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Departments of Internal Medicine (M.A., S.M., A.G., A.F., N.F., M.B.) and Biochemical, Physiological, and Nutritional Sciences (H.M., E.B.A.), Section of Physiology and Human Nutrition; Department of Clinical and Experimental Medicine and Pharmacology (L.M., F.P., A.B., D.A., F.S.), Section of Pharmacology; and Department of Obstetrical and Gynecological Sciences (R.D., M.L.C., F.Co., F.Ca.), University of Messina, 98125 Messina, Italy; and Department of Medical Physiopathology (C.L.), La Sapienza University, 00161 Rome, Italy
Address all correspondence and requests for reprints to: Prof. Francesco Squadrito, Section of Pharmacology, Department of Clinical and Experimental Medicine and Pharmacology, Torre Biologica, 5th Floor, AOU Policlinico "G. Martino," Via C. Valeria Gazzi, 98125 Messina, Italy. E-mail: Francesco.Squadrito{at}unime.it.
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Abstract |
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Objective: Our objective was to assess the effects of genistein administration (54 mg/d) on some predictors of cardiovascular risk in osteopenic, postmenopausal women.
Design and Setting: We conducted a randomized, double-blind, placebo-controlled trial at three Italian university medical centers.
Intervention: After a 4-wk stabilization on a standard isocaloric, fat-reduced diet, participants were randomly assigned to receive genistein (n = 198) or placebo (n = 191) daily for 24 months. Both intervention and placebo contained calcium and vitamin D3.
Outcome Measures: Blood lipid profiles, fasting glucose and insulin, homeostasis model assessment for insulin resistance, fibrinogen, soluble intercellular adhesion molecule-1, soluble vascular cellular adhesion molecule-1, F2-isoprostanes, and osteoprotegerin at baseline and after 12 and 24 months of treatment were measured.
Results: Compared with placebo, genistein significantly reduced fasting glucose and insulin as well as homeostasis model assessment for insulin resistance after both 12 and 24 months of treatment. By contrast, genistein administration did not affect blood lipid levels although fibrinogen, F2-isoprostanes, soluble intercellular adhesion molecule-1, and soluble vascular cellular adhesion molecule-1 decreased significantly compared with placebo after 24 months. Serum osteoprotegerin was higher in the genistein group compared with placebo. At 24 months, the genistein group showed no change in endometrial thickness compared with placebo. Most treatment-related adverse events were moderate and composed of gastrointestinal side effects [genistein, n = 37 (19%); placebo, n = 15 (8%)].
Conclusions: These results suggest that 54 mg genistein plus calcium, vitamin D3, and a healthy diet was associated with favorable effects on both glycemic control and some cardiovascular risk markers in a cohort of osteopenic, postmenopausal women.
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Introduction |
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Epidemiological data suggest that women ingesting high amounts of phytoestrogens and plant-derived diphenolic compounds, which are structurally and functionally similar to estradiol, have less CAD and breast and uterine cancer and fewer vasomotor symptoms compared with those who consume Western diets (9). Furthermore, phytoestrogens have in vitro antineoplastic effects via the inhibition of both cellular proliferation as well as angiogenesis, properties that could be protective against development of certain types of cancer (10). However, the American Heart Association has stated that neither soy protein nor isoflavones have significant benefit with regard to cardiovascular health (11), even though this conclusion was based primarily on the lack of effect on plasma lipids and lipoproteins. Other cardiovascular markers, though not as widely tested for in typical office or laboratory settings, must also be considered when examining the efficacy of new therapies for CAD.
Genistein is an isoflavone found in low concentrations in soybeans and elevated amounts in certain soy-derived food, whereas genistin, the glucoside form of the aglycone genistein, is much more abundant in the unprocessed soybean. Data suggest that genistein might have a potential preventive role in reducing CAD (12). Genistein, acting as a natural selective estrogen receptor (ER) modulator, may also positively affect the cardiovascular system without the harmful estrogenic side effects in breast and uterine tissue. This safety profile may be a direct consequence of greater genistein affinity for the ERß found to be more abundant in both bone and endothelial tissue of arteries, whereas ER
exists at higher densities in the reproductive tissues (13). In experimental models, genistein has been shown 1) to enhance the dilator response to acetylcholine of atherosclerotic arteries (14); 2) to relax rat arteries by a nitric oxide-dependent mechanism (15); and 3) to reduce infarct size in an experimental model of myocardial ischemia/reperfusion injury (16).
