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Article

Consumption of Soy Protein Reduces Cholesterol Absorption Compared to Casein Protein Alone or Supplemented with an Isoflavone Extract or Conjugated Equine Estrogen in Ovariectomized Cynomolgus Monkeys¹

Wake Forest University School of Medicine, Department of Pathology, Section on Comparative Medicine, Winston-Salem, NC 27157-1040

²To whom correspondence should be addressed.

	ABSTRACT

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Dietary intake of soy protein is associated with reductions in plasma cholesterol. Isoflavones are thought to be active components of soy and responsible for the beneficial effects because of their structural similarities to estrogen. The purposes of this study were to determine if i) soy protein or a semipurified soy extract, rich in isoflavones, is responsible for improving the lipid profile and ii) altered intestinal cholesterol metabolism is one mechanism for hypocholesterolemic effects. Ovariectomized adult female cynomolgus monkeys (40) were assigned to groups fed diets containing i) casein-lactalbumin (CAS) ii) intact soy protein (SOY), iii) CAS plus an isoflavone-rich semipurified soy extract similar in isoflavone content as SOY (ISO) or iv) CAS plus conjugated equine estrogen (CEE) for 20 wk. Cholesterol absorption was determined using the fecal isotope ratio method. Bile acid excretion was measured using the 3-hydroxysteroid dehydrogenase assay. The SOY group had significantly lower total- and VLDL + LDL-cholesterol compared to the other three groups and significantly higher HDL-cholesterol compared to the CAS and CEE groups. Cholesterol absorption was significantly lower in the SOY group compared to the other groups, but bile acid excretion was not significantly affected. The hypocholesterolemic effect of soy protein appears to be mediated in part by decreased cholesterol absorption. The semipurified soy extract, rich in isoflavones, added to casein protein did not have lipid-lowering effects. Other components of soy such as saponins, phytic acid or the amino acid composition may be involved in the hypocholesterolemic effects seen in this study.

KEY WORDS: • cholesterol absorption • cynomolgus monkeys • isoflavones • menopause • soy protein.

	INTRODUCTION

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Coronary heart disease (CHD)⁴ is the leading cause of morbidity and mortality in the United States. Elevated levels of plasma total (TC) and LDL cholesterol (LDLC) have long been implicated as primary risk factors for CHD (LRC 1984 ). LDL is the major cholesterol-carrying lipoprotein in humans and is thought to be a principal contributor to atherosclerotic lesion formation (Davis et al. 1991 , Mukhin et al. 1991 ). In support of this, a reduction in LDLC by pharmacologic means is associated with a significant reduction in the incidence of CHD (Frick et al. 1987 , LRC 1984 ). Reductions in TC and LDLC by dietary means may be preferable to pharmacological means to avoid unforeseen side effects.

Estrogen replacement therapy (ERT) is associated with a 50% reduction in CHD risk in postmenopausal women (Barrett-Connor and Bush 1991 , Stampfer and Colditz 1991 ). Although the benefits to ERT use are many, risks and side effects is also associated with ERT use. Risk of breast and endometrial cancers are increased and side effects and complications are documented with the addition of a progestin (Colditz et al. 1995 , Grady et al. 1995 , Hulka 1994 ). Additionally, the HERS trial recently reported no overall benefit of combined continuous hormone replacement therapy (HRT) in women with pre-existing CHD (Hulley et al. 1998 ). Compliance with HRT use is low due to the previously mentioned reasons, as well as personal reasons expressed by many women (Bush et al. 1983 , Derby et al. 1993 , Hemminski et al. 1991 , Nabulsi et al. 1993 ). Alternative therapies to HRT are necessary to reduce the risk of disease in the many postmenopausal women not receiving HRT.

The dietary intake of soy protein and soy-based food products has been linked with a reduction in CHD. CHD mortality and morbidity in Asian countries are substantially lower than in Western countries (Robertson et al. 1977 , Thom et al. 1992 ). Adlercreutz (1990) has suggested that this may be due to the considerably higher intake of soy protein in Asian countries. Animal studies have shown that the replacement of casein protein with soy protein decreased atherosclerotic lesion formation in both rabbits and nonhuman primates (Anthony et al. 1997 , Huff et al. 1982 ). Previous clinical studies, again replacing dietary animal protein with intact soy protein, have shown significant improvements in CHD risk factors, particularly TC, LDLC and triglycerides in humans and laboratory animals (Anderson et al. 1995 , Carroll 1991 ).

