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Department of Pathology/Comparative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27157 and * The Cancer Research Center of Hawaii, Honolulu, HI 96813
3To whom correspondence should be addressed. E-mail: madams{at}wfubmc.edu.
ABSTRACT
The cardiovascular effects of dietary soy on men or adult male experimental animals have received little attention. We determined the effects of long-term (31 mo) consumption of a commercially available soy protein concentrate containing experimentally varied concentrations of isoflavones on the development of atherosclerosis and vascular reactivity in adult male monkeys. The monkeys were fed atherogenic diets that differed only in the source of protein: Control (n = 30), casein and lactalbumin; low-isoflavone soy (n = 30), a mixture of unmodified soy protein isolate and isoflavone-depleted soy protein isolate containing 0.94 mg of isoflavones/g protein; and high-isoflavone soy (n = 31), unmodified soy protein isolate containing 1.88 mg of isoflavone/g protein. Plasma LDL cholesterol was reduced, whereas HDL cholesterol and apolipoprotein A-1 (P < 0.05) were increased in both groups that consumed soy protein. Atherosclerosis (mean plaque size in the coronary arteries) was reduced by 
34% (P < 0.05) in both groups fed soy protein. There were no effects of dietary soy on endothelium-dependent or -independent reactivity of coronary arteries. The results indicate that long-term consumption of soy protein containing a modest amount of isoflavones inhibits the early progression of coronary artery atherosclerosis without affecting endothelium-dependent or -independent arterial function.
KEY WORDS: • soy isoflavones • atherosclerosis • monkeys • vascular reactivity
The U.S. FDA’s approval of a cardiovascular health claim for nutritional products containing 25 g of soy protein has contributed to widespread use of soy supplements. However, although consumption of this amount of soy protein contributes to modest improvements in plasma lipoprotein profiles in some subjects, there are no data addressing its effects on clinical cardiovascular endpoints such as incidence of coronary heart disease or atherosclerosis progression in human subjects. Similarly, although estrogenic isoflavones are postulated to be among the components of soy with cardioprotective activity, there are no data on the effects of isoflavones in amounts found in 25 g of soy protein on coronary heart disease risk. Although results of a recent prospective observational study found no association between phytoestrogen intake, as assessed by questionnaire, and risk of coronary heart disease in Dutch women (1), soy intake was not adequately accounted for in that study, and soy consumption in that population was quite uncommon. Furthermore, median daily phytoestrogen consumption was <1% of the amount contained in 25 g of soy protein. Thus, the relevance of these data is questionable at best. More relevant, perhaps, are the findings of Zhang et al. (2) in a prospective cohort study of 65,000 Chinese women with a median intake of soy protein of 7.36 g/d. In this population, there was a clear inverse dose-response relation between soy intake and coronary heart disease risk and an adjusted relative risk of 0.25 (95% CI = 0.10 to 0.63) for the highest quartile of soy protein intake compared with the lowest quartile. There are no similar data addressing the existence of a similar relation in men.
Similarly, the effects of soy consumption on indices of endothelium-dependent and endothelium-independent vascular reactivity in men have received little attention. A randomized, double-blind trial demonstrated a decline in flow-mediated (endothelium-dependent) peripheral vasodilatory function in healthy 50- to 75-y-old men who had consumed isoflavone-containing soy protein supplements for 3 mo compared with men who had consumed a casein placebo (3). Also, a previous study by our group found a tendency toward a decline in endothelium-dependent vasodilatory function of coronary arteries in male monkeys fed isoflavone-replete soy protein for 6 mo compared with monkeys fed isoflavone-deficient soy protein (4). These findings conflict with those from studies of women and female monkeys, in which isoflavone-containing soy supplements seemed to improve vascular reactivity or have no effect (3–7).
Data from numerous studies with experimental animals have demonstrated a lipoprotein-independent inhibitory effect of soy-based diets, compared with animal protein-based diets, on the development of atherosclerotic plaques (8–12). These studies suggest that the cardiovascular benefit of soy consumption for human beings may exceed that predicted by its modest effects on plasma lipoproteins. Previous studies with monkeys found an inhibitory effect of isoflavone-containing dietary soy protein on atherosclerosis in juvenile male and ovariectomized female monkeys, an effect that was partially lost when the isoflavones were removed from the soy protein by alcohol washing (13,14). It remains unclear whether the inhibitory effects of soy or isoflavones on atherosclerosis extend to adult male monkeys, whether phytoestrogenic isoflavones can be consumed for long periods by adult males without adverse effects, and what amount of soy isoflavones is optimal for inhibiting atherosclerosis progression.
We determined the effects of the consumption of soy protein containing 2 concentrations of isoflavones on the development of atherosclerotic plaques and vascular reactivity in coronary arteries of adult male monkeys.
