Controlled substitution of soy protein for meat protein: effects on calcium retention, bone, and cardiovascular health indices in postmenopausal women.

Abstract

In a controlled feeding study, the effects of substituting 25 g soy protein for meat on calcium retention and bone biomarkers were determined. Postmenopausal women (n = 13) ate two diets that were similar, except that, in one diet, 25 g high-isoflavone soy protein (SOY) was substituted for an equivalent amount of meat protein (control diet), for 7 wk each in a randomized crossover design. After 3 wk of equilibration, calcium retention was measured by labeling the 2-d menu with (47)Ca, followed by whole-body counting for 28 d. Urinary calcium and renal acid excretion were measured at wk 3, 5, and 7. Biomarkers of bone and cardiovascular health were measured at the beginning and end of each diet. Calcium was similarly retained during the control and SOY diets (d 28, percent dose, mean +/- pooled sd: 14.1 and 14.0 +/- 1.6, respectively). Despite a 15-20% lower renal acid excretion during the SOY diet, urinary calcium loss was unaffected by diet. Diet also did not affect any of the indicators of bone or cardiovascular health. Substitution of 25 g high isoflavone soy protein for meat, in the presence of typical calcium intakes, did not improve or impair calcium retention or indicators of bone and cardiovascular health in postmenopausal women.

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Controlled Substitution of Soy Protein for Meat Protein:
Effects on Calcium Retention, Bone, and Cardiovascular
Health Indices in Postmenopausal Women
Zamzam K. (Fariba) Roughead, Janet R. Hunt, LuAnn K. Johnson, Thomas M. Badger, and
Glenn I. Lykken
United States Department of Agriculture (Z.K.R., J.R.H., L.K.J.), Agricultural Research Service, Grand Forks Human
Nutrition Research Center, Grand Forks, North Dakota 58202-9034; Arkansas Children’s Nutrition Center (T.M.B.),
Department of Pediatrics and Physiology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72202; and
Physics Department (G.I.L.), University of North Dakota, Grand Forks, North Dakota 58202
In a controlled feeding study, the effects of substituting 25 g
soy protein for meat on calcium retention and bone biomar-
kers were determined. Postmenopausal women (n ? 13) ate
two diets that were similar, except that, in one diet, 25 g high-
isoflavonesoyprotein(SOY)wassubstitutedforanequivalent
amount of meat protein (control diet), for 7 wk each in a ran-
domizedcrossoverdesign.After3wkofequilibration,calcium
retention was measured by labeling the 2-d menu with47Ca,
followed by whole-body counting for 28 d. Urinary calcium
and renal acid excretion were measured at wk 3, 5, and 7.
Biomarkers of bone and cardiovascular health were mea-
sured at the beginning and end of each diet. Calcium was
similarly retained during the control and SOY diets (d 28,
percent dose, mean ? pooled SD: 14.1 and 14.0 ? 1.6, respec-
tively).Despitea15–20%lowerrenalacidexcretionduringthe
SOY diet, urinary calcium loss was unaffected by diet. Diet
also did not affect any of the indicators of bone or cardiovas-
cular health. Substitution of 25 g high isoflavone soy protein
for meat, in the presence of typical calcium intakes, did not
improveorimpaircalciumretentionorindicatorsofboneand
cardiovascular health in postmenopausal women. (J Clin En-
docrinol Metab 90: 181–189, 2005)
I
reservoir and aids the kidneys and lungs in the tight regu-
lation of the systemic hydrogen ion concentration. Dietary
practices that lead to chronic production of acid ash, such as
diets high in meat, are hypothesized to tap into this alkali
reservoir and cause a gradual dissolution of bone mineral (1,
2) and, as such, are considered a risk for hypercalciuria and
osteoporosis (3–5). Although the sulfur amino acids in ani-
mal proteins, such as meat, are thought to cause hypercal-
ciuria, the high phosphorus content of these proteins has
been found to negate this effect (6). Many staple plant pro-
teins,suchaswheatandrice,havesulfuraminoacidcontents
that are similar to or higher than those in meats (7), but the
coexisting alkalis are thought to reduce the dietary acid load
(8). Furthermore, the increased ammoniagenesis observed
with higher protein intake may partly neutralize the acid
production (9). Therefore, the net effect of a protein source
on calcium balance is determined by many coexisting factors
in the protein and in the whole diet and is therefore difficult
to predict. Observational studies have indicated positive
(10–12), negative (5, 13–15), or no association (16) between
animal protein intake and bone health.
