Protein consumption is an important predictor of lower limb bone mass in elderly women

¹ From the School of Medicine and Pharmacology, University of Western Australia, Perth, Australia (AD, IMD, AFMI, and RLP); the Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Nedlands, Australia (AD, IMD, SSD, and RLP); the Western Australian Institute of Medical Research, Perth, Australia (AD, IMD, AFMI, and RLP); the School of Exercise, Biomedical and Health Science, Edith Cowan University, Perth, Australia (AD); and the School of Public Health, Curtin University of Technology, Perth, Australia (SSD)

² Supported by the Healthway Health Promotion Foundation of Western Australia, the Australasian Menopause Society, and the National Health Medical Research Council Project (grant no. 254627).

³ Address reprint requests and correspondence to A Devine, School of Medicine and Pharmacology, University of Western Australia, 1st Floor C Block, Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Nedlands, Western Australia 6009. E-mail: adevine{at}cyllene.uwa.edu.au.

ABSTRACT  
BACKGROUND:: The effect of protein intake on bone density is uncertain, and evidence exists for beneficial effects of both low and high protein intakes.

OBJECTIVE:: The objective was to study the relation between protein consumption and bone mass in elderly women with allowance for other lifestyle factors affecting bone metabolism.

DESIGN:: We conducted a cross-sectional and longitudinal study of a population-based sample of 1077 women aged 75 ± 3 y. At baseline, protein consumption was measured with a food-frequency questionnaire, and bone mass and structure were measured by using quantitative ultrasound of the heel. One year later, hip bone mineral density (BMD) was measured by using dual-energy X-ray absorptiometry.

RESULTS:: Subjects consumed a mean (±SD) of 80.5 ± 27.8 g protein/d (1.19 ± 0.44 g protein/kg body wt). Regression analysis showed a positive correlation between protein intake and qualitative ultrasound of the heel and BMD after adjustment for age, body mass index, and other nutrients. The dose-response effect was best characterized by protein consumption expressed in tertiles, such that subjects in the lowest tertile (<66 g protein/d) had significantly lower qualitative ultrasound of the heel (1.3%) and hip BMD (2.6%) than did the subjects in the higher tertiles (>87 g protein/d).

CONCLUSION:: These data suggest that protein intakes for elderly women above current recommendations may be necessary to optimize bone mass.

Key Words: Protein • elderly women • bone density • dietary intake

INTRODUCTION  
A variety of nutrients have been shown to have significant relations with bone mineral density (BMD) (1-3). Separating the interactive effects of different nutritional components is a complex task because food intake is associated with and modified by other nutrients as well as by factors such as age, lifestyle, environment, and heredity. Research suggests that, in addition to calcium, macronutrients including protein may affect bone density (4). Certainly a beneficial effect of protein on clinical outcomes and bone density after hip fracture has been reported (5, 6), and a mechanism of protein action on increasing the concentration of insulin-like growth factor I has been suggested (6).

In epidemiologic studies, high protein intakes have been associated with both positive and negative effects on calcium balance and bone density (1, 7-9). Several studies have shown that a high-protein diet is associated with high BMD (10-14), whereas other studies, in younger women, have shown no effect (15, 16) or a negative effect (17). Most reports support a positive relation between protein intake and bone density and changes in bone density. However, few of these studies have examined the effect of dietary protein in women aged >70 y.

The effects of dietary protein in older women remain uncertain, especially because the responses of skeletal and muscle tissue may be affected by aging. In Australia and the United States, the daily adult dietary requirements for protein are considered to be 0.75 g/kg (18) and 0.8 g/kg (19), respectively. Generally, both Americans and Australians consume moderate to high amounts of protein, but subgroups of the population, including the elderly, may consume diets low in protein (20, 21). To improve our knowledge base in this area, this study examines the effect of protein consumption, in relation to carbohydrate, fat, and calcium consumption, on bone density to ascertain whether protein has an independent association with bone mass in elderly Australian women.

SUBJECTS AND METHODS  
Subjects
Women (n = 1077) were recruited by using a population-based approach in which a random selection of women aged >70 y who were registered to vote received a letter inviting them to join the study. More than 98% of women of this age are registered to vote, and 18% of the women we approached responded. Of the responders, approximately one-third came in to the study, one-third changed their minds, and one-third were excluded because they were receiving pharmaceutical agents that act on bone, including calcium supplements, or because they had significant current illness. Although the subjects entering the study were weighted in favor of those in higher socioeconomic categories (22), they did not differ from the whole population in the use of health resources (23). All subjects were enrolled in a 5-y trial of the effects of calcium supplementation on fracture outcome, and they received 1.2 g calcium carbonate/d or a matched placebo. Data collected at baseline and at year 1 of the study were available for data analysis.

