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Effects of Isolated Isoflavonoids on Lipids, Lipoproteins, Insulin Sensitivity, and Ghrelin in Postmenopausal Women

Departments of Obstetrics and Gynecology (E.N., A.T., K.L., O.Y.) and Medicine (M.T.), Helsinki University Central Hospital, FIN-00029 HUS Helsinki, Finland; and Department of Obstetrics and Gynecology (E.N.), Jorvi Hospital, FIN-02740 Espoo, Finland

Address all correspondence and requests for reprints to: Dr. Eini Nikander, Department of Obstetrics and Gynecology, Helsinki University Central Hospital, P.O. Box 140, FIN-00029 HUS Helsinki, Finland. E-mail: eini.nikander{at}pp.fimnet.fi.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The low cardiovascular risk in Asian women has been thought to result from high isoflavonoid intake. In a double-blind, randomized, placebo-controlled trial, we studied the effects of isolated isoflavonoids (114 mg/d) on lipids, lipoproteins, insulin sensitivity, and ghrelin in 56 nondiabetic postmenopausal women with a history of breast cancer. Isoflavonoid or placebo tablets were given for 3 months, and the treatment regimens crossed over after a 2-month washout period.

The concentrations of total cholesterol, high- and low-density lipoprotein cholesterol, triglycerides, apolipoproteins B and A1, and lipoprotein (a) were not affected by isoflavonoids. However, during the isoflavonoid regimen, women with low-density lipoprotein cholesterol level above the median (4.20 mmol/liter) showed a rise [0.65 ± 0.60 (SD) mmol/liter], which was statistically different from the fall during the placebo regimen (–0.45 ± 0.67 mmol/liter, P = 0.009).

Isoflavonoids did not affect insulin sensitivity as assessed by an oral 2-h glucose tolerance test (75 g). Changes in ghrelin levels differed (P = 0.048) during the isoflavonoid (–7.1 ± 151 µmol/liter) and placebo regimens (+47.9 ± 198 µmol/liter).

In conclusion, we found no effects of isolated isoflavonoids on lipids, lipoproteins, or insulin sensitivity in postmenopausal women, implying no vascular benefit. Isoflavonoids may reduce ghrelin levels and thus hunger and weight.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
HIGH INTAKE OF dietary phytoestrogens has been suggested to account for the lower rates of cardiovascular diseases (CVDs) in Asia than in Western countries (1, 2). Isoflavonoids are phytoestrogens present in soybeans, concomitantly with soy protein, and they structurally and functionally resemble estradiol (3). Indeed, isoflavones such as genistein and daidzein bind weakly to estrogen receptor- and more strongly to estrogen receptor-ß, and they may possess organ-specific estrogenic and antiestrogenic effects (4, 5).

Because estrogen, alone or in combination with different progestins [hormone therapy (HT)], exerts beneficial effects on circulating lipid and lipoprotein levels (6), it is possible that phytoestrogens may also have these effects. Indeed, according to one metaanalysis, the intake of soy protein (mean of 47 g/d) with isoflavonoids was associated with reductions in total cholesterol (9.3%), low-density lipoprotein (LDL) cholesterol (12.9%), and triglycerides (10.5%), whereas the levels of high-density lipoprotein (HDL) cholesterol showed no significant change (7). The changes in total cholesterol and LDL cholesterol were directly related to the initial serum cholesterol concentrations (7). The benefits of soy protein on the serum lipid profile may appear to resemble those of HT (6), but unfortunately the data are far from being uniform (8, 9, 10, 11, 12). The discrepancies may be derived in part from differences in study populations or phytoestrogen regimens used in various trials (8, 9, 10, 11, 12).

