile acid metabolism in rats. Groups of 8 male Wistar rats were fed a semipurified diet containing 15% cellulose, freeze-dried raw potatoes, or freeze-dried cooked potatoes for four weeks. Serum liver cholesterol, and cholesterol, fecal steroid excretion were determined. Serum cholesterol was liver higher cholesterol was significantly and significantly lower in response to the cooked potato diet Total compared to the cellulose or raw potato diet. steroid excretion was significantly higher in rats fed the cooked potato diet (26.31 mg/d) compared to those fed the cellulose or raw potato diet (14.27 mg/d and 16.81 mg/d, respectively). Daily total bile acid excretion was significantly different among the three groups, with highest excretion seen in rats fed cooked potatoes. High daily excretion of lithocholic acid and hyodeoxycholic acid was observed in rats fed cooked potatoes. These results suggest that changes in the cecal microflora and in the production, pool size, and excretion of chenodeoxycholic acid and its derivatives may be responsible for alterations in cholesterol and bile acid metabolism observed with resistant starch feeding. 0 1997 I3xviuscim.xhc. KEY WORDS: cholesterol bile acids resistant starch
' To whom correspondence should be addressed at Department of Family and Consumer Sciences, Ball State University, Muncie, IN 47306, Telephone: (765)285-5959, FAX: (765)285-2314, E-mail: [email protected]
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INTRODUCTION Resistant starch escapes digestion in the small intestine and is available, to some extent, for fermentation by bacteria in the large intestine. Resistant starch results from a variety of circumstances, including starch enclosed in food structures(RS,), ungelatinized starch (RS,), and starch which has undergone retrogradation (RS,) (1). In the gastrointestinal tract, resistant starch mimics many of the effects of soluble fibers, increasing transit time and fermenting to short chain fatty acids (SCFA) in the large intestine, resulting in lowered fecal pH (Z-4). Whether resistant starch affects cholesterol metabolism in a manner similar to soluble fiber is a topic of considerable controversy. Consumption of soluble fibers has been shown to reduce serum cholesterol in both rats and humans (5-7). Similar cholesterollowering effects have been noted in rats fed various forms of resistant starch (3,8,9). Much of the previous research in humans has focused on the effects of diets rich in amylose, which contains appreciable levels of RS. Reiser et al. (10) and Behall et al. (11) reported significant reductions of serum cholesterol in adults fed high-amylose cornstarch compared to fructose and low-amylose cornstarch. In contrast, a recent study by Heijnen et al. (12) found no differences in serum cholesterol levels of adult subjects whose diets were supplemented with glucose, raw high-amylose cornstarch (RS,), or retrograded high-amylose cornstarch (RS,). Several mechanisms have been postulated for soluble fibers' cholesterol-lowering effect. These include: increased viscosity of intestinal contents (13,14); binding of bile acids by soluble fiber (7,13); insolubilization of bile acids as a result of SCFA production (9); and alterations in hepatic lipid metabolism, thought to be related to absorption of SCFA's (7,15). The gel-forming capacity of RS is lower than that of many soluble fibers; viscosity is probably not a major factor in the hypocholesterolemic effect of RS (16). Due to its helical structure, RS complexes bile acids in vitro (17). Several studies have reported the effects of RS on intestinal production of SCFA's. In rats, RS feeding results in increased concentration of cecal SCFA (9,18,19), reduced cecal pH (3,9,18,19), and increased arterial and portal vein concentrations of SCFA's (9). In humans, consumption of a diet rich in RS has been reported to increase total breath hydrogen, an indirect measure of RS fermentation (20-22); decrease fecal pH (4); increase fecal SCFA excretion (4); and increase venous SCFA's (4,23). The purpose of this study was to investigate the effects of resistant starch on cholesterol concentrations and steroid excretion in the rat. Two types of RS, ungelatinized starch from raw potatoes and retrograded starch in the form of cooked and cooled potatoes, were examined.
