The Journal of Nutrition Vol. 128 No. 11 November 1998, pp. 1884-1889

Dietary Soy Protein Isolate, Compared with Casein, Reduces Atherosclerotic Lesion Area in Apolipoprotein E-Deficient Mice^1,2

Weihua Ni, Yasuyuki Tsuda, Masanobu Sakono^*, and Katsumi Imaizumi³

Laboratory of Nutrition Chemistry, Department of Food Science and Technology, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan and ^* Laboratory of Nutrition Chemistry, Department of Biological Resource Sciences, Faculty of Agriculture, Miyazaki University, Miyazaki 889-2192, Japan

	ABSTRACT

Abstract Introduction Methods Results Discussion References

The objective of this study was to compare the effects of dietary soy protein isolate and casein on atherosclerotic lesion development in apolipoprotein (apo) E-deficient mice. Male C57BL/6J apoE-deficient mice (9-10 wk old) in groups of 6-9 were used in a series of feeding studies. In the first experiment, mice were fed purified diets containing cholesterol (1 g/100 g) and cholate (0.25 g/100 g) for 6 wk; soy protein isolate or casein was used as the protein source. Although serum total cholesterol concentration did not differ between groups, the lesion area of the thoracic aorta in the soy protein isolate group was lower than that of the casein group (P < 0.01). In the second and third experiments, mice were fed the same purified diet as in Experiment 1, only without supplementation of cholesterol and cholate for 24 and 9 wk, respectively. In each of these two experiments, serum total cholesterol concentrations again did not differ between soy protein isolate- and casein-fed groups. Serum homocysteine concentrations did not differ between groups in Experiment 3. Dietary soy protein isolate, compared with casein, lowered the thoracic aorta lesion area (Experiment 2; P < 0.001) and the percentage of the aortic arch inner surface covered by lesions (P < 0.05). In the final experiment, mice were fed the cholesterol-free diets containing ethanol-extracted soy protein isolate or casein plus the soy protein ethanol extracts for 9 wk. There were no differences in serum total cholesterol concentration or thoracic aorta lesion areas between the two groups. These results indicate that the antiatherogenic effect of native soy protein isolate cannot be explained by its effect on serum lipids or homocysteine and suggest that both the protein component and the ethanol extracts of the soy protein isolate may contribute to the antiatherogenic effect of the native soy protein isolate.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Soy protein preparations can reduce serum total cholesterol concentration in humans (Anderson et al. 1995, Potter 1996) as well as experimental animals, compared with animal proteins (Beynen 1992), and thus may reduce the risk of atherosclerosis (Anderson et al. 1995, Czarnecki and Kritchevsky 1992, Potter 1996). During the past 20 years, a variety of components such as soy protein amino acids and peptides, isoflavones, saponins, phytic acid, fibers and protease inhibitors have been implicated in the hypocholesterolemic effect of soy protein preparations [reviewed by Anderson et al. (1995) and Potter (1996)]. However, the results remain inconclusive. Besides hypocholesterolemic effects, soy isoflavones have been reported to modify certain metabolic processes associated with atherosclerotic lesion development, such as inhibiting endothelial cell proliferation and macrophage expression of cytokines (Raines and Ross 1995). In addition, Morita et al. (1997) recently showed that the methionine content of dietary protein is positively correlated with serum total cholesterol concentration in rats. Matthias et al. (1996) reported that high dose dietary supplementation of L-methionine induced arteriosclerosis-like alterations in the aorta of rats.

In an effort to evaluate the beneficial effects of soy protein preparations on atherosclerosis, investigators have used mainly rabbits (Czarnecki and Kritchevsky 1992). In previous experiments, we showed that dietary soy protein isolate, compared with casein, resulted in a reduced intimal thickening in all sections of the thoracic aorta in exogenously hypercholesterolemic rats maintained on a high cholesterol diet for 6 mo (Sakono et al. 1997). To further elucidate the antiatherogenic effects and mechanism(s), better animal models are required to fully reproduce human atherosclerotic lesions. Recently, a series of transgenic murine models were generated in several laboratories (Breslow 1993). ApoE-deficient mice are currently regarded as one the most appropriate animal models for human atherosclerosis because they develop severe hypercholesterolemia and atherosclerotic lesions that are similar in distribution and appearance to those observed in humans (Nakashima et al. 1994, Zhang et al. 1992). Using this animal model, we compared the effects of soy protein preparations and casein on the serum lipids and homocysteine concentrations and development of atherosclerotic lesions. In addition, the effects on lesion development of removal of isoflavones, saponins and other components from soy protein preparation by ethanol extraction were also assessed.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Animals and diets.  C57BL/6J apoE-deficient mice (n = 10 male and 10 female) engineered at the University of North Carolina (Chapel Hill, NC) (Zhang et al. 1992) were purchased from Jackson Laboratory (Bar Harbor, ME) in 1994. Mice were bred and maintained at the Laboratory of Animal Experiments in Kyushu University School of Medicine (Fukuoka, Japan ). Six to nine male mice (9-10 wk old, weight 15-18 g) in each group were housed individually in a temperature-controlled room at 22-25°C with a 12-h light:dark cycle and given free access to food and nonionized water throughout the experimental period.