N ADDITION TO providing structural framework and
locomotion, the skeletal system serves as a buffering
Very few controlled feeding studies have tested the rela-
tive calciuric effect of animal vs. plant protein food sources,
in the context of a mixed diet (17–19). In a short-term feeding
study (12 d), under conditions of equalized phosphorus and
low calcium (400 mg), higher net renal acid excretion and
urinary calcium loss were observed from diets containing
meat vs. soy protein (19). However, in a recent controlled
feeding study of high- vs. low-meat diets of much longer
duration (8 wk), in which phosphorus content of the diets
was not manipulated and calcium content mimicked typical
intakes (?600 mg/d), no increase in urinary calcium excre-
tion or change in whole-body calcium retention was ob-
served in postmenopausal women (20). Similar calcium re-
tention occurred despite the higher sulfur amino acid intake
and the higher urinary sulfate and renal acid excretion dur-
ing the high-meat diet. Although these findings (20), com-
bined with those from earlier controlled feeding studies (17,
18), indicate that a moderate increase in meat intake does not
impair calcium retention, three short-term (3- to 6-month)
supplementation studies suggest that incorporation of soy
protein isolate in the diet may improve calcium homeostasis
(21–23). Although these studies point to a component in soy
proteinisolate,suchasisoflavones,asanenhancerofcalcium
absorption and/or utilization, the phytate and oxalate con-
tent of soy may reduce the intestinal absorption of calcium
(24) and other minerals (25, 26); thus, the net effect of regular
consumption of soy protein on calcium homeostasis is not
known.
The primary objective of this carefully controlled feeding
study was to determine the effects of daily substitution of
First Published Online October 13, 2004
Abbreviations: CONTROL, Control diet; GFR, glomerular filtration
rate; HDL, high-density lipoprotein; IGF-IBP3, IGF-I binding protein-3;
iPTH, intact PTH; LDL, low-density lipoprotein; NS, not significant;
SOY, 25-g high-isoflavone soy protein (diet).
JCEM is published monthly by The Endocrine Society (http://www.
endo-society.org), the foremost professional society serving the en-
docrine community.
0021-972X/05/$15.00/0
Printed in U.S.A.
The Journal of Clinical Endocrinology & Metabolism 90(1):181–189
Copyright © 2005 by The Endocrine Society
doi: 10.1210/jc.2004-0393
181
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high-isoflavone, intact soy protein for meat protein, in a
typical mixed diet, on calcium retention and bone metabo-
lisminhealthypostmenopausalwomen.Becausetheamount
of soy protein (25 g) was selected to conform with the Food
andDrugAdministration(FDA)-approvedhealthclaim(27),
the secondary objective was to test the effects of this dietary
practice on indicators of cardiovascular health.
Subjects and Methods
Subjects
Postmenopausal women were recruited through public advertising
(television, newspapers) and by direct mailings. The women were se-
lected after an interview and blood analysis and qualified to enter the
study if they were age 50–75 yr; at least 3 yr since last menses; had FSH
more than 40 IU/liter; were nonsmokers; had no apparent underlying
disease; had normal bone mineral density (femoral neck T score ? ?2.5)
as determined by dual-energy x-ray absorptiometry (Hologic Delphi
QDR, Bedford, MA); had normal thyroid, liver, and kidney functions;
were willing to discontinue any nutritional supplements as soon as their
applications were received; and did not regularly use any medications
(except for hormone replacement therapy).
The study was approved by the University of North Dakota Radio-
active Drug Research Committee and Institutional Review Board, and
by the United States Department of Agriculture Radiological Safety
Office. The study was explained verbally and in writing by the inves-
tigators, and written informed consent was given by each woman.
