Journal of Gerontology: MEDICAL SCIENCES
Cite journal as: J Gerontol A Biol Sci Med Sci. 2010 October;65A(10):1115–1122
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Advance Access published on July 6, 2010
insulin sensitivity and cardiovascular function ( 1 ). How-
ever, weight loss increases the rate of bone mineral density
(BMD) loss ( 2 ). Moderately higher protein (22% – 29% of
energy) or higher protein (>30% of energy) diets are popu-
lar for weight loss, in part, because they may help preserve
lean body mass ( 3 , 4 ). The impact of dietary protein on bone
remains controversial. Some researchers observed that in-
adequate or excessive protein intakes adversely affected
BMD ( 5 , 6 ), whereas adequate protein intake helped mini-
mize BMD loss in older persons ( 7 ). Other researchers re-
ported no association between protein intake and bone ( 8 ).
Some ( 9 , 10 ), but not all ( 7 ), researchers also found that ani-
mal protein, compared with plant protein, was more detri-
mental to bone due to increased urinary calcium excretion.
Data are limited on the potential impact of dietary protein
on bone during weight loss. In premenopausal women and
similarly aged men, ingestion of an energy-restricted diet
with 25% versus 12% energy from protein diminished the
loss of bone mineral content after 6 months ( 11 ). Overweight
EIGHT loss is recommended for overweight and
obese adults to promote health, including improved
participants who consumed energy-restricted diets with
27% versus 16% of energy from protein for 4 months did
not experience adverse changes in markers of bone turnover
( 4 ). In middle-aged men and women who consumed an
energy-restricted diet with 34% protein for 12 weeks, markers
of bone resorption (urinary deoxypyridinoline) and bone
formation (plasma osteocalcin) increased, indicating higher
bone turnover ( 12 ). The changes in these markers were
blunted when the protein was dairy based compared with an
isonitrogenous diet from mixed protein sources ( 12 ). Re-
cent research ( 13 ) suggested that achieving a higher protein
intake (1.4 vs 0.8 g·kg − 1 ·day − 1 ), mainly from dairy, attenu-
ated BMD loss after a 4-month period of energy restriction –
induced weight loss.
Collectively, these data document that weight loss and
the quantity and source of dietary protein may infl uence
BMD and bone turnover markers. The paucity of data, the
contradiction of results, and the popularity and potential en-
hanced effi cacy of higher protein intake during energy re-
striction and weight loss underscore the need for additional
research on the impact of protein quantity and source on
Protein Intake, Weight Loss, and Bone Mineral Density in
Wayne W. Campbell and Minghua Tang
Department of Foods and Nutrition, Purdue University, West Lafayette, Indiana .
Address correspondence to Wayne Campbell, PhD, Department of Foods and Nutrition, Purdue University, 700 West State Street,
West Lafayette, IN 47907. Email: email@example.com
Background. Higher protein diets are promoted for effective weight loss. Striated tissues in omnivorous diets contain
high-quality protein, but limited data exist regarding their effects on bone.
Methods. To examine the effects of energy restriction – induced weight loss with higher protein omnivorous diets ver-
sus lower protein vegetarian diets on bone mineral density in overweight postmenopausal women, two randomized con-
trolled feeding studies were conducted. In Study 1, 28 women consumed 750 kcal/day energy defi cit diets with 18%
energy from protein via lacto-ovo vegetarian sources (normal protein, n = 15) or 30% energy from protein with 40% of
protein from lean pork (higher protein, n = 13, omnivorous) for 12 weeks. In Study 2, 54 women consumed their habitual
diet (control, n = 11) or 1,250 kcal/day diets with 16% energy from nonmeat protein sources ( n = 14) or 26% energy from
protein, including chicken ( n = 15) or beef ( n = 14) for 9 weeks.
