The Impact of Dietary Protein on Calcium Absorption and Kinetic Measures of Bone Turnover in Women
http://www.100md.com
《临床内分泌与代谢杂志》
Abstract
Although high-protein diets induce hypercalciuria in humans, the source of the additional urinary calcium remains unclear. One hypothesis is that the high endogenous acid load of a high-protein diet is partially buffered by bone, leading to increased skeletal resorption and hypercalciuria. We used dual stable calcium isotopes to quantify the effect of a high-protein diet on calcium kinetics in women. The study consisted of 2 wk of a lead-in, well-balanced diet followed by 10 d of an experimental diet containing either moderate (1.0 g/kg) or high (2.1 g/kg) protein. Thirteen healthy women received both levels of protein in random order. Intestinal calcium absorption increased during the high-protein diet in comparison with the moderate (26.2 ± 1.9% vs. 18.5 ± 1.6%, P < 0.0001, mean ± SEM) as did urinary calcium (5.23 ± 0.37 vs. 3.57 ± 0.35 mmol/d, P < 0.0001, mean ± SEM). The high-protein diet caused a significant reduction in the fraction of urinary calcium of bone origin and a nonsignificant trend toward a reduction in the rate of bone turnover. There were no protein-induced effects on net bone balance. These data directly demonstrate that, at least in the short term, high-protein diets are not detrimental to bone.
Introduction
MORE THAN 80 yr ago, Sherman (1) documented that humans fed a high-protein diet developed hypercalciuria. A recent meta-analysis of 26 controlled human intervention studies shows that for every 25-g increase in dietary protein, there is a 0.8 mmol rise in urinary calcium (2). The source of the additional urinary calcium excreted during a high-protein diet is unknown.
A popular hypothesis is that the additional calcium excreted in the urine results, in part, from changes in bone homeostasis. This hypothesis is supported by some but not all studies. It is well established that dietary protein increases endogenous acid production (3). In response to the acid load, bone may be called upon as a reservoir of alkali, and, as a consequence, bone calcium is mobilized. If true, then the long-term consequence of buffering by bone would be increased bone resorption and a decline in bone mineral density (BMD). Consistent with this hypothesis, osteoclast-mediated bone resorption is increased in an acid environment when compared with studies done at neutral or alkaline pH (4, 5, 6, 7). Most balance studies (2) have reported that intestinal calcium absorption is unaffected by dietary protein, suggesting that the source of the extra urinary calcium resulting from a high-protein diet was not of intestinal origin. At odds with the bone hypothesis are a large number of epidemiological studies showing that a high-protein diet is associated with a high, not a low BMD, as might be predicted (8, 9, 10, 11, 12, 13, 14, 15, 16, 17).
Because osteoporosis is a major public health problem, clarifying the impact of dietary protein on the skeleton is important. Therefore, we employed dual stable calcium isotopes to determine the relative contributions of the skeleton and the intestine to the rise in urinary calcium during the first week of a high-protein diet.
Subjects and Methods
Study design
The protocol consisted of two cycles involving 2 wk of an adjustment diet, followed by 10 d of an experimental diet. For the adjustment period, subjects modified their diets to contain moderate amounts of protein, sodium, calcium, and caffeine. During the 10-d experimental period, subjects received all food from the Yale General Clinical Research Center (GCRC) metabolic kitchen. These diets contained tightly controlled levels of calcium, sodium, phosphorus, and one of two levels of dietary protein: moderate (1.0 g/kg) or high (2.1 g/kg). The 2-wk adjustment period and 10-d experimental cycle were repeated again until all subjects received both levels of protein in random order. Fasting blood and urine samples were collected on the mornings of d 0 and 4 of each experimental period. During the morning of d 4, subjects received an iv dose of 42Ca. Three oral doses of 44Ca were administered with each meal during that day. For the next 6 d, blood and urine samples were collected for measurements of 42Ca and 44Ca.
Subjects
Ten healthy young women between 20–40 yr of age (29 ± 6 yr, mean ± SD) and three healthy postmenopausal women between 55–70 yr (61 ± 7 yr, mean ± SD) participated in the study. Body weight averaged 65 ± 2 and 65 ± 6 kg and body mass index averaged 23 ± 1 and 25 ± 2 kg/m2 in the young and older women, respectively. Exclusion criteria included medications known to affect calcium metabolism, amenorrhea, pregnancy, smoking, eating disorders, diabetes, renal or gastrointestinal disease, bone disease, nephrolithiasis, or intensive daily physical exercise. Subjects suspended all vitamin and mineral supplements during the entire study.
The racial background of the subjects was either Caucasian or Asian because these are the groups of women at highest risk for osteoporosis. Subjects continued their usual activities at home, school, and work during the study. The study was approved by Investigational Review Boards at Yale University (New Haven, CT), the University of Connecticut (Storrs, CT), and Johns Hopkins University (Baltimore, MD). Informed consent was obtained from each participant.
Diets
The experimental and adjustment diets are similar to those described in previous reports (18, 19). During the first week of the adjustment period, subjects were instructed to select diets containing approximately 1.2 g protein/kg, 20 mmol calcium, and 100 mmol sodium. This diet was provided to them by the GCRC’s metabolic kitchen during the second week of the adjustment period to completely control nutrient intake. Subjects consumed adequate energy for weight maintenance. Caffeine-containing beverages were limited to one a day, and alcohol was not permitted.
During the 10-d experimental period, subjects reported daily to the GCRC to receive their meals and record their body weight. Energy intake ranged from 0.13 to 0.15 MJ/kg (30–35 kcal/kg) and was adjusted with simple sugars and fats to maintain body weight. We asked that the subjects consume all the food and beverage (and nothing other than the food and beverage) that was provided to them. Distilled water was provided ad lib.
All experimental diets were individually calculated to contain one of two levels of protein, whereas the levels of other nutrients were fixed (20 mmol calcium, 100 mmol sodium, and 32–39 mmol phosphorus). The macronutrient and mineral composition of the experimental diets was calculated from the U.S. Department of Agriculture Handbook No. 8 and manufacturer’s information. The primary sources of calcium in the experimental diets were dairy foods and a chewable calcium carbonate (Tums, GlaxoSmithKline, Pittsburgh, PA). The increase in protein from the control to the high-protein diet was accomplished by adding both animal and vegetable sources of protein. Renal net acid excretion (RNAE) was estimated by the method of Frassetto et al. (20). RNAE (mEq/d) = 62.1 x (protein in g/potassium in mEq) –17.9. Using their equation, the estimated RNAE resulting from our medium-protein diet was 40.5 ± 2.6 and high-protein diet was 109.4 ± 6.5 (P < 0.0001).
Nonisotopic sample collection and analyses
Fasting blood collections were obtained at d 0 and 4 of each 10-d experimental period for measures of PTH, total and ionized calcium, phosphorus, and creatinine. A timed 24-h urine was collected on d –1 and d 3 for measurement of calcium, phosphorus, sodium, and creatinine. The tubular reabsorption for calcium (TRCa) was calculated from a fasting 2-h morning urine collection (0700–0900 h) and a midpoint blood sample (0800 h) using serum creatinine and ionized calcium and urine calcium and creatinine. The mean d 4 TRCa on the medium diet was 0.978 ± 0.006 and on the high diet 0.976 ± 0.003 (not significant). All nonisotopic assays were performed as reported (18).
