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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2002;22:1852.)
© 2002 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Lipoprotein Response to Diets High in Soy or Animal Protein With and Without Isoflavones in Moderately Hypercholesterolemic Subjects

Alice H. Lichtenstein; Susan M. Jalbert; Herman Adlercreutz; Barry R. Goldin; Helen Rasmussen; Ernst J. Schaefer; Lynne M. Ausman

From the Cardiovascular Nutrition Laboratory and Lipid Metabolism Laboratory (A.H.L., S.M.J., H.R., E.J.S., L.M.A.), JM HNRC Human Nutrition Research Center on Aging at Tufts University, Boston, Mass; Folkhälsan Research Center and the Department of Clinical Chemistry (H.A.), University of Helsinki, Finland; and Department of Community Medicine, Tufts University School of Medicine, Boston, Mass (B.R.G.).

Correspondence to Dr. Alice H. Lichtenstein, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, 711 Washington Street, Boston, MA 02111. E-mail: Lichtenstein{at}hnrc.tufts.edu

	Abstract

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Objective— The objective of this study was to assess the independent effect of soy relative to common sources of animal protein and soy-derived isoflavones on blood lipids.

Methods and Results— Forty-two subjects with LDL cholesterol levels 3.36 mmol/L were fed each of four diets in randomized order for 6 weeks per phase. Diets contained a minimum of 25 g animal protein or isolated soy protein/4.2 MJ, with each containing trace amounts or 50 mg of isoflavones/4.2 MJ. Soy protein had a modest effect on total, LDL and HDL cholesterol, and triglyceride concentrations (-2%, P=0.017; -2%, P=0.042; +3%; P=0.034, -11%, P<0.001, respectively). Soy protein had no significant effect on plasma lipids in individuals with LDL cholesterol <4.14 mmol/L and significantly reduced total and LDL cholesterol and triglyceride concentrations in individuals with LDL cholesterol 4.14 mmol/L (-4%, P=0.001; -5%, P=0.003; -15%, P<0.001, respectively). No significant effect of isoflavones on plasma lipid levels was observed either constituent to the soy protein or supplemental to the animal protein.

Conclusions— Although potentially helpful when used to displace products containing animal fat from the diet, the regular intake of relatively high levels of soy protein (>50 g/day) had only a modest effect on blood cholesterol levels and only in subjects with elevated LDL cholesterol levels (4.14 mmol/L). Soy-derived isoflavones had no significant effect.

Key Words: soy protein • isoflavones • animal protein • saturated fat • diet • triglyceride • LDL cholesterol • HDL cholesterol • vegetable protein • cardiovascular disease

	Introduction

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In October of 1999, the Food and Drug Administration authorized the use of a health claim relating intakes of at least 25 g of soy protein per day to reduced risk of developing coronary heart disease.¹ Much of the support for this health claim seems to have been based on a meta-analysis published in 1995.² After comprehensively reviewing the available literature at the time, the authors of that study concluded that soy protein, when compared with animal protein, reduced LDL cholesterol levels by 7% in individuals with initial total cholesterol levels between 3.31 and 6.59 mmol/L, 10% in individuals with initial total cholesterol levels between 6.70 and 8.61 mmol/L, and 24% in individuals with initial total cholesterol levels greater than 8.66 mmol/L. A significant decrease in triglyceride and a nonsignificant increase in HDL cholesterol levels also were noted. Whether the changes were attributable to the soy protein per se or another soy-derived factor, potentially constitutive isoflavones, could not be assessed. Notably, 77% of the studies included in the meta-analysis had 95% confidence intervals that encompassed zero. Since that time, a number of studies have re-examined the effect of soy protein and/or isoflavones on blood lipid levels. The results of these studies are variable yet consistently less positive than the original assessment of the earlier literature.² Changes in LDL or non–HDL cholesterol levels attributable to the substitution of 25 to 50 g of soy protein for animal protein range from a null to a 5% decline in individuals with moderately elevated total cholesterol levels.^3–9 Some work has demonstrated that the potential benefit of soy protein may be dependent on whether it is ingested with the constituent isoflavones,⁴ whereas other work does not support this observation.^10–13

