Supplemental Material can be found at:
http://jn.nutrition.org/content/suppl/2009/10/21/jn.109.10637
7.DC1.html
The Journal of Nutrition
Nutrient Requirements and Optimal Nutrition
A High Mixed Protein Diet Reduces Body Fat
without Altering the Mechanical Properties of
Bone in Female Rats1–3
Kathleen M. Pye,4 Andrew P. Wakefield,5 Harold M. Aukema,5 James D. House,5 Malcolm R. Ogborn,5
and Hope A. Weiler4*
4
School of Dietetics and Human Nutrition, McGill University, Macdonald Campus, Ste-Anne-de-Bellevue H9X 3V9, Quebec, Canada;
and 5Department of Human Nutritional Sciences, University of Manitoba, Winnipeg R3T 2N2, Manitoba, Canada
Abstract
Long-term consumption of high-protein (HP) diets at 35% of energy is postulated to negatively influence bone health.
Previous studies have not comprehensively examined the biochemical, physical, and biomechanical properties of bone
metabolism, mass, and strength in rats. Adult female Sprague-Dawley rats (n = 80) were randomized to receive for 4, 8, 12,
or 17 mo a normal-protein (NP) control diet (15% of energy) or a HP diet (35% of energy). Diets were balanced for calcium
because the protein sources were rich in calcium. At each time point, measurements included weight, body composition,
and bone mass using dual-energy X-ray absorptiometry, mechanical strength at the mid-diaphysis of femur and tibia,
microarchitecture of femurs using microcomputerized tomography and serum osteocalcin, carboxy-terminal crosslinks of
type I collagen (CTX), insulin-like growth factor-1 (IGF-1), leptin, and adiponectin. Effects of diet, time, and their interaction
were tested using factorial ANOVA. The HP diet resulted in lower body weight, total body, and abdominal fat and higher
lean mass. Serum leptin and adiponectin were greater in HP-fed than in NP-fed rats, but IGF-1 did not differ between the
groups. Whereas the HP diet resulted in higher relative bone mineral content (g/kg) in the femur, tibia, and vertebrae, serum
osteocalcin and CTX and bone internal architecture and biomechanical strength were unaffected. In conclusion, HP diets at
35% of energy lower body fat content without hindering the mechanical and weight-bearing properties of bone. J. Nutr.
139: 2099–2105, 2009.
Introduction
Since the implementation of the dietary reference intakes,
protein has been expressed in terms of the acceptable macronutrient distribution range (AMDR),6 defined as the level at which
the consumption of a specific source of energy provides an
acceptable nutrient value while concurrently reducing incidence
of disease (1). For protein, the lower and upper AMDR ranges
for adults are currently set at 10 and 35%, respectively (2).
There is presently limited experimental affirmation to deem the
1
Supported by a grant from the Canadian Institutes for Health Research and a
Canadian Foundation for Innovation Grant and Canada Research Chair (H.W.).
2
Author disclosures: K. M. Pye, A. P. Wakefield, H. M. Aukema, J. D. House,
M. R. Ogborn, and H. A. Weiler, no conflicts of interest.
3
Supplemental Table 1 and Figure 1 are available with the online posting of this
paper at jn.nutrition.org.
6
Abbreviations used: AMDR, acceptable macronutrient distribution range;
BMC, bone mineral content; BMD, bone mineral density; BW, body weight;
CTX, carboxy-terminal crosslinks of type I collagen; DRI, dietary reference
intakes; DXA, dual-energy X-ray absorptiometry; FM, fat mass; HP, high-protein
diet; IGF-1, insulin-like growth factor 1; LBM, lean body mass; NP, normal-protein
diet; mCT, microcomputerized tomography.
* To whom correspondence should be addressed. E-mail: hope.weiler@mcgill.
ca.
35% upper limit AMDR for protein as safe with respect to longterm human health (2). Because high-protein (HP) diets at or
above AMDR are widely utilized as a means for weight loss and
control (3), this lack of evidence is particularly worrisome (4).