Results. Study 1: With weight loss (normal protein − 11.2%, higher protein − 10.1%), bone mineral density was not
signifi cantly changed in normal protein ( − 0.003 ± 0.003 g/cm 2 , − 0.3%) but decreased in higher protein ( − 0.0167 ± 0.004
g/cm 2 , − 1. 4%, group-by-time p < .05). Study 2: The control, nonmeat, chicken, and beef groups lost 1.5%, 7.7%, 10.4%,
and 8.1% weight and 0.0%, 0.4%, 1.1%, and 1.4% bone mineral density, respectively. The change of bone mineral density
was signifi cant for chicken and beef compared with the control (group-by-time, p < .05). Markers of calcium metabolism
and bone homeostasis in blood and urine were not changed over time or differentially affected by diet.
Conclusion. Consumption of higher protein omnivorous diets promoted decreased bone mineral density after weight
loss in overweight postmenopausal women.
Key Words: Protein — Weight loss — Bone mineral density .
Received January 15 , 2010 ; Accepted April 23 , 2010
Decision Editor: Luigi Ferrucci, MD, PhD
CAMPBELL AND TANG
BMD and markers of bone formation and resorption. The
purposes of this research are to assess the infl uence of higher
protein intake and the inclusion of striated tissue in the diet
(ie, an omnivorous diet) during energy restriction on BMD
and markers of bone turnover in overweight and moderately
obese postmenopausal women, a population at high risk for
osteoporosis, from two controlled feeding studies.
Study 1 and Study 2: Ethics and Protocol Registration
The Purdue University Institutional Review Board
approved the protocols and procedures for both studies. All
participants provided written informed consent and received
Study 1: Experimental Design
This 13-week protocol included a 1-week baseline and a
12-week dietary intervention with energy restriction. The
participants were randomly assigned to one of two energy-
restricted diets that differed in macronutrient distribution.
Testing was conducted at baseline and postintervention.
Study 1: Experimental Participants
The exclusion criteria were men; aged <20 years; body
mass index <25 and >37 kg/m 2 at screening; clinically
abnormal kidney, liver, or heart function; clinically abnor-
mal protein or hematological status; diagnosed diabetes
mellitus or receiving insulin replacement therapy; and
smoker. Fifty-four women were eligible and started the
intervention; 46 women completed the study ( Supplementary
Figure 1 ). Data from all 46 women were used to assess the
effects of higher protein intake on body composition and
satiety ( 3 ). The current analyses utilized data from the 28
postmenopausal women (higher protein: n = 13, normal
protein: n = 15) to avoid potential confounding effects of
menstrual status on weight loss – induced changes in bone.
Study 1: Energy-Restricted Diet Intervention
All participants consumed a diet with 750 kcal/day less
than their estimated energy requirement and contained ei-
ther the recommended dietary allowance of 0.8 g·kg − 1 ·day − 1
(normal protein group) or 175% of the recommended di-
etary allowance (1.4 g·kg − 1 ·day − 1 , higher protein group)
for protein based on the participants ’ screening body
masses. The higher protein diet contained 30% of energy
from protein, 45% carbohydrate, and 25% fat; the normal
protein diet contained 18% protein, 57% carbohydrate,
and 25% fat.
The participants were counseled to follow 7-day menus
with specifi ed quantities of typical and brand-specifi c food
items to purchase and consume. These menus were void of
animal fl esh foods (ie, striated tissues). The higher protein
group was provided with portioned quantities of cooked
pork (loin, ham, or Canadian bacon) products comprising
40% of their total protein intake, whereas the normal pro-
tein group was given portioned quantities of milk compris-
ing 13% of their total protein intake. Thus, the higher protein
diet was omnivorous, and the normal protein diet was lacto-
ovo vegetarian. This study was not designed to compare
sources of protein (meat vs dairy). Milk was given to the
normal protein group to equalize the interactions and con-
tact time with the higher protein group. The higher protein
and normal protein diets had comparable amounts of pro-
tein from dairy: 42 and 36 g/day, respectively, based on a
typical 1,550 kcal/day diet. All participants consumed daily
a multivitamin/mineral tablet (Centrum; Wyeth Consumer
Healthcare, Madison, NJ) and two calcium citrate tablets
(400 mg calcium/tablet consumed in the morning and eve-
ning, total 800 mg calcium/day). For more detailed descrip-
tions of the dietary intervention, see Leidy and colleagues ( 3 ).