Besides lipids, phytoestrogens could also affect insulin sensitivity, which, when reduced, is an independent risk factor for CVDs (13). Insulin sensitivity is largely genetically determined (14), but, additionally, it is regulated by a number of steroid and peptide hormones (15). A fall in insulin sensitivity is accompanied by estrogen deficiency after natural menopause (16). The recently detected hormone ghrelin is synthesized in the stomach (17, 18) in response to insulin-induced hypoglycemia (19). After reaching the brain through circulating plasma, ghrelin stimulates hunger sensation and increases appetite (20). There are some data to suggest that isoflavones together with soy protein improve insulin sensitivity in ovariectomized cynomolgus monkeys (21) and also in postmenopausal women with type 2 diabetes (22) but not in healthy postmenopausal women (23). No data exist on the concomitant effects of phytoestrogens on lipids, insulin sensitivity, and ghrelin in nondiabetic postmenopausal women. Therefore, we studied the effects of isolated isoflavonoids on lipids, lipoproteins, insulin sensitivity, and ghrelin in nondiabetic postmenopausal women with a history of breast cancer.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Subjects and study design

With permission of the local ethics committee, we studied 64 postmenopausal women who had been treated for breast cancer more than 6 months earlier (Table 1). These volunteers received written and verbal information on the purpose and procedures of the study, and informed consent was obtained from all of them. Each woman was devoid of any metastasis at recruitment, which took place between September 1, 1999 and October 10, 2000. All women had incapacitating hot flashes and other climacteric symptoms; at least 6 months had elapsed after their last menstruation; and menopausal status was confirmed by a serum level of FSH exceeding 30 U/liter. The women were not using HT, tamoxifen, statins, natural products with presumed estrogenic activity, drugs possibly affecting climacteric symptoms, or substances affecting the metabolism or absorption of phytoestrogens (e.g. antibiotics during the previous 3 months). None had a history of a thromboembolic or hepatic event. Before the diagnosis of breast cancer, 25 (39.1%) of the 64 women had used some form of HT. Eight women took antihypertensive drugs (Table 1).

Following a double-blind crossover technique, the women were treated in computer-randomized order with either isoflavonoids or a similar-looking placebo. Randomization was carried out in blocks of 20 subjects. Each treatment lasted 3 months, and the treatment phases were interrupted by a 2-month washout period. Isoflavonoid tablets and similar-looking placebo tablets were to be taken every 12 h (three tablets) with a glass of water. Isoflavonoid tablets (Bonette, Novomed, Helsinki, Finland, 19 mg of isoflavonoids) consisted of glycitein (11 mg, 58%), daidzein (7 mg, 36%) and genistein (1 mg, 6%) (24).

The women visited the research center immediately before and on the last day of each treatment period. General and pelvic examinations were performed and appropriate blood samples were collected. In addition, an oral 2-h glucose tolerance test (75 g) was carried out at baseline and at 3 months of both treatments in the first 20 subjects.

During the study the women were encouraged to lead normal lives with no changes in dietary habits, alcohol consumption, or physical activity, which were all recorded by means of questionnaires before and at the end of each treatment period. They kept weekly diaries concerning their general health, possible side effects, bleeding, and use of antibiotics or other concomitant drugs. Compliance with use of the study medication was confirmed by checking diaries and analyzing serum levels of the isoflavonoids daidzein, genistein, and equol, as reported previously (24).

Laboratory methods

Blood samples were collected after an overnight fast immediately before the start of the regimen and on the last day of each treatment period. Blood glucose and insulin levels were assessed immediately. For other assessments serum was separated by centrifugation and kept frozen at –20 C until assayed. To eliminate the impact of interassay variation, the assessments were carried out in a minimum number of assays.

Serum total cholesterol and triglyceride concentrations were determined by enzymatic colorimetric methods (Roche, Basel, Switzerland). LDL cholesterol and HDL-2 cholesterol were separated from serum by sequential ultracentrifugation. The total HDL fraction was obtained by the heparin-manganese chloride precipitation method. Levels of apolipoprotein B and apolipoprotein A1 were determined by immunochemical methods (Orion Diagnostica, Espoo, Finland). The concentrations of lipoprotein (a) were determined by an immunoturbidimetric method (WAK Chemie, Steinbach, Germany). All these lipids were assessed in every subject except for LDL and HDL-2 cholesterol, which could be assessed in only the first 20 subjects.

