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The Journal of Nutrition Vol. 127 No. 3 March 1997, pp. 470-477
Copyright ©1997 by the American Society for Nutritional Sciences

Cholesterol-Lowering Effects of Soybean, Potato and Rice Proteins Depend On Their Low Methionine Contents In Rats Fed a Cholesterol-Free Purified Diet1

Tatsuya Morita2, Akira Oh-hashi, Kaori Takei*, Michiyoshi Ikai, Seiichi Kasaoka, and Shuhachi Kiriyama*, 3

Azusawa Laboratories, Health Science Laboratories, Yamanouchi Pharmaceutical Co., Ltd. Tokyo 174, Japan and * Laboratory of Nutritional Biochemistry, Department of Bioscience and Chemistry, Faculty of Agriculture, Hokkaido University, Sapporo 060, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
LITERATURE CITED

ABSTRACT

Rats were fed cholesterol-free purified diets containing casein, rice (RP), potato (PP) or soybean (SP) proteins having different amounts of methionine (25.9, 21.3, 16.2 and 10.9 g methionine/kg, respectively). Each protein was fed at 250 g/kg diet for 14 d. Growth rates of rats were the same in all groups. Serum total cholesterol concentrations were lower in rats fed SP, PP and RP than in those fed casein. Fecal bile acid plus neutral steroid excretion was significantly higher in rats fed the RP, PP and SP diets compared with those fed casein. There was a significant negative correlation between serum cholesterol concentration and fecal total steroid excretion (r = -0.490, P = 0.01). However, a stronger positive correlation was observed between serum cholesterol concentration and dietary methionine concentration (r = 0.674, P = 0.0003) or methionine:glycine ratios (r = 0.656, P = 0.0005). In a separate experiment in rats fed diets containing amino acid mixtures simulating the RP, PP and SP diets, serum total cholesterol concentrations were lower than in rats fed simulated casein. Fecal total steroid excretion was the same in all groups. A strong correlation was found between serum cholesterol concentration and dietary methionine concentration (r = 0.743, P = 0.0002) or the methionine:glycine ratio (r = 0.685, P = 0.0009) in rats fed the amino acid mixtures. Finally, we examined the hypocholesterolemic effects of 250 g SP or casein/kg diet with or without supplementation with 0.3 g/100 g sodium taurocholate (TC). Supplementation with TC did not alter the hypocholesterolemic effect of SP. These results support the view that RP, PP and SP lower serum cholesterol concentration in a similar manner.

Key words: serum cholesterol, soybean protein, rice protein, potato protein, rats.

INTRODUCTION

Numerous attempts have been made to explain the mechanisms by which soybean protein exerts hypocholesterolemic effects in experimental animals. Huff and Carroll (1980) reported that in rabbits fed a cholesterol-free purified diet soybean protein increased fecal excretion of bile acids relative to casein. This finding has been reproduced in a number of studies using rats fed a cholesterol-free purified diet (Beynen et al. 1986, Nagata et al. 1982) and is considered to be one of the main mechanisms of hypocholesterolemic activity of soybean protein in rats as well as rabbits.

Further studies showed that an amino acid mixture simulating soybean protein also demonstrated hypocholesterolemic effects without influencing fecal bile acid excretion in rats (Nagata et al. 1981) and rabbits (Huff et al. 1977) compared with an amino acid mixture simulating casein, although the hypocholesterolemic effect of the corresponding amino acid mixture was weaker than that of native soybean protein (Potter 1995). These results suggest that a mechanism other than the enhancement of bile acid excretion may be a factor in the hypocholesterolemic effect of soybean protein and also suggest the possiblity of different mechanisms for the hypocholesterolemic effects of the native protein and its corresponding amino acid mixture.

On the basis of studies of the metabolites of methionine, Sugiyama et al. (1986) reported that the methyl group of methionine is responsible for the cholesterol-elevating effect of methionine in rats. Our previous study (Saeki and Kiriyama 1990) also showed that the addition of 0.22% methionine to a 25% soybean protein diet, when the methionine concentration of this diet was equivalent to that of the 25% casein diet, increased plasma cholesterol to the levels of rats fed the casein diet. In contrast, Moundras et al. (1995) reported that when rats were fed soybean protein at a suboptimal level (13%), serum cholesterol concentration was significantly higher than that in rats fed a 13% casein diet, and this higher cholesterol level was counteracted by supplementation of the diet with 0.4% methionine.