Study 2: Experimental Design
This 11-week protocol included a 2-week baseline and a
9-week dietary intervention. The participants were ran-
domly assigned to a nonintervention control group (CON-
TROL) or one of three energy-restricted groups (CARB,
CHICKEN, BEEF). Testing was conducted at baseline and
Study 2: Experimental Participants
The exclusion criteria were men; age <50 or >80 years;
<2-year postmenopausal; body mass index <25 and >34 kg/m 2
at screening; clinically abnormal kidney, liver, or heart func-
tions; clinically abnormal protein or hematological status;
diagnosed diabetes mellitus or receiving insulin replace-
ment therapy; smoker; and use of antiinfl ammatory steroid
medications. Fifty-seven women who were eligible started
the protocol and were randomized to one of the four groups.
Fifty-four women completed the study (CONTROL n = 11;
CARB n = 14; CHICKEN n = 15; and BEEF n = 14;
Supplementary Figure 2 ).
Study 2: Diet Intervention
The women in the three energy-restricted groups were
counseled to consume the same 1,000 kcal/day lacto-ovo
vegetarian basal diet (5-day fi xed rotation menu consisting
of three meals plus two snacks) and were provided with 250
kcal/day of shortbread cookies and sugar-coated chocolates
(CARB), cooked breast meat chicken (CHICKEN; plus 10
g butter to match the total and saturated fat contents of the
beef), or beef tenderloin (BEEF). Thus, the CARB diet was
vegetarian, and the CHICKEN and BEEF diets were om-
nivorous. Details of the counseling and food preparation
procedures are published ( 14 ). The 1,250 kcal/day diet of
the CARB group was designed to contain 58% of energy
PROTEIN, WEIGHT LOSS, AND BONE MINERAL DENSITY
from carbohydrate, 16% protein, and 26% fat, whereas the
CHICKEN and BEEF group ’ s diets contained 48% carbo-
hydrate, 26% protein, and 26% fat. The CONTROL partici-
pants continued to consume their usual diets and did not
purposefully alter their eating behavior or change body
mass. At baseline and post, each participant ’ s energy and ma-
cronutrient intakes were estimated from 3-day food records
(Nutritionist Pro software; First databank, San Bruno, CA).
Each woman was requested to stop using nutritional
supplements that were not recommended by a physician,
nonprescription medications, and alcohol 3 weeks prior to
and throughout the study. Twenty-nine of the 54 women
who completed the study used calcium supplements (8
CARB, 9 CHICKEN, 8 BEEF, and 4 CONTROL) during
the study. For more details of the dietary intervention, see
Mahon and colleagues ( 14 ).
Study 1 and Study 2: Body Composition Measures
Each participant ’ s fasting-state mass and height were
measured, and body mass index (kg/m 2 ) was calculated.
Body composition, whole body BMD (g/cm 2 ), bone min-
eral content (g), and bone area (cm 2 ) were measured using a
dual-energy x-ray absorptiometer (GE LUNAR Prodigy
with EnCORE software version 5.60, Madison, WI). The
participant was asked to lie still on an x-ray table for less
than 30 min while the measurements were being taken.
Blood and Urine Sampling and Analyses
For Study 1, fasting-state venous blood samples taken
postintervention were analyzed for blood urea nitrogen us-
ing a photometric assay (Chemistry Immuno Analyzer
aU5700; Olympus, Center Valley, PA) by MidAmerica
For Study 2, fasting-state venous blood samples were
collected at baseline and postintervention, processed to
obtain plasma and serum, and aliquots stored at − 20°C.
Measurements of serum osteocalcin (marker of bone forma-
tion), bone alkaline phosphatase (marker of bone forma-
tion), parathyroid hormone (marker of calcium homeostasis),
and insulin-like growth factor 1 (IGF-1, associated with
BMD in older humans) were made. The analytical methods
used were osteocalcin — enzyme-linked immunosorbent
assays (ELISA), Quidel Corp., San Diego, CA; bone alka-
line phosphatase — ELISA, Quidel Corp.; parathyroid
hormone — immunoradiometric assay, Nichols Institute,
San Juan Capistrano, CA; and IGF-1 — radioimmunoassay,
Diagnostics Systems Laboratories, Webster, TX.
