Feb 20, 1998 - Human Nutrition and Metabolism ... The major dietary isoflavonoids, genistein and daidzein, have estrogen-like activity and are classed ..... Anthony, M. S., Clarkson, T. B., Hughes, C. L. Jr., Morgan, T. M. & Burke, G. L..
Human Nutrition and Metabolism
Supplementation with Isoflavonoid Phytoestrogens Does Not Alter Serum Lipid Concentrations: A Randomized Controlled Trial in Humans1,2 Jonathan M. Hodgson,3,4 Ian B. Puddey, Lawrence J. Beilin, Trevor A. Mori and Kevin D. Croft University of Western Australia Department of Medicine and the Western Australian Heart Research Institute, Royal Perth Hospital, Perth, WA 6001 Australia ABSTRACT Isoflavonoids are a class of flavonoids that are derived in the human diet mainly from soybeanbased foods. The major dietary isoflavonoids, genistein and daidzein, have estrogen-like activity and are classed as phytoestrogens. Because estrogens can lower serum LDL cholesterol and raise HDL cholesterol, the objective of this study was to determine if isoflavonoids could improve serum lipids in healthy subjects. Forty-six men and 13 postmenopausal women not receiving hormone replacement therapy completed a randomized, double-blind, placebo-controlled trial of two-way parallel design and 8 wk duration. One tablet containing 55 mg of isoflavonoids (predominantly in the form of genistein) or one placebo tablet was taken daily with the evening meal. Subjects maintained their usual diet and physical activity, which were unchanged throughout the intervention. Measurement of isoflavonoids and their metabolites in 24-h urine samples provided an assessment of compliance and of isoflavonoid metabolism. Serum total, LDL, HDL and HDL subclass cholesterol, triglycerides and lipoprotein (a) were assessed at baseline and during the last week of intervention. After adjustment for baseline values, no significant differences in postintervention serum lipid and lipoprotein (a) concentrations between groups were identified. Further adjustment for age, gender and weight change did not alter the results. In addition, changes in urinary isoflavonoids were not significantly correlated with changes in serum lipids and lipoprotein (a). Therefore, this study does not support the hypothesis that isoflavonoid phytoestrogens can improve the serum lipids, at least in subjects with average serum cholesterol concentrations. J. Nutr. 128: 728–732, 1998. KEY WORDS: • isoflavonoids • phytoestrogens • genistein • cholesterol • lipoproteins • humans
Isoflavonoids are a class of flavonoids that are constituents of particular legumes. In the human diet, isoflavonoids are derived mainly from soybeans and soybean-based foods, but they can also be derived from other legumes. The major dietary isoflavonoids are the glycosides of genistein and daidzein. Genistein and daidzein are phytoestrogens that have been shown to have estrogenic or anti-estrogenic activity in vitro (Setchell and Adlercreutz 1988). These compounds may also have estrogenic activity in vivo (Cassidy et al. 1994, Lu et al. 1996). Postmenopausal estrogen replacement has been shown to decrease total and LDL cholesterol (Newnham 1993, Tikkanen 1996) and lipoprotein (a) [Lp(a)] (Kim et al. 1994, Shewmon et al. 1994), increase HDL cholesterol (Muesing et al. 1992) and alter HDL subfraction composition (Muesing et al. 1992, Walsh et al. 1994). The synthetic anti-estrogen, tamoxifen, has also been shown to beneficially alter serum lipid and Lp(a) concentrations (Love et al. 1991, Shewmon et al. 1994).