Blood samples for assay of glucose and insulin were drawn before and at 1 and 2 h after the intake of glucose (75 g). Blood glucose concentrations were determined by the hexokinase method (Roche Diagnostics GmbH, Mannheim, Germany), the coefficients of intra- and interassay variation being between 2 and 4%. Serum insulin levels were quantified by double-antibody solid-phase RIA (Phadeseph Insulin RIA, Kabi Pharmacia Diagnostics, Uppsala, Sweden). The detection limit of the assay was 2.5 mU/liter. The interassay coefficients of variation was 6% at 11, 3% at 39, and 5% at 101 mU/liter. Cross-reaction with C-peptide was less than 0.1%.

One aliquot of the serum samples collected for lipid determination was used for the assessment of ghrelin concentrations by competitive RIA (Phoenix Pharmaceuticals, Inc., Belmont, CA). The intra- and interassay coefficients of variation were both less than 10%.

Concentrations of isoflavonoids (daidzein, genistein, and equol) were assessed by time-resolved fluoroimmunoassays as reported previously (24).

Statistical analysis

The data are presented as mean ± SD. The possibilities of a period effect and of treatment-period interaction were investigated by the paired t test. Neither was detected. Because no carryover effect was discovered, comparison of the two different treatments (isoflavonoid vs. placebo) was carried out by repeated-measures ANOVA using a general linear model. Differences between baseline and posttreatment values were analyzed by the paired t test. Correlations between variables were calculated by means of Pearson’s parametric correlation analysis. Baseline levels were compared by using the unpaired t test (normally distributed data). P < 0.05 was considered significant. According to a large metaanalysis, phytoestrogens reduced total cholesterol and LDL cholesterol by 10–13% (7), and therefore our study group of 62 women was calculated to give an 80% power to detect this difference. Because the levels of equol, which is exclusively produced by intestinal bacteria from precursor isoflavonoids of dietary origin, may determine the health effects of soy isoflavonoids (25), we also carried out a subgroup analysis based on levels of equol at 3 months of isoflavonoid use (equol producer = equol concentration above 83 nmol/liter and nonequol producer <40 nmol/liter (25). Statistical analyses were performed by using SPSS 10.0 or 11.0 statistical package (SPSS Institute, Inc., Chicago, IL).

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Of the 64 screened women, two were excluded; one because her serum FSH level was less than 30 U/liter and the other because she had taken a course of antibiotics within 3 months of the study. Of the 62 randomized women, 32 were to start with phytoestrogens and 30 with placebo. Six women discontinued the trial during the first treatment regimen, four in the isoflavonoid group (two because of stomach ache, one for personal reasons, and one because of recurrence of breast cancer) and two in the placebo group (one because of lack of effect and one because of vaginal bleeding). Thus, 56 of the randomized 62 women (90.3%) completed the study (Table 1). The age of these women was 54 ± 6 yr of age, and the time since menopause was 5.3 ± 5.5 yr.

The possibilities of a period effect and treatment-period interaction were tested, and no carryover effect was found. Therefore, all the data were pooled to form a single isoflavonoid group and a single placebo group.

None of the measured variables differed at baseline between the isoflavonoid and placebo groups. Weight at baseline correlated inversely with the levels of total HDL cholesterol (Pearson’s correlation coefficient = –0.327, P = 0.014) and HDL-2 cholesterol (Pearson’s correlation coefficient = –0.340, P = 0.010). Smoking was associated with a trend toward elevated levels of triglycerides (1.43 ± 0.69 vs. 1.14 ± 0.43 mmol/liter, P = 0.071), and smokers (n = 13, body mass index = 25.8 ± 2.8 kg/m²) had higher levels of ghrelin (1039.2 ± 456.7 µmol/liter) than nonsmokers (n = 43, body mass index = 26.4 ± 3.5 kg/m²) (800.7 ± 336.9 µmol/liter, P = 0.045).

