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KEY WORDS:
calcium
meat protein
postmenopausal
osteoporosis
renal acid
1
Supported primarily by the U.S. Department of Agriculture, with additional
support from the North Dakota Beef Commission.
2
Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does
not imply its approval to the exclusion of other products that may also be suitable.
The U.S. Department of Agriculture, Agricultural Research Service, Northern
Plains Area, is an equal opportunity/affirmative action employer and all agency
services are available without discrimination.
3
To whom correspondence should be addressed.
E-mail: froughea@gfhnrc.ars.usda.gov.
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ABSTRACT Calcium balance is decreased by an increased intake of purified proteins, although the effects of
common dietary sources of protein (like meat) on calcium economy remain controversial. We compared the effects
of several weeks of controlled high and low meat diets on body calcium retention, using sensitive radiotracer and
whole body scintillation counting methodology. Healthy postmenopausal women (n 15) consumed diets with
similar calcium content (600 mg), but either low or high in meat (12 vs. 20% of energy as protein) for 8 wk each,
in a randomized crossover design. After 4 wk of equilibration of each diet, calcium retention was measured by
extrinsically labeling the 2-d menu with 47Ca, followed by whole body scintillation counting for 28 d. Urinary and
blood indicators of bone metabolism were also determined for each diet. Calcium retention was not different during
the high and low meat dietary periods (d 28, mean pooled SD: 17.1 and 15.6%, 0.6%, respectively; P 0.09).
An initially higher renal acid excretion in subjects consuming the high meat compared with the low meat diet
decreased significantly with time. The diets did not affect urinary calcium loss or indicators of bone metabolism.
In conclusion, under controlled conditions, a high meat compared with a low meat diet for 8 wk did not affect
calcium retention or biomarkers of bone metabolism in healthy postmenopausal women. Calcium retention is not
reduced when subjects consume a high protein diet from common dietary sources such as meat. J. Nutr. 133:
1020 1026, 2003.
4
Abbreviations used: BMI, body mass index; cAMP, cyclic adenosine monophosphate; GFR, glomerular filtration rate; IGF-1, insulin-like growth factor 1;
PTH, parathyroid hormone.
TABLE 1
Ingredient differences and nutrient composition of controlled
high and low meat diets consumed by healthy
postmenopausal women for 8 wk each in a
crossover design1,2
High meat
Diet ingredient differences, g/d
Pork
Turkey breast
Beef round
Ham
Chicken breast
Pasta, cooked
Rice, cooked
Pretzels
Croissant
Bagel
Chow mein noodles
Oven-fried potatoes
Margarine, soft
Nutrient composition
Protein, % of energy
g/kg body weight
g/d
Fat, % of energy
Carbohydrate, % of energy
Calcium, mg
Phosphorus, mg
Sodium, mg
Potassium, mg
Sulfur, mg
31
30
117
34
40
20
1.62 0.15
117
31
50
596 19
1679 108
3601 278
2887 339
1789 125
Low meat
70
39
8
5
18
8
26
4
12
0.94 0.09
68
31
60
617 20
1266 90
3243 158
2249 290
1222 55
scintillation counting (29), with adjustments to disregard 47Sc activity. The 47Ca isotope was obtained by neutron activation (University
of Missouri, Columbia, MO) of stable 46Ca (as calcium carbonate,
30.89% enriched; Oak Ridge National Research Laboratory, TN).
The custom-made scintillation counter (30) detects gamma emissions
with 32 crystal NaI(T1) detectors (10 10 41 cm each), arranged
in two planes above and below a bed.
After 4 wk of dietary equilibration, the 2-d menu was labeled with
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 (31), both diets were designed with
milk as the primary source of calcium. For each meal, the tracer was
mixed with milk and allowed to equilibrate overnight. The specific
activity (ratio of 47Ca to elemental calcium) was constant for all
meals for each individual. The energy provided by the radiolabeled
meals was constant between the two diets for each participant. All
labeled meals were consumed at the research center.
The initial total body activity was determined from the whole
body count 13 h after the first labeled meal (before any isotope was
excreted), divided by the fraction of the total activity in the first
meal. Whole body calcium retention was monitored for 28 d. Activity
was corrected to the midpoint of the 2nd d of labeled meals and
adjusted for background and minor fluctuations in the measurement
of a 47Ca standard distributed in water (32). The precision of the
whole body counting measurements was 1.4%.