In addition, there may be similar beneficial effects of estrogen on LDL and HDL cholesterol concentrations in men (Berglund et al. 1996, Eriksson et al. 1989, Ohshige et al. 1996). Beneficial effects of estrogen on serum lipids have generally been shown in largely normolipidemic subjects (Eriksson et al. 1989, Kim et al. 1994, Love et al. 1991, Muesing et al. 1992, Ohshige et al. 1996, Shewmon et al. 1994), at least by Western standards. However, on average, Westernized populations have raised total cholesterol concentrations and increased risk of cardiovascular disease (Keys 1970). Decreases in serum cholesterol concentrations, even within subjects with average total cholesterol concentrations (Ç5.5 mmol/L), can reduce cardiovascular disease risk (Sacks et al. 1996). Recently there has been interest in determining whether isoflavonoids alter serum lipids (Anderson et al. 1995, Anthony et al. 1996). Hypocholesterolemic effects of soy proteins have been demonstrated in experimental animals and human subjects (Carroll 1991, Potter 1995), and it has been suggested that isoflavonoids could be partly responsible (Anderson et al. 1995). In rhesus monkeys, soy protein–containing isoflavonoids significantly lowered plasma LDL and VLDL cholesterol, and raised HDL cholesterol in comparison with soy protein with isoflavonoids removed (Anthony et al. 1996). However, soy proteins utilized in human studies have often had negligible isoflavonoid content (Sirtori et al. 1997). The effects of isofla-
1 Supported by grants from Novogen Limited and the MSD Research Foundation, and by a Program grant from the National Health and Medical Research Council. 2 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 USC section 1734 solely to indicate this fact. 3 J.M.H. was supported by an Athelstan and Amy Saw Medical Research Fellowship from the University of Western Australia. 4 To whom correspondence should be addressed.
0022-3166/98 $3.00 q 1998 American Society for Nutritional Sciences. Manuscript received 27 August 1997. Initial review completed 19 November 1997. Revision accepted 23 December 1997. 728
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vonoid supplementation on serum lipids in humans therefore remain unclear. In this study, healthy men and postmenopausal women with serum lipid concentrations approximating the mean of the general population were recruited. The objective was to determine if dietary supplementation with purified isoflavonoids, at an intake level achievable by dietary means, could improve serum lipids in this group.
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of the food records. Food intake data were analysed using Xyris, a nutrient analysis program based on NUTTAB 1993 food composition tables (Xyris, Brisbane, Australia). Body weight was measured at baseline, at the end of intervention and at 2-wk intervals during the supplementation period. Biochemical analyses. During the last week of baseline and the final week of intervention, blood was taken from each subject after an overnight fast. Serum lipids and other biochemical measurements were performed in the Department of Clinical Biochemistry at Royal Perth Hospital. Total cholesterol and triglycerides were analyzed enzymatically on a Roche Diagnostics Cobas Mira analyzer (Basel, Switzerland) with the use of reagents from Trace Scientific (Perth, Australia). HDL cholesterol was analyzed on a heparin-manganese chloride supernatant, HDL3 cholesterol was measured by a single precipitation procedure (Gidez et al. 1982) and HDL2 cholesterol was calculated by difference (total HDL cholesterol 0 HDL3 cholesterol). The Friedewald formula (Friedewald et al. 1972), converted to SI units, was used to calculate LDL cholesterol. Lipoprotein (a) was measured in serum by using a latex-enhanced nephelometric method. Measurements were performed on the Behring Nephelometer (Behring Werke, Marburg, Germany) by using the Behring N-Latex Lp(a) method (Behring Diagnostics, Sydney, Australia). DAKO rabbit antihuman antibodies (DAKO Corporation, Carpitera, CA) were used instead of Behring antibodies. A 24-h urine collection was performed at baseline and during the final week of intervention. Urinary isoflavonoids, including biochanin A, genistein, formononetin, daidzein, and the daidzein metabolites equol and O-desmethylangolensin (O-DMA) were measured by using HPLC according to the method of Franke et al. (1995). Statistics. Statistical analyses were performed using SPSS software (Chicago IL). The independent-samples t test was used to compare means of baseline values. Levene’s test for equality of variances was used, and if variances were unequal, the independent-samples t test, in which variances are not assumed to be equal, was used to compare means of baseline values. Results are presented as means { SEM. Pearson’s correlation coefficient (r) was used to determine the degree and direction of association between two variables. ANOVA was used to compare postintervention differences between active and placebo groups after adjustment for baseline values. Adjustment for co-variates such as age, gender and weight change was also performed with the use of this technique.
