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See corresponding editorial on page 733.
The effect of soy protein and soy isoflavones on calciummetabolism in postmenopausal women: a randomized crossoverstudy1–3 Lisa A Spence, Elaine R Lipscomb, Jo Cadogan, Berdine Martin, Meryl E Wastney, Munro Peacock, andConnie M Weaver ABSTRACT
established osteopenia. Moreover, soy protein had no effect on Background: Evidence suggests that soy isoflavones act as estro-
bone turnover or bone mineral density (BMD) in ovariectomized gen agonists and have beneficial skeletal effects, but the effects on calcium metabolism in humans are not known.
Clinical studies of the effect of soy isoflavones have been of Objective: This study tested whether soybean isoflavones, soy pro-
short duration, involved relatively few subjects, and examined tein, or both alter calcium metabolism in postmenopausal women.
markers of bone turnover and BMD. Studies in perimenopausal Design: Calcium metabolism in 15 postmenopausal women was
and postmenopausal women found no loss (11) or an increase studied by using metabolic balance and kinetic modeling in a ran- (12) in lumbar spine BMD in women consuming soy protein at 40 domized, crossover design of three 1-mo controlled dietary inter- g/d containing 80 –90 mg isoflavones/d in comparison to women ventions: soy protein isolate enriched with isoflavones (soy-plus taking soy protein devoid of isoflavones (11), whey protein (11), diet), soy protein isolate devoid of isoflavones (soy-minus diet), and or casein-based milk protein (12), all of which produced a de- a casein-whey protein isolate (control diet).
crease in lumbar spine BMD. In a randomized, double-blind, Results: There was no significant difference between the diets in net
placebo-controlled, 12-mo trial, hormone replacement therapy acid excretion (P ҃ 0.12). Urinary calcium excretion was signifi-cantly (P  0.01) less with consumption of either of the soy diets and genistein (54 mg/d) were both effective in increasing femur (soy-plus diet: 85 Ȁ 34 mg/d; soy-minus diet: 80 Ȁ 34 mg/d) than and spine BMD in 90 healthy postmenopausal women (13). In with consumption of the control diet (121 Ȁ 63 mg/d), but fractional contrast, a study in postmenopausal women supplemented with calcium absorption was unaffected by treatment. Endogenous fecal 150 mg isoflavones twice daily for 6 mo resulted in no significant calcium was significantly (P  0.01) greater with consumption of changes in calcaneus BMD (14). Soy protein isolate supple- the soy-minus diet than with consumption of the other diets. Total mented with isoflavones showed no effect in early postmeno- fecal calcium excretion, bone deposition and resorption, and calcium pausal women after 9 mo (15) or in postmenopausal women after retention were not significantly affected by the dietary regimens.
12 mo (16). Furthermore, a landmark 4-y multicenter trial Conclusions: The lower urinary calcium seen with the consumption
showed that ipriflavone had no effect on BMD in postmeno- of an isolated soy protein than with that of an isolated milk protein was not associated with improved calcium retention. This finding The variable results in these studies and the lack of under- reinforces the importance of evaluating all aspects of calcium me- standing of the mechanism by which soy isoflavones affect bone tabolism. Soy isoflavones did not significantly affect calcium leave the relation between soy isoflavones and bone resorption Am J Clin Nutr 2005;81:916 –22.
unanswered. These varied clinical results may be related to thelack of control of the dietary factors that affect bone loss, ie, KEY WORDS
dietary sodium, calcium, and protein. Studies on the effect of isoflavones, calcium absorption, calcium kinetics, urinary calcium isoflavones on calcium metabolism are necessary to determinewhether calcium metabolism is perturbed by soy protein or soyisoflavones. This requires a controlled feeding study and calcium INTRODUCTION
The estrogenic properties of soy isoflavones (1, 2) and the 1 From the Department of Foods and Nutrition, Purdue University, West efficacy of the synthetic isoflavone ipriflavone in reducing bone Lafayette, IN (LAS, ERL, JC, BM, and CMW), Modeling Services, Ltd, loss in rats (3) and humans (4) suggest that soy isoflavones may Dalesford, New Zealand (MEW), and the Indiana University Medical Center reduce bone loss in postmenopausal women. However, evidence General Clinical Research Center, Indianapolis, IN (MP).
of the efficacy of soy isoflavones as an alternative to postmeno- Supported by Indiana Soybean Board, PHS P50-AT000477, and NIH pausal estrogen replacement therapy is conflicting. Comparable Minority Research Grant on Aging AG 16274.