See page 1743

The data to date examining the effect of soy protein and isoflavones on blood lipid levels are conflicting, and the more recent work is not consistent with some earlier assessments. Nonetheless, there has been a proliferation of soy protein–containing foods in the marketplace, most of which claiming or implying that the consumption of such products will reduce the risk of developing cardiovascular disease. This study was designed to assess the independent effect of soy protein relative to common sources of animal protein (meat and dairy), distinct from the fatty acid profile and isoflavones and, likewise, the independent effect of soy isoflavones, distinct from protein type and fatty acid profile, in a controlled trial in individuals at elevated risk of developing cardiovascular disease.

	Methods

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This study protocol was approved by the Human Investigation Review Committee of New England Medical Center and Tufts University.

Subjects
Forty-two subjects over the age of 50 years with LDL cholesterol levels greater than 3.36 mmol/L were recruited for this study from the greater Boston area. Subjects were not taking medications known to affect blood lipid levels, and all females were postmenopausal. After randomization but before the start of the study, 5 subjects (2 females, 3 males) dropped out, 4 because of time/employment constraints and 1 because of a change in medical status. During or at the end of the first phase, 9 subjects (1 female, 8 males) dropped out, 1 because of a change in employment, 3 because of a change in medical status, 3 because of a change in medical status of family member, and 2 because of a dislike of study food. At the end of second phase, 1 female dropped out because of a dislike of study food. Characteristics of the study subjects at the time of screening are shown in Table 1. Subjects that dropped out were replaced.

View this table: [in this window] [in a new window]   Table 1. Characteristics of the Subjects at the Time of Screening for the Study

Experimental Design
Study subjects were provided with each of four experimental diets; soy protein depleted of isoflavones (soy/-), soy protein enriched in isoflavones (soy/+), animal protein with no added isoflavones (animal/-), and animal protein with added isoflavones (animal/+), for periods of 42 days each according to a 2x2 factorial design. All food and drink were provided to the subjects in containers appropriate for either microwave or conventional ovens, obviating the need to transfer food before consumption. Body weight was maintained throughout the study period. The mean caloric intake (mean±SD) of the female subjects was 9.25±1.00 MJ/day and of male subjects was 11.85±2.97 MJ/day. After day 35 of each diet phase, blood samples were obtained on 3 separate days in the fasting state and on 1 day 4 hours after the dinner meal.

Diets
The animal/- diet was designed to mimic a nonoptimal diet of a hypercholesterolemic subject. The other diets were designed to be similar in total fat, carbohydrate, protein, fatty acid profile, fiber, and cholesterol content. This was achieved by substituting isolated soy protein for animal protein (two thirds of total protein, 10% of energy; 25 g soy protein/4.2 MJ) and adjusting the fatty acid profile of the diet using commonly available fats and oils. For the animal/- and animal/+ diets, the variable protein component was contributed by dairy and meat. Isoflavones, in the form of a powdered concentrate (Archer Daniels Midland Company), were mixed into different food items of the animal/+ diet. For the soy/- and soy/+ diets, the variable protein component was contributed by specially prepared batches of isolated soy protein, one depleted (0.12 mg aglycone/g protein) and one enriched (1.96 mg aglycone/g protein) in isoflavones (Protein Technologies). The mean soy intake of the female subjects was 55±6 g/day and of male subjects was 71±18 g/day. The isoflavone intake of the female subjects was 108±12 mg/day and of male subjects was 139±35 mg/day (Table 2). Chemical analysis of food homogenates was performed by Covance Laboratories, Inc; dietary isoflavones analysis was performed in the laboratory of Dr. Patricia A. Murphy (Iowa State University, Ames, IA).