SUBJECTS AND METHODS Subjects. Fifty-nine healthy subjects, 46 men and 13 postmenopausal women not receiving hormone replacement therapy, aged between 35 and 69 y, were recruited from the general population. There were 22 men and 7 women in the placebo group (mean age 57.0 y), and 24 men and 6 women in the isoflavonoid group (mean age 54.3 y). Subjects excluded from the study were current smokers or exsmokers who had stopped smoking for less than 6 mo; those whose usual alcohol intake exceeded four standard drinks per day (ú40 g alcohol/d); diabetics on medication; those with any history of heart, liver or renal disease; those with systolic blood pressure õ125 mm Hg; those taking medication for dyslipidemia or hypertension or any medication that might influence serum lipids concentrations or blood pressure; and vegetarians or individuals whose usual diet included more than one soy-containing meal per week. The project was approved by the Royal Perth Hospital Ethics Committee, and all participants gave informed written consent. Experimental design. The study was a randomized, double-blind, placebo-controlled trial of two-way parallel design and 8 wk duration. Baseline assessments were performed in the last 2 wk of a 4-wk runin phase; randomization performed by a third party observer occurred at the end of run-in phase. Subjects were assigned to take either one tablet containing 55 mg of isoflavonoids or one placebo tablet daily with their evening meal, throughout the intervention. The isoflavonoid tablets provided by Novogen (North Ryde, Sydney, Australia) were derived from subterranean clover (Trifolium subterraneum) and contained the following (per tablet in mg): 16 biochanin A, 30 genistein, 8 formononetin and 1 daidzein. Compliance was assessed by tablet counts at 2-wk intervals and by measurement of isoflavonoids in 24-h urine samples at baseline and postintervention. Food intake and body weight assessment. Usual food intake, alcohol consumption and physical activity were assessed by questionnaire at baseline and monitored by a dietitian throughout the intervention to ensure minimal changes of these potential confounders throughout the study. Food intake was assessed by 24-h diet records completed on two week days and one day of the weekend. Instruction was given on the use of the Salter digital food scales (Tonbridge, England) and the recording of all foods and beverages consumed over a 24-h period on a standardized food record form to ensure accuracy
RESULTS Weight and dietary intake. There was no significant difference between placebo and isoflavonoid groups in mean weight at baseline (84.1 { 2.2 vs. 82.9 { 2.5 kg, respectively), and no significant difference between groups in postintervention weight after adjustment for baseline values. In addition, there
TABLE 1 Baseline and postintervention daily energy and macronutrient intakes for subjects in the placebo and isoflavonoid groups1 Placebo Characteristic Energy, kJ Protein, g Total fat, g Saturated, g Monounsaturated, g Polyunsaturated, g Cholesterol, mg Carbohydrate, g Sugar, g Starch, g Fiber, g Alcohol, g
Isoflavonoid
Baseline 9640 102 80 32 29 13 342 267 117 147 28 17
{ { { { { { { { { { { {
433 4 4 2 2 1 37 14 6 10 2 4
Post 9365 99 76 31 27 12 284 259 114 143 30 19
{ 523 { 5 { 6 { 3 { 2 { 1 { 24 { 15 { 8 { 9 { 2 { 5
Baseline 9405 98 79 31 28 13 274 264 112 148 28 14
{ 473 { 5 { 5 { 3 { 2 { 1 { 25 { 15 { 9 { 9 { 2 { 4
1 Values are means { SEM. For placebo group, n Å 29; for isoflavonoid group, n Å 30.
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Post 9278 95 85 33 32 13 289 253 104 146 26 10
{ 504 { 5 { 6 { 3 { 2 { 1 { 28 { 14 { 9 { 9 { 2 { 3
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TABLE 2 Effects of an 8-wk intervention, involving dietary supplementation with isoflavonoids (55 mg/d) or placebo, on urinary isoflavonoid excretion1,2 Placebo
Isoflavonoid
Baseline
Post
Baseline
Post
mg/d Biochanin A Genistein Formononetin Daidzein Equol O-DMA4 Total
0.01 0.13 0.01 0.14 0.00 0.00 0.28
{ { { { { { {
0.01 0.14 0.01 0.063 0.00 0.00 0.085
0.02 0.19 0.05 0.43 0.00 0.04 0.74
{ { { { { { {
0.02 0.09 0.03 0.16 0.00 0.04 0.26
0.00 0.41 0.00 0.79 0.36 0.12 1.67
{ { { { { { {
0.00 0.14 0.00 0.183 0.36 0.09 0.605
0.14 5.63 0.20 3.32 0.32 0.72 10.33
{ { { { { { {
0.05 0.77 0.06 0.47 0.32 0.21 1.33
1 Values are means { SEM. For placebo group, n Å 29; for isoflavonoid group, n Å 30. 2 Baseline and postintervention 24-h urinary excretion of isoflavonoids and metabolites in subjects in the placebo and isoflavonoid groups are
presented. 3 P õ 0.05 for difference between placebo and isoflavonoid groups in baseline daidzein excretion. 4 O-desmethylangolensin. 5 P õ 0.05 for difference between placebo and isoflavonoid groups in baseline total isoflavonoid excretion.
