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Nutritional Epidemiology

A Positive Association of Lumbar Spine Bone Mineral Density with Dietary Protein Is Suppressed by a Negative Association with Protein Sulfur^1,2

³ Division of Nutritional Sciences, and ⁴ Kinesiology and Community Health, University of Illinois at Urbana-Champaign, Urbana, IL 61801

^* To whom correspondence should be addressed. E-mail: elevans{at}uiuc.edu.

	ABSTRACT

TOP ABSTRACT Introduction Methods Results Discussion LITERATURE CITED

 
Dietary protein is theorized to hold both anabolic effects on bone and demineralizing effects mediated by the diet acid load of sulfate derived from methionine and cysteine. The relative importance of these effects is unknown but relevant to osteoporosis prevention. Postmenopausal women (n = 161, 67.9 ± 6.0 y) were assessed for areal bone mineral density (aBMD) of lumbar spine (LS) and total hip (TH) using dual X-ray absorptiometry, and dietary intakes of protein, sulfur-containing amino acids, and minerals using a USDA multiple-pass 24-h recall. The acidifying influence of the diet was estimated using the ratio of protein:potassium intake, the potential renal acid load (PRAL), and intake of sulfate equivalents from protein. aBMD was regressed onto protein intake then protein was controlled for estimated dietary acid load. A step-down procedure assessed potential confounding influences (weight, age, physical activity, and calcium and vitamin D intakes). Protein alone did not predict LS aBMD (P = 0.81); however, after accounting for a negative effect of sulfate (β = –0.28; P < 0.01), the direct effect of protein intake was positive (β = 0.22; P = 0.04). At the TH, protein intake predicted aBMD (β = 0.18; P = 0.03), but R² did not improve with adjustment for sulfate (P = 0.83). PRAL and the protein:potassium ratio were not significant predictors of aBMD. Results suggest that protein intake is positively associated with aBMD, but benefit at the LS is offset by a negative impact of the protein sulfur acid load. If validated experimentally, these findings harmonize conflicting theories on the role of dietary protein in bone health.

	Introduction

TOP ABSTRACT Introduction Methods Results Discussion LITERATURE CITED

It has been proposed that bone demineralization is promoted by a mild but chronic dietary acid load characteristic of the Western diet (7). This acid load can be characterized by nutrient intake as the estimated net endogenous acid production (NEAP),⁵ calculated using a ratio of protein:potassium intake (8,9) or using a function of protein, calcium, potassium, magnesium, and phosphorus intake known as the potential renal acid load (PRAL) (3,10). These methods assume the sulfur content of protein is a fixed ratio; however, it is acknowledged that the estimation of actual sulfur intakes improves estimations of dietary acid load (11,12), because actual methionine and cysteine contents vary according to protein source. Currently, nutrient databases are available that account for variation in sulfur-containing amino acids (13).

The primary aim of this study was to elucidate the role of dietary protein in bone health status as estimated by dual X-ray absorptiometry (DXA) measures of areal bone mineral density (aBMD). Diet record analysis was used to estimate intakes of total protein and sulfate from amino acids for calculation of the dietary acid load. We anticipated that although dietary protein would be positively related to aBMD, this relationship would be suppressed by a negative association of aBMD with the dietary acid load related to protein intake.

	Methods

TOP ABSTRACT Introduction Methods Results Discussion LITERATURE CITED

 
    Participants. Our sample consisted of 161 postmenopausal women (67.9 ± 6.0 y) from Champaign County, IL participating in an ongoing study of the relation between physical activity, gait ability, and self-efficacy. Subjects were recruited using local media advertising, churches, senior centers, and health facilities. Women with neurological illness or severe orthopedic or cognitive limitations preventing physical testing for the broader study were excluded. Cross-sectional data were used for our analysis. Study participants provided written, informed consent; all study procedures met ethical standards of and were approved by the Institutional Review Board of the University of Illinois at Urbana-Champaign.

    DXA. For bone measures, women changed into medical clothing or wore light-weight clothing and removed all jewelry and other clothing except underwear. Lumbar spine (LS) and total hip (TH) aBMD were measured by DXA using a Hologic QDR 4500A bone densitometer (software version 11.2). LS scans included lumbar vertebrae L1–L4. Short- and long-term accuracy of the densitometer were verified by scanning a manufacturer's hydroxyapatite spine phantom of a known density. All DXA scans were performed by an Illinois state licensed X-ray technologist and analyzed by the same investigator trained in scan analysis by Hologic (E. M. Evans ). Precision for DXA aBMD measures of interest are 1–1.5% in our laboratory with CV% calculated from duplicate scans of both young adults and postmenopausal women.