Moreover, genistein therapy in healthy postmenopausal women increases the ratio of nitric oxide to endothelin (17), improves flow-mediated endothelium-dependent vasodilation (17), and has a favorable effect on some cardiovascular risk markers (18). In those studies, isolated genistein administration exhibited a uniform bioavailability and pharmacokinetic behavior. Nevertheless, a potential limitation of these trials was their relatively short duration with respect to the outcome measures and the small number of patients initially enrolled.
Therefore, to improve the field of understanding of the multiple actions of genistein in humans, we investigated the effects of pure genistein administration (54 mg/d for 24 months) on specific predictors of cardiovascular risk in osteopenic, postmenopausal women (n = 389) in a randomized, double-blind, placebo-controlled trial.
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Patients and Methods |
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The trial was designed to assess the effects of purified genistein, isolated from soy, on bone mineral density (BMD) and cardiovascular safety (Clinical Trials.gov identifier: NCT 00355953).
After the ethical committee approved the study, patients were recruited from osteoporosis and menopause centers at the University of Messina (Messina, Italy) and at the Medical Physiopathology department at the University "La Sapienza" (Rome, Italy). All participants gave written informed consent.
Over 2000 postmenopausal women were initially screened. Approximately 1390 were identified under the inclusion/exclusion criteria for the trial and were then referred to these centers as potential participants. Finally, 575 women met the study inclusion criteria. Of that number, 186 women refused to participate, thus generating the final study number of 389 women (Fig. 1
). Randomization was performed by using a computer-generated randomization sequence. All women were between 49–67 yr of age, were 12 months beyond menopause at baseline, in good general health, had a BMD at femoral neck of less than 0.795 g/cm2, had not undergone surgically induced menopause, and had FSH levels of more than 50 IU/liter and a serum 17ß-estradiol level of no more than 100 pmol/liter. At the beginning of the study, a complete family history, physical examination, and laboratory evaluation (chemistry and hematology panel) were performed at the Department of Internal Medicine of the University of Messina.
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Diet
An isocaloric fat-restricted diet was offered to the 389 patients, composed of 25–30% energy intake from fat, less than 10% of energy intake from saturated fatty acids, 55–60% energy intake from carbohydrates, 15% energy intake from proteins, cholesterol intake of less than 300 mg/d, and fiber intake of at least 35 g/d. Caloric energy intake and expenditure was based on body size and calculated using the Harris-Benedict equation. This isocaloric fat-restricted diet was provided to study participants for the stabilization period of 4 wk to ensure the same energy consumption by all subjects and avoid any interference relating to lipid profiles when beginning the study. The intake of soy products, legumes, or other nutrient supplements was completely prohibited. Moreover, the previous isoflavone intake for study participants before randomization, assessed by a food-frequency questionnaire, was 1–2 mg/d. This intake has been shown to be typical of Western populations (9). The diet was continued throughout the study, and compliance was reinforced by a nutritionist. In addition, genistein serum levels, dietary intake, and body mass index (BMI) in the follow-up period were also evaluated in all postmenopausal women included in the trial.
Treatments
After the 4-wk stabilization on the standard, isocaloric fat-reduced diet, participants in the study were randomly assigned to receive the phytoestrogen genistein (n = 198; 54 mg/d in two tablets) (Laboratori Plants, Messina, Italy) or placebo (n = 191). Previously, this intake of phytoestrogen was described to have biological effects (19). The purity of genistein was at least 98% (data not shown). Placebo and genistein tablets appeared exteriorly similar. All tablets contained the following other components: 500 mg calcium carbonate and 400 IU vitamin D per tablet.
