The incidence of coronary heart disease and other vascular diseases related to abnormalities in plasma lipid metabolism have increased in Taiwan in recent years (International Lipid Information Bureau Taiwan 1994). Increased blood lipid and serum cholesterol concentrations contribute to the etiology of cardiovascular diseases (Anderson et al. 1990). Lipids, carbohydrates and dietary fiber are factors that affect lipid and cholesterol metabolism (Grundy and Denke 1990). Both the American Heart Association and the American Dietetics Association suggest that the ratio of carbohydrates in the diet should be increased, especially by the intake of foods rich in complex carbohydrates. Rice is the staple food in many Asian cultures and also the main source of carbohydrates. Earlier studies mainly concerned the effects of dietary fiber (Sacks et al. 1975), and although the total fiber concentration in polished rice is low, polished rice (Cheng 1993, Cheng et al. 1994) and rice bran oil have been shown to lower serum cholesterol (Nicolosi et al. 1991). Therefore, there may be factors other than dietary fiber that influence serum cholesterol. Polished rice contains 2 g enzyme resistant starch (RS)⁴/100 g, which is not easily digested by intestinal enzymes, and RS in cornstarch has been shown to reduce serum cholesterol in rats (De Deckere et al. 1992, Morand et al. 1992). Moreover, many researchers believe that RS is fermented in the large intestine to produce short-chain fatty acids (SCFA), which may reduce serum cholesterol. Morand et al. (1992) and Marsono et al. (1993) have reported the effects of RS on plasma lipids in rats and pigs, respectively. Chen et al. (1984) reported that dietary propionate reduced cholesterol accumulation in both serum and liver of cholesterol-fed rats. Thus propionate, a metabolic product of fiber fermentation, may mediate some of the hypocholesterolemic effects of certain soluble plant fibers.

The objective of this research was to investigate the effect of RS in cooked-stored polished rice (CSPR) diets, with and without exogenous cholesterol, on lipid metabolism in rats. This study was also designed to determine whether fermentation in the hindgut of rats fed antibiotics (Nebacitin) influences lipid metabolism in rats.

MATERIALS AND METHODS

Table 1. Composition of the cook-stored polished rice (CSPR) diets1 [View Table]

Table 2. Concentrations of triglyceride, total cholesterol, HDL cholesterol and LDL cholesterol in the serum of rats fed cooked-stored polished rice (CSPR) diets with or without 7% Nebacitin and with or without 1% cholesterol1 [View Table]

The raw polished rice was obtained by polishing the brown rice for 25 s in a polisher (Kuang Tien Co., Taichung, Taiwan). The rice was cooked in an electric cooker for 30 min at 100°C, dried in a hot dryer at 60°C for 16 h and cooled. The same process was repeated five times. The rice was then ground and sifted using a 0.42-mm diameter mesh analytical sifter. Finally it was stored in a refrigerator at 6°C as retrograded cooked-stored polished rice.

The RS content was measured according to Berry's method (1986) on precisely weighed samples. Pancreatic -amylase was added for 16 h at 37°C followed by four volumes of absolute ethanol for 1 h, and the sample was centrifuged (15,000 × g) for 10 min. The supernatant was then skimmed and the sediment dissolved with 2 mol/L KOH. Acetic acid was added to adjust the pH to 4.5. Amyloglucosidase was added, and the sample was incubated for 90 min at 65°C followed by centrifugation (1200 × g) for 3 min. The supernatant was skimmed and measured by the glucose oxidase assay, and the RS content was then calculated.

During the 4-wk feeding period, feces were collected once every 2 d and stored in a freezer at 20°C until analyzed. Food was withheld for 12 h during wk 4. The rats were anesthetized with 50 g/L sodium pentobarbital and dissected. Blood was collected from the abdominal aorta, incubated at room temperature for 45 min, and centrifuged at 4000 × g for 15 min. The serum was stored in a freezer at 70°C. The livers were excised, rinsed in 9 g/L NaCl, sealed tightly and stored at 70°C until the assays were performed.

