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Effect of Dietary Protein Supplements on Calcium Excretion in Healthy Older Men and Women

Bone Metabolism Laboratory (B.D.-H., L.S., G.E.D.) and Metabolic Research Unit, Nutrition Services Department (H.R.), Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts 02111; and New England Research Institutes (S.S.H.), Watertown, Massachusetts 02472

Address all correspondence and requests for reprints to: Bess Dawson-Hughes, M.D., Bone Metabolism Laboratory at the Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging, Tufts University, 711 Washington Street, Boston, Massachusetts 02111. E-mail: bess.dawson-hughes{at}tufts.edu.

	Abstract

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Currently there is no consensus on the impact of dietary protein on calcium and bone metabolism. This study was conducted to examine the effect of increasing protein intake on urinary calcium excretion and to compare circulating levels of IGF-I and biochemical markers of bone turnover in healthy older men and women who consumed either a high or a low protein food supplement for 9 wk. Thirty-two subjects with usual protein intakes of less than 0.85 g/kg·d were randomly assigned to daily high (0.75 g/kg) or low (0.04 g/kg) protein supplement groups. Isocaloric diets were maintained by advising subjects to reduce their intake of carbohydrates. Selected biochemical measurements were made at baseline and on d 35 and either d 49 or 63. Changes in urinary calcium excretion in the two groups did not differ significantly over the course of the study. The high protein group had significantly higher levels of serum IGF-I (P = 0.008) and lower levels of urinary N-telopeptide (P = 0.038) over the period of d 35–49 or 63. We conclude that increasing protein intake from 0.78 to 1.55 g/kg·d with meat supplements in combination with reducing carbohydrate intake did not alter urine calcium excretion, but was associated with higher circulating levels of IGF-I, a bone growth factor, and lowered levels of urinary N-telopeptide, a marker of bone resorption. In contrast to the widely held belief that increased protein intake results in calcium wasting, meat supplements, when exchanged isocalorically for carbohydrates, may have a favorable impact on the skeleton in healthy older men and women.

	Introduction

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DIETARY PROTEIN HAS several effects on the way calcium is handled, but there is little information from protein intervention studies describing the impact of protein on bone mass. In the one available intervention study, elderly patients with acute hip fractures had decreased rates of bone loss from the contralateral hip over the year after their fracture when supplemented for 6 months with 20 g/d protein compared with controls (1).

There have been several shorter-term intervention studies assessing the effects of protein on serum IGF-I levels, biochemical markers of bone turnover, and urinary calcium excretion, and there is little consistency in their findings. For example, IGF-I has either increased (1, 2, 3) or remained unchanged (4); biochemical markers of bone resorption have increased (5), remained unchanged (1, 4), or decreased (3); and urinary calcium excretion has increased (3, 5, 6, 7, 8) or remained unchanged (3, 4, 9). Variability in the results of these intervention studies may be related to the populations studied, the starting protein intakes of the subjects, their calcium intakes, the overall acid/base balance of the diet, the duration of the protein intervention period, and the type of protein used (purified vs. food based, and animal vs. plant). Observational studies have given an equally mixed picture of the impact of dietary protein on bone mass, change in bone mass, and fracture incidence.

This study was conducted to evaluate the impact of increasing protein intake from meat (in combination with lowering carbohydrate intake) for 9 wk on urinary calcium excretion in healthy older men and women. We also compared serum IGF-I levels and markers of bone turnover in the high and low protein groups after d 35 and either d 49 or 63 on the food supplements.

