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Department of Biological Resources, Faculty of Agriculture, Ehime University, Matsuyama 790-8566, Japan and * Department of Hygiene, Kinki University School of Medicine, Osaka 589-8511, Japan
2To whom correspondence should be addressed. E-mail: ebihara{at}agr.ehime-u.ac.jp.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-hydroxylase activity. In intact rats, biliary bile acid flux into the small intestine was significantly greater in those fed the HACS diet than in those fed the CS diet. Thus, the hypocholesterolemic effect of HACS appears to be mediated by accelerated fecal excretion of bile acids and increases in the intestinal pool and biliary flux of bile acids, and not by cecal fermentation products.
KEY WORDS: • high amylose cornstarch • cecectomy • plasma cholesterol • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). By the standard method of the American Association of Cereal Chemists for determining the level of dietary fiber (DF) (2
), RS is an insoluble DF (3
). RS that escapes hydrolysis in the small intestine enters into the large intestine, where it is fermented (4
). In this regard, RS possesses physiological properties similar to those of DF (5
).
RS is currently divided into four categories [physically inaccessible starch (RS1), resistant granules and high amylose starches (RS2), retrograded starches (RS3) and chemically modified starches (RS4)] (6
). High amylose cornstarch (HACS) contains RS2, ungelatinized starch granules that are highly resistant to digestion by -amylase until gelatinized. Several studies have shown that HACS reduces serum cholesterol and triglyceride concentrations in rats (7
–9
) and hamsters (10
). The mechanism of the hypocholesterolemic effect of HACS remains unclear, but several are possible: 1) increased fecal excretion of bile acids and sterols; 2) increase in the intestinal pool and biliary production of bile acids; and 3) increased synthesis of fermentation products that affect hepatic cholesterol synthesis (11
). One of the fermentation products, propionic acid, may lower plasma cholesterol levels (11
). Fermentation products of sugar-beet fiber incubated with cecal bacteria reduced plasma cholesterol concentration in rats (12
). The cecum is a site of vigorous microbial activity in rats, where undigested food residues are fermented, yielding various products.
The aim of the present study was to determine the contribution of cecal fermentation products to the hypocholesterolemic effect of HACS. Rats with cecal resections are suitable as experimental model to test whether the hypocholesterolemic effect of HACS is modified by cecal fermentation products.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Normal cornstarch (CS; Nisshoku Cornstarch Y) and HACS (Nisshoku High Amylose Starch) were purchased from Nihon Shokuhin Kako (Tokyo, Japan). The amylose concentrations of CS and HACS were 26 and 68 g/100 g, respectively. The DF concentrations of CS and HACS as determined by the method of the AOAC (13
) were 0.5 and 19.3 g/100 g, respectively.
Animals and diets.
This study was approved by the Laboratory Animal Care Committee of Ehime University. Rats were maintained in accordance with the Guidelines for the Care and Use of Laboratory Animals of Ehime University.
Four-wk-old, male Wistar rats (Japan SLC, Hamamatsu, Japan) weighing 60–80 g (Experiment 1), and 5-wk-old, male Wistar rats weighing 100–120 g (Experiment 2) were housed individually in screen-bottomed, stainless steel cages in a room maintained at 23 ± 1°C with a 12-h light:dark cycle (light, 0700–1900 h). Rats were acclimated by feeding a commercial solid diet (MF, oriental Yeast, Osaka, Japan) for 7 d. Body weight and food intake were recorded daily for each rat in the morning before replacing the food.
Experiment 1.
After acclimation, rats were divided into two groups of 12 and 16 rats. Rats were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg/kg body; Nembutal, Abbott Laboratories, North Chicago, IL). Sixteen rats were cecectomized. The cecum was surgically removed by the method of Lambert (14
). Twelve rats were sham-operated. Cecectomized and sham-operated rats were each divided into two groups, one of which was allowed free access to the CS diet (CS/Sham and CS/Cecectomy groups) and the other was allowed free access to the HACS diet (HACS/Sham and HACS/Cecectomy groups) for 21 d. The compositions of the CS and HACS diets are shown in Table 1
.
