Table 3.

Protein and bone health in children and adolescents

Macronutrient	Reference	Study description	Population description	Number of subjects	End points	Results
RCTs
Protein	Ballard et al. 2006 [143]	This study investigated whether 6 months of protein supplementation in conjunction with a strength and conditioning training program improves vBMD, bone geometry, and total body BMC.	Sex: male and female Age: 18–25 years Race: not specified Location: South Dakota, USA Year(s): not specified	68	Data are shown for the protein supplemented group (n = 36)	Mean change, protein group (n = 36)			P
					4 % site
					Total vBMD (mg/cm3)	0.20			NS
					Trabecular vBMD (mg/cm3)	−0.50			NS
					Total area (cm2)	5.0			NS
					20 % site
					Cortical vBMD (mg/cm3)	2.4			NS
					Cortical area (cm2)	1.7			NS
					Cortical thickness (mm)	0.05			NS
					Periosteal circumference (mm)	−0.20			NS
					Endosteal circumference (mm)	−0.50			NS
					Polar SSI (mm3)	57			NS
					Total body
					BMC (g)	−3.5			NS
					Bone area (cm2)	−3.9			NS
					Leg
					BMC (g)	1.3			NS
					Arm
					BMC (g)	5.7			NS
					Data presented above are least-squares means determined by ANCOVA while controlling for initial height and weight and baseline bone value.
Prospective studies
Protein	Alexy et al. 2005 [144]	This study examined whether the long-term dietary protein intake and diet net acid load are associated with bone status in children. In a prospective study design, long-term dietary intakes were calculated from 3-day weighed dietary records that were collected yearly over the 4-year period before a one-time bone analysis using pQCT.	Sex: male and female Age: 6–18 years Race: white Location: Dortmund, Germany Year(s): 1998–1999 subcohort of the DONALD study	229	Data are shown for the overall group (N = 229)	Protein (g/day)
						ß		ßstand	r 2	P
					Forearm
					Periosteal circumference (mm2)	0.07		0.17	0.03	<0.01
					Cortical area (mm2)	0.42		0.27	0.04	<0.01
					BMC (mg/mm)	0.46		0.26	0.03	<0.01
					Polar SSI (mm3)	1.83		0.29	0.06	<0.01
					• Data presented above are results from stepwise multiple regression and after adjustment for age, sex, and energy intake. • ßstand is the standardized parameter estimate. • Children with a higher dietary PRAL had significantly less cortical area (P < 0.05) and BMC (P < 0.01). • Long-term calcium intake had no significant effect on any bone variable.
Protein	Bounds et al. 2005 [146]	This study aimed to identify factors related to children’s bone mineral indexes at age 8 years, and to assess bone mineral indexes in the same children at ages 6 and 8 years. Children’s dietary intake and BMC were assessed as part of a longitudinal study from ages 2 months to 8 years.	Sex: male and female Age: 6 years (baseline) and 8 years (follow-up) Race: white Location: Knoxville, TN Year(s): not specified	52	Data are shown for the overall group (N = 229)	Protein intake (g) over ages 2–8 years
						r			P
					Total body
					BMC at age 8 years	0.37			≤0.05
						ß		Partial R 2		P
					BMC model 1	(+) 2.40		0.08		<0.01
					Data presented above show the Pearson’s correlation coefficient (r) relating protein intake over ages 2–8 years, representing 27 days of dietary data. BMC model 1 (R 2 = 0.69, F = 20.7, P < 0.01)
Protein	Vatanparast et al. 2007 [147]	This mixed-longitudinal study investigated the influence of protein intake on bone mass measures in young adults, considering the influence of calcium intake through adolescence. Dietary intake was assessed via serial 24-h recalls carried out at least once yearly.	Sex: male and female Age: 8–21 years during phase I of the study; 17–29 years for phase II Race: majority Caucasian Location: Saskatoon, Saskatchewan, Canada Year(s): 1991–1997 (phase I); 2003–2006 (phase II); participating in the University of Saskatchewan Pediatric Bone Mineral Accrual Study	133	Data are shown for the overall group (N = 133) and a subgroup (n = 44)	Protein intake (g)
						Regression coefficient		Partial R 2		P
					Total body (N = 133)
					BMC	NS		—		NS
					BMC net gain	0.11		0.21		0.02
					Total body (n = 44)
					BMC	0.21		0.33		0.04
					BMC net gain	0.21		0.37		0.02
					The net gain of total body BMC and the net gains of height and weight from age of peak height velocity to early adulthood were entered into the model. Variables in the multiple regression model (stepwise) were sex, current height, weight, physical activity level, protein intake, vegetable and fruit intake, and periadolescence intakes of vegetables and fruit, protein, and physical activity. Protein intake predicted total body BMC net gain in all subjects. In females at periadolescence or early adulthood with adequate calcium intake(>1000 mg/day) (n = 44), protein intake positively predicted total body BMC and BMC net gain.