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METHODS AND MATERIALS The experimental protocol was reviewed and approved by the Purdue University Animal Care and Use Committee. Animals and Diets: Twenty-four male Wistar rats (Harlan-Sprague Dawley, Inc., Indianapolis, initially weighing 94-118 were IN), grams, systematically randomized by weight and placed in one of three groups, each group with an average body weight of 106 grams. Rats were housed individually in wire-bottomed cages with a 12 hour 1ight:dark cycle. They were fed a commercial laboratory diet (Rodent Laboratory Chow @, Ralston Purina Co., St. Louis, MO) for three days, then given free access to experimental diets and water for 28 days. One of three semipurified experimental diets, containing 15% (150 grams per kg diet) cellulose, raw freeze-dried ground potatoes, or cooked freeze-dried ground potatoes, was fed to each group (Table 1). Body weight was recorded weekly, feed consumption was monitored during the third week, and feces were collected daily during the final three days of the experiment. At the end of the experiment, following a 12 hour fast, the rats were sacrificed by decapitation. Feces, serum, and liver samples were frozen (-80°C) prior to analysis. TABLE 1 Composition of Diets Component
Sucrose Casein Corn oil Mineral mix' Vitamin mixX Polysaccharide"
Cellulose (g/kg diet) 450 250 100 40 10 150
Raw Potato (g/kg diet)
Cooked Potato (g/kg diet)
450 250 100 40 10 150
450 250 100 40 10 150
'AINmineral mixture, ICN Pharmaceutical, Cleveland, OH "AIN-76a vitamin mixture, ICN Pharmaceutical, Cleveland, OH "Cellulose, raw potato, or cooked potato Fecal Bile Acids and Neutral Steroid Analysis: Feces from the three day collection period were pooled for each animal, lyophilized, and ground in a Wiley Mill (Arthur H. Thomas co., Philadelphia, PA) to 20 mesh. Internal standards, 58-cholanic acid (Sigma Chemical Co., St Louis, MO) for acidic steroids and 5acholestane (Sigma Chemical Co.) for neutral steroids, were added to 0.5 g aliquots of feces. Samples were refluxed with methanol:chloroform for 4 hours at 65'C, followed by extraction of
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the steroids into methanol:chloroform (1:l). Bile acids were decongugated by incubation with choloylglycine hydrolase (Sigma Chemical Co.) at 37°C for four hours. Samples were brought to pH 11 using aqueous NaOH and saponified at 110°C for four hours. Neutral steroids were then extracted into petroleum ether, derivitized with Tri-Sil Z@ (Pierce, Rockford, IL), and analyzed on a Hewlett Packard (Cincinnati, OH) model 5890 gas chromatograph with a 30 m DB 1701 capillary column (J&W Scientific, Ranch0 Cordova, CA). Bile acids were extracted from the sample with diethyl ether and ethyl acetate, following addition of HCl to bring sample pH to 1. Bile acids were purified using CI8 Sep-PakB cartridges (Millipore Waters, Milford, MA) attached to nylon syringe filters (Nalgene, Rochester, NY), methylated using dimethoxypropane (Aldrich Chemical Co., Milwaukee, WI) and HCl, silylated with Tri-Sil Z@, and analyzed by gas chromatography. Determination of serum and liver cholesterol: Serum and liver samples were analyzed using the method of Rude1 and Morris (24). Statistical analysis: Data were analyzed by the Statistical Analysis System (SAS Institute Inc., 1985) using one-way ANOVA with Fisher's Least Significant Difference test. Differences were considered significant at pcO.05. Values are presented as the mean + the standard error of the mean (SEM).
RESULTS Results of weight gain, feed:weight gain ratio, serum and liver cholesterol, dry fecal weight, and total steroid excretion are shown in Table 2. Weight gain and feed consumption per gram of weight gain were not significantly different among the three groups. Serum cholesterol was significantly higher in rats fed cooked potatoes (99 mg/dl) compared to those fed cellulose (80 mg/dl) or raw potatoes (84 mg/dl). Both concentration and total content of liver cholesterol were significantly lower in rats fed the cooked potato diet compared to those fed the cellulose or raw potato diet. Dry fecal weight was significantly different among the three groups, with the highest fecal weight observed in animals fed the cellulose diet and the lowest fecal weight in animals fed the cooked potato Daily total steroid excretion was significantly higher in diet. rats fed the cooked potato diet (26.31 mg/d) compared to those fed the cellulose or raw potato diet (14.27 mg/d and 16.81 mgld, respectively).
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TABLE 2 Comparison of Weight Gain, Feed:Weight Gain Ratio, Dry Fecal Weight, Serum Cholesterol, Liver Cholesterol, and Total Steroid Excretion'," Cellulose Weight Gain (g/d) Feed:Weight Gain (g/g) Dry Fecal Weight
Raw Potato
Cooked Potato
7.9
f 0.3
7.1
_+ 0.6
8.0
+ 0.4
3.0
t 0.1
3.1
* 0.3
2.8
+ 0.2
12.3
t 0.6"
3.9
+ 0.3b
2.3
& 0.2"
(g/d) Serum Chol (mg/dl) Liver Chol (mg/g) Total Liver Chol (mg) Total Ster Excr (q/d)
80.42 + 1.4Zb
84.06 t 4.13b
99.48 f 3.20"
3.17 f 0.14"
3.35 * 0.14"
2.58 f O.Ogb
* 3.41"
51.45 ? 4.05"
38.38 + 1.3Eb
14.27 + 0.34b
16.81 * 0.41b
26.31 + 1.06"
47.8
*Values represent mean t standard error of the mean, n=8. 'Data analyzed by one-way ANOVA with the least significant difference. Values in the same row with the same superscript letter are not significantly different (p>O.O5). Table 3 provides data on fecal neutral steroid excretion. Total neutral steroid excretion was significantly higher in response to the raw potato and cooked potato diets compared to the cellulose diet. Similarly, concentrations and daily excretion of total fecal neutral steroids and cholesterol were significantly higher in rats fed the raw potato and cooked potato diets than in rats fed the cellulose diet. While fecal cholestanol concentration was similar in the three groups, concentrations of other cholesterol metabolites were not. Fecal concentration and daily excretion of cholestanone was significantly higher with raw potato feeding, while cooked potato feeding resulted in significantly higher fecal concentration and daily excretion of coprostanol. Patterns of fecal bile acid excretion are shown in Table 4. Daily total bile acid excretion was significantly different among the three groups; the highest excretion was seen in rats fed cooked potatoes. Fecal concentrations and daily excretion of lithocholic acid and hyodeoxycholic acid were significantly higher with feeding of the cooked potato diet. Concentration of P-muricholic was not significantly different among the groups. Although fecal concentration of deoxycholic acid was significantly higher in rats fed cooked potatoes, daily excretion, which takes differences in fecal weight into account, was similar in rats fed cellulose or cooked potatoes.
