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Effects of Soy Isoflavones on Markers of Bone Turnover in Premenopausal and Postmenopausal Women¹

Department of Food Science and Nutrition (K.E.W., A.M.D., B.E.M.-D., X.X., M.S.K.), University of Minnesota, St. Paul, Minnesota 55108; Aging Study Unit (R.M.), Department of Veterans Affairs Medical Center, Stanford University School of Medicine, Palo Alto, California 94304; and Department of Obstetrics and Gynecology (W.R.P.), University of Rochester, Rochester, New York 14642.

Address correspondence and requests for reprints to: Mindy S. Kurzer, Department of Food Science and Nutrition, University of Minnesota, 1334 Eckles Avenue, St. Paul, Minnesota 55108. E-mail: mkurzer{at}umn.edu

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Soy isoflavones are hypothesized to exert hormonal effects in women and thus may play a role in bone metabolism throughout life. In 2 randomized, cross-over studies, 14 pre- and 17 postmenopausal women were given 3 soy protein isolates containing different amounts of isoflavones [control, 0.13; low isoflavone (low-iso), 1.00; and high-iso, 2.01 mg/kg body wt·day, averaging 8, 65, and 130 mg/day, respectively], for over 3 months each. Food records, blood samples, and 24-h urine collections were obtained throughout the studies. The endpoints evaluated included plasma or serum concentrations of bone-specific alkaline phosphatase, osteocalcin, insulin-like growth factor-I (IGFI), IGF binding protein-3 (IGFBP3), and urine concentrations of deoxypyridinoline cross-links and carboxy-terminal telopeptide of type I collagen. In premenopausal women, IGFI and IGFBP3 concentrations were increased by the low-iso diet, and deoxypyridinoline cross-links was increased by both the low- and high-iso diets during certain phases of the menstrual cycle. In postmenopausal women, bone-specific alkaline phosphatase was decreased by both the low- and high-iso diets, and there were trends toward decreased osteocalcin, IGFI, and IGFBP3 concentrations with increasing isoflavone consumption. Although soy isoflavones do affect markers of bone turnover, the changes observed were of small magnitude and not likely to be clinically relevant. These data do not support the hypothesis that dietary isoflavones per se exert beneficial effects on bone turnover in women.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
DATA FROM the National Health and Nutrition Examination Survey III indicate that the prevalence of reduced bone mineral density (BMD), defined as one or more SDs below the mean of young women, is high in women in the United States. According to National Health and Nutrition Examination Survey III, 55% of women who are 50–59 yr of age have reduced BMD, and the number rises to 69% and 88% for women in their 60s and 70s, respectively (1). Among women 65 or older, 29.3% have osteoporosis, defined as BMD greater than 2.5 SDs below the mean of young women (2). Researchers have estimated that the incidence of hip fractures in North America will be over 669,000 by 2025 (3).

One of the primary causes of osteoporosis, and subsequent fractures, is the reduction in endogenous estrogen concentrations that occurs at menopause. Hormone replacement therapy (HRT) is accepted by many to be the best method to maintain BMD after menopause and is one of several options for the treatment of osteoporosis (4). However, because of patients’ concerns about the side effects and safety of HRT, the interest in natural alternatives remains. Of particular interest are phytoestrogens such as soy isoflavones, compounds that exert estrogenic activity on several tissues and are thus being investigated as possible alternatives to HRT (5).

Studies investigating the effects of dietary isoflavones on bone are extremely limited, although substantial research efforts have focused on ipriflavone, a synthetic isoflavone that has been shown to reduce the rate of bone loss in postmenopausal women (6, 7, 8). The effects of dietary isoflavones on bone have been investigated in animal models and in one human trial. Ovariectomized (OVX) rats treated with the isoflavones genistein or daidzein have shown increased dry femoral weight (9, 10), increased BMD (11, 12), increased rate of bone formation (12), and decreased rate of resorption (11), compared with untreated OVX rats. OVX rats given isoflavone-rich isolated soy protein (ISP) have shown increased BMD (13) or no change in BMD (14), and no difference in markers of bone turnover (13, 14), compared with OVX rats given either casein or isoflavone-depleted ISP; whereas cynomolgous monkeys consuming ISP showed increased bone turnover, compared with casein-fed animals (15). Postmenopausal women consuming 40 g of ISP containing 90 mg isoflavones per day showed a small (but statistically significant) increase in BMD in the lumbar spine, compared with baseline, unlike women consuming casein or 40 g of ISP containing 56 mg isoflavones per day (16).