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Experiment 2.
After acclimation, intact rats were divided into two groups of 10 and were allowed free access to the CS or HACS diets (Table 1)
for 21 d. At the end of the experiment, fed rats were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg/kg body); a mid-line laparotomy was performed and the bile duct was exposed and ligated distally. The bile duct was then cannulated with a PE-10 polyethylene tube (Clay Adams, Parsippany, NJ), and bile was collected into a preweighed tube that had been cooled on ice for 30 min. The bile volume was determined gravimetrically.
Chemical analyses.
The concentrations of triglyceride and phospholipids in the plasma were determined enzymatically with commercial diagnostic kits (Triglyceride E-Test Wako and Phospholipids C-Test Wako, Wako Pure Chemical Industries, Osaka, Japan).
Plasma lipoproteins were isolated by ultracentrifugation by the method of Hatch (16
) with the slight modification described below. Briefly, for VLDL separation, 0.3 mL of the 1.006 kg/L NaCl density solution was added to 0.6 mL of serum into polycarbonate tube of the Beckman TL-100.2 rotor (Beckmann Instruments, Palo Alto, CA). Ultracentrifugation was performed in a Beckman TL-100 ultracentrifuge at 100,000 x g for 2.5 h at 12°C, and VLDL were removed from the top. Then, the middle layer in the tube (0.15 mL) was removed, and the bottom layer in the tube (0.6 mL) was transferred to another tube. For LDL separation, 0.3 mL of 1.006 kg/L NaBr density solution was added to the tube, mixed, ultracentrifuged as above and LDL removed from the top. The middle layer in the tube (0.15 mL) was removed, and the bottom layer in the tube (0.6 mL) was transferred to another tube. For HDL separation, 0.3 mL of 1.478 kg/L NaBr density solution was added to the bottom 0.6 mL in the tube, mixed, centrifuged at 100,000 x g for 4 h at 12°C, and HDL removed from the top. All NaBr density solutions contained 0.5 mL of 0.5 mol/L Na2EDTA. The concentration of cholesterol in the lipoprotein fractions was determined enzymatically with a commercial kit (Cholesterol E-Test Wako, Wako Pure Chemical Industries).
The concentrations of apolipoproteins (apoA-I, A-IV, B and E) were estimated by rocket immunoelectrophoresis according to the method of Laurell (17
) with the slight modification described below. Briefly, 2 µL of serum diluted to an appropriate concentration with electrophoresis buffer containing Triton X-100 (10 g/L buffer) was applied to the agarose gel (10 g/L, SeaKem LE agarose, Marine Colloids Division, FMC, Lockland, ME) plate containing antiserum (125 µL of anti-apo A-I, 150 µL of anti-apo A-IV, 150 µL of anti-apo B or 300 µL anti-apo E/9 mL agarose gel solution) and subjected to electrophoresis in 0.0148 mol/L Barbital-0.075 mol/L Tris-glycine buffer (pH 8.8) containing Triton X-100 (1 g/L buffer) at 8.4 V/cm, 14–16°C for 3 h for apo A-I and E or for 4 h for apo A-IV.
The concentration of liver total lipids was determined gravimetrically after extraction by the method of Folch et al. (18
). The concentrations of liver triglyceride and total cholesterol were also measured. Lipids were extracted from 500 mg liver with chloroform:methanol (2:1, v/v) according to the method of Folch et al. (18
). After extraction, the volume of the lipid solution was adjusted to 20 mL with the same solution of chloroform:methanol (2:1, v/v). This extract (1 mL) was dried under a nitrogen stream, and the residue obtained was mixed with 100 µL of isopropyl alcohol containing 100 g Triton X-100/L (Wako Pure Chemical Industries). This mixture (30 µL) was mixed with 3 mL of aqueous enzyme solution according to the standard procedure of the assay kits (Triglyceride E-Test Wako and Cholesterol E-Test Wako, Wako Pure Chemical Industries), and the triglyceride and cholesterol concentrations were determined colorimetrically. In a preliminary study, 30 µL of isopropyl alcohol containing 100 g Triton X-100/L did not affect the enzymatic reactions (data not shown).