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TABLE 3 Neutral Steroid Excretion.'l' Neutral Steroid (mg/g feces) Coprostanol Cholesterol Cholestanol Coprostanone Cholestanone TNS" cone TNS" (w/day)
Cellulose 0.03 0.22 0.00 0.02 0.23 0.50 6.15
+ + + + f + f
O.Olb 0.04" 0.00 O.Olb 0.05' o.loc 0.35b
Raw Potato
Cooked Potato
0.17 1.03 0.03 0.12 2.22 3.57 13.92
3.30 2.07 0.04 0.39 0.97 6.77 15.57
+ + * f f f *
O.Ogb 0.05b 0.03 0.06b 0.16" 0.17b 0.2ga
f + + f + + &
0.50" 0.29" 0.02 0.03" 0.16b 0.89" 0.79"
'Values represent mean f standard error of the mean, n=8 'Data analyzed by one-way ANOVA with the least significant difference. Values in the same row with the same superscript letter are not significantly different (p>O.O5). "TNS = Total neutral steroids. TABLE 4 Bile Acid Excretion.'*' Bile Acid (mg/g feces)
Cellulose
Raw Potato
Cooked Potato
Lithocholic cr.-Muricholic Deoxycholic Cholic Chenodeoxycholic Hyodeoxycholic P-Muricholic TBA" TBA*' (mg/d)
0.06 0.02 0.31 0.01 0.01 0.18 0.07 0.66 8.12
0.18 0.14 0.15 0.06 0.05 0.05 0.11 0.74 2.89
0.81 f 0.06= 0.15 * 0.04a 1.45 + 0.16"
f f + + + f + f +
0.01' O.Olb 0.02b O.Olb O.OOb 0.02b 0.00 0.04b 0.17b
? + f + + f + + +
0.02b 0.03" 0.04b 0.02" 0.02a O.Olb 0.02 O.llb 0.17'
,"," 2.15 + 0.18" 0.11 + 0.03 4.67 f 0.38a 10.74 + 0.36"
'Values represent mean f standard error of the mean, n=8 'Data analyzed by one-way ANOVA with the least significant difference. Values in the same row with the same superscript letter are not significantly different (p>O.O5). "TBA = Total bile acids. DISCUSSION Because caloric intake can affect cholesterol metabolism, body weight and feed intake were monitored during the study. Weight gain was not significantly different among the groups. Although feeding of some soluble fibers may reduce weight gain in rats (25,261, our study and others (3,9,27) have not seen similar effects with resistant starch feeding. Because cellulose is neither digested nor
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STARCH AND STEROLS
fermented, one would expect higher values for the feed:weight gain ratio with the cellulose diet. While the ratio was higher in cellulose-fed rats compared to rats fed raw or cooked potatoes, it did not reach significance: this is most likely due to the small contribution (15%) these components made to the diet. Dry fecal weight provides a measure of the amount of material unavailable for digestion or fermentation in the gastrointestinal tract. Cellulose feeding resulted in the highest fecal weight. The higher fecal weight in rats fed raw potatoes adds support to our assumption that the raw potatoes, high in ungelatinized starch, would be unavailable for digestion or fermentation. The lower fecal weight in rats fed cooked potatoes suggests the majority of starch was digestible or fermentable. These findings agree with those of Calvert et al. (2), who found increased fecal weight and fecal starch in rats fed raw potato starch compared to cooked potato starch. Several studies examining the effects of resistant starch from purified sources on serum cholesterol suggest it, like soluble fiber, lowers serum cholesterol (3,8,28). De Deckere et al. (3) reported a reduction of serum cholesterol in rats fed a diet containing retrograded compared to nonretrograded modified amylose cornstarch. Similar results were observed by Morand et al. in rats fed amylase-resistant cornstarch (28). A few studies have reported no change in serum cholesterol with resistant starch feeding. In a study described by Verbeek et al. (271, serum cholesterol levels were not significantly different in rats fed diets containing a modified maize starch rich in RS, or a common maize starch low in RS,. Heijnen et al. (12), in a study of normolipidemic men and women, reported no significant difference in serum cholesterol following dietary supplementation with raw high amylose cornstarch (RS,),retrograded high amylose cornstarch (RS,),or glucose. In the present study, serum cholesterol levels were not significantly different between the raw potato-fed (RS,) and cellulose-fed groups, a finding consistent with previous research. However, the increased serum cholesterol level observed in rats fed cooked potatoes (RS,) contrasts sharply with the results of others. The reason for this lack of agreement is not clear, although several possibilities exist, including differences in the amount of resistant starch fed, the native source of resistant starch, and the extent of purification/modification of the native starch source. Liver measure of cholesterol cholesterol content, a accumulation, typically decreased with feeding of is hypocholesterolemic substances, including soluble fiber (5,29). In the present study, reduced liver cholesterol, coupled with increased serum cholesterol, was observed in rats fed cooked potatoes. These findings contrast those of Younes et al. (9) who reported reduced plasma cholesterol with no reduction of liver cholesterol following resistant starch feeding. While the increase in serum cholesterol in the group fed cooked potatoes was unexpected, the reduction in liver cholesterol level may have been due to increased bile acid synthesis to offset high fecal bile acid losses. Data from the current study, coupled with recent work by Morand et al. (28) and