Measurements
Both fasting glucose and insulin were measured in serum collected after an overnight fast using routine methods (normal range 65–110 mg/dl and 3–35 µUI/ml, respectively). Homeostasis model assessment for insulin resistance (HOMA-IR) was then calculated using the following formula: fasting glucose (mg/dl) x fasting insulin (µIU/ml)/22.5 (18).
Fibrinogen was analyzed in serum samples using routine methods (normal range 125–250 mg/dl). Soluble intercellular adhesion molecule (sICAM-1) and soluble vascular cellular adhesion molecule (sVCAM-1) were determined using commercially available ELISA kits (Diaclone Research, Besancon, France; normal range, 0.25–8 and 1.56–50 ng/ml, respectively; sensitivity, <0.1 and <0.6 ng/ml, respectively). Standard curves for each were obtained by spiking serum samples with known quantities of sICAM-1 and sVCAM-1. F2-isoprostanes were measured in urine collected from the first void in the morning according to the manufacturer’s protocol (Oxford Biomedical Research, Oxford, MI). Total serum cholesterol, high-density lipoprotein (HDL) cholesterol, lipoprotein A, and triglycerides were measured by routine enzymatic methods, and low-density lipoprotein (LDL) cholesterol was calculated in according to Friedewald’s formula: total cholesterol (mg/dl) – HDL cholesterol (mg/dl) – triglycerides (mg/dl)/5. Osteoprotegerin (OPG) (normal range, 2.5–4.5 pmol/liter in postmenopausal women) was measured by an ELISA (Pantec s.r.l., Turin, Italy). Genistein levels were measured in plasma samples by a time-resolved fluorometric assay following the manufacturer’s instructions (TR-FIA test; Labmaster, Turku, Finland) (20). Briefly, 200 µl of 100 mM acetate buffer (pH 5.0) containing 0.2 U/ml ß-glucuronidase and 2 U/ml sulfatase was added to 200 µl serum. Samples were then incubated overnight at 37 C. After incubation, free genistein was extracted twice with 1.5 ml diethyl ether by mixing for 3 min; 200 µl assay buffer was added to each sample to achieve a concentration corresponding to the original genistein concentration in plasma. Then, a 20-µl aliquot of this solution was used for time-resolved fluoroimmunoassay. The fluorescent signal was read using a Perkin-Elmer (Norwalk, CT) Victor 1420 multilabel counter.
Clinic visits
The women were questioned about any symptoms at clinic visits every 3 months. Standard clinical evaluations and laboratory analyses, including hematological, renal, and liver function tests were performed every 6 months. Ultrasonographic endometrial thickness was evaluated at baseline and again after 12 and 24 months. The endometrial thickness, at sagittal incidence, was measured from one basal layer to the other. If the endometrial thickness was at least 8 mm or if uterine bleeding was present during the treatment, then hysteroscopic examination and endometrial biopsy were performed. All unfavorable and unintended clinical effects were considered adverse effects and were evaluated by the investigators with respect to severity, duration, seriousness, and relation to the study drug and outcome. We specifically checked all postmenopausal women included in the trial for gastrointestinal symptoms, breast tenderness, vasomotor symptoms, depression, irritability, insomnia, and vaginal bleeding. This evaluation was conducted using a checklist.
Statistics
The primary efficacy data were analyzed according to the intention-to-treat principle on femoral neck and lumbar BMD. These analyses included all 389 postmenopausal women in whom BMD was measured at baseline. Although subjects were expected to drop out during the study and, thus, not all subjects would have BMD measurements after 1 and 2 yr of treatment, characteristics of individuals who withdrew from the study were investigated. Moreover, we imputed missing data for patients with at least an interim value by carrying the last observation forward. The significance of the between-group differences was then assessed by a repeated-measures analysis, which included fixed effects for treatment, visit, and their interaction.
Finally, linear regression analysis was used to detect the relationship between genistein plasma levels and outcome measures. Statistical analysis was performed using SPSS, Inc. for Windows release 6.0 (SPSS Inc., Chicago, IL). Before analysis, all analysis endpoints were tested for normality using the Kolmogorov-Smirnov test, normal probability plots, box and whiskers plots, and plots of raw scores against normal percentile plots. These analyses did not reveal any pronounced deviations from normality, and all analyses were conducted on raw scores.