MATERIALS AND METHODS
Animals and diets. Adult male cynomolgus monkeys (Macaca fascicularis; n = 91) were imported from Indonesia (Institut Pertanian Bogor). Monkeys consumed a moderately atherogenic soy-free diet (Table 1) for a 5-mo pretreatment period and were then assigned to treatment groups using a stratified randomization scheme that matched groups for pretreatment plasma total cholesterol and HDL cholesterol concentrations. For an additional 31 mo, monkeys were fed moderately atherogenic diets (Table 1) differing only in the source of protein. The source of protein (casein and lactalbumin) for the control group (n = 30) was the same as that for the soy-free pretreatment diet; for the low-isoflavone soy group (n = 30), it was a mixture of unmodified soy protein isolate and alcohol-washed (isoflavone-depleted) soy protein isolate containing 0.94 mg of isoflavones/g protein (an amount approximating a human intake of 75 mg/d); and for the high-isoflavone soy group (n = 31), it was unmodified soy protein isolate containing 1.88 mg of isoflavone/g protein (an amount approximating a human intake of 150 mg/d). Monkeys lived in social groups of 4 individuals. All procedures involving animals were approved by the Institutional Animal Care and Use Committee of Wake Forest University School of Medicine.
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Serum isoflavones. Analysis of serum isoflavones was carried out at mo 31 using LC photo-diode array electrospray MS slightly modified from a previously established method to include equol in the panel of isoflavonoids (genistein, dihydrogenistein, daidzein, dihydrodaidzein, glycitein, O-desmethylangolensin) and isotopically labeled internal standards (23,24).
Quantitative coronary angiography. Near the end of the treatment period, the monkeys were sedated with ketamine hydrochloride (10–15 mg/kg, i.m.) and butorphanol (0.025 mg/kg, i.m.). A custom-designed 3F (tapered to 1.8F) catheter was inserted into the left femoral artery and advanced into the left main coronary artery under fluoroscopic guidance. To determine endothelium-dependent and -independent coronary artery responses, serial 2-min infusions of 1) 5% dextrose in water, 2) acetylcholine (10–8, 10–7, and 10–6 mol/L, estimated final concentration in the coronary artery), 3) 5% dextrose in water, and 4) nitroglycerin (15 g/min) were performed as previously described (25,26). Quantitative coronary angiography (QCA Plus, Sanders Data Systems) was also done using techniques described previously (25,26). Films were analyzed by an operator who was unaware of the monkey’s treatment group.
Necropsy and atherosclerosis assessment. At the end of the treatment period, monkeys were anesthetized deeply with pentobarbital (30 mg/kg, i.v.), and the cardiovascular system was flushed with normal saline. After ligation of the vena cava and pulmonary arteries, the heart was excised. The heart and coronary arteries were perfusion-fixed via the aorta with 4% paraformaldehyde at a pressure of 100 mm Hg for 1 h. The heart was then immersed in 4% paraformaldehyde. After fixation, 5 serial tissue blocks were cut from each of the left circumflex, left anterior descending, and right coronary arteries. One section from each block was stained with Verhoeff-van Gieson’s stain, sections were projected, and the cross-sectional area occupied by intimal lesion (plaque size) was measured using a digitizer. Plaque size was expressed as the mean of these 15 sections of the 3 coronary arteries.
Data analysis. To satisfy the linearity, homogeneity, and normality assumptions of parametric statistical methods, reduce skewness, and equalize group variances, atherosclerosis data underwent square-root transformation before analysis. ANOVA, repeated-measures ANOVA, analysis of covariance, and multiple regression were used. Duncan’s new multiple range test was used for post hoc comparisons. Differences with P < 0.05 were considered significant.
RESULTS
Lipoproteins. At the end of the treatment period (mo 31), the plasma LDL cholesterol concentration was lower in the soy-treated groups than in the control group (Table 2) and plasma HDL cholesterol and apoA-I were greater in the treated groups than controls (Table 2). Plasma apoA-II, apoB, apoE, or lipoprotein(a) did not differ among the groups (data not shown).
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DISCUSSION
The principal finding was that the development of atherosclerotic plaques was inhibited in adult male monkeys by long-term consumption of diets rich in soy protein and containing the human equivalent of either 75 or 150 mg of isoflavones/d. Additionally, these results were accounted for statistically in large part by reductions in plasma LDL cholesterol and increases in plasma HDL cholesterol. Previous studies with monkeys utilized juvenile male monkeys or ovariectomized female monkeys. In juvenile males, atherosclerotic plaque size was reduced by 90% relative to a casein-consuming control group in those monkeys fed a soy protein isolate containing the human equivalent of 120 mg/d isoflavones (13). The reason for the difference in the magnitude of the effect observed in that study and the present study is unclear, but could relate either to the difference in the ages of the monkeys or the fact that in the previous study, an atherogenic diet was fed for only 14 mo. In a previous study with ovariectomized female monkeys fed atherogenic diets containing a soy protein isolate with the human equivalent of 120 mg isoflavones/d for 36 mo, there was a reduction in plaque size of 25% (27). An important difference between that study and the present study is that the control group was fed an isoflavone-depleted soy protein isolate. In another study of ovariectomized monkeys fed a soy-based diet containing the human equivalent of 120 mg isoflavones/d for only 20 wk, there was a reduction in arterial LDL accumulation of 50% (14). Thus, although there are substantial differences in the designs of the 3 studies, the available evidence suggests that the effects of isoflavone-containing soy protein isolates on the development of atherosclerosis in adult male and ovariectomized female monkeys are similar and are accounted for statistically largely by reductions in plasma LDL cholesterol and increases in plasma HDL cholesterol.