Standard procedures (Merck Chemical, Darmstadt, Germany) were used to determine serum triglyceride (Merck procedure 14354), serum total cholesterol (Merckotest 14366), serum HDL cholesterol (Merckotest 14210), serum LDL cholesterol (Merckotest 14992) and bile acids (Merckotest 14352). Short-chain fatty acids were measured by gas-liquid chromatography using serum samples after ethanolic extraction (Demigné and Rémésy 1982). Frozen liver was thawed and lipids were then extracted according to the Folch et al. (1957) method. The liver lipid extract was according to the method of Soloni (1971), compared with a triolein standard solution, and used to determine the liver triglyceride content. Liver cholesterol was measured by an enzymatic assay (Carlson and Goldfarb 1977).

Total neutral sterols and bile acids were extracted according to the method of Roscoe and Fahrenbach (1963). An aliquot of the extract was saponified, and neutral sterols were removed by extracting the saponified, dried extract with hexane. The method of Collings et al. (1979) was used to determine fecal total neutral steroid concentration and fecal bile acids.

Statistical analysis was performed by two-way ANOVA using SAS software (SAS Institute, Cary, NC), and the differences among groups were compared by Duncan's multiple range test (Ott 1988). Differences of P  0.05 were regarded as significant. The hepatic triglyceride and total cholesterol concentrations, fecal RS contents, fecal neutral steroids concentrations and butyrate concentrations were logarithmically transformed before ANOVA. Untransformed means and SEM are presented.

RESULTS

In rats fed Nebacitin (RN, CRN), serum total cholesterol and triglyceride concentrations were greater than in the R and CR groups, regardless of dietary cholesterol concentration (Table 2). Adding Nebacitin did not influence serum HDL cholesterol concentration. In rats fed the CRN diet, serum LDL cholesterol concentration was greater than in the R, RN and CR groups. Liver weights of rats fed nebacitin (RN, CRN) were comparable to those of the R and CR groups, respectively (Table 3). The rats fed Nebacitin and cholesterol (CRN) had significantly higher liver total cholesterol content and concentration than the R, RN and CR groups (Table 3).

Table 4. Weight, moisture, crude fat and crude protein in the feces of rats fed cooked-stored polished rice (CSPR) diets with or without 7% Nebacitin and with or without 1% cholesterol1 [View Table]

Table 3. Weight, triglyceride and total cholesterol concentrations in the liver of rats fed cooked-stored polished rice (CSPR) diets with or without 7% Nebacitin and with or without 1% cholesterol1 [View Table]

The Nebacitin-fed groups (RN, CRN) had higher fecal wet weight, dry weight, moisture and crude protein concentrations, and lower fecal crude fat concentration than the R and CR groups, respectively (Table 4). The Nebacitin-fed groups (RN, CRN) had significantly higher fecal RS concentration and excretion (Table 5) and neutral steroid excretion than the Nebacitin-free groups (R, CR) (Table 6). The rats fed Nebacitin and cholesterol (CRN) had significantly higher bile acid concentration and excretion than the R, RN and CR groups (Table 6). The Nebacitin-fed groups (RN, CRN) had lower serum SCFA concentration than the Nebacitin-free groups (R, CR) (Table 7).

Table 5. Resistant starch contents of diet and feces in rats fed cooked-stored polished rice (CSPR) diets with or without 7% Nebacitin and with or without 1% cholesterol1 [View Table]

Table 6. Fecal total neutral steroids and total bile acid excretions and contents in rats fed cooked-stored polished rice (CSPR) diets with or without 7% Nebacitin and with or without 1% cholesterol1 [View Table]

Table 7. Concentration of short-chain fatty acids in the serum of rats fed cooked-stored polished rice (CSPR) with or without 7% Nebacitin and with or without 1% cholesterol1 [View Table]

DISCUSSION

The RS cannot be completely digested by enzymes in the small intestine. In this study, little of the RS was excreted into the feces, indicating that most of the RS in the CSPR diets was fermented by bacteria in the intestines of rats not fed Nebacitin. Under these dietary conditions, the addition of Nebacitin to the diet did not affect weight gain in rats, perhaps because the RS products fermented in the large intestine can provide energy (metabolizable energy of about 8.36 kJ/g; Livesey 1990).