The level of cholesterol 7-hydroxylase activity in the liver was determined according to the method of Ogishima and Okuda (19
) with the slight modification described below. Briefly, the mixture (0.4 mL) containing 0.1 mmol/L EDTA, 20 mmol/L cysteamine, 5 mmol/L MgCl2, 10 mmol/L glucose 6-phosphate, 1 mmol/L NADP, 1 U of 6-phosphate dehydrogenase and microsome (0.4–0.6 mg protein) in 100 mmol/L potassium phosphate buffer (pH 7.4) was incubated at 37°C for 10 min. The enzyme reaction was terminated by the addition of 0.05 mL of sodium cholate (60 g/L). Cholesterol oxidase (0.5 U) dissolved in 0.02 mL of 0.1 mol/L phosphate buffer (pH 7.4) was then added and the mixture was incubated at 37°C for an additional 25 min to convert 7-hydroxycholesterol to 7-hydroxy-4-cholestene-3-one. The product was extracted with hexane and assayed by normal-phase HPLC using a S5W column (4.6 x 250 mm, Phase Separation, Baltimore, MD).
Steroids were extracted from the digestive contents (small intestine, cecum and colon) and feces by a mixture of chloroform/methanol (1:1, v/v) at 70°C for 60 h (20
). The concentration of total bile acids in these samples was determined enzymatically by the 3-dehydrogenase assay method of Sheltaway and Losowsky (21
) using taurocholic acid as a standard. Measurements of fecal cholesterol and coprostanol were performed as described previously (22
).
The moisture level of the cecal contents was determined as the difference between the wet mass and the dry mass of the cecal contents after freeze-drying. The pH of the cecal contents was measured immediately after removal with a compact pH meter using a sampling sheet (Model C-1, Horiba, Tokyo, Japan; calibrated at 20°C). Measurement of cecal organic acids was performed as described previously (22
).
Hepatic mRNA.
Total RNA was isolated from the liver according to the method described by Chomczynski and Sacchi (23
), and 15 µg of total RNA was subjected to Northern blot hybridization. The cDNA fragments used in the synthesis of RNA probes were as follows: rat LDL-receptor cDNA, a fragment corresponding to +2521 to +2874 (Sawady Technology, Tokyo, Japan); rat HMG-CoA reductase cDNA, a fragment corresponding to +576 to +960 (Sawady Technology). The cDNA fragments were subcloned to pGEM-T Easy Vector (Promega, Madison, WI) and RNA probes were synthesized with the Dig RNA labeling kit (SP6/T7) (Roche Molecular Biochemicals, Tokyo, Japan) and used for hybridization. The RNA probe of human ß-actin was used as a normalization standard. The specific hybridization was detected by the Dig Luminescent Detection kit (Roche Molecular Biochemicals) and the membrane was exposed to X-ray film at room temperature for 30 min.
Statistical analyses.
In Experiment 1, treatment effects (starch and cecectomy) were analyzed by two-way ANOVA, using a computer software package (StatView version 4.5, Abacus Concepts, Berkeley, CA). Duncan’s new multiple range post-hoc test was used to determine significant differences among groups. Groups in Experiment 2 were compared by Student’s t test. Differences were considered to be significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The body mass gain, food intake and plasma triacylglycerol concentration were not affected by cecectomy or dietary HACS (Table 2
). The digestibility of HACS was significantly lower in cecectomized rats (-17%), but that of CS was not. The plasma total cholesterol concentrations in sham-operated rats (-19%) and cecectomized rats (-10%) were significantly reduced by dietary HACS. Plasma LDL and HDL cholesterol concentration in cecectomized rats (-36 and -18%) were significantly reduced by dietary HACS.
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). The liver triacylglycerol concentration was not affected by dietary HACS but it was by cecectomy. The level of 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase) mRNA was not affected by cecectomy but it was affected by dietary starch.
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). The plasma apo A-IV concentration in rats fed the CS diet was significantly increased by cecectomy (+44%). The concentrations of plasma apo B and E were not affected by cecectomy or dietary HACS.