Excessive protein consumption may deleteriously affect renal
(5) and bone health (6,7). Acting as a buffer system, bone aids in
the modulation of acid-base homeostasis (8) through the release
of calcium (9). In theory, elevated protein consumption is
acidogenic (6), resulting in increased bone resorption (10). An
observational study in women suggests that HP diets are
associated with higher bone resorption (11), while 2 others
suggest that HP diets of animal sources resulted in higher risk of
fracture of the forearm (12) or hip fracture (13). This was
confirmed by observations in healthy rats (14,15). Conversely,
other HP studies in both healthy rodents (9,16,17) and elderly
adults (18) did not show a negative relationship, highlighting the
uncertainty surrounding the effect of HP consumption on bone.
To fully understand the effect of high protein intakes on bone,
it is necessary to comprehensively investigate the physiological,
anthropometrical, biochemical, and biomechanical parameters
of bone during short- to long-term HP consumption. Because
many protein sources such as milk powder are rich in calcium, it
0022-3166/08 $8.00 ã 2009 American Society for Nutrition.
Manuscript received March 3, 2009. Initial review completed April 9, 2009. Revision accepted August 12, 2009.
First published online September 16, 2009; doi:10.3945/jn.109.106377.
2099
Downloaded from jn.nutrition.org by guest on November 4, 2012
required to arrive at this conclusion. Our objective in this study was to examine the long-term effect of a HP diet on bone
is important to control for dietary calcium intakes. Therefore, it
was the purpose of this study to comprehensively examine the
effect of long-term elevated protein consumption at the current
AMDR upper range of 35% on bone and body composition.
This research represents an arm of a larger study designed to
address renal and bone consequences of a high protein diet in
Sprague-Dawley rats, a rodent strain appropriate for such
analyses (19).
Methods and Materials
TABLE 1
Diet composition of NP and HP diets
NP
(15% AMDR)
HP
(35% AMDR)
g/kg diet
Wheat (14.6% protein)
Barley (10.8% protein)
Low ash poultry meal (67.4% protein)
Pork meal (57.4% protein)
Dried egg albumen (74.6% protein)
Skim milk powder (34.1% protein)
Sucrose
Corn starch
Vitamin mix (AIN-93VX)1
Mineral mix (AIN-93M without Ca, P)1
Calcium carbonate
Sodium phosphate monobasic
Dicalcium phosphate
Potassium chloride
Sodium chloride
Lactose
Canola oil
Lard
1
246.0
61.0
32.0
22.0
35.0
78.0
166.0
262.0
10
132
0.14
0
9.38
5.6
6.3
29.0
13.0
12.0
246.0
61.0
32.0
22.0
250.0
140.0
80.0
127.0
10
133
0
0.74
1.49
1.9
2.1
0
13.0
6.0
Based on the AIN-93 for maintenance (20).
Dicalcium phosphate (0.9 g/100 g), potassium chloride (0.6 g/100 g), and sodium
chloride (0.6 g/100 g) were added to the NP diet.
3
Sodium phosphate monobasic (0.07 g/100 g) and dicalcium phosphate (0.2 g/100 g),
potassium chloride (0.2 g/100 g), and sodium chloride (0.2 g/100 g) were added to the
HP diet to balance Ca, P, K, and Na levels.
2
2100
Pye et al.
Whole body composition and osseous mineralization analysis.
Rats were scanned in the anesthetized state using isoflurane gas (Baxter
International) at their respective month using DXA (Hologic 4500A
QDR version 11.2, Hologic). Measurements of body weight, body fat
(BF), and lean body mass (LBM) were made using small animal software.
To examine if the effect of the HP diet on BF was generalized or confined
to a specific region, abdominal fat mass was measured as a proxy for
visceral fat (24,25) and defined as the region from just below the last rib
to the top of the iliac crest of the pelvic region. We also measured bone
mineral content (BMC) and density (BMD) using the small animal
software for whole body and high resolution regional scans (right
femora, tibia, and the lumbar vertebrae 1–4). Data were expressed as
absolute values and BMC was expressed relative to body weight (g/kg).