were no differences in total energy or macronutrient intakes between placebo and isoflavonoid groups at baseline and no differences between groups in the postintervention daily energy and nutrient intake after adjustment for baseline values. The baseline and postintervention energy and macronutrient intakes for the two groups are presented in Table 1. Isoflavonoids. Unadjusted baseline and postintervention 24-h excretion of isoflavonoids and metabolites is presented in Table 2. After adjustment for baseline values, there were significantly greater urinary concentrations of biochanin A (P õ 0.05), genistein (P õ 0.0001), formononetin (P õ 0.05), daidzein (P õ 0.0001) and O-DMA (P õ 0.01) in the isoflavonoid group compared with the placebo group. The magnitude of the difference was larger for genistein and daidzein than for other compounds. Lipids and lipoproteins. The unadjusted baseline and postintervention lipid and Lp(a) concentrations are presented in Table 3. After adjustment for baseline values, there were no significant differences between placebo and isoflavonoid groups in postintervention lipid and Lp(a) concentrations. Further adjustment for age, weight change and change in nutrient intakes did not alter the outcome. Analyses of baseline adjusted postintervention differences in serum lipids and Lp(a) performed in subjects with baseline serum total cholesterol concentrations above the 50th percentile (5.3 mmol/L) also showed no differences between groups. Correlations between changes in urinary isoflavonoids or metabolites and serum lipid and Lp(a) concentrations within the isoflavonoid group did not show any significant associations. DISCUSSION Dietary supplementation with 55 mg/d of isoflavonoids did not significantly alter serum lipid and Lp(a) concentrations. An estrogen-like effect of isoflavonoids was the primary proposed mechanism for lipid lowering. Therefore subjects with low circulating estrogen concentrations, men and postmenopausal women not receiving hormone replacement therapy, were recruited. An isoflavonoid intake of 55 mg/d was proposed to have effects on serum lipid concentrations similar to those of estrogen. Concentrations of estrogen that have been shown to im-
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prove serum lipids have been equivalent to about 1.5–2 mg/d of estradiol (Tikkanen 1996). Genistein and daidzein have estrogenic activity (Setchell and Adlercreutz 1988), which may be of the order of 1000–10,000 times less than that of estradiol (Messina et al. 1994). However, in populations with high intakes of isoflavonoids, such as the Japanese, plasma concentrations of isoflavonoid phytoestrogens (Adlercreutz et al. 1993) may be around 1000–10,000 times that of circulating concentrations of estradiol 17-b (Genuth 1986) in men and postmenopausal women. An isoflavonoid intake of 55 mg/d is also representative of an upper level of intake achievable by dietary means. The Japanese population, who consume large amounts of soy products, are estimated to have a mean isoflavonoid intake of Ç30 mg/d (Messina 1995), including about 20 mg/d genistein. A greater part of the isoflavonoid content of the active supplement (46 mg) was genistein and biochanin A (which is converted directly to genistein). Our conclusions must therefore be limited to possible effects of genistein. When ingested by humans, isoflavonoids undergo acidic and enzymatic hydrolysis and demethylation to yield the aglycones, genistein and daidzein. Biochanin A is converted to genistein and formononetin is converted to daidzein. Genistein and daidzein may then be further metabolized by gut flora (Setchell and Adlercreutz 1988). The metabolic products of genistein metabolism in humans have not been clearly demonstrated. The two main products of daidzein metabolism appear to be equol and O-DMA (Kelly et al. 1993). The isoflavonoids and their metabolites are unlikely to be further metabolized once they have been absorbed. A more detailed discussion of isoflavonoid metabolism is given by Joannou et al. (1995) and Kurzer and Xu (1997). Greater urinary excretions of genistein and daidzein were observed in the isoflavonoid group: 5.22 and 2.53 mg/d, respectively. However, these increases were considerably less than the increase in the intake of biochanin A plus genistein (46 mg/d) and formononetin plus daidzein (9 mg/d). The subjects taking the active tablet therefore excreted 11% of the daily increase in genistein plus biochanin A, and 28% of the daily increase in daidzein plus formononetin. These results are similar to those of Xu et al. (1994) who found that the bioavailability for genistein was 9%, and for daidzein was 21%.