	Subjects and Methods

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Subjects

A total of 33 healthy men and women age 50 yr and older were enrolled. All subjects completed the study. The protocol was approved by the investigation review board at Tufts University, and written informed consent was obtained from each subject. Telephone prescreening was used to identify subjects with usual dietary protein intakes of 0.85 g/kg or less and calcium intakes less than 700 mg/d and to determine general eligibility. No subject was taking estrogen, glucocorticoids, or insulin, and none had a history of a disorder known to alter calcium or bone metabolism. Screening evaluation included dual energy x-ray absorptiometry hip scans, and blood and urine tests. Subjects were excluded if they had a femoral neck z-score less than -2.0, an abnormal renal or liver function test, or a 24-h urinary calcium level above 300 mg/d. One subject assigned to the low protein group was excluded from the analysis because he was believed to have abnormal bone metabolism. He had very high urinary excretion of N-telopeptide (1004 nm bone collagen equivalents on d 63, compared with the next highest value of 360 nm bone collagen equivalents). This individual also had an elevated urinary calcium excretion during the study.

Experimental design

Subjects were randomly assigned to high or low protein intakes throughout this 63-d study. Fasting blood was drawn, and 24-h urine collections were returned during screening (2 wk before enrollment) and on d 35, 49, and 63. During the last month (d 35–63), subjects took 800 mg/d supplemental calcium for 2 wk and placebo for 2 wk, in random sequence. At the end of the study, there was no urinary calcium or PTH response to the high calcium intake. This led to the discovery that the calcium triphosphate and placebo pills, prepared by a local compounding pharmacy, did not disintegrate until they had been exposed to acid for 50 min. Hence, the high calcium portion of the experiment is invalid, and we report only the low calcium intake results on d 35 and d 49 or 63, with half of the subjects from each treatment group measured on each of these 2 d.

Diets and food supplements

Subjects maintained their usual low protein diets and consumed a multivitamin and a food supplement daily during the study. Subjects assigned to the high protein group chose from a menu of meat supplements, each containing 0.75 g protein/kg body weight. Subjects assigned to the low protein group consumed a food supplement that was isocaloric with the protein supplements; it consisted mainly of carbohydrates (e.g. rice or pasta salad) and contained small amounts of fat in the form of olive oil for palatability. Subjects were weighed each week and were counseled by the research dietitian to modify their intake of carbohydrates as needed to maintain weight within 1.5 kg of their initial weight.

Food supplements were cooked, wrapped in daily portion sizes, and frozen. They could be eaten at any time of day. Subjects were limited to no more than two caffeine-containing beverages per day. Subjects were given diaries in which to record whether they had eaten the food supplements and taken their multivitamins.

Dietary assessments

Dietary intakes of protein, calcium, phosphorus, magnesium, and total energy over the preceding 2 months were assessed on the screening visit and on d 63 with use of the Fred Hutchinson Food Frequency Questionnaire (10). The questionnaires were self-administered on site and reviewed for completeness by a dietitian.

Bone mineral density and content

Screening femoral neck bone mineral density measurements were made with a GE Lunar Prodigy scanner (Lunar Corp., Madison, WI) with a precision of 1.66% (11). Total body bone mineral content (BMC), nonfat soft tissue, and fat were measured at the beginning and end of the study, with precisions of 1.12%, 0.77%, and 0.94%, respectively (11).

Biochemical measurements

Blood was drawn after an 8-h fast. Serum 25-hydroxyvitamin D was measured with RIA kits from Diasorin (Stillwater, MN), serum PTH was determined with immunoradiometric assay kits from Nichols Institute Diagnostics (San Juan Capistrano, CA), serum IGF-I and osteocalcin were measured with RIA kits from Nichols Institute Diagnostics, and serum N-telopeptide levels were determined with competitive inhibition ELISA kits from Ostex International (Seattle, WA). Serum and urinary creatinine were measured by colorimetry with a Cobas Mira chemistry analyzer (Roche, Bellville, NJ). Urinary sodium and potassium were measured by direct current plasma emission spectroscopy with a Spectraspan 6 (Beckman Instruments, Palo Alto, CA). The intra- and interassay coefficients of variation are 2.7% and 6.8%, respectively. Urinary nitrogen was measured with a model FP-2000 nitrogen/protein determinator (LECO, St. Joseph, MI). This instrument employs a Dumas combustion method and detection using a thermal conductivity cell. It measures nitrogen with a precision of 15 ppm. Samples from individual subjects were batched for the 25-hydroxyvitamin D, PTH, osteocalcin, IGF-I, N-telopeptide, sodium, and potassium analyses.