Many investigations have focused on the isoflavone components of soy protein, genistein and daidzein, also referred to as phytoestrogens because of their estrogenic activities. These isoflavones are thought to be the active components of soy protein and responsible for many of its beneficial effects. Anthony et al. (1996) reported that consumption of intact soy protein resulted in a significant decrease in TC and LDLC plus very low density lipoprotein (VLDL) cholesterol (V + LDLC) in female rhesus macaques when compared to a diet containing soy protein with isoflavones removed. They concluded that the isoflavones may, in fact, be responsible for the lipid-lowering effects of soy protein.

Mechanisms for the hypocholesterolemic action of soy have yet to be identified. Possible mechanisms of action include a decrease in the intestinal absorption of dietary cholesterol or bile acids, changes in the hepatic metabolism of cholesterol and lipoproteins or both (Potter 1998 ). The protein and saponin components of soy may be responsible for its intestinal effects, while isoflavones may be involved in LDL receptor activity regulation. In addition, an increase in bile acid and neutral steroid secretion may decrease hepatic cholesterol reserves and increase hepatic LDL receptor activity. A recent report in hamsters confirmed an increase in fecal bile acid excretion and a reduction in hepatic cholesterol content with soy consumption compared to dietary casein (Wright and Salter 1998 ). The purpose of this study was to examine intestinal cholesterol absorption and bile acid excretion in ovariectomized cynomolgus monkeys consuming a moderately high-fat and moderately high-cholesterol diet containing either: i) casein-lactalbumin as the protein source, ii) soy as the protein source, iii) casein-lactalbumin as the protein source with the addition of a semipurified soy extract, rich in isoflavones or iv) casein-lactalbumin as the protein source with the addition of conjugated equine estrogens.

	MATERIALS AND METHODS

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Study population and diet composition.

Adult female cynomolgus monkeys (Macaca fascicularis) were imported directly from the Indonesian Primate Center (Bogor, Indonesia). All 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. Following release from quarantine, monkeys were fed a moderately atherogenic casein-lactalbumin diet (0.07 mg/kJ). Baseline plasma samples were taken after 4 and 5 wk. Monkeys were subsequently ovariectomized and assigned to one of four treatment groups (n = 10 per group) based on TC and HDL cholesterol (HDLC) concentrations.

Monkeys were fed treatment diets for 5 mo. All diets were designed to be identical in composition except for protein type and isoflavone or estrogen content (Table 1). Three of the diets, as well as the baseline diet, contained casein-lactalbumin as a protein source while the fourth diet contained soy protein isolate as the protein source (SOY). The three casein-lactalbumin diets contained either no additives (CAS), the addition of a semipurified soy extract, rich in the isoflavones genistein and daidzein (ISO), or conjugated equine estrogens (CEE; Premarin^®; Wyeth-Ayerst, Philadelphia, PA; equivalent to a woman’s dose of 0.625 mg/d). The chemical composition of the soy protein isolate fed to the monkeys in this study was 87% protein, 4.2% moisture, 4.6% fat and 4.2% ash. The semipurified soy extract added to the casein-lactalbumin protein was made from an alcohol extract of a soy protein isolate. The composition of the soy extract was 68.9% total isoflavones (43.7% genistein, 21.8% daidzein, 3.4% glycitein), 1.8% protein, 0.9% moisture, 0.4% fat, and 0.1% ash. Isoflavones were present in the aglycone form. Phytosterols were found in the soy protein isolate at a concentration of 13 mg/100 g protein (dry weight); however ß-sitosterol was not present. Additionally, saponins have not been identified in either compound.

Plasma genistein, daidzein and equol concentrations were determined by ESA Inc. (Chelmsford, MA) on a subsample (n = 8 per group) from the CAS, ISO and SOY groups. The CoulArray® Model 5600 8-channel HPLC system was used as previously described (Gamache and Acworth 1998 ) with a 150 x 3.0 mm i.d., 3 µm, C18 MD-150 column (ESA Inc.). Isocratic elution with a water/methanol/acetonitrile, 68:25:7 (v/v/v), mobile phase containing 0.2 M sodium acetate buffer (pH 4.8) was used with a flow rate of 0.6 mL/min, column temperature of 42°C and detector potentials of 340, 470, 500, 530, 560, 620, 680, 760 (mV vs. Pd). Serum preparation was adopted from a described HPLC-MS procedure (Coward et al. 1996 ) modified by using estriol 3-(ß-glucuronide) as an internal standard. Mean plasma isoflavone concentrations, measured 2-h post-feeding at least 9 wk after starting treatment, were comparable between the ISO and SOY groups (genistein: CAS—not detectable, ISO 86.0 ± 26.0 nmol/L, SOY 110.1 ± 23.6 nmol/L; daidzein: CAS—not detectable, ISO 80.8 ± 25.9 nmol/L, SOY 92.3 ± 18.5 nmol/L; equol: CAS < 20 nmol/L, ISO 540.2 ± 110.8 nmol/L; SOY 361.7 ± 52.8 nmol/L).