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TABLE 3 Effects of an 8-wk intervention, involving dietary supplementation with isoflavonoids (55 mg/d) or placebo, on lipid and lipoprotein (a) concentrations1,2 Placebo
Isoflavonoid
Baseline
Post
Baseline
Post
mmol/L Total cholesterol LDL cholesterol HDL cholesterol HDL2 cholesterol HDL3 cholesterol Triglycerides
5.07 3.24 1.21 0.53 0.68 1.26
{ { { { { {
0.15 0.12 0.053 0.04 0.03 0.114
5.07 3.34 1.20 0.46 0.74 1.18
{ { { { { {
0.15 0.13 0.05 0.03 0.03 0.10
5.41 3.52 1.07 0.47 0.60 1.84
{ { { { { {
0.23 0.18 0.043 0.03 0.03 0.214
5.35 3.51 1.05 0.39 0.66 1.82
{ { { { { {
0.23 0.20 0.04 0.03 0.03 0.21
mg/L Lp(a)5
163 { 46
163 { 44
280 { 45
264 { 44
1 Values are means { SEM. For placebo group, n Å 29; for isoflavonoid group, n Å 30. 2 Baseline and postintervention values are shown for subjects in the placebo and isoflavonoid groups. 3 P õ 0.05 for difference between placebo and isoflavonoid groups in baseline HDL cholesterol concentrations. 4 P õ 0.05 for difference between placebo and isoflavonoid groups in baseline triglyceride concentrations. 5 Lipoprotein (a).
There was considerable variability in urinary isoflavonoid concentrations in the isoflavonoid group. This result suggests that there was variability in isoflavonoid metabolism and absorption between individuals, which is consistent with previous findings (Morton et al. 1994). However, we found no significant correlations between changes in urinary isoflavonoids and serum lipids and Lp(a) concentrations in the isoflavonoid group. Although we found no effect of isoflavonoids on serum lipids in a healthy population, the results do not eliminate the possibility of a hypolipidemic effect of isoflavonoids in hypercholesterolemic subjects. Studies that have shown decreases in serum LDL cholesterol with soy protein in humans have generally involved hypercholesterolemic subjects (Descovich et al. 1980, Gaddi et al. 1987 and 1991, Verrillo et al. 1985). In a meta-analysis of human trials with a soy protein intervention focusing on lipid effects, the factor most strongly associated with decreases in LDL cholesterol concentrations was the initial cholesterol concentration of the study population (Anderson et al. 1995). In our study, analyses of postintervention lipid differences in those subjects with higher (ú5.3 mmol/L) baseline serum total cholesterol concentrations did not demonstrate any significant differences between groups. However, it remains possible that if isoflavonoids have a cholesterol-lowering effect, little or no activity may be seen in normocholesterolemic subjects. In conclusion, supplementation with Ç55 mg of isoflavonoids per day does not significantly alter serum lipid or Lp(a) concentrations in healthy middle-aged subjects with serum lipid concentrations approximating the mean of the general population. Because a large percentage of the supplement used was in the form of genistein, the results indicate that at least this isoflavonoid does not influence serum lipids and Lp(a) significantly. These results do not support the hypothesis that isoflavonoid phytoestrogens lower LDL cholesterol and Lp(a) and raise HDL cholesterol in subjects with average serum lipid concentrations. ACKNOWLEDGMENTS The authors thank Nardia Langley for assistance with monitoring of diets, Penny Rogers and Mary-Ann Powell for nursing help, Lyn
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McCahon, Tina Bermingham and Natasha Morton for technical assistance and Valerie Burke for her help with statistical analyses.