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-hydroxylase activity was greater in the cecectomized rats than in the sham-operated rats (+70% in rats fed the CS diet and + 56% in rats fed the HACS diet) (Table 5
). The bile acid pool in the small intestine was significantly greater in rats fed the HACS diet than in rats fed the CS diet (+156% in sham-operated rats and + 44% in cecectomized rats). However, the bile acid pool in the colon was not affected by cecectomy or dietary HACS. In the sham-operated rats, the bile acid pool in the cecum was significantly increased 188% by dietary HACS. The dry weight of the fecal output was significantly increased by dietary HACS, especially in the cecectomized rats (+154%). Fecal moisture was not affected by cecectomy or dietary HACS. In cecectomized rats, the fecal excretion of bile acids was significantly increased by dietary HACS (+37%). The fecal excretion of coprostanol was reduced by cecectomy. In cecectomized rats, fecal excretion of cholesterol was significantly increased by the HACS diet (+72%).
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). The moisture in the cecal contents was not affected by the diet. The concentrations of acetic, n-butyric and total short-chain fatty acids in the cecal contents were not affected by the diet. However, the concentrations of propionic, succinic and lactic acids in the cecal contents were significantly increased by dietary HACS (+98, +788 and +733%).
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The bile flow and bile acid flux from the liver to the intestine, which was calculated from the bile flow and biliary bile acid concentration, in intact rats fed the HACS diet were significantly greater than those in rats fed the CS diet, although the biliary bile acid concentration did not differ between the two groups (Table 7
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Kasaoka et al. (9
) reported that the plasma cholesterol concentration decreased as the dietary level of HACS increased in rats fed diets containing 100, 200 or 400 g HACS/kg, and suggested that the hypocholesterolemic effect of HACS is due to the reduction in energy intake. In the present study, however, the starch-derived energy intakes during the experimental periods taking into consideration digestibility, did not differ significantly among the 4 groups and were 1030 ± 50, 1071 ± 33, 983 ± 42 and 925 ± 29 kJ in the CS/Sham group, CS/Cecectomy group, HACS/Sham group and HACS/Cecectomy group, respectively. Body mass gain also was not affected by cecectomy or HACS diet feeding.
Nishimura et al. (24
) reported that the cecum is necessary for the hypocholesterolemic action of a highly fermentable sugar-beet fiber. Hara et al. (12
) reported that the fermentation products of sugar-beet fiber by cecal bacteria lower the plasma cholesterol concentration in rats. Propionate, a product of fermentation in the large intestine, has been reported to inhibit cholesterol synthesis in vitro in rat hepatocytes (25
). The cecal propionic acid concentration and wet weight of the cecal contents in the HACS/Sham group were significantly greater than those in the CS/Sham group. Therefore, an increased propionic acid concentration and its effect on hepatic cholesterol synthesis may be one of the mechanisms of the hypocholesterolemic effect of HACS. In this study, the plasma total cholesterol concentration in the HACS/Sham group tended to be lower (P = 0.19) than that in the HACS/Cecectomy group, which suggests that the fermentation products in the large intestine play a role in the hypocholesterolemic action of HACS. However, it has been pointed out that in vivo, the effect of propionate might not be sufficient to reduce the activity of HMG-CoA reductase, the rate-limiting enzyme in hepatic cholesterol synthesis (25
). In contrast, Sacquet et al. (7
) reported that amylomaize starch containing 65–75 g amylose/100 g lowered the plasma cholesterol concentration in germ-free rats. In the present study, the HMG-CoA reductase mRNA concentration did not differ between the two groups. These results suggest that the hypocholesterolemic effect of HACS is not mediated by the fermentation products in the large intestine. Also, most investigators currently believe that propionate does not inhibit cholesterol synthesis (26
,27
). In the sham-operated rats, the coprostanol/cholesterol ratio in the feces was significantly higher in those fed the HACS diet than in those fed the CS diet (0.235 vs. 0.690, P < 0.05), suggesting that HACS may alter the relative proportion of cecal bacterial species.