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.

Blood was sampled at baseline and at 20 wk of treatment. Monkeys were food deprived for 18 h prior to blood sample collection. Vacutainer tubes containing EDTA were used for collections after animals were sedated with ketamine (10 mg/kg). Blood was immediately put on ice until centrifugation at 1500 x g for 30 min at 4°C. TC, HDLC and triglyceride concentrations were determined using enzymatic methods on the COBAS FARA II analyzer (Roche Diagnostic Systems, Somerville, NJ), with protocols and reagents supplied by Boehringer Mannheim (BM Cholesterol HP 236691, BM Triglycerides/GB; Indianapolis, IN). HDLC concentrations were determined using a modification of the heparin-maganese precipitation procedure as described previously (Burstein and Samaille 1960 ). The 2 mol/L of MnCl₂ was used rather than the 1 mol/L of MnCl₂ to facilitate the complete precipitation of LDL, portions of which are resistant to precipitation in certain hyperlipoproteinemic monkeys. Analyses for TC, HDLC and triglycerides are in full standardization with the Centers for Disease Control-National Heart, Lung and Blood Institute Standardization Program. Apoprotein B-containing lipoprotein cholesterol (V + LDLC) was calculated as the difference between TC and HDLC.

Cholesterol absorption and bile acid excretion.

Intestinal cholesterol absorption was measured using the fecal isotope ratio method (Borgstrom 1969 , Quintao et al. 1971 , Rudel et al. 1994 ). The dose consisted of [7(n)-³H]cholesterol and ß-[4-¹⁴C]sitosterol in a known amount and a ratio of ~5:1. Both components were purified by HPLC to 98% purity. Monkeys were individually caged, and the dose was fed to each monkey on an apple slice. The afternoon meal was fed immediately following administration of the dose in order to mix with a typical meal in the gut. Feces were collected quantitatively over a 4-d period and homogenized. The percentage of cholesterol absorbed was calculated using the ratio of ³H to ¹⁴C in the fed dose and in the recovered feces as follows: cholesterol absorption = (ratio in dose - ratio in feces)/ratio in dose.

Endogenous fecal bile acid excretion was measured as reported by Schiller et al. (1990) . Carboxyl [¹⁴C] chenodeoxycholic acid was added as an internal standard. Briefly, feces were weighed, homogenized and saponified in alcoholic KOH. The neutral steroids were extracted with petroleum ether. The lower aqueous phase was dried down and reconstituted in methanol. One aliquot of sample was counted to determine recovery of the internal standard, and a second aliquot was used to measure total bile acids using the 3-hydroxysteroid dehydrogenase assay described by Turley and Dietschy (1978) .

Cholesterol absorption measures were not determined on three monkeys from the ISO group and one monkey from the CEE group due to severe diarrhea during feces collection. Bile acid excretion was not determined on nine additional monkeys due to problems with feces collection.

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 of the data were performed if unequal variances were found among groups. Pearson product-moment correlations were used to assess relationships among variables. Significant difference was based on a P-value 0.05.

	RESULTS

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Plasma lipids.

No differences were found among treatment groups in any of the baseline lipid variables. (Table 2). After 20 wk of treatment, the SOY group had significantly lower plasma TC and V + LDLC concentrations compared to the three casein-lactalbumin protein groups (Table 3). Additionally, the SOY group had a significantly higher HDLC concentrations than the CAS and CEE groups. The HDLC concentration in the ISO group was not significantly different from any of the other three groups. The ISO and CEE groups did not differ significantly from the CAS group in TC, V + LDLC or the TC/HDLC ratio. The lower TC and higher HDLC in the SOY group was reflected in a significantly lower TC/HDLC ratio than in the CAS group. Body weights and triglyceride concentrations were not affected by any treatments. Although the CAS group was fed the same diet during baseline and treatment periods, dramatic differences in lipid concentrations were found. The baseline measurements were taken after only 4 or 5 wk of consuming the diet but before ovariectomy, while treatment measurements were taken after 20 wk, suggesting both an effect of the ovariectomy as well as an effect of duration of dietary treatment.

The SOY group had significantly lower percentage dietary cholesterol absorption compared to the CAS, ISO or CEE groups (Fig. 1 ), which did not differ from one another. There were borderline significant correlations between percentage cholesterol absorption and TC (r = 0.31; P = 0.067) and V + LDLC (r = 0.29; P = 0.092), although P-values for correlations in each treatment group were > 0.1. There were no treatment effects on bile acid excretion among the groups. Data for bile acid excretion are reported with one value removed from the ISO group because it was almost two SD above the mean. Dropping this value had no effect on statistical results.