The primary purpose of this study was to assess the effects of soy isoflavone consumption on plasma or serum concentrations of markers of bone formation [bone-specific alkaline phosphatase (BAP) and osteocalcin (OC)], urine concentrations of markers of bone resorption [deoxypyridinoline cross-links (DPD) and carboxy-terminal telopeptide of type I collagen (CTX)], as well as plasma concentrations of insulin-like growth factor-I (IGFI) and IGF binding protein-3 (IGFBP3), in healthy pre- and postmenopausal women. In a randomized cross-over design, free-living subjects consumed three soy protein isolates that differed only in isoflavone content, for over 3 months each.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Study design

This research was part of two studies designed to investigate the effects of soy isoflavone consumption on reproductive hormones in premenopausal (17) and postmenopausal women (18). The study protocols were approved by the University of Minnesota Institutional Review Board: Human Subjects Committee, and subjects signed a written consent before participation. Both studies used a randomized cross-over design consisting of three diet periods separated by washouts of approximately 3 weeks. Premenopausal women began each of the three diet periods on day 2 of their menstrual cycle and continued each diet period for 3 menstrual cycles plus 9 days. To approximate the average length of the premenopausal study diet periods, postmenopausal women completed three 93-day diet periods. All subjects were free-living and consumed their habitual diets supplemented with one of three ISP beverage powders during each diet period. Subjects and lab personnel were blinded as to which ISP the subjects were consuming.

Subjects

Potential subjects were selected from the general population in the Minneapolis-St. Paul, Minnesota area after a phone questionnaire, interview, and health screen. Exclusionary criteria included strict vegetarian, high fiber, high soy, or low fat diets; regular consumption of vitamin and mineral supplementation greater than the Recommended Dietary Allowances; athleticism; cigarette smoking; antibiotic or hormone use within 6 months; history of chronic disorders (including endocrine or gynecological diseases); regular use of medication known to interfere with study endpoints (including aspirin); body mass index less than 18 or greater than 35 kg/m²; weight changes of more than 4.5 kg within the previous year, and inability to abstain from alcoholic beverages during the study. Additional exclusionary criteria for premenopausal women included current pregnancy or lactation and irregular menstrual cycles; and for postmenopausal women, menstrual bleeding within 12 months, hysterectomy or oophorectomy, and FSH concentration less than 25 IU/L. Twenty women were enrolled in the premenopausal study (mean age, 26.5 ± 4.7 yr) and 23 women in the postmenopausal study (mean age, 57.1 ± 5.9 yr).

Soy supplement

Subjects consumed their habitual diets, with detailed instructions to minimize phytoestrogen consumption and avoid alcoholic beverages, as described previously (17, 18). Their usual diets were supplemented with each of three ISP powders differing only in concentration of isoflavones (Supro Brand Isolated Soy Protein, Protein Technologies International, St. Louis, MO). The three ISP powders provided 0.15 ± 0.01 (control); 1.01 ± 0.04 [low-isoflavone (low-iso)]; and 2.01 ± 0.03 (high-iso) mg total isoflavones/kg body weight·day (10 ± 1.1, 64 ± 9.2, and 128 ± 16 mg isoflavones/day, respectively) in the premenopausal study, and 0.11 ± 0.01 (control); 0.99 ± 0.01 (low-iso); and 2.00 ± 0.02 (high-iso) mg total isoflavones/kg BW·day (7±1.1, 65 ± 11, and 132 ± 22 mg isoflavones/day, respectively) in the postmenopausal study, expressed as aglycone units. The isoflavone content of each ISP was evaluated by high-performance liquid chromatography (Dr. Patricia Murphy, Food Science and Human Nutrition Department, Iowa State University, Ames, IA) using a method described previously (19). The specific nutrient and isoflavone composition of the ISPs has been described previously (17, 18). The ISP beverage powders were provided to subjects in daily dose packets and kept refrigerated or frozen before consumption. The average daily nutrient contribution of the ISP powders was 1.21 Mega Joules (MJ) (290 kcal), 53 g protein, 15 g carbohydrate, and 1.9 g fat in the premenopausal study; and 1.46 MJ (349 kcal), 63 g protein, 21 g carbohydrate, and 1.9 g fat in the postmenopausal study. The ISPs contained no cholesterol or fiber.