3 Address reprint requests and correspondence to CM Weaver, Depart- bone-sparing effects of 17␤-estradiol and soy protein isolate (5), ment of Foods and Nutrition, Purdue University, 700 W State Street, West genistein (6), and daidzein (6, 7) have been reported in a young Lafayette, IN 47907-2059. E-mail: weavercm@purdue.edu.
ovariectomized rat model, whereas a study in adult ovariecto- mized rats (8) found no benefit of soy isoflavones in reversing Accepted for publication November 12, 2004.
Am J Clin Nutr 2005;81:916 –22. Printed in USA. 2005 American Society for Clinical Nutrition SOY ISOFLAVONES AND CALCIUM METABOLISM IN POSTMENOPAUSAL WOMEN kinetics. Because many subject characteristics and dietary fac- include composition of protein isolates. Final diet composition tors affect bone loss, we thought it important to compare the was directly analyzed for nutrient content. Kilocalorie adjust- effect of soy protein with and without soy isoflavones with that ments were made on an individual basis by using foods that of milk protein in an otherwise constant diet on bone metabolism would not contribute significant calcium, sodium, or protein (eg, in the same postmenopausal women in a crossover study.
fruit and hard candy) to the diet, so that each subject couldmaintain her weight during the intervention. Deionized waterwas allowed ad libitum. All foods and beverages were delivered SUBJECTS AND METHODS
to the free-living subjects at their home or workplace twice a Subjects
The first 7 d of each 28-d intervention was a period of equil- Fifteen healthy, community-dwelling, postmenopausal white ibration to the diet. Days 8 –28 made up the metabolic balance women were recruited through advertisements including flyers period, in which all urine and feces were collected. Completeness and posters. The exclusion criteria included existence of endo- of collections was monitored and corrected for by using mea- crine, gastrointestinal, bone, liver, or kidney disease; participa- surements of urinary creatinine concentrations and of a fecal tion in energy-restricted diets; and sensitivity to soy or milk marker, polyethylene glycol (PEG). Excreta sample collection protein. Excluded medications included hormone replacement containers were delivered to and collected from the subjects on a therapy, drugs for treatment of osteoporosis, thiazide diuretics, daily basis at their home or workplace. On days 8 and 15 of each and steroids. At baseline, a fasting blood sample, urine and fecal phase, subjects were admitted to the General Clinical Research samples, and height and weight data were obtained along with Center (GCRC) at the Indiana University School of Medicine in questionnaires on general health, nutrition, and physical activity Indianapolis for the oral and intravenous administration of ra- dioisotope, respectively. To determine calcium kinetics, 10 ␮Ci 45Ca was given orally and intravenously, after an overnight fast, Study design
with a breakfast meal consisting of the test protein at approxi- The study design was a blinded, randomized, crossover inter- mately one-third of the daily intake (Ȃ13 g) and calcium at vention to measure calcium balance and calcium kinetics along approximately one-third of the daily intake (Ȃ300 mg). Before with urinary sulfate, net acid excretion, and renal function. Each isotopic administration of 45Ca, a catheter with a heparin lock subject, blinded to the intervention and serving as her own con- was inserted into the forearm of the subject and a baseline blood trol, was studied 3 times under 3 different dietary interventions: sample was collected. Timed blood collections occurred at 60, soy protein enriched with isoflavones (soy-plus diet), soy protein 120,180, 240, 300, 360, 420, 540, 720, and 1440 min after the oral void of isoflavones (soy-minus diet), and casein-whey (control isotopic administration and at 5, 10, 20, 60, 90, 120, 150, 180, diet). The 3-phase design allowed comparisons of soy isofla- 240, 300, 420, 600, and 1440 min and 36, 48, 72, 96 h, 6, 8, 10, vones, soy protein, and casein-whey.
12, and 14 d after the intravenous administration.