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Younes et al. (9) indicating increased 3-hydroxy-3-methylglutaryl (HMG) CoA reductase activity in rats fed resistant starch, suggest resistant starch exerts an effect on liver cholesterol metabolism similar to that seen with soluble fiber feeding (30,31). Increased excretion of neutral steroids may be one mechanism by which the hypocholesterolemic effect of soluble fibers is achieved (26,32). In the present study, total neutral steroid excretion was higher in the two potato-fed groups compared to the group fed cellulose, suggesting mediation directly, or indirectly, by consumption of resistant starch. These findings do not agree with a study by Verbeek et al. (27), who reported decreased fecal neutral steroids in rats fed high levels of resistant starch compared to rats fed low levels of resistant starch. Of the individual neutral steroids, daily excretion of coprostanol and cholestanone, metabolites of cholesterol resulting from bacterial conversion, were significantly higher in rats fed cooked potatoes and raw potatoes, respectively. These results support the theory that resistant starch, like soluble fibers, may alter intestinal microflora, enhancing bacterial transformation of cholesterol and neutral steroid excretion. Increased fecal bile acid excretion has also been considered as a possible mechanism for soluble fibers' hypocholesterolemic effect. Typically, fibers which lower serum cholesterol also increase fecal acid while bile excretion, fibers which do not have hypocholesterolemic effects generally do not increase excretion of fecal bile acids (13,30-32). In the present study, total bile acid excretion was significantly different among the three groups; highest bile acid excretion was seen in response to the cooked potato diet, while the raw potato diet resulted in the lowest levels of fecal bile acids. Differences in total fecal bile acid excretion among groups were due mainly to lithocholic acid (LC) and hyodeoxycholic acid (HDC), two products of bacterial transformation of chenodeoxycholic acid; rats fed cooked potatoes had higher daily excretions of these two bile acids (1.86 mg LC, 4.95 mg HDC) compared to rats fed cellulose (O-74 mg LC, 2.21 mg HDC) or raw potatoes (0.70 mg LC, 0.20 mg HDC). Story (33) observed increases in the pool size of chenodeoxycholic acid and its derivatives in rats fed oat bran, a soluble fiber. Work by Matheson and Story (25) indicates greater pool sizes of three chenodeoxycholic derivatives in rats fed soluble fiber compared to rats fed cellulose. Others have noted that increases in the chenodeoxycholic acid pool result in reductions in cholesterol absorption (34) and bile acid reabsorption (35) from the gastrointestinal tract. In a study by Abadie et al. (17), undigested starch increased excretion of chenodeoxycholic acid and its metabolites in mice and hamsters; the proposed mechanism was binding of bile acids by starch. Based on the findings of the current study and those of other studies, it is proposed that the cooked potato diet may exert its effects on cholesterol and bile acid metabolism in the rat by altering synthesis, pool size, and/or excretion of chenodeoxycholic acid and its derivatives. The results of the present study indicate both raw and cooked
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potatoes affect steroid metabolism in the rat, the response to cooked potato feeding being the more striking of the two. Rats fed the cooked potatoes exhibited an unusual combination of cholesterol metabolism indices - increased serum cholesterol, reduced liver cholesterol, and increased excretion of neutral sterols and bile acids - which have not been previously reported with either dietary or pharmacological intervention. While the mechanism responsible for the rise in serum cholesterol with cooked potato feeding is not clear, alterations in microbial flora and in production, pool size and excretion of chenodeoxycholic acid and its derivatives seem the most likely explanation for the decrease in liver cholesterol and alterations in bile acid and neutral steroid excretion seen with raw and cooked potato feeding. Although not examined in the present study, alterations in microbial flora appear to be related to the nature and amount of fermentable substrate available in the cecum (36). What has yet to be determined is the underlying mechanisms for the modification in chenodeoxycholic acid metabolism. Work by our lab and others suggest hepatic cholesterol and bile acid synthesis is regulated by the relative hydrophobicity of the bile acid pool which, in turn, can be influenced by soluble fiber (25,3739). Gaining a more complete picture of pool sizes of bile acids and synthesis rates of cholesterol and bile acids may yield more conclusive information on the role of resistant starch in steroid metabolism. ACKNOWLEDGEMENTS This work was supported in part by the Indiana Elks and the Purdue Agricultural Research Program (paper # 15,363). The authors are indebted to Dr. Judith Marlett for her valuable discussions regarding this project. REFERENCES 1.
Englyst HN, Kingman SM, Cummings JH. Classification and measurement of nutritionally important starch fractions. Eur J Clin Nutr 1992;46(Suppl 2):S33-50.
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Anderson JW, Deakins DA, Brideges SR. Soluble fiber: hypcholesterolemic effects and proposed mechanisms. In: Kritchevsky D, Bonfield C, Anderson JW, eds. Dietary Fiber: Chemistry, Physiology, and Health Effects, New York: Plenum, 1990: 339-64.
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Gallaher DD, Hassel CA, Lee K, Gallaher CM. Viscosity and fermentability as attributes of dietary fiber responsible for the hypocholesterolemic effect in hamsters. J Nutr 1993;123:244-52.
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Story JA, Furumoto EJ. Dietary fiber and bile acid metabolism. In: Kritchevsky D, Bonfield C, Anderson JW, eds. Dietary.Fiber: Chemistry, Physiology, and Health Effects, New York: Plenum, 1990: 365-74.
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Weursch, P. Starch in human nutrition. In: Bourne GJ, ed. Nutritional Value of Cereal Products, Beans and Starches, Amsterdam: Karger, 1989: 199-256.
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Abadie C, Hug M, Kubi C, Gains N. Effect of cyclodextrins and
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undigested starch on the loss of chenodeoxycholate in the faeces. Biochem J 1994;299:725-30. 18.