    Dietary intake and estimation of acid load. Intake was assessed using the USDA multiple-pass 24-h dietary recall method (14,15). Participants completed an interview with researchers to screen for missed foods, portion size clarification, and recall completeness. Diet records were analyzed for total energy, protein, methionine, cysteine, and micronutrients of interest for calculation of estimated NEAP: potassium, calcium, magnesium, and phosphorus. Nutrient analysis was performed using Nutritionist Pro version 2.3.1 (First Data Bank).

Protein sulfur load was calculated as mEq/d using intakes of methionine and cystine divided by their molecular weights, as described by Frassetto et al. (8).

(Eq. 1)

The PRAL of the diet was estimated according to the method of Remer et al. (3,10):

(Eq. 2)

The protein:potassium ratio estimation of NEAP was calculated according to the method of Frasetto et al. (8):

(Eq. 3)

    Statistical analyses. Distributions were assessed for normality and outliers using the Shapiro-Wilk statistic in conjunction with box plot outlier labeling (16). Correlations for energy intake, protein, protein sulfur, minerals of interest, vitamin D, aBMD, and body composition (weight, fat mass, and lean mass) were calculated for descriptive purposes.

Statistical tests followed the prescriptions of MacKinnon et al. (17) for modeling suppression and mediation effects. Mediation effects, more commonly described as intermediate endpoints in epidemiology, describe a situation where all or part of the influence of X on Y is transmitted through a 3rd, intermediate variable. Modeling these effects is similar statistically, but different conceptually from confounding variables, in which a spurious association between 2 variables is explained by a 3rd, nonintermediate variable that presumably causes both X and Y. A suppressor effect is a special case of an intermediate endpoint in which X has a direct association with Y and an indirect association, transmitted through a 3rd variable, which is opposite in sign to the direct effect (17). For example, our present hypothesis posits that protein intake will be positively associated with aBMD (the direct effect), but that a 3rd, intermediate variable, the acid load associated with protein intake, will be negatively associated with aBMD, thereby suppressing the association of protein intake with aBMD unless the model is statistically controlled for the intermediate variable.

Sulfate, PRAL, and the protein:potassium calculation were entered individually as the 2nd block of a regression equation containing dietary protein to predict aBMD of the LS and TH. The change in R² was observed between blocks 1 and 2, indicating the improvement in the model conferred by the inclusion of respective estimates of diet acid load. A step-down regression procedure was then applied to examine the potentially confounding influences of body weight, age, physical activity, and calcium and vitamin D intakes. Because the interaction of dietary protein and calcium intakes have been reported to impact bone health (1,18), a protein by calcium interaction term was also tested for statistical control. At each step, covariates yielding P > 0.1 were removed from the model. Covariates were tested before and after the addition of sulfate to the model to ascertain any confounding influences. The variance inflation factor was observed as a test of multicolinearity.

Where change in R² was significant, unique protein effects and sulfur-mediated protein effects were estimated and tested for significance. The "Indirect" mediation macro for SPSS by Preacher and Hayes (19) provided bootstrapped variance estimates for the indirect or sulfur-mediated effect using 10,000 resamples with replacement. To describe the individual contributions of protein and related sulfur intakes, scatter plots and least squares regression lines were produced using sample Z-scores (difference between observation and sample mean divided by SD) of aBMD and protein adjusted for sulfur, then of aBMD and sulfur, adjusted for protein (Fig. 1). Additionally, mean LS aBMD was compared for subjects split across the median for both total protein and sulfur from protein (Fig. 2). SPSS 14.0 was used in all analyses.

View larger version (12K): [in this window] [in a new window]   FIGURE 1  Scatter plot with least squares regression line and mean 95% CI of LS aBMD (g/cm2) with protein, adjusted for associated sulfur intake (A) and with sulfur from protein, adjusted for total protein intake (B) in postmenopausal women (n = 161). Values are sample Z-scores, adjusted by ordinary multiple regression. Regressions were also controlled for body weight. For aBMD on protein, β = 0.21; P = 0.04. For aBMD on sulfur, β = –0.28; P < 0.01.

View larger version (35K): [in this window] [in a new window]   FIGURE 2  Descriptive representation of median LS aBMD in 161 postmenopausal women with intakes above (high) and below (low) median intakes of protein and sulfur from protein.

	Results

TOP ABSTRACT Introduction Methods Results Discussion LITERATURE CITED

At the TH, step-down regression removed weekly energy expended in physical activity (P = 0.95), vitamin D (P = 0.33), and the calcium x protein interaction (P = 0.26) terms, but retained age (P < 0.01), weight (P < 0.01), and calcium intake (P = 0.02) as covariates. Protein was a significant (P = 0.03), positive (β = 0.18) predictor of TH aBMD; however, no improvement in R² was observed with the addition of PRAL (P = 0.87), estimated NEAP using the protein:potassium ratio (P = 0.29), or sulfate (P = 0.83) to the model. The largest variance inflation factor observed was 2.1.