Ionized calcium and phosphorus concentrations as well as pH were
measured in serum using a Nova analyzer (Model 11, Nova Biomedical;
CV ,1.6%). To quantify bone formation and bone resorption, serum
concentrations of both osteocalcin (Metra Osteocalcin, Quidel; CV =
6.8%) and carboxy-terminal crosslinks of type I collagen (CTX)
(Ratlaps, Nordic Bioscience Diagnostics; CV = 8.2%) were quantified
using rat-specific ELISA.
Insulin-like growth factor 1 (IGF-1), leptin, and adiponectin were
measured as they relate to bone and body composition. Serum leptin (SPI
BIO Leptin, Cayman Chemical; CV = 4.8%) and adiponectin (ALPCO
Diagnostics; CV = 3.1%) were assessed using specific enzyme immunoassays while serum IGF-1 (IDS; CV = 3.2%) was measured with a ratspecific ELISA.
Bone microarchitecture analysis. Microcomputerized tomography
(mCT) was used at the 17-mo time point to assess the microarchitecture
of the left femora of rats fed either of the 2 diets. A total of 17 femurs
(NP: n = 7; HP: n = 10) were measured at the Center for Bone and
Periodontal Research at McGill University (Montreal, Canada) using a
small rodent mCT (Skyscan version 1072, SKYSCAN). To standardize
settings for all bones, a source of 80 kV and 124 mA, a 0.5-mm filter, a
253 zoom, a 11.25-mm pixel size, a time of 7.5 s, and a rotation of 0.9
were used. All femurs were analyzed just under the femoral neck and
assessed for trabecular measurements using the Skyscan ‘shrink wrap’
option to define bone contours and internal porosity.
Bone biomechanical analysis. Prior to biomechanical testing, all
femurs and tibias were measured using a digital caliper for total length,
mid-diaphysis, and 1/3 proximal diaphysis (26). Bone strength was
assessed in right femurs and tibias using the 3-point flexure method at the
mid-diaphysis (Instron version 5544). Bones were warmed to 378C for
5 min in a saline bath to mimic physiological conditions (27) then mounted
on the jig with a base span of 14 mm for femurs and 12 mm for tibias. For
right femurs, a 5N load was applied at a preloading rate of 15 mm/min
until contact with the mid-diaphysis, at which point the rate continued at
1 mm/min (28). Left femurs underwent similar 3-point flexure testing but
were broken at the proximal 1/3 diaphysis point to represent a higher
cancellous:cortical ratio. Measurements for flexure load at yield and
load at break were obtained. Flexure load at yield (n) is defined as the
corresponding load at the point in which permanent bone deformation
instigates (29). Load at break (n), also referred to as the ultimate
strength, is the final force recorded at the ultimate fracture (29). As the
femoral neck is the bone site with the highest incidence of fracture (30),
previously broken right femora were tested for femoral neck strength to
represent a loading-based fracture (31). The femoral diaphysis was
potted in dental resin and after 40 min mounted on the base of the
Instron. A breaking rate of 0.5 mm/min was applied to the medial region
of the femoral head as previously described (30). Following biomechan-
Downloaded from jn.nutrition.org by guest on November 4, 2012
Animals and experimental design. Eighty female Sprague-Dawley
rats (University of Manitoba) were randomized to receive 4, 8, 12, or 17
mo of a normal-protein (NP) control (15% AMDR) or HP (35%
AMDR) diet. Rats consumed all diets ad libitum, commencing at 70 d to
initiate dietary consumption at sexual maturity but prior to achievement
of peak bone mass (Table 1). These time points were chosen to represent
physiological periods of importance to bone health in females: acquisition of peak bone mass (baseline to mo 4), maintenance of bone and
just prior to natural menopause (mo 8), early postmenopause (mo 12),
and advanced aging (mo 17). The diets were based on the AIN-93 diet
for maintenance (20) with changes made to macronutrient values to
accommodate the HP diet. All micronutrients were standardized
between the 2 diets, matching the nutrient requirements for rat growth
and maintenance (21). To simulate a representative mixed HP diet
typical of humans, the protein composition contained one-third plantand two-thirds animal-based protein sources (22). Rats were fed and
housed in pairs and weight was measured at baseline, weekly for the
entirety of the study, and at termination. During the last week of study
prior to the 4, 8, and 12 mo time points, blood was collected between
0800 and 1000 from the saphenous vein to obtain serum that was stored
at –808C until analysis. Rats then underwent a 3-d metabolic balance for
measuring food intake followed by dual-energy X-ray absorptiometry
(DXA) scanning to measure body composition, bone mass, and then
necropsy. Due to the large size of the rats at 17 mo relative to the
metabolic cages, the 3-d metabolic balance for food intake was not
conducted. Following necropsy, femurs and tibias were excised, cleaned,
wrapped in saline-soaked gauze, and stored at 2808C. After the
completion of the study, all samples were shipped on dry ice from the
University of Manitoba to McGill University for analysis. All procedures
were reviewed and approved by the University of Manitoba Animal Care
Committee and in accordance with the Canadian Council on Animal
Care Guidelines (23).