The composition of the basal diet, based on the AIN-93G formulation (Reeves et al. 1993), is summarized in Table 1. As a source of dietary fat, olive oil was used instead of soybean oil, because our preliminary experiments showed that olive oil induced higher serum cholesterol concentration in rats than did soybean oil (unpublished observations). As a source of dietary protein, we used soy protein isolate that contained (g/100 g) protein 90.5, fat 0.3, ash 4.3, and water 4.9 (Fujipro R, Fuji Oil, Osaka, Japan), casein that contained (g/100 g) protein 88.7, fat 0.8, ash 2.0, and water 8.5 (Wako Pure Chemicals, Osaka, Japan) or ethanol-extracted soy protein isolate. To remove isoflavones and saponin, soy protein isolate was treated with 70% ethanol, according to the method described by Kudou et al. (1991). From 100 g soy protein isolate, 143 mg isoflavones were removed, which were composed of the following (g/100 g): genistein, 15.2; genistin, 52.1; daidzein, 8.1 and daidzin, 24.5, when measured by HPLC on a CAPCELL PAK C18 AG120 column (250 × 4.6 mm, Shiseido, Tokyo, Japan) using a Waters 600 multisolvent delivery system, Waters 486 UV detector and Waters 741 data module (Millipore Corporation, Milford, MA) according to the method as described (Kudou et al. 1991). The ethanol extract was freeze-dried and kept at 4°C.

Experiment 1.  Two groups of 6 mice were fed the basal diet with soy protein isolate or casein as the protein source for 6 wk. To accelerate atherosclerosis, the diet was supplemented with 1 g/100 g cholesterol (Nacalai Tesque, Kyoto, Japan) and 0.25 g/100 g sodium cholate (Nacalai Tesque) (Plump et al. 1992).

Experiment 2.  Two groups of 8 or 9 mice were fed the basal diet without cholesterol and cholate for 24 wk.

Experiment 3.  Two groups of 8 or 9 mice were fed the same diets as in Experiment 2 for 9 wk.

Experiment 4.  Two groups of 6 mice were fed the basal diet with ethanol-extracted soy protein isolate or casein plus the ethanol extract of the soy protein isolate as the protein source for 9 wk. The number of mice used for determination of aortic lesion is shown in the figure legends. All aspects of the experiment were approved by Kyushu University Animal Policy and Welfare Committee.

Tissue preparation and morphometric determination of atherosclerosis.  In each feeding study, after food was withheld for 6 h from 0600 to 1200 h, the mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (5 mg/100 g body weight) and killed by withdrawing blood from the right ventricle. The aorta was perfused with 50 mL PBS via a cannula inserted in the right ventricle, allowing unrestricted efflux from an incision in the vena cava. Perfusion was continued with 50 mL of a 10% neutral formalin buffer solution at pH 7.4 (Wako Pure Chemicals), and the heart and aorta with its main branches were dissected entirely to the iliac bifurcation; the bulk of the fat and tissue adhering to the adventitia was dissected from the aorta as much as possible in situ.. The aorta was cut transversely at the distal end of the thoracic aorta; the position of the cut was identified by drawing a line along the aortic root. (Fig. 1). The heart was cut transversely at the level of the tips of atria; the ascending aorta and descending aorta were then divided into six parts. Part 6 included the end of the curvature of the aorta. The tissues were preserved in a 20% neutral formalin buffer solution at pH 7.4 (Wako Pure Chemicals) and embedded in paraffin; serial tissue cross sections (5 µm thick) were then prepared from the proximal end of each of six parts. Sections were stained with Van Gieson elastic fiber stain as described (Sakono et al. 1997). The intimal area was measured by capturing the image using a video camera mounted on an Olympus IX70 light microscope (Olympus Optical, Tokyo, Japan) and analyzed by NIH image/68k 1.57 software (National Institute of Health, Bethesda, MD) on a power Macintosh computer. The intima area of 10-15 consecutive cross sections from the proximal end of each of the six parts was measured, and the data were expressed as the mean intimal areas of each part.

