OSTEOPOROSIS and low bone mass (osteopenia) affect almost 44 million men and women older than 50 years in the United States, and this number is expected to grow to 52 million by 2010. 1 The health problem is reaching near epidemic proportions. In response, The Institute of Medicine revised and released the recommended dietary allowances, RDAs (known collectively as the dietary reference intakes, DRIs), for the bone-related nutrients (calcium, phosphorus, magnesium, fluoride, and vitamin D) before the other nutrients. The DRIs reflect both state-of-the-art understanding and the substantial gaps in the scientific data base for nutritional factors that influence skeletal health. The DRIs cite the extensive number of human and animal studies addressing the role of dietary calcium and vitamin D in acquiring and maintaining skeletal mass through the life span. In contrast, our understanding of how other dietary components, such as protein, affect the skeleton is still limited. In this review, we will focus on how dietary protein influences calcium metabolism and how those processes may affect bone health.
Our capacity to measure changes in skeletal homeostasis has improved considerably in the last decade. We now have better ways to noninvasively assess rates of skeletal formation and resorption, to quantify bone mass and measure calcium homeostasis. Using these tools, a complex picture of protein's effect on calcium and bone metabolism is emerging. It is frequently suggested that a high protein diet is detrimental to bone health, particularly if the protein is from animal sources. However, recent epidemiological and clinical data do not necessarily support these hypotheses. This review will develop the hypothesis that dietary protein is an important regulator of calcium metabolism and that high protein diets may be less detrimental than previously thought.
HOW MUCH PROTEIN AND CALCIUM DO WE CONSUME?
The mean protein intake for adult men and women in the United States and the percentage of individuals consuming each level of protein are summarized 2 in Table 1. For purposes of this review, we have identified low, medium, high, and very high protein diets in comparison to the RDA as defined in Table 1. The most current RDA for protein remains at 0.8 g protein/kg in the adult. 3 Between 15% and 25% of adult men (20–59 years) and 5% and 10% of adult women (20–59 years) consume more than double the RDA for protein. As seen from Table 1, dietary protein generally declines, when expressed in absolute grams/day, over the age of 60 years.
The majority of adult men and women in the United States consume less than 100% of the 1989 RDA for calcium (800 mg for men and women older than 25 years). 2 Therefore, when considering the impact of dietary protein on calcium metabolism, it should be kept in mind that most Americans consume a diet rich in protein, but poor in calcium.
DIETS MODERATE IN PROTEIN DO NO HARM
There is uniform agreement that at moderate levels of protein intake (roughly 100%–150% of the RDA or 0.8–1.2 g protein/kg), measures of calcium and skeletal metabolism remain normal. 4–8 Approximately 30% to 50% of adults in the United States have protein intakes that are considered moderate. Using a 4-day experimental model where nutrients were controlled, we have also documented normal calcium homeostasis when healthy adults consumed a diet containing 1.0 g protein/kg and 20 mmol calcium. 4 Measures of the PTH-1-α-hydroxylase axis and urinary calcium excretion were within the normal ranges under these moderate conditions.
HIGH DIETARY PROTEIN INCREASES URINARY CALCIUM
Eighty years ago, Sherman 9 first observed that feeding an all-meat diet to humans increased urinary calcium. There was tremendous interest during the 1970s and 1980s in the relationship between dietary protein and calcium metabolism and approximately 30 human studies addressing this topic were published during those 2 decades. It is now clearly established that increasing dietary protein increases urinary calcium. We have reviewed data from 26 clinical intervention trials in adult humans where the diet was controlled, dietary protein was manipulated (150 g protein and less), and urinary calcium was measured (Fig 1). 4,5,10–33 Despite varied experimental designs, most studies reported a positive relationship between protein intake and urinary calcium and the overall association is strong (P < .001, r = 0.7). The linear regression equation from Figure 1 is as follows:
where urine calcium is expressed as mmol/d and dietary protein is g/d, slope = 3.208E-02, and y-intercept is 1.501. These data clearly establish that dietary protein is an important regulator of urinary calcium excretion, at least as important as dietary calcium. 16,34
Because protein and phosphorus are typically found together in foods, a high protein diet (typically from meat) generally also means a high phosphorus diet and phosphorus is hypocalciuric. Because of the phosphorus effect, those studies that use meat (as opposed to purified proteins) as a way of increasing dietary protein may not observe the increase in urinary calcium during a high meat diet 5,17,35,36 (although there are divergent studies on this issue). 4,22,26,28,30,37 Nevertheless, it is fair to say that high phosphorus intakes (typically concurrent with the high protein intake), tends to blunt, but not clearly ameliorate the rise in urinary calcium in response to high protein diets. 38
HIGH PROTEIN DIETS: HARMFUL TO THE SKELETON?