Isotope administration and sample analysis
On d 4 of the experimental diet, oral 44Ca was administered in three divided doses, delivered with each meal in proportion to the calcium content of the meal. Dividing the oral dose corrects for potential differences in calcium bioavailability between meals. Each oral calcium isotope was equilibrated in milk for 8–24 h. Immediately after breakfast, the subject received an iv infusion of 42Ca, administered over a 5-min interval. The IV line was then flushed with saline. Immediately after the IV infusion, eight blood samples were collected at 5, 10, 20, 40, 60, 120, 240, and 480 min. All urine was then collected in acid-washed containers for the next 34 h in pools of 8, 12, and 14 h. Three spot urine samples were collected daily by the subject beginning the evening of d 5 and continuing until the end of d 9.
Calcium isotope ratios were measured using either a quadrupole (THQ, Finnigan, Bremen, Germany) or magnetic sector (Triton TI, Finnigan, Bremen, Germany) thermal ionization mass spectrometer. A ratio was made between each administered calcium tracer (42Ca and 44Ca) and another naturally occurring calcium isotope (48Ca or 43Ca). All isotopes were corrected for isotopic fractionation by normalizing the data to the 43/48Ca or 48/43Ca. Fractional calcium absorption was determined as the ratio of the cumulative oral tracer recovery to the cumulative IV tracer recovery in the 34-h urine collection obtained postdosing. True calcium absorption was calculated as the product of fractional calcium absorption and the calculated calcium intake.
Calcium kinetic measurements were determined using a multicompartmental model of calcium kinetics (21, 22, 23). After the first 24 h, the clearance of the oral and IV calcium tracer parallel one another and urine samples can be used to monitor the disappearance of the IV tracer. Rates of bone calcium deposition (Vo+) and resorption (Vo–) were then determined after accounting for urinary and endogenous fecal calcium losses. Net bone balance was determined as the difference between Vo+ and Vo–. The relative contribution of the recent diet and bone to urinary calcium excretion was calculated by the method of Welch et al. (24). Urinary calcium derived from the recent diet (Vou) is the product of dietary intake and the percentage of oral isotopic calcium recovered in the urine. Urinary calcium derived from bone (Vbu) is the difference between urinary calcium and Vou.
Statistical analyses
All values are presented as mean ± SEM (unless otherwise specified). A Student’s t test was initially used to compare the younger with the older women, and because there were no important differences in calcium homeostasis, the two age groups were pooled for all subsequent analyses. A paired Student’s t test was then used to evaluate differences in all biochemical outcomes between the two levels of protein at baseline and at d 4. The mean dietary intakes were evaluated between the two levels of protein using a paired Student’s t test. A probability level of P < 0.05 was statistically significant.
Results
Subjects remained healthy throughout the experiment. The average nutrient composition of the experimental diets is presented in Table 1. As shown in Table 2, the average urinary sodium on d 4 of the moderate- and high-protein diets was not different and indicative of good compliance.
Baseline measures (d 0) of serum and urine metabolites did not differ between the two diets (Table 2). Aside from urinary calcium, the diets induced no changes in calcium indices that we measured at d 4 (Table 2). By d 4, urinary calcium in every subject increased during the high-protein diet (Fig. 1), rising from 3.57 ± 0.35 during the moderate to 5.23 ± 0.37 mmol/d during the high-protein diet (P < 0.0001).
There were no statistically significant differences in kinetic measures of bone formation, resorption, or balance between the diets (Table 3). However, there was a nonsignificant trend toward lower bone turnover (both formation and resorption) during the high-protein diet in comparison with the moderate control protein. There was a significant increase during the high-protein diet in the relative fraction of urinary calcium of dietary origin and a significant decrease in the relative fraction of urinary calcium from bone origin compared with the moderate protein diet (Table 3). All subjects were in negative calcium balance during all interventions.
Percent intestinal calcium absorption increased during the high-protein diet (26.2 ± 1.9%) in comparison with the moderate (18.5 ± 1.6%, P < 0.0001), representing a mean increase of 7.7% (raw increase) or 42% (relative increase; Fig. 1). Because dietary calcium was fixed at 20 mmol/d, the intestinal absorption on the high-protein diet was 1.54 ± 0.20 mmol/d higher than on the moderate diet. The mean increase in urinary calcium between the two diets was 1.66 ± 0.25 mmol/d.
Discussion
We used dual stable calcium isotopes to quantify the contributions of bone and intestine to the increment in urinary calcium observed during a high-protein diet. High-protein diets (in comparison with the moderate, control protein diet) resulted in a significant increase in intestinal calcium absorption that paralleled the increase in urinary calcium excretion. These effects were evident in all subjects and were not influenced by age across the age range studied. The increase in true calcium absorption during the high-protein diet was accompanied by a significant decrease in the fraction of calcium from nondietary (bone) origin. Therefore, at least acutely, the increase in urinary calcium from a high-protein diet is explained by increases in intestinal calcium absorption with no contribution from bone resorption. Finally, no matter what level of protein was ingested, all women were in negative calcium balance while consuming the 20 mmol calcium, suggesting that 20 mmol calcium is not adequate to maintain bone balance.
A regression equation based on data from 26 published human studies (that included a wide variety of protein sources, subjects, and study design) predicts that a 58-g increase in dietary protein would result in a 1.86 mmol rise in urinary calcium (2). Consistent with this prediction, the addition of 58 g of protein to the diets of our subjects increased urinary calcium by 1.66 mmol.
The dual stable calcium isotope method we used measures true intestinal calcium absorption. Although we did not measure fecal calcium and cannot directly quantify endogenous excretion of calcium, we can estimate endogenous calcium excretion based on published equations (25). Intestinal calcium secretion can itself be influenced by calcium absorption and dietary phosphorus. During the high-protein diet, when absorption efficiency is increased, one would expect that both dietary calcium and endogenously secreted calcium would be absorbed more efficiently, so there would be less calcium in the feces. We have calculated, on the basis of published equations (25), that the 7.7% raw increase in intestinal calcium absorption would result in a decrease of 0.2 mmol/d in endogenous fecal calcium excreted during the high-protein diet. However, the high-protein diet contained 3.2 mmol more phosphorus than the medium-protein diet. Again, on the basis of the work of Heaney and Recker (25), this difference in dietary phosphorus would lead to an additional 6 mg (0.2 mmol) calcium in the intestinal tract during the high-protein diet. Thus, these two countervailing effects negate each other because the increased absorption of secreted calcium is off set by the dietary phosphorus-induced increase in endogenous fecal calcium. Furthermore, it should be kept in mind that these calculated changes are relatively small in relation to the large change in urinary calcium between the diets of 1.66 mmol.
The 42% relative increase (or 7.7% raw increase) in intestinal calcium absorption (18.5 vs. 26.2%) in the current study accounts for 1.54 of the 1.66 mmol rise in urinary calcium or approximately 93%. The difference in intestinal calcium absorption between the control and the high-protein diet in our study is large and probably explains why human intervention studies generally report an increase in urinary calcium in response to a high-protein diet despite variability in experimental design, subjects, types of diets, and nutrient composition (2).