View larger version (31K): [in this window] [in a new window]   Fig 1. Line drawing of arteries in mice. The aortic arch was separated into six parts as shown.

In Experiment 3, the percentage of the aortic surface covered by lesions was determined using an en face preparation according to the method described by Paigen et al. (1987) with a slight modification (Tangirala et al. 1995). Briefly, after perfusion fixation as described above, the aorta and its main branches were dissected from the aortic valve to the iliac bifurcation (Fig. 2). The adventitia was removed as much as possible in situ, and the aorta was opened longitudinally as described (Tangirala et al. 1995); in addition, the origins of the brachiocephalic artery, the left common carotid artery and the left subclavian artery were opened. Other minor branching arteries were cut off and the aorta was stained by Sudan IV as described (Tangirala et al. 1995). Finally, the aorta was laid open and fixed on a piece of glass slide by cyanoacrylate adhesives (Toa Gousei, Tokyo, Japan). Each aorta was evaluated for percentage of lesion coverage by direct image capture using a Canon FTb camera (Connon, Tokyo, Japan), attached to a copy stand. The lesion area was determined using NIH image/68k 1.57 software on a power Macintosh computer.

View larger version (94K): [in this window] [in a new window]   Fig 2. Photographs showing the gross appearance of the atherosclerotic lesions in apolipoprotein (apo) E-deficient mice that were fed soy protein isolate (right photograph) and casein (left) diets for 9 wk. Samples were stained with Sudan IV. Lesion areas were measured separately at the end of the curvature of the aortic arch as indicated by arrows.

Analysis of serum lipid and homocysteine.  Serum lipid levels were determined by commercially available kits (Cholesterol C Test, Triglyceride G Test and Phospholipid B Test, all from Wako Pure Chemicals). Total homocysteine was determined in serum using reversed-phase HPLC as described (Araki and Sako 1987).

Statistical analysis.  Serum lipid levels and lesion size data in Experiment 3 were analyzed by Student's t test. Data of intimal areas were analyzed by two-way ANOVA followed by post-hoc test (Fisher's protected least significant difference test). Differences were considered significant at P < 0.05.

	RESULTS

Abstract Introduction Methods Results Discussion References

Food intake, growth and serum lipids.  In each feeding study, there were no significant differences in food intake and final body weight between the soy protein isolate-fed group and casein-fed mice (Table 2). Neither dietary protein nor ethanol treatment of soy protein significantly affected serum lipid concentrations with one exception; in Experiment 4, there was a difference in serum phospholipid concentrations between the ethanol-extracted soy protein isolate-fed group and the group fed casein plus the extracts. As expected, the high cholesterol diet resulted in higher concentrations of serum total cholesterol and triacylglycerols than did the cholesterol-free diet.

Histological findings.  The lesions ranged from multilayered foam cell deposits to fibrous plaques and advanced plaques with cholesterol cleft deposition in the intima (Experiment 4; Fig. 3). The severity of the lesions depended on the location within the thoracic aorta, with smaller lesion areas in the distal thoracic aorta. As reported by others (Nakashima et al. 1994), these mice develop the entire spectrum of lesion types, which are similar to those in humans. In these studies, it was difficult to qualitatively evaluate the dietary effect on the type of lesion because the lesion types were quite variable even within the same group. Accordingly, the effect was evaluated in each part of the thoracic aorta as lesion area as described in Materials and Methods. When mice were fed the high cholesterol diet for 6 wk (Experiment 1; Fig. 4), the lesion area in the soy protein isolate-fed group was significantly lower than that of the casein-fed group (P < 0.01); the lesion areas in parts 1 and 3 were lower in mice fed soy protein isolate than in those fed casein (P < 0.05). The effect of dietary soy protein isolate on the lesion area was also observed when mice were fed a cholesterol-free diet for 24 wk (P < 0.001) (Experiment 2; Fig. 5). The lesion areas in parts 4 and 5 were lower in mice fed soy protein isolate than in those fed casein (P < 0.05).