For Study 2, 24-hour urine collections were made at base-
line and postintervention. Aliquots were stored at − 20°C and
N-telopeptide cross-links, free deoxypyridinoline (markers
of bone resorption rate), creatinine, calcium, phosphorous,
and total nitrogen were measured. The analytical methods
used were N-telopeptide cross-links — ELISA, Ostex Interna-
tional, Seattle, WA; free deoxypyridinoline — ELISA, Quidel
Corp.; creatinine — Cobas Mira with Jaffe Kinetic assay,
Roche Diagnostics, Indianapolis, IN; calcium — Cobas Mira
with Arsenazo III assay, Roche Diagnostics; phosphorous —
Cobas Mira with ammonium molybdate assay, Roche Diag-
nostics; and total nitrogen — Leco model FP-528, Leco,
St. Joseph, MI.
Potential Renal Acid Load Estimations
Participants ’ micronutrient intakes were estimated using
Nutrition Data System for Research 2008 software (Univer-
sity of Minnesota Nutrition Data System for Research). Po-
tential renal acid load (PRAL, mEq/day) of the diets was
estimated as follows ( 15 ):
[0.021 K (mg)] [0.02 Mg (mg)] [0.013 Ca (mg)].
For Study 1, PRAL was evaluated using representative
menus from the normal protein and higher protein diets at
three energy levels (1,200, 1,500, and 1,700 kcal/day). For
Study 2, PRAL was estimated for the BEEF, CHICKEN,
and CARB diets.
PRAL[0.49 protein (g)] [0.037 P (mg)]
Data were analyzed using PROC GLM (SAS version
9.1.2; SAS Institute Inc, Cary, NC). Repeated measures
ANOVA was performed to access the main effects of group
and time, and the group-by-time interaction. In Study 1,
t tests were used for post hoc analyses to detect differences
within (paired t test; pre vs post) and between (independent
t test) the normal protein and higher protein groups. In
Study 2, Dunnett ’ s test of multiple comparisons was used
for post hoc analyses to detect differences between treat-
ment groups and the control group. Paired t test was used to
examine the difference within groups (pre vs post). Data are
presented as mean ± SEM . Statistical signifi cance was
accepted at p < .05.
Study 1: Participant Characteristics and Body
There was no difference at baseline or differential change
over time in height, body mass, body mass index, fat mass,
and fat-free mass between the normal protein and higher
protein groups ( Table 1 ).
Study 1: Diet Composition, Protein Intake, and PRAL
Energy intake was lower during the intervention than base-
line for both groups ( p < .001; Supplementary Table 1 ). As
designed, during energy restriction, the normal protein group
consumed less protein, more carbohydrate, and the same fat
compared with the higher protein group. The absolute amount
CAMPBELL AND TANG
of protein (g/day) consumed during the 12-week intervention
was constant for the normal protein and higher protein groups,
but the relative protein intakes (grams per kilogram per day)
increased as body mass loss occurred. At intervention Week
1, protein intakes for the normal protein and higher protein
groups were 0.81 ± 0.03 and 1.39 ± 0.04 g·kg − 1 ·day − 1 , respec-
tively. Postintervention, the protein intakes were 0.91 ± 0.04
and 1.55 ± 0.04 g·kg − 1 ·day − 1 , respectively. The group-specifi c
protein intakes were supported by differential blood urea ni-
trogen postintervention (normal protein 12.6 ± 0.9 and higher
protein 16.5 ± 0.8 mg/dL; p < .05). The sources of dietary
protein are presented in Supplementary Table 2 . During en-
ergy restriction, total calcium intake (diet + supplement) was
>2,000 mg/day for both groups.
At 1,200 – 1,700 kcal/day energy intakes, PRAL ranged
from 6.4 – 13.7 and 29.1 – 42.2 mEq/day for the normal pro-
tein and higher protein groups, respectively.
Study 1: Bone Parameters
At baseline, total body BMD, bone mineral content, and
bone area were not different between the normal protein
and higher protein groups ( Table 1 ). After weight loss, BMD
was not changed in normal protein ( – 0.3% ± 0.2%) but de-
creased in higher protein ( − 1.4% ± 0.4%, p < .01; Figure 1 ,
group-by-time p < .05). Bone mineral content and bone area
were not changed after weight loss, independent of diet.
