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Nutrient Metabolism

Intact Dietary Soy Protein, but Not Adding an Isoflavone-Rich Soy Extract to Casein, Improves Plasma Lipids in Ovariectomized Cynomolgus Monkeys^{1 ,2}

³To whom correspondence and reprint requests should be addressed.

	ABSTRACT

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The dietary consumption of soy protein has been linked to a reduction in coronary heart disease and improvements in a number of related risk factors. Recent investigations have focused on isoflavone components of soy protein. The purpose of this study was to examine plasma lipids and lipoproteins, particularly LDL, with the intake of intact soy protein or casein-lactalbumin diets with and without a semipurified extract of soy, rich in isoflavones. Sixty ovariectomized cynomolgus monkeys were assigned to one of three groups fed diets containing the following: 1) casein-lactalbumin as the protein source (CAS; n = 20); 2) CAS plus a semipurified extract of soy, rich in isoflavones (ISO; n = 20); or 3) intact soy protein (SOY; n = 20) for 12 wk. Lipoproteins were fractionated by combined ultracentrifugation and HPLC. Isolated LDL particles were further subfractionated by dividing the LDL peak into three fractions for compositional analyses. The SOY group had significantly lower plasma total cholesterol, VLDL plus IDL cholesterol and LDL cholesterol, and significantly less HDL cholesterol than the CAS group. LDL particles from the SOY group had a significantly less cholesteryl ester than the CAS group. The semipurified extract of soy, rich in isoflavones, added to casein-lactalbumin protein did not have the same effects as intact soy protein on plasma lipids and lipoproteins. Other components of soy protein, either alone or in combination with isoflavones, may be involved in the effects seen in this study.

KEY WORDS: • cynomolgus monkeys • isoflavones • lipoproteins • menopause • soy protein

	INTRODUCTION

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Coronary heart disease (CHD)⁴ is the leading cause of death among women in the United States. Although evidence suggests that estrogen replacement therapy (ERT) reduces the risk of CHD, many women are unwilling to use ERT due to the increased risk of cancer and the unwanted side effects of the therapy (Cauley et al. 1990 , Nabulsi et al. 1993 ). Additionally, a recent paper from the Heart and Estrogen/progestin Replacement Study (HERS), a prospective, double-blind placebo-control trial, reported that oral conjugated equine estrogen plus medroxyprogesterone acetate did not reduce the rate of CHD events compared with placebo in postmenopausal women with established coronary artery disease (Hulley et al. 1998 ). Thus, the development of alternative therapies to ERT that reduce CHD risk without the added cancer risks and side effects is necessary.

High LDL cholesterol (LDLC) concentration is a primary risk factor for CHD (Lipid Research Clinics Program 1984 ). LDL particles are composed primarily of cholesteryl ester, with smaller amounts of protein, phospholipid, triglyceride and free cholesterol. Epidemiologic research suggests that small, dense LDL particles may be atherogenic (Austin et al. 1988 , Campos et al. 1992 , Crouse et al. 1986 ). Alternatively, more recent evidence suggests that a predominance of larger particles is associated with an increased risk of CHD in both humans (Campos et al. 1995 , Tallis et al. 1995 ) and nonhuman primates (Parks et al. 1990 ). ERT has been found to increase the clearance and production of both large and small LDL (Campos et al. 1997 ). The greatest effect was seen in the clearance of the large LDL particles, resulting in a lower number of large LDL particles and an overall reduction in mean LDL particle size. Thus, the clinical finding of smaller LDL particle size in both women (Seed and Crook 1994 , Wakatsuki et al. 1998 , Walsh et al. 1991 ) and monkeys (Manning et al. 1996 , Wagner et al. 1996 ) with ERT may be the result of a beneficial metabolism of LDL particles.