View larger version (35K): [in this window] [in a new window]   Figure 1. Effect of 20 wk of casein-lactalbumin protein (CAS), casein-lactalbumin protein plus semipurified isoflavone extract (ISO), soy protein (SOY), and casein-lactalbumin protein plus conjugated equine estrogen (CEE) on cholesterol absorption and bile acid (BA) excretion in ovariectomized adult cynomolgus monkeys. Values are means ± SEM. Bars with different letters differ, P 0.05.;10>

	DISCUSSION

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Significant reductions in TC and LDLC have been found in studies of both animals and humans when soy protein is substituted for casein-lactalbumin in the diet (Anderson et al. 1995 , Carroll 1991 , Carroll and Kurowska 1995 ). The mechanism of this hypolipidemic effect is still unclear. A decrease in intestinal cholesterol absorption and an increase in bile acid excretion, mediated possibly by the amino acid or peptide components, saponins or isoflavones found in soy protein, have been suggested as possible mediators for the lipid-lowering effect of soy protein (Huff and Carroll 1980 , Nagata et al. 1982 , Potter 1995 , Potter 1998 , Sugano et al. 1988 ). This paper reported on intestinal cholesterol absorption and bile acid excretion with both intact soy protein and a semipurified soy extract, rich in isoflavones, added to the casein-lactalbumin protein.

Consumption of soy protein isolate reduced cholesterol absorption in the present study, whereas a semipurified soy extract, rich in isoflavones, added to a casein-lactalbumin protein diet did not. Reductions in cholesterol absorption, associated with reduced plasma or serum cholesterol, have been seen in male rabbits (Huff and Carroll 1980 ) and rats (Nagata et al. 1982 ) fed soy protein isolates compared to casein protein. Huff and Carroll (1980) fed both a low-fat, cholesterol-free diet and a high-fat, high-cholesterol diet to New Zealand White rabbits for 42 d and found that soy protein isolate consumption significantly reduced total serum cholesterol and cholesterol absorption, as well as the level of fecal neutral steroids in the groups consuming soy protein isolate compared to casein protein. An additional group fed nonpurified diet did not differ from the two groups fed soy. Cholesterol is predominately carried by HDL particles in rabbits fed a "typical" nonpurified diet; however, increases in LDLC concentrations occur in rabbits fed an atherogenic diet. The authors did not report HDLC or LDLC concentrations in this report; however, we assume that the increase in total cholesterol with casein consumption compared to soy or nonpurified diet was due to an increase in LDLC. HDLC also predominates in rats fed a "typical" nonpurified diet. However, rats are fairly resistant to diet-induced increases in LDLC. Nagata and co-workers (1982) reported that feeding rats soy diets for 1 mo significantly reduced serum total cholesterol and intestinal cholesterol absorption compared to casein feeding. Rats typically respond to dietary cholesterol feeding by down-regulating cholesterol synthesis and up-regulating bile acid synthesis, leading to very little change in serum total cholesterol. Soy protein feeding, with the infusion of both intravenous and intragastric labeled cholesterol, increased fecal excretion of acidic steroids, suggesting that bile acid synthesis was up-regulated, although the authors suggested that cholesterol synthesis was not significantly affected. Additionally, Vahouny and co-workers (1984) performed timed lymph collections from cannulation of the left thoracic lymphatic duct in male rats and found a more rapid absorption of cholesterol and oleic acid with casein feeding compared to soy protein isolate feeding. These studies in rabbits and rats suggest that soy protein isolate consumption compared to casein consumption reduces cholesterol absorption in species in which plasma cholesterol is carried predominately in the HDL particles. Additionally, results from the soy-fed animals do not differ significantly from chow-fed animals, suggesting that the effects seen in these studies may actually be due to hypercholesterolemic actions of casein.

Conversely, a study in Golden Syrian hamsters fed a diet containing 0.01% cholesterol and soy protein isolate reported a reduction in plasma VLDLC levels compared to casein protein, but no significant effect on fecal cholesterol (Wright and Salter 1998 ). Hamsters have cholesterol associated predominately with HDL, but LDLC usually responds to dietary cholesterol consumption. The mechanism for an increase in LDLC with cholesterol consumption is not yet known. The authors did, however, find significant increases in fecal cholesterol with increasing amounts of soy protein isolate.