Study 2: Participant Characteristics and Body Composition
There were no differences in age, height, body mass,
body mass index, or body composition among the four
Table 1. Study 1: Participant Characteristics and Body Composition at Baseline and Post*
Parameter Group BaselinePost Change
Age † (y) Normal protein
60 ± 3
51 ± 2
164 ± 2
163 ± 2
80.0 ± 2.9
82.5 ± 4.2
30.0 ± 0.9
30.8 ± 1.1
35.6 ± 1.8
36.8 ± 2.5
41.8 ± 1.3
43.2 ± 1.8
1.165 ± 0.028
1.183 ± 0.025
2606 ± 93
2523 ± 119
2234 ± 49
2123 ± 64
Weight ‡ (kg)
71.0 ± 2.6
74.3 ± 4.0
26.4 ± 0.8
27.7 ± 1.1
28.7 ± 1.8
30.2 ± 2.6
39.7 ± 1.1
41.6 ± 1.7
1.161 ± 0.029
1.165 ± 0.024
2574 ± 96
2517 ± 130
2212 ± 49
2144 ± 70
− 9.1 ± 0.8
− 8.2 ± 0.5
− 3.4 ± 0.3
− 3.1 ± 0.2
− 6.9 ± 0.6
− 6.6 ± 0.5
− 2.2 ± 0.4
− 1.6 ± 0.3
− 0.003 ± 0.003
− 0.017 ± 0.004
− 32 ± 24
− 6 ± 18
− 22 ± 20
21 ± 20
Body mass index ‡ (kg/m 2 )
Fat mass ‡ (kg)
Fat-free mass ‡ (kg)
Total bone mineral density § (g/cm 2 )
Total bone mineral content (g)
Total bone area (cm 2 )
Notes : *Mean ± SEM ; normal protein, n = 15; higher protein, n = 13.
† Signifi cant difference between groups, p = .007.
‡ Signifi cant change over time (pre to post), p < .001.
§ Signifi cant group-by-time interaction, p < .01. p Values of paired Student ’ s t test for change over time in the normal protein and higher protein groups are 0.216
and 0.003, respectively.
Delta BMD g/cm2
normal proteinhigher protein
Figure 1. Study 1: Changes in total body BMD. * p < .01. BMD: bone min-
groups at baseline ( Table 2 ). Compared with CONTROL,
the CARB, CHICKEN, and BEEF groups lost body mass,
fat mass, and fat-free mass and lowered body mass index.
Study 2: Diet Composition, Protein Intake, and PRAL
At baseline, energy, protein, carbohydrate, fat, and cal-
cium intakes were not different among the four groups
( Supplementary Table 3 ). During the intervention, energy
intake was lower in the three energy-restricted groups com-
pared with CONTROL. Protein intake was lower in the
CARB (0.73 ± 0.12 g protein·kg − 1 ·day − 1 ) but not different
in the CHICKEN (0.99 ± 0.08 g protein·kg − 1 ·day − 1 ) and
BEEF (0.90 ± 0.09 g protein·kg − 1 ·day − 1 ) compared with
CONTROL (0.89 ± 0.11 g protein·kg − 1 ·day − 1 ). These pro-
tein intake results were supported by 24-hour urinary total
PROTEIN, WEIGHT LOSS, AND BONE MINERAL DENSITY
nitrogen excretion, which was lower for CARB, but not
CHICKEN and BEEF compared with CONTROL: 4.8 ±
0.3, 8.3 ± 0.3, 8.6 ± 0.9, and 7.8 ± 0.7 g nitrogen·kg − 1 ·day − 1 ,
respectively ( 14 ). The sources of dietary protein among the
three energy-restricted groups are provided in Supplementary
Table 2 . Average dietary calcium intake among all partici-
pants was 732 ± 46 mg/day.