Thus, recent studies of rats fed cholesterol-free purified diet have focused on the methionine content as well as the amino acid composition of soybean protein rather than changes in fecal steroid excretion as causative factors of hypocholesterolemia. However, few studies have been done to verify the importance of the methionine content of other dietary proteins.

Our aim in this study was to examine whether it is possible to uniformly explain the role of methionine in hypocholesterolemic effects of some dietary proteins in rats fed cholesterol-free purified diets. We used four different proteins: casein, soybean protein (SP),4 potato protein (PP) and rice protein (RP) that contain graded levels of methionine, and examined their effects on serum cholesterol concentrations. In addition, we reexamined the involvement of bile acid metabolism in the hypocholesterolemic effect of SP.

MATERIALS AND METHODS

Proteins. A crude rice protein fraction (moisture content, 50%) extracted from rice grits of Oryza sativa L. Japonica (cv. Todoroki) with 0.0875 mol NaOH/L was supplied by Shimada Chemical Co. (Niigata, Japan). This fraction contained 368 g protein·kg dry matter-1 and was used as starting material for the production of alkaline-extracted RP. Portions of crude protein (750 g) were each mixed with 10 L of NaOH solution (0.0875 mol/L, pH 12.5-13.5), and the mixture was stirred with a motor-driven propeller shaft at room temperature (~23°C) for 2 h. The mixture was centrifuged at 500 × g for 2 min, and supernatant was collected. The pH of the supernatant was adjusted to 6.0 with HCl (1 mol/L). Care was exercised to keep the pH >= 5.5 in order to avoid formation of prolamin salts and consequent loss by solubilization. The precipitate was allowed to settle for 1 h at 4°C. After removing most of the supernatant, the precipitate was collected by centrifuging at 500 × g for 2 min, and the packed fraction was washed twice with 5 L of distilled water to remove NaCl. Finally, the packed fraction was washed twice with 5 L of ethanol (99%) and air-dried at room temperature. From 750 g starting material, 80-90 g RP was obtained with a protein recovery of 50-60%. This rice protein preparation contained 122.6 mg N/g. Potato protein (122.6 mg N/g) obtained from potato juice by steam coagulation after adjustment of pH to 5.0-5.5 was supplied by UnicoopJapan (Tokyo, Japan). Casein (125.5 mg N/g) and SP (126.2 mg N/g) were purchased from New Zealand Dairy Board (Wellington, New Zealand) and Fuji Oil (Osaka, Japan), respectively. The moisture levels of casein and SP were 12 and 13%, respectively.

Table 1. Chemical composition of potato protein and rice protein

[View Table]

Serum cholesterol concentration is affected by other factors present in protein fractions such as carbohydrate (Carroll and Hamilton 1975, Neves et al. 1980). Therefore, we used four different protein sources in which the protein concentration was >80 g/100 g dry matter. Each protein preparation resulted in maximum growth rates when fed to rats at a concentration of 250 g/kg diet (data not shown).

Chemical analyses. Protein content was determined by the Kjeldahl method (Miller and Houghton 1945) using the N-to-protein conversion factor of 6.25. Moisture was determined as the loss in weight after drying at 105°C for 24 h. Ash content was determined by the direct ignition method (550°C overnight). Total dietary fiber content was measured by the method of Prosky et al. (1988). Lipids were extracted with chloroform:methanol (2:1, v/v) by the method of Folch et al. (1957), and crude lipid content was measured gravimetrically after removal of the solvent. Carbohydrate content was calculated by subtracting protein, ash, water, lipids and total dietary fiber mass from the total mass of the protein preparation. The results of our chemical analyses for PP and RP are shown in Table 1.

Amino acid composition (with the exception of tryptophan) was determined using an amino acid autoanalyzer (Beckman 120C, Schiller Park, IL) according to the method of Chang et al (1986). Tryptophan content was determined by HPLC (Lambda-Max Model LC Spectrometer, Waters, Tokyo, Japan) after protein hydrolysis under alkaline conditions. The amino acid compositions of casein, SP, RP and PP are shown in Table 2.