Of 15 women enrolled, two dropped out on the second day of the
study (one because of transportation problems and the other without
providing a reason). The remaining 13 (all white, six using hormone
replacement therapy) were age 59.9 ? 5.0 yr (mean ? sd; range, 52–69),
with a body mass index of 26.0 ? 5.0 kg/m2; range, 19.7–38.5; median,
24.8; and femoral neck bone mineral density, 0.742 ? 0.093 (T-score
range,?2.33to?0.56).Asestimatedfrom3-dfoodrecords,theircalcium
and protein intakes before the study were 857 ? 336 mg/d and 76 ? 23
g/d, respectively.
Diets
Registered dietitians planned two experimental diets using ordinary
foods in a 2-d menu cycle (Table 1). The subjects consumed both diets
for 7 wk, in a randomized crossover design, with a 2-wk break during
which they consumed their self-selected diets but continued to take the
multivitamin provided (Table 1). The weighed control diet (CONTROL)
and 25-g high-isoflavone soy protein (SOY) diets provided (mean ? sd)
similar amounts of protein (15% of energy; 1.32 ? 0.19 and 1.33 ? 0.17
g/kg body weight) but contained 170 and 55 g/d meat, respectively
(Table 1). The amount of calcium in the diet was chosen to be similar to
typical intakes by postmenopausal women in the United States (28).
Although the diets were planned with similar amounts of calcium, the
TABLE 1. Ingredient difference and nutrient composition of controlled high- and low-meat diets consumed by healthy postmenopausal
women for 7 wk each in a crossover designa,b
CONTROLSOY
Diet ingredient difference (g/d)
d 1:
Soy protein isolate (added to banana bread)c
Mandarin oranges
Beefd
Soy protein isolate (added to beef casserole)c
Chickend
White dinner roll
Soy protein isolate (added to brownie)c
d 2:
Soy protein isolate (added to peanut butter)c
Hamd
Soy protein isolate (added to gingerbread)c
Chickend
Soy protein isolate (added to cornbread)c
Tub margarine
Nutrient composition
Protein (% of energy)
Sulfur amino acids (g)
Fat (% of energy)
Saturated fat
Monounsaturated fat
Polyunsaturated fat
Cholesterol (mg)
Carbohydrate (% of energy)
Dietary fiber (g)
Calcium (mg)
Potassium (mg)
Sodium (mg)
Phosphorus (mg)
Magnesium (mg)
Phytate (mg)
?10
?10
?55
?10
?55
?5
?10
?5
?60
?15
?60
?10
?1
1515
3.0
30
7
13
7
220
55
19
2.7
30
7
13
7
157
55
19
690 (670 ? 7)
2617 (2584 ? 290)
3657 (3450 ? 152)
1505 (1596 ? 87)
306
1673
746 (754 ? 17)
2303 (2587 ? 339)
3504 (3323 ? 268)
1533 (1638 ? 104)
321
2246
aAll values are based on energy intake of 9.2 MJ (2200 kcal). Values for protein, carbohydrate, fat, cholesterol, and minerals were calculated
from U.S. Department of Agriculture food composition data. Data in parentheses represent analyzed values for minerals (mean ? SD).
bA chewable multivitamin tablet containing 100% daily value (DV) for vitamins A, C, D, E, B6, B12, thiamin, riboflavin, niacin, folic acid,
and pantothenic acid was taken daily by the participants.
cThe soy protein isolate contained 3.72 mg isoflavones/g protein (2.28 mg aglycone/mg protein; 1.22, 0.87, 0.19 mg/g protein of genistein,
daidzein, and glycitein, respectively).
dThe protein content of beef, chicken, and ham was calculated from U.S. Department of Agriculture, Nutrient Database for Standard
Reference, Release 14 (Nutrient Data Laboratory home page, http//www.nal.usda.gov/fnic/foodcomp) and was 22.03, 19.35, and 23.09 (g/100
g), respectively.