Statistical analyses

Characteristics of the subjects in the high and low protein groups were compared using two-sample t tests (continuous variables) and ² tests (categorical variables). Differences between protein groups with regard to changes in laboratory values were examined using repeated measures analysis of covariance. Correlation coefficients were compared using Fisher’s z statistic. Analyses were conducted with SPSS for Windows (version 11.5, SPSS, Inc., Chicago, IL). All results are the mean ± SD unless otherwise stated. All P values are two-sided, and P < 0.05 was considered to indicate statistical significance.

	Results

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The clinical characteristics of the 32 subjects in the high and low protein groups are shown in Table 1. The groups were similar in age, sex distribution, weight, and other characteristics.

View larger version (24K): [in this window] [in a new window]   FIG. 1. Mean (±SEM) serum IGF-I, 24-h urinary N-telopeptide, and serum osteocalcin levels in the low () and high () protein groups. Differences in the two protein groups were significant for IGF-I (P = 0.008) and N-telopeptide (NTX; P = 0.038), but not for osteocalcin (P = 0.795).

Serum osteocalcin and PTH levels did not differ significantly in the two protein groups. As expected, the high protein intake group had significantly greater urinary excretion of nitrogen and potassium than the low protein group (Table 5).

Mean total body BMC and lean and fat tissue weights were similar in the two protein groups at entry (Table 1), and changes in the two groups did not differ significantly (high protein group change, 30.6 ± 56.9 g; low protein group change, 22.9 ± 79.6 g; P = 0.757). However, within the high protein group, total body BMC increased significantly over the 9-wk period (P = 0.049) in contrast to the low protein group (P = 0.268). Within the low protein group, nonfat soft tissue weight decreased significantly over the study interval (P = 0.022), but it did not change significantly in the high protein group (P = 0.179). Total body fat tissue increased significantly in both groups (P < 0.03).

	Discussion

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It has often been observed that protein induces calciuria (5, 6, 7, 8), and that this occurs in relation to its acid-producing load (12). Other components of the diet also influence acid-base balance; carbohydrate, like protein, increases the potential renal acid load (13), and fruits and vegetables lower it by providing potential alkali (13). It is therefore not surprising that calciuria has not been a consistent finding in intervention studies in which food sources of protein were tested in subjects on isocaloric diets (3, 4). In our study meat supplements had no significant effect on urinary calcium excretion in healthy older subjects who maintained isocaloric diets by reducing their intake of carbohydrate. The high protein group did not report any change in their intake of fruits and vegetables, as indicated by their stable intakes of potassium and magnesium. Nonetheless, urinary potassium excretion was higher in the high than in the low protein group during the study, probably because of the potassium present in the meat supplements, although we cannot rule out the possibility that the high protein group also increased their fruit and vegetable intake despite their report of no change. In the high protein group, there was some suggestion that urinary calcium excretion first rose and then declined, indicating that perhaps some adaptation may have occurred. There is other evidence that the meat supplements did not induce losses of bone calcium. The meat group had lower levels of the bone resorption marker, N-telopeptide, than the low protein group, and they had a gain in total body BMC during the study. Additionally, there was lack of a significant correlation of urinary N-telopeptide with urinary calcium excretion in the high protein group in contrast to the low protein group.