Two reports have been published on studies in swine with similar results (Beynen et al. 1990 , Kim et al. 1978 ). Beynen and coworkers (1990) reported that swine fed soy protein isolate and 2 g of cholesterol per day had a significantly higher fecal total neutral steroid output and a significantly higher ileal output of both neutral steroids and cholesterol compared to casein-fed swine. Ileal output was determined via sampling from a surgically attached re-entrant ileo-cecal cannula. These data suggest that cholesterol absorption was reduced with soy protein consumption. A second study in swine reported reductions in serum cholesterol and cholesterol absorption, although the reduction in cholesterol absorption was not statistically significant, with only three animals per group (Kim et al. 1978 ). Swine in this study were fed textured soy protein and ~1 g of cholesterol/d. In swine, as in humans, cholesterol is carried predominately by LDL particles, and LDLC is sensitive to diet-induced elevations. Reductions in plasma cholesterol due to decreases in intestinal cholesterol absorption and increases in excretion of cholesterol and bile acids were found in swine, suggesting that intestinal metabolism of cholesterol mediates the hypocholesterolemic effects of soy consumption.

Soy protein diets significantly reduced cholesterol absorption compared to the casein protein diets in rats. However, no difference was found between groups consuming amino acid mixture equivalents of soy and casein protein (Nagata et al. 1982 ). These results suggest that the amino acid components of soy may not be mediating the reduction in intestinal cholesterol absorption, and thus reductions in plasma cholesterol levels. Additionally, one study found that soy protein feeding compared to casein protein feeding did not reduce lymphatic cholesterol absorption in fasted rats given an intragastric infusion of a lipid test meal (Vahouny et al. 1984 ). These data suggest that soy protein directly affects intestinal lipid processing, similarly to dietary fiber, and does not mediate an adaptive process in the intestinal mucosal cells. A component of soy, such as the peptide components or saponins, may bind dietary cholesterol during transit through the gastrointestinal system, thus reducing absorption. A recent report by Nagaoka and co-workers (1999) suggests that soy peptides may inhibit both cholesterol absorption and the reabsorption of bile acids.

There were no significant treatment effects on bile acid excretion in the present study. Although we would not expect increases in bile acid excretion with consumption of casein-lactalbumin diets containing either the isoflavone extract or CEE because there were no reductions in plasma cholesterol, we might have expected an increase in bile acid excretion with soy consumption. Recently, Wright and Salter (1998) reported an increase in bile acid excretion in hamsters fed intact soy protein compared to animals fed casein protein. They also reported a significant correlation between soy intake and bile acid excretion. In an earlier study, Nagata et al. (1982) also found increases in fecal steroid excretion in rats with dietary soy protein compared to casein, but found no significant effects from consumption of amino acid mixtures of soy and casein protein. Duane (1999) reported a significant increase in fecal neutral sterol output with textured vegetable protein feeding, but only a tendency for an increase in fecal acidic sterol output (P = 0.10) in eight normocholesterolemic men living on a metabolic ward. The lack of an effect of soy protein on bile acid excretion in both the present study and the study by Duane (1999) may be due to lack of statistical power, as the sample sizes were small.

Isoflavones have been proposed as the bioactive component of soy protein since they are very similar in structure to estrogen and estrogen therapy has such a profound effect on CHD risk. None of the previously mentioned studies investigated isoflavone action on cholesterol absorption or bile acid excretion. However, our results suggest that a semipurified soy extract, rich in isoflavones, added to casein-lactalbumin protein is not alone sufficient to mediate improvements in lipids in moderately hypercholesterolemic ovariectomized cynomolgus monkeys. Additionally, two recent studies in humans reported no effect on plasma lipids and lipoproteins by the addition of a daily isoflavone tablet to the diet (Hodgson et al. 1998 , Nestel et al. 1997 ).

CEE added to a casein-lactalbumin protein diet did not have significant effects on plasma lipids, cholesterol absorption or bile acid excretion. Similar results have been published previously (Adams et al. 1997 , Manning et al. 1996 ) For instance, when CEE was added directly to a casein diet fed to ovariectomized cynomolgus monkeys, no significant differences were found compared to the control group in TC, LDLC or HDLC concentrations (Manning et al. 1996 ). However, researchers found significantly smaller LDL particles which were rich in protein and triglyceride and poor in cholesteryl ester and apolipoprotein E. Monkeys in the CEE group also had a 50% lower hepatic cholesterol content than a group consuming casein-lactalbumin without additives. CEE added to the diet has also been found to decrease atherosclerosis without significant decreases in plasma cholesterol (Adams et al. 1997 ). It has been suggested that only 25 to 50% of the beneficial effects of estrogen on CHD are due to changes in plasma HDLC and LDLC, leaving 50 to 75% due to other mechanisms (Barrett-Connor and Bush 1991 ). Estrogen’s beneficial effects may occur through interactions directly with the arterial wall (Wagner et al. 1997 ).