View this table: [in this window] [in a new window]   Table 2. Nutrient Composition of Each of the Experimental Diets as Determined by Chemical Analysis of Food

Biochemical Analysis
Blood samples were collected and analyzed as previously described.^14–17 Plasma genistein and daidzein, in both the fasting and nonfasting serum samples, were measured by time-resolved fluoroimmunoassay using a recently validated method.¹⁸

Statistical Analysis
Before the analysis, descriptive statistics and graphs (PROC UNIVARIATE and PROC MEANS; SAS) were used to summarize the overall effects of diets. When violations of the basic testing assumptions were noted, appropriate transformations of the data were used. A two-way analysis of variance (PROC GLM; SAS) with main effects of dietary protein type and isoflavone content (minimal or supplemented) with subject as a repeated measure was performed for each outcome measure. In a further post-hoc analysis to determine whether the response to the dietary protein or isoflavone content was dependent on the degree of hypercholesterolemia after consuming the animal/- diet, the PROC GLM was repeated for each group.

	Results

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In response to an increase in dietary isoflavone intake, both fasting and nonfasting plasma isoflavone levels were increased dramatically (Table 3). Twenty-five grams of soy protein/4.2 MJ resulted in a small but significant reduction in total and LDL cholesterol levels of 0.13 mmol/L (-2%) and 0.10 mmol/L (-2%; P=0.017 and P=0.042, respectively), and an increase in HDL cholesterol levels (0.03 mmol/L; +3%; P=0.034; Table 4; Figure 1). The change in HDL cholesterol was attributable to the HDL₃ subfraction. The sum of these changes resulted in a lower (more favorable) total cholesterol/HDL cholesterol ratio. The greatest effect of soy protein was on triglyceride levels, lowering the mean value by 0.18 mmol/L (11%; P<0.001). For the most part, the effect of dietary protein on VLDL cholesterol (P=0.052), apo B (P=0.003), and apo A-I levels mirrored those of triglyceride, LDL, and HDL cholesterol levels, respectively. There was little change in the LDL cholesterol/apo B or HDL cholesterol/apo A-I ratios as a result of varying protein type, suggesting little change in the composition of the HDL and LDL particles.

View this table: [in this window] [in a new window]   Table 3. Fasting Plasma Isoflavone Levels at the End of Each Dietary Phase

View this table: [in this window] [in a new window]   Table 4. Fasting Serum Lipid, Lipoprotein and Apolipoprotein Levels at the End of Each Diet Phase

View larger version (22K): [in this window] [in a new window]   Percent change in serum lipid levels after subjects consumed (A) diets containing soy protein compared with diets containing animal protein and (B) diets without compared with diets with soy-derived isoflavones. Solid bars, all subjects (n=42); diagonal striped bars, subjects with LDL <4.14 mmol/L (n=20); cross-hatched bars, subjects with LDL 4.14 mmol/L (n=22). The small letters indicate the significance of either the protein or isoflavone effect from the two-way ANOVA (a=P<0.05; b=P<0.01; c=P <0.001).

Soy-derived isoflavones, either as a constituent part of the soy protein or added to the animal protein–containing diet, had little independent effect on the parameters assessed (Table 4; Figure 1).

In light of previous work suggesting the hypocholesterolemic effect of soy protein was greater in individuals with higher LDL cholesterol levels,⁴ the data were further analyzed to determine whether the response was related to the degree of hypercholesterolemia. The benefit attributable to soy protein with respect to total cholesterol was limited to those subjects with LDL cholesterol 4.14 mmol/L and was still modest (Table 5; Figure 1). In those subjects, soy protein resulted in total and LDL cholesterol levels that were 0.28 mmol/L (-4%) and 0.26 mmol/L (-5%) lower (P=0.001 and P=0.003, respectively). In contrast, there was no significant effect of soy protein on HDL cholesterol levels in these individuals but a small effect in individuals with LDL cholesterol levels <4.14 mmol/L (+3%), an effect that was restricted to the HDL₃ subfraction. The significant effect of soy protein on triglyceride levels was greater in individuals with LDL cholesterol levels 4.14 mmol/L than <4.14 mmol/L (-15% and -7%, P<0.001 and P=0.014, respectively). No significant effect of soy protein on the LDL cholesterol/apo B and HDL cholesterol/apo A ratios was observed.