Fecal bile acid excretion in sham-operated and cecectomized rats fed the HACS diet was significantly greater than that in the respective rats fed the CS diet. In the sham-operated and cecectomized rats, the plasma total cholesterol concentration was significantly lower in rats fed the HACS diet than in rats fed the CS diet. From these results, increased fecal excretion of bile acid may be a mechanism of the hypocholesterolemic effect of HACS. The fecal excretion of bile acids and the level of cholesterol 7-hydroxylase activity were significantly greater in the cecectomized rats fed HACS than in the sham-operated rats, which would require enhanced bile acid synthesis after loss of the reservoir action on the intestinal contents of the cecum by cecectomy.
The small intestinal and cecal bile acid pool sizes in rats fed the HACS diet were greater than those in their counterparts fed the CS diet. Moundras et al. (28
) showed that the hypocholesterolemic effect of guar gum was mediated by an increase in the intestinal pool of bile acids. An enlargement of the bile acid pool will increase portal bile acid concentration, which may affect cholesterol metabolism in the liver, including decreases of cholesterol synthesis and VLDL secretion. Therefore, an increase in the intestinal pool of bile acids may be another possible mechanism of the hypocholesterolemic effect of HACS.
The bile acid pool size and fecal excretion of bile acids were increased by HACS, yet there was no significant increase in cholesterol 7-hydroxylase activity. Moriceau et al. (29
) reported that a marked enlargement of the intestinal pool of bile acids in rats fed guar gum may depend on the accelerated enterohepatic cycling of bile acids. There is more than one pathway for bile acid synthesis (30
). This synthesis is dependent in part on the sterol 27-hydroxylase pathway whose nutritional regulation remains poorly understood (31
). Therefore, the above-mentioned discrepancy may be explained in part by the accelerated enterohepatic cycling of bile acids and by the contribution of bile acid pathways other than the sterol 7-hydroxylase pathway to bile acid synthesis
Apo A-I and apo B are the principal proteins in HDL and LDL, respectively. Apo B plays a major role in the recognition of cellular receptors in the catabolism of LDL (32
). Numerous studies have indicated that measurement of apo A-I and apo B is useful in assessing the risk for cardiovascular disease (32
–34
). It has been reported that they are more specific and sensitive biochemical markers of cardiovascular disease risk than HDL cholesterol and LDL cholesterol concentrations (35
). In the present study, both HDL and LDL cholesterol concentrations were reduced by dietary HACS. The concentration of apo-A-I was lowered by dietary HACS, but that of apo B was not. The amount of cholesterol per LDL particle generally does not change much. The reason for this discrepancy is unknown. Apo E plays a major role in systemic cholesterol metabolism by serving as a ligand for the removal of cholesterol-laden plasma lipoproteins by hepatic receptors, and is able to protect against atherosclerosis. However, HACS did not favorably modify the concentration of apo E and the level of LDL-receptor mRNA.
The data from rat studies on the hypocholesterolemic effect of HACS (7
,9
,36
,37
) have not been confirmed in human studies (38
). Differences between the responses in humans and rats may be due to differences in lipid metabolism between the two species, and to the greater HACS intake in the latter, which is not achievable in humans.
In conclusion, HACS has a hypocholesterolemic effect in rats fed a cholesterol-free diet, and this effect is most likely mediated through enlargement of the bile acid pool in the intestine and increased fecal excretion of bile acid, and not through fermentation of HACS to produce additional propionate in the large intestine. HACS may enlarge the bile acid pool size by the acceleration of the enterohepatic cycling of bile acids and by the contribution of bile acid pathways other than the sterol 7-hydroxylase pathway to bile acid synthesis, which would increase fecal bile acid excretion.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: apo, apolipoprotein; CS, cornstarch; DF, dietary fiber; HACS, high amylose cornstarch; HMG-CoA, 3-hydroxy-3-methylglutaryl CoA; RS, resistant starch.
Manuscript received 17 December 2001. Initial review completed 8 March 2002. Revision accepted 2 June 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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