It is often suggested that a decrease in renal tubular calcium reabsorption explains, in part, the hypercalciuria observed on a high-protein diet. This was not the case in our experiment because TRCa was unchanged on the two diets (medium diet, 0.978 ± 0.006; high diet, 0.976 ± 0.003; P = not significant).
The majority of the balance studies in humans have not reported a change in calcium absorption in response to dietary protein (2). However, there are at least three balance studies that showed an increase in calcium absorption as dietary protein is increased (26, 27, 28). In general, balance studies should be interpreted with caution because the method is highly dependent on quantifying fecal calcium loss, which is difficult to measure accurately.
Two isotopic studies found no association between dietary protein intake and calcium absorption (29, 30). Heaney (29) reported that, in a longitudinal study of a large group of Roman Catholic nuns (mean age 48.7 ± 7.0 yr), there was no relationship between intestinal calcium absorption and usual intake of dietary protein. The mean protein intake in his study was 62 g (or approximately 1 g/kg), and their calcium intake averaged 17.6 mmol/d. Dawson-Hughes found similar results in adults over the age of 65 yr (30). Although these studies employed isotopic measures of calcium absorption, a method generally considered more accurate than a balance approach, dietary protein was not experimentally manipulated. It is possible that the large interindividual variability in intestinal calcium absorption and the multitude of other dietary factors that can also affect calcium absorption may explain why the relationship between protein intake and intestinal calcium absorption was not observed in these two studies.
In general, the evidence that dietary protein influences intestinal calcium transport in animals is strong. In rodents, several studies have shown that increasing dietary protein increases intestinal calcium absorption and shifts the route of calcium excretion from the intestinal tract to the urine (31, 32, 33).
The mechanism that underlies the rise in intestinal calcium absorption in response to dietary protein is unknown. Dietary protein and amino acids are gastric acid and gastrin secretagogues (34, 35). Gastric acid helps to solubilize dietary calcium and could potentially improve its bioavailability, although the literature on this topic is contradictory (36, 37, 38). Alternatively, the peptide fragments (e.g. casein phosphopeptides) that are released during dairy protein digestion may aid in solubilizing calcium, making it more available for absorption (39, 40, 41, 42).
Although not statistically significant, there was a trend toward decreased bone turnover (Table 2) during the high-protein diet. The high-protein diet was associated with a significant decrease in the fraction of urinary calcium that was of bone origin. We calculated RNAE by the method of Frassetto et al. (20) to estimate the potential renal acid load generated by the two experimental diets. The two are highly correlated (20). By these calculations, the high-protein diet generated 2.7 times more fixed acid than did the control diet (RNAE in mEq/d was 109.4 ± 6.5 high protein vs. 40.5 ± 2.6 medium protein; P < 0.0001). Despite this, there was no effect on bone resorption.
Our experimental intervention of 1 wk was perhaps too short to detect significant changes in bone homeostasis despite the large change in intestinal calcium absorption. Moreover, because of the variability in rates of bone turnover, larger sample sizes may be needed to detect significant changes in this parameter even with a paired study design. However, the acute increase in intestinal calcium absorption and trend toward decreased bone turnover observed in our study may help explain the epidemiological studies that show higher BMD associated with high protein intakes (8, 9, 10, 11, 12, 13, 14, 15, 16, 17). Clearly, longer term intervention studies are needed to answer the question.
In a recent isotopic intervention study of 8 wk in length, Roughead et al. (43) evaluated calcium retention using sensitive radiotracer and whole-body scintillation counting in 15 postmenopausal women as they consumed moderate and high-protein diets. Calcium retention did not differ between the two levels of protein, although there was a nonsignificant trend toward better retention during the high-protein diet. Taken together, the two isotopic intervention studies [ours and that of Roughead et al. (43)] support the conclusion that high-protein diets, at least in the short and intermediate term, do not result in increased bone loss. If anything, both studies showed nonsignificant trends toward lower bone turnover and better calcium retention during high-protein diets.
Our finding that the average bone balance in our subjects was negative by 2.5 mmol/d (no matter what level of dietary protein was consumed) was unanticipated. No subject was in positive bone balance on any intervention, even when intestinal calcium absorption was highest during the high-protein diet. Clearly, 20 mmol dietary calcium is not enough to maintain calcium balance in our healthy female adults, both young and old. We selected 20 mmol calcium because it was the recommended daily allowance at the time the study was designed, and it approximates the calcium intake of many adult women. In the most recent version of the Dietary Reference Intakes, recommendations are higher, 25–30 mmol, depending on age (44). It would be important to determine whether the current higher recommended intakes for calcium would restore bone balance.
In summary, we used dual stable calcium isotopes to evaluate the acute impact of dietary protein on bone kinetics in a diet-controlled, cross-over study. The majority of the rise in urinary calcium in response to an increase in dietary protein was due to an increase in intestinal calcium absorption. Our short-term study needs to be extended to longer term intervention trials to fully evaluate the impact of dietary protein on bone turnover. Our study does not support the hypothesis that increased dietary protein is acutely harmful to bone.
Footnotes
This work was supported by Grant DK52128-03 from the National Institutes of Health (NIH) RO1, by The Ethel F. Donaghue Women’s Health Investigator Program at Yale University, and by Yale’s GCRC (NIH MO1-RR00125).
First Published Online November 16, 2004
Abbreviations: BMD, Bone mineral density; GCRC, General Clinical Research Center; RNAE, renal net acid excretion; TRCa, tubular reabsorption for calcium; Vbu, urinary calcium derived from bone; Vo+, bone calcium deposition; Vo–, bone calcium resorption; Vou, urinary calcium derived from the recent diet.
Received February 5, 2004.
Accepted September 9, 2004.
References
Sherman H 1920 Calcium requirement of maintenance in man. J Biol Chem 44:21–27
Kerstetter JE, O’Brien KO, Insogna KL 2003 Low protein intake: the impact on calcium and bone homeostasis in humans. J Nutr 133:855S–861S
Barzel US, Massey LK 1998 Excess dietary protein can adversely affect bone. J Nutr 128:1051–1053
Bushinsky DA 1996 Metabolic alkalosis decreases bone calcium efflux by suppressing osteoclasts and stimulating osteoblasts. Am J Physiol 271:F216–F222
Arnett TR, Spowage M 1996 Modulation of the resorptive activity of rat osteoclasts by small changes in extracellular pH near the physiological range. Bone 18:277–279
Bushinsky DA, Parker WR, Alexander KM, Krieger NS 2001 Metabolic, but not respiratory, acidosis increases bone PGE(2) levels and calcium release. Am J Physiol Renal Physiol 281:F1058–F1066
Sebastian A, Harris ST, Ottaway JH, Todd KM, Morris Jr RC 1994 Improved mineral balance and skeletal metabolism in postmenopausal women treated with potassium bicarbonate. N Engl J Med 330:1776–1781.