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Garcia-Diez F, Garcia-Mediavilla V, Bayon JE, Gonzalez-Gallego J. Pectin feedina influences fecal bile acid excretion, hepatic bile acid and cholesterol synthesis and serum cholesterol in rats. J Nutr 1996;126:1766-71.
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Ponz de Leon M, Carulli N, Loria P, Zirone F. The effect of chenodeoxycholic acid (CDCA) on cholesterol absorption. Gastroenterology 1979;77:223-30.
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Accepted for publication September 11,
1997.
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EFFECTS OF RESISTANT POTATO STARCH ON CHOLESTEROL AND BILE ACID METABOLISM IN THE RAT JoCarol Chezem, Ph.D., R.D.', Emily Furumoto, Ph.D., and Jon Story, Ph.D. Department of Foods and Nutrition, Purdue University, West Lafayette, IN
ABSTRACT This study examines the effects of two types of resistant starch (RS), raw starch from uncooked potatoes and retrograded starch in the form of cooked and cooled potatoes, on cholesterol and bile acid metabolism in rats. Groups of 8 male Wistar rats were fed a semipurified diet containing 15% cellulose, freeze-dried raw potatoes, or freeze-dried cooked potatoes for four weeks. Serum liver cholesterol, and cholesterol, fecal steroid excretion were determined. Serum cholesterol was liver higher cholesterol was significantly and significantly lower in response to the cooked potato diet Total compared to the cellulose or raw potato diet. steroid excretion was significantly higher in rats fed the cooked potato diet (26.31 mg/d) compared to those fed the cellulose or raw potato diet (14.27 mg/d and 16.81 mg/d, respectively). Daily total bile acid excretion was significantly different among the three groups, with highest excretion seen in rats fed cooked potatoes. High daily excretion of lithocholic acid and hyodeoxycholic acid was observed in rats fed cooked potatoes. These results suggest that changes in the cecal microflora and in the production, pool size, and excretion of chenodeoxycholic acid and its derivatives may be responsible for alterations in cholesterol and bile acid metabolism observed with resistant starch feeding. 0 1997 I3xviuscim.xhc. KEY WORDS: cholesterol bile acids resistant starch
' To whom correspondence should be addressed at Department of Family and Consumer Sciences, Ball State University, Muncie, IN 47306, Telephone: (765)285-5959, FAX: (765)285-2314, E-mail: [email protected]
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INTRODUCTION Resistant starch escapes digestion in the small intestine and is available, to some extent, for fermentation by bacteria in the large intestine. Resistant starch results from a variety of circumstances, including starch enclosed in food structures(RS,), ungelatinized starch (RS,), and starch which has undergone retrogradation (RS,) (1). In the gastrointestinal tract, resistant starch mimics many of the effects of soluble fibers, increasing transit time and fermenting to short chain fatty acids (SCFA) in the large intestine, resulting in lowered fecal pH (Z-4). Whether resistant starch affects cholesterol metabolism in a manner similar to soluble fiber is a topic of considerable controversy. Consumption of soluble fibers has been shown to reduce serum cholesterol in both rats and humans (5-7). Similar cholesterollowering effects have been noted in rats fed various forms of resistant starch (3,8,9). Much of the previous research in humans has focused on the effects of diets rich in amylose, which contains appreciable levels of RS. Reiser et al. (10) and Behall et al. (11) reported significant reductions of serum cholesterol in adults fed high-amylose cornstarch compared to fructose and low-amylose cornstarch. In contrast, a recent study by Heijnen et al. (12) found no differences in serum cholesterol levels of adult subjects whose diets were supplemented with glucose, raw high-amylose cornstarch (RS,), or retrograded high-amylose cornstarch (RS,). Several mechanisms have been postulated for soluble fibers' cholesterol-lowering effect. These include: increased viscosity of intestinal contents (13,14); binding of bile acids by soluble fiber (7,13); insolubilization of bile acids as a result of SCFA production (9); and alterations in hepatic lipid metabolism, thought to be related to absorption of SCFA's (7,15). The gel-forming capacity of RS is lower than that of many soluble fibers; viscosity is probably not a major factor in the hypocholesterolemic effect of RS (16). Due to its helical structure, RS complexes bile acids in vitro (17). Several studies have reported the effects of RS on intestinal production of SCFA's. In rats, RS feeding results in increased concentration of cecal SCFA (9,18,19), reduced cecal pH (3,9,18,19), and increased arterial and portal vein concentrations of SCFA's (9). In humans, consumption of a diet rich in RS has been reported to increase total breath hydrogen, an indirect measure of RS fermentation (20-22); decrease fecal pH (4); increase fecal SCFA excretion (4); and increase venous SCFA's (4,23). The purpose of this study was to investigate the effects of resistant starch on cholesterol concentrations and steroid excretion in the rat. Two types of RS, ungelatinized starch from raw potatoes and retrograded starch in the form of cooked and cooled potatoes, were examined.