The use of isoflavonoids led to significant rises in the levels of daidzein (a rise of 1059.6 ± 782.5 nmol/liter, 106-fold rise), genistein (403.8 ± 275.7 nmol/liter, 20-fold rise), and equol (39.9 ± 78.3 nmol/liter, 19-fold rise), whereas the placebo regimen had no effect (24). The isoflavonoid regimen had no effect on blood pressure or body weight.

The isoflavonoid regimen did not affect the levels of various lipids (Table 2). However, during the isoflavonoid regimen, women with prestudy LDL cholesterol levels above the median (4.20 mmol/liter) showed a rise in LDL cholesterol (0.65 ± 0.60 mmol/liter), which was statistically different from the change during the placebo regimen (fall of 0.45 ± 0.67 mmol/liter, P = 0.009). The change in LDL cholesterol was not correlated with the baseline LDL cholesterol level.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
We studied the effects of isolated isoflavones on lipids, lipoproteins, and insulin sensitivity in postmenopausal women who had a history of breast cancer. There is no epidemiological or other evidence that women with a history of breast cancer would have any inherent abnormality in lipids, lipoproteins, or insulin sensitivity, and therefore we believe that these women (of normal weight) were representative of healthy postmenopausal women in this regard. This is also supported by the levels of lipids and lipoproteins, which fell within the normal range for Finnish postmenopausal women (26). Three women had earlier used tamoxifen, which can affect serum lipids (27), but as a result of discontinuation of tamoxifen treatment 5 months to 4 yr before our trial, we feel that the effect of tamoxifen had fully vanished. We decided to give isolated isoflavones (114 mg/d) at a dose that clearly exceeds the amount of phytoestrogens present in a typical Asian diet (20–80 mg/d) (2, 28) but that is comparable with dosages used in some other studies (11, 12). This dosage raised the levels of isoflavonoids in our subjects (24), and even though no relief of hot flashes or other climacteric symptoms was seen (24), we are confident that the dosage of phytoestrogens was adequate. This is supported by a beneficial effect of our regimen on bone resorption markers (29). Lipid levels and insulin sensitivity respond well within 6 wk to 3 months to HT (6, 30, 31), and therefore, if the effects of phytoestrogens mimic those of estrogen, 3 months’ exposure to phytoestrogens is likely to be long enough to reveal any clinically significant effects on lipids and insulin sensitivity.

Of the large number of lipids and lipoproteins possibly playing a role in the onset of CVD (32), we assessed the eight lipid variables that have most commonly been studied in association with the use of postmenopausal HT (6) and that, most likely, are among the most reliable predictors of the risk of CVD (16). In a subgroup of subjects, we also studied the effect of isoflavonoids on insulin resistance, which is often associated with abnormalities in the concentrations of lipids and lipoproteins (13). To the best of our knowledge, there are no previous data on the concomitant effects of isoflavonoids on lipids and insulin in nondiabetic postmenopausal women. However, it is known that the phytoestrogen regimen we used does not affect the levels of C-reactive protein, a vascular inflammatory marker (33). Because ghrelin is associated with insulin resistance (18), we also measured its levels.

It is clear from our study that isoflavonoid supplementation does not affect the serum levels of total cholesterol, LDL, HDL, or HDL-2 cholesterol, triglycerides, apolipoprotein A1, apolipoprotein B, or lipoprotein (a) in Finnish postmenopausal women. Assuming that lipid abnormalities contribute to CVDs (34) and that phytoestrogen intake should protect against CVDs (35), our findings may appear surprising. However, others have also reported the lack of effect of isolated isoflavonoid supplementation of 2–6 months on serum lipoproteins in controlled studies (8, 9, 11, 36, 37). In fact, there are only two studies that hint at a beneficial effect of isoflavonoids on lipids: one study on 12 women who used isoflavonoids for only 4 wk (38) or another one lacking a placebo arm (39). In contrast, the intake of isoflavonoids (34–132 mg) and soy protein jointly for 6 wk to 3 months has been accompanied by falls in LDL cholesterol levels (6–10%) (10, 12, 40, 41), although no improvement was seen in vascular function (10). Moreover, there appears to be an association between falls in lipid levels and the amount of isoflavonoids with soy protein in some (41, 42, 43) but not all studies (28, 44, 45).