Freudenheim JL, Johnson NE, Smith EL 1986 Relationships between usual nutrient intake and bone-mineral content of women 35–65 years of age: longitudinal and cross-sectional analysis. Am J Clin Nutr 44:863–876
Teegarden D, Lyle RM, McCabe GP, McCabe LD, Proulx WR, Michon K, Knight AP, Johnston CC, Weaver CM 1998 Dietary calcium, protein, and phosphorus are related to bone mineral density and content in young women. Am J Clin Nutr 68:749–754
Lacey JM, Anderson JJ, Fujita T, Yoshimoto Y, Fukase M, Tsuchie S, Koch GG 1991 Correlates of cortical bone mass among premenopausal and postmenopausal Japanese women. J Bone Miner Res 6:651–659
Geinoz G, Rapin CH, Rizzoli R, Kraemer R, Buchs B, Slosman D, Michel JP, Bonjour JP 1993 Relationship between bone mineral density and dietary intakes in the elderly. Osteoporos Int 3:242–248
Cooper C, Atkinson EJ, Hensrud DD, Wahner HW, O’Fallon WM, Riggs BL, Melton 3rd LJ 1996 Dietary protein intake and bone mass in women. Calcif Tissue Int 58:320–325
Chiu JF, Lan SJ, Yang CY, Wang PW, Yao WJ, Su LH, Hsieh CC 1997 Long-term vegetarian diet and bone mineral density in postmenopausal Taiwanese women. Calcif Tissue Int 60:245–249
Lau EM, Kwok T, Woo J, Ho SC 1998 Bone mineral density in Chinese elderly female vegetarians, vegans, lacto-vegetarians and omnivores. Eur J Clin Nutr 52:60–64
Hannan MT, Tucker KL, Dawson-Hughes B, Cupples LA, Felson DT, Kiel DP 2000 Effect of dietary protein on bone loss in elderly men and women: the Framingham Osteoporosis Study. J Bone Miner Res 15:2504–2512
Kerstetter JE, Looker AC, Insogna KL 2000 Low protein intake and low bone density. Calcif Tissue Int 66:313
Promislow JH, Goodman-Gruen D, Slymen DJ, Barrett-Connor E 2002 Protein consumption and bone mineral density in the elderly : the Rancho Bernardo Study. Am J Epidemiol 155:636–644
Kerstetter JE, Caseria DM, Mitnick ME, Ellison AF, Gay LF, Liskov TA, Carpenter TO, Insogna KL 1997 Increased circulating concentrations of parathyroid hormone in healthy, young women consuming a protein-restricted diet. Am J Clin Nutr 66:1188–1196
Kerstetter JE, O’Brien KO, Insogna KL 1998 Dietary protein affects intestinal calcium absorption. Am J Clin Nutr 68:859–865
Frassetto LA, Todd KM, Morris Jr RC, Sebastian A 1998 Estimation of net endogenous noncarbonic acid production in humans from diet potassium and protein contents. Am J Clin Nutr 68:576–583
Neer R, Berman M, Fisher L, Rosenberg R 1967 Multicompartmental analysis of calcium kinetics in normal adult males. J Clin Invest 46:1364–1379
Abrams SA, Esteban NV, Vieira NE, Sidbury JB, Specker BL, Yergey AL 1992 Developmental changes in calcium kinetics in children assessed using stable isotopes. J Bone Miner Res 7:287–293
Yergey AL, Abrams SA, Vieira NE, Eastell R, Hillman LS, Covell DG 1990 Recent studies of human calcium metabolism using stable isotopic tracers. Can J Physiol Pharmacol 68:973–976
Welch TR, Abrams SA, Shoemaker L, Yergey AL, Vieira N, Stuff JE 1995 Precise determination of the absorptive component of urinary calcium excretion using stable isotopes. Pediatr Nephrol 9:295–297
Heaney RP, Recker RR 1994 Determinants of endogenous fecal calcium in healthy women. J Bone Miner Res 9:1621–1627
Kaneko K, Masaki U, Aikyo M, Yabuki K, Haga A, Matoba C, Sasaki H, Koike G 1990 Urinary calcium and calcium balance in young women affected by high protein diet of soy protein isolate and adding sulfur-containing amino acids and/or potassium. J Nutr Sci Vitaminol (Tokyo) 36:105–116
Lutz J, Linkswiler HM 1981 Calcium metabolism in postmenopausal and osteoporotic women consuming two levels of dietary protein. Am J Clin Nutr 34:2178–2186
Walker RM, Linkswiler HM 1972 Calcium retention in the adult human male as affected by protein intake. J Nutr 102:1297–1302
Heaney RP 2000 Dietary protein and phosphorus do not affect calcium absorption. Am J Clin Nutr 72:758–761
Dawson-Hughes B, Harris SS 2002 Calcium intake influences the association of protein intake with rates of bone loss in elderly men and women. Am J Clin Nutr 75:773–779
Bennett T, Desmond A, Harrington M, McDonagh D, FitzGerald R, Flynn A, Cashman KD 2000 The effect of high intakes of casein and casein phosphopeptide on calcium absorption in the rat. Br J Nutr 83:673–680
Orwoll E, Ware M, Stribrska L, Bikle D, Sanchez T, Andon M, Li H 1992 Effects of dietary protein deficiency on mineral metabolism and bone mineral density. Am J Clin Nutr 56:314–319
Calvo MS, Bell RR, Forbes RM 1982 Effect of protein-induced calciuria on calcium metabolism and bone status in adult rats. J Nutr 112:1401–1413
Schulte-Frohlinde E, Schmid R, Schusdziarra V 1993 Effect of a postprandial amino acid pattern on gastric acid secretion in man. Z Gastroenterol 31:711–715
DelValle J, Yamada T 1990 Amino acids and amines stimulate gastrin release from canine antral G-cells via different pathways. J Clin Invest 85:139–143
Recker RR 1985 Calcium absorption and achlorhydria. N Engl J Med 313:70–73
Wood RJ, Serfaty-Lacrosniere C 1992 Gastric acidity, atrophic gastritis, and calcium absorption. Nutr Rev 50:33–40
Serfaty-Lacrosniere C, Wood RJ, Voytko D, Saltzman JR, Pedrosa M, Sepe TE, Russell RR 1995 Hypochlorhydria from short-term omeprazole treatment does not inhibit intestinal absorption of calcium, phosphorus, magnesium or zinc from food in humans. J Am Coll Nutr 14:364–368
Ferraretto A, Signorile A, Gravaghi C, Fiorilli A, Tettamanti G 2001 Casein phosphopeptides influence calcium uptake by cultured human intestinal HT-29 tumor cells. J Nutr 131:1655–1661
Erba D, Ciappellano S, Testolin G 2002 Effect of the ratio of casein phosphopeptides to calcium (w/w) on passive calcium transport in the distal small intestine of rats. Nutrition 18:743–746
Kitts DD, Yuan YV, Nagasawa T, Moriyama Y 1992 Effect of casein, casein phosphopeptides and calcium intake on ileal 45Ca disappearance and temporal systolic blood pressure in spontaneously hypertensive rats. Br J Nutr 68:765–781
Hansen M, Sandstrom B, Jensen M, Sorensen SS 1997 Casein phosphopeptides improve zinc and calcium absorption from rice-based but not from whole-grain infant cereal. J Pediatr Gastroenterol Nutr 24:56–62
Roughead ZK, Johnson LK, Lykken GI, Hunt JR 2003 Controlled high meat diets do not affect calcium retention or indices of bone status in healthy postmenopausal women. J Nutr 133:1020–1026
Standing Committee on the Scientific Evaluation of Dietary Reference Intakes, Food and Nutrition Board, Institute of Medicine 1999 Dietary reference intakes for calcium, phosphorus, magnesium, vitamin D and fluoride. Washington, DC: National Academy Press(Jane E. Kerstetter, Kimbe)
Although high-protein diets induce hypercalciuria in humans, the source of the additional urinary calcium remains unclear. One hypothesis is that the high endogenous acid load of a high-protein diet is partially buffered by bone, leading to increased skeletal resorption and hypercalciuria. We used dual stable calcium isotopes to quantify the effect of a high-protein diet on calcium kinetics in women. The study consisted of 2 wk of a lead-in, well-balanced diet followed by 10 d of an experimental diet containing either moderate (1.0 g/kg) or high (2.1 g/kg) protein. Thirteen healthy women received both levels of protein in random order. Intestinal calcium absorption increased during the high-protein diet in comparison with the moderate (26.2 ± 1.9% vs. 18.5 ± 1.6%, P < 0.0001, mean ± SEM) as did urinary calcium (5.23 ± 0.37 vs. 3.57 ± 0.35 mmol/d, P < 0.0001, mean ± SEM). The high-protein diet caused a significant reduction in the fraction of urinary calcium of bone origin and a nonsignificant trend toward a reduction in the rate of bone turnover. There were no protein-induced effects on net bone balance. These data directly demonstrate that, at least in the short term, high-protein diets are not detrimental to bone.