While it is clear that increasing dietary protein increases urinary calcium, the question remains, where does the additional urinary calcium originate? Three potential sources are the diet, intestine, and bone, or any combination thereof. In most of the experiments shown in Figure 1, dietary calcium is well controlled and, therefore, diet can be easily ruled out as a source. The second option that changes in intestinal calcium absorption underlie high-protein-diet–induced hypercalciuria has generally been considered unlikely. Most human balance studies report no difference in calcium absorption when dietary protein is altered 5,6,12–16,19,22 (although there are a few exceptions). 11,20,39 Consequently, it is thought that the additional urinary calcium excreted in response to a high protein diet results from increased bone resorption. The long term consequence of “leaching” calcium from bone would be a reduction in bone mass and increased risk for fragility fractures. Figure 2 summarizes a widely held traditional view on how dietary protein affects calcium homeostasis and skeletal metabolism. The strength of the data supporting this formulation are reviewed below.
Dietary protein, because it is rich in the sulfur-containing amino acids, clearly increases endogenous acid production. 40 The richer the protein source is in sulfur amino acids, the more fixed acid it generates. The endogenous acids are produced by the following reaction 41:
It is argued that the acid load generated by a high protein diet reduces tubular calcium reabsorption leading to hypercalciuria. 41 In addition, it is thought that, particularly in older individuals, buffering of some of the fixed acid load in bone leads to a loss of skeletal mass. Theoretically, increasing dietary protein from 75 to 125 g would increase urine calcium (and decrease calcium balance) by 60 mg daily, which equates to 1% to 2% bone loss annually in an adult. Should the increase in dietary protein persist for a decade, then the calculated 10% to 20% loss in bone would be significant. Conversely, ingesting foods that generate alkali should result in a reduction of net acid excretion and decreased urine calcium excretion. 40 Net acid excretion can be measured in a 24-hour urine and is well known to parallel dietary protein. The potential renal acid load (PRAL) of a diet can be estimated from the composition of the diet using established formulas. 42–44 Depending on the composition of the diets, high protein diets generally produce a greater PRAL than low or moderate protein diets.
Nonetheless, the scientific data supporting the conclusion that the skeleton is called upon to buffer the fixed acid generated by a high protein diet is largely indirect. Acutely exposing cultured calvariae in vitro to an acidic environment causes the release of bone calcium because of simple physicochemical dissolution. If exposed chronically, a low pH in the media increases the activity of osteoclasts (the cells that break down bone) and decreases osteoblastic activity (the cells that stimulate bone formation). 41,45 Likewise, exposing calvariae to alkalotic conditions decreases calcium efflux from bone, probably by decreasing osteoclastic resorption and increasing osteoblastic activity. 46
The human studies regarding acid-base manipulation and the skeleton are consistent with the cell studies. For example, supplementation with potassium bicarbonate in humans improved calcium balance and reduced indirect makers of bone resorption. 24,47 This observation was confirmed, most recently, by Maurer and colleagues, 48 who showed that supplementing healthy men and women with potassium and sodium bicarbonate improved calcium retention and reduced markers of bone resorption.
While it seems clear that pharmacologic manipulation of acid-base status both in vitro and in vivo can affect calcium homeostasis, it remains less certain whether the considerably less extreme alterations induced by a typical diet can produce the same effect. A number of factors tend to ameliorate the potential deleterious effect of fixed acid generated by a high protein diet. We eat our high protein diets over a course of an entire day, 12 to 15 hours, and so the acid generating foods are “dribbled in” rather than “dumped in.” Human diets are complex, containing both acid and alkaline generating capacities. The capacity of the renal and respiratory systems to buffer an acid load is substantial. Given all of these factors, several critical questions remain. In those adults that chronically consume a high protein diet, is there enough endogenous acid produced by that diet to damage the skeleton? Are there data to show that bone mineral density (BMD) declines or bone resorption increases under high dietary protein conditions, as would be predicted? To date, it has been difficult to definitively show, in well-controlled intervention studies, that dietary protein affects the skeleton. In one short and one intermediate-length intervention study in humans using isotopic methods to quantitate calcium kinetics, dietary protein did not affect calcium retention or bone turnover. 36,49 Additionally, there are several large, cross-sectional 50–58 and longitudinal studies 8,59–61 suggesting that BMD is higher in those who consume a high protein diet. These studies are reviewed in the next section.