Body composition and bone mass. Both diet (P # 0.0001)
and time (P # 0.0001) affected LBM and HP rats had ~12%
more LBM as a percentage of body weight than NP controls
(data not shown). Similarly, HP rats had a greater absolute LBM
(P # 0.0001) and the content increased over time (P = 0.05)
(Table 2). At both the whole body and regional level at all ages,
HP rats had ~29% less fat content than NP-fed rats and fat
content increased over time (P # 0.0001).
HP rats had ~7% less whole body BMC (12.7 6 2.6 g) than
NP-fed rats (13.6 6 2.1 g; P = 0.03) but not following
adjustment for weight (NP, 31.4 6 2.9; HP, 31.7 g/kg; P =
0.54). Similarly, diet did not affect whole body BMD (g/cm2;
data not shown). All of these variables consistently increased
over time (P = 0.0004, P # 0.0001, P = 0.005; data not shown).
Although not in regional measurements of BMC, HP
increased weight-adjusted BMC in the femur (P = 0.002), tibia
(P = 0.009), and lumbar vertebra (P = 0.02) (Table 3). All
variables were affected by time. Time, but not diet, affected
BMD in the femur (P # 0.0001) and lumbar vertebra (P =
0.002). In the tibia, BMD was affected by an interaction between
diet and time (P = 0.046), although post hoc analysis did not
reveal differences among the groups across time (Table 3).
ical testing, mid-diaphysis cancellous (outer to the inner endosteum) and
cortical (outer to the inner periosteum) diameters were measured using
the caliper at the break site.
Statistical methods. The sample size was calculated as 8 rats per group
to detect a 10% change in femoral BMC with a = 0.05 and a power =
0.80. The number of rats per group studied was 10 per group to account
for unrelated morbidity over the long-term study.
Differences between the diet groups were tested using diet and time
(4, 8, 12, or 17 mo) as fixed effects in a 2-way factorial ANOVA
including diet 3 time interactions (SAS version 9.1). Because there were
only 2 diets, post hoc testing was not required. Post hoc testing for
significant effects of time or diet 3 time interactions (indicated where
applicable) were assessed using the all pairwise Bonferroni’s test with a
P , 0.05. As mCT was only performed at mo 17, measurements were
assessed using a t test with a P , 0.05. Normality was assessed using the
Kolmogorov-Smirnov test and equal variances examined using the
Levene test. Data that failed assumptions for normality were transformed prior to ANOVA testing; square root was used for tibia BMC and
BMC/kg and log was used for lean and fat mass. All results are expressed
as mean 6 SD.
Serum biochemistry. Diet did not affect calcium, phosphorus,
or blood pH. Phosphorus decreased (mo 4, 2.2 6 0.1; mo 8,
1.9 6 0.3; mo 12, 1.8 6 0.2 mmol/L; P = 0.001) and pH increased (mo 4, 7.49 6 0.05; mo 8, 7.47 6 0.05; mo 12, 7.57 6
0.06; P # 0.001) over time but there were no diet 3 time
interactions affecting these variables.
