The study involved a double blind, parallel-group randomized controlled trial design to compare the independent and combined effects of isoflavones and exercise training. The setting was the community of Saskatoon, SK, Canada, and involved one center. Participants were randomized on a 1:1:1:1 basis to four unique groups after exclusion criteria were applied: Group 1 was exercise training (combined weight training and walking) plus isoflavone placebo (Ex); Group 2 was exercise training placebo (flexibility program) plus isoflavone therapy (Iso; 165 mg total isoflavone/d or 105 mg aglycone equivalent/d); Group 3 was exercise training plus isoflavone therapy (ExIso); and Group 4 was exercise training placebo plus isoflavone placebo (control). A program combining resistance training and walking was chosen because mixed exercise programs seem to have the most benefit on BMD.4 A higher dose of isoflavone than most other studies was chosen as isoflavones are most effective at higher doses for increasing BMD.20 Randomization was done with a computer-generated allocation schedule through our research pharmacy, which was independent from the rest of the study staff. Randomization was performed using a fixed block size of eight (using a permuted block design with a computer random number generator). Isoflavone and placebo were administered in a double-blind fashion in the form of tablets that were identical in taste, color, and appearance. They were prepacked in bottles and placed into opaque study kits that were sequentially numbered for each woman according to the randomization schedule. Instructions for either the actual exercise program or the placebo exercise program were also placed into each kit. The allocation sequence was concealed from the research assistant enrolling and assessing participants. Corresponding kits were opened only after the enrolled participants completed all baseline assessments and it was time to allocate the intervention. Although the participants could not be blinded to the exercise assignment, they were blinded to the hypothesis that the resistance exercise combined with walking would be superior to the placebo-flexibility exercises. All researchers and those involved in outcome assessment (ie, the individuals performing exercise tests, bone densitometry/sonography, medical examinations, and data analysis) were blinded to group assignment. Exercise tests were run by a research assistant blinded to group assignments. Training was supervised by two additional research assistants who were not involved in any other aspect of the study and who were blinded to whether subjects were on isoflavone or placebo. Another research assistant was in charge of all data entry, during which she remained blinded to group allocations (groups were coded). Our study statistician performed a blinded statistical analysis. The study was approved by the Biomedical Research Ethics Board at the University of Saskatchewan and all participants signed an informed consent form before participating. The study complied with the World Medical Association Declaration of Helskinki—Ethical Principles for Medical Research Involving Human Subjects. We adhered to the Consolidated Standards of Reporting Trials (CONSORT) guidelines for reporting on randomized clinical trials21, 22 including trials with factorial designs.23 This trial was registered with clinicaltrials.gov (http://clinicaltrials.gov/show/NCT00204425. Effect of Exercise Training and Soy-Based Nutritional Supplementation on Prevention of Osteoporosis).
Of 1175 participants who were recruited, 351 eligible postmenopausal women were stratified as either 1 to 9 years postmenopause or over 9 years postmenopause then randomized into four groups, as described above in Study Design (see Fig. 1 for flowchart of participants). Participants were stratified this way because loss of BMD is most rapid in the early years postmenopause and then the rate of loss levels off.24 Participants were recruited from November 2004 to January 2006 and all had completed the intervention by June 2008. Participants in all intervention groups received supplemental calcium and vitamin D (1200 mg and 800 IU, respectively) each day. All participants were postmenopausal, as indicated by questionnaire about their last menstrual period. If women reported that they were less than 2 years postmenopause, menopausal status was verified by determining levels of follicle stimulating hormone and luteinizing hormone. Exclusion criteria were the following: previous fragility fractures (defined as fractures resulting from minimal trauma); having taken bisphosphonates, hormone replacement therapy, selective estrogen receptor modulators (Rolaxofene), parathyroid hormone, or calcitonin within the past 12 months; currently taking corticosteroids or thiazide diuretics; Crohn's disease; Cushing's disease; kidney disease, allergy to soy; severe osteoarthritis; currently involved in vigorous exercise training (defined as jogging or resistance training for more than 20 minutes per session, more than twice per week); planning to travel outside of Saskatoon for an extended period during the study; osteoporosis (lumbar spine or proximal femur BMD 2.5 SD below the young adult mean (ie, T-score of −2.5 or lower); current or previous breast cancer; and current or previous endometrial cancer. All exclusion criteria were determined by questionnaire except osteoporosis, breast cancer, and endometrial cancer, which were evaluated by bone densitometry, mammography, and ultrasound, respectively. Participants who developed osteoporosis or started taking medications for low BMD during the study were removed from the study. This was required for ethical reasons (ie, women who developed osteoporosis were referred to their family physicians for treatment). Participants were recruited via newspaper advertisements and posters from the city of Saskatoon.