Analyses. The subjects provided total 48-h urine collections during wk 3, 5 and 8 of each dietary period. Blood samples were drawn
at wk 4 and 8 of each dietary period. Calcium in the urine and
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1021
ROUGHEAD ET AL.
1022
y 1ek1t 2ek2t
where y represents 47Ca retention as a percentage of the administered
dose, t represents the time (in h) and coefficients 1 and 2 represent
the turnover of the radiotracer (%) at rates k1 and k2, respectively.
Because of the early delay in isotope elimination related to the
gastrointestinal transit of unabsorbed isotope, calcium retention data
for d 25 were not included in the model. The percentage of 47Ca
absorbed was separately estimated from the y-intercept of the linear
portion (d 9 28) of a semilogarithmic retention plot [ln (% 47Ca
retained) vs. time].
Diet and sequence effects were evaluated using repeated-measures
ANOVA followed by Tukeys contrasts (41). Variances in the data
were expressed as pooled SD from the ANOVA. When data were not
normally distributed, they were logarithmically transformed and both
transformed and geometric means are reported. Using two-tailed
probabilities, P 0.05 was considered significant. The diet sequence
did not significantly affect any of the reported variables.
RESULTS
Whole body retention and intestinal absorption of calcium. Calcium retention was not adversely affected by the
high meat diet; rather, calcium retention was similar, if not
slightly improved, compared with that of the low meat diet
(Fig. 1, Table 2). As indicated by the two-component exponential model, about 74% of the calcium tracer was cleared
rapidly from the body. This represented early elimination of
the unabsorbed isotope as well as short-term endogenous urinary and fecal excretion, with biological half-lives of 1.7 and
1.5 d, associated with the high and low meat diets, respectively
(Table 2). The remaining calcium tracer was eliminated less
rapidly with biological half-lives of 56 and 40 d in subjects
consuming the high compared with the low meat diets, respectively (P 0.04; Table 2). Thus, loss of calcium tracer
from the body was slightly but significantly greater in subjects
consuming the low meat compared with the high meat diet.
Other measures from the exponential model of calcium retention were not affected by diet (Table 2).
Whole body calcium retention was not affected by diet at
any of the weekly time points tested. On d 28, 17.1 and 15.6%
(0.6) of the calcium tracer was retained from the high and
low meat diets, respectively (P 0.09; Table 2, Fig. 1).
Because the tendency for a slight difference in retention while
consuming the two diets was apparent as early as 5 d after
isotope administration (Fig. 1A), this possible divergence in
the calcium retention observed on d 28 cannot be clearly
attributed to differences in excretory and absorption pathways.
Percentage calcium absorption was not different in subjects consuming the high compared with low meat diets (29.9
vs. 28.4%, Table 2), with about 178 and 175 mg calcium
absorbed daily, respectively. Calcium retention also was not
different in the two diets in women using hormone replacement therapy (n 6). Although the mean calcium retention
in subjects consuming the two diets tended to be slightly lower
in these women, compared with the women not using hormone replacement (n 9), this difference was not significant
(d 28: 14.4 and 18.0%, 2.0, respectively; P 0.08). At least
12 subjects per group would have been needed (with 90%
power) to detect a 5 percentage point difference in calcium
retention between the hormone replacement therapy users and
nonusers.
Biomarkers of bone metabolism. Diet did not affect biomarkers of bone formation (serum bone-specific alkaline
phosphatase, osteocalcin and IGF-1) or of bone resorption
(serum tartrate-resistant acid phosphatase, urinary N-telopeptides) (Tables 3 and 4). Several other indices of bone and
mineral metabolism, such as intact PTH (and urinary cAMP,
an indicator of PTH secretion), estradiol, 25-hydroxy-vitamin
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acid-digested diet aliquots (33) was determined by inductively coupled argon plasma emission spectrophotometry. Mean (SD) measurements were 98 4% of certified values for calcium in a standard
reference material (Typical Diet, 1548b, U.S. National Institute of
Standards and Technology).