LITERATURE CITED Adlercreutz, H., Markkanen, H. & Watanabe, S. (1993) Plasma concentrations of phyto-oestrogens in Japanese men. Lancet 342: 1209–1210. Anderson, J. W., Johnstone, B. M. & Cook-Newell, M. E. (1995) Meta-analysis of the effects of soy protein intake on serum lipids. N. Engl. J. Med. 333: 276–282. Anthony, M. S., Clarkson, T. B., Hughes, C. L. Jr., Morgan, T. M. & Burke, G. L. (1996) Soybean isoflavones improve cardiovascular risk factors without affecting the reproductive system of peripubertal rhesus monkeys. J. Nutr. 126: 43–50. Berglund, L., Carlstrom, K., Stege, R., Gottleib, C., Eriksson, M., Angelin, B. & Henriksson, P. (1996) Hormonal regulation of lipoprotein (a) levels: effects of parenteral administration of estrogen or testosterone in males. J. Clin. Endocrinol. Metab. 81: 2622–2627. Carroll, K. K. (1991) Review of clinical studies on cholesterol-lowering response to soy protein. J. Am. Diet. Assoc. 91: 820–827. Cassidy, A., Bingham, S. & Setchell, K.D.R. (1994) Biological effects of a diet of soy protein rich in isoflavones on the menstrual cycle of premenopausal women. Am. J. Clin. Nutr. 60: 333–340. Descovich, G. C., Ceredi, C., Gaddi, A., Benassi, M. S., Mannino, G., Colombo, L., Cattin, L., Fontana, G., Senin, U., Mannarino, E., Caruzzo, C., Bertelli, E., Fragiacomo, C., Norseda, G., Sirtori, M. & Sirtori, C. R. (1980) Multicentre study of soybean protein diet for outpatient hypercholesterolaemic patients. Lancet II: 709–712. Eriksson, M., Berglund, L., Rudling, M., Henriksson, P. & Angelin B. (1989) Effects of estrogen on low density lipoprotein metabolism in males: short-term and long-term studies during hormonal treatment of prostatic carcinoma. J. Clin. Invest. 84: 802–810. Franke, A. A., Custer, L. J., Cerna, C. M. & Narla, K. (1995) Rapid HPLC analysis of dietary phytoestrogens from legumes and from human urine. Proc. Soc. Exp. Biol. Med. 208: 18–26. Friedewald, W. T., Levy, R. I. & Fredrickson, D. S. (1972) Estimation of the concentration of low density lipoprotein in plasma without use of the preparative ultracentrifuge. Clin. Chem. 18: 499–502. Gaddi, A., Ciarrocchi, A., Matteuci, A., Rimondi, S., Ravaglia, G., Descovich, G. C. & Sirtori, C. R. (1991) Dietary treatment for familial hypercholesterolemia—differential effects of dietary soy protein according to the apolipoprotein E phenotypes. Am. J. Clin. Nutr. 53: 1191–1196. Gaddi, A., Descovich, G. C., Noseda, G., Fragiacomo, C., Nicolini, A., Montanari, G., Vanetti, G., Sirtori, M.,Gatti, E. & Sirtori, C. R. (1987) Hypercholesterolaemia treated by soybean protein diet. Arch. Dis. Child. 62: 274–278. Genuth, S. M. (1986) The reproductive glands. In: Physiology (Berne, R. M., ed.), pp. 960–971. Mosby, Washington, DC. Gidez, L. I., Miller, G. J., Burstein, M., Slagle, S. & Eder, H. A. (1982) Separation and quantitation of subclasses of human plasma high density lipoproteins by a single precipitation procedure. J. Lipid Res. 23: 1206–1223. Huff, M. W., Hamilton, R.M.G. & Carroll, K. K. (1977) Plasma cholesterol levels in rabbits fed low fat, cholesterol-free, semi-purified diets: effects of dietary
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