View larger version (127K): [in this window] [in a new window]   Fig 3. Photomicrographs showing tissue cross sections corresponding to each of the six parts of the aortic arch in Figure 1 in an apolipoprotein (apo) E-deficient mouse fed casein plus ethanol extracts of soy protein isolate (Experiment 4; bars = 200 µm).

View larger version (50K): [in this window] [in a new window]   Fig 4. The intima area in apolipoprotein (apo) E-deficient mice fed diets containing 1 g/100 g cholesterol plus 0.25 g/100 g sodium cholate with either soy protein isolate (SPI) or casein as the protein source for 6 wk (Experiment 1). Bars are means ± SEM, n = 6. The lesion area was affected by diet (P < 0.01) and the part of the thoracic aorta (P < 0.01). *Significantly different from casein-fed group, P < 0.05.

View larger version (57K): [in this window] [in a new window]   Fig 5. The intima area inapolipoprotein (apo) E-deficient mice fed a cholesterol-free diet with either soy protein isolate (SPI) or casein as protein sources for 24 wk (Experiment 2). Bars are means ± SEM, n = 5 or 6. The lesion area was affected by diet (P < 0.001) and the part of the thoracic aorta (P < 0.001). *Significantly different from casein-fed group, P < 0.05.

Aortic lesions also were evaluated using the en face preparations as described in Materials and Methods (Experiment 3; Fig. 2). Lesions were observed throughout the aorta with some predilection for the proximal thoracic aorta with its main branches and the origins of main branches of abdominal aorta in both casein- and soy protein isolate-fed groups. Quantitative analysis showed that the percentage of the inner surface at the aortic arch covered by lesions in soy protein isolate-fed mice was significantly less than that in casein-fed animals (Table 3). No significant differences were found in the whole aorta or other parts of the aorta (without the arch).

Finally, the effect of removal of isoflavones from soy protein isolate on the lesion area of the thoracic aorta was measured in mice fed a cholesterol-free diet for 9 wk (Experiment 4). The lesion area in mice fed diets with ethanol-treated soy protein isolate was not different from that in mice fed diets with casein plus ethanol extracts (Fig. 6).

View larger version (41K): [in this window] [in a new window]   Fig 6. The intima area in apolipoprotein (apo) E-deficient mice fed a cholesterol-free diet with either ethanol-extracted soy protein isolate (SPI-EE) or casein plus ethanol extracts of soy protein (Casein + EE) as the protein source for 9 wk (Experiment 4). Bars are means ± SEM, n = 6. The lesion area was affected by the part of the thoracic aorta (P < 0.001), but was not affected by diet.

Serum homocysteine.  Serum homocysteine concentrations did not differ due to protein source in mice fed a cholesterol-free diet for 9 wk (Experiment 3; Table 3). The concentration was in the range of that in rats reported by Matthias et al. (1996).

	DISCUSSION

Abstract Introduction Methods Results Discussion References

We have evaluated atherosclerotic lesions by quantifying the lesion area in the tissue cross sections of six segments from the entire thoracic aorta and the extent of atherosclerotic surface area in the whole aorta. Tangirala et al. (1995) reported a correlation between lesions in the aortic origin and in the entire aorta, but others have observed substantial variability at the aortic origin (Palinski et al. 1994). To increase the accuracy of the measurement, we extended the section area studied to the whole thoracic aorta. We found that soy protein isolate, compared with casein, slightly but significantly reduced the atherosclerotic lesion area in the thoracic aorta. In agreement with Nakashima et al. (1994), specific predilection sites for lesions were observed in apoE-deficient mice fed soy protein isolate as well as casein. The lesions in the mice fed both proteins were composed of typical multilayered foam cells with cholesterol cleft deposition in the intima and typical fibrous plaques with necrotic cores (Fig. 3). These lesions were similar to those reported by others who fed apoE-deficient mice high fat diets (Nakashima et al, 1994, Plump et al, 1992). However, in this study, it was not clear whether the lesions were less fibrous in the soy protein isolate-fed group than in casein-fed group.