The major findings in this study are that soy protein diet decreased both plasma cholesterol concentrations and intestinal cholesterol absorption. The addition of a semipurified soy extract, rich in isoflavones, to a casein-lactalbumin protein did not improve plasma lipids or reduce cholesterol absorption. Furthermore, the addition of CEE to a casein-lactalbumin protein diet did not improve plasma lipids or affect intestinal cholesterol absorption. A bioactive component of soy protein other than, or in addition to, isoflavones such as the saponins, phytic acid, protein components, amino acid composition of the soy protein or a protein-isoflavone interaction may be involved in the lipid-lowering effects. The reduction in CHD risk with soy protein consumption may be mediated, at least in part, by a reduced intestinal absorption of cholesterol; however, the component of soy protein responsible for this action has yet to be discovered.

	ACKNOWLEDGMENTS

 
The authors thank Mary Anthony, Vickie Hardy and Kirsten Williford for their technical assistance. Soy protein and isoflavones were provided by Protein Technologies International (St. Louis, MO).

	FOOTNOTES

 
¹ Supported in part by grants PO1 HL 45666 (JKW, JDW) from the National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, and T32 RR07009 (KAG) from the National Institutes of Health.

³ Current address: Protein Technologies International, St. Louis, MO 63188.

⁴ Abbreviations used: CAS, casein-lactalbumin protein; CEE, CAS plus conjugated equine estrogen; CHD, coronary heart disease; ERT, estrogen replacement therapy; HDLC, high density lipoprotein (HDL) cholesterol; HRT, hormone replacement therapy; ISO, CAS plus isolated isoflavone extract; LDLC, low density lipoprotein (LDL) cholesterol; SOY, soy protein isolate; TC, total plasma cholesterol; V + LDLC, very low density plus LDL cholesterol.

Manuscript received June 18, 1999. Initial review completed July 23, 1999. Revision accepted November 16, 1999.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Adams M. R., Register T. C., Golden D. L., Wagner J. D., Williams J. K. Medroxyprogesterone acetate antagonizes inhibitory effects of conjugated equine estrogens on coronary artery atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 1997;17:217-221[Abstract/Free Full Text]

2. Adlercreutz H. Western diet and Western diseases: Some hormonal and biochemical mechanisms and associations. Scand. J. Lab. Clin. Invest. 1990;50(Suppl 201):3-23

3. Anderson J. W., Johnstone B. M., Cook-Newell M. E. Meta-analysis of the effects of soy protein intake on serum lipids. N. Engl. J. Med. 1995;333:276-282[Abstract/Free Full Text]

4. Anthony M. S., Clarkson T. B., Bullock B. C., Wagner J. D. Soy protein versus soy phytoestrogens in the prevention of diet-induced coronary artery atherosclerosis of male cynomolgus monkeys. Arterioscler. Thromb. Vasc. Biol. 1997;17:2524-2531[Abstract/Free Full Text]

5. Anthony M. S., Clarkson T. B., Hughes C. L. Jr, Morgan T. M., Burke G. L. Soybean isoflavones improve cardiovascular risk factors without affecting the reproductive system of peripubertal rhesus monkeys. J. Nutr. 1996;126:43-50

6. Barrett-Connor E., Bush T. L. Estrogen and coronary heart disease in women. J. Am. Med. Assoc. 1991;265:1861-1867[Abstract]

7. Beynen A. C., West C. E., Spaaij C.J.K., Huisman J., Van Leeuwen P., Schutte J. B., Hackeng W.H.L. Cholesterol metabolism digestion rates and postprandial changes in serum of swine fed purified diets containing either casein or soybean protein. J. Nutr. 1990;120:422-430

8. Borgstrom B. Quantification of cholesterol absorption in man by fecal analysis after the feeding of a single isotope-labeled meal. J. Lipid Res. 1969;10:331-337[Abstract]

9. Burstein M., Samaille J. Sur un dosage rapide du cholesterol liè aux -et aux ß-lipoprotèines du sèrum. Clin. Chim. Acta. 1960;5:609[Medline]

10. Bush T. L, Cowan L. D., Barrett-Connor E., Criqui M. H., Karon J. M., Wallace R. B., Tyroler H. A., Rifkind B. M. Estrogen use and all-cause mortality. Preliminary results from the Lipid Research Clinics program follow-up study. J. Am. Med. Assoc. 1983;249:903-906[Abstract]

11. Carroll K. K. Review of clinical studies on cholesterol-lowering response to soy protein. J. Am. Dietetic Assoc. 1991;91:820-827[Medline]

12. Carroll K. K., Kurowska E. M. Soy consumption and cholesterol reduction: Review of animal and human studies. J. Nutr. 1995;125:594S-597S