Introduction
MORE THAN 80 yr ago, Sherman (1) documented that humans fed a high-protein diet developed hypercalciuria. A recent meta-analysis of 26 controlled human intervention studies shows that for every 25-g increase in dietary protein, there is a 0.8 mmol rise in urinary calcium (2). The source of the additional urinary calcium excreted during a high-protein diet is unknown.
A popular hypothesis is that the additional calcium excreted in the urine results, in part, from changes in bone homeostasis. This hypothesis is supported by some but not all studies. It is well established that dietary protein increases endogenous acid production (3). In response to the acid load, bone may be called upon as a reservoir of alkali, and, as a consequence, bone calcium is mobilized. If true, then the long-term consequence of buffering by bone would be increased bone resorption and a decline in bone mineral density (BMD). Consistent with this hypothesis, osteoclast-mediated bone resorption is increased in an acid environment when compared with studies done at neutral or alkaline pH (4, 5, 6, 7). Most balance studies (2) have reported that intestinal calcium absorption is unaffected by dietary protein, suggesting that the source of the extra urinary calcium resulting from a high-protein diet was not of intestinal origin. At odds with the bone hypothesis are a large number of epidemiological studies showing that a high-protein diet is associated with a high, not a low BMD, as might be predicted (8, 9, 10, 11, 12, 13, 14, 15, 16, 17).
Because osteoporosis is a major public health problem, clarifying the impact of dietary protein on the skeleton is important. Therefore, we employed dual stable calcium isotopes to determine the relative contributions of the skeleton and the intestine to the rise in urinary calcium during the first week of a high-protein diet.
Subjects and Methods
Study design
The protocol consisted of two cycles involving 2 wk of an adjustment diet, followed by 10 d of an experimental diet. For the adjustment period, subjects modified their diets to contain moderate amounts of protein, sodium, calcium, and caffeine. During the 10-d experimental period, subjects received all food from the Yale General Clinical Research Center (GCRC) metabolic kitchen. These diets contained tightly controlled levels of calcium, sodium, phosphorus, and one of two levels of dietary protein: moderate (1.0 g/kg) or high (2.1 g/kg). The 2-wk adjustment period and 10-d experimental cycle were repeated again until all subjects received both levels of protein in random order. Fasting blood and urine samples were collected on the mornings of d 0 and 4 of each experimental period. During the morning of d 4, subjects received an iv dose of 42Ca. Three oral doses of 44Ca were administered with each meal during that day. For the next 6 d, blood and urine samples were collected for measurements of 42Ca and 44Ca.
Subjects
Ten healthy young women between 20–40 yr of age (29 ± 6 yr, mean ± SD) and three healthy postmenopausal women between 55–70 yr (61 ± 7 yr, mean ± SD) participated in the study. Body weight averaged 65 ± 2 and 65 ± 6 kg and body mass index averaged 23 ± 1 and 25 ± 2 kg/m2 in the young and older women, respectively. Exclusion criteria included medications known to affect calcium metabolism, amenorrhea, pregnancy, smoking, eating disorders, diabetes, renal or gastrointestinal disease, bone disease, nephrolithiasis, or intensive daily physical exercise. Subjects suspended all vitamin and mineral supplements during the entire study.
The racial background of the subjects was either Caucasian or Asian because these are the groups of women at highest risk for osteoporosis. Subjects continued their usual activities at home, school, and work during the study. The study was approved by Investigational Review Boards at Yale University (New Haven, CT), the University of Connecticut (Storrs, CT), and Johns Hopkins University (Baltimore, MD). Informed consent was obtained from each participant.
Diets
The experimental and adjustment diets are similar to those described in previous reports (18, 19). During the first week of the adjustment period, subjects were instructed to select diets containing approximately 1.2 g protein/kg, 20 mmol calcium, and 100 mmol sodium. This diet was provided to them by the GCRC’s metabolic kitchen during the second week of the adjustment period to completely control nutrient intake. Subjects consumed adequate energy for weight maintenance. Caffeine-containing beverages were limited to one a day, and alcohol was not permitted.
During the 10-d experimental period, subjects reported daily to the GCRC to receive their meals and record their body weight. Energy intake ranged from 0.13 to 0.15 MJ/kg (30–35 kcal/kg) and was adjusted with simple sugars and fats to maintain body weight. We asked that the subjects consume all the food and beverage (and nothing other than the food and beverage) that was provided to them. Distilled water was provided ad lib.
All experimental diets were individually calculated to contain one of two levels of protein, whereas the levels of other nutrients were fixed (20 mmol calcium, 100 mmol sodium, and 32–39 mmol phosphorus). The macronutrient and mineral composition of the experimental diets was calculated from the U.S. Department of Agriculture Handbook No. 8 and manufacturer’s information. The primary sources of calcium in the experimental diets were dairy foods and a chewable calcium carbonate (Tums, GlaxoSmithKline, Pittsburgh, PA). The increase in protein from the control to the high-protein diet was accomplished by adding both animal and vegetable sources of protein. Renal net acid excretion (RNAE) was estimated by the method of Frassetto et al. (20). RNAE (mEq/d) = 62.1 x (protein in g/potassium in mEq) –17.9. Using their equation, the estimated RNAE resulting from our medium-protein diet was 40.5 ± 2.6 and high-protein diet was 109.4 ± 6.5 (P < 0.0001).
Nonisotopic sample collection and analyses
Fasting blood collections were obtained at d 0 and 4 of each 10-d experimental period for measures of PTH, total and ionized calcium, phosphorus, and creatinine. A timed 24-h urine was collected on d –1 and d 3 for measurement of calcium, phosphorus, sodium, and creatinine. The tubular reabsorption for calcium (TRCa) was calculated from a fasting 2-h morning urine collection (0700–0900 h) and a midpoint blood sample (0800 h) using serum creatinine and ionized calcium and urine calcium and creatinine. The mean d 4 TRCa on the medium diet was 0.978 ± 0.006 and on the high diet 0.976 ± 0.003 (not significant). All nonisotopic assays were performed as reported (18).