Recent reports have linked the dietary intake of soy protein and soy-based food products with a reduction in CHD and improvements in a number of related risk factors. Asian women, who consume primarily soy protein, have a lower CHD mortality than women eating a Western diet (Beaglehole 1990 , Thom et al. 1992 ). Soy protein has also been found to reduce the extent of atherosclerosis in rabbits (Huff and Carroll 1982 ), transgenic mice (Kirk et al. 1998 ) and cynomolgus monkeys (Anthony et al. 1997 ). The replacement of dietary animal protein with intact soy protein in both laboratory animals and humans lowers plasma total cholesterol (TC), LDLC and triglycerides (Anderson et al. 1995 , Carroll 1991 ). The magnitude of change in LDLC in humans was related to initial cholesterol concentration (Anderson et al. 1995 ). Effects on plasma HDL cholesterol (HDLC) concentrations, however, are inconsistent. Improvements in HDLC with soy protein intake may also be related to initial plasma HDLC concentrations (Sirtori et al. 1985 ) and may be more likely to occur in postmenopausal women (Baum et al. 1998 , Potter et al. 1998 ). Thus, beneficial effects of soy on lipoprotein concentrations may be influenced by initial cholesterol concentrations as well as gender and physiologic state.

Many investigations have sought to identify the active components of soy protein. Anthony et al. (1996) reported that consumption of intact soy protein significantly decreased LDLC plus VLDL cholesterol (VLDLC) and TC in female rhesus macaques compared with a diet containing soy protein with isoflavones removed. The authors concluded that the isoflavones may be responsible for the lipid-lowering effects of soy protein. Epidemiologic and experimental data suggest that soy protein intake reduces the risk of breast cancer and does not seem to harm the endometrium (Boring et al. 1994 ). As such, isoflavones may be an alternative to ERT (Clarkson et al. 1998 , Lichtenstein 1998 ).

Soy protein seems to have its greatest and most consistent effect on LDLC. The question addressed in this study is whether a semipurified extract of soy, rich in isoflavones, mediates this effect. The purpose of this study was to examine changes in plasma lipids and lipoproteins in monkeys consuming a moderately high fat and moderately high cholesterol diet containing one of the following: 1) casein-lactalbumin as the protein source, 2) casein-lactalbumin as the protein source with the addition of a semipurified extract of soy, rich in isoflavones, or 3) intact soy protein.

	MATERIALS AND METHODS

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Study population and diet composition.

Adult female cynomolgus monkeys (Macaca fascicularis) (n = 60) were imported directly from the Indonesian Primate Center (Bogor, Indonesia). The monkeys were quarantined and fed a standard nonpurified diet [15% Primate Diet (W); Harlan Teklad, Madison, WI] for 3 mo after arriving at the Comparative Medicine Clinical Research Center at the Wake Forest University School of Medicine. After release from quarantine, monkeys were fed a moderately atherogenic casein-lactalbumin diet (CAS, 0.07 mg cholesterol/kJ) for 2 mo and subsequently ovariectomized and assigned to one of three treatment groups (n = 20 per group) on the basis of TC and HDLC concentrations.

Treatment diets were designed to be identical in composition except for protein type and isoflavone content (Table 1). Two of the diets contained casein-lactalbumin as a protein source, whereas the third diet contained soy as the protein source (SOY). The two casein-lactalbumin diets contained either no additives (CAS) or the addition of a semipurified extract of soy, rich in genistein and daidzein (ISO). Monkeys were fed 504 kJ/(kg body weight·d). All diets included cholesterol (0.07 mg/kJ) to generate TC concentrations of ~6.5–7.8 mmol/L and had a nutrient breakdown (as % of energy) for protein, fat and carbohydrate of 19.4, 42.4 and 38.2%, respectively.

All procedures involving animals were conducted in compliance with state and federal laws, standards of the U.S. Department of Health and Human Services and guidelines established by the Institutional Animal Care and Use Committee. Ovariectomies were performed while monkeys were anesthetized with ketamine hydrochloride (15 mg/kg) and butorphanol (0.05 mg/kg).

Plasma lipids, lipoproteins, and apoproteins.

Blood was sampled at baseline and after 12 wk of treatment. Monkeys were deprived of food for 18 h before blood sample collection. Vacutainer tubes containing EDTA were used for collections after monkeys were sedated with ketamine hydrochloride (10 mg/kg). Blood was immediately put on ice until centrifugation at 1500 x g for 30 min at 4°C. Baseline TC, HDLC and triglyceride concentrations were determined using enzymatic methods on the COBAS FARA II analyzer (Roche Diagnostic Systems, Somerville, NJ). HDLC concentrations were determined using a modification of the heparin-maganese precipitation procedure as described previously (Burstein and Samaille 1960 ). Baseline lipid measurements and body weights were taken after 4 and 5 wk, respectively, of consuming the CAS diet and before ovariectomies.