Isoflavone or placebo was administered orally in tablets. Isoflavone (Novasoy; Archer Daniels Midland, Inc., Decatur, IL, USA) was administered at a dose of 165 mg per day, containing 105 mg aglycone equivalents per day (35 mg aglycone equivalents taken three times per day). This isoflavone supplement contained genistin, daidzin, and glycitin in a ratio of 1:1:0.2. The placebo tablets contained dicalcium phosphate, magnesium stearate, and sorbitol. Analysis of placebo tablets indicated a minimal amount of isoflavone (0.001 mg per tablet). Contents of the isoflavone and placebo tablets were verified by testing in an independent laboratory (Douglas Laboratories, Pittsburgh, PA, USA). The exercise training intervention consisted of weight training combined with walking. The exercise training placebo consisted of a home-based flexibility program (a type of training that has no effect on bone mineral). Exercise training involved strength (resistance, weight) training twice a week and brisk walking four times per week. The strength training and walking were combined two days per week, at which time exercise sessions were fully supervised. On 2 other days of the week, participants were required to perform walking exercise on their own. Exercises during strength training included: hack squat, hip abduction, adduction, flexion, and extension (on a multihip machine), hamstrings curl, quadriceps (knee) extension, back extension, abdomen flexion, bench press, lat-pull down, shoulder press, biceps curl, and triceps extension (presses). Two sets of eight repetitions for each exercise were done at intensities corresponding to approximately 80% of 1-RM (1 repetition maximum; ie, the maximal amount of weight a participant could lift one time) for hack squat and bench press and at about 8-RM (ie, the maximal amount of weight that could be lifted eight times) for the other exercises. The walking program involved 20 to 30 minutes of brisk walking per session at an intensity corresponding to 70% of age-predicted maximal heart rate (220–age). Women were instructed on how to take their radial pulse for a 15-second count to ensure they were exercising at the proper intensity. Intensity and duration of exercise were increased progressively on an individual basis. Pedometers were given to the women to track walking distance and to encourage compliance. The exercise placebo groups were given a home-based flexibility program that involved stretching exercises for all major muscle groups, requiring 20 to 30 minutes of stretching 4 days per week. The exercise placebo groups were telephoned periodically to assess compliance to the flexibility program (and to decrease the difference in amount of attention given to the exercise and exercise placebo groups). Duration of the interventions was 2 years.
Compliance with the isoflavone and placebo, and the calcium and vitamin D supplement were assessed with logs. Compliance with the exercise program was assessed by attendance at the supervised exercise sessions and logs for the exercises done at home. Participants were surveyed after the study to assess the effectiveness of our blinding by asking if they thought they were on the isoflavone supplement, the placebo, or were not sure.
All outcome measurements were made at baseline, 1 year, and 2 years. Primary outcomes were lumbar spine and proximal femur BMD. Height and mass were measured by a standard stadiometer and a calibrated scale, and recorded to the nearest 0.1 cm and 0.1 kg, respectively, and used to determine body mass index (BMI, kg/m2). Waist circumference was measured at the superior border of the iliac crest (National Institutes of Health protocol).25 Blood pressure was measured using a mercury sphygmomanometer, with the cuff placed over the bare arm about 2 cm above the elbow after the participant was in a comfortable seated position for 5 minutes. Blood pressure was recorded to the nearest 2 mmHg. If systolic blood pressure was greater than 144 or diastolic blood pressure greater than 94, then blood pressure was measured again after the participant remained seated for 5 minutes. In these cases, the second blood pressure measurement was recorded.