Urinary ammonium was determined colorimetrically (34)
(Raichem; Hemagen Diagnostics, San Diego, CA). Titratable acidity
was determined in undiluted urine by titrating to pH 7.40 with 0.1
mol/L NaOH. Free organic acids were measured by the method of
Van Slyke and Palmer (35) as modified by Lemann et al. (36).
Urinary sulfates were determined turbidometrically (37). Enzymelinked immunoassays were used to determine serum bone-specific
alkaline phosphatase (Metra Biosystems, Mountain View, CA), estradiol (Abbott Laboratories, Abbott Park, IL) and urinary cyclic
adenosine monophosphate (cAMP; Biomedical Technology,
Shoughton, MA). Serum tartrate-resistant acid phosphatase activity
was determined using -naphthylphosphate and diazotized-2-amino5-chlorotoluene as substrates (38). Creatinine clearance was calculated from serum and urinary creatinine, which were measured using
alkaline picric acid (39). Serum parathyroid hormone (PTH), calcitonin, osteocalcin (Diasorin, Stillwater, MN) and 25-hydroxy vitamin D (INSTAR, Stillwater, MN) were determined by radioimmunoassay. Serum insulin-like growth factor 1 (IGF-1; Diagnostic
Systems Laboratory, Webster, TX) and urinary N-telopeptides (Ostex, Seattle, WA) were determined by enzyme-linked immunosorbant assays. Plasma-ionized calcium was measured with an electrode
(Nova 8 Electrolyte Analyzer, Waltham, MA) (40).
Statistics. Individual 47Ca retention data were modeled with a
two-component exponential equation:
TABLE 2
Whole body retention of a calcium radiotracer (47Ca)
administered to healthy postmenopausal women at the 4-wk
midpoints of controlled high and low meat diets consumed
for 8 wk each in a crossover design1,2
Variable
1, % of dose
2, % of dose
ln (T1), d
ln (T2), d
Low meat
74.5
25.7
0.51
(1.7, 1.02.8)
4.0
(56.2, 26352)
74.0
26.0
0.38
(1.5, 1.02.8)
3.7*
(40, 24112)
28.6
21.5
18.9
17.1
29.9
26.8
20.6
17.8
15.6
28.4
Pooled
SD
3.1
2.9
0.3
0.4
3.4
2.4
2.1
2.1
2.7
was lower after 3 wk when women consumed the high compared with the low meat diet (5.68 and 6.02), this difference
decreased with time, from 0.34 to 0.02 and 0.03 pH units at 3,
5 and 8 wk, respectively (P 0.05; Table 4). Similarly, a
greater urinary titratable acidity when women consumed the
high compared with the low meat diet was moderated by time
with differences of 15.6, 11.1 and 4.9 mEq/d, at 3, 5 and 8 wk
(P 0.05; Table 4), respectively. This continually diminishing difference suggests that further adaptation may have occurred beyond 8 wk. Urinary free organic acid excretion,
representing compounds such as citric, lactic and acetic acids,
was marginally, but not significantly, higher (P 0.07) when
women consumed the high meat diet (Table 4), and this
difference was also smallest at 8 wk.
As expected, the higher exogenous substrates provided by
the high compared with the low meat diet (Table 1) significantly increased urinary ammonium, sulfate, creatinine and
phosphorus at all time points (Table 4). The high compared
with the low meat diet increased creatinine clearance (1.38
and 1.21 mL/s, respectively, P 0.05; Table 4), without
affecting blood creatinine concentrations (80 9 mol/L).
This likely reflected a difference in the substrate load, rather
than in the glomerular filtration rate (GFR) of these healthy
women (see Discussion).
Despite differences in urinary acidity and excreta, urinary
calcium loss between 3 and 8 wk was unaffected by diet.
Urinary calcium was 2.45 and 2.38 0.88 mmol/d in subjects
consuming the high and low meat diets, respectively, without
detectable change over time (Table 4).
DISCUSSION
These results indicate that high vs. low meat diets consumed for several weeks did not reduce whole body calcium
retention (Table 2, Fig. 1) or adversely affect biochemical
indicators of bone turnover (Tables 3 and 4). Although the
slightly greater retention when women consumed the high
meat diet (significantly longer biological half-life; Table 2, Fig.