PRAL for BEEF and CHICKEN diets were 5.4 and 6.9
mEq/day, respectively. PRAL for CARB diet was − 5.5
Study 2: Bone Parameters
At baseline, BMD, bone mineral content, and bone area
were not different among the four groups ( Table 2 ). After
intervention, BMD decreased in CHICKEN ( − 1.1 ± 0.3%)
and BEEF ( − 1.4 ± 0.2%) from baseline and was not
changed in CARB ( − 0.4 ± 0.3%) and CONTROL (0.0 ±
0.3%; Figure 2 , group-by-time p < .05). The BMD changes
in the BEEF and CHICKEN groups were different from
CONTROL. Bone mineral content and bone area were not
changed over time or differentially infl uenced by diet.
Among the energy-restricted participants, the change
in BMD was related to the change in 24-hour urinary total
nitrogen excretion ( R 2 = 0.137, p < .05; Figure 3 ) but not
related to the change in body mass.
Study 2: Markers of Calcium Metabolism and Bone
The blood and urinary markers of calcium metabolism
and bone turnover were not different among groups at base-
line except serum parathyroid hormone or changed over
time ( Supplementary Table 4 ).
Table 2. Study 2: Participant Characteristics at Baseline and Post*
Age (y) CONTROL
54 ± 5
59 ± 2
60 ± 2
58 ± 2
163 ± 2
164 ± 1
162 ± 1
164 ± 1
79.8 ± 3.4
75.9 ± 2.4
76.2 ± 2.8
81.0 ± 2.5
30.1 ± 1.1
28.4 ± 0.9
29.1 ± 1.1
30.1 ± 0.8
35.3 ± 2.9
33.5 ± 2.0
32.9 ± 1.9
35.4 ± 2.1
44.5 ± 1.0
42.4 ± 0.8
43.3 ± 1.2
45.5 ± 0.9
1.166 ± 0.028
1.111 ± 0.026
1.143 ± 0.024
1.140 ± 0.026
2542 ± 88
2377 ± 81
2242 ± 76
2463 ± 78
2170 ± 48
2140 ± 44
2130 ± 41
2165 ± 43
Body mass † (kg)
79.1 ± 3.7
70.3 ± 2.4
67.3 ± 2.6
74.4 ± 2.6
29.8 ± 1.2
26.3 ± 0.9
26.1 ± 1.0
27.6 ± 0.9
35.9 ± 3.2
29.6 ± 2.0
27.3 ± 1.7
31.1 ± 2.2
44.5 ± 0.9
40.7 ± 0.7
41.0 ± 1.1
43.3 ± 0.8
1.166 ± 0.028
1.107 ± 0.026
1.131 ± 0.024
1.126 ± 0.025
2548 ± 95
2354 ± 88
2445 ± 81
2480 ± 84
2176 ± 53
2127 ± 49
2152 ± 45
2205 ± 47
− 1.2 ± 0.4
− 5.6 ± 0.5 ‡
− 7.9 ± 0.7 ‡
− 6.6 ± 0.7 ‡
− 0.3 ± 0.2
− 2.1 ± 0.2 ‡
− 3.0 ± 0.3 ‡
− 2.5 ± 0.3 ‡
0.6 ± 1.1
− 3.9 ± 0.4 ‡
− 5.6 ± 0.6 ‡
− 4.3 ± 0.6 ‡
0.0 ± 0.3
− 1.7 ± 0.3 ‡
− 2.3 ± 0.3 ‡
− 2.2 ± 0.3 ‡
− 0.0000 ± 0.004
− 0.0038 ± 0.003
− 0.0123 ± 0.003 ‡
− 0.0145 ± 0.003 ‡
7 ± 21
− 23 ± 20
3 ± 18
18 ± 19
6 ± 21
− 12 ± 20
23 ± 18
40 ± 19
Body mass index † (kg/cm 2 )
Fat mass † (kg)
Fat-free mass † (kg)
Total bone mineral density † (g/cm 2 )
Total bone mineral content (g)
Total bone area (cm 2 )
Notes : *Mean ± SEM ; BEEF = moderate protein beef group ( n = 14), CHICKEN = moderate protein chicken group ( n = 15), CARB = lower protein, lacto-ovo
vegetarian group ( n = 14), and CONTROL = nonintervention control group ( n = 11).
† Signifi cant group-by-time interaction p < .05.
‡ Signifi cant difference compared with control p < .05.