Table 2. Amino acid compositions of casein, soybean, potato and rice proteins

[View Table]

Care of animals. This study was approved by Animal Use Committees of Yamanouchi Pharmaceutical Co. and Hokkaido University, and animals were maintained in accordance with the guidelines for the care and use of laboratory animals of Yamanouchi Pharmaceutical Co. and Hokkaido University.

Male Sprague-Dawley rats (Shizuoka Laboratory Animal Center, Hamamatsu, Japan) were housed individually in screen-bottomed, stainless steel cages in a room maintained at 23 ± 1°C and lighted from 0600-1800 h. After acclimation to a starch-based diet (Table 3) with 250 g casein/kg diet for 1 wk, rats were divided into groups on the basis of body weight and allowed free access to experimental diets and water. Body weight and food intake were recorded each morning before replenishing the diet.

Table 3. Composition of casein-starch diet

[View Table]

Experiment 1: Effects of various proteins on lipid metabolism in rats. Six-wk-old rats weighing 205-228 g were divided into 4 groups of 6 standardized for body weight. Each group was fed freely for 14 d one of the respective test diets (casein, SP, PP and RP diets). The composition of each test diet was the same as the casein-starch diet (Table 3) except for protein source. Feces were collected for the last 3 d of the experimental period. Food was removed from cages 2-3 h prior to killing rats with Nembutal (pentobarbital sodium, Abbott Laboratories, North Chicago, IL). Blood was collected from the abdominal aorta, and serum was prepared and stored at -20°C. After blood collection, livers were perfused with ice-cold NaCl solution (0.154 mol/L). The microsomal fraction was prepared (Horio et al. 1989) and suspended in 0.05 mol Tris-HCl/L buffer (pH 7.4). Microsomal protein was determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard. Cholesterol-7alpha hydroxylase activity (EC 1.14.13.17) was measured according to the method of Horio et al.(1989). The remaining portions of livers were stored frozen at -50°C until the lipid content was measured.

Aortic blood serum was used for determination of cholesterol. Total and HDL cholesterol concentrations were measured colorimetrically with commercially available kits (Cholesterol C-test and HDL-cholesterol test, Wako Pure Chemical Industries, Tokyo, Japan). The concentration of (VLDL + LDL) cholesterol was calculated as the difference between total and HDL cholesterol.

Liver total cholesterol was measured with the commercial kit mentioned. Lipids were extracted from 500 mg liver with chloroform:methanol (2:1, v/v) according to the method of Folch et al. (1957). After lipid extraction, the lipid solution volume was adjusted to 20 mL with the same solution. From this extract, 1 mL was dried under a nitrogen stream, and the residue obtained was mixed with 200 µL isopropyl alcohol containing 10% Triton X-100 (Wako Pure Chemical Industries, Tokyo, Japan). From this mixture, 20 µL was mixed with 3 mL of aqueous enzyme solution according to the standard procedure of the assay kit, and cholesterol concentration was determined colorimetrically. In a preliminary study, 20 µL of isopropyl alcohol containing 100 g Triton X-100/L did not affect the enzymatic reactions (data not shown).

Total fecal steroids were extracted with a mixture of chloroform:methanol (1:1, v/v) at 70°C for 60 h (Eneroth et al. 1968). Total fecal bile acids were determined enzymatically by the 3alpha -hydroxysteroid dehydrogenase assay (EC 1.1.1.50) method of Sheltawy and Losowsky (1975) using lithocholic acid as a standard. The analysis of fecal neutral sterols was determined using a gas-liquid chromatograph (GC-14B, Shimadzu, Kyoto, Japan) equipped with flame-ionization detecter. For separation, a glass column with silicone SE-30 as stationary phase and chromosorb W AW-DMCS as support (2.6 mm i.d. × 2.1 m, Shinwa Chemical, Kyoto, Japan) was used at 270°C. Carrier gas was nitrogen at a flow rate of 50 mL/min. 5alpha -Cholestane was used as the internal standard. In this experiment, we defined neutral sterols as the sum of cholesterol and coprostanol.

Experiment 2: Effects of amino acid mixtures simulating native proteins on serum cholesterol concentrations in rats. Amino acid mixtures simulating casein, SP, PP and RP were prepared using crystalline L-amino acids (Ajinomoto Co., Tokyo, Japan) according to the amino acid composition of the corresponding protein sources (Table 2). The diets containing amino acid mixtures were isonitrogenous with the 250 g casein/kg diet. Half of the aspartic and glutamic acids were replaced by the respective amides because we did not have any direct measurement of asparagine or glutamine concentration in each protein.