Study procedures

Fasting body weight, in a hospital gown, was obtained every 7–14 days. Skinfold thickness measurements were evaluated at triceps, biceps, suprailiac, and subscapular sites on the subject’s nondominant side, at the beginning of the study and at the end of each diet period. The same Registered Dietitian took measurements twice at each site, to the nearest 0.1 mm, with a skinfold caliper (Cambridge Scientific Instruments, Ltd., Cambridge, MD). Using age- and gender-specific equations, body density was calculated, and a predictive equation was used to determine percent body fat (20).

To assess any changes in dietary intake, 3-day food records were obtained twice during each menstrual cycle (both follicular and luteal phases) in the premenopausal study and once per month during the postmenopausal study.

In the premenopausal study, the day of ovulation was determined using a home urinary LH test (OvuQUICK Self-Test, Quidel, San Diego, CA) and daily basal body temperature, as previously described (17). Fasting blood samples were obtained every other day, at the same time (± 30 min), from the seventh day after the LH surge in the second menstrual cycle until the end of the diet period. Blood was collected in heparinized tubes, and plasma was separated within 30 min, placed in aliquots, and frozen at -70 C until analysis. Complete urine collections were obtained every day during the third menstrual cycle of each diet period. The 24-h urine samples were collected in plastic bottles containing 1 g ascorbic acid/L, separated into aliquots, and frozen at -20 C until analysis.

After the conclusion of the premenopausal study, the day of ovulation was precisely identified by a reproductive endocrinologist (W. R. Phipps) using OvuQUICK results, plasma hormones, urinary LH, and basal body temperature charts, as described previously in greater detail (17). The menstrual cycle was then divided into four phases, each with a distinct hormone profile: early follicular (EF; days 2 and 4), midfollicular (MF; days 7 and 9), periovulatory (PO; ovulation -3, -1, and +1), and midluteal (ML; ovulation +5, +7, and +9). Samples from the PO or ML phases were excluded for one subject who had anovulatory cycles during two diet periods. To allow adaptation to each diet, only samples collected during the third menstrual cycle were used. One plasma sample per subject per diet from each of the four phases was selected for analysis of bone turnover endpoints. One urine sample per subject per diet from each EF and MF phase and two samples from each PO and ML phase during the third menstrual cycle were selected for analysis.

In the postmenopausal study, fasting blood samples were obtained on day 1 of the study and on days 36–38, 64–66, and 92–94 of each diet period, at the same time each morning (± 30 min). Blood was collected into Vacutainer tubes containing heparin (plasma) or Serum Separater Tube (SST) with gel clot activator (serum), and plasma or serum was separated within 30 min, placed into aliquots, and frozen at -70 C until analysis. Complete 24-h urine collections were obtained for 3 consecutive days before day 1 of the study and on days 35–37, 63–65, and 91–93 of each diet period. Urine was collected and handled in the same manner as in the premenopausal study. To allow adaptation to each diet, samples collected on days 35–37 of each diet were excluded, and urine collections from days 63–65 and 91–93 were combined into 3-day pools to reduce day-to-day variability and the number of analyses.

Analytical methods

Plasma samples were analyzed for OC, IGFI, and IGFBP3. Serum samples, available only in the postmenopausal study, were analyzed for BAP. All samples from an individual subject were analyzed in duplicate in the same daily batch. OC, IGFI, and IGFBP3 were determined by two-site immunoradiometric assay using ¹²⁵I-labeled antibody (OC and IGFBP3) or peptide (IGFI) (Diagnostic Systems Laboratories, Inc., Webster, TX). BAP was determined by an enzyme-linked immunosorbent assay (Metra Biosystems, Mountain View, CA). Intraassay variabilities were 4.7, 0.6, 1.4, and 6.1%, and interassay variabilities were 8.0, 5.0, 7.0, and 8.7% for OC, IGFI, IGFBP3, and BAP, respectively.