Isotope administration and sample analysis
On d 4 of the experimental diet, oral 44Ca was administered in three divided doses, delivered with each meal in proportion to the calcium content of the meal. Dividing the oral dose corrects for potential differences in calcium bioavailability between meals. Each oral calcium isotope was equilibrated in milk for 8–24 h. Immediately after breakfast, the subject received an iv infusion of 42Ca, administered over a 5-min interval. The IV line was then flushed with saline. Immediately after the IV infusion, eight blood samples were collected at 5, 10, 20, 40, 60, 120, 240, and 480 min. All urine was then collected in acid-washed containers for the next 34 h in pools of 8, 12, and 14 h. Three spot urine samples were collected daily by the subject beginning the evening of d 5 and continuing until the end of d 9.
Calcium isotope ratios were measured using either a quadrupole (THQ, Finnigan, Bremen, Germany) or magnetic sector (Triton TI, Finnigan, Bremen, Germany) thermal ionization mass spectrometer. A ratio was made between each administered calcium tracer (42Ca and 44Ca) and another naturally occurring calcium isotope (48Ca or 43Ca). All isotopes were corrected for isotopic fractionation by normalizing the data to the 43/48Ca or 48/43Ca. Fractional calcium absorption was determined as the ratio of the cumulative oral tracer recovery to the cumulative IV tracer recovery in the 34-h urine collection obtained postdosing. True calcium absorption was calculated as the product of fractional calcium absorption and the calculated calcium intake.
Calcium kinetic measurements were determined using a multicompartmental model of calcium kinetics (21, 22, 23). After the first 24 h, the clearance of the oral and IV calcium tracer parallel one another and urine samples can be used to monitor the disappearance of the IV tracer. Rates of bone calcium deposition (Vo+) and resorption (Vo–) were then determined after accounting for urinary and endogenous fecal calcium losses. Net bone balance was determined as the difference between Vo+ and Vo–. The relative contribution of the recent diet and bone to urinary calcium excretion was calculated by the method of Welch et al. (24). Urinary calcium derived from the recent diet (Vou) is the product of dietary intake and the percentage of oral isotopic calcium recovered in the urine. Urinary calcium derived from bone (Vbu) is the difference between urinary calcium and Vou.
Statistical analyses
All values are presented as mean ± SEM (unless otherwise specified). A Student’s t test was initially used to compare the younger with the older women, and because there were no important differences in calcium homeostasis, the two age groups were pooled for all subsequent analyses. A paired Student’s t test was then used to evaluate differences in all biochemical outcomes between the two levels of protein at baseline and at d 4. The mean dietary intakes were evaluated between the two levels of protein using a paired Student’s t test. A probability level of P < 0.05 was statistically significant.
Results
Subjects remained healthy throughout the experiment. The average nutrient composition of the experimental diets is presented in Table 1. As shown in Table 2, the average urinary sodium on d 4 of the moderate- and high-protein diets was not different and indicative of good compliance.
Baseline measures (d 0) of serum and urine metabolites did not differ between the two diets (Table 2). Aside from urinary calcium, the diets induced no changes in calcium indices that we measured at d 4 (Table 2). By d 4, urinary calcium in every subject increased during the high-protein diet (Fig. 1), rising from 3.57 ± 0.35 during the moderate to 5.23 ± 0.37 mmol/d during the high-protein diet (P < 0.0001).
There were no statistically significant differences in kinetic measures of bone formation, resorption, or balance between the diets (Table 3). However, there was a nonsignificant trend toward lower bone turnover (both formation and resorption) during the high-protein diet in comparison with the moderate control protein. There was a significant increase during the high-protein diet in the relative fraction of urinary calcium of dietary origin and a significant decrease in the relative fraction of urinary calcium from bone origin compared with the moderate protein diet (Table 3). All subjects were in negative calcium balance during all interventions.
Percent intestinal calcium absorption increased during the high-protein diet (26.2 ± 1.9%) in comparison with the moderate (18.5 ± 1.6%, P < 0.0001), representing a mean increase of 7.7% (raw increase) or 42% (relative increase; Fig. 1). Because dietary calcium was fixed at 20 mmol/d, the intestinal absorption on the high-protein diet was 1.54 ± 0.20 mmol/d higher than on the moderate diet. The mean increase in urinary calcium between the two diets was 1.66 ± 0.25 mmol/d.
Discussion
We used dual stable calcium isotopes to quantify the contributions of bone and intestine to the increment in urinary calcium observed during a high-protein diet. High-protein diets (in comparison with the moderate, control protein diet) resulted in a significant increase in intestinal calcium absorption that paralleled the increase in urinary calcium excretion. These effects were evident in all subjects and were not influenced by age across the age range studied. The increase in true calcium absorption during the high-protein diet was accompanied by a significant decrease in the fraction of calcium from nondietary (bone) origin. Therefore, at least acutely, the increase in urinary calcium from a high-protein diet is explained by increases in intestinal calcium absorption with no contribution from bone resorption. Finally, no matter what level of protein was ingested, all women were in negative calcium balance while consuming the 20 mmol calcium, suggesting that 20 mmol calcium is not adequate to maintain bone balance.
A regression equation based on data from 26 published human studies (that included a wide variety of protein sources, subjects, and study design) predicts that a 58-g increase in dietary protein would result in a 1.86 mmol rise in urinary calcium (2). Consistent with this prediction, the addition of 58 g of protein to the diets of our subjects increased urinary calcium by 1.66 mmol.
The dual stable calcium isotope method we used measures true intestinal calcium absorption. Although we did not measure fecal calcium and cannot directly quantify endogenous excretion of calcium, we can estimate endogenous calcium excretion based on published equations (25). Intestinal calcium secretion can itself be influenced by calcium absorption and dietary phosphorus. During the high-protein diet, when absorption efficiency is increased, one would expect that both dietary calcium and endogenously secreted calcium would be absorbed more efficiently, so there would be less calcium in the feces. We have calculated, on the basis of published equations (25), that the 7.7% raw increase in intestinal calcium absorption would result in a decrease of 0.2 mmol/d in endogenous fecal calcium excreted during the high-protein diet. However, the high-protein diet contained 3.2 mmol more phosphorus than the medium-protein diet. Again, on the basis of the work of Heaney and Recker (25), this difference in dietary phosphorus would lead to an additional 6 mg (0.2 mmol) calcium in the intestinal tract during the high-protein diet. Thus, these two countervailing effects negate each other because the increased absorption of secreted calcium is off set by the dietary phosphorus-induced increase in endogenous fecal calcium. Furthermore, it should be kept in mind that these calculated changes are relatively small in relation to the large change in urinary calcium between the diets of 1.66 mmol.
The 42% relative increase (or 7.7% raw increase) in intestinal calcium absorption (18.5 vs. 26.2%) in the current study accounts for 1.54 of the 1.66 mmol rise in urinary calcium or approximately 93%. The difference in intestinal calcium absorption between the control and the high-protein diet in our study is large and probably explains why human intervention studies generally report an increase in urinary calcium in response to a high-protein diet despite variability in experimental design, subjects, types of diets, and nutrient composition (2).
It is often suggested that a decrease in renal tubular calcium reabsorption explains, in part, the hypercalciuria observed on a high-protein diet. This was not the case in our experiment because TRCa was unchanged on the two diets (medium diet, 0.978 ± 0.006; high diet, 0.976 ± 0.003; P = not significant).