13. Colditz G. A., Hankinson S. E., Hunter D. J., Willett W. C., Manson J. E., Stampfer M. J., Hennekens C., Rosner B., Speizer F. E. The use of estrogens and progestins and the risk of breast cancer in postmenopausal women. N. Engl. J. Med. 1995;332:1589-1593[Abstract/Free Full Text]

14. Coward L., Kirk M., Albin N., Barnes S. Analysis of plasma isoflavones by reversed-phase HPLC-multiple reaction ion monitoring-mass spectrometry. Clin. Chim. Acta. 1996;247:121-142[Medline]

15. Davis M. J., Krikler D. M., Katz D. Atherosclerosis: Inhibition or regression as therapeutic possibilities. Br. Heart J. 1991;65:302-310[Free Full Text]

16. Derby C. A., Hume A. L., Barbour M. M., McPhillips J. B., Lasater T. M., Carleton R. A. Correlates of postmenopausal estrogen use and trends through the 1980s in two southeastern New England communities. Am. J. Epidemiol. 1993;137:1125-1135[Abstract/Free Full Text]

17. Duane W. C. Effects of soybean protein and very low dietary cholesterol on serum lipids, biliary lipids, and fecal sterols in humans. Metabolism 1999;48:489-494[Medline]

18. Frick M. H., Elo O., Haapa K., Heinonen O. P., Heinsalmi P., Helo P., Huttunen J. K., Kaitaniemi P., Koskinen P., Manninen V., Mäenpäää H., Mälkönen M., Mänttäri M., Norola S., Pasternack A., Pikkarainen J., Romo M., Sjöblom T., Nikkilä E. A. Helsinki Heart Study: primary-prevention trial with gemfibrozil in middle-aged men with dyslipidemia. Safety of treatment, changes in risk factors, and incidence of coronary heart disease. N. Engl. J. Med. 1987;317:1237-1245[Abstract]

19. Gamache P. H., Acworth I. N. Analysis of phytoestrogens and polyphenols in plasma, tissue, and urine using HPLC with colormetric array detection. Proc. Soc. Exp. Biol. 1998;217:274-280[Abstract]

20. Grady D., Gebretsadik T., Kerlikowske K., Ernster V., Petitti D. Hormone replacement therapy and endometrial cancer risk: a meta-analysis. Obstet. Gynecol. 1995;85:304-313[Abstract]

21. Hemminski E., Brambilla B.D.J., McKinlay S. M., Posner J. G. Use of estrogens among middle-aged Massachusetts women. Ann. Pharmacother. 1991;25:418-422[Abstract]

22. Hodgson J. M., Puddey I. B., Beilin L. J., Mori T. A., Croft K. D. Supplementation with isoflavonoid phytoestrogens does not alter serum lipid concentrations: A randomized controlled trial in humans. J. Nutr. 1998;128:728-732[Abstract/Free Full Text]

23. Huff M. W., Carroll K. K. Effects of dietary protein on turnover, oxidation, and absorption of cholesterol and on steroid excretion in rabbits. J. Lipid Res. 1980;21:546-548[Abstract]

24. Huff M. W., Roberts D., Carroll K. K. Long-term effects of semipurified diets containing casein or soy protein isolate on atherosclerosis and plasma lipoproteins in rabbits. Atherosclerosis 1982;41:327-336[Medline]

25. Hulka B. S. Links between hormone replacement therapy and neoplasia. Fertil. Steril. 1994;62(Suppl. 2):168.S-175S[Medline]

26. for the HERS Research GroupHulley S., Grady D., Bush T., Furberg C., Herrington D., Riggs B., Vittinghoff E. Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women. J. Am. Med. Assoc. 1998;280:605-613[Abstract/Free Full Text]

27. Kim D. N., Lee K. T., Reiner J. M., Thomas W. A. Effects of a soy protein product on serum and tissue cholesterol concentrations in swine fed high-fat, high-cholesterol diets. Exp. Mol. Pathol. 1978;29:385-399[Medline]

28. Lipid Research Clinics Program The Lipid Research Clinics Coronary Primary Prevention Trial results. II. The relationship of reduction in incidence of coronary heart disease to cholesterol lowering. J. Am. Med. Assoc. 1984;251:365-374[Abstract]

29. Manning J. M., Campos G., Edwards I. J., Wagner W. D., Wagner J. D., Adams M. R., Parks J. S. Effects of hormone replacement modalities on low density lipoprotein composition and distribution in ovariectomized cynomolgus monkeys. Atherosclerosis 1996;121:217-230[Medline]