Under each phase of the study, subjects consumed a controlled Written informed consent was obtained from all subjects. The diet containing the assigned protein for 28 d with a washout protocol was approved by the Human Institutional Review period of ͧ4 wk between phases. The metabolic diets contained Boards and the Radiation Safety Committees at Purdue Univer- Ȃ1100 mg calcium/d, 40 g test protein isolates/d (protein isolates sity, Indiana University-Purdue University Indianapolis, and in powder form were incorporated into baked goods and bever- ages as the only variable of diet), and 40 –50 g protein/d fromother sources (animal and vegetable protein) along with 1800 – Chemical analysis
2000 kcal/d. Test proteins were supplied by The Solae Company(St Louis, MO): the soy protein products with isoflavones were Daily fecal and urine samples were collected in acid-washed made with SUPRO SOY, a soy protein isolate; the soy protein containers. Two 24-h urine samples collected at various times products with trace isoflavones were made with soy protein iso- during days 15–28 of each metabolic period were used for mea- late that had undergone alcohol extraction; and the milk protein surement of net acid excretion (NAE) and sulfate concentrations.
products were made with milk protein isolate. Test proteins were These samples were collected under mineral oil and a 5% (wt: handled in the same manner in recipes used for each of the 3 vol) solution of thymol in isopropanol. Urine for mineral analysis dietary treatments. The energy needs of each subject were esti- was acidified with 1% (by vol) HCl and stored at Ҁ40 °C for mated to allow weight maintenance according to baseline food future analysis. Nonacidified urine aliquots for measurement of records. Macronutrient content was designed to approximate NAE and sulfate were stored at Ҁ20 °C for future analyses. Fecal recommended guidelines of the American Dietetic Association samples were homogenized with deionized water and concen- of 50 – 60% energy from carbohydrates, 30% of energy from fat, trated HCl using a laboratory stomacher (Tekmar Co, Cincinnati, and 10 –20% of energy from protein. The diet provided an array OH); they were then treated in a drying oven at 50 °C for a of fruit, vegetables, pasta, rice, breads, dairy products, fish, poul- minimum of 24 h, ashed in a muffle furnace at 600 °C for 96 h, try, and beef. Vitamin D was taken at 400 IU/d through a sup- and diluted in 1N HCl for total calcium analysis and 45Ca. Both plement consumed from 2 wk before the study throughout the urine and fecal samples were further diluted with LaCl (0.5%)- study periods. A 7-d diet cycle was designed to be constant in HCl (0.5N). Dietary composites for each day of the 7-d cycle daily kilocalories, protein, fat, fiber, magnesium, phosphorus, were collected every 4 mo over the 2.5-year study. Diet, serum, and sodium to prevent possible confounding effects by these urine, and feces were analyzed for total calcium by using atomic nutrients on calcium metabolism. Diets were formulated based absorption spectrophotometry (5100 PC; Perkin-Elmer, Nor- on NUTRITIONIST IV NUTRIENT ANALYSIS software (ver- walk, CT). Serum, urine, and feces were analyzed for 45Ca ac- sion 4.1; First Databank Division, San Bruno, CA) modified to tivity by using beta scintillation counting (Beckman LS 6500; Beckman Instruments Inc, Fullerton, CA). Counts were adjusted Characteristics of the subjects and reference population Urine samples were analyzed for sulfate concentrations with the use of a turbidimetric technique (18). Urinary NAE was measured by titration (Model 290 Acid/Base Auto Titrator; Den-ver Instrument Co, Arvada, CO) (19).
Amino acid analysis was performed on the soy and milk pro- tein isolates by using ion exchange chromatography with ninhy- drin as a derivatization agent (Beckman System 7300; Beckman Serum isoflavone concentrations were analyzed in the Com- prehensive Cancer Center Mass Spectrometry Shared Facility at the University of Alabama at Birmingham with the use of 1 x៮ Ȁ SD (all such values).
reversed-phase HPLC-electrospray ionization and a PE-Sciex 2 Subjects included women who had natural menopause (n ҃ 13) and API III triple quadrupole mass spectrometer (Sciex, Concord, those who had surgical menopause (n ҃ 2).