	Discussion

TOP ABSTRACT Introduction Methods Results Discussion LITERATURE CITED

 
Our findings suggest that, within the range of intake reported in our sample, increasing dietary protein is beneficial to aBMD of the LS and TH of postmenopausal women but that this benefit is suppressed at the LS by the dietary acid load associated with sulfur-containing amino acids. Neither the PRAL nor estimated NEAP using the protein:potassium ratio contributed to the prediction of aBMD at either site. The observed regression coefficients are small but clinically meaningful. A participant consuming mean levels of sulfate but protein at +1 SD would be predicted to have 3.2% (95% CI = 0.12–6.4) additional LS aBMD above the sample mean. Conversely, a subject consuming mean protein but +1 SD sulfate would be predicted to have 4.3% (–7.4 to –1.2) lower LS aBMD. Although further research is necessary to validate these cross-sectional data, the observed differences support hypotheses that improving intake of low-sulfate protein sources, or, alternatively, improving protein intake with a corresponding reduction in the dietary acid load, may be beneficial in osteoporosis prevention.

Protein deficiency is detrimental to bone health (21). Observational studies of bone health tend to promote a positive view of protein intakes > 0.8 g·kg^–1·d^–1 (22–26), although not all studies are favorable (27–29). Increasing protein intake elevates calcium losses in urine; in many studies, fecal calcium measures indicated no apparent compensation in calcium absorption, promoting the view that urinary calcium must reflect mineral lost from bone (30). More recently, however, Kerstetter et al. (2) have demonstrated not only increased calcium absorption but a reduction in urinary calcium of bone origin in subjects consuming 2.1 g·kg^–1·d^–1 protein compared with 1.0 g·kg^–1·d^–1 in a kinetic study using dual stable calcium isotopes. In the same study, no net differences in bone mineral mass were observed between levels of protein intake; however, there was a trend toward reduced bone turnover.

In light of the influence of protein on urinary calcium excretion, Dawson-Hughes and Harris (1,31) suggest that increased protein intake is beneficial provided that calcium intake is sufficient to support bone growth and urinary losses. Skov et al. (32) and unpublished data (M. Thorpe, D. Layman, P. Kris-Etherton, and E. Evans, researchers) from our laboratory demonstrate benefits of protein greater than recommended levels to bone mineral mass, in the presence of adequate calcium, during weight loss. In the present study, the interaction of calcium and protein intake was not significant at the LS (P = 0.77) or TH (P = 0.26).

Proposed mechanisms whereby dietary protein may enhance bone health include providing substrate for collagen deposition and increasing circulating levels of insulin-like growth factor-1, a known growth factor for bone (1). A recent prospective study by Alexy et al. (33) demonstrated improved bone health in youth with increasing protein intake. Conversely, bone status was negatively associated with the diet acid load, estimated by the PRAL, which includes estimation of the acidifying effect of dietary protein. The authors noted that this index of dietary acid load does not directly assess methionine and cysteine content of individual proteins but assumes a fixed proportion of sulfur to protein. The young, growing population is distinct in several important ways from the older population of this study, but the results illustrate a similar concept: protein is positively associated with bone mineral mass but also contributes to an acid load with negative ramifications for bone.

Total protein and protein sulfur are highly and intuitively correlated, but the actual ratio depends on protein source. Methionine is an essential amino acid and deficiency causes adverse health outcomes. However, our results suggest that the addition of lower sulfate protein to a diet that is already adequate in all essential amino acids may be beneficial in osteoporosis prevention; further research is necessary to test this hypothesis. Soy is implicated as a protein source with a low sulfur:protein ratio (13), estimated by Massey (34) at 39.8 mEq sulfur/100 g protein, compared with a mid-range 54.8 mEq sulfur/100 g protein for milk and 59.4 mEq sulfur/100 g protein for beef, and a higher 73.0 mEq sulfur/100 g protein for pork and 82.2 mEq sulfur/100 g protein for oatmeal. Experiments have tested the impact of soy protein, with and without isoflavones, on calcium balance with mixed results (35–38). Alternatively increased protein from all sources in connection with enhanced intake of alkalizing nutrients may be beneficial. It has been shown that a more alkaline diet is associated with improved bone density (39–43) and that supplementation of potassium bicarbonate or potassium citrate attenuates bone turnover (44,45). Jajoo et al. (46) demonstrated that replacing some cereal, an acidifying diet component, with more base-producing fruits and vegetables resulted in reduced levels of parathyroid hormone, bone resorption, and calcium excretion relative to controls.