The serum leptin concentration was ~35% lower in rats that
consumed the HP diet (0.9 6 0.7 nmol/L) compared with those
that consumed the NP diet (1.4 6 0.8 nmol/L; P = 0.003;
Food intake and growth. Although food intake relative to
body weight did not differ by diet (NP, 38.4 6 9.4; HP, 37.4 6 7.7
g/kg; P = 0.61), rats at 12 mo (32.6 6 8.8 g/kg) had lower intakes
than at 4 (40.9 6 5.9 g/kg; P = 0.005) and 8 mo (40.3 6 8.2 g/kg;
P = 0.009). Overall, HP rats had ~10% lower whole body weight
than their NP-fed counterparts (P = 0.006; Table 2) despite no
differences in body or tail length. Although rats increased in body
weight (P # 0.0001), length (P = 0.01), and tail length over time
(P # 0.0001; Table 2), there were no interactions with diet.
TABLE 2
Anthropometric variables in rats after 4, 8, 12, or 17 mo of consuming the NP or HP diet1
P-value
Body length, cm
Tail length, cm
Body weight, g
LBM, g
Whole BF, g
Abdominal BF, g
1
Mo
NP
HP
All
Time
Diet
4
8
12
17
4
8
12
17
4
8
12
17
4
8
12
17
4
8
12
17
4
8
12
17
41.9 6 1.1
42.7 6 1.2
42.9 6 0.7
42.1 6 1.3
19.0 6 0.6
19.7 6 0.9
20.2 6 0.62
20.5 6 1.28
354.4 6 36.7
397.1 6 62.5
489.8 6 56.9
541.9 6 108.0
253.9 6 21.1
263.1 6 19.2
290.5 6 21.1
287.8 6 35.2
85.5 6 29.7
119.7 6 48.1
194.3 6 61.5
248.0 6 86.5
23.7 6 8.5
31.6 6 12.4
57.5 6 17.4
65.4 6 20.6
42.1 6 0.8
42.8 6 0.6
42.8 6 1.1
41.8 6 1.1
19.2 6 0.6
20.4 6 0.4
20.1 6 0.57
20.2 6 0.61
333.2 6 33.2
376.5 6 36.0
449.4 6 64.5
460.7 6 104.9
265.7 6 20.9
280.1 6 13.1
300.0 6 31.0
295.4 6 32.6
56.5 6 22.5
77.9 6 37.2
142.3 6 48.8
163.1 6 84.0
17.8 6 5.5
23.6 6 9.1
39.9 6 16.3
44.5 6 24.1
42.0 6 1.0ab
42.7 6 1.0ab
42.8 6 0.9b
41.9 6 1.2a
19.1 6 0.6a
20.0 6 0.7b
20.1 6 0.6b
20.3 6 0.9b
343.8 6 35.8a
386.8 6 50.7a
469.4 6 62.7b
494.9 6 111.1b
259.8 6 21.3a
271.6 6 18.2a
295.3 6 26.3b
292.2 6 33.0b
71.0 6 29.6a
98.8 6 47.0a
168.3 6 60.3b
198.9 6 93.1b
21.1 6 7.8a
27.8 6 11.4a
48.7 6 18.7b
52.6 6 24.5b
0.01
0.95
#0.0001
0.43
#0.0001
0.006
0.05
#0.001
#0.001
#0.001
#0.001
#0.001
Data are mean 6 SD, n = 10 except n = 7 for NP at mo 17. For all rats, means without a common letter differ, P , 0.05.