30. Mukhin D. N., Orekhov A. N., Andreeva E. R., Schindeler E. M., Sminov V. N. Lipids in cells of atherosclerotic and uninvolved human aorta. III. Lipid distribution in intimal sublayers. Exp. Mol. Pathol. 1991;54:22-30[Medline]

31. for the ARIC InvestigatorsNabulsi A. A., Folsom A. R., White A. Association of hormone-replacement therapy with various cardiovascular risk factors in postmenopausal women. N. Engl. J. Med. 1993;328:1069-1075[Abstract/Free Full Text]

32. Nagaoka S., Miwa K., Eto M, Kuzuya Y., Hori G., Yamamoto K. Soy protein peptic hydrolysate with bound phospholipids decreases micellar solubility and cholesterol absorption in rats and Caco-2 cells. J. Nutr. 1999;129:1725-1730[Abstract/Free Full Text]

33. Nagata Y., Ishiwaki N., Sugano M. Studies on the mechanism of antihypercholesterolemic action of soy protein and soy protein-type amino acid mixtures in relation to the casein counterparts in rats. J. Nutr. 1982;112:1614-1625

34. Nestel P. J., Yamashita T., Sasahara T., Pomeroy S., Dart A., Komesaroff P., Owen A., Abbey M. Soy isoflavones improve systemic arterial compliance but not plasma lipids in menopausal and perimenopausal women. Arterioscler. Thromb. Vasc. Biol. 1997;17:3392-3398[Abstract/Free Full Text]

35. Potter S. M. Overview of proposed mechanisms for the hypocholesterolemic effect of soy. J. Nutr. 1995;125:606S-611S

36. Potter S. M. Soy protein and cardiovascular disease: The impact of bioactive components in soy. Nutr. Rev. 1998;56:231-235[Medline]

37. Quintao E., Grundy S. M., Ahrens E. H. An evaluation of four methods for measuring cholesterol absorption by the intestine of man. J. Lipid Res. 1971;12:221-232[Abstract]

38. Robertson T. L., Kato H., Rhoads G. G., Kagan A., Marmot M., Syme S. L., Gordon T., Worth R. M., Belsky J. L., Dock D. S., Miyanishi M., Kawamoto S. Epidemiologic studies of coronary heart disease and stroke in Japanese men living in Japan, Hawaii, and California. Incidence of myocardial infarction and death from coronary heart disease. Am. J. Cardiol. 1977;39:239-243[Medline]

39. Rudel L., Deckelman C., Wilson M., Scobey M., Anderson R. Dietary cholesterol and downregulation of cholesterol 7-hydroxylase and cholesterol absorption in African green monkeys. J. Clin. Invest. 1994;93:2463-2472

40. Schiller L. R., Bilhartz L. E., Santa Ana C. A., Fordtran J. S. Comparison of endogenous and radiolabeled bile acid excretion in patients with idiopathic chronic diarrhea. Gastroenterology 1990;98:1036-1043[Medline]

41. Stampfer M. J., Colditz G. A. Estrogen replacement therapy and coronary heart disease: a quantitative assessment of the epidemiologic evidence. Prev. Med. 1991;20:47-63[Medline]

42. Sugano M., Yamada Y., Yoshida K., Hashimoto Y., Matsuo T., Kimoto M. The hypocholesterolemic action of the undigested fraction of soybean proteins in rats. Atherosclerosis 1988;72:115-122[Medline]

43. Thom, T. J., Epstein, F. H, Feldman, J. J., Leaverton, P. E. & Wolz, M. (1992) Total Mortality and Morbidity from Heart Disease, Cancer and Stroke from 1950 to 1987 in 27 Countries. National Institutes of Health publication no. 02–3088. Bethesda, MD: National Institutes of Health, National Heart, Lung, and Blood Institute.

44. Turley S. D., Dietschy J. M. Re-evaluation of the 3-hydroxysteroid dehydrogenase assay for total bile acids in bile. J. Lipid Res. 1978;19:924-928[Abstract]

45. Vahouny G. V., Chalcarz W., Satchithanandam S., Adamson I., Klurfeld D. M., Kritchevsky D. Effect of soy protein and casein intake on intestinal absorption and lymphatic transport of cholesterol and oleic acid. Am. J. Clin. Nutr. 1984;40:1156-1164[Abstract/Free Full Text]

46. Wagner J. D., Zhang L., Adams M. R. Effect of estrogens on arterial LDL metabolism. Forte T. M. eds. Hormonal, Metabolic and Cellular Influences on Cardiovascular Disease in Women 1997:153-174 Futura Publishing Company, Inc Armonk, NY.

47. Wright S. M., Salter A. M. Effects of soy protein on plasma cholesterol and bile acid excretion in hamsters. Comp. Biochem. Physiol. 1998;119B:247-254

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