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METHODS AND MATERIALS The experimental protocol was reviewed and approved by the Purdue University Animal Care and Use Committee. Animals and Diets: Twenty-four male Wistar rats (Harlan-Sprague Dawley, Inc., Indianapolis, initially weighing 94-118 were IN), grams, systematically randomized by weight and placed in one of three groups, each group with an average body weight of 106 grams. Rats were housed individually in wire-bottomed cages with a 12 hour 1ight:dark cycle. They were fed a commercial laboratory diet (Rodent Laboratory Chow @, Ralston Purina Co., St. Louis, MO) for three days, then given free access to experimental diets and water for 28 days. One of three semipurified experimental diets, containing 15% (150 grams per kg diet) cellulose, raw freeze-dried ground potatoes, or cooked freeze-dried ground potatoes, was fed to each group (Table 1). Body weight was recorded weekly, feed consumption was monitored during the third week, and feces were collected daily during the final three days of the experiment. At the end of the experiment, following a 12 hour fast, the rats were sacrificed by decapitation. Feces, serum, and liver samples were frozen (-80°C) prior to analysis. TABLE 1 Composition of Diets Component
Sucrose Casein Corn oil Mineral mix' Vitamin mixX Polysaccharide"
Cellulose (g/kg diet) 450 250 100 40 10 150
Raw Potato (g/kg diet)
Cooked Potato (g/kg diet)
450 250 100 40 10 150
450 250 100 40 10 150
'AINmineral mixture, ICN Pharmaceutical, Cleveland, OH "AIN-76a vitamin mixture, ICN Pharmaceutical, Cleveland, OH "Cellulose, raw potato, or cooked potato Fecal Bile Acids and Neutral Steroid Analysis: Feces from the three day collection period were pooled for each animal, lyophilized, and ground in a Wiley Mill (Arthur H. Thomas co., Philadelphia, PA) to 20 mesh. Internal standards, 58-cholanic acid (Sigma Chemical Co., St Louis, MO) for acidic steroids and 5acholestane (Sigma Chemical Co.) for neutral steroids, were added to 0.5 g aliquots of feces. Samples were refluxed with methanol:chloroform for 4 hours at 65'C, followed by extraction of
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the steroids into methanol:chloroform (1:l). Bile acids were decongugated by incubation with choloylglycine hydrolase (Sigma Chemical Co.) at 37°C for four hours. Samples were brought to pH 11 using aqueous NaOH and saponified at 110°C for four hours. Neutral steroids were then extracted into petroleum ether, derivitized with Tri-Sil Z@ (Pierce, Rockford, IL), and analyzed on a Hewlett Packard (Cincinnati, OH) model 5890 gas chromatograph with a 30 m DB 1701 capillary column (J&W Scientific, Ranch0 Cordova, CA). Bile acids were extracted from the sample with diethyl ether and ethyl acetate, following addition of HCl to bring sample pH to 1. Bile acids were purified using CI8 Sep-PakB cartridges (Millipore Waters, Milford, MA) attached to nylon syringe filters (Nalgene, Rochester, NY), methylated using dimethoxypropane (Aldrich Chemical Co., Milwaukee, WI) and HCl, silylated with Tri-Sil Z@, and analyzed by gas chromatography. Determination of serum and liver cholesterol: Serum and liver samples were analyzed using the method of Rude1 and Morris (24). Statistical analysis: Data were analyzed by the Statistical Analysis System (SAS Institute Inc., 1985) using one-way ANOVA with Fisher's Least Significant Difference test. Differences were considered significant at pcO.05. Values are presented as the mean + the standard error of the mean (SEM).
RESULTS Results of weight gain, feed:weight gain ratio, serum and liver cholesterol, dry fecal weight, and total steroid excretion are shown in Table 2. Weight gain and feed consumption per gram of weight gain were not significantly different among the three groups. Serum cholesterol was significantly higher in rats fed cooked potatoes (99 mg/dl) compared to those fed cellulose (80 mg/dl) or raw potatoes (84 mg/dl). Both concentration and total content of liver cholesterol were significantly lower in rats fed the cooked potato diet compared to those fed the cellulose or raw potato diet. Dry fecal weight was significantly different among the three groups, with the highest fecal weight observed in animals fed the cellulose diet and the lowest fecal weight in animals fed the cooked potato Daily total steroid excretion was significantly higher in diet. rats fed the cooked potato diet (26.31 mg/d) compared to those fed the cellulose or raw potato diet (14.27 mg/d and 16.81 mgld, respectively).
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TABLE 2 Comparison of Weight Gain, Feed:Weight Gain Ratio, Dry Fecal Weight, Serum Cholesterol, Liver Cholesterol, and Total Steroid Excretion'," Cellulose Weight Gain (g/d) Feed:Weight Gain (g/g) Dry Fecal Weight
Raw Potato
Cooked Potato
7.9
f 0.3
7.1
_+ 0.6
8.0
+ 0.4
3.0
t 0.1
3.1
* 0.3
2.8
+ 0.2
12.3
t 0.6"
3.9
+ 0.3b
2.3
& 0.2"
(g/d) Serum Chol (mg/dl) Liver Chol (mg/g) Total Liver Chol (mg) Total Ster Excr (q/d)
80.42 + 1.4Zb
84.06 t 4.13b
99.48 f 3.20"
3.17 f 0.14"
3.35 * 0.14"
2.58 f O.Ogb
* 3.41"
51.45 ? 4.05"
38.38 + 1.3Eb
14.27 + 0.34b
16.81 * 0.41b
26.31 + 1.06"
47.8
*Values represent mean t standard error of the mean, n=8. 'Data analyzed by one-way ANOVA with the least significant difference. Values in the same row with the same superscript letter are not significantly different (p>O.O5). Table 3 provides data on fecal neutral steroid excretion. Total neutral steroid excretion was significantly higher in response to the raw potato and cooked potato diets compared to the cellulose diet. Similarly, concentrations and daily excretion of total fecal neutral steroids and cholesterol were significantly higher in rats fed the raw potato and cooked potato diets than in rats fed the cellulose diet. While fecal cholestanol concentration was similar in the three groups, concentrations of other cholesterol metabolites were not. Fecal concentration and daily excretion of cholestanone was significantly higher with raw potato feeding, while cooked potato feeding resulted in significantly higher fecal concentration and daily excretion of coprostanol. Patterns of fecal bile acid excretion are shown in Table 4. Daily total bile acid excretion was significantly different among the three groups; the highest excretion was seen in rats fed cooked potatoes. Fecal concentrations and daily excretion of lithocholic acid and hyodeoxycholic acid were significantly higher with feeding of the cooked potato diet. Concentration of P-muricholic was not significantly different among the groups. Although fecal concentration of deoxycholic acid was significantly higher in rats fed cooked potatoes, daily excretion, which takes differences in fecal weight into account, was similar in rats fed cellulose or cooked potatoes.