Written informed consent was obtained from all subjects. The Human Rights Committee of the University of Western Australia approved the study.

Anthropometry and dietary intake
At baseline, weight and height were measured while participants were wearing light clothes and no shoes. Body mass index (BMI; in kg/m²) was calculated. At the start of the study, each subject completed a self-administered, semiquantitative food-frequency questionnaire (FFQ) developed by the Anti Cancer Council of Victoria (ACCV) (24, 25), from which information on the daily dietary intakes of energy, carbohydrate, protein, fat, and calcium was derived. The dietary calcium was from food alone and did not include the amount from any supplement.

Bone density
At baseline, a quantitative ultrasound (QUS) measurement of the calcaneus of the left foot was taken twice in all subjects with an Achilles Ultrasound machine (Lunar Corp, Madison, WI). The manufacturer's quality-assurance methods were employed. The averaged measurement of broadband ultrasound attenuation (BUA), speed of sound (SOS), and stiffness were used in these analyses. The CVs for BUA and SOS in our laboratory were 1.59% and 0.43%, respectively.

Bone mineral density (BMD) was measured at the hip on an Acclaim QDR 4500A fan-beam densitometer (Hologic, Waltham, MA) at 12 mo. The CV at the hip was 1.2% (26).

Statistical analysis
All statistical procedures were performed with SPSS for WINDOWS software (version 11.5; SPSS Inc, Chicago, IL). The homogeneity of variance and normality assumptions were checked by visual inspection of residuals from analysis of variance models and by using Kolmogorov-Smirnov goodness-of-fit test, respectively. For the BMD data at year 1, treatment with calcium or placebo was entered into the model as a covariate.

Dietary effects on measures of bone density were examined by using linear regression and multiple regression analysis after control for age, BMI, and calcium treatment. A generalized linear model (GLM; SPSS Inc) procedure was used to calculate the estimated means of the BMD and QUS variables for the lowest, middle, and highest protein groups after adjustment for age and BMI. All statistical tests were two-tailed, and probability values of < 5% were considered significant.

RESULTS  
The baseline characteristics of the entire cohort are shown in Table 1. The average daily dietary intake of calcium was 955 ± 347 mg. Intakes ranged from 231 to 2345 mg/d. Sixty-two percent of the subjects had a calcium intake <1000 mg/d. On average, the subjects consumed 80.5 ± 27.8 g protein/d, which is almost twice the 45 g/d recommended in Australia for women aged >55 y. The energy derived from protein was 19.0 ± 2.9%. The average amount of protein consumed was 1.19 ± 0.44 g · kg body wt ^–1 · d^–1 and ranged between 0.31 and 4.51 g · kg body wt^–1 · d^–1. Most (84%) of the population had an intake that was above the recommended requirement of 0.75 g protein · kg body wt^–1 · d^–1. On average, the women had fat and carbohydrate intakes that were 64.5 ± 24.5 and 192 ± 59 g, respectively, which yielded 33.0 ± 5.3% and 43.2 ± 5.5% of energy from fat and carbohydrate, respectively.

View this table:
TABLE 1. Demographic data, macronutrient intakes, quantitative ultrasound, and bone mineral density of participants¹

 
The relation between the macronutrients was explored, and a high degree of correlation existed, especially between protein, fat, and carbohydrate (Table 2). Calcium intake was strongly related to protein intake. Calcium was also related to fat and carbohydrate intake (Table 2).

View this table:
TABLE 2. Correlation between dietary macronutrients and BMI

 
Association between dietary intake, BMD, and QUS
In this study, the BUA measurements of heel QUS and total hip BMD were weakly associated negatively with age (r = –0.104, P < 0.01, and r = –0.120, P < 0.001, respectively). All heel QUS and hip DXA measurements—eg, those for BUA (r = 0.335, P < 0.001) and total hip BMD (r = 0.490, P < 0.001)—were positively correlated with BMI. In the cross-sectional study, each of the macronutrients was positively associated with the BUA measure of heel QUS at the calcaneus (fat: r = 0.102, P < 0.01; protein: r = 0.136, P < 0.001; carbohydrate: r = 0.098, P < 0.01; calcium: r = 0.092, P < 0.01). In the prospective study, the macronutrients were positively associated with all hip DXA BMD measurements (eg, total hip and fat: r = 0.092, P < 0.01; protein: r = 0.138, P < 0.001; carbohydrate: r = 0.094, P < 0.01; calcium: r = 0.096, P < 0.01).