182
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analyzed values for calcium were slightly higher in the SOY diet than
theCONTROL(754?17vs.670?7mgcalcium/2200kcal,respectively),
reflecting the calcium content of the soy protein isolate. The isoflavone
content of the isolate was 2.28 mg aglycone/G protein (product:
IB1.2UN30CA, lot no. 038A-02; Solae Company, St. Louis, MO) (Table
1). Phytate content of the diets was calculated from food composition
tables (29) (Table 1). To maintain body weights, energy intakes were
adjusted by proportionally changing the amounts of all foods. The
energy intakes were similar during the two dietary periods (2216 ? 271
and 2189 ? 300 kcal for CONTROL and SOY, respectively). Amounts of
coffee, tea, and artificially sweetened, noncola, carbonated beverages
(limited to two total servings daily), salt, and pepper were individual-
ized and kept constant. City water and chewing gum were consumed
as desired. Participants were given a list of approved over-the-counter
medications and mouth products. The food items were prepared in
oven-/microwave-oven-safe containers. Participants consumed the
food quantitatively, using spatulas and rinse bottles, eating one meal at
the research center on weekdays and the remaining foods elsewhere.
Calcium retention measurements with47Ca
Dietary calcium retention was measured with a47Ca radiotracer and
whole-body scintillation counting (30) with adjustments to account for
47Sc activity. The47Ca isotope was obtained by neutron activation (Uni-
versity of Missouri, Columbia, MO) of stable46Ca (as calcium carbonate,
30.89% enriched; Oak Ridge National Research Laboratory, Oakridge,
TN). The custom-made scintillation counter (31) detects ?-emissions
with 32 crystal NaI(T1) detectors (10 ? 10 ? 41 cm each), arranged in
two planes above and below a bed on which the subjects lie.
After3wkofdietaryequilibration,allthemealsinthe2-dmenuwere
labeled with a total of 148 kBq (4 ?Ci)47Ca (?4 ?g elemental calcium).
Because of the concern that ingested calcium from some dietary sources
may not form a common absorptive pool (32), both diets were designed
withmilkastheprimarysourceofcalcium.Foreachmeal,thetracerwas
mixed with milk and allowed to equilibrate overnight. The specific
activity (ratio of47Ca to elemental calcium) was constant for all meals
for each individual during both dietary periods. The energy provided
by the radiolabeled meals was constant during each 2-d administration.
All labeled meals were consumed at the research center.
The initial total body activity was determined from the whole-body
count,1–3hafterthefirstlabeledmeal(beforeanyisotopewasexcreted),
divided by the fraction of the total activity that was in the first meal.
Whole-body calcium retention was monitored for 28 d. Activity was
corrected to the midpoint of the 2 d of labeled meals and adjusted for
background and minor fluctuations in the measurement of a47Ca stan-
darddistributedinwater(33).Theprecisionofthewhole-bodycounting
measurements was 1.4%.
Analyses
The subjects provided total 48-h urine collections during wk 0, 3, 5,
and 7 of each dietary period. Fasting blood samples were drawn at the
beginning (wk 1) and end (wk 7) of each dietary period. For homocys-
teine and serum lipids, fasting blood samples were collected twice at the
beginning and end of the study, separated by a few days, and the values
were averaged. Calcium in the urine and acid-digested diet aliquots (34)
was determined by inductively coupled argon plasma emission spec-
trophotometry. Mean (?sd) measurements were 98 ? 4% of certified
values for calcium in a standard reference material (Typical Diet, 1548b,
United States National Institute of Standards and Technology).
Urinary ammonium was determined colorimetrically (35) (Raichem,
Hemagen Diagnostics, San Diego, CA). Titratable acidity was deter-
mined in undiluted urine by titrating to pH 7.40 with 0.1 n NaOH. Free
organic acids were measured by the Van Slyke and Palmer (36) method
as modified by Lemann et al. (37). Urinary sulfates were determined
turbidometrically (38). ELISAs were used to determine bone-specific
alkaline phosphatase (Metra Biosystems, Mountain View, CA) and es-
tradiol (Abbott Laboratories, Abbott Park, IL). The inter- and intraassay
variabilities were 5.2 and 5.0%, respectively, for bone-specific alkaline
phosphataseand6.2and6.7%,respectively,forestradiol.Serumtartrate-
resistant acid phosphatase activity was determined using ?-naphth-
ylphosphate and diazotized-2-amino-5-chlorotoluene as substrates (39).