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Results |
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The baseline characteristics of the postmenopausal women participating in our study are shown in Table 1. No statistically significant difference was observed among the different groups. Additionally, all the groups had a similar age and BMI (Table 1). Characteristics of individuals who withdrew from the study before completion (n = 85) were also compared with the baseline characteristics of subjects who completed the study; this post hoc comparison was made between the two treatment groups. We did not find any inference that could affect potential outcomes of interest. Moreover, 28 of 48 individuals (58%) in the genistein group and 18 of 37 individuals (48%) in the placebo group withdrawing from the study completed a 1-yr follow-up. In these noncompleters with 1-yr assessments, there was no evidence that variables differed between completers and noncompleters.
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Administration of 54 mg/d of genistein purified from soy resulted in a marked increase in plasma levels of the phytoestrogen, whereas placebo treatment did not affect the circulating levels of genistein (Table 2). Genistein administration significantly reduced fasting blood glucose at 12 months (genistein = –2.28 ± 0.94%; placebo = 2.60 ± 0.29%; P < 0.001) and 24 months (genistein = –3.19 ± 0.90%; placebo = 2.52 ± 0.38%; P < 0.001). Fasting insulin was also reduced at 12 months (genistein = –7.77 ± 0.27%; placebo = 9.78 ± 0.16%; P < 0.001) and 24 months (genistein = –8.24 ± 0.30%; placebo = 10.10 ± 0.20%; P < 0.001). HOMA-IR showed a similar pattern at 12 months (genistein = –9.23 ± 0.96%; placebo = 12.80 ± 0.41%; P < 0.001) and 24 months (genistein = –10.70 ± 0.88%; placebo = 13.06 ± 0.50%; P < 0.001) (Table 2 and Fig. 2).
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; Fig. 3), F2-isoprostanes were markedly lower (genistein = –13.00 ± 0.39%; placebo = 5.60 ± 0.43%; P < 0.001) (Table 2; Figure 4), sICAM-1 decreased dramatically (genistein = –10.54 ± 0.23%; placebo 4.87 ± 0.24%; P < 0.001) (Table 2; Fig. 3), and sVCAM-1 trended sharply downward (genistein = –9.63 ± 0.15%; placebo = 3.10 ± 0.23%; P < 0.001) (Table 2 and Fig. 3) vs. placebo.
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). Linear regression analysis showed a significant negative correlation between genistein plasma levels and fasting blood glucose (r= –0.224; P < 0.0001), fasting insulin (r = –0.153; P = 0.0040), fibrinogen (r = –0.308; P < 0.001), sICAM-1 (r = –0.379; P < 0.0001), sVCAM-1 (r = –0.256; P < 0.0001), and urinary F2-isoprostanes (r = –0.701; P < 0.0001) and a positive correlation with OPG (r = 0.219; P < 0.001) serum levels.
Levels of total, LDL, and HDL cholesterol, lipoprotein A, and triglycerides were similar among the two different groups (Table 3) at baseline. Genistein or placebo did not significantly modify the total, LDL, and HDL cholesterol levels, lipoprotein A levels, and triglycerides after either 12 or 24 months of therapy (Table 3).
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Genistein and placebo were generally well tolerated and ingested with a high degree of compliance. There were no significant changes in routine biochemistry, liver function, or hematology results. The daily administration of 54 mg of the genistein did not cause any significant change in the endometrial thickness (Table 4).
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). In total, 37 (19%) genistein recipients and 15 (8%) placebo recipients discontinued therapy because of adverse events (P = 0.002), all of which were gastrointestinal. No patient who remained in the study experienced an adverse event. All the adverse events related to gastrointestinal discomfort (Table 4). |
Discussion |
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There is a lack of information about the effect of isolated phytoestrogens, such as genistein, on cardiovascular risk factors, and therefore the results of the present study need to be analyzed in light of the available literature data. Most previous studies have used mixed isoflavones or phytoestrogens with different dosage regimens, soy foods, or supplements as the active treatment arm. The quality and amount of genistein varied widely in all of these previous studies.