CAMPBELL AND TANG
These results indicate that dietary protein is an important
factor infl uencing BMD changes in conjunction with weight
loss in postmenopausal women and that energy-restricted
diets with higher amounts of dietary protein, mainly from
animal fl esh sources, promoted greater BMD loss than
lower protein diets void of animal fl esh foods. Past research
showed that a higher protein diet either did not infl uence
BMD ( 11 , 12 ) or helped preserve BMD ( 13 ) after weight
loss. In one study ( 11 ), no signifi cant BMD loss occurred in
participants consuming either a higher protein (25% of en-
ergy from protein) or a normal protein diet for 6 month.
Another 12-week study ( 12 ) with participants consuming a
higher protein (34% of energy from protein), energy-
restricted diet from different sources showed no change in
BMD from baseline or interactions with diet. A recent study
( 12 ) found that whole body BMD was higher by 1.6% in the
higher protein (30% of energy from protein) group com-
pared with a normal protein diet, after 4-month of weight
loss followed by 8-month of weight maintenance. These
Delta BMD g/cm2
CONTROL CARB CHICKENBEEF
Figure 2. Study 2: Changes in total body BMD. * p < .05: Change of bone
mineral density in CHICKEN and BEEF were different from CONTROL.
CONTROL: nonintervention control group, CARB: lower protein lacto-ovo
vegetarian group, CHICKEN: higher protein chicken group, BEEF: higher
protein beef group, and BMD: bone mineral density.
-8 -6-4 -20246
Delta 24-h urinary Nitrogen Excretion, g/d
Delta BMD g/cm2
Figure 3. Study 2: Correlation between the change in whole body bone
mineral density and the change in 24-hour urinary nitrogen excretion. BMD:
bone mineral density .
investigators ( 11 – 13 ) indicated that dairy was the predomi-
nant source of protein, although the sources of protein were
not quantifi ed. In the current research, participants in Study
1 and Study 2 assigned to the higher protein diets consumed
36% and 24% of protein from dairy, respectively, but BMD
loss still occurred. Neither study was intended to compare
protein from meat versus dairy. We are not able to distin-
guish between the effect of protein source and protein quan-
tity on BMD loss in the current studies.
The combination of higher protein and calcium intakes
might promote bone mineral accrual in older people ( 16 ).
With regard to weight loss, participants who consumed a
high dairy high calcium (2,400 mg Ca/day) diet apparently
had slower bone turnover (assessed using blood markers of
bone formation and resorption) than those who consumed a
comparable high protein diet from mixed protein sources
(500 mg Ca/day) ( 12 ). In Study 1, all the participants con-
sumed calcium supplements. Despite calcium intakes
>2,000 mg/day, the higher protein group lost BMD after
weight loss, whereas the normal protein group did not. In
Study 2, although about one half of the participants con-
sumed calcium supplements, this use did not infl uence the
BMD responses to weight loss. Collectively, higher calcium
intakes from supplements do not appear to prevent or infl u-
ence the loss of BMD when overweight and obese post-
menopausal women lose weight while consuming a higher
protein omnivorous diet.
A common explanation for bone loss with a higher pro-
tein diet is elevated endogenous acid production ( 9 ). In-
creased bone resorption may occur to help balance
endogenous acidity, which may result in a negative calcium
balance and higher risk of osteoporosis ( 10 ). In the current
research, the higher protein intakes of the omnivorous
groups translated into higher dietary PRALs, which could
theoretically promote reduced BMD. This interpretation
should be drawn with caution. Although the BMD losses of
the higher protein groups were comparable (Study 1, higher
protein − 1.4%; Study 2, CHICKEN − 1.1%, BEEF − 1.4%),
the PRALs were fi vefold higher in Study 1 than in Study 2.
Although speculative, these observations support the notion
that humans with normal kidney function possess the meta-
bolic capacity to buffer diet-related acidity without the mo-
bilization of bone ( 17 ), and PRAL is not a signifi cant factor
with regard to BMD changes with weight loss.