Creatinine clearance was calculated from serum and urinary creatinine,
which were measured using alkaline picric acid (40). RIAs were used to
determineserumTSH(TSH,T3,T4)(AbbottLaboratories).Theintra-and
interassay variabilities were 4.2 and 5.4%, respectively, for TSH and 3.64
and 4.23%, respectively, for T4. RIAs were also used for intact PTH
(iPTH), osteocalcin, and 25-hydroxyvitamin D (Diasorin, Stillwater,
MN). The intra- and interassay variabilities were 3.6 and 3.4%, respec-
tively, for iPTH; 4.3 and 11.9%, respectively, for osteocalcin; and 8.2 and
8.6%, respectively, for 25-hydroxyvitamin D. Serum IGF-I and IGF-I
binding protein-3 (IGF-IBP3) (Diagnostic Systems Laboratory, Webster,
TX) and urinary N-telopeptides (Ostex, Seattle, WA) were determined
by ELISAs. The intra- and interassay variabilities were 7.1 and 5.4%,
respectively, for IGF-I; 9.6 and 11.4% for IGF-IBP3; and 8.0 and 5.1% for
N-telopeptides.Plasmaionizedcalciumwasmeasuredwithanelectrode
(41) (8?Electrolyte Analyzer, Nova, Waltham, MA). The inter- and
intraassay variabilities for this assay were 2.0 and 3.0%, respectively.
Serum cholesterol fractions and triglycerides were determined using an
automated procedure (Cobas Mira, Roche Diagnostic Systems, Inc.,
Sommerville, NJ). The inter- and intraassay variabilities were 1.0 and
1.2%, respectively, for total cholesterol; 2.1 and 2.6%, respectively, for
high-density lipoprotein (HDL) fraction; and 1.0 and 3.0%, respectively,
for triglycerides. Serum homocysteine was analyzed by fluorescence
polarization immunoassay using an automated procedure (Abbott Lab-
oratories). The inter- and intraassay variabilities were 2.2 and 5.2%,
respectively.
Urine samples from wk 3 were analyzed for the unconjugated isofla-
vones (aglycones) and for total isoflavone content as previously de-
scribed (42, 43). Briefly, for excreted aglycones, samples were extracted
twice with 5 ml diethyl ether, and the organic layers were evaporated
to dryness at 55 C under nitrogen. For conjugated isoflavones, samples
were enzymatically hydrolyzed with Helix pomatia (sulfatase/glucuron-
idase, 100/1000 U) at 37 C for 3 h. All samples were then extracted twice
with 5 ml diethyl ether, and the organic layers were evaporated to
dryness at 55 C under nitrogen. Dried extracts were reconstituted in 0.5
ml of a solvent containing a known amount of biochanin A and injected
into the LC-MS system to determine the aglycone concentrations using
conditions reported previously (1, 2). All samples were analyzed in
triplicate, and the results were expressed as nanograms/milligrams
creatinine after normalization with biochanin A. Genistein (5,7,4?-
trihydroxyisoflavone) and daidzein (7,4?-dihydroxyisoflavone) were
purchased from Indofine Chemical Company, Inc. (Belle Mead, NJ).
Sulfatase type V (aryl-sulfate sulfohydrolase) from Helix pomatia with
reported sulfatase activity of 15–40 U/mg and glucuronidase activity of
400–600 U/mg was purchased from Sigma Chemical Company (St.
Louis, MO).
Statistics
Individual47Ca retention data were modeled with a two-component
exponential equation, y ? ?1e?k1t? ?2e?k2t, where y represents47Ca
retention as a percentage of the administered dose, t represents the time
in hours, and coefficients ?1and ?2represent the fractional biological
turnover of the radiotracer, expressed as percent of dose, at rates k1and
k2, respectively. The calcium retention data for d 2–5 were not included
in the model because they primarily represent the delay in elimination
oftheunabsorbedisotope.Thepercentageof47Cainitiallyabsorbedwas
separately estimated from the y-intercept of the linear portion (d 9–25)
of a semilogarithmic retention plot of percent47Ca retained vs. time.