To identify the nutrient with the strongest correlation with bone structural measurements, carbohydrate, fat, protein, and calcium were entered in a stepwise multiple regression analysis with heel QUS or hip DXA as the dependent variable (Table 3). For the BUA and the DXA BMD analysis, protein intake was the only nutrient to enter the model after control for age, BMI, and (for the prospective hip analysis only) calcium treatment group. An interactive effect between protein and BMI was entered into the model but was not significant at any of the sites. The effect of the calcium treatment group was not significant and was not considered further in the analysis.

View this table:
TABLE 3. Regression equations for the dietary predictors of bone mineral density and quantitative ultrasound¹

 
Protein intake
To study the dose-response relation between protein intake and bone structure, GLM with adjustment for age and BMI was used to determine the effect of protein intake, classified into tertiles, on heel QUS and hip BMD. Subjects in the low-protein (<66 g protein/d) tertile had significantly lower heel BUA than did subjects in the high-protein (>87 g protein/d) tertile (Figure 1). A similar association was seen with DXA BMD at all hip sites. Those in the low-protein tertile had significantly lower BMD than did those in the high-protein tertile (Figure 2). When moderate and high protein intakes (66 g protein/d) were combined into a single group, the mean BMD at all sites of the hip and the measurements of BUA and stiffness were significantly higher than those in the low-protein tertile [total hip: 819 and 798 mg/cm², respectively (P < 0.002); femoral neck: 698 and 679 mg/cm², respectively (P < 0.002); trochanter: 644 and 625 mg/cm², respectively (P < 0.002); intertrochanter: 961 and 937 mg/cm², respectively (P < 0.01); BUA: 101.0 and 99.6 db/MHz, respectively (P < 0.005); and stiffness: 71.1% and 69.5% of the mean in healthy young adults, respectively (P < 0.025)].

View larger version (17K):
FIGURE 1.. Between-subjects effects on broadband ultrasound attenuation (BUA) were determined by using ANOVA based on estimated marginal means adjusted for baseline age of 75 y and BMI (in kg/m²) of 27. Groups with different lower-case letters are significantly different, P < 0.05 (Bonferroni correction).

 

View larger version (28K):
FIGURE 2.. Between-subjects effects on bone mineral density (BMD) of the hip sites were determined by using ANOVA based on estimated marginal means adjusted for baseline age of 75 y and BMI (in kg/m²) of 27. Groups with different lower-case letters are significantly different, P < 0.05 (Bonferroni correction). PTN, protein.

 

DISCUSSION  
This epidemiologic study examined the association of nutrient intake with BMD in a large population of elderly women living in the community. Subjects with low protein intakes had 2.5–3.0% lower hip BMD and 1.3–2.2% lower calcaneus BUA and stiffness measurements than did those with moderate and high protein intakes. These results extend findings from other studies of postmenopausal women that suggest beneficial effects of higher protein intake on BMD (3, 12, 13, 27, 28). The cutoffs for protein intakes derived from the tertile analysis reflect those used in other studies (12). The Framingham Osteoporosis Study was able to show that the 2 lowest quartiles of protein intake (<67 g/d) were associated with greater bone loss at the femoral neck than was the highest quartile (>84 g/d), even though no cross-sectional relation between baseline BMD and protein intake was identified (12).

A protective effect of protein on reducing fracture risk has been reported (29-32). In an observational case-control study, higher intakes of protein have been shown to reduce the risk of hip fracture in men and women aged 50–69 y, but not in older men and women (30). In some studies, a high protein intake has been related to increased fracture risk (33-36). Sellmeyer reported that women aged >65 y who consumed a diet with a high ratio of animal to vegetable protein had significantly greater femoral neck bone loss and a rate of hip fracture three times that in the women who consumed a low ratio of animal to vegetable protein (33).

In this study, all but a small proportion (12%) of subjects consumed more than the recommended requirement of 0.75 g protein · kg body wt^–1 · d^–1 (37). The protein intake from this population-based study of elderly women reflects intakes reported previously from other samples of older women (38-40). The dietary intake of older Australian women is generally higher in fat and protein and lower in carbohydrate than is recommended (41). In this study, the dietary intakes of calcium were higher than those reported nationally (42). Alternatively, the FFQ method may overestimate the actual protein and calcium intakes in this age group (25). Although the FFQ method is routinely used for large population studies such as this, a 4-d weighed diet record or multiple 24-h recalls are more reliable and precise when dietary intakes are estimated. In the current study, a high intake of protein was associated with a high calcium intake that was due to the consumption of dairy products—food sources that are calcium and protein dense. The effect of protein independent of calcium can be determined only in a prospective trial, but these data support the concept that higher intakes of protein may result in higher bone mass after calcium intake is accounted for.

