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
established osteopenia. Moreover, soy protein had no effect on
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
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
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,
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
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
unanswered. These varied clinical results may be related to thelack of control of the dietary factors that affect bone loss, ie,
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
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: email@example.com.
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
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,
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 –
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
Ȁ SD (all such values).
reversed-phase HPLC-electrospray ionization and a PE-Sciex
Subjects included women who had natural menopause (n
҃ 13) and
API III triple quadrupole mass spectrometer (Sciex, Concord,
those who had surgical menopause (n
Canada). To measure isoflavones in dietary samples, methanol
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
-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
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.
Ȁ SD (all such values).
Subject characteristics are shown in Table 1
. The study popu-
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
Calcium balance and kinetic modeling variables during each diet in postmenopausal women1
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
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-
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).
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
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 25 mIU/mL in-
calcium balance observed in this study is common in postmeno-
dicate stable menopausal status (28). The sex hormone– binding
pausal women. The study diets provided 1100 mg calcium/d,
Biomarkers of bone turnover, hormonal markers, and serum total isoflavones at baseline and during each diet in postmenopausal women1
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).
Soy-plus, soy protein isolate enriched with isoflavones; soy-minus, soy protein isolate devoid of isoflavones; control, casein-whey protein isolate.
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.
SOY ISOFLAVONES AND CALCIUM METABOLISM IN POSTMENOPAUSAL WOMEN
Although we lacked the power to determine small differences
in calcium retention by using the metabolic balance approach,
1. Markiewica L, Garey J, Adlercreutz H, Gurpide E. In vitro bioassays of
given the large variation in fecal calcium, it is more likely that the
non-steroidal phytoestrogens. J Steroid Biochem Mol Biol 1993;45:
soy treatments are not beneficial to bone, despite the difference
2. Mayr U, Butsch A, Schneider S. Validation of two in vitro test systems
in sulfur amino acid load. For no changes in calcium retention to
for estrogenic activities with zearalenone, phytoestrogens and cereal
occur when urinary calcium excretion is reduced, endogenous
extracts. Toxicology 1992;74:135– 49.
fecal calcium losses must increase, as was observed during the
3. Cecchini MG, Fleisch H, Muhlbauer RC. Ipriflavone inhibits bone re-
soy-minus treatment and as has been observed elsewhere with
sorption in intact and ovariectomized rats. Calcif Tissue Int 1997;61:
increases in dietary phosphorus (44, 45). Increasing dietary pro-
4. Gennari C, Adami S, Agnusdei D, et al. Effect of chronic treatment with
tein blunts the calciuretic effect of the protein by increasing
ipriflavone in postmenopausal women with low bone mass. Calcif Tis-
intestinal calcium excretion (46, 47) without an effect on calcium
balance. Studies targeted only at understanding urinary calcium
5. Arjmandi BH, Alekel L, Hollis BW, et al. Dietary soybean protein
losses are unable to determine the benefits of a treatment. In
prevents bone loss in an ovariectomized rat model of osteoporosis. J Nutr
support of our findings, whole-body 47Ca retention, a sensitive
technique of whole-body retention that is not affected by fecal
6. Ishida H, Uesugi T, Hirai K, Toda T, Nukaya H, Yokotsuka K, Tsuji K.
Preventative effects of the plant isoflavones, daidzein and genistein, on
variation or complete collection, did not differ significantly be-
bone loss in ovariectomized rats fed a calcium-deficient diet. Biol Pharm
tween high- and low-meat diets in postmenopausal women (48).
Although these results may be affected by the nature of the
7. Fanti P, Monier-Faugere MC, Geng Z, et al. The phytoestrogen genistein
proteins substituted for meat—ie, high-sulfur amino acid cereal
reduces bone loss in short term ovariectomized rats. Osteoporosis Int
proteins could also be calciuric (49)—these women’s diets
8. Picherit C, Bennetau-Pelissero C, Chanteranne B, et al. Soybean isofla-
achieved a 16% difference in urinary sulfate load. Urinary 47Ca
vones dose-dependently reduce bone turnover but do not reverse estab-
excretion was initially greater on the high-meat diet, but it
lished osteopenia in adult ovariectomized rats. J Nutr 2001;131:723– 8.
9. Jayo Mj, Anthony MS, Register TC, Rankin SE, Vest T, Clarkson TB.
Our study cannot exclude the possibility that soy isoflavones
Dietary soy isoflavones and bone loss: a study in ovariectomized mon-
increase calcium retention at high doses or that soy isoflavones
keys. J Bone Miner Res 1996;11:S228 (abstr).
10. Lees CJ, Ginn TA. Soy protein isolate diet does not prevent increased
are site specific or more effective immediately after menopause,
cortical bone turnover in ovariectomized macaques. Calcif Tissue Int
when bone turnover is higher. Other studies using similar or
higher doses of isoflavones have produced mixed results. Wan-
11. Alekel L, St Germain A, Peterson CT, Hanson K, Stewart JW, Toda T.
gen et al (28) provided dietary isoflavones at 65 mg/d and 132
Isoflavone-rich soy protein isolate attenuates bone loss in the lumbar
mg/d and found no clinically relevant effects on bone biomarkers
spine of perimenopausal women. Am J Clin Nutr 2000;72:844 –52.