HIGH PROTEIN DIETS: GOOD FOR THE SKELETON?
The results of cross-sectional studies evaluating the association of dietary protein and bone density are relatively consistent. There are at least 9 such studies where BMD is the primary outcome showing that a high protein diet is associated with a high BMD (not a low BMD, as might be predicted). 50–58 There are few studies showing no association, 62,63 but only one study showing a negative association. 64
For example, using the NHANES III data base we found, in 1882 non-Hispanic white women aged 50 years and older, that after adjusting for age and body weight, the higher the protein intake, the higher is the hip BMD (Fig 3). 55 When the analysis was restricted to women with intakes of calcium greater than 800 mg/d, the relationship between dietary protein and hip bone density was still observed suggesting that the effect of protein was not due to concurrently low intakes of dietary calcium. Consistent with these data, Munger et al found, in a prospective observational study, that 55-year-old to 69-year-old women consuming the highest amounts of protein, particularly animal protein, had the lowest risk of hip fracture. 65
Several long-term longitudinal studies have evaluated the relationship between typical protein intake and BMD in adults. Almost all have found that subjects (generally older men and women) consuming the highest protein intakes also showed the least amount of bone loss. 8,59–61 For example, Hannan and colleagues 60 studied 615 participants in the Framingham Osteoporosis Study over a 4-year period of time. In this elderly group, higher protein intake was significantly associated with lower rates of bone loss at the hip and spine. Persons in the highest quartile for protein intake showed the lowest rates of bone loss (Fig 4). The bone sparing effect persisted after all known potentially confounding variables were controlled. These findings are consistent with earlier work of Freudenheim, who reported that a high protein intake was associated with better preservation of bone density at the wrist in 35-year-old to 65-year-old women. 59 Dawson-Hughes et al, in a longitudinal intervention study, examined the effect of usual protein intake on BMD in 342 older adults. Subjects were either randomized to taking 12.5 mmol of calcium (as calcium citrate malate) with vitamin D daily or a placebo. The subject's typical protein intake was divided into tertiles. In the calcium supplemented group, increasing dietary protein exerted a positive effect on total body BMD. 8 There is consensus in the longitudinal studies that higher protein intakes are associated with favorable skeletal outcomes.
Many clinical intervention studies have used a calcium balance approach to measure protein's effects on calcium homeostasis. The limitation of such an approach is the relative insensitivity of balance studies to small changes in absorption. In particular, a critical component of balance studies is the measurement of fecal calcium. It is very difficult to accurately quantitate fecal calcium excretion given the differences in intestinal transit time and variation caused by dietary changes.
Isotopic studies are more sensitive than balance studies, but have not been extensively used to assess protein's effect on calcium in part because of the expense, specialized equipment, and expertise required. However, recently there have been 2 isotopic studies specifically designed to evaluate the impact of dietary protein on calcium retention and indices of bone metabolism. Roughead et al, 36 using isotopic calcium tracers, compared the effects of a high and low meat diet on body calcium retention. Fifteen healthy postmenopausal women consumed both high and low meat diets for 8 weeks in a randomized, cross-over study design. At the end of this time period, there was no statistically significant difference in 47Ca retention when subjects consumed a diet containing 12% versus 20% of the energy as protein. There were also no differences in measures of bone turnover. In fact, there appeared to be a slight trend (albeit not significant) toward improved 47Ca calcium retention on the high meat diet. Clearly, over the 8-week duration of this study, a high meat diet was not detrimental to bone.