A recent review suggests that moderate intakes of protein (range: 1.0–1.5 g protein/kg) do not alter bone and calcium homeostasis (43). The effects of various protein intakes on calcium homeostasis in young women were studied, and it was found that high protein diets led to hypercalciuria because of increases in the absorption of intestinal calcium (44). After consumption of a moderately low-protein diet (0.7 g/kg) for 4 d, secondary hyperparathyroidism developed because of reduced intestinal calcium absorption (45). Similar results were found in men and postmenopausal women (43, 46) and in subjects with idiopathic hypercalciuria and calcium nephrolithiasis (46).

Protein-dosing studies show that serum parathyroid hormone rises when protein intakes approximate 0.7–0.8 g/kg but not when protein intakes approximate 0.9–1.0 g/kg (47). These data suggest a threshold effect in which changes in calcium homeostasis occur in healthy women when protein intakes fall below 0.9 g/kg in the presence of a calcium-sufficient diet (47). Changes in bone turnover that have been observed in young women consuming different amounts of dietary protein may in part explain the effects of protein on bone (48). Diets high in protein (ie, 2.1 g/kg) were associated with increased rates of bone resorption without an increase in markers of bone formation. These data suggest that increased urinary calcium excretion observed in these young women may be from bone resorption (20%) as well as from intestinal absorption (80%) (49). A recent study comparing the short-term effects of high (2.1 g/kg) or moderate (1.0 g/kg) protein intakes on calcium kinetics in younger women confirmed these previous findings (50). Whereas urinary calcium excretion and calcium absorption were increased in the high-protein group, sensitive isotopic kinetics determined that the high-protein diet caused a reduction in the proportion of urinary calcium derived from bone, although only a nonsignificant trend toward reduced bone resorption was reported (50). Moreover, diets low in protein (ie, 0.7 g/kg) do not influence bone resorption rates but have been shown to reduce intestinal calcium absorption. This may explain an increase in parathyroid hormone concentrations and low bone density (48) and also an increase in fracture risk in those with habitually low protein intakes. Our findings support the hypothesis that persons who consume a low-protein diet have low calcium absorption that over a lifetime leads to a low bone mass. In our population-based study, the intake that significantly determined a low bone mass was that of < 66 g protein/d (0.84 g protein/kg body wt), which is higher than that determined by Kerstetter et al (14), who reported an association of protein intake with femur BMD in postmenopausal women.

One study has examined the effects of increasing protein in the diets of postmenopausal women by adding meat to the diet (51). In a randomized crossover design, subjects consumed a diet with either low or high meat content for a total of 8 wk. Isotopic calcium tracers and whole-body scintillation were used to measure calcium retention after a 4-wk period of equilibration for each diet. There was no difference in calcium retention, urinary calcium losses, or markers of bone turnover between the subjects consuming the 2 diets. A confounding factor of this study reported by Sebastian (52) suggests that the net endogenous acid production of the 2 diets was similar because of substitution of cereal grains for meat in the low-meat diet. This difference in the composition of the diet may explain the lack of effect seen between the 2 diets. A study by Roughead et al (53) examined the difference between meat and soy protein and was unable to show a difference in calcium retention or markers of bone turnover, including IGF-I. Although the soy diet reduced renal acid excretion, the urinary calcium excretion did not differ significantly between the 2 diets.

Studies have shown an effect of increased IGF-I with dietary protein supplements in elderly hip fracture patients (6), young girls (54), and older men (55, 56) and women (55). Therefore, dietary protein intake above habitual intakes seemingly has the ability to increase IGF-I concentrations to some degree. This may mean that habitual intakes of protein are inadequate. Low IGF-I concentrations may in part reflect poor nutritional status of the person, and bone loss could be associated with reductions in IGF-I concentrations that are due to nutritional deficiencies.

Our data support the concept that protein intake has a beneficial effect on bone mass. These data suggest that a protein intake >66 g protein/d (>0.84 g protein/kg body wt) is required in elderly women to maximize bone mass. Therefore, recommendations for protein intake may need to be reconsidered in elderly women.

ACKNOWLEDGMENTS  
AD, IMD, and RLP were responsible for the study design. AD, IMD, RLP, SSD, and AFMI were responsible for the data analysis and writing of the manuscript. AD was responsible for the supervision of the data collection. All authors approved the manuscript. AD, IMD, AFMI, SSD, and RLP have no affiliations with the funding bodies for this study.

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