Body composition was assessed with dual-energy X-ray absorptiometry (DXA). BMD and bone mineral content of the whole body, lumbar spine (L1–L4 vertebrae), and proximal femur (including the femoral neck, trochanter, Ward's, and total hip) were measured by DXA in array mode (QDR Discovery Wi; Hologic, Inc., Bedford, MD, USA) using QDR software for Windows XP (QDR Discovery, Hologic, Inc.). The coefficients of variation for these measures from our laboratory were 0.5% for the whole body, 0.7% for the lumbar spine, and 1.0% for the proximal femur. Lean tissue and fat mass were assessed from the whole-body scans. In our laboratory, the coefficients of variation for these measurements are 0.5%, and 3%, respectively. Hip structural analysis as described by Beck and colleagues19 was used to assess structural characteristics of three regions of the proximal femur from the DXA scans: the narrow neck region, which is located across the narrowest segment of the femoral neck; the intertrochanteric region along the bisector of the neck-shaft angle; and the femoral shaft, which is located 2 cm distal to the midpoint of the lesser trochanter. For each region the distribution of the bone mass across the bone is extracted, then subperiosteal width (SPW), bone cross-sectional area (CSA), which is equivalent to cortical area, cross-sectional moment of inertia (CSMI), and BMD (grams of bone divided by bone area) were measured. Section modulus (Z), a measure of bone bending strength, is determined by dividing the CSMI by one-half of the SPW. Like bone mineral content, CSA measures the amount of bone within the cross-section but expresses the quantity in terms of cortical equivalent surface area (important for axial bone strength) rather than mineral mass. The coefficients of variation for narrow neck, intertrochanteric, and femoral shaft regions, respectively, were as follows: BMD (1.7%, 1.3%, and 1.3%); SPW (5.3%, 1.8%, and 1.2%); CSA (2.6%, 2.2%, and 1.8%); CSMI (7.2%, 4.3%, and 3.7%); and Z (3.5%, 3.4%, and 2.1%). BMD and geometric measurements of multiple areas of the proximal femur were included as outcomes because each has been associated with fracture risk.26, 27
Ultrasound measurements were made over the distal radius and tibial shaft using a multisite bone sonometer (Sunlight, Omnisense, 7000S; BeamMed Ltd., Petah Tikva, Israel). This gives a measurement of bone speed of sound, which reflects the architecture and density of the bone.18 The coefficient of variation for this measurement in our laboratory is 1.5%.
Overnight fasting blood samples were drawn into Vacutainer serum separator tubes for the analysis of glucose, total cholesterol, HDL cholesterol, LDL cholesterol, and triacylglycerides. Blood was centrifuged at 2160 × g for 10 minutes at 20°C. An LX20 Beckman Coulter analyzer (Beckman Coulter Canada, Inc., Mississauga, ON, Canada) was used to analyze glucose, total cholesterol, HDL cholesterol, and triacylglycerides by enzymatic kits. LDL cholesterol concentrations were calculated using the Friedwald formula.
Analog film mammograms were collected at a breast cancer screening clinic. Quality control with a phantom was performed weekly. A technician, certified through the Canadian Association of Medical Radiation Technologists, reviewed the films. Mammograms were assessed for cancers, cysts, and any other abnormalities.
Transvaginal ultrasonography was used to assess endometrial thickness.28 High-resolution Ultramark 9 and ATL HDI 5000 ultrasound machines (Advanced Technologies Laboratories, Bothell, WA, USA) with 5-MHz to 90-MHz multifrequency convex array transducers were used to acquire imaging data. Endometrial thickness was measured as the distance from the anterior stratum basalis–myometrial junction to the posterior stratum basalis–to the myometrial junction, in the mid-sagittal plane. The transverse and sagittal planes of section that represented the largest dimensions of the fundal aspect of the endometrium were used for all measurements.