Meta-analysis of clinical trials that focused on serum lipids definitely shows that soy isoflavones have little effect (21). Products that contain a high amount of genistein, however, appear to be effective for increasing BMD (22), reducing menopausal symptoms (23), and affecting some cardiovascular markers (18) .
Several studies suggest that soy has beneficial effects on diabetes mellitus (24, 25). Jayacopal and co-workers (26) reported that phytoestrogen supplementation for 12 wk significantly lowers mean values of fasting insulin and insulin resistance in postmenopausal women with type 2 diabetes. Furthermore, it has been indicated that some positive soy-induced metabolic effects might be influenced by the menopausal status (27). However, in these studies, it was not clear whether the favorable effects of soy could be related to its isoflavone content. Interestingly, another study showed that the consumption of isoflavones genistein and daidzein was associated with lower BMI, fasting insulin concentrations, and insulin response to an oral glucose load, demonstrating the beneficial effects of isoflavones on excess body weight and hyperinsulinemia, which are typical and well-known cardiovascular risk factors (28). Indeed, our study agrees with the findings of lower fasting serum glucose, fasting insulin, and HOMA-IR in postmenopausal women taking 54 mg genistein compared with placebo, suggesting the potential value of genistein as an antidiabetic agent.
So far, the key mechanisms by which genistein affects glucose metabolism have not been fully elucidated, although several hypotheses have been suggested (29, 30, 31). Recently genistein has been also shown to activate both peroxisome proliferator-activated receptors (PPAR), which are nuclear receptors that participate in cellular glucose homeostasis and blood lipids regulation (32).
However, in contrast with positive effects on glucose metabolism, this study found no effects of genistein compared with placebo on lipid profiles in postmenopausal women. The evidence for a beneficial role and the potential mechanism(s) of this soy isoflavone on blood lipoproteins in humans remain controversial (33). It could be hypothesized that the different biological effects of genistein on human metabolism strongly depend on the concentration of genistein present and/or the levels of ERs, PPAR
, PPAR, and ß-adrenergic activity within that particular tissue studied (32, 34). Circulating levels of F2-isoprostanes also increase in several disease states involving oxidative stress, including atherosclerosis and diabetes mellitus (35, 36). It is likely, then, that strong antioxidants might provide a significant protective role in the prevention of CAD. Phytoestrogens, in general, have been shown to possess strong antioxidant activity, having the ability to prevent oxidative modification of LDL in the arterial wall and possibly attenuate the development of atherosclerosis (37). In contrast with in vitro studies, data from interventional studies have failed to show an antioxidant effect from isoflavone supplements (38, 39). We, however, observed lower F2-isoprostanes in postmenopausal women taking 54 mg genistein compared with placebo as previously shown with soy-containing food with high isoflavone content (40).
The association between OPG, a cytokine that inhibits osteoclastogenesis, and cardiovascular risk is of particular interest. There is a strong association between serum OPG levels and diabetes, CAD, and cardiovascular mortality (41). In our study, genistein-treated women had higher OPG levels after 12 and 24 months compared with placebo. This finding suggests that genistein might be cardioprotective in light of recent experimental data that showed that OPG can inhibit vessel calcification and endothelial apoptosis (42, 43). High levels of serum fibrinogen are also an important risk factor for CAD. These levels tend to increase in women after menopause (44, 45). We observed a decrease in fibrinogen after 24 months of therapy with genistein, thus suggesting another potential cardiovascular benefit of this phytoestrogen.
Cell adhesion molecules, such as ICAM-1 and VCAM-1, play an important role in the development of atherosclerosis. Elevated serum levels of these molecules are seen in CAD (46). The decrease in ICAM-1 and VCAM-1 production in response to administration of genistein may result from an inhibition of protein tyrosine kinase-dependent signal transduction pathways (47). These findings are compatible with the results of this study showing that at 24 months of therapy with genistein, sICAM-1 and sVCAM-1 were reduced.
The data obtained in the present study are highly suggestive that genistein might play a preventive role in the development of CAD in osteopenic, postmenopausal women.