A second calcium isotopic study was conducted by our research group and initial results from that study have been reported. 49 In this acute study, 13 adult women consumed both a medium protein (1 g protein/kg) and a high protein (2.1 g protein/kg) diet in random order for a week. All experimental diets were designed to contain 20 ± 0 mmol calcium and 100 ± 0 mmol sodium. The phosphorus intake on the low protein diet averaged 35 ± 1 mmol, whereas on the high protein diet it was 38 ± 0 mmol. Using dual stable calcium isotopes at the end of each intervention, we measured bone formation, resorption, balance, and calcium absorption. As expected, when subjects consumed the high protein diet, they developed hypercalciuria. There was no change in kinetic measures of bone formation, bone resorption, or bone balance between the medium and the high protein diets. This study provides direct evidence that the increase in urinary calcium excretion in response to a high protein diet is not due to an increase in bone resorption. Rather, an increase in intestinal calcium absorption explains almost all of the increment in urinary calcium following a high protein diet (as detailed in the next section titled “Intestinal Calcium Absorption”). In both isotopic studies, 36,49 calcium intake was controlled (600–800 mg) and close to the average consumed by US women, albeit lower than current recommendations. 66
Taken together, the 2 isotopic studies 36,49 do not support the traditional hypothesis that a high protein diet in adults adversely affects calcium retention or skeletal homeostasis. These 2 studies, the first to use isotopic methodology in well-controlled dietary intervention, directly address the effects of dietary protein on bone. Isotopic methods of assessing calcium homeostasis are considered by many to be the gold standard because of their sensitivity. The acute increase in intestinal calcium absorption from the high protein diets 49 may not persist, because there was no change in intestinal calcium absorption at 8 weeks in the Roughead study, 36 thereby raising the question of intestinal adaptation over time.
Protein supplementation studies
In several clinical trials where hip fracture patients were supplemented with protein, there was a reduction in the degree of bone loss 67,68 and a significant improvement in the rate of recovery. 69–71 For example, Schurch et al 69 studied 82 patients (mean age = 80.7 ± 7.4 years) who had recently sustained an osteoporotic hip fracture. These patients had self-selected very low protein intakes (approximately 40 g). The administration of additional 20 g of protein per day was associated with significant attenuation of proximal femur bone loss in the fractured hip. 1 year, bone loss rates were 50% lower in the protein-supplemented individuals than in those that received an isocaloric control supplement. Interestingly, those patients that received the protein supplements spent less time in the rehabilitation ward than those who received the control supplement.
POTENTIAL MECHANISMS TO EXPLAIN WHY DIETARY PROTEIN IS BONE PROTECTIVE
There are at least 3 mechanisms by which dietary protein could exert a positive effect on bone: via an improvement in intestinal calcium absorption, an increase in insulin-like growth-factor 1 (IGF-1), or protein's supportive role in bone protein matrix, or any combination of these 3 factors. Data bearing on each mechanism are reviewed below.
Intestinal calcium absorption
As noted above, we have found that a high protein diet induces an acute increase in intestinal calcium absorption while kinetic measure of bone turnover remains unaltered. We initially studied intestinal calcium absorption in 7 young women as they consumed a high (2.1 g/kg) and low protein (0.7 g/kg) diet for 4 days. 28 Since then, using the same protocol, we have studied 13 additional healthy women (10 young and 3 postmenopausal). The results in all 20 of these study subjects are summarized 72 in Figure 5. Most notably, calcium absorption during the low protein diet averaged 18.4% ± 1.3%, significantly lower than during the high protein diet: 26.3 ± 1.5% (P < .0001, paired t-test). The change in intestinal calcium absorption from low to high protein (8%) explains ~80% of the change in urinary calcium excretion between the low and high protein diets (3.4 to 5.4 mmol) since dietary calcium was held constant in this study at 20 mmol. Therefore, the conclusion based on balance studies that dietary protein does not affect intestinal calcium absorption, is likely to be incorrect (at least acutely). It is possible that balance studies could not detect intestinal calcium absorption differences because of the very high interindividual variability in basal rates of intestinal calcium absorption and the relative insensitivity of the methodology. Our paired design controlled for the former limitation and the use of stable calcium isotopic methodology represent an improvement in sensitivity vis a vis the standard balance approach.