Diet and sequence effects were evaluated for the parameter estimates
in the calcium retention models, calcium retention on d 7, 14, 21, and 28,
and initial calcium absorption. Urinary and blood measurements (wk 3,
5, and 7) were analyzed by using repeated-measures ANOVA followed
byTukey’scontrasts(44).Variancesinthedatawereexpressedaspooled
sd from the ANOVA. Sequence effects were evaluated before testing for
diet and time effects and were found to be significant (P ? 0.1) only for
serum creatinine, homocysteine, low-density lipoprotein (LDL), total
HDL, and HDL-3. For these variables, the mixed model was modified
to use the diet ? time ? sequence interaction as the error term for the
primary effects of diet and time. When data were highly skewed, they
were logarithmically transformed so that the distribution would more
closely approximate a normal distribution. For these, geometric means
are reported in addition to the mean and pooled sd of the transformed
data. The urinary isoflavone data were highly variable and were there-
fore ranked before ANOVA, and median and range of values are re-
Roughead et al. • Calcium Retention from Soy Protein vs. MeatJ Clin Endocrinol Metab, January 2005, 90(1):181–189
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ported. Using two-tailed probabilities, P ? 0.05 was considered
significant.
Results
Whole-body retention and intestinal absorption of calcium
Substitution of 25 g soy protein for meat protein did not
affect the efficiency of calcium retention at any of the weekly
time points tested (Table 2 and Fig. 1). By d 28, 14.0 and 14.1
(percent dose, ?1.6) of the calcium tracer was retained from
the CONTROL and SOY diets, respectively (Table 2, Fig. 1).
Asindicatedbythetwo-componentexponentialmodel,with
both diets, more than 75% of the tracer was eliminated rap-
idly (biological half-life, 1.5 d), indicating the excretion of the
unabsorbed isotope and early endogenous losses. The re-
maining tracer (?20%) was eliminated less rapidly, with a
biological half-life of 45–49 d (Table 2). Despite the higher
phytate content of the SOY diet (by 573 mg/d), the initial
absorption of calcium, expressed as percent dose, was also
similar between the two diets [mean ? pooled sd, 26.1 vs.
27.0% ? 7.0; not significant (NS); Table 2]. Calcium retention
was not different between the two diets, at any time points
tested, in women using hormone replacement therapy (n ? 6).
Urine composition
Urinary pH was higher with the SOY, compared with
CONTROL, by an average of about 0.15 pH units across all
time points tested (overall mean, 6.33 vs. 6.19, respectively;
P ? 0.0001; Table 3) and decreased with time on both diets
(P ? 0.002). Titratable acidity was also lower by wk 3 of the
SOY diet, and this difference was sustained throughout the
diet period (overall mean, 41.5 vs. 53.4 mEq/d for SOY and
CONTROL, respectively; P ? 0.03; Table 3). Diet did not
affect ammonium excretion at any of the time points tested
(overall mean, 37.6 vs. 43.9 mEq/d for SOY and CONTROL,
respectively, NS; Table 3), at least partially reflecting the
similar nitrogen content of the two menus. However, renal
acid excretion, defined as the sum of ammonium and titrat-
able acidity, was consistently lower during the SOY dietary
period (overall mean, 97.2 vs. 79.1 mEq/d; P ? 0.0001 for
CONTROL and SOY diets, respectively; Table 3). Urinary
free organic acid (representing compounds such as citric,
acetic, and lactic acids) was similar during the two dietary
periods. Urinary sulfate excretion was initially lower during
the SOY than the CONTROL diet at wk 3, but this difference
abated by wk 7 because of a gradual decrease during the
CONTROL and an increase during the SOY dietary period
(diet ? time interaction, P ? 0.01, Table 3). Diet did not affect
creatinine clearance measured at the end of each dietary
period (1.33 vs.1.27 ml/sec for CONTROL and SOY diets,
respectively, NS; Table 3).
Despite the lower urine pH, and approximately15–20%
lower renal acid excretion during the SOY diet, urinary
calcium excretion was similar between the two diets at all
time points tested (overall mean, 3.53 and 3.48 mmol/d,
NS, for the CONTROL and SOY diets, respectively; Table
3). No correlation between renal acid and urinary calcium
excretion was detected. Urinary calcium, phosphorus, and
oxalate excretion slightly increased, and urinary pH de-
creased, over time during both dietary periods (P ? 0.002;
Table 3).