Although soy has modest effects on plasma LDL cholesterol in both human beings and monkeys, favorable effects on HDL cholesterol are more pronounced in monkeys (13,14,27). This may limit the relevance of these findings to human subjects. On the other hand, because it is unclear how the elevation in HDL cholesterol is mediated and whether an antiatherogenic process is involved, it is possible that this elevation in HDL may represent an epiphenomenon and, therefore, effects of soy on atherosclerosis may be mediated through other pathways. In addition, although treatment with soy protein isolates, but not isoflavone concentrates, has favorable influences on plasma lipoproteins in human beings and monkeys, studies with nonprimate animal models indicate, albeit indirectly, that both the isoflavone and peptide components of soy have atheroprotective activities that are independent of plasma lipoproteins (8–12). Therefore, it remains possible that the effects of soy on the development of atherosclerosis and coronary heart disease in human populations may exceed those predicted by its modest effects on plasma lipoproteins. Supporting the hypothesis that increased soy consumption results in a substantial reduction in risk of coronary heart disease are the findings of a prospective cohort study of 65,000 Chinese women with a median intake of soy protein of 7.36 g/d. In this population, there was a clear inverse dose-response relation between soy intake and coronary heart disease risk and an adjusted relative risk of 0.25 (95% CI = 0.10 to 0.63) for the highest quartile of soy protein intake compared with the lowest quartile (2).
In previous studies with monkeys, atheroprotection was achieved by the consumption of soy containing the human equivalent of 120 mg isoflavones/d or more (13,14,27). Our findings suggest that substantial atheroprotection can be achieved with as little as 75 mg/d and that there is no further benefit associated with a higher dose. However, it also is possible that isoflavones have no role in soy-induced inhibition of atherosclerosis progression. Supporting this possibility is the finding that there was no relation between plasma isoflavone concentrations and atherosclerotic plaque size. However, the answer to this question is obscured by the fact that studies aimed at assessing the role of isoflavones in inhibiting atherosclerosis relied on a comparison of the effects of an unmodified commercially available soy protein isolate to those of this same isolate, which had been washed with ethanol to remove the isoflavones. Because ethanol washing may have effects other than removing isoflavones, this approach may have unintended confounding consequences. For example, other soy components with potential atheroprotective activity, e.g., saponins, are also ethanol-soluble and removed by ethanol washing. In addition, the physical characteristics of the protein components such as tertiary structure and solubility are likely to be altered by the extraction process. Furthermore, Gianazza et al. (28) showed that ethanol washing changes the molecular size distribution of the peptide components of the resulting protein isolate. The fact that one peptide size fraction, i.e., the 7S fraction (ß-conglycinin) of soy protein, has a potent isoflavone-independent atheroinhibitory influence (12), whereas another, the 11S fraction (glycinin), has no effect modifications such as those induced by ethanol, could greatly influence the atheroprotective activity of soy protein isolates. The findings of Giannaza et al. (28) also showed that the 7S fraction is degraded extensively in some soy protein isolates commonly used in dietary soy supplements. Whether these degradation products have atheroinhibitory activity is uncertain. Thus, substantial work is still required to identify the relative roles of isoflavone, protein, or other components of soy responsible for its atheroprotective activity.
A limited body of data indicates that consumption of isoflavone-containing soy protein may have adverse effects on endothelium-mediated arterial function of male, but not female, humans and monkeys (3–7). It is important to note that these earlier studies employed relatively short-term soy consumption (3–6 mo). The data presented here indicate no effects of long-term (31 mo) soy consumption. More studies are warranted to clarify the reason for this apparent contradiction.
We conclude that long-term consumption of soy protein containing a relatively modest amount of isoflavones inhibits the early progression of coronary artery atherosclerosis, whereas it has no effect on endothelium-mediated arterial function.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the technical assistance of Laurie J. Custer in conducting isoflavone analyses. Soy products were provided by the Solae Company, St. Louis, MO.
FOOTNOTES
1 Supported by grants HL45666 and CA71789 from the National Institutes of Health. 
2 Supplemental Tables 1 and 2 are available as Online Supporting Material with the online posting of this paper at www.nutrition.org.
Manuscript received 10 August 2005. Initial review completed 24 August 2005. Revision accepted 10 September 2005.
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