However, additional randomized clinical studies in postmenopausal women with a high risk of CAD are needed to affirm genistein as a treatment for this disease state. Moreover, our data are in disagreement with a previous study that showed that soy isoflavone-enriched foods had no effect on lipid and glucose markers (48). The discrepancy between our results and those by Hall and co-workers (48) might be due to different study designs and treatment components. Hall et al. (48) used a crossover intervention, a relatively small number of enrolled patients (n = 117), a shorter observational period (8 wk), and a different treatment. In contrast, we used a well defined dose of genistein that caused documented and stable increases in the serum level of the isoflavone.
Finally, 54 mg/d of genistein over 24 months was not associated with any significant adverse effect on the uterus as shown by no changes in endometrial thickness. However, caution is still necessary, and safety concerns must be addressed, especially in patients at high risk of endometrial and/or breast cancer. Moreover, whether the safety results found in our study can be extended to isoflavones as a class is still unclear and deserves further study. Indeed, we observed a moderate number of gastrointestinal side effects in the genistein-treated women. These side effects were also observed in the placebo arm at a lower rate. It is possible that calcium carbonate may elicit a similar response and that genistein exacerbates the effect. Collectively, the data presented here suggest that genistein might be of therapeutic value for the prevention of CAD in osteopenic, postmenopausal women.
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Footnotes |
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Author Disclosure: M.A., H.M., L.M., F.P., A.B., D.A., S.M., R.D., M.L.C., A.G., A.F., N.F., F.Co., F.Ca., C.L., M.B., E.B.A., and F.S. have nothing to declare.
First Published Online May 22, 2007
1 M.A., H.M., and L.M. contributed equally to this article. 
Abbreviations: BMD, Bone mineral density; BMI, body mass index; CAD, coronary artery disease; ER, estrogen receptor; HDL, high-density lipoprotein; HOMA-IR, homeostasis model assessment for insulin resistance; HRT, hormone replacement therapy; LDL, low-density lipoprotein; OPG, osteoprotegerin; PPAR, peroxisome proliferator-activated receptor; sICAM-1, soluble intercellular adhesion molecule; sVCAM-1, soluble vascular cellular adhesion molecule.
Received October 20, 2006.
Accepted May 11, 2007.
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References |
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(PPAR) as a molecular target for the soy phytoestrogen genistein. J Biol Chem 278:962–967 stimulation improves lipoprotein composition in healthy moderately obese subjects. Metabolism 55:26–34[CrossRef][Medline]vß3-induced, NF-
B-dependent survival factor for endothelial cells. J Biol Chem 275:20959–20962This article has been cited by other articles:
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  A. Bitto, D. Altavilla, A. Bonaiuto, F. Polito, L. Minutoli, V. Di Stefano, D. Giuliani, S. Guarini, V. Arcoraci, and F. Squadrito Effects of aglycone genistein in a rat experimental model of postmenopausal metabolic syndrome J. Endocrinol., March 1, 2009; 200(3): 367 - 376. [Abstract] [Full Text] [PDF]
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 P. Villa, B. Costantini, R. Suriano, C. Perri, F. Macri, L. Ricciardi, S. Panunzi, and A. Lanzone The Differential Effect of the Phytoestrogen Genistein on Cardiovascular Risk Factors in Postmenopausal Women: Relationship with the Metabolic Status J. Clin. Endocrinol. Metab., February 1, 2009; 94(2): 552 - 558. [Abstract] [Full Text] [PDF]
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 I. Rudkowska Functional foods for cardiovascular disease in women Menopause Int, June 1, 2008; 14(2): 63 - 69. [Abstract] [Full Text] [PDF]
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 T. M. Cho, N. Peng, J. T. Clark, L. Novak, S. Roysommuti, J. Prasain, and J. M. Wyss Genistein Attenuates the Hypertensive Effects of Dietary NaCl in Hypertensive Male Rats Endocrinology, November 1, 2007; 148(11): 5396 - 5402. [Abstract] [Full Text] [PDF]
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, P < 0.05 vs. baseline; 
, P < 0.001 vs. baseline; #, P < 0.001 vs. placebo), fasting insulin (