Dietary protein-induced increases in IGF-1 may stimu-
late bone formation. Serum IGF-1 was higher in participants
who consumed higher protein provided from milk ( 18 , 19 ),
and IGF-1 concentration was positively associated with
dairy protein ( 20 ). The specifi city of dairy-based proteins to
increase IGF-1 is not fi rmly established because other re-
searchers found no association between dairy protein intake
and IGF-1 ( 21 ). Meat protein intake has ( 21 ) and has not
( 20 ) been positively associated with IGF-1. In Study 2, se-
rum IGF-1 was not changed over time in all four groups,
which suggests that IGF-1 may not have infl uenced the
PROTEIN, WEIGHT LOSS, AND BONE MINERAL DENSITY
differential BMD responses to weight loss among the dif-
ferent energy-restricted diets.
The novel observation that the change in BMD was nega-
tively correlated with the change in 24-hour urinary total
nitrogen excretion suggests that older women who increase
their total protein intake during the period of energy restric-
tion may be at higher risk for BMD loss, whereas a reduc-
tion in total protein intake may help preserve BMD. Future
studies should document how much protein a person regu-
larly consumes before starting a weight loss program
and whether women who usually consume a higher protein
diet do not experience greater BMD loss with energy
Results from research when both bone turnover markers
and BMD were measured are inconsistent. In one study
( 22 ), bone turnover was increased, but BMD was unchanged.
In another study ( 23 ), C-telopeptide of Type I collagen
(bone resorption marker) was increased when weight loss
was achieved using either energy restriction or exercise, but
hip BMD was decreased in the energy-restricted group only.
In Study 2, the post-weight loss reductions in BMD ob-
served in the BEEF and CHICKEN groups occurred with-
out any apparent changes in bone turnover markers. In terms
of assessing the risk of osteoporosis, BMD data obtained
from dual-energy X-ray absorptiometry scans are more rel-
evant than bone turnover markers because the technique has
less variation ( 24 ), is non-invasive and is a direct measure of
changes in bone. The combination of increased bone turn-
over markers with decreased BMD indicates higher risk of
osteoporosis than any of them alone ( 24 ).
Strengths of the current studies include using randomized
repeated measures experimental designs, using blood urea
nitrogen or 24-hour urinary total nitrogen excretion as inde-
pendent markers to confi rm differential protein intakes and
using a control group in Study 2. One limitation is the short-
term length of the interventions and the possibility that the
BMD changes refl ect transient bone remodeling ( 25 ). Fu-
ture longer term interventions are needed to fully under-
stand the impacts of dietary protein on bone in conjunction
with weight loss. Another limitation is the possibility that
BMD changes after weight loss are not physiological but
may be an artifact because the dual-energy x-ray absorpti-
ometer lacks sensitivity when body weight and composition
are changed ( 26 ). However, within the two current studies,
the lower and higher protein groups lost comparable
amounts of body mass and fat mass, yet only the higher
protein groups reduced BMD signifi cantly. Thus, the dif-
ferential BMD responses attributable to dietary protein were
measurable despite possible instrument insensitivity. A du-
al-energy x-ray absorptiometer uses bone mineral content
and bone area to calculate BMD. A potential confounder of
dual-energy x-ray absorptiometry measurement is body wa-
ter, which may alter with higher protein diets ( 27 ). Change
in body water may affect the precision of bone mineral
content measurement and as a result affect BMD.
In summary, a higher protein energy-restricted diet with
protein predominantly from animal fl esh sources promoted
total body BMD loss in overweight and obese postmeno-
pausal women during weight loss compared with a lower pro-
tein energy-restricted diet void of animal fl esh foods. Our
results suggested that for postmenopausal women, choosing
a higher protein omnivorous diet for effective weight loss,
may decrease BMD and increase the risk of osteoporosis.
Study 1 was supported by the National Pork Board. Study 2 was supported
by the Cattlemen ’ s Beef Board and the National Cattlemen ’ s Beef Associa-
tion and National Institutes of Health (grant number M01 RR00750).
S upplementary M aterial
Supplementary material can be found at: http :// biomed . gerontologyjournals
. org /
The abstract of Study 1 was presented at the Experimental Biology
Conference 2009. The abstract of Study 2 was presented at the Obesity
Society ’ s Annual Scientifi c Meeting 2006.
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