Six-wk-old rats weighing 205-228 g were divided into 4 groups of 5 standardized for body weight. Each group was fed freely for 14 d one of the respective amino acid diets simulating casein (casein-AA), SP (SP-AA), PP (PP-AA) and RP (RP-AA). The composition of each diet was the same as the casein-starch diet (Table 3) except for nitrogen sources. Feces were collected for the last 3 d of the experimental period. At the end of experiment, lipid metabolism was studied as described above.

Experiment 3: Influence of dietary taurocholate supplementation on cholesterol-lowering effect of SP. In a preliminary study, we confirmed that supplementation of 0.3 g sodium taurocholate (TC)/100 g diet to the 250 g SP/kg diet increased the gastrointestinal bile acid pool (small intestine + cecum) threefold compared with the unsupplemented basal diet (33.16 ± 4.6 and 94.85 ± 8.2 µmol total bile acids, respectively). Consequently, this experiment was planned on the premise that if enhancement of bile acid excretion is the main causative factor for the hypocholesterolemic activity of SP, this activity might disappear when the bile acid pool size was enlarged by the addition of TC to the diet.

Five-week-old rats weighing 170-185 g were divided into 4 groups of 6 standardized for body weight. Each group was fed freely for 10 d one of the respective test diets (casein, SP, TC-supplemented casein, and TC-supplemented SP). In this experiment, sucrose was used as the sole carbohydrate source, and other dietary ingredients were the same as in the casein-starch diet (Table 3) except for protein source and supplementation of TC. Supplementation of TC to 3 g/kg diet was accomplished by replacing an equal weight of TC-free diet with TC. At the end of the experimental period lipid metabolism was examined as described above.

Statistical analyses. Data from Experiments 1 and 2 were analyzed by one-way ANOVA, and data from Experiment 3 were analyzed by two-way ANOVA. Significant differences among means were separated by using Duncan's multiple range test (Shibata 1974). A 5% level of probability was used to define differences as significant. The correlations among serum cholesterol concentration, fecal bile acid excretion, cholesterol-7alpha hydroxylase activity, and dietary methionine concentration and dietary methionine:glycine ratio were analyzed by linear regression (Snedecor and Cochran 1967).

RESULTS

Experiment 1: Effects of various proteins on lipid metabolism in rats. Although the mean food intake of rats fed the PP diet was significantly lower than those fed the other diets, there were no significant differences in body weight gain among the dietary groups (Table 4). Serum cholesterol concentrations in rats fed the SP, PP and RP diets were significantly lower than in rats fed the casein diet. This was accompanied by several differences in the distribution of cholesterol among serum lipoproteins. Mean serum HDL cholesterol concentration was lower in rats fed the SP and PP diets but not the RP diet, while LDL + VLDL cholesterol concentration was lower in rats fed the RP diet as well as the SP and PP diets compared with those fed the casein diet.

Table 4. Body weight gain, food intake, liver and serum cholesterol concentrations, total liver lipids and activities of hepatic cholesterol 7 alpha -hydroxylase in rats fed the casein, soybean, potato and rice protein diets for 14 d (Experiment 1)1

[View Table]

Mean relative liver weights of rats fed the SP, PP and RP diets were significantly lower than in rats fed the casein diet. Feeding the PP diet significantly lowered the concentration of liver total lipids relative to the casein diet, but the SP and RP diets had no effect on liver total lipids. The mean concentration of liver cholesterol was significantly lower in rats fed the PP and RP diets, but not SP diet, compared with those fed the casein diet.

The activity of hepatic cholesterol 7alpha -hydroxylase was expressed per mg microsomal protein (specific activity) or per total hepatic microsomal protein (total activity). Mean specific activity in rats fed the SP, PP and RP diets was significantly higher than that of rats fed the casein diet. Mean specific activity of the RP-fed group was also higher than that of the SP- and PP-fed groups. Total activity in rats fed the PP and RP diets was also significantly higher than that in rats fed the casein diet, but the difference between the rats fed the SP and casein diets was not significant.