High dietary protein does not impair bone
2101
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Results
TABLE 3
DXA variables in rats after 4, 8, 12, or 17 mo of consuming the NP or HP diet1
P-value
Mo
Femur
BMC, g
BMC, g/kg
BMD, g/cm2
Tibia
BMC, g
BMD, g/cm2
Lumbar vertebrae
BMC, g
BMC, g/kg
BMD, g/cm2
1
HP
Diet
0.54
0.70
0.69
0.57
1.53
1.35
1.15
1.08
0.397
0.336
0.326
0.453
6 0.08
6 0.06
6 0.10
6 0.06
6 0.20
6 0.20
6 0.16
6 0.16
6 0.030
6 0.020
6 0.044
6 0.051
0.55
0.69
0.72
0.56
1.64
1.55
1.27
1.24
0.424
0.348
0.328
0.432
6
6
6
6
6
6
6
6
6
6
6
6
0.07
0.10
0.08
0.09
0.22
0.17
0.08
0.16
0.038
0.025
0.033
0.041
0.54 6 0.07a
0.70 6 0.08b
0.71 6 0.09b
0.56 6 0.08a
1.58 6 0.21b
1.45 6 0.21b
1.21 6 0.14a
1.17 6 0.17a
0.411 6 0.036b
0.342 6 0.023a
0.327 6 0.038a
0.441 6 0.045c
#0.001
0.66
#0.001
0.002
#0.001
0.55
4
8
12
17
4
8
12
17
4
8
12
17
0.37
0.70
0.69
0.40
1.04
1.03
0.85
0.75
0.237
0.266
0.269
0.284
6 0.06
6 0.06
6 0.10
6 0.05
6 0.15
6 0.32
6 0.14
6 0.12
6 0.028
6 0.033
6 0.066
6 0.021
0.40
0.70
0.72
0.37
1.18
1.25
0.92
0.82
0.256
0.280
0.259
0.247
6
6
6
6
6
6
6
6
6
6
6
6
0.05
0.06
0.08
0.05
0.12
0.54
0.12
0.12
0.029
0.036
0.015
0.021
0.38 6 0.05a
0.70 6 0.08b
0.71 6 0.09b
0.38 6 0.05a
1.11 6 0.15d
1.14 6 0.45c
0.88 6 0.13b
0.79 6 0.12a
0.246 6 0.029
0.273 6 0.034
0.264 6 0.022
0.262 6 0.048
#0.001
0.62
#0.001
0.009
0.09
0.63
4
8
12
17
4
8
12
17
4
8
12
17
0.65
0.70
0.69
0.65
1.84
1.79
1.43
1.23
0.322
0.336
0.326
0.304
6 0.07
6 0.06
6 0.10
6 0.08
6 0.19
6 0.22
6 0.31
6 0.26
6 0.019
6 0.020
6 0.044
6 0.022
0.66
0.70
0.72
0.62
1.98
1.88
1.62
1.39
0.327
0.348
0.328
0.304
6
6
6
6
6
6
6
6
6
6
6
6
0.06
0.10
0.08
0.10
0.23
0.36
0.19
0.25
0.018
0.024
0.033
0.035
0.66 6
0.70 6
0.71 6
0.63 6
1.91 6
1.83 6
1.53 6
1.32 6
0.324 6
0.342 6
0.327 6
0.304 6
0.026
0.81
#0.001
0.02
0.002
0.47
0.06ab
0.08ab
0.09b
0.09a
0.22b
0.30b
0.27a
0.26a
0.018ab
0.023b
0.038ab
0.031a
Data are mean 6 SD, n = 10 except n = 7 for NP at mo 17. For all rats, means without a common letter differ, P , 0.05.
Biomechanical testing. Diet did not alter any femoral or tibia
biomechanical parameters at mid-diaphysis, whereas time significantly affected femoral load at break (Table 4). Similarly, diet
did not affect any of the biomechanical parameters at the
femoral head or 1/3 diaphysis (data not shown). There were no
Pye et al.
Time
4
8
12
17
4
8
12
17
4
8
12
17
Supplemental Table 1). Rats at mo 12 (1.7 6 1.0 nmol/L) had
higher leptin concentrations than at mo 4 (0.9 6 0.4 nmol/L; P =
0.003) and mo 8 (1.0 6 0.5 nmol/L; P = 0.009). Serum
adiponectin concentrations in rats fed the HP diet (0.13 6 0.05
mmol/L) were ~33% lower than in NP-fed rats (0.19 6 0.06
mmol/L; P # 0.0001). However, HP consumption did not alter
concentrations of IGF-1. Serum concentrations of adiponectin
or IGF-1 did not change over time.
Diet did not alter the bone biomarkers osteocalcin and CTX
(Supplemental Table 1). Unlike osteocalcin, concentrations of
CTX decreased over time in both dietary groups (P = 0.003).
2102
All
interactions noted in any of the parameters measured. Rats fed
the HP diet had lower tibia cortical diameters (0.75 6 0.08 mm)
compared with those fed the NP diet (0.80 6 0.07 mm; P =
0.006), but this variable did not change over time.
mCT analysis. Femurs from the 17 mo time point were analyzed
for trabecular, whole, and interpolated cortical bone specifics.