Protein	Zhang et al. 2010 [148]	This study assessed the association between protein intakes and bone mass accrual in girls who participated in a 5-year study including 2 years of milk supplementation (intervention groups only) and 3 years of follow-up study.	Sex: female Mean age: 10.1 years Race: Chinese Location: Beijing Year(s): 1999–2004	757	Data are shown for the overall group (N = 757)	Average protein intake
						ß			P value
					Total body
					Bone area	–			NS
					BMC	−1.92			0.02
					Proximal forearm
					Bone area	−9.11			<0.01
					BMC	−10.2			<0.01
					Distal forearm
					Bone area	–			NS
					BMC	−4.82			<0.01
					• Data presented above (ß) represent the percentage change in the dependent variable associated with intake of protein after controlling for baseline bone mass and pubertal development, age and physical activity, survey time, group, and clustering by schools. Protein, among other nutrients, was included in an initial model and flowed by backward elimination with P < 0.01 as the standard for retention, exclusion by the regression model. • When protein intake was considered according to animal or plant food sources, protein from animal foods, particularly meat, had significant negative effects on BMC accrual at the proximal and distal forearm (P < 0.05).
Protein	Remer et al. 2011 [145]	The aim of the study was to examine whether the association of long-term dietary acid load and protein intake with children’s bone status can be confirmed using approved urinary biomarkers and whether these diet influences may be independent of potential bone-anabolic sex steroids. Data were collected in 197 healthy children during the 4 years preceding proximal forearm bone analyses by pQCT.	Sex: male and female Age: 6–18 years Race: white Location: Dortmund, Germany Year(s): 1998–1999 subcohort of the DONALD study	197	Data are shown for the overall group (N = 197)	Urinary uN			Urinary PRAL
						ß	P		ß	P
					Forearm
					BMC (mg/mm) [log 10]	0.03	<0.01		−0.02	0.03
					Cortical area (mm2) [log 10]	0.02	<0.01		−0.02	0.03
					Polar SSI (mm3) [log 10]	0.02	<0.01		−0.01	NS
					Periosteal circumference (mm)	0.50	0.03		0.02	NS
					BMD (mg/cm3)	5.40	NS		−8.70	NS
					• Data presented above are from multivariate regression models showing independent associations of both long-term protein intake (as uN) and PRAL as explanatory variables with forearm bone variables. Data are adjusted for age, sex, pubertal stage, forearm muscle area, forearm length, and urinary calcium. • Data show that 1 Z-score variation in uN leads to an average 7.2 % increase in BMC and a 4.7 % increase in cortical area as well as SSI. A 1 Z-score uN corresponds to 0.28-g protein intake/kg body wt, implying that an additional 1-g protein intake/kg body wt may lead to an average increase of 26 % in BMC and 17 % in cortical area and SSI. A 1-g protein intake/kg body wt is associated with an average increase of 1.8-mm periosteal circumference.