Raines and Ross (1995) proposed that isoflavones in soy protein preparations may be beneficial in preventing atherosclerosis because isoflavones can inhibit cell adhesion, alter growth factor activity and inhibit cell proliferation in tissue cultures. Anthony et al. (1996) showed that soybean isoflavones improved cardiovascular risk factors in rhesus monkeys by feeding diets containing native soy protein from which most of the isolfavones and saponins were removed. As far as we know, there are no direct reports showing that the ethanol extracts containing isoflavones and saponins exert beneficial effects. Thus, in this experiment, we attempted to examine directly the role of ethanol extracts in atherosclerosis development. The results showed that mice fed diets containing ethanol-treated soy protein isolate had lesion areas similar to those found in mice fed diets with casein plus ethanol extracts. These results suggest that the antiatherogenic effects of soy protein isolate observed in this study cannot be attributed solely to the isoflavones and/or saponins in the ethanol extracts. In an unpublished study, we found that mice fed soy protein-type amino acid mixtures had less aortic lesion area than did mice fed casein-type amino acid mixtures, and mice fed casein-containing diets supplemented with arginine, compared with those fed diets containing casein, had reduced the aorta lesion area near the aortic sinus. It is therefore likely that the protein portion of intact soy protein isolate lowered the atherosclerotic lesion area in concert with the ethanol extracts.

In this study, a reduction of serum total cholesterol concentration was not observed in soy protein isolate-fed mice, compared with those fed casein. This is in contrast to a number of animal (Beynen 1992, Potter 1996) and human studies (Anderson 1995) in which soy protein, soy protein isolate or other soy protein products exerted hypocholesterolemic actions, compared with casein-based diets. Recently it was reported that soy isoflavones lowered serum cholesterol concentration in monkeys (Anthony et al. 1996). However, Gooderham et al. (1996) reported that although soy protein supplementation to a typical Western type diet can increase plasma concentrations of isoflavones in normal men, this may not necessarily be sufficient to counter heart disease risk factors such as high plasma total cholesterol and platelet aggregation. In our unpublished observations, diets containing ethanol-extracted soy protein isolate, compared with the intact soy protein isolate, resulted in greater serum total cholesterol concentration and lower abundances of mRNA for LDL receptor and cholesterol 7 hydroxylase. Thus, the hypocholesterolemic action of isoflavones and/or other ethanol extracts seems to vary with species and strains of animals. In any event, it is likely that the hypocholesterolemic action observed in animals or humans involves uptake of lipoproteins through hepatic lipoprotein receptors, as proposed by Sirtori et al. (1995) because apoE is the ligand for LDL and remnants of triglyceride-rich lipoproteins (Mahley 1988).

Because serum cholesterol concentration did not differ between groups, dietary soy protein isolate must lower atherosclerotic lesion development in the thoracic aorta via a mechanism other than the lowering of serum cholesterol. Soy protein isolate differs from casein in the proportion of methionine (14 vs. 31 g/kg protein) and arginine (84 vs. 38 g/kg protein). In light of the recently clarified role of these amino acids in atherogenesis, it was expected that the high proportion of methionine in casein would result in more homocysteine, which would lead to the development of atherosclerotic lesions (Duell et al. 1997, Nehler et al. 1997, Toborek et al. 1995). In fact, oral administration of high doses of methionine to normotensive and hypertensive rats was reported to result in a marked elevation of serum homocysteine (34.2 and 61.0 µmol/L for normotensive and hypertensive rats, respectively) and arteriosclerosis-like alterations of the aorta (Matthias et al. 1996). In this study, casein-fed mice had serum concentrations of homocysteine similar to those in mice fed soy protein isolate, and the concentrations were in the range of those (~3.8-8.6 µmol/L) from rats fed diets without supplemental methionine (Matthias et al. 1996). Therefore, the level of homocysteine in the mice in this experiment may not be sufficient to induce atherosclerotic lesions. Besides methionine, dietary arginine has been shown to markedly reduce atherogenesis in a number of studies, probably through formation of nitric oxide (Böger et al. 1996, Cooke et al. 1992). Our preliminary experiments (unpublished data) showed that supplementation of arginine to a casein-based diet, which is equivalent to the content of the soy protein isolate diet, lowered lesion development. Therefore, further studies are required to elucidate the nature of the mechanism of the antiatherogenic effects of soy protein isolate.

In summary, this study confirmed that soy products reduce atherosclerotic lesion development, indicating that the antiatherogenic effect of native soy protein isolate cannot be explained by its effect on serum lipids or homocysteine; this suggests that both the protein fraction and ethanol extracts of soy protein isolate may contribute to the antiatherogenic effect of the native soy protein isolate.

	FOOTNOTES

Manuscript received 22 January 1998. Initial reviews completed 23 March 1998. Revision accepted 13 July 1998.

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