All this may imply that isoflavonoids affect lipids and lipoproteins but only in the presence of soy protein. This is supported by data from a study on cynomolgus monkeys, which responded to isoflavonoids with falls in lipid levels but only if they received isoflavonoids concomitantly with soy protein (46). The mechanism by which soy protein may bring about the effects of isoflavonoids on lipids is not known, but it may facilitate the transport of isoflavonoids in blood or their entry into the target organs, such as liver or muscle cells. Thus, dietary isoflavonoids in conjunction with soy protein may affect lipids and perhaps contribute to vascular effects. We should also take into account the possibility that isoflavonoids can affect directly the cholesterol efflux from endothelial cells, which is a risk factor of CVD, and may change independently of the concomitant levels of lipids in blood (47). Of course we must acknowledge a number of other factors, such as differences in race, genetic background, environment, and lifestyle, all of which may contribute to the varying results on lipid and lipoprotein concentrations after phytoestrogen intake. And finally, we would like to mention that HT-induced favorable changes in lipids and lipoproteins were not predictive for the occurrence of CVD events in two large randomized trials [Heart and Estrogen/Progestin Replacement Study, Women’s Health Initiative (WHI)], suggesting that the significance of these changes may have been overestimated (48, 49)

There is plenty of evidence that postmenopausal HT can improve insulin sensitivity (30, 31, 50), although the data are not uniform (51, 52). Ideally, insulin sensitivity should be measured by means of a clamp technique (53) or the minimal model (54), but there is general consensus that even basal levels of glucose or insulin (55) are sensitive enough to detect insulin resistance of clinical significance (56). Therefore, we are confident that an oral 2-h glucose tolerance test (75 g) would be efficient enough to detect clinically significant changes in insulin resistance. We expected to see an improvement in insulin sensitivity during isoflavone intake in our postmenopausal nondiabetic women because previous data imply a hypoinsulinemic effect of soy protein rich in isoflavones (22, 57). However, isoflavonoids failed to affect the glucose-insulin balance in our normoinsulinemic subjects. It remains to be seen whether more elaborate techniques to assess insulin sensitivity (clamp, the minimal model), more prolonged trials, and/or different phytoestrogen regimens will yield different results. Moreover, as with lipids, an effect of phytoestrogens on insulin sensitivity may require the concomitant presence of soy protein.

Ghrelin has been implicated in both mealtime hunger and the long-term regulation of body weight (58). The levels of ghrelin rise shortly before a meal and fall shortly afterward and have been correlated with insulin sensitivity in some (18, 59) but not all studies (60). In our study the changes in ghrelin levels were significantly different during the isoflavonoid and placebo regimens, as the result of a rise in ghrelin levels in the placebo group. This rise could be a consequence of an increase in age of 3–8 months during our study because circulating ghrelin concentrations correlate positively with age (61). Thus, isoflavonoids may have prevented the age-dependent rise in ghrelin levels in our subjects. However, much more data are needed before we can speculate whether the unchanging level of ghrelin during isoflavonoid intake is clinically significant or whether it was a random finding in our study.

In summary, we found no effects of isolated isoflavonoids (114 mg/d) on lipids, lipoproteins, or insulin sensitivity in postmenopausal women. This suggests no vascular benefit of isoflavonoids mediated by the circulating levels of lipids and lipoproteins.

	Footnotes

 
This work was supported by grants from the research funds of Jorvi Hospital and Helsinki University Central Hospital and the Juho Vainio Foundation.

Abbreviations: CVD, Cardiovascular disease; HDL, high-density lipoprotein; HT, hormone therapy; LDL, low-density lipoprotein.

Received December 30, 2003.

Accepted March 29, 2004.

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Top Abstract Introduction Subjects and Methods Results Discussion References

 

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