After 12 wk of treatment, plasma samples were collected; cholesterol was measured enzymatically on whole plasma and on lipoproteins separated on a Superose 6 column (Amersham Pharmacia Biotech, Piscataway, NJ; Carroll and Rudel 1983 ). LDL subclassifications were determined using nuclear magnetic resonance spectroscopy (NMR; Otvos et al. 1992 ). The spectra for the LDL subcomponents reported by Otvos et al. (1992) were modified slightly for monkey plasma. Size distributions are defined as LDL1 being the smallest particle and LDL6 being the largest. Mean LDL particle size was determined as a weighted mean diameter.

Lipoproteins were fractionated on a subset of monkeys (n = 16/group) at 12 wk of treatment by combined ultracentrifugation and HPLC (Parks and Gebre 1991 ). The LDL eluting material was pooled into three fractions, with LDL1 being the largest and LDL3 being the smallest size fraction. Measurements of lipoprotein total and free cholesterol and triglyceride concentrations were determined enzymatically (Auerbach et al. 1991 ). Total protein was determined by the method of Lowry et al. (1951) . Apoproteins B and E were determined on the total LDL and three subfractions, as well as plasma, by ELISA (Koritnik and Rudel 1983 , Sorci-Thomas et al. 1989 ). Average LDL particle size was determined as retention time of the LDL peak on a Superose 6 HPLC column (Amersham Pharmacia Biotech; Carroll and Rudel 1983 ). LDL fatty acid cholesteryl ester content was also determined using gas-liquid chromatography after lipid extraction using the method of Bligh and Dyer (1959) .

Data analysis.

Data are presented as the mean ± SEM. Statistical analyses were performed using BMDP Statistical Software (Version 7.0; BMDP, Los Angeles, CA). One-way ANOVA was used to detect differences among treatment groups and Duncan's Multiple Range post-hoc test was used to determine specific group differences. Log transformations were performed if unequal variances were found among groups and P-values from these analyses are reported. Significance was based on a value of P 0.05.

	RESULTS

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Plasma lipids and lipoproteins.

No significant differences were found among groups in baseline TC (CAS, 6.02 ± 0.5; ISO, 5.92 ± 0.4; SOY, 5.93 ± 0.3 mmol/L) and HDLC (CAS, 1.33 ± 0.07; ISO, 1.56 ± 0.14; SOY 1.40 ± 0.12 mmol/L) concentrations. Body weights (CAS, 2.87 ± 0.09; ISO, 2.90 ± 0.10; SOY, 2.87 ± 0.09 kg) and estimated ages (CAS, 15.7 ± 0.38; ISO, 15.2 ± 0.74; SOY 15.2 ± 0.65 y) were also not significantly different among groups.

After 12 wk of treatment, the SOY group had significantly lower TC, VLDLC + IDLC, and LDLC concentrations and significantly higher HDLC concentrations than either casein protein–fed group (Table 2). The ISO group did not differ significantly from the CAS group in any of these variables. The lower TC and higher HDLC in the SOY group were reflected in a 55% lower TC:HDLC ratio than in the CAS or ISO groups (P < 0.01). LDL molecular weight was significantly lower (P < 0.01) in the SOY group than in the CAS and ISO groups. Plasma concentrations of apoprotein B tended to be lower in the SOY group than in either casein-fed group (P = 0.11). Triglyceride concentrations and body weights were not affected by either treatment.

Consistent with LDL molecular weight values measured by HPLC, the SOY group had a significantly smaller mean LDL particle size than the CAS or ISO groups (Table 3). This size difference was due primarily to significantly less cholesterol in the LDL5 and LDL6 particles. Also, consistent with results from HPLC analysis, the SOY group had a significantly lower LDLC concentration than the casein groups by NMR analysis. The correlation coefficient for LDLC concentrations determined by NMR vs. HPLC column chromatography was 0.88 (P < 0.001); for LDL size, it was 0.78 (P < 0.001).

(Table 4

(Table 5

(Table 6

	DISCUSSION

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Important to the association between soy protein intake and CHD risk factors is identification of the component or components that may be exerting protective effects. Previous studies have suggested that the isoflavones are the cardioprotective component of soy (Anthony et al. 1996 , Balmir et al. 1996 , Cassidy et al. 1995 , Pelletier et al. 1995 ). Anthony and co-workers (1996) found that the intake of intact soy protein improved CHD risk factors in peripubertal rhesus monkeys compared with the intake of an alcohol-extracted soy protein. The authors suggested that isoflavones, which had been removed by alcohol extraction, were the necessary components of soy and mediated the reduction in CHD risk. In this study, the addition of a semipurified extract of soy, rich in isoflavones, to a casein protein–based diet had no effect on TC, LDLC, or VLDLC + IDLC, suggesting that some other component of soy is responsible.