The majority of the balance studies in humans have not reported a change in calcium absorption in response to dietary protein (2). However, there are at least three balance studies that showed an increase in calcium absorption as dietary protein is increased (26, 27, 28). In general, balance studies should be interpreted with caution because the method is highly dependent on quantifying fecal calcium loss, which is difficult to measure accurately.
Two isotopic studies found no association between dietary protein intake and calcium absorption (29, 30). Heaney (29) reported that, in a longitudinal study of a large group of Roman Catholic nuns (mean age 48.7 ± 7.0 yr), there was no relationship between intestinal calcium absorption and usual intake of dietary protein. The mean protein intake in his study was 62 g (or approximately 1 g/kg), and their calcium intake averaged 17.6 mmol/d. Dawson-Hughes found similar results in adults over the age of 65 yr (30). Although these studies employed isotopic measures of calcium absorption, a method generally considered more accurate than a balance approach, dietary protein was not experimentally manipulated. It is possible that the large interindividual variability in intestinal calcium absorption and the multitude of other dietary factors that can also affect calcium absorption may explain why the relationship between protein intake and intestinal calcium absorption was not observed in these two studies.
In general, the evidence that dietary protein influences intestinal calcium transport in animals is strong. In rodents, several studies have shown that increasing dietary protein increases intestinal calcium absorption and shifts the route of calcium excretion from the intestinal tract to the urine (31, 32, 33).
The mechanism that underlies the rise in intestinal calcium absorption in response to dietary protein is unknown. Dietary protein and amino acids are gastric acid and gastrin secretagogues (34, 35). Gastric acid helps to solubilize dietary calcium and could potentially improve its bioavailability, although the literature on this topic is contradictory (36, 37, 38). Alternatively, the peptide fragments (e.g. casein phosphopeptides) that are released during dairy protein digestion may aid in solubilizing calcium, making it more available for absorption (39, 40, 41, 42).
Although not statistically significant, there was a trend toward decreased bone turnover (Table 2) during the high-protein diet. The high-protein diet was associated with a significant decrease in the fraction of urinary calcium that was of bone origin. We calculated RNAE by the method of Frassetto et al. (20) to estimate the potential renal acid load generated by the two experimental diets. The two are highly correlated (20). By these calculations, the high-protein diet generated 2.7 times more fixed acid than did the control diet (RNAE in mEq/d was 109.4 ± 6.5 high protein vs. 40.5 ± 2.6 medium protein; P < 0.0001). Despite this, there was no effect on bone resorption.
Our experimental intervention of 1 wk was perhaps too short to detect significant changes in bone homeostasis despite the large change in intestinal calcium absorption. Moreover, because of the variability in rates of bone turnover, larger sample sizes may be needed to detect significant changes in this parameter even with a paired study design. However, the acute increase in intestinal calcium absorption and trend toward decreased bone turnover observed in our study may help explain the epidemiological studies that show higher BMD associated with high protein intakes (8, 9, 10, 11, 12, 13, 14, 15, 16, 17). Clearly, longer term intervention studies are needed to answer the question.
In a recent isotopic intervention study of 8 wk in length, Roughead et al. (43) evaluated calcium retention using sensitive radiotracer and whole-body scintillation counting in 15 postmenopausal women as they consumed moderate and high-protein diets. Calcium retention did not differ between the two levels of protein, although there was a nonsignificant trend toward better retention during the high-protein diet. Taken together, the two isotopic intervention studies [ours and that of Roughead et al. (43)] support the conclusion that high-protein diets, at least in the short and intermediate term, do not result in increased bone loss. If anything, both studies showed nonsignificant trends toward lower bone turnover and better calcium retention during high-protein diets.
Our finding that the average bone balance in our subjects was negative by 2.5 mmol/d (no matter what level of dietary protein was consumed) was unanticipated. No subject was in positive bone balance on any intervention, even when intestinal calcium absorption was highest during the high-protein diet. Clearly, 20 mmol dietary calcium is not enough to maintain calcium balance in our healthy female adults, both young and old. We selected 20 mmol calcium because it was the recommended daily allowance at the time the study was designed, and it approximates the calcium intake of many adult women. In the most recent version of the Dietary Reference Intakes, recommendations are higher, 25–30 mmol, depending on age (44). It would be important to determine whether the current higher recommended intakes for calcium would restore bone balance.
In summary, we used dual stable calcium isotopes to evaluate the acute impact of dietary protein on bone kinetics in a diet-controlled, cross-over study. The majority of the rise in urinary calcium in response to an increase in dietary protein was due to an increase in intestinal calcium absorption. Our short-term study needs to be extended to longer term intervention trials to fully evaluate the impact of dietary protein on bone turnover. Our study does not support the hypothesis that increased dietary protein is acutely harmful to bone.
Footnotes
This work was supported by Grant DK52128-03 from the National Institutes of Health (NIH) RO1, by The Ethel F. Donaghue Women’s Health Investigator Program at Yale University, and by Yale’s GCRC (NIH MO1-RR00125).
First Published Online November 16, 2004
Abbreviations: BMD, Bone mineral density; GCRC, General Clinical Research Center; RNAE, renal net acid excretion; TRCa, tubular reabsorption for calcium; Vbu, urinary calcium derived from bone; Vo+, bone calcium deposition; Vo–, bone calcium resorption; Vou, urinary calcium derived from the recent diet.
Received February 5, 2004.
Accepted September 9, 2004.
References
Sherman H 1920 Calcium requirement of maintenance in man. J Biol Chem 44:21–27
Kerstetter JE, O’Brien KO, Insogna KL 2003 Low protein intake: the impact on calcium and bone homeostasis in humans. J Nutr 133:855S–861S
Barzel US, Massey LK 1998 Excess dietary protein can adversely affect bone. J Nutr 128:1051–1053
Bushinsky DA 1996 Metabolic alkalosis decreases bone calcium efflux by suppressing osteoclasts and stimulating osteoblasts. Am J Physiol 271:F216–F222
Arnett TR, Spowage M 1996 Modulation of the resorptive activity of rat osteoclasts by small changes in extracellular pH near the physiological range. Bone 18:277–279
Bushinsky DA, Parker WR, Alexander KM, Krieger NS 2001 Metabolic, but not respiratory, acidosis increases bone PGE(2) levels and calcium release. Am J Physiol Renal Physiol 281:F1058–F1066
Sebastian A, Harris ST, Ottaway JH, Todd KM, Morris Jr RC 1994 Improved mineral balance and skeletal metabolism in postmenopausal women treated with potassium bicarbonate. N Engl J Med 330:1776–1781.