Resistant starch is fermented by the gut microflora into SCFA in the large intestines (Demigné and Rémésy 1982). Chen et al. (1984) reported that the propionate generated by bacterial fermentation of fiber reduced cholesterol accumulation in both serum and liver of cholesterol-fed rats. The hypocholesterolemic effect of propionate may be related to altered hepatic cholesterol synthesis. Nishina and Freedland (1990) reported that the effect of propionate on lipid metabolism is apparently limited to inhibition of de novo fatty acid synthesis. Moundras et al. (1994) suggested that the propionate-enhanced fecal bile acid excretion seems not to be the only mechanism by which propionate lowers plasma cholesterol, because acetate is also effective.

Some investigators (Jonnalagadda et al. 1993, Morgan et al. 1993) contend that dietary fiber can reduce the absorption of exogenous cholesterol and increase the excretion of fecal steroids, which may thus reduce serum cholesterol. The RS in the CSPR diets had components similar to those of dietary fiber (Sacquet et al. 1983), which could therefore reduce the absorption of exogenous cholesterol. Alterations in large bowel steroid metabolism may be involved in rats fed both bile acids and cholesterol, because feeding bile acids raises plasma cholesterol in rats fed cholesterol (Beynen et al. 1986). If there were more primary bile acids in the stool, then rats, which are coprophagic (Jackson and Topping 1993), could be exposed to more bile acids, leading to an effect similar to the feeding of cholate in the diet. Beynen et al. (1986) showed clearly that only when cholesterol is fed with bile acids do plasma cholesterol concentrations approach those found in humans.

Because the RS in the CSPR diets had components similar to those of dietary fiber, it could combine with bile acids (Topping 1991), thus reducing the amount of bile acids absorbed into the liver through the enterohepatic circulation. The RS could increase the excretion of bile acids.

The cholesterol-free groups (R, RN) had lower hepatic cholesterol concentrations than the cholesterol-fed groups (CR, CRN). When the rats were fed cholesterol-free diets, the influence of Nebacitin was not apparent. In rats fed cholesterol-containing diets, lipid metabolism was altered and an increase in hepatic cholesterol concentration due to Nebacitin was evident. Moundras et al. (1994) suggested that a 40-50% rise in bile acid excretion is required to significantly depress plasma cholesterol and to induce hydroxymethylglutaryl CoA reductase and cholesterol 7-hydroxylase.

In this study, the RS reduced serum triglyceride concentration. Vahouny et al. (1988) reported that the dietary fiber reduces lipid absorption and decreases triglyceride concentration. Because RS can generate SCFA by fermentation, some researchers (Suzuki and Kajuu 1983) have pointed out that SCFA can inhibit the synthesis of hepatic triglycerides and reduce serum lipids. Nishina and Freedland (1990) have shown that the propionate can inhibit the activity of pyruvate dehydrogenase in liver and thus reduce the synthesis of fatty acids. Moreover, when antibiotics (Nebacitin) were added to the CSPR diet to inhibit fermentation, the serum triglyceride concentration was greater than in R and CR groups, which did not receive Nebacitin (P < 0.05).

In summary, the results presented here indicate that hepatic cholesterol concentration was greater in rats fed Nebacitin only when the diet also contained cholesterol. Therefore, dietary Nebacitin alters lipid metabolism in rats, and some effects are most pronounced in rats that are also fed cholesterol.