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The Journal of Nutrition Vol. 128 No. 5 May 1998,
pp. 839-842
Division of Nutritional Sciences and the Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL 61801
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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The ability of alcohol extract of isolated soy protein to contribute to the hypochoesterolemic effect mediated by the intake of soy protein was tested in gerbils. Gerbils were assigned to five different groups (n = 8) and provided experimental diets for 28 d. Diets contained either casein or alcohol-washed isolated soy protein (ISP). The ISP diet was provided alone, or supplemented with one of three different levels of an alcohol extract of isolated soy protein contributing either 2.1, 3.6 or 6.2 mg isoflavones/g protein. Gerbils fed all of the soy-based diets had significantly lower (P < 0.05) total cholesterol, LDL + VLDL cholesterol, and apolipoprotein B concentrations than those fed casein. The addition of the alcohol extract to ISP did not reduce serum cholesterol concentrations any further, but reduced hepatic apolipoprotein A-I mRNA levels (P < 0.05) compared with casein- and ISP-fed groups. Levels of apolipoprotein E mRNA were not affected by diet. These data suggest that in gerbils, consumption of an isoflavone-containing extract does not contribute to the hypocholesterolemic effect of alcohol-extracted soy, but may influence lipid metabolism by altering gene expression for lipid-related genes.
KEY WORDS: apolipoproteins · cholesterol · gerbils · isoflavones · soy protein
High concentrations of LDL cholesterol and apolipoprotein B (Apo B)6 in blood pose a risk to human health (Grundy 1995 Soy is a commonly consumed plant protein, and many investigations have been undertaken to elucidate the mechanism by which soy intake reduces LDL cholesterol concentrations. It has been suggested that specific soy components including isoflavones, amino acid content, peptides, saponins, phytic acid, fiber and trypsin inhibitors may contribute to the hypocholesterolemic effect (Potter 1995 Interest in the potential role of isoflavones as an important hypocholesterolemic agent in soy has been raised as a result of their estrogenic potential (Anthony et al. 1996 This study was designed to examine the effect of isoflavones on cholesterol metabolism. Diet components were obtained that allowed the preparation of essentially isoflavone-free and isoflavone-supplemented soy protein diets. These diets were provided to Mongolian gerbils, a rodent model used because of its sensitivity to dietary cholesterol and its LDL-dominant blood lipid profile. Graded doses of isoflavone-containing soy extract were provided to gerbils, blood lipid parameters assessed and their effect on hepatic apolipoprotein gene expression was tested.
Animals and diets.
All animal protocols were approved by the University of Illinois Laboratory Animal Care Advisory Committee. Adult Mongolian gerbils (Meriones unguiculatus; Harlan HSD:MON, Indianapolis, IN) that weighed a mean of 60 g were housed individually in plastic shoebox cages in an environmentally controlled room at 23°C with an alternating 12-h light:dark cycle. Upon arrival, gerbils were fed powdered nonpurified diet rodent chow, (Purina, St. Louis, MO) for 1 wk to acclimate them to our facility and the powdered diets. Gerbils were randomly assigned to one of five dietary treatment groups (n = 8 per group). Diets were similar except for the source of protein and the amount of alcohol extract added (Tables 1 and 2). The protein source was casein or an alcohol-washed, essentially isoflavone-free isolated soy protein Supro 670IF (ISP, Protein Technologies International, St. Louis, MO), or ISP supplemented with one of three levels of an isoflavone-containing alcohol extract of ISP (Protein Technologies International; ISP+, ISP++, ISP+++). The concentration of isoflavones (aglycone-form) was 0.05 mg/g in ISP and 11.4 mg/g in the soy protein extract. This extract contains other organic compounds including saponins, phospholipids and phenolic acids. Compositions of the ISP and extract are detailed in Table 2. The three levels of supplementation were designed to provide 2.1, 3.6 or 6.2 mg total isoflavones/g protein, based on the isoflavone concentration in unextracted soy protein of 2.1 mg/g protein. Thus, the levels of isoflavones provided in these diets represent an amount that could be consumed in an all-soy diet (2.1 mg/g) to three times that amount. These levels of isoflavones are physiological rather than pharmacological. Quantitation of isoflavone concentrations was performed by HPLC by the manufacturer. All gerbils received 5.6 g diet/d for a 28-d period. At the end of 28 d, gerbils were killed by decapitation after anesthetization. Trunk blood was collected without anticoagulant to isolate serum, and liver was obtained for isolation of total RNA.
Cholesterol and apolipoprotein assays.
Northern analysis.
Northern analysis was performed essentially as described (Shay and Cousins 1993 Statistics.