Canada). To measure isoflavones in dietary samples, methanol 3 Ranges (all such values). The reference data for height, weight, and extraction of the isoflavones from the freeze-dried dietary sam- BMI were taken from the Third National Health and Nutrition Examination ple was followed by HPLC analysis (21). The total isoflavone Survey database (1988 –1994) representing non-Hispanic white women aged content was measured and then converted mathematically to 50 – 69 y (26). The reference data for bone measurements were from refer- Radioimmunoassays, immunoradiometric assay, and enzyme immunoassays—specifically, enzyme-linked immunosorbent weight and BMI were greater in the study population than in the assays—were used to measure biochemical markers of bone turnover and hormonal concentrations in the serum and urine.
The average analytic nutrient content of the dietary compos- Serum 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D were ites is shown in Table 2. The diet was designed to approximate
measured by using radioimmunoassays (DiaSorin Inc, Stillwa- the recommended calcium intake for postmenopausal women, ter, MN), as were estrone-sulfate and sex hormone– binding which is 1200 mg/d. Diets were designed to maintain constant globulin (Diagnostic Systems Laboratories Inc, Webster, TX).
nutrients in each treatment with the exception of the test protein Serum osteocalcin was measured by using a radioimmunoassay and corresponding amino acid composition. The amount of total developed at the Indiana University-Purdue University India- sulfur amino acid content in the milk was 18.7% greater than that napolis General Clinical Research Center. Parathyroid hormone in the soy protein isolate. Mean (Ȁ SD) cystine and methionine was measured using an immunoradiometric assay (Nichols In- concentrations were 0 g/100 g protein and 3.48 Ȁ 0.05 g/100 g stitute Diagnostics, San Juan Capistrano, CA). Both estradiol and estrone were measured by using enzyme immunoassays (Diag- Analytical nutrient composition of the 3 diets per day N-telopeptides of type I collagen, serum bone alkaline phospha- tase (BAP), and serum follicle-stimulating hormone were mea-sured by using an enzyme-linked immunosorbent assay, the OSTEOMARK test (Osttex International, Seattle, WA), the Me-tra test (Quidel Mountain View, Santa Clara, CA), and the AC- TIVE test (Diagnostic Systems Laboratories Inc), respectively.
Statistical analysis and kinetic data analysis
SAS software (version 6.0; SAS Institute Inc, Cary, NC) was used for all statistical analyses. Data were analyzed by account- ing for dietary intervention, order of intervention by time, and subject based on the crossover design. Group mean differences 1 Soy-plus, soy protein isolate enriched with isoflavones; soy-minus, were determined by using analysis of variance with Tukey’s soy protein isolate devoid of isoflavones; control, casein-whey protein iso- grouping at P  0.05. Calcium kinetic data were analyzed by late. Average nutrient values represent triplicate analysis of data from each using WinSAAM software (a Windows program of Simulation, day of the 7-d menu cycle with each of the 3 diet types. The individual protein Analysis, and Modeling; version 2.2.1; National Institutes of isolates contained 67.15– 69.6% protein, ͨ 1% fat, and moisture and ash Health, Bethesda, MD) (22, 23) and a compartmental model (24) including 583– 658 mg sodium/100 g, 821 mg potassium/100 g, 583– 658 mg for all subjects under each dietary intervention.
calcium/100 g, 149 –158 mg magnesium/100 g, 111 mg chloride/100 g, and1030 –1090 mg phosphorus/100 g for soy and 66.4 – 68.7% protein, ͨ1% fat,and moisture and ash including 359 –386 mg sodium/100 g, 639 mg potas- sium/100 g, 759 – 807 mg calcium/100 g, 369 –382 mg magnesium/100 g,111 mg chloride/100 g, and 910 –980 mg phosphorus/100 g for milk.
All subjects completed all 3 phases of the study intervention.
2 x៮ Ȁ SD (all such values).
Subject characteristics are shown in Table 1. The study popu-
3 Total isoflavones were 82 mg/d by a method of the Association of lation was representative of the non-Hispanic white female US Official Analytical Chemists performed by Nestle Purina Analytical Lab (St population in this age group with respect to age at menopause, height, total femur BMD, and lumbar spine BMD (25). Body SOY ISOFLAVONES AND CALCIUM METABOLISM IN POSTMENOPAUSAL WOMEN TABLE 3
Calcium balance and kinetic modeling variables during each diet in postmenopausal women1
1 All values are x៮ Ȁ SD. Values in the same row with different superscript letters are significantly different, P  0.01 (ANOVA with Tukey’s grouping).