Two recent studies examining isoflavone intake in humans have reported a lack of effect on plasma lipids and lipoproteins (Hodgson et al. 1998 , Nestel et al. 1997 ). However, Nestel et al. (1997) did find a significant improvement in systemic arterial compliance with dietary isoflavone intake, despite the lack of effect on plasma lipids. Participants were excluded from both studies if they were vegetarians or included soy-containing meals in their diets, suggesting either that isoflavones are not the active component of soy protein or that some basal amount of soy protein must be consumed in addition to isoflavones for improvements in lipids and lipoproteins to occur. These studies were conducted among marginally hypercholesterolemic individuals; previous research suggests that improvements in lipids and lipoproteins due to soy protein may be restricted to hypercholesterolemic individuals. The authors did not present data on the association between baseline cholesterol and subsequent changes with isoflavone intake. Hodgson and co-workers (1998) reported similar results when data were analyzed only in subjects with TC concentrations > 5.3 mmol/L; however, the data were not reported.

Results from these studies, as well as this study, suggest that isoflavones alone do not improve plasma lipid and lipoprotein variables or impart the same CHD health benefits as intact soy protein. However, a study from Balmir and co-workers suggests that in rats and hamsters, soy protein and isoflavone extracts from soy reduce TC and LDLC but have no effect on HDLC or triglycerides (Balmir et al. 1996 ). The authors concluded that intact soy protein has beneficial effects on plasma lipids; however, they further concluded that the isoflavone extract of soy protein also has a lipid-lowering effect. Caution must be exercised in extrapolating study results from animals to humans because there are conflicting results in the literature that may be species specific.

In this study, the consumption of intact soy protein by ovariectomized monkeys had beneficial effects on HDL as well as apoprotein B–containing lipoproteins. Significant changes in HDLC are not found consistently with soy protein consumption (Anderson et al. 1995 ). However, gender differences in response to soy protein intake have been reported. Anthony et al. (1996) reported improvements in TC, apoprotein B lipoproteins and the TC:HDLC ratio in both male and female monkeys fed intact soy protein compared with those fed alcohol-extracted soy protein, whereas an increase in HDLC was seen only in females and a reduction in triglycerides was seen only in males. Gender differences were also seen in other lipid variables because female monkeys had lower LDL molecular weight and lipoprotein(a) concentration, and higher apoprotein AI and AII concentrations when fed intact soy protein. No changes in these variables were found in males. Additionally, significant increases in HDLC were found in postmenopausal women when isolated soy protein was substituted for a diet enriched with animal protein (Baum et al. 1998 , Potter et al. 1998 ). On the other hand, a previous study from the same laboratory reported no change in HDLC concentrations in mildly hypercholesterolemic men when isolated soy protein rather than casein was the protein source (Bakhit et al. 1994 ). Results from the above studies, as well as this study, suggest that intact soy protein has a greater effect on HDLC concentrations in females, particularly postmenopausal females, than in males. The mechanism for the effects on HDLC and the reasons for the differential gender effects are unknown. It has been postulated that the effect may be mediated by the interaction between isoflavones and estrogen receptors (Potter 1998 ). Our data seem to rule out this hypothesis because there was no effect on HDLC concentrations when a semipurified extract of soy, rich in isoflavones, was added to the casein-lactalbumin diet.

An inverse relationship is typically found between HDLC and triglyceride concentrations (Havel 1988 and 1990 ). We did not see this reciprocal relationship in this study; HDLC concentrations were significantly higher in the SOY group compared with the CAS group, but triglyceride concentrations were not affected by treatment. The independence of the regulation of HDLC and triglyceride concentrations has been reported previously. For example, Baum and co-workers (1998) reported a significant increase in HLDC concentrations and no change in triglyceride concentrations with soy protein consumption in postmenopausal women. The mechanisms for the increase in HDLC are unknown; however, the data suggest that the two lipids may be regulated independently of one another.