All data were analyzed by one-way ANOVA by using SAS software (SAS version 6, SAS Institute, Cary, NC). A P value < 0.05 was considered significant. After significance was determined by ANOVA, post hoc tests were made using least significant difference (Montgomery 1984 Gerbils consuming the soy-based diets had significantly lower total cholesterol concentrations (Table 3; P < 0.05) than those fed casein. This reduction in cholesterol was largely due to a significant reduction in the LDL + VLDL cholesterol fraction (P < 0.05). Serum Apo A-I and Apo B concentrations reflected the concentrations of the HDL and LDL + VLDL cholesterol fractions, respectively. Serum Apo A-I and the HDL cholesterol concentrations increased in a dose-dependent manner in the ISP, ISP+, and ISP++ groups, but this trend was not followed in the ISP+++ group. The Apo A-I and HDL cholesterol concentrations were significantly higher in the ISP++ group than in gerbils fed casein, ISP or ISP+++ (P < 0.05). Serum concentrations of Apo B and LDL + VLDL cholesterol were reduced (P < 0.05) in all soy-fed groups compared with the casein-fed group. Concentrations of Apo B were lowest (P < 0.05) in gerbils fed the ISP+++ diet. These blood lipid responses were replicated in a second study that used a similar number of gerbils fed the same five diets (data not shown).
All gerbils fed soy protein had reduced total serum and LDL cholesterol concentrations compared with those fed a casein-based diet. The addition of isoflavone-containing extract to alcohol-extracted soy protein did not further reduce levels. These results are consistent with Balmir et al. (1996) The authors wish to thank John Erdman for helpful discussion pertaining to this project, and Protein Technologies International for generously providing protein and extract for these studies.
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
), including the risk of coronary heart disease, the leading cause of death in the United States (Grundy 1994). In 1981, Levy suggested that high levels of HDL cholesterol and apolipoprotein A-I (Apo A-I) are associated with a reduced incidence of coronary heart disease (Levy 1981). Studies in both animal models and humans have shown that cholesterol levels may decrease when plant protein is substituted for animal protein (Anderson et al. 1995, Carroll 1991, Erdman and Fordyce 1989).
). Nevertheless, the exact mechanisms by which soy intake decreases cholesterol levels in blood are still unknown.
, Balmir et al. 1996, Potter 1995). Isoflavones have structural similarities with estrogen, which has hypocholesterolemic properties (Bierman and Glomset 1992, Stampfer et al. 1985). Estrogen may affect the metabolism of apolipoproteins, which are constituents of HDL and LDL (Deeley et al. 1985). The most abundant isoflavones in soybeans are genistein and daidzein (Naim et al. 1973); however, emphasis has been focused on genestein as a nonprotein factor that may alter cholesterol blood lipid profiles (Akiyama, et al. 1987, Linassier et al. 1990, Okura et al. 1988). Debate exists regarding the effect of isoflavones on cholesterol concentrations. Although the meta-analysis of Anderson et al. (1995) suggested that up to 60% of the hypocholesterolemic effect of soy observed in humans may be attributed to isoflavones, others have questioned this value, demonstrating hypocholesterolemic effects when low isoflavone soy products were given to humans (Sirtori et al. 1997).
MATERIALS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
View this table:
Table 1.
Basal diet composition
View this table:
Table 2.
Protein source and extract composition
(HDL) and 
(LDL + VLDL) lipoprotein concentrations were separated using heparin-bound agarose columns (LDL Direct, Isolabs, Akron, OH). Total, HDL, and LDL + VLDL cholesterol concentrations were measured enzymatically following the method of Allain et al. (1974) by using a commercially available cholesterol reagent (Sigma Diagnostics, St. Louis, MO). Serum Apo A-I and Apo B were measured by a turbidimetric immunoassay as described by Rifai and King (1986) by using commercially available Apo A-1 and Apo B reagent kits (RAI, San Diego, CA).
). Liver RNA was isolated by using an Ultraspec-II kit (Biotecx Laboratories, Houston, TX) following the manufacturer's suggested protocol, and 15 µg of each RNA sample was size-separated by MOPS/formaldehyde electrophoresis and capillary blotted to Hybond-N+ nylon membranes (Amersham, Arlington Heights, IL). Blots were hybridized to [32-P]-dCTP-labeled cDNAs corresponding to the Apo A-I and Apo E mRNAs and the 28S rRNA. Ethidium bromide staining of gels verified equivalent loading of ±20% between samples. After washing of blots, hybridization was visualized first by exposure to Kodak X-AR film (Eastman Kodak, Rochester, NY), and then to a phoshorimage screen (Molecular Dynamics, Sunnyvale, CA). Exposed phosphorimaging screens were then quantitated by phosphorimager, with normalization of Apo A-I and Apo E values to the 28S rRNA.
).
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
View this table:
Table 3.
Serum cholesterol and apolipoprotein A-I and B concentrations in gerbils fed casein- or isolated soy protein-based diets with or without added soy protein extract1
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Fig 1.