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TABLE 3 Neutral Steroid Excretion.'l' Neutral Steroid (mg/g feces) Coprostanol Cholesterol Cholestanol Coprostanone Cholestanone TNS" cone TNS" (w/day)
Cellulose 0.03 0.22 0.00 0.02 0.23 0.50 6.15
+ + + + f + f
O.Olb 0.04" 0.00 O.Olb 0.05' o.loc 0.35b
Raw Potato
Cooked Potato
0.17 1.03 0.03 0.12 2.22 3.57 13.92
3.30 2.07 0.04 0.39 0.97 6.77 15.57
+ + * f f f *
O.Ogb 0.05b 0.03 0.06b 0.16" 0.17b 0.2ga
f + + f + + &
0.50" 0.29" 0.02 0.03" 0.16b 0.89" 0.79"
'Values represent mean f standard error of the mean, n=8 'Data analyzed by one-way ANOVA with the least significant difference. Values in the same row with the same superscript letter are not significantly different (p>O.O5). "TNS = Total neutral steroids. TABLE 4 Bile Acid Excretion.'*' Bile Acid (mg/g feces)
Cellulose
Raw Potato
Cooked Potato
Lithocholic cr.-Muricholic Deoxycholic Cholic Chenodeoxycholic Hyodeoxycholic P-Muricholic TBA" TBA*' (mg/d)
0.06 0.02 0.31 0.01 0.01 0.18 0.07 0.66 8.12
0.18 0.14 0.15 0.06 0.05 0.05 0.11 0.74 2.89
0.81 f 0.06= 0.15 * 0.04a 1.45 + 0.16"
f f + + + f + f +
0.01' O.Olb 0.02b O.Olb O.OOb 0.02b 0.00 0.04b 0.17b
? + f + + f + + +
0.02b 0.03" 0.04b 0.02" 0.02a O.Olb 0.02 O.llb 0.17'
,"," 2.15 + 0.18" 0.11 + 0.03 4.67 f 0.38a 10.74 + 0.36"
'Values represent mean f standard error of the mean, n=8 'Data analyzed by one-way ANOVA with the least significant difference. Values in the same row with the same superscript letter are not significantly different (p>O.O5). "TBA = Total bile acids. DISCUSSION Because caloric intake can affect cholesterol metabolism, body weight and feed intake were monitored during the study. Weight gain was not significantly different among the groups. Although feeding of some soluble fibers may reduce weight gain in rats (25,261, our study and others (3,9,27) have not seen similar effects with resistant starch feeding. Because cellulose is neither digested nor
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STARCH AND STEROLS
fermented, one would expect higher values for the feed:weight gain ratio with the cellulose diet. While the ratio was higher in cellulose-fed rats compared to rats fed raw or cooked potatoes, it did not reach significance: this is most likely due to the small contribution (15%) these components made to the diet. Dry fecal weight provides a measure of the amount of material unavailable for digestion or fermentation in the gastrointestinal tract. Cellulose feeding resulted in the highest fecal weight. The higher fecal weight in rats fed raw potatoes adds support to our assumption that the raw potatoes, high in ungelatinized starch, would be unavailable for digestion or fermentation. The lower fecal weight in rats fed cooked potatoes suggests the majority of starch was digestible or fermentable. These findings agree with those of Calvert et al. (2), who found increased fecal weight and fecal starch in rats fed raw potato starch compared to cooked potato starch. Several studies examining the effects of resistant starch from purified sources on serum cholesterol suggest it, like soluble fiber, lowers serum cholesterol (3,8,28). De Deckere et al. (3) reported a reduction of serum cholesterol in rats fed a diet containing retrograded compared to nonretrograded modified amylose cornstarch. Similar results were observed by Morand et al. in rats fed amylase-resistant cornstarch (28). A few studies have reported no change in serum cholesterol with resistant starch feeding. In a study described by Verbeek et al. (271, serum cholesterol levels were not significantly different in rats fed diets containing a modified maize starch rich in RS, or a common maize starch low in RS,. Heijnen et al. (12), in a study of normolipidemic men and women, reported no significant difference in serum cholesterol following dietary supplementation with raw high amylose cornstarch (RS,),retrograded high amylose cornstarch (RS,),or glucose. In the present study, serum cholesterol levels were not significantly different between the raw potato-fed (RS,) and cellulose-fed groups, a finding consistent with previous research. However, the increased serum cholesterol level observed in rats fed cooked potatoes (RS,) contrasts sharply with the results of others. The reason for this lack of agreement is not clear, although several possibilities exist, including differences in the amount of resistant starch fed, the native source of resistant starch, and the extent of purification/modification of the native starch source. Liver measure of cholesterol cholesterol content, a accumulation, typically decreased with feeding of is hypocholesterolemic substances, including soluble fiber (5,29). In the present study, reduced liver cholesterol, coupled with increased serum cholesterol, was observed in rats fed cooked potatoes. These findings contrast those of Younes et al. (9) who reported reduced plasma cholesterol with no reduction of liver cholesterol following resistant starch feeding. While the increase in serum cholesterol in the group fed cooked potatoes was unexpected, the reduction in liver cholesterol level may have been due to increased bile acid synthesis to offset high fecal bile acid losses. Data from the current study, coupled with recent work by Morand et al. (28) and