In this study, reductions in both the LDL particle molecular weight and diameter were found in the group consuming soy protein. A reduction in particle size has previously been thought to result in a more atherogenic particle. However, hypertriglyceridemia may confound these results because triglyceride concentrations are inversely associated with LDL particle size (Capell et al. 1996 , Coresh et al. 1993 , Crouse et al. 1985 ). More recent studies have shown that large LDL particles may be more atherogenic than smaller particles in the normotriglyceridemic state (Campos et al. 1995 , Parks et al. 1990 , Tallis et al. 1995 ). Additionally, in both humans and monkeys, both reduced LDL particle size and decreased atherosclerotic risk occur with ERT administration (Manning et al. 1996 , Seed and Crook 1994 , Wagner et al. 1996 , Wakatsuki et al. 1998 , Walsh et al. 1991 ). In this study, we found smaller LDL particles in the presence of normal triglyceride concentrations in monkeys consuming soy protein, suggesting a reduction in atherosclerotic risk. Additionally, the smaller LDL particles from the soy group had significantly less cholesteryl ester than the LDL particles from the casein group, which may be responsible for decreased atherosclerotic risk.

A decrease in intestinal cholesterol absorption and an increase in bile acid excretion, mediated possibly by the protein or saponin components of soy, have been suggested as possible mechanisms for the lipid-lowering effects of soy protein (Huff and Carroll 1980 , Nagata et al. 1982 , Potter 1995 and 1998 , Sugano et al. 1988 ). Additionally, hepatic clearance of plasma lipoprotein particles may also be a factor in reducing cholesterol levels (Khosla et al. 1991 , Lovati et al. 1991 ). Both an increase in LDL receptor mRNA levels in mononuclear cells (Baum et al. 1998 ) and an increase in LDL receptor activity (Lovati et al. 1987 , Sirtori et al. 1984 ) have been found with soy consumption. Mechanisms for a decrease in LDL particle size and cholesteryl ester content due to soy protein may involve both intestinal cholesterol metabolism and effects on the hepatic LDL receptor. If cholesterol absorption is decreased and bile acid excretion is increased due to dietary soy protein, hepatic cholesterol content would tend to be decreased. VLDL particles produced under this condition would contain less cholesteryl ester, resulting in LDL particles with less cholesteryl ester, and thus smaller particles without increased plasma triglycerides. Additionally, increased LDL receptor number and activity, as well as an apoprotein E enrichment of the LDL particle, would enhance clearance of larger LDL particles.

The major findings in this study are the following: 1) soy protein improves the lipid profile, reduces LDL particle size and alters the composition of the LDL particles; and 2) the addition of a semipurified extract of soy, rich in isoflavones, to casein-lactalbumin protein does not result in an atheroprotective lipid and lipoprotein profile as does intact soy protein. Additionally, the similar plasma isoflavone concentrations in the ISO and SOY groups suggest that isoflavones were absorbed equally by monkeys in the two groups. We are confident in reporting that isoflavones added to casein-lactalbumin protein do not produce the beneficial effects on plasma lipids and lipoproteins seen with intact soy protein. Our conclusions are limited by the fact that a portion of the extract is yet unidentified. When these components are identified, they can be tested for effects on lipids and lipoproteins. Components of soy protein other than isoflavones may affect cholesterol metabolism; these include the saponins, phytic acid, amino acid composition of the soy protein or a protein-isoflavone interaction. The health benefit of soy protein is clear; the components responsible for such benefits are yet to be determined.

	ACKNOWLEDGMENTS

 
The authors thank Zhang Li, Martha Wilson and Vickie Hardy for technical assistance.

	FOOTNOTES

 
¹ Supported in part by grants PO1 HL 45666 (J.K.W. and J.D.W.) from the National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD and T32 RR07009 (K.A.G.) from the National Institutes of Health.

² Soy protein and isoflavones were provided by Protein Technologies International, St. Louis, MO.

⁴ Abbreviations used: CAS, casein-lactalbumin protein; CHD, coronary heart disease; ERT, estrogen replacement therapy; HDLC, HDL cholesterol; ISO, casein-lactalbumin protein plus isolated isoflavone extract; LDLC, LDL cholesterol; NMR, nuclear magnetic resonance; SOY, soy protein isolate; TC, total plasma cholesterol; VLDLC, VLDL cholesterol.

Manuscript received January 29, 1999. Initial review completed March 5, 1999. Revision accepted May 5, 1999.

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