Significantly greater amounts of feces were excreted by rats fed the SP, PP and RP diets compared with rats fed the casein diet (Table 5). Fecal total bile acid excretion by rats fed the SP, PP and RP diets was significantly higher than that of rats fed the casein diet (P < 0.05), with total bile acid excretion by rats fed PP being greater than that of all other groups. The excretion of cholesterol and coprostanol in rats fed the SP and PP diets was significantly higher than that of rats fed the casein diet, while the RP diet did not increase neutral sterol excretion relative to the casein diet. Thus, total steroid excretion (bile acids + cholesterol + coprostanol) was significantly greater by rats fed the SP, RP and PP diets compared with that by rats fed the casein diet, with total excretion by rats fed PP being greater than that of all other groups.

Table 5. Fecal excretion in rats fed the casein, soybean, potato and rice protein diets for 14 d (Experiment 1)1

[View Table]

Siginificant negative correlations were observed between serum cholesterol concentration and the fecal excretion of total steroids (r = -0.490, P = 0.0151) or bile acids (r = -0.489, P = 0.0153) (Table 6). A significant positive correlation was also observed between fecal bile acid excretion and the specific activity of hepatic cholesterol 7alpha -hydroxylase (r = 0.548, P = 0.0056). However, stronger correlations were observed between serum cholesterol concentrations and the dietary methionine concentration (r = 0.708, P = 0.0003) or methionine:glycine ratios (r = 0.656, P = 0.0005). The lysine:arginine ratio in the diets also showed a significant correlation with serum cholesterol concentrations (r = 0.442, P = 0.0306).

Table 6. Linear regression analyses among various factors influencing serum cholesterol concentrations in rats fed the respective protein diets for 14 d (Experiment 1)

[View Table]

Experiment 2: Effects of amino acid mixtures simulating parent proteins on serum cholesterol concentrations in rats. There were no significant differences in food intakes and body weight gains among rats fed the respective amino acid diets for 14 d (Table 7). The cholesterol-lowering effects of the SP, PP and RP diets were clearly reproduced in their corresponding amino acid diets relative to casein-AA diet. Serum cholesterol concentrations in rats fed the SP-AA, PP-AA and RP-AA diets were significantly lower than those in rats fed the casein-AA diet and the differences among SP-AA, PP-AA and RP-AA diets were not significant. Differences in HDL cholesterol paralleled those of total cholesterol concentrations among the test diet groups. The only significant difference in (LDL + VLDL) cholesterol was observed between the SP-AA and casein-AA diet groups.

Table 7. Body weight gain, food intake, liver and serum cholesterol concentrations, total liver lipids and activities of hepatic cholesterol 7alpha -hydroxylase in rats fed amino acid diets simulating casein, soy, potato and rice proteins for 14 d (Experiment 2)1

[View Table]

Although there were no significant differences in relative liver weights among the dietary groups, liver total lipids in rats fed the SP-AA and PP-AA diets were significantly higher than those in rats fed the RP-AA and casein-AA diets. Liver cholesterol in rats fed the SP-AA diet was also higher than that in rats fed the casein-AA diet. These results were quite different from the results obtained in rats fed the intact protein diets (Experiment 1) where total liver lipids as well as liver cholesterol in rats fed the SP, PP and RP diets tended to be lower than those in rats fed the casein diet. Differences in hepatic cholesterol 7alpha -hydroxylase activities among the amino acid diet groups were not significant.

Fecal weight as well as fecal bile acid excretion were comparable among the groups tested. Excretion of cholesterol and coprostanol differed among groups, but there were no significant differences in the excretion of total neutral sterols (cholesterol + coprostanol). Total steroid excretion (total bile acids + total neutral sterols) was also comparable in the groups (Table 8).

Table 8. Fecal steroid excretion in rats fed amino acid diets for 14 d (Experiment 2)1

[View Table]

In contrast to the results of Experiment 1, the results with amino acid diets showed no significant correlations between serum cholesterol concentration and fecal total steroid excretion or fecal bile acid excretion (Table 9). However, there were significant correlations between serum cholesterol concentrations and diet methionine concentration (r = 0.743, P = 0.0002) or methionine:glycine ratios (r = 0.685, P = 0.0009). The lysine:arginine ratio in the diets also significantly correlated with serum cholesterol concentration (r = 0.536, P = 0.0148).