Freudenheim JL, Johnson NE, Smith EL 1986 Relationships between usual nutrient intake and bone-mineral content of women 35–65 years of age: longitudinal and cross-sectional analysis. Am J Clin Nutr 44:863–876
Teegarden D, Lyle RM, McCabe GP, McCabe LD, Proulx WR, Michon K, Knight AP, Johnston CC, Weaver CM 1998 Dietary calcium, protein, and phosphorus are related to bone mineral density and content in young women. Am J Clin Nutr 68:749–754
Lacey JM, Anderson JJ, Fujita T, Yoshimoto Y, Fukase M, Tsuchie S, Koch GG 1991 Correlates of cortical bone mass among premenopausal and postmenopausal Japanese women. J Bone Miner Res 6:651–659
Geinoz G, Rapin CH, Rizzoli R, Kraemer R, Buchs B, Slosman D, Michel JP, Bonjour JP 1993 Relationship between bone mineral density and dietary intakes in the elderly. Osteoporos Int 3:242–248
Cooper C, Atkinson EJ, Hensrud DD, Wahner HW, O’Fallon WM, Riggs BL, Melton 3rd LJ 1996 Dietary protein intake and bone mass in women. Calcif Tissue Int 58:320–325
Chiu JF, Lan SJ, Yang CY, Wang PW, Yao WJ, Su LH, Hsieh CC 1997 Long-term vegetarian diet and bone mineral density in postmenopausal Taiwanese women. Calcif Tissue Int 60:245–249
Lau EM, Kwok T, Woo J, Ho SC 1998 Bone mineral density in Chinese elderly female vegetarians, vegans, lacto-vegetarians and omnivores. Eur J Clin Nutr 52:60–64
Hannan MT, Tucker KL, Dawson-Hughes B, Cupples LA, Felson DT, Kiel DP 2000 Effect of dietary protein on bone loss in elderly men and women: the Framingham Osteoporosis Study. J Bone Miner Res 15:2504–2512
Kerstetter JE, Looker AC, Insogna KL 2000 Low protein intake and low bone density. Calcif Tissue Int 66:313
Promislow JH, Goodman-Gruen D, Slymen DJ, Barrett-Connor E 2002 Protein consumption and bone mineral density in the elderly : the Rancho Bernardo Study. Am J Epidemiol 155:636–644
Kerstetter JE, Caseria DM, Mitnick ME, Ellison AF, Gay LF, Liskov TA, Carpenter TO, Insogna KL 1997 Increased circulating concentrations of parathyroid hormone in healthy, young women consuming a protein-restricted diet. Am J Clin Nutr 66:1188–1196
Kerstetter JE, O’Brien KO, Insogna KL 1998 Dietary protein affects intestinal calcium absorption. Am J Clin Nutr 68:859–865
Frassetto LA, Todd KM, Morris Jr RC, Sebastian A 1998 Estimation of net endogenous noncarbonic acid production in humans from diet potassium and protein contents. Am J Clin Nutr 68:576–583
Neer R, Berman M, Fisher L, Rosenberg R 1967 Multicompartmental analysis of calcium kinetics in normal adult males. J Clin Invest 46:1364–1379
Abrams SA, Esteban NV, Vieira NE, Sidbury JB, Specker BL, Yergey AL 1992 Developmental changes in calcium kinetics in children assessed using stable isotopes. J Bone Miner Res 7:287–293
Yergey AL, Abrams SA, Vieira NE, Eastell R, Hillman LS, Covell DG 1990 Recent studies of human calcium metabolism using stable isotopic tracers. Can J Physiol Pharmacol 68:973–976
Welch TR, Abrams SA, Shoemaker L, Yergey AL, Vieira N, Stuff JE 1995 Precise determination of the absorptive component of urinary calcium excretion using stable isotopes. Pediatr Nephrol 9:295–297
Heaney RP, Recker RR 1994 Determinants of endogenous fecal calcium in healthy women. J Bone Miner Res 9:1621–1627
Kaneko K, Masaki U, Aikyo M, Yabuki K, Haga A, Matoba C, Sasaki H, Koike G 1990 Urinary calcium and calcium balance in young women affected by high protein diet of soy protein isolate and adding sulfur-containing amino acids and/or potassium. J Nutr Sci Vitaminol (Tokyo) 36:105–116
Lutz J, Linkswiler HM 1981 Calcium metabolism in postmenopausal and osteoporotic women consuming two levels of dietary protein. Am J Clin Nutr 34:2178–2186
Walker RM, Linkswiler HM 1972 Calcium retention in the adult human male as affected by protein intake. J Nutr 102:1297–1302
Heaney RP 2000 Dietary protein and phosphorus do not affect calcium absorption. Am J Clin Nutr 72:758–761
Dawson-Hughes B, Harris SS 2002 Calcium intake influences the association of protein intake with rates of bone loss in elderly men and women. Am J Clin Nutr 75:773–779
Bennett T, Desmond A, Harrington M, McDonagh D, FitzGerald R, Flynn A, Cashman KD 2000 The effect of high intakes of casein and casein phosphopeptide on calcium absorption in the rat. Br J Nutr 83:673–680
Orwoll E, Ware M, Stribrska L, Bikle D, Sanchez T, Andon M, Li H 1992 Effects of dietary protein deficiency on mineral metabolism and bone mineral density. Am J Clin Nutr 56:314–319
Calvo MS, Bell RR, Forbes RM 1982 Effect of protein-induced calciuria on calcium metabolism and bone status in adult rats. J Nutr 112:1401–1413
Schulte-Frohlinde E, Schmid R, Schusdziarra V 1993 Effect of a postprandial amino acid pattern on gastric acid secretion in man. Z Gastroenterol 31:711–715
DelValle J, Yamada T 1990 Amino acids and amines stimulate gastrin release from canine antral G-cells via different pathways. J Clin Invest 85:139–143
Recker RR 1985 Calcium absorption and achlorhydria. N Engl J Med 313:70–73
Wood RJ, Serfaty-Lacrosniere C 1992 Gastric acidity, atrophic gastritis, and calcium absorption. Nutr Rev 50:33–40
Serfaty-Lacrosniere C, Wood RJ, Voytko D, Saltzman JR, Pedrosa M, Sepe TE, Russell RR 1995 Hypochlorhydria from short-term omeprazole treatment does not inhibit intestinal absorption of calcium, phosphorus, magnesium or zinc from food in humans. J Am Coll Nutr 14:364–368
Ferraretto A, Signorile A, Gravaghi C, Fiorilli A, Tettamanti G 2001 Casein phosphopeptides influence calcium uptake by cultured human intestinal HT-29 tumor cells. J Nutr 131:1655–1661
Erba D, Ciappellano S, Testolin G 2002 Effect of the ratio of casein phosphopeptides to calcium (w/w) on passive calcium transport in the distal small intestine of rats. Nutrition 18:743–746
Kitts DD, Yuan YV, Nagasawa T, Moriyama Y 1992 Effect of casein, casein phosphopeptides and calcium intake on ileal 45Ca disappearance and temporal systolic blood pressure in spontaneously hypertensive rats. Br J Nutr 68:765–781
Hansen M, Sandstrom B, Jensen M, Sorensen SS 1997 Casein phosphopeptides improve zinc and calcium absorption from rice-based but not from whole-grain infant cereal. J Pediatr Gastroenterol Nutr 24:56–62
Roughead ZK, Johnson LK, Lykken GI, Hunt JR 2003 Controlled high meat diets do not affect calcium retention or indices of bone status in healthy postmenopausal women. J Nutr 133:1020–1026
Standing Committee on the Scientific Evaluation of Dietary Reference Intakes, Food and Nutrition Board, Institute of Medicine 1999 Dietary reference intakes for calcium, phosphorus, magnesium, vitamin D and fluoride. Washington, DC: National Academy Press(Jane E. Kerstetter, Kimbe)