Northern analysis of hepatic apolipoprotein A-I (Apo A-I) mRNA/28S rRNA concentrations in gerbils fed casein- and isolated soy protein-based diets with or without genestein-containing soy protein extract. Top: representative phosphorimage of hybridized blot illustrating Apo A-I mRNA and 28S rRNA concentrations from the five treatment groups. Legend: 1, casein-fed; 2, isolated soy protein-fed; 3, ISP + 1.2 mg genestein/g protein; 4, ISP + 2.0 mg genestein/g protein; 5, ISP + 3.5 mg genestein/g protein. Bottom: Relative ratios of Apo A-I/28S RNAs from the five treatment groups. The mean value of the CAS group was set at 1.0. All other mean values were normalized to the CAS group. Values (n = 8 per group) represent means ± SEM. Values without common superscripts are significantly different at P < 0.05 as determined by ANOVA. Legend: CAS, casein-fed; ISP, isolated soy protein-fed; ISP +, ISP + 1.2 mg genestein/g protein; ISP++, ISP + 2.0 mg genestein/g protein; ISP+++, ISP + 3.5 mg genestein/g protein.
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Fig 2.
Northern analysis of hepatic apolipoprotein E (Apo E) mRNA/28S rRNA concentrations in gerbils fed casein- and isolated soy protein-based diets with or without genestein-containing soy protein extract. Top: representative phosphorimage of hybridized blot illustrating Apo E mRNA and 28S rRNA concentrations from the five treatment groups. Legend: 1, casein-fed; 2, isolated soy protein-fed; 3, ISP + 1.2 mg genestein/g protein; 4, ISP + 2.0 mg genestein/g protein; 5, ISP + 3.5 mg genestein/g protein. Bottom: Relative ratios of Apo E/28S RNAs from the five treatment groups. The mean value of the CAS group was set at 1.0. All other mean values were normalized to the CAS group. Values (n = 8 per group) represent averages ± SEM. No differences at P < 0.05 were determined by ANOVA. Legend: CAS, casein-fed; ISP, isolated soy protein-fed; ISP+, ISP + 1.2 mg genestein/g protein; ISP++, ISP + 2.0 mg genestein/g protein; ISP+++, ISP + 3.5 mg genestein/g protein.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
who reported no differences in blood lipid variables in rats and hamsters fed high or low isoflavone-containing soy protein diets. However, our results are different than those of Anthony et al. (1996) who reported differences in blood lipid variables in monkeys fed high and low phytoestrogen-containing soy diets. It is likely that species differences played a large role in these differences. It is also important to recognize that Anthony et al. (1996) utilized unextracted soy protein source containing phytoestrogens in addition to alcohol-extracted protein. Further, their diets were supplemented with methionine. All of the soy protein utilized in our study was alcohol-extracted, there was no supplemental methionine, and an isoflavone-containing alcohol extract was added at three different concentrations. To account for the differences in the blood lipid variables reported in this study and the study of Anthony et al. (1996), we would suggest that an alcohol-soluble component in soy, able to mediate an effect on lipid parameters, may have been affected by the extraction process. The reduction in serum lipid variables that this component produces may be in addition to the reduction in total cholesterol provided by alcohol-extracted soy protein. It may also be that the hypolipidemic effect of the alcohol extract of soy may be more readily observed in high methionine diets.
-cholesterol and apolipoprotein A-I concentrations in the ISP, ISP+, and ISP++ groups, but this trend was not observed in the ISP+++ group, suggesting a plateau or maximum may have been reached at the concentration of extract provided in the ISP++ diet. In contrast, no dose-dependent relationship was observed in Apo A-I mRNA levels. In fact, relative concentrations for the Apo A-I mRNA were generally lower in the groups provided alcohol extract. This apparent discrepancy between circulating Apo A-I concentrations and hepatic gene expression may be due to differences in turnover rates of circulating lipoprotein particles or increased delivery of intestinally derived Apo A-I to the serum. The fact that hepatic Apo A-I mRNA concentrations were reduced in gerbils provided an isoflavone-containing alcohol extract demonstrates that this component has the ability to regulate gene expression in vivo and perhaps alter lipid metabolism in ways that are not directly reflected in the concentration or distribution of the circulating lipoproteins. These effects may be specific to certain genes because differences were observed for Apo A-I mRNA concentrations, whereas no differences were observed for Apo E mRNA, another hepatic lipoprotein-encoding mRNA of similar relative abundance to Apo A-I. If it is the isoflavones that are directing changes in gene expression, this may be occurring by enhancement or antagonism of estrogen-regulated mechanisms.
reported that soybean, potato and rice proteins all produced a hypocholesterolemic effect in rats. They reported that dietary methionine and methionine/glycine ratios were the best predictors of hypocholesterolemia among all variables tested. Soy protein is characterized by its low methionine content, and this factor is likely a strong contributor to the cholesterol-reducing effect of soy.
ACKNOWLEDGMENTS
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FOOTNOTES |
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Manuscript received 10 September 1997. Initial reviews completed 11 November 1997. Revision accepted 14 January 1998.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results
Discussion
References
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