1) was of questionable practical importance, it emphasizes that
calcium was at least as well retained in subjects consuming the
high compared with the low meat diet, and that sufficient
TABLE 3
Blood biomarkers of bone metabolism in healthy postmenopausal women consuming
controlled high and low meat diets for 8 wk each in a crossover design1
Biomarker
Tartrate-resistant acid phosphatase, nkat/L
Bone-specific alkaline phosphatase, U/L
Serum ionized Ca,2 mmol/L
ln (intact PTH),3 pmol/L
Calcitonin, mol/L
ln (25-OH vitamin D),4 nmol/L
Osteocalcin, g/L
Insulin-like growth factor 1, g/L
ln (estradiol),5 pmol/L
High meat
Low meat
72
18.1
1.21
0.32
(1.4, 0.73.5)
13.4
4.63
(103, 51210)
4.01
12.7
4.84
(127, 53300)
73
18.3
1.21
0.35
(1.4, 0.53.4)
11.9
4.64
(104, 68268)
3.94
12.8
4.81
(123, 65254)
Pooled
SD
4
1.0
0.02
0.23
3.6
0.16
0.9
1.0
0.40
1 Values are arithmetic means or (geometric means, ranges) and pooled SD from measurements at wk 4 and 8 of each dietary period, except for
serum IGF-1, which was measured only at midpoint of each dietary period (wk 4), n 15. None of the variables was affected by diet.
2 To convert the values for calcium to mg/L, multiply by 40.08.
3 To convert PTH to pg/mL, multiply by 9.5.
4 To convert 25-OH vitamin D to ng/mL, multiply by 0.401.
5 To convert the values for estradiol to pg/mL, multiply by 0.272.
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Retention, % dose
d7
d 14
d 21
d 28
Absorption,3 % dose
High meat
1023
ROUGHEAD ET AL.
1024
TABLE 4
Renal acid excretion and urinary biomarkers of bone metabolism in healthy postmenopausal women consuming
controlled high and low meat diets for 8 wk each in a crossover design1,2
High meat
Calcium, mmol/d
Phosphorus, mmol/d
Sodium, mmol/d
Potassium, mmol/d
Ammonium, mmol/d
ln (sulfate), mmol/d
wk 3
wk 5
wk 8
wk 3
wk 5
wk 8
2.61
22.6
117.3
42.2
42.1
3.1
(22, 1230)
8.7
5.68a
40.2a
2.40
(11, 522)
1.11
(3, 19)
3.82
(46, 22143)
76.6
2.25
19.3
116.6
43.9
40.6
3.1
(22, 1532)
9.2
5.88ab
37.4ab
2.33
(10, 626)
0.92
(3, 15)
3.71
(41, 18116)
72.8
2.47
20.3
120.9
45.6
42.5
3.1
(22, 1230)
8.8
1.38
5.89ab
35.4ab
2.26
(10, 323)
1.01
(3, 17)
3.79
(44, 27103)
71.5
2.43
15.2
109.0
32.4
32.3
2.6
(14, 917)
7.9
6.02b
24.6c
1.96
(7, 215)
1.16
(3, 125)
3.92
(50, 13128)
63.4
2.22
15.2
100.6
31.7
32.7
2.7
(15, 822)
8.1
5.90ab
26.3c
2.15
(9, 417)
0.76
(2, 0.210)
3.90
(49, 29143)
59.7
2.5
19.2
100.0
30.9
35.0
2.6
(14, 523)
7.8
1.21
5.92ab
30.5bc
2.23
(9, 342)
1.03
(3, 114)
3.83
(46, 2785)
64.5
Pooled
SD
Diet effect
0.88
5.7
15.9
5.5
6.3
0.2
NS
0.001
0.0001
0.0001
0.0001
0.0001
1.0
0.18
0.23
7.4
0.56
0.0001
0.05
0.01
0.0001
NS
0.47
NS
0.33
NS
13.4
0.001
1 Values are arithmetic means or (geometric means, ranges) and pooled SD, n 15. To convert the values to mg/d, multiply by 40.08 for calcium,
by 31.0 for phosphorus, by 23.0 for sodium, by 39.0 for potassium, and by 0.131 for hydroxyproline. To convert the values for ammonium to g/d,
multiply by 18.0; to convert the values for creatinine to g/d, multiply by 0.113.
2 Urinary measurements are from total 48-h urine composites collected during wk 3, 5 and 8 of each dietary period [except creatinine clearance,
which was measured at the end of each dietary period (wk 8)].