All urine samples were analyzed for DPD by a competitive binding enzyme immunoassay (Metra Biosystems). Urine samples from the postmenopausal study were analyzed for CTX by a competitive binding enzyme immunoassay (Diagnostic Systems Laboratories, Inc.). All urine samples for each subject were analyzed in one batch, and results were corrected for urinary creatinine, which was determined by colorimetric assay (Metra Biosystems). Intraassay variabilities were 1.3 and 7.6%, and interassay variabilities were 7.2 and 12.1% for DPD and CTX, respectively.

Food records, collected throughout the studies, were analyzed by a Registered Dietitian using Nutritionist IV for Windows, Version 4.0 (1995 First Databank, The Hearst Corporation, San Bruno, CA). For each 3-day food record, averages were calculated for energy, protein, carbohydrate, fat, monounsaturated fatty acids, polyunsaturated fatty acids, saturated fatty acids, cholesterol, dietary fiber, and all known essential micronutrients.

Statistical analysis

Statistical analyses were performed using the Statistical Analysis System, Version 6.12 (SAS Institute, Inc., Cary, NC). In both studies, repeated-measures ANOVA was performed on anthropometric and food record endpoints, controlling for subject and diet.

In the premenopausal study, the effect of menstrual cycle phase on bone turnover endpoints was evaluated using repeated-measures ANOVA, controlling for subject, diet period, diet, and average calcium intake. Values were averaged across the three diets for OC, IGFI, and IGFBP3, because of nonsignificant phase-by-diet interactions. Analyses were performed separately within each diet for DPD, because of a significant phase-by-diet interaction. Because of unequal variance between menstrual cycle phases, the effects of diet on bone turnover endpoints were examined within each menstrual cycle phase separately, using repeated-measures ANOVA, controlling for subject, diet period, diet, and average calcium intake.

In the postmenopausal study, repeated-measures ANOVA was performed to evaluate the effects of the three diets on bone turnover endpoints, controlling for subject, diet period, average calcium intake, and time of collection (days 64–66 vs. days 92–94). No significant interactions were seen between diet and time of collection. Paired t tests were used for comparisons between baseline bone turnover endpoints and values during each of the three diets.

All results are expressed as mean ± SD or as least-squares mean ± SE, to account for imbalance in the event of missing data. Significance was considered at P < 0.05.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Subject characteristics for women completing the 2 studies are summarized in Table 1. Six of the 20 premenopausal subjects and 4 of the 23 postmenopausal subjects dropped out because of difficulties complying with the study protocol. In addition, 1 subject in the postmenopausal study was excluded a priori from all bone related analyses because of her use of alendronate sodium, and 1 subject was excluded from all analyses when her plasma hormones revealed that she was not postmenopausal. Thus, 14 premenopausal subjects and 17 postmenopausal subjects were included in the final data analyses. As reported previously, there were no significant changes in body weight, body mass index, or percent body fat in either group (17, 18).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The purpose of this research was 2-fold. First, we evaluated the effect of the menstrual cycle on IGFI, IGFBP3, and markers of bone turnover, after plasma and urinary hormone data were used to carefully define the phases of each menstrual cycle. Second, we evaluated the effects of soy isoflavone consumption on IGFI, IGFBP3, and markers of bone turnover in 14 premenopausal and 17 postmenopausal women.

Studies evaluating the effect of menstrual cycle phase on OC, IGFI, IGFBP3, and DPD have been reported, but they are challenging to interpret because of differences in the definition of menstrual cycle phases. Our finding of increased concentrations of DPD in the EF phase, when compared with the MF and PO phases, is consistent with recent work by Zittermann et al. (21), who observed increased DPD in the EF phase, compared with samples obtained near ovulation. In contrast, others have reported no change in DPD across the menstrual cycle (22, 23) or lower DPD during the follicular phase, when compared with the early luteal phase (24). Our findings of lower concentrations of IGFI in the EF (compared with the ML and MF) phases are consistent with previous studies (25, 26), although there have been reports of unchanged IGFI concentrations across the menstrual cycle (27, 28). These results indicate that controlling for menstrual cycle phase may be necessary to observe changes of small magnitude in studies of IGFI and DPD. Our observations of unchanged OC and IGFBP3 concentrations across the menstrual cycle are consistent with previous reports (22, 23, 24, 26, 27, 28, 29).