12. Potter SM, Baum JA, Teng H, Stillman RJ, Shay NF, Erdman JW. Soy
or hormones. Similarly, BMD was not significantly affected after
protein and isoflavones: their effects on blood lipids and bone density in
supplementing postmenopausal women with 150 mg isoflavones
postmenopausal women. Am J Clin Nutr 1998;68(suppl):1375S–9S.
twice daily for 6 mo (14) or with 99 mg isoflavones/d for 12 mo
13. Morabito N, Crisafulli A, Vergara C, et al. Effects of genistein and
(16). On the other hand, isolated soy protein enriched with soy
hormone-replacement therapy on bone loss in early postmenopausal
isoflavones increased BMD at the lumbar spine in perimeno-
women: a randomized double-blind placebo-controlled study J BoneMiner Res 2002;17:1904 –12.
pausal and postmenopausal women after 9 and 6 mo, respectively
14. Hsu CS, Shen WW, Hsueh YM, Yeh SL. Soy isoflavone supplementa-
tion in postmenopausal women: effects on plasma lipids, antioxidant
Controlled feeding studies that combine balance and kinetic
enzyme activities and bone density. J Reprod Med 2001;46:221– 6.
tracer analysis are useful in quantitating changes in calcium metab-
15. Gallagher JC, Satpathy R, Rafferty K, Haynatzka V. The effect of soy
olism in response to short-term treatment. To effect changes in bone
protein isolate on bone metabolism. Menopause 2004;11:290 – 8.
mass, calcium balance must first be perturbed. In this study, cal-
16. Kreijkamp-Kaspers S, Kok L, DE Grobbee, et al. Effect of soy protein
containing isoflavones on cognitive function, bone mineral density, and
cium absorption, bone turnover, and bone balance were not affected
plasma lipids in postmenopausal women: a randomized controlled trial.
by protein type or the presence of isoflavones. The greater calciuria
with the control (milk) diet than with the soy diets and the associ-
17. Alexandersen P, Toussaint A, Christiansen C, et al. Ipriflavone in the
ated changes in sulfate excretion are consistent with results of other
treatment of postmenopausal osteoporosis. JAMA 2001;285:1482– 8.
studies using purified proteins to simulate protein from animal and
18. Ma RSW, Chan JCM. Endogenous sulphuric acid production: a method
of measurement by extrapolation. Clin Biochem 1973;6:82–7.
vegetable sources. However, net calcium retention was not im-
19. Chan JCM. The rapid determination of urinary titratable acid and am-
proved by the reduction in urinary calcium, which suggests that it is
monium and evaluation of freezing as a method of preservation. Clin
important to evaluate overall calcium metabolism rather than to rely
exclusively on urinary calcium for predicting consequences to
20. Zumwalt RW, Absheer JS, Kaiser FE, Gehrke CW. Acid hydrolysis of
bone. Long-term studies to ascertain the effectiveness of soy isofla-
proteins for chromatographic analysis of amino acids. J AOAC 1987;70:147–51.
vones in reducing bone loss in postmenopausal women are not
21. Barnes S, Coward L, Kirk M, Sfakianos J. HPLC-mass spectrometry
likely to be successful with the type and dose used in our study.
analysis of isoflavones. Exp Biol Med 1998;217:254 – 62.
22. Greif P, Wastney M, Linares O, Boston R. Balancing needs, efficiency,
We thank Stephen Barnes at the University of Alabama at Birmingham, in
and functionality in the provision of modeling software: a perspective of
whose laboratory and with the assistance of the laboratory research staff the
the NIH WinSAAM project. Adv Exp Med Biol 1998;445:3–20.
23. Berman M, Weiss MF. SAAM manual. DHEW Publication no. (NIH)
CMW was responsible for study design; LAS, ERL, JC, and MP were
78-180. Washington, DC: Government Printing Office, 1978.
responsible for data collection; LAS, ERL, BM, and MEW were responsible
24. Wastney ME, Ng J, Smith D, Martin BR, Peacock M, Weaver CM.
for data analysis; and LAS, ERL, and CMW were responsible for writing the
Differences in calcium kinetics between adolescent girls and young
manuscript. None of the authors had any financial or personal conflicts of
women. Am J Physiol 1996;271:R208 –16.
25. Kleerekoper M, Nelson DA, Peterson EL, et al. Reference data for bone
mass, calciotropic hormones, and biochemical markers of bone remod-
37. Schuette SA, Hegsted M, Zemel MB, Linkswiler HM. Renal acid, uri-
eling in older (55–75) postmenopausal white and black women. J Bone
nary cyclic AMP, and hydroxyproline excretion as affected by level of
protein, sulfur amino acid, and phosphorus intake. J Nutr 1981;111:
26. National Center for Health Statistics. Tables 6, 9, and 15. In: NHANES
anthropometric reference data, United States, 1988-1994. Internet:
38. Schuette SA, Zemel MB, Linkswiler HM. Studies on the mechanism of
protein-induced hypercalciuria in older men and women. J Nutr 1980;
Anthropometric%20Measures.htm (accessed October 2003).