View this table: [in this window] [in a new window]   Table 5. Fasting Serum Lipid, Lipoprotein, and Apolipoprotein Levels at the End of Each Diet Phase

When the data were analyzed on the basis of LDL cholesterol subgroup, soy-derived isoflavones had no significant effect on blood lipid parameters measured with one exception, total cholesterol levels in the LDL 4.14 mmol/L group (Table 5; Figure 1). When this effect was assessed on the basis of lipoprotein cholesterol levels, VLDL, LDL, and HDL, the difference did not remain significant.

	Discussion

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The current study was designed to assess, under controlled conditions, the independent effect of protein type, distinct from fatty acid profile or presence of soy-derived isoflavones and, likewise, the independent affect of isoflavones, distinct from protein type or fatty profile, on plasma lipid levels. Despite providing soy protein at levels that were at least twice that of the daily minimum intake on which the current health claim is based, the response in individuals with moderately elevated LDL cholesterol levels (<4.14 mmol/L; mean total cholesterol 5.69 mmol/L) was null, and in the group with more severely elevated LDL, cholesterol levels (4.14 mmol/L; mean total cholesterol 7.19 mmol/L) were about half that which would have been predicted on the basis of the data used to formulate the health claim.² These observations are actually consistent with or exceeded that which would be predicted from more recently published data^2–9 and much of the earlier work.^19–27 Little independent effect of isoflavones on blood lipid levels was observed.

The most striking effect of soy protein, relative to animal protein, was the resultant lower plasma triglyceride levels. This observation was somewhat unexpected. Although it had originally been reported that soy protein resulted in a reduction in triglyceride levels,² the observation had not been corroborated by the more recent studies^3–8 with the exception of one.⁹ In the current study, the lower triglyceride levels observed after the subjects consumed the soy-containing diets were greater in the individuals with the higher LDL cholesterol levels. The effect persisted in the nonfasting state (data not shown). The change in triglyceride levels was not accompanied by a reciprocal response in HDL cholesterol levels, as is frequently observed. Whether a decrease in triglyceride levels translates independently to a decrease in cardiovascular disease risk is a topic of considerable interest at this time.^28,29

Notable was the large variability in plasma isoflavone levels observed among the study subjects both in the fasting and nonfasting state. A likely explanation is interindividual variation in the gut microflora among subjects that determine the metabolism and bioavailability of isoflavones before absorption rather than poor dietary compliance.³⁰ The distribution of the isoflavones genistein, daidzein, and glycitein was 59, 30, and 11% in the soy/+ diet and 52, 40, and 8% in the animal/+ diet, respectively. The pharmacokinetics of genistein and daidzein, the two major soy-derived isoflavones, are somewhat different, although the bioavailabilities are similar with the exception of urinary excretion rates.³¹ The elimination half-life of daidzein has been estimated to be 7.4 hours and of genistein 5.7 hours. Although the rate of urinary excretion of daidzein is greater than genistein, the ratio of the areas under the plasma concentration versus time curves have been reported to equal the ratio of the concentrations of the respective isoflavones in the isoflavone-containing meal.³¹ Hence, it is unlikely that the differences in the proportion of the individual isoflavones in the two preparations used in the study had a major impact on the outcome.

There was no significant relationship between circulating isoflavone levels and changes in LDL cholesterol or triglyceride levels. The amounts of dietary isoflavones previously reported to have a significant effect on LDL cholesterol levels have been at or below those provided to our subjects.^4,32 Hence, even the patients considered low absorbers in the current study should have absorbed adequate amounts of isoflavones to achieve a biological effect were there one to be manifested. Howes et al³³ also reported that supplementation of postmenopausal females with dietary isoflavones, specifically derived from purified extract of red clover, did not significantly alter total, LDL or HDL cholesterol, or triglyceride levels. They did report that there was an inverse correlation between both urinary genistein and O-desmethylangolensin excretion (an isoflavone metabolite) and plasma triglyceride levels. The primary isoflavones in red clover are biochanin A and formononetin, with only small amounts of genistein and daidzein.