The dietary intervention did not affect any of the parameters
measured at both the trabecular and whole bone (Table 5). An
image of this analysis can be viewed in Supplemental Figure 1.
Discussion
Attributable to growing concern, a thorough understanding of
the physiological outcomes of long-term HP consumption on the
renal system and bone remains a fundamental goal in setting safe
dietary recommendations. The aim of this study was to examine
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BMC, g/kg
NP
TABLE 4
Mid-diaphysis 3 point flexure mechanical testing variables in rats after 4, 8, 12, or 17 mo of
consuming the NP or HP diet1
P-value
Mo
Femur
Load at break, n
Flexure load at yield, n
Tibia
Load at break, n
Flexure load at yield, n
4
8
12
17
4
8
12
17
151.8
148.9
159.7
171.1
13.7
13.6
15.6
16.1
6
6
6
6
6
6
6
6
HP
30.3
31.2
25.6
39.5
1.6
2.6
4.2
4.2
74.1 6 15.9
86.8 6 24.6
93.5 6 30.8
67.8 6 34.0
9.3 6 1.3
10.2 6 0.9
9.9 6 2.3
8.4 6 1.4
138.3
166.2
172.9
175.1
14.2
15.9
16.9
16.3
6
6
6
6
6
6
6
6
24.6
25.9
44.0
33.0
2.6
2.9
4.7
4.7
87.4 6 22.4
113.0 6 13.0
83.9 6 62.5
80.2 6 27.0
9.7 6 1.5
10.8 6 2.6
9.4 6 1.8
9.5 6 2.9
All
Time
Diet
145.0 6 27.7a
157.1 6 29.4ab
166.0 6 35.1ab
173.4 6 34.8b
14.0 6 2.1
14.6 6 2.9
16.2 6 3.7
16.3 6 4.4
0.05
0.48
0.18
0.24
0.12
0.17
0.21
0.45
81.1 6 20.2
100.0 6 23.4
88.9 6 47.3
75.8 6 29.2
9.5 6 1.4
10.5 6 1.9
9.7 6 2.1
9.1 6 2.4
For all rats, means with superscripts without a common letter differ, P , 0.05.
the effect of a high mixed protein (35% of energy) diet on body
composition and bone in female rats from young adult age well
into aging.
The effectiveness of HP diets as a means of weight and fat
reduction has been demonstrated in previous studies (4). As was
anticipated, rat fed HP diets had lower measurements for body
weight and fat mass in addition to higher LBM. Given that BMI
and fat mass are inversely related to bone resorption (32) and
positively related to bone mass (33,34), they are regarded as a
safeguard for bone (35). While in this study the HP diet resulted
in lower whole body BMC, areas susceptible to fracture
maintained BMC and BMD. Similarly, Mardon et al. (9)
observed no reduction in femoral BMD following 21 mo of
26% HP diet in rats. Furthermore, femur, tibia, and spine BMC
were 10–13% higher in HP-fed rats when adjusted for body
weight. This suggests that even though fat mass following the
HP diet was lower, there was no compromise to bone. This is
further confirmed by no difference between groups in the bone
turnover markers and bone strength testing.
It has been postulated that HP consumption coupled with
adequate calcium intakes attenuate losses of mineral from bone
(36). In this study, HP and NP diets were equal in calcium
(0.50%) and phosphorus (0.3%) content at recommended
amounts (21). Two human trials of HP diets combined with
adequate calcium through dairy products demonstrate protection against bone resorption (37) and bone loss despite elevated
urinary calcium (36). The hypercalciuria likely reflects increased
intestinal calcium absorption rather than bone resorption as
demonstrated in a 10-d study of HP diets in women (38). In our
study, bone formation, resorption, BMD, and mCT data
indicated no significant differences between the dietary groups.
Collectively, these findings suggest that when provided with
adequate or high calcium intakes, bone loss ascribed to high
protein consumption may be lessened.