To our knowledge, this is the first study to report the effects of soy isoflavone consumption on IGFI, IGFBP3, and markers of bone turnover in humans. The effects seemed to be different in premenopausal and postmenopausal women. In premenopausal women, IGFI and IGFBP3 concentrations were increased by the low-iso diet, but not by the high-iso diet, during the PO and EF phases, respectively. DPD concentrations were increased by both the low- and high-iso diets; however, this increase was observed only in the EF phase. In postmenopausal women, IGFI concentrations were decreased by the high-iso diet, BAP was decreased by both the low-iso and high-iso diets, and there were trends toward decreased OC and IGFBP3 by the high-iso diet.

It is interesting to note that, in the postmenopausal study, all three diets increased BAP, OC, and IGFI (when compared with baseline concentrations), although the increases in OC and IGFI were not statistically significant at the highest dose of isoflavones. This effect is not likely to be attributable to the small increase in calcium consumption between the baseline and the diet periods, nor is it likely to be caused by the increased consumption of protein during the study diets, given that a recent study in premenopausal women consuming protein in similar quantities reported no significant changes in OC, BAP, or N-telopeptide (30).

It is difficult to compare our results with those of the few previously published studies performed in postmenopausal women or animal models. Though we found decreased formation markers and no effects on resorption markers in the postmenopausal women, studies in OVX rats have reported that isoflavones result in increased (12) or unchanged (13, 14) markers of bone formation and decreased (11) or unchanged (11, 12, 13, 14) markers of bone resorption. Related studies have shown that 600 mg/day ipriflavone, a synthetic isoflavone, results in decreased (7, 31) or unchanged (6, 7, 8) markers of bone formation and decreased markers of bone resorption (6, 7, 8, 31) in postmenopausal women. Decreased concentrations of markers of bone turnover are also observed in women treated with HRT (32).

Although the effects observed in this study are interesting, they are not likely to be clinically important. Despite the relatively high doses of soy isoflavones consumed in this study, the magnitude of effects on IGFI, IGFBP3, markers of bone formation (BAP, OC), and markers of bone resorption (DPD and CTX) were quite small. Long-term effects on BMD of such small alterations are not known. In addition, although studies have shown that increased rates of bone turnover are associated with lower BMD (33, 34, 35), recent work indicates that interventions that alter bone turnover markers do not necessarily predict changes in BMD for individual women (36). The failure of isoflavones to show a clinically significant alteration in bone remodeling after 3 months suggests that a long-term benefit on BMD is unlikely. In conclusion, although consumption of soy isoflavones resulted in statistically significant changes in IGFI, IGFBP3, and bone turnover markers, the magnitude of these changes was quite small and not of clinical significance. Thus, our data do not support the hypothesis that dietary isoflavones per se exert beneficial effects on bone turnover in women.

	Acknowledgments

 
The soy protein isolate powders were generously donated by Protein Technologies International. Immunoassay kits were generously donated by Metra Biosystems and Diagnostic Systems Laboratories, Inc. We thank the study volunteers for dedication and hard work, as well as the staff of the Clinical Research Center, University of Minnesota. We also thank Leah Holloway (Geriatric Research, Education, and Clinical Center, Veterans Affairs Medical Center, Stanford University School of Medicine) for analytical expertise. We are grateful to Dr. Will Thomas (Department of Biostatistics, University of Minnesota) for statistical advice.

	Footnotes

 
¹ This research was supported by NIH Grant CA-66016, General Clinical Research Center Grant M01-RR-00400 from the National Center for Research Resources, and Minnesota Agricultural Experiment Station Project 18-34.

Received March 29, 2000.

Revised May 15, 2000.

Accepted May 24, 2000.

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