In this study increasing protein intake as meat from 0.78 to 1.6 g/kg·d was associated with 25% higher serum IGF-I levels. This is consistent with other evidence that dietary protein can increase circulating IGF-I levels in older populations. Arjmandi et al. (3) observed significant increases in serum IGF-I with the addition of 40 g soy and milk protein in older women with basal protein intakes of 0.71–0.86 g/kg·d. In another study adult men and women with basal protein intakes of about 65 g/d, when supplemented with three extra servings of milk per day (containing 27 g protein), had significant increases in serum IGF-I (2). Schurch et al. (1) administered 20 g protein daily for 6 months to elderly acute hip fracture patients with a usual mean protein intake of 0.75 g/kg·d and observed an 80% increase in serum IGF-I levels. In contrast, Roughhead et al. (4) reported no change in serum IGF-I levels after increasing dietary protein intake from 0.94 to 1.62 g/kg·d for 8 wk in postmenopausal women. An explanation for the varied effects of protein on serum IGF-I may lie in the different starting protein intake levels in these studies. In classical experiments, Isley et al. (14) found that fasting for 5 d lowered serum IGF-I levels by about 65% in healthy young subjects and that refeeding normal calories and graded amounts of protein resulted in progressive recovery of IGF-I levels with increasing protein intake up to a level of 1.0 g/kg·d. It is unknown whether the same plateau exists in older subjects, but the lack of increase in older women with starting intakes of 0.94 g/kg·d (4) suggests that it may be similar.

The higher protein intake was associated with lower indexes of bone resorption, as indicated by a lower 24-h urinary N-telopeptide level. With the observed higher IGF-I levels, one might have expected to see an increase in bone formation, based on studies in which exogenous IGF-I was administered to postmenopausal women (15) and osteoporotic men (16), but we saw no significant group difference in serum osteocalcin levels. Other studies report divergent effects of protein on markers of bone turnover. Kerstetter et al. (5) observed higher levels of urinary N-telopeptide in subjects on high protein diets compared with low protein diets; however, this study in young subjects had short diet intervention periods of only 4 d. In a metabolic study with 8-wk diet periods, Roughhead et al.(4) found no change in markers of bone formation or resorption after increasing dietary protein from 0.94 to 1.62 g/kg·d. Similarly, Schurch et al. (1) found no significant changes in bone turnover over 6 months, although there were trends toward higher serum osteocalcin and lower urinary pyridinoline levels in the hip fracture patients supplemented with 20 g/d protein. Arjmandi et al. (3) noted a significant decrease in urinary deoxypyridinoline levels with soy, but not with milk, protein supplements and no change in bone-specific alkaline phosphatase, a marker of bone formation, in either group. The increase in IGF-I may have contributed to the stabilization of total body nonfat soft tissue, an index of muscle mass, in the high protein group, but caution should be taken not to overinterpret this short-term study.

Serum PTH levels did not differ after 1–2 months of supplementation in our two protein groups, leading us to conclude that the observed differences in circulating IGF-I and N-telopeptide levels in our two groups were independent of PTH. In another study serum PTH levels rose in the first 4 d of protein loading (5), but serum PTH levels did not change significantly in any of the other longer-term IGF-I (15, 16) or protein (1, 4) intervention studies.

A limitation of this study is that baseline measurements were not available for the items in Table 5; however, the two groups agreed well on clinical characteristics, dietary profiles, calcium excretion, and bone mineral density, making it likely that they also agreed on the other measurements.

In conclusion, this study did not confirm the perception that increased dietary protein results in urinary calcium loss. The constellation of findings that meat supplements containing 55 g/d protein, when exchanged for carbohydrate did not significantly increase urinary calcium excretion and were associated with higher levels of serum IGF-I and lower levels of the bone resorption marker, N-telopeptide, together with a lack of significant correlation of urinary N-telopeptide with urinary calcium excretion in the high protein group (in contrast to the low protein) point to the possibility that higher meat intake may potentially improve bone mass in many older men and women.

	Footnotes

 
This material is based on work supported by the U.S. Department of Agriculture, under Agreement 58-1950-9001. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture.

Abbreviation: BMC, Bone mineral content.

Received August 21, 2003.

Accepted December 4, 2003.

	References

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Dawson-Hughes, B. Articles by Dallal, G. E. Search for Related Content PubMed PubMed Citation Articles by Dawson-Hughes, B. Articles by Dallal, G. E. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB CALCIUM COMPOUNDS CALCIUM, ELEMENTAL PARATHYROID HORMONE Medline Plus Health Information Dietary Proteins