TABLE 2. Whole-body retention of a calcium radiotracer (47Ca),
measured by whole-body scintillation counting, as affected by daily
substitution of SOY for an equivalent amount of CONTROL for 7
wk in healthy postmenopausal womena,b
CONTROL
77.4
22.7
0.43
[1.5]
3.8
[44.7]
24.4
18.2
15.9
14.1
26.1
SOY
78.5
21.6
0.43
[1.5]
3.9
[49.4]
23.5
17.7
15.6
14.0
27.0
Pooled SD
3.0
2.9
0.09
P
?1(% dose)
?2(% dose)
ln (T1)
[Days]c
ln (T2)
[Days]c
Retention @ d 7 (%)
Retention @ d 14 (%)
Retention @ d 21 (%)
Retention @ d 28 (%)
Absorption (% of dose)d
NS
NS
NS
0.13NS
4.8
2.3
1.7
1.6
7.0
NS
NS
NS
NS
NS
aMean ? SD, n ? 12.
bAtwo-componentexponentialmodelfitthedata(R2of0.97to0.99
for models of individuals on each diet). ?1and ?2represent the per-
centage of the isotope having a biological half-life of T1and T2, re-
spectively. One subject’s data could not be modeled using the two-
component exponential equation.
cGeometric means.
dIntestinal calcium absorption, as percentage of dose, was esti-
mated by using the linear portion (d 9–25 after47Ca administration)
of a semilogarithmic retention plot [ln(% retention vs. time)] and
extrapolating back to the time of tracer administration.
FIG. 1. A, Whole-body calcium retention, as percent of dose (mean ?
SEM, n ? 13) over time. Calcium retention was similar between the
CONTROL and SOY diets at all weekly time points tested (see Table
2). B, The two component exponential models that fit the data for 12
of the 13 subjects. These group models (y ? 77.4 e?0.0196t? 22.7
e?0.0007tfor high-meat and y ? 78.5 e?0.0191t? 21.6 e?0.0007tfor
CONTROL, where t is expressed in hours) use the mean coefficients
from the modeled retention curves for each individual. Because of the
variability in the early elimination of the isotope, calcium retention
data for the first 2–5 d were omitted before the modeling of the data.
The retention curves, modeled separately for individual on each diet,
had coefficients of determination (R2) of 0.97–0.99.
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Biomarkers of soy intake
Urinary isoflavone concentrations were used as a confir-
matory marker of soy protein intake. The mean (?sem) total
urinary isoflavone concentrations were 100.41 ? 11.39
?mol/mg creatinine in the soy-consuming women and 7.5?
1.30 ?mol/mg creatinine when no added soy protein was
consumed. Based on our previous experience, the total uri-
nary isoflavone excretion was consistent with the amount of
soy consumed (42, 43). Furthermore, none of the women in
this study were considered equol producers, because the
mean equol excretion rate during the soy intake periods was
13.69 ? 4.46 ?mol/d (range, 1.22–9.49).
Biomarkers of bone metabolism
Substitution of soy protein for meat did not affect bone
metabolism as indicated by specific blood biomarkers of
bone formation (serum bone-specific alkaline phosphatase,
osteocalcin, and IGF-I) or of bone resorption (serum tartrate-
resistantacidphosphatase)(Table4).Severalotherbloodand
urinary indices of bone and mineral metabolism, iPTH, 25-
hydroxyvitamin D (Table 4), cortisol, plasma zinc, magne-
sium, calcium, phosphorus, ionized calcium, ionized mag-
nesium (data not shown), and urinary N-telopeptide and
hydroxyproline excretion, were also unaffected by dietary
treatments(Table3).Dietdidnotchangeanyofthemeasured
indicators of thyroid function (Table 4). The subjects were
replete with vitamin D as indicated by serum 25-hydroxyvi-
tamin D at wk 1 of both dietary periods (71.8 and 68.2 nmol/
liter for CONTROL and SOY, respectively). Although this
studywasconductedduringOctober–MarchinGrandForks,
ND (latitude, 47.5° N), the vitamin D status of the subjects
was successfully maintained and even slightly increased
with a daily vitamin D3supplement of 20 ?g/d (Table 4).