Cross-sectional studies
Protein	Hoppe et al. 2000 [149]	The objective of the study was to identify associations between dietary factors and total body bone measurements in a random sample of healthy Danish children.	Sex: male and female Age: 10 years Race: Danish, otherwise unspecified Location: Hvidovre, Denmark Year(s): 1997–1998, from the Copenhagen Cohort Study on Infant Nutrition and Growth	105	Data are shown for the overall group (N = 105)	Protein (g/day)
						Pearson’s r			P
					Total body
					Bone area (cm2)	0.31			<0.01
					BMC (g)	0.33			<0.01
					• The data above are unadjusted. • In the multiple linear regression analysis including height, weight, sex, energy intake, and bone-related nutrients in the model, dietary protein was not significantly associated with bone area or BMC. After backward elimination, in which height, weight, and sex were forced to stay in the model, dietary protein was positively associated with bone area (P < 0.05). • Inclusion of pubertal stages in the analyses did not alter the bone area or BMC outcomes.
Protein	Iuliano-Burns et al. 2005 [150]	This cross-sectional study assessed monozygotic and dizygotic male twin pairs to test the following hypotheses: (1) associations between bone mass and dimensions and exercise are greater than between bone mass and dimensions and protein or calcium intakes; and (2) exercise or nutrient intake are associated with appendicular bone mass before puberty and axial bone mass during and after puberty.	Sex: male Age: 7–20 years Race: not specified Location: Melbourne, Australia Year(s): 1997–2001	112 (56 twin pairs)	Data are shown for the overall group (N = 112)	Differences in protein
						Univariate		Size adjusted		All lifestyle and size adjusted
						ß	P	ß	P	ß	P
					Differences in BMC (g)
					Total body	3.5	NS	1.3	NS	1.3	NS
					Arms	0.8	<0.05	0.7	<0.05	0.8	<0.05
					Legs	1.6	NS	0.3	NS	0.3	NS
					Lumbar spine	0.0	NS	0.0	NS	0.0	NS
					Differences in BMC (%)
					Total body	0.3	<0.05	0.2	<0.05	0.1	NS
					Arms	0.4	<0.05	0.4	<0.01	0.4	<0.05
					Legs	0.3	NS	0.1	NS	0.1	NS
					Lumbar spine	0.1	NS	0.1	NS	0.2	NS
					Differences in bone dimensions (mm)
					Cortical thickness	0.0	NS	0.0	NS	0.0	NS
					Periosteal diameter	0.0	NS	0.0	NS	0.0	NS
					Endosteal diameter	0.0	NS	0.0	NS	−0.0	NS
					Differences in bone dimensions (%)
					Cortical thickness	0.2	NS	0.1	NS	0.4	NS
					Periosteal diameter	0.1	NS	0.0	NS	0.0	NS
					Endosteal diameter	−0.0	NS	−0.1	NS	−0.3	NS
					• Data presented above are ß coefficients for within-pair differences in protein versus (1) within-pair differences in BMC and bone dimensions, (2) within-pair differences in size-adjusted BMC and bone dimensions, and (3) when all within-pair differences in protein, calcium, exercise duration, and size are included in the regression equation. • A 1-g difference in protein intake was associated with a 0.8-g (0.4 %) difference in arm BMC (P < 0.05). These relationships were present in peripubertal and postpubertal pairs but not in prepubertal pairs. Exercise during growth appears to have greater skeletal benefits than variations in protein or calcium intakes, with the site-specific effects evident in more mature twins.