Table 9. Linear regression analyses among various factors influencing serum cholesterol concentrations in rats fed the respective amino acid diets for 14 d (Experiment 2)

[View Table]

Experiment 3: Influence of bile acid supplementation on cholesterol-lowering effect of SP. Feeding SP significantly increased body weight gain and food intake compared with feeding casein (Table 10). Mean serum cholesterol concentration of rats fed the SP and TC-supplemented SP diets was significantly lower than that of rats fed the casein and TC-supplemented casein diets. Supplementation of TC itself did not affect serum cholesterol concentrations in either casein or SP groups.

Table 10. Body weight gain, food intake and liver and serum cholesterol concentrations in rats fed the casein and soybean protein diets with or without 3 g sodium taurocholate/kg diet supplementation for 10 d (Experiment 3)1

[View Table]

Relative mean liver weight was comparable in the four groups. Supplementation of TC caused significantly greater hepatic cholesterol concentration in rats fed both casein and SP diets. However, mean hepatic cholesterol concentration in the SP group was significantly lower than that in casein group as observed in Experiment 1, even when TC was supplemented to 3 g/kg in casein and SP diets.

DISCUSSION

These experiments showed that the cholesterol-lowering effects of SP, RP and PP were correlated with their methionine concentration or methionine:glycine ratios (Experiment 1). The effects of SP, PP and RP were reproduced in the experiment with AA mixture diets (Experiment 2) and strong correlations between serum cholesterol concentration and the methionine concentration or methionine:glycine ratio in the mixtures were observed. The ratio of lysine:arginine also correlated with serum cholesterol as reported by Kritchevsky et al. (1982).

Enhancement of fecal steroid excretion is proposed as a possible mechanism for the effects of SP in both rats and rabbits fed cholesterol-free purified diets. The increased fecal steroid excretion would lead to an increased hepatic conversion of cholesterol to bile acids and activation of cholesterol 7alpha -hydroxylase, causing an elevation in LDL receptor expression and reducing serum cholesterol concentrations. This may be true in rabbits (Huff and Carroll 1980, Kurowska et al. 1993), but caution must be exercised in extrapolating to rats, since there are species-specific differences in the distribution of cholesterol in the lipoprotein fractions between rats and rabbits. In rats, most of the serum cholesterol (60-80%) is transported in HDL and only 5-10% in LDL (Day et al. 1979). In rabbits, 50% of serum cholesterol is carried by HDL, 35% by LDL and 15% by VLDL (Brattsand 1976). Also, rats do not possess lipid transfer protein.

Several observations suggest that increased fecal steroid excretion is a primary mediator of the hypocholesterolemic effects of SP. Although a number of studies have shown enhanced fecal bile acid excretion in rats fed cholesterol-free purified diets containing 200-250 g SP/kg diet, the increase was only 50 to 100% greater than in rats fed casein diet (Nagata et al. 1981, Sautier et al. 1986). In contrast, cholestyramine at a concentration of only 8 g/kg diet increased fecal bile acid excretion fivefold, but did not lower serum cholesterol concentrations (Younes et al. 1995). In additon, Huff et al. (1963) reported 30 years ago that 2 g cholestyramine/kg diet normalized plasma cholesterol concentrations in rats fed a cholesterol-enriched diet. Again, this treatment failed to lower plasma cholesterol concentrations in rats fed a cholesterol-free diet even though fecal bile acid excretion was greatly enhanced. They suggested that increased hepatic de novo cholesterol synthesis might have counteracted the anticipated reduction of plasma cholesterol.

Most bile acids (90-95%) secreted into the intestinal lumen are reabsorbed in the ileum. Ileal resection in rats increases fecal steroids, but the cholesterol-lowering effects of SP remains (Saeki et al 1987) in rats with ileectomy. In addition, plasma cholesterol concentrations in rats with ileectomy were similar to those found in sham-operated rats fed either casein or SP diets. This was also the case for rats with jejunectomy.

Experiment 3 showed that TC supplementation of the SP diet did not compromise the cholesterol-lowering effect of SP, even though the bile acid pool size was enlarged threefold in rats fed the TC-supplemented SP diets compared with that in rats fed the usual SP diet. Taking these findings into consideration, a high rate of bile acid excretion is effective only in rats fed a cholesterol-enriched diet and is not always sufficient to elicit a cholesterol-lowering effect in rats fed a cholesterol-free purified diet. Therefore, methionine contents in the dietary proteins would be more responsible for cholesterol-lowering effects of RP, PP and SP rather than the enhancement of fecal steroid excretion.