Measurements to determine effectiveness of intervention implementation
Strength in the lower body was assessed by determining the 1-RM during the hack squat; whereas upper body strength was determined by the 1-RM during the bench press, as described.29 Dynamic balance was measured as time taken to perform backward tandem walking (ie, toe to heel) over a distance of 6 m on a board that was 10 cm in width and raised about 4 cm off the ground. Number of errors (ie, number of times the participant stepped off the walking board) during the test was also recorded as a measure of dynamic balance. This test is sensitive to the effects of exercise training.30 Walking speed was assessed by timing walking over an 80-m course at a fast pace.31 The coefficients of variation for squat strength, bench press strength, dynamic balance (backward tandem walking time), and walking time over 80 m, were 31.3%, 8.2%, 19.3%, and 4.5%, respectively.
Uncontrolled intervention factors
A food frequency questionnaire, modified to include only Canadian fortification levels, was used to assess individual calcium, vitamin D, and total calories, and isoflavone intake (Block 98.2 FFQ; Block Dietary Data Systems, Berkeley, CA, USA). This food frequency questionnaire has been validated for assessment of isoflavones.32 The food frequency questionnaire was filled out on computer sheets by filling in bubbles beside appropriate food items, with pencil. This computer sheet was then sent for computer scanning to determine nutrient values (Nutrition Quest, Berkeley, CA, USA; www.nutritionquest.com). Physical activity levels outside of the training program were assessed by questionnaire.33 An arbitrary physical activity score is derived from this questionnaire based on frequency of physical activities performed in a typical week and classified as mild, moderate, and strenuous in intensity. This questionnaire has good reliability (test-retest correlation of 0.62–0.74) in middle-age to older adults.33, 34 Validity of this questionnaire has been confirmed in middle-age to older adults by correlating the physical activity score with treadmill time (r = 0.57) and maximal aerobic capacity (r = 0.56).34 We have previously shown that older men (59–76 years of age) who perform poorly on tests of muscular strength and power also have lower amounts of activities classified as “strenuous” from this questionnaire.35
Adverse events were collected on adverse event forms throughout the trial. Adverse events were probed for each time research assistants had contact with participants. This included a description of the adverse event, its relationship to the intervention (not related, unlikely, possibly, probably, definite), whether it was serious (ie, resulted in death, life-threatening, required hospitalization, or resulted in persistent disability) or nonserious, and its intensity (mild, moderate, severe, life-threatening). Endometrial and breast cancers, coronary heart disease, stroke, and pulmonary embolism were assessed for 2 years after the intervention, by questionnaire.
BMD variables from the standard DXA analyses (ie, total hip, trochanter, femoral neck, Wards, lumbar spine, and whole-body BMD) were analyzed by a three-factor multivariate analysis of variance (MANOVA), with between-group factors for exercise (exercise groups versus non-exercise groups) and isoflavone (isoflavone groups versus placebo groups) and a within-subjects factor of time (baseline versus 1 year versus 2 years). When a significant interaction was detected from this MANOVA, univariate tests were performed. When a significant interaction was detected from univariate tests, a Bonferroni post hoc test was used to determine differences between means. There were unequal subject numbers assessed for the remaining dependent variables; therefore, each of these were analyzed by separate three-factor analyses of variance, rather than MANOVA, with between-group factors for exercise and isoflavone, and a within-subjects factor of time. A Bonferroni post hoc test was used to assess differences between means when main effects or interactions were found. All analyses were done using Statistica version 7 (Statsoft, Chicago, IL, USA). Missing observations were assumed to be missing completely at random. Data were collected at baseline, 1 year, and 2 years. The 1-year data were used to account for any missing data at the 2-year mark (ie, data were carried forward). To verify that year 1 data were an appropriate substitute for year 2 data, all analyses were also run using only participants who completed all testing time points (ie, those that dropped out between years 1 and 2 were excluded). Data were analyzed on an intent-to-treat basis; ie, an attempt was made to follow up participants that did not adhere to the exercise or supplementation. Our intent-to-treat analysis included women who were removed during the study when they developed low BMD or were treated for low BMD. To evaluate this effect on our results, we also ran all analyses excluding these women. Adverse events across groups were compared by chi-square analysis. Baseline data are presented as means (SD). All other data are presented as absolute change scores and their 95% confidence intervals. Significance was set at alpha = 0.05.