Biomarkers of cardiovascular health
Daily substitution of soy protein for meat protein did not
affect serum homocysteine concentration or the lipid profile
(LDL, HDL, total cholesterol, and triacylglycerol) (Table 5).
Diet also did not change any of the measured hemostatic
indicators, such as fibrinogen, protime, and partial throm-
boplastin time (data not shown).
Discussion
Effects of meat vs. soy protein on calcium homeostasis
The results of this carefully controlled feeding study in-
dicate that a daily substitution of 25 g intact, high isoflavone
soy protein for an equivalent amount of meat protein for
several weeks, in a mixed diet with typical calcium content,
does not improve or impair calcium homeostasis in healthy
postmenopausalwomen.Thisconclusionissupportedbythe
whole-body calcium retention data (Table 2 and Fig. 1) and
by a host of biomarkers of bone metabolism (Tables 3 and 4).
The design of this study was optimized for comparison of
calcium retention by assuring sufficient statistical power (12
subjects were needed to detect a difference of 2.5 percentage
pointsincalciumretentioninacrossoverdesign;90%power;
? ? 0.05; residual sd ? 1.5). The measurements were made
TABLE 3. Urine composition of total 48-h composites as affected by a daily substitution of SOY for an equivalent amount of CONTROL
for 7 wk in healthy postmenopausal women
CONTROL SOY
Pooled
SD
Diet
P value
Time
P value
Wk 3
6.25
28.2
Wk 5
6.17
25.9
Wk 7
6.13
25.1
Wk 3
6.41
19.9
Wk 5
6.29
20.4
Wk 7
6.30
20.2
pH
Titratable acidity
(mEq/d)
Ammonium
(mmol/d)
Renal acid excretion
(mEq/d)a
ln (free organic acid)
(mEq/d)
Sulfate (mmol/d)b
ln (calcium)
[mg/d (mmol/d)]
Phosphorus
[mg/d (mmol/d)]
Oxalate
[mg/d (?mol/d)]
Creatinine
[g/d (mmol/d)]
ln (N-telopeptide),
nM BCE/mM
creatinine
Hydroxyproline
[mg/d (?mol/d)]
Creatinine clearance
(ml/sec)
0.12 0.0001 0.002
8.10.03 NS
43.541.9 46.135.035.5 42.25.8 NS NS
70.9 66.369.455.056.462.310.50.0001 NS
1.82 [6.17]1.94 [6.96]1.57 [4.81] 1.22 [3.39] 1.63 [5.10]1.75 [5.75] 0.56 NSNS
17.0x
4.86 (3.25)
[130.0]
964.9 (31.2)
15.3x,y
4.81 (3.09)
[123.6]
866.8 (28.0)
16.8x
5.06 (3.97)
[158.8]
1167.5 (37.7)
14.3x
4.86 (3.22)
[128.7]
874.4 (28.2)
15.4x,y
4.88 (3.32)
[132.6]
896.8 (29.0)
16.4x
5.05 (3.93)
[157.2]
1097.5 (35.4)
1.7
0.22
0.01
NS
0.03
0.001
173.2NS0.0001
26.2 (291) 24.5 (272) 28.3 (314)21.4 (238) 24.2 (269) 27.5 (305) 3.6NS 0.0004
1.10 (9.7) 1.11 (9.8)1.14 (10.1) 0.98 (8.6) 1.01 (8.9) 1.07 (9.5) 0.12 0.003NS
3.08 [21.8]3.20 [25.0] 0.24NS
0.108 (107.9) 0.105 (104.5) 17NS
1.38 1.37 0.16 NS
Data are means with pooled SD from ANOVA (n ? 13). P values are from ANOVA. Data in brackets are geometric means.
aDefined as sum of titratable acidity and ammonium excretion. BCE, Bone collagen equivalents.
bSignificantly affected by diet ? time interaction. Means without a common superscript (x, y) are significantly different as determined by
Tukey’s contrasts (P ? 0.05).
Roughead et al. • Calcium Retention from Soy Protein vs. MeatJ Clin Endocrinol Metab, January 2005, 90(1):181–189
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