We have no direct explanation for the mechanism of the effect of methionine on cholesterol metabolism. However, Oda et al. (1991 and 1993) have shown that, compared with a casein diet, the SP diet decreased the expression of hepatic apolipoprotein A-I mRNA and thereby led to a reduction of apolipoprotein A-I and HDL secretion from the liver in rats fed a cholesterol-free diet. These reductions were restored by the supplementation of methionine to a SP diet. When we consider that more than 50% of serum cholesterol is carried by HDL and that the degrees of reduction in serum cholesterol in rats fed the RP, PP and SP diets generally were the highest in HDL fractions, it is reasonable to assume that the amounts of methionine in the diets play a central role in cholesterol-lowering effects through the regulation of Apo A-I secretion from the liver into blood circulation. If this is the only mechanism for the cholesterol-lowering effects of RP, PP and SP evidenced here, it would be natural to expect the higher hepatic cholesterol concentrations in rats fed the SP, PP and RP diets because their lower concentrations of methionine relative to casein could lower the secretion rate of HDL cholesterol from the liver. However, RP, PP and SP diets did not increase the liver cholesterol concentrations. In contrast, a tendency to increase liver cholesterol concentration was observed in rats fed the SP-AA, PP-AA and RP-AA diets compared with casein-AA. In fact, others also have reported that the level of hepatic cholesterol showed no correlation with the degree of hypocholesterolemia induced by a dietary amino acid mixture simulating SP (Nagata et al. 1981).

These different responses of hepatic cholesterol concentration to the native protein and amino acid diets might be due to the presence or absence of the enhancement of fecal steroid excretion. In this connection, one hypothesis is that although the enhancement of fecal steroid excretion itself cannot be a primary factor for the cholesterol-lowering effects of RP, PP and SP as described above, these effects might be exerted through the increased fecal excretion of steroids and more importantly, the decreased secretion of cholesterol from the liver to blood circulation.

In contrast to methionine which is hypercholesterolemic in rats fed a cholesterol-free diet (Saeki and Kiriyama 1990), Sugiyama et al. (1993) clearly showed that supplementation of 2.0% glycine to a 250 g casein/kg diet significantly reduced serum cholesterol concentrations compared with unsupplemented diet, indicating that dietary glycine is hypocholesterolemic. However, a mechanism by which the methionine:glycine ratio could regulate serum cholesterol is still unclear.

FOOTNOTES

1   The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
2   To whom correspondence should be addressed.
3   Present address: Otsuma Women's University, Tokyo 102, Japan.
4   Abbreviations used: casein-AA, amino acid mixture simulating casein; PP, potato protein; PP-AA, amino acid mixture simulating potato protein; RP, rice protein; RP-AA, amino acid mixture simulating rice protein; SP, soybean-AA, amino acid mixture simulating soybean protein; TC, sodium taurocholate.

Manuscript received 14 June 1996. Initial reviews completed 13 August 1996. Revision accepted 12 November 1996.

LITERATURE CITED

0022-3166/97 $3.00 ©1997 American Society for Nutritional Sciences



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T. Morita, S. Kasaoka, K. Hase, and S. Kiriyama
Oligo-L-Methionine and Resistant Protein Promote Cecal Butyrate Production in Rats Fed Resistant Starch and Fructooligosaccharide
J. Nutr., July 1, 1999; 129(7): 1333 - 1339.
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W. Ni, Y. Tsuda, M. Sakono, and K. Imaizumi
Dietary Soy Protein Isolate, Compared with Casein, Reduces Atherosclerotic Lesion Area in Apolipoprotein E-Deficient Mice
J. Nutr., November 1, 1998; 128(11): 1884 - 1889.
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T. Morita, S. Kasaoka, A. Oh-hashi, M. Ikai, Y. Numasaki, and S. Kiriyama
Resistant Proteins Alter Cecal Short-Chain Fatty Acid Profiles in Rats Fed High Amylose Cornstarch
J. Nutr., July 1, 1998; 128(7): 1156 - 1164.
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C. Tovar-Palacio, S. M. Potter, J. C. Hafermann, and N. F. Shay
Intake of Soy Protein and Soy Protein Extracts Influences Lipid Metabolism and Hepatic Gene Expression in Gerbils
J. Nutr., May 1, 1998; 128(5): 839 - 842.
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