3 Significant diet time interaction (P 0.05). Values followed by a common letter are not significantly different as determined by Tukeys
contrasts (P 0.05). NS, not significant.
4 Because urinary creatinine excretion was higher when subjects consumed the high meat diet, the cAMP and N-telopeptide data were tested with
and without correction for urinary creatinine, with no change in the statistical results.
5 BCE, bone collagen equivalents.
statistical power existed to detect a more meaningful difference had it occurred. Prospective power analysis estimated
that 12 subjects were needed to detect a difference of 5
percentage points in the percentage absorption (pooled SD of
2.5, 90% power, 0.05, two-tailed test). Data from 14
subjects did not show the hypothesized difference.
The calciuretic effect of dietary protein has been partially
attributed to an increased GFR (17,42). Increased protein
intake from isolated protein sources has been repeatedly reported to increase GFR (10,11,13,42,43) by 10 20%, as measured by creatinine clearance, a magnitude similar to the
present results with meat as the protein source (Table 4).
However, creatinine clearance is not a direct measure of GFR,
given that small quantities of creatinine are reabsorbed or
secreted in the renal tubules, and because a stable intake and
endogenous production of creatinine is assumed (39). The
latter assumption cannot be applied in the present study because meat was an exogenous source of creatinine. The assumption also may not be applicable with an increased intake
of isolated proteins, which may increase endogenous creatinine production (44,45). Unfortunately, previous studies of
increased GFR with increased intake of isolated proteins
(10,11,13,42,43) did not report total creatinine excretion. In
the present study, the apparent greater creatinine clearance
associated with the high meat diet was proportional to a
comparable increase in urinary creatinine excretion, without
affecting urinary calcium (Table 4).
The calciuretic effect of protein has also been related to an
increase in renal acid load, which in this study adapted over
time. The potential renal acid load when women consumed
the high meat diet was estimated to be about twice that when
women consumed the low meat diet (60 vs. 30 mEq/d) using
the method of Remer and Manz (46), which assumes constant
absorption of dietary minerals and electrolytes (without allowing for adaptation over time). The higher dietary acid load
during the high meat dietary period was reflected in the higher
initial renal acid excretion; however, this difference abated
between 3 and 8 wk (Table 4). Furthermore, despite this early
difference in urinary acid excretion and the continuing greater
excretion of sulfate and ammonium, urinary calcium excretion
was not different at any time point tested. Similarly, in previous studies, although addition of meat to the diet has been
associated with hypercalciuria in studies of 3 (47) or 15 d (48),
Spencer and co-workers (15) have reported adaptation that
almost completely reversed an initial hypercalciuria in a single
subject studied for 72 d. Urinary calcium excretion by postmenopausal women, measured during the last 18 d of 7-wk
controlled diets (in a crossover design), does not differ between high and low meat diet periods (14). The present results
concurred with these latter studies (14,15) and suggest that if
early hypercalciuria occurred in response to the high meat
diet, it was reversed by adaptation within 3 wk, despite a more
gradual adaptation in acid excretion (Table 4). In contrast to
meat protein, with increased intakes of isolated protein sources
for 75 (42) or 95 d (10), no adaptation in renal acid excretion
or hypercalciuria was observed. This difference in adaptation
may reflect differences in the protein source.
This studys results were consistent with previous observations that coingested phosphorus offsets the hypercalciuric
effect of protein, possibly through a PTH-mediated mechanism (49), and that net calcium balance is not reduced (14
16,18,50). Our calcium retention results were in contrast to
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Creatinine, mmol/d
Creatinine clearance, mL/s
pH3
Titratable acidity,3 mEq/d
ln (free organic acid), mEq/d
Low meat
ACKNOWLEDGMENTS
We gratefully acknowledge the insightful reviews of the study
protocol by Bess Dawson-Hughes and Robert Heaney. We are also
thankful for the invaluable assistance of the staff at Grand Forks
Human Nutrition Research Center, Carol Zito, Emily Nielsen,
Brenda Hanson, Debbie Krause, Angela Scheett, Jackie Nelson and
Sandra Gallagher. We thank Steve Morris of Missouri Research
Reactor. We are deeply indebted to the study participants.
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