If increasing dietary protein improves intestinal calcium absorption without stimulating bone breakdown, the availability of more calcium, particularly in a protein-sufficient state may favor new bone formation. At the very least, the improvement in intestinal calcium absorption induced with increasing dietary protein may help to counteract the fall in the efficiency of calcium absorption that occurs with aging. This would suggest that menopausal women, in whom impaired intestinal calcium absorption is common, would benefit from an adequate intake of dietary protein (as well as sufficient calcium). Others have agreed that adequate protein intake is essential for bone health in older adults. 73
Insulin-like growth factor 1
IGF-1 has emerged as a key mediator of bone growth. 74 The principal regulator of skeletal metabolism, on a day to day basis is parathyroid hormone (PTH). Parathyroid hormone has complex effects on mineral homeostasis. However, one of PTH's prominent effects, at least when the skeleton is exposed to the hormone in transient daily bursts, is to stimulate bone growth. 75–78 This anabolic effect of PTH requires IGF-1. 79,80 In fact, PTH induces the expression of IGF-1 in bone, and IGF-1 is thought to be a key mediator of PTH-dependent bone growth. 81 IGF-1 also functions in the kidney to stimulate the renal transport of inorganic phosphate and the production of 1,25-dihydroxy vitamin D. 82
Dietary protein is an important regulator of circulating IGF-1 levels. 83 A low protein diet decreases the production and action of IGF-1, whereas protein supplements or a high protein diet increase serum IGF-1 levels. 84 Serum IGF-1 levels are positively correlated with BMD in adult men and women. 85–89 It is possible, therefore, that a high protein diet contributes to bone by increasing circulating IGF-1 levels. 83,90
Protein as a structural component
Bone consists of an organic matrix (or osteoid) in which salts of calcium and phosphate are deposited and, in combination with hydroxyl ions, form special crystals called hydroxyapatite. The matrix is primarily collagen protein fibers. Therefore, protein is an important structural component of bone, accounting for approximately half of bone volume. Because the adult skeleton is constantly remodeling itself, adequate dietary protein is required to provide substrate for adequate new bone formation.
THE REMAINING QUESTIONS
Paradoxically, when fracture is the principal outcome, high-protein intakes are associated with higher rates of fracture in most epidemiologic studies 91–95 (except for Munger et al, 65 as noted above). If a high protein diet is associated with high BMD as most of the epidemiological data show and as most of the clinical trials support, it is very difficult to explain why a high protein intake would be associated with an increased risk for fracture. Perhaps protein intake is tracking with an unmeasured risk factor for osteoporotic fracture. For example, because protein foods tend to be relatively expensive, perhaps it is a surrogate for socioeconomic status. It is known that fracture risk increases with socioeconomic status. Explaining this apparent paradox is critical to a full understanding of how dietary protein affects the skeleton.
Do the short-term and intermediate-length studies of Kerstetter et al 49 and Roughead et al 36 accurately predict the long-term effects on mineral metabolism of a high protein diet? The answer is not known. The fact that the majority of the epidemiological data show a higher incidence of fracture in those consuming the most protein, suggests that the question is worth pursuing in a longitudinal study. Such a study would be difficult and expensive to execute properly, but it is the only way that this important question can be answered.
SUMMARY AND CONCLUSIONS
Available evidence indicates that diets moderate in protein (in the approximate range of 1.0–1.5 g protein/kg) are associated with normal calcium metabolism and presumably are sufficient for bone health. Based on our work and that of others, this appears to be true in men, young women, and postmenopausal women. 5,6,96 Approximately 40% to 50% of adults in the United States consume dietary protein that could be considered moderate. Between 15% and 25% of adult men and between 5% and 10% of adult women (20–59 years) are consuming exceptionally high protein diets, more than 1.6 g protein/kg. Are these diets detrimental to skeletal health? Based on available evidence, our answer to that question is “probably not.” Clearly, humans become hypercalciuric during high protein diets but, at least acutely, this is solely due to an increase in intestinal calcium absorption. 49 At an intermediate time point (2 months), it appears that there is still no evidence for a detrimental effect on bone. 36 There is a wide variety of epidemiological and clinical data showing that dietary protein is associated with improved bone and plausible cellular mechanisms for such a relationship exist.
Bone is complex tissue that changes slowly. As such, it is difficult to design and conduct well-controlled nutrition studies in humans to quantitate the effect of one nutrient on bone. Whereas calcium and vitamin D remain our most important bone-related nutrients, there are other nutrients or food components that may also play a role in skeletal health. 97 Given the rising incidence of osteoporosis in our culture and the clear impact that dietary protein has on calcium metabolism, it is imperative that we gain a better understanding of the complex interplay between dietary protein and the skeleton.
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