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Younes et al. (9) indicating increased 3-hydroxy-3-methylglutaryl (HMG) CoA reductase activity in rats fed resistant starch, suggest resistant starch exerts an effect on liver cholesterol metabolism similar to that seen with soluble fiber feeding (30,31). Increased excretion of neutral steroids may be one mechanism by which the hypocholesterolemic effect of soluble fibers is achieved (26,32). In the present study, total neutral steroid excretion was higher in the two potato-fed groups compared to the group fed cellulose, suggesting mediation directly, or indirectly, by consumption of resistant starch. These findings do not agree with a study by Verbeek et al. (27), who reported decreased fecal neutral steroids in rats fed high levels of resistant starch compared to rats fed low levels of resistant starch. Of the individual neutral steroids, daily excretion of coprostanol and cholestanone, metabolites of cholesterol resulting from bacterial conversion, were significantly higher in rats fed cooked potatoes and raw potatoes, respectively. These results support the theory that resistant starch, like soluble fibers, may alter intestinal microflora, enhancing bacterial transformation of cholesterol and neutral steroid excretion. Increased fecal bile acid excretion has also been considered as a possible mechanism for soluble fibers' hypocholesterolemic effect. Typically, fibers which lower serum cholesterol also increase fecal acid while bile excretion, fibers which do not have hypocholesterolemic effects generally do not increase excretion of fecal bile acids (13,30-32). In the present study, total bile acid excretion was significantly different among the three groups; highest bile acid excretion was seen in response to the cooked potato diet, while the raw potato diet resulted in the lowest levels of fecal bile acids. Differences in total fecal bile acid excretion among groups were due mainly to lithocholic acid (LC) and hyodeoxycholic acid (HDC), two products of bacterial transformation of chenodeoxycholic acid; rats fed cooked potatoes had higher daily excretions of these two bile acids (1.86 mg LC, 4.95 mg HDC) compared to rats fed cellulose (O-74 mg LC, 2.21 mg HDC) or raw potatoes (0.70 mg LC, 0.20 mg HDC). Story (33) observed increases in the pool size of chenodeoxycholic acid and its derivatives in rats fed oat bran, a soluble fiber. Work by Matheson and Story (25) indicates greater pool sizes of three chenodeoxycholic derivatives in rats fed soluble fiber compared to rats fed cellulose. Others have noted that increases in the chenodeoxycholic acid pool result in reductions in cholesterol absorption (34) and bile acid reabsorption (35) from the gastrointestinal tract. In a study by Abadie et al. (17), undigested starch increased excretion of chenodeoxycholic acid and its metabolites in mice and hamsters; the proposed mechanism was binding of bile acids by starch. Based on the findings of the current study and those of other studies, it is proposed that the cooked potato diet may exert its effects on cholesterol and bile acid metabolism in the rat by altering synthesis, pool size, and/or excretion of chenodeoxycholic acid and its derivatives. The results of the present study indicate both raw and cooked
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potatoes affect steroid metabolism in the rat, the response to cooked potato feeding being the more striking of the two. Rats fed the cooked potatoes exhibited an unusual combination of cholesterol metabolism indices - increased serum cholesterol, reduced liver cholesterol, and increased excretion of neutral sterols and bile acids - which have not been previously reported with either dietary or pharmacological intervention. While the mechanism responsible for the rise in serum cholesterol with cooked potato feeding is not clear, alterations in microbial flora and in production, pool size and excretion of chenodeoxycholic acid and its derivatives seem the most likely explanation for the decrease in liver cholesterol and alterations in bile acid and neutral steroid excretion seen with raw and cooked potato feeding. Although not examined in the present study, alterations in microbial flora appear to be related to the nature and amount of fermentable substrate available in the cecum (36). What has yet to be determined is the underlying mechanisms for the modification in chenodeoxycholic acid metabolism. Work by our lab and others suggest hepatic cholesterol and bile acid synthesis is regulated by the relative hydrophobicity of the bile acid pool which, in turn, can be influenced by soluble fiber (25,3739). Gaining a more complete picture of pool sizes of bile acids and synthesis rates of cholesterol and bile acids may yield more conclusive information on the role of resistant starch in steroid metabolism. ACKNOWLEDGEMENTS This work was supported in part by the Indiana Elks and the Purdue Agricultural Research Program (paper # 15,363). The authors are indebted to Dr. Judith Marlett for her valuable discussions regarding this project. REFERENCES 1.
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Matheson HB, Colon IS, Story JA. Cholesterol 7a-hydroxylase activity is increased by dietary modification with psyllium hydrocolloid, pectin, cholesterol and cholestyramine in rats. J'Nutr 1995;125:454-58.
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Accepted for publication September 11,
1997.