Higher BW can improve bone quality through biomechanical
loading, in addition to the release of vital fat-related hormones
that effect bone (39). As an adipocyte-secreted hormone, leptin
aids in the maintenance of body weight (40) and appetite (32).
Leptin also modulates bone turnover (41) by inhibiting bone
formation (34). Similarly, another adipose-derived hormone,
adiponectin, typically is inversely associated with BMD (42) and
bone mass (43). Although HP-fed rats in this study had lower
leptin and adiponectin in agreement with lower fat mass, BMD
was not altered. Because this is, to the authors’ knowledge, one
of the first HP analyses to measure both adipose hormones,
further analyses are required to better understand this lack of
association with bone.
As a known bone-promoting factor (35), production of IGF1 is in turn stimulated by the consumption of protein (44). Low
protein intake can significantly reduce circulating IGF-1,
resulting in a possible detriment to bone (45). In this study,
IGF-1 was unaffected by HP consumption, matching the lack of
TABLE 5
mCT variables in rats after 17 mo of consuming the
NP or HP diet1,2
Whole BV, mm3
Whole BV/TV, %
Whole BS, mm2
Whole BS/BV, m21
Trab. BV, mm3
Trab. BV/TV, %
Trab. BS, mm2
Trab. BS/BV, m21
Trab. n., m21
Trab. Sp., m
NP
HP
P-value
44.6 6 5.0
63.3 6 5.2
439.2 6 63.7
9.9 6 1.0
8.4 6 2.6
25.9 6 7.4
56.3 6 21.3
33.3 6 4.5
2.0 6 0.5
0.60 6 0.11
46.2 6 5.9
64.1 6 4.8
450.4 6 47.9
9.9 6 1.6
9.0 6 1.8
27.3 6 5.3
43.9 6 13.9
32.3 6 3.7
2.1 6 0.4
0.60 6 0.08
0.56
0.74
0.68
0.97
0.58
0.66
0.57
0.66
0.79
0.98
Data are mean 6 SD, n = 10 (HP) and 7 (NP), P , 0.05.
BV, Bone volume; BV/TV, bone volume:tissue volume; BS, bone surface; BS/TV,
bone surface density; BS/BV, bone surface:volume; Trab. BV, trabecular bone volume;
Trabecular BV/TV, trabecular bone volume:tissue volume; Trab. BS, trabecular bone
surface; Trab. BS/BV, trabecular bone surface:bone volume, Trab. n, trabecular
number; Tr. Sp., trabecular spacing.
1
2
High dietary protein does not impair bone
2103
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1
4
8
12
17
4
8
12
17
NP
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
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nature of the diets. A similar argument could be made for this
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mimic that commonly consumed by humans (22). In keeping
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concluded no detrimental effect to bone (47,48) as opposed to
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Densitometry alone provides very limited indication of total
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HP consumption neither improved nor hindered bone stability at
any of these sites that represent different cancellous:cortical
ratios. Aside from the slight tendency for lower BMD in tibias, no
other assessments were altered. Thus, tibia integrity was not
significantly altered by HP consumption.
This study did present several limitations. Measurements for
food intake and serum pH, calcium, and phosphorus were not
obtained at mo 17 for practical reasons. The mCT measurements
for mo 4, 8, and 12 were also unavailable. Additionally, only a
sample size of 7 was available for a dietary group in the mo 17
analysis. Although this is below the estimated sample size of 8
per group, the reported results consistently indicate a lack of
dietary effect on bone using 3 standard measurements for bone:
DXA, mCT, and bone strength.
Previously, research investigating the effect of an elevated
protein diet at 35% AMDR upper limit on bone was inconsistent. Because the adoption of HP diets for weight loss and
maintenance has become increasingly popular over the past
decades, it is crucial that researchers set standards of protein that
are safe for public consumption. In this study, sexually mature
female Sprague-Dawley rats were randomized to receive either a
normal 15% or a high 35% of energy mixed protein diet
throughout their adult lifespan. The high mixed protein consumption was associated with reduced weight, fat mass, and
higher LBM, but no deleterious effects to bone. Therefore, it can
be concluded that a mixed HP diet containing adequate calcium
levels at the current upper 35% of energy can be deemed safe in
respect to long-term bone health in rats.
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