Protein	Chevalley et al. 2008 [151]	This study analyzed the relationship between physical activity levels and protein compared with calcium intake on BMC in healthy prepubertal boys.	Sex: male Age: 6.5–8.5 years Race: white Location: Geneva, Switzerland Year(s): recruitment period 1999–2000, otherwise unspecified	232	Data are shown for the overall group (N = 232)	Correlation with protein intake (g/day)
						r	P		ßAdjusted		P
					BMC (g)
					Radial metaphysis	0.26	<0.01		0.20		0.01
					Radial diaphysis	0.21	<0.01		0.12		NS
					Total radius	0.27	<0.01		0.20		0.01
					Femoral neck	0.20	<0.01		0.19		0.03
					Total hip	0.18	<0.01		0.12		NS
					Femoral diaphysis	0.23	<0.01		0.19		0.03
					Lumbar spine	0.24	<0.01		0.22		<0.01
					Data presented above are from univariate (r) and multivariate (ßAdjusted) analyses; the latter takes into account the respective contribution of physical activity, protein, and calcium intakes.
Protein	Esterle et al. 2009 [152]	This study explored dietary calcium sources and nutrients possibly associated with lumbar bone mineralization and calcium metabolism in adolescent girls and evaluated the possible influence of a genetic polymorphic trait associated with adult-type hypolactasia.	Sex: female Age: 12–22 years Race: Caucasian Location: France Year(s): not specified	192	Data are shown for the postmenarchal group only (n = 142)	Protein from milk			Protein from other foods
						Adj R 2	ßstand	P	Adj R 2	ßstand	P
					Lumbar spine BMC (g)
					Absolute	0.61	0.14	<0.01	0.61	<0.01	NS
					Adjusted	0.60	0.13	0.02	0.60	<0.01	NS
					• Data presented above are from multivariate linear analyses. • Absolute protein intake is expressed in g/days. • Adjusted protein intake for weight, years after menarche, and vertebral area is expressed in g/kg/days. • Girls with milk intakes <55 mL/day had significantly lower BMC compared to girls consuming >260 mL/day. • Neither BMC nor milk consumption was associated with −13,910 LCT polymorphism.
Protein	Ekbote et al. 2011 [153]	The aim of this study was to examine lifestyle factors as determinants of total body BMC and bone area in Indian preschool children.	Sex: male and female Age: 2–3 years Race: Indian Location: Pune, India Year(s): 2009	71	Data are shown for the overall group (N = 71)	Protein (g/days)
						Normal		Malnourished		All
						r	P	r	P	r	P
					Total body
					Bone area	0.65	<0.01	0.57	<0.01	0.58	<0.05
					BMC	0.62	<0.01	0.44	<0.05	0.55	<0.01
					Data presented above are from Pearson’s correlation coefficients correlating protein intake and bone among normal children, malnourished children, and all (normal and malnourished children combined).
Protein	Libuda et al. 2011 [154]	This study examined relevant nutrients that are supposed to have an impact on bone parameters and compared their effect sizes with those of known predictors of bone development.	Sex: male and female Median age: 8.1 years Race: white Location: Dortmund, Germany Year(s): 1998–1999 subcohort of the DONALD study	107	Data are shown for the overall group (N = 107)	Protein (g/MJ)
						ß	ßstand		R 2		P
					Forearm
					Polar SSI (mm3)	–	–		–		NS
					Periosteal circumference (mm)	–	–		–		NS
					BMC (mg/mm)	1.49	0.11		0.01		NS
					Cortical area (mm2)	1.37	0.11		0.01		NS
					• Data presented above are from stepwise linear regression analyses, considering muscle area, BMI standard deviation scores, body fat %, age, sex, and rostenediol, as well as intakes of protein, calcium, vitamin D, and PRAL. • Of all nutrients considered, only protein showed a trend for an association with BMC (P = 0.073) and cortical area (P = 0.056) in stepwise linear regression models. • None of the other dietary variables were associated with bone parameters. • The protein effect did not differ between sexes.

Adj adjusted, ANCOVA analysis of covariance, BMC bone mineral content, BMD bone mineral density, DONALD Dortmund Nutritional and Anthropometric Longitudinally Designed Study, NS not significant, pQCT peripheral quantitative computed tomography, PRAL potential renal acid load, RCT randomized controlled trial, SSI stress–strain index, uN urinary nitrogen, vBMD volumetric bone mineral density