It is conceptually important that the positive association in this sample between protein and LS aBMD, controlling for sulfur, was almost perfectly negated by the negative association of sulfate equivalents from methionine and cysteine and aBMD. This suppressor effect indicates that any study evaluating the association between protein intake and bone mineral status without controlling for actual sulfur content of protein may observe no significant correlation despite real positive and negative protein effects. A neutral combined influence of protein and sulfur on aBMD prediction would mask true effects in both studies hypothesizing detrimental acid load effects to bone and studies hypothesizing beneficial effects of dietary protein. This suppressor effect might explain the discordance of reports concerning the impact of dietary protein or acid load on bone health. Likewise, it is possible that uncontrolled influences of the total dietary acid load might explain conflicting reports. If true, this would suggest that future studies of associations between protein intake and bone density must account for the dietary acid load to produce unbiased results.

Our results suggest that direct estimation of sulfur from amino acids may perform better than the protein:potassium ratio or PRAL in studies of acid-base balance and bone mineral mass. These constructs were developed and validated for the estimation of the acidifying influence of dietary intake (3,8–10). However, because each estimates the acidifying effect of protein as a fixed ratio of total protein intake, they may not be well suited to the investigation of bone, where the negative acid effect seems to be opposed by an anabolic protein effect. Indeed, our results suggest that the negative and positive effects of protein may neatly cancel out one another at the LS, leaving the estimated influence on bone of total protein, as used in the protein:potassium ratio and PRAL, neutral despite otherwise reliable estimations of dietary acid load. The PRAL is also a function of calcium and phosphorus intake, which have well-established influences on the calcium economy (47). These factors may further confound the influence of the PRAL on bone. In light of these complex associations, it may be advisable to use direct intakes of protein, amino-acid sulfur (the acidifying agent of protein), and other relevant minerals such as potassium in lieu of estimated NEAP when calcium balance or bone mineral mass are investigated.

It is not clear why this suppressive association of sulfur intake would describe aBMD at the LS and not at the TH, although differences in the response of these 2 sites are commonly reported (48–50). The change in R² at the LS indicated a small likelihood of a type I error or inappropriate rejection of the null hypothesis that sulfur intake does not predict aBMD. Conversely, a post hoc power analysis of the regression at the hip was performed and indicated a power of 0.74 to detect an improvement in R² as large as that observed at the spine. Though it is not entirely improbable that our regression failed to detect a true difference at the hip, it is very possible that a real difference between protein associations at the hip and spine explain divergent findings. Differences in observations between these measurement sites may relate to differing levels of trabecular compared with cortical bone content; specifically, the ratio of trabecular:cortical bone is greater at the spine than at the hip. It has been theorized that trabecular bone, being more metabolically active, may be more sensitive to dietary intervention, at least in the short term (51,52). However, a post hoc analysis of the trochanter subregion, with a greater proportion of trabecular bone, did not mimic the findings at the LS in this sample (data not shown). Alternatively, it is possible that the weight-bearing load at the hip during ambulation may blunt the effects of dietary factors.

This study is not without limitations; the constraints of cross-sectional data are recognized. This research is performed in the context of a broader body of literature suggesting causal connections between protein intake and bone mineral mass (31,32), sulfate intake and acid-base balance (53,54), and acid base balance and bone demineralization (4–6,44,55). The results are theoretically consistent and harmonize apparently conflicting research; however, this observational study offers no basis for new causal inference and is best interpreted as a rationale for additional investigation.

In conclusion, protein intake is positively associated with aBMD in postmenopausal women, but this association is suppressed by a negative association of sulfur from amino acids at the LS. This observation may reconcile reports of positive impacts of dietary protein on bone health with reports of a negative impact of the acid load from sulfur-containing amino acids. At the TH, dietary protein intake is positively associated with aBMD and may not exhibit the same negative association with sulfur from protein as observed at the spine. These results highlight the need to evaluate actual sulfur contents of varying dietary protein sources rather than assuming a fixed ratio of sulfur:protein. Future research in this line of inquiry should evaluate the role of dietary protein in preserving bone health in populations at higher risk for fracture such as the elderly.

	FOOTNOTES

 
¹ Supported by National Institute on Aging Grant R01 AG20118 to Edward McAuley.

² Author disclosures: M. Thorpe, M. C. Mojtahedi, K. Chapman-Novakofski, E. McAuley, and E. M. Evans, no conflicts of interest.

⁵ Abbreviations used: aBMD, areal bone mineral density; DXA, dual X-ray absorptiometry; LS, lumbar spine; NEAP, net endogenous acid production; PRAL, potential renal acid load; TH, total hip.

Manuscript received 21 August 2007. Initial review completed 13 September 2007. Revision accepted 9 October 2007.

	LITERATURE CITED

TOP ABSTRACT Introduction Methods Results Discussion LITERATURE CITED

 

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