2 Soy-plus, soy protein isolate enriched with isoflavones; soy-minus, soy protein isolate devoid of isoflavones; control, casein-whey protein isolate.
3 Direct analytical results. n ҃ 15.
4 Derived results from kinetic modeling. Because of incomplete collection of tracer data, n ҃ 14, 14, and 13 for the soy-plus, soy-minus, and control diets, protein for the milk and 1.19 Ȁ 0.07 g/100 g protein and 1.74 Ȁ globulin concentrations in the study population were above nor- 0.22 g/100 g protein for the soy, respectively.
Dietary treatment had no significant effect on fasting biomar- Calcium kinetics
kers of bone turnover or hormonal markers with the exception of The dietary interventions had no significant effect on calcium serum osteocalcin and serum estrone sulfate (Table 4). Serum
absorption, total fecal calcium, calcium retention, bone deposi- osteocalcin was significantly greater (P  0.05) with the soy- tion, bone resorption, or bone balance. Urinary calcium was minus diet than with the control diet, whereas the concentrations significantly lower with the soy-minus and soy-plus diets than of serum osteocalcin were intermediate with the soy-plus diet.
with the control diet. Endogenous fecal calcium was signifi- Serum estrone sulfate was significantly (P  0.05) greater with cantly greater in the soy-minus diet than in the other 2 dietary the soy-minus diet than with the other 2 dietary interventions.
treatments. The mean values for observed and model calculated Serum isoflavones were significantly (P  0.01) greater during variables under each dietary intervention are shown in Table 3.
the soy-plus diet than during the other 2 dietary treatments.
At day 28 of each dietary treatment, several variables differed Urinary sulfate, net acid excretion, and renal function
significantly from their baseline values (P  0.05) (Table 4).
BAP was significantly less at baseline than with the soy-minus Urinary sulfate was 20% (P  0.05) higher in subjects receiv- diet, but baseline BAP did not differ significantly from concen- ing the control diet than in those receiving the soy diets (4.5 Ȁ 1.1 trations with the other 2 dietary treatments. Serum 25- and 3.6 Ȁ 0.97 mEq/d, respectively), which is reflective of the hydroxyvitamin D was significantly lower at baseline than with 18% greater sulfur amino acid content of the milk protein powder each dietary treatment because of supplementation with vitamin in the control diet. There was no significant treatment effect on D during the intervention. Estrone sulfate was significantly net acid excretion (40.2 Ȁ 19 mEq/d for milk and 38.6 Ȁ 11 greater at baseline than with the control diet and significantly lower at baseline than with the soy-minus diet, but baselineestrone sulfate did not differ significantly from that with the Biomarkers of bone turnover and hormonal markers
The baseline measures for the biochemical markers of bone turnover, serum BAP and osteocalcin, and urinary cross-linkedN-telopeptides of type I collagen and the markers of calcium DISCUSSION
status, parathyroid hormone, 25-hydroxyvitamin D, and 1,25- Calcium absorption, fecal calcium, and calcium retention in dihydroxyvitamin D fell within normal ranges for postmeno- postmenopausal women did not differ significantly with con- pausal women (27) and are shown in Table 4. The hormonal
sumption of any of the 3 diets. The type and concentration of soy status of these subjects was representative of postmenopausal protein isolates and isoflavones used appears not to affect bone women with decreased concentrations of estradiol and estrone deposition, resorption, or balance in postmenopausal women and increased concentrations of follicle-stimulating hormone.
who were beyond the phase of rapid bone loss. The negative Follicle-stimulating hormone concentrations Π25 mIU/mL in- calcium balance observed in this study is common in postmeno- dicate stable menopausal status (28). The sex hormoneРbinding pausal women. The study diets provided 1100 mg calcium/d, TABLE 4
Biomarkers of bone turnover, hormonal markers, and serum total isoflavones at baseline and during each diet in postmenopausal women1
1 BAP, bone alkaline phosphatase; NTx, crosslinked N-telopeptides of type I collagen; BCE, bone collagen equivalents; PTH, parathyroid hormone; FSH, follicle-stimulating hormone; SHBG, sex hormone– binding globulin; 25(OH)D, 25-hydroxyvitamin D; 1,25(OH) D, 1,25-dihydroxyvitamin D. n ҃ 15. Values in the same row with different superscript letters are significantly different (repeated-measures ANOVA with Tukey’s grouping): P  0.05 or P  0.01 (serumtotal isoflavones).