There are a number of factors that may explain the large variability in responses attributable to soy protein on cholesterol levels. Of the studies reporting dramatic decreases in LDL cholesterol levels that are attributable to soy protein, the majority studied familial hypercholesterolemic subjects and used what may have been a unique textured lecithinated soy protein preparation.^34–40 Some component(s) of the soybean, lost during the manufacturing process, may have also contributed to the outcome observed. Replacing animal with vegetable protein can shift the fatty acid profile of the diet from saturated to unsaturated fatty acids, a change that may be difficult to adequately assess when relying on a patient’s self-reported dietary data. Frequently, the relative effect of dietary supplements containing soy protein was assessed relative to similar supplements containing casein. A unique amino acid profile of casein relative to soy protein may have influenced the outcome of the studies.

Another potential explanation for the discrepancy in results of the current study and the positive ones previously reported may be the presence of the soybean isoflavones. However, for the most part, no significant effect of isoflavones was observed in any of the lipid or apolipoprotein levels measured, regardless of whether the isoflavones were consumed as a constituent component of the soy protein or added in the isolated form to the animal protein–containing diet. This lack of an independent effect is consistent with that reported for isoflavones given in supplement form.^10–13 The data published to date on soy protein with constituent isoflavones are more complicated. Some work has reported no effect of soy protein per se but reported a decrease in LDL cholesterol levels only when the soy protein contained isoflavones,⁴ no effect of either soy protein with or without isoflavones,¹⁰ a similar effect of soy protein with either 65 mg and 132 mg isoflavones relative to trace amounts of isoflavones, or no significant effect of soy protein with 65 mg of isoflavones but a significant effect of soy protein with 93 mg of isoflavones.⁴¹ The levels of soy-derived isoflavones provided in the current study are comparable with or exceeded those previously investigated.

A limitation of the current study is that using a single source and type of soy protein created a unique scenario. However, because the intent of the investigation was to assess the independent effect of each soy protein and soy-derived isoflavones, it was critical to control for potential confounding by other soybean constituents that may be present as a result of variations in processing techniques.⁴² There was a slight difference in the relative proportions of genistein, daidzein, and glycitein between the diets with soy protein with constituent isoflavones and animal protein with added isoflavones. However, at this point there is no evidence to suggest that this discrepancy would account for the differences observed. The magnitude of the dietary perturbations and dose of soy protein and isoflavones were extreme and not necessarily representative of an ad libitum scenario. However, this investigation was designed to maximize the ability to observe a positive effect, and the results need to be interpreted taking the aforementioned into consideration.

Our results suggest that the daily consumption of relatively high levels of soy protein (>50 g/day), at least double the level that would allow foods to qualify for a health claim, would be predicted to confer little benefit to normocholesterolemic subjects or those with borderline high LDL cholesterol levels (3.36 mmol/L). At these high levels of soy protein intake, patients with LDL cholesterol levels of 4.14 mmol/L realized a small benefit, roughly comparable with that predicted to result from the consumption of a diet enriched in soluble fiber.⁴³ As with other dietary interventions, to obtain this benefit, high levels of soy protein intake would have to be maintained on a relatively consistent basis. Many products currently on the market profess to contain soy protein but per serving contain levels just at or below that allowable for a health claim (6.25 g). On the basis of the current study it is concluded that, although helpful when used to displace products containing animal fat from the diet, hypercholesterolemic patients and the general public should be cautioned against an overreliance on the casual use of soy protein–containing foods or the use of isolated isoflavones to control serum lipid levels.

	Acknowledgments

 
This work was supported by Grant HL 58008 from the National Institutes of Health, Bethesda, MD, Grant 96-35200-3250 from the USDA/CSREES/NRICGP, Washington, DC, and a cooperative contract from the US Department of Agriculture under agreement No. 58-1950-9-001. The authors thank the staff of the Metabolic Research Unit for the expert care provided to the study subjects and gratefully acknowledge the cooperation of the study subjects, without whom this investigation would not have been possible. They also thank both Archer Daniels Midland Company, Decatur, Ill, for their generous gift of the isoflavone concentrate and Protein Technologies, St. Louis, Mo, for their generous gift of isolated soy protein depleted and enriched in isoflavones.

Received July 9, 2002; accepted August 1, 2002.

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