27. Kress BC. Appendix iv: Laboratory values of importance for calcium
39. Lutz J. Calcium balance and acid-base status of women as affected by
metabolism and metabolic bone disease. In: Favas MJ, ed. Primer on the
increased protein intake and by sodium bicarbonate ingestion. Am J Clin
metabolic bone diseases and disorders of mineral metabolism. 4th ed.
Philadelphia: Lippincott Williams & Wilkins, 1999:476 – 8.
40. Hegsted M, Schuette SA, Zemel MB, Linkswiler HM. Urinary calcium
28. Wangen KE, Duncan AM, Merz-Demlow BE, et al.Effects of soy isofla-
and calcium balance in young men as affected by level of protein and
vones on markers of bone turnover in premenopausal and postmeno-
phosphorus intake. J Nutr 1981;111:553– 62.
pausal women. J Clin Endocrinol Metab 2000;85:3043– 8.
41. Schuette SA, Linkswiler HM. Effects on Ca and P metabolism in humans
29. Selby C. Sex hormone binding globulin: origin, function, and clinical
by adding meat, meat plus milk, or purified proteins plus Ca and P to a
significance. Ann Clin Biochem 1990;27:532– 41.
low protein diet. J Nutr 1982;112:338 – 49.
42. Sebastian A, Frassetto LA, Sellmeyer DE, Merriam RL, Morris RC.
30. Heaney RP, Weaver CM, Fitzsimmons ML. Soybean phytate content:
Estimation of the net acid load of the diet of ancestral preagricultural
effect on calcium absorption. Am J Clin Nutr 1991;53:745–7.
Homosapiens and their hominid ancestors. Am J Clin Nutr 2002;76:
31. Martin BR, Weaver CM, Heaney RP, Packard PT, Smith DL. Calcium
absorption from three salts and CaSO4-fortified bread in postmeno-
43. Eastell R, Dewanjee MK, Riggs BL. Comparison of polyethylene glycol
pausal women. J Agric Food Chem 2002;50:3874 – 6.
and chromium-51 chloride as nonabsorbable stool markers in calcium
32. Heaney RP, Dowell MS, Rafferty K, Bierman J. Bioavailability of the
balance studies. Bone Miner 1989;6:95–105.
calcium in fortified soy imitation milk with some observations on the
44. Spencer H, Kramer L, Osis D. Do protein and phosphorus cause calcium
method. Am J Clin Nutr 2000;71:1166 –9.
33. Writing Group for the Women’s Health Initiative Investigators. Risk and
45. Hunt JR, Gallagher SK, Johnston LK, Lykken GI. High- vs low-meat
benefits of estrogen plus progestin in healthy postmenopausal women.
diets: effects on zinc absorption, iron status, and calcium, copper, iron,
Principal results from the Women’s Health Initiative randomized con-
magnesium, manganese, nitrogen, phosphorus and zinc balance in post-
trolled trial. JAMA 2002;288:321–33.
menopausal women. Am J Clin Nutr 1995;62:621–32.
34. Heaney RP. Estrogen-calcium interactions in the postmenopause: a
46. Heaney RP. Excess dietary protein may not adversely affect bone. J Nutr
quantitative description. Bone Miner 1990;11:67– 84.
35. Riggs BL, Khosla S, Melton LJ III. A unitary model for involutional
47. Heaney RP, Recker RR. Effects of nitrogen, phosphorus, and caffeine on
osteoporosis: estrogen deficiency causes both type I and type II osteo-
calcium balance in women. J Lab Clin Med 1982;99:46 –55.
porosis in postmenopausal women and contributes to bone loss in aging
48. Roughead ZK, Johnson LK, Lykken GI, Hunt JR. Controlled high meat
men. J Bone Miner Res 1998;13:763–73.
diets do not affect calcium retention or indices of bone status in healthy
36. Breslau NA, Brinkley L, Hill KD, Pak CYC. Relationship of animal
postmenopausal women. J Nutr 2003;133:1020 – 6.
protein-rich diet to kidney stone formation and calcium metabolism.
49. Sebastian A. Low versus high meat diets: effects on calcium metabolism.
J Clin Endocrinol Metab 1988;66:140 – 6.
Subject: Attachments: From: Fourways Equine Clinic <> Your Trainer says “Your horse bleeds!” An explanation – Exercised Induced Pulmonary Haemorrhage. By Dr. Willem LF van Wyk BSc, BVSc Wellness Program Exercise induced pulmonary haemorrhage (EIPH) is a common condition seen often in race Launched! horses where the horse bleeds into the airways following/during wo
Parker & Waichman, LLP Attorneys at Law Viagra Information Guide The Case Against Viagra: In a recent study, US researchers describe seven patients who developed nonarteritic ischemic optic neuropathy (NAION), an eye ailment that can result in permanent vision loss, after taking Viagra (sildenafil) for erection difficulties. Combined with past reports, this Manufacturer: s