2 Soy-plus, soy protein isolate enriched with isoflavones; soy-minus, soy protein isolate devoid of isoflavones; control, casein-whey protein isolate.
3 x៮ Ȁ SD (all such values).
4 Assay cannot detect concentrations 5 pg/mL.
5 x៮ (all such values).
which was not sufficient to maintain calcium balance. The rec- (control) and soy diets on NAE in our study, possibly because of ommended calcium intake for this population is 1200 mg/d, insufficient power to detect small differences, although others whereas 1500 mg/d has been suggested for postmenopausal have reported increases in both urinary sulfate and NAE on high women who are not receiving hormone replacement therapy.
protein and animal protein diets in younger subjects (36 – 41).
According to the calcium retention of this study population, 1500 Net endogenous acid production calculated by using the method mg calcium/d may have been warranted to obtain a positive of Sebastian et al (42) estimated that the net endogenous acid production of the soy isolate was approximately one-half that of Results from the analysis of hormones and biochemical mark- the milk isolate, at 6.26 and 11.70 mEq/d, respectively.
ers of bone turnover were consistent with the results of the cal- Urinary calcium with the soy diets, regardless of isoflavone cium kinetic analysis. We found no evidence of the effects of content, was lower than that with the milk (control) diet. Reduced dietary treatment on hormones or biochemical markers of bone urinary calcium excretion was likely due to the lower content of turnover, except for serum osteocalcin and estrone sulfate, and sulfur-containing amino acids found in soy protein than in milk the effects on them likely have little clinical significance.
and to the effect of that lower content on the acid-base balance.
In designing this study, we postulated that fractional calcium This urinary conservation was not reflected in calcium retention, absorption could have varied either because of differing bioavail- and the discrepancy may have been due to the high variation in ability of calcium from meals containing different protein pow- fecal calcium, which drives the variation seen in calcium reten- ders or because of an estrogen-like enhancement of calcium tion. One factor that can influence the calcium balance values is absorption due to adaptation to the soy isoflavones. Calcium the degree of compliancy of free-living subjects with excreta fractional absorption was also similar between whole soybeans collection. Laboratory analysis of weekly pooled fecal samples and milk (30) and between tofu and milk (31). In contrast, cal- indicated an average recovery rate of Ȃ80%. Comparison of cium fractional absorption from calcium-fortified soy milk was PEG recovery with chromium-51 chloride hexahydrate recovery 75% of that from cow milk (32). Estrogen has been reported to in balance studies has indicated that stool recovery measured increase calcium absorption, and estrogen replacement therapy with PEG was 81%, whereas that measured with 51Cr was 95% administered to postmenopausal women can return calcium ab- (43). This suggests that PEG recovery may appear lower than the sorption to premenopausal amounts (33–35); however, in the actual stool recovery, and thus collection compliancy may be present study, no estrogen-like enhancement from isoflavones greater than the sensitivity of the PEG assay can detect. Treat- ment effects tested with different PEG compliance percentages The 18% difference in the sulfur amino acid content of the found no difference in calcium retention even when data below protein isolate powders was reflected in the significantly greater 80% compliance were eliminated. On the basis of 80% power urinary sulfate excretion with the control diet than with the soy with an ␣ of 0.05 and an SD for calcium retention of 205 mg (the protein diets. The higher urinary sulfate excretion with the con- mean for all treatments taken from balance methods) in the trol diet is an indication that the acid-generating potential of the present study, a sample size of 180 would have been needed to milk protein was higher than that of the soy protein-powder.
observe a difference of approximately 40 mg in calcium retention There was no significant difference in the effects of the milk between dietary treatments, as was reported for urinary calcium.
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