Bone, 13, 343-350, (1992) Printed in the USA. All rights reserved.

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8756-3282192 $5.00 + .OO 0 1992 Pergamon Press Ltd.

REVIEW

The Effects of Dietary Protein Insufficiency and Excess on Skeletal Health E. S. ORWOLL Chief, Endocrinology and Metabolism, Department of Veterans Affairs, Portland VA Medical Center, Associate Professor of Medicine, Oregon Health Sciences University, Portland, OR, U.S.A. Address correspondence and reprints: Eric S. Orwell, M.D., Medical Services (11 lP), Portland VA Med Center, P.O. Box 1034, Portland, OR 91201, U.S.A.

Key Words: Nutrition-Skeleton-Dietary

protein.

Introduction Nutritional deprivation is obviously of signal importance in disadvantaged populations. It can also assume a major role in the health of individuals living in selected segments of affluent societies. For instance, nutrition in the elderly may be limited by several factors, and in younger persons nutritional knowledge may be limited and “fast-food” or fad diet habits may constrict nutrient intake. In contrast, the diets of other segments in affluent societies may be over-replate with some substances, and a variety of disorders of dietary indulgence have been described (obesity, atherosclerosis, renal insufficiency). As with other nutrients, evidence for disorders caused by both insufficient and excessive dietary protein have been reported, and in both instances there may be implications for skeletal health.

Nutritional Protein Requirements in Humans The paleontological record suggests that humanoid evolution has been characterized by nutritional variation, and hence humans maintain the “versatility of the omnivore” of most primates (Eaton 1985). Whereas fruits and vegetables apparently made up a large portion of the human diet until several million years ago, meat constituted a major fraction of food intake (up to 50%) after tool making began. In the last 50,000 years, the dawn of agriculture (and associated changes in population density and the availability of animal food sources) has had a major effect on human dietary intake, resulting in a markedly decreased animal protein intake. Nevertheless, this shift to agriculture occurred only a short time ago in evolutionary terms, and the modem human genome is thus probably more a product of a variable, but in general protein-rich, dietary environment. Dietary protein intake clearly affects the rates at which tissues synthesize and metabolize endogenous proteins. The available information suggests that protein intakes below maintenance result in a reduction in both protein synthesis and catabolism, so that there is obvious adaptation to low protein intakes (Garlick 1991; Pellett 1990; Reeds 1984). This adaptation is influenced by energy balance. In the presence of low energy availability, the need to utilize endogenous protein for fuel is increased, and thus a negative protein balance is reached much more quickly as dietary protein availability is reduced. The protein intake re-

quired for the achievement of nitrogen balance can increase by 50% in the face of a reduction in calorie intake from 57 to 45 Cal/kg (Munro & Crim 1980). In turn, energy requirements (and hence protein balance) are dependent on activity levels. Hence, the sufficiency of protein nutrition is defined in the context of energy requirements and total energy intake. In the presence of adequate calories, the conservation of endogenous nitrogen is quite efficient in adults (Munro & Crim 1980; Waterlow 1986), and active young men can remain in nitrogen balance with protein intakes of less than 50 g/day (Waterlow 1986). In childhood the demands of growth increase dietary protein needs and make adaptation more difficult (Pellett 1990; Munro & Crim 1980). In children any adaptation to protein deprivation eventually occurs at the expense of growth, and protein malnutrition results in reductions in height, weight, and whole body and muscle protein. When protein nutrition is restricted in experimental animals, skeletal protein synthesis is reduced at least in proportion to that in other tissues (Reeds 1984). Current recommended allowances for high quality (good digestibility, optimal amino acid content, etc.) dietary protein range from about 2 g/kg/day in infants to about 1 g/kg/d in adolescents (Pellett 1990; Munro & Crim 1980; Subcommittee on the Tenth Edition of the RDAs 1989). In adults, the recommended allowance declines (because of the elimination of growth requirements) and is currently 0.75 g/kg/d. These recommendations are based upon studies that suggest that in young men-there is no convincing information that young nonpregnant women differ substantially in their requirements-a mean intake of 0.61 g/kg/d of high-quality protein achieves nitrogen equilibrium. To insure that individual variation is considered, allowances have been increased to two standard deviations above that mean (or 0.75 g/kg/d). In the elderly, there is considerable controversy concerning the adequacy of the usual adult recommendations for protein nutrition (Young 1984; Uauy et al. 1978; Munro et al. 1987; Gersovitz et al. 1982; Morley 1986; Morley et al. 1988). Conflicting studies are difficult to resolve in the face of potential differences in energy intake, activity, and medications in the study populations. However, it is apparently true that whereas energy requirements decline with age (by 20%-30% from age 21 to 75 years), protein requirements do not (Young 1984; Pellett 1990). Hence, in the elderly the proportional requirement for protein may increase considerably. In some older individuals, total energy intake may be quite low, and protein nutrition deficient (Surgeon General’s Report on Nutrition and Health 1988). This is particularly true in those with chronic disorders that may increase protein requirements. 343

E. S. 01~011: Dietary protein and the skeleton

344

Average protein intakes in the U.S. and other industrialized countries are above (140%-160%) these recommended daily allowances, and well above (200%) the mean requirement for nitrogen balance (Subcommittee on the Tenth Edition of the RDAs 1989). Protein intakes above those to maintain nitrogen equilibrium (or a positive nitrogen balance during growth) result in excess amino acids entering the energy pathways, with nitrogen being excreted as urea. This occurs without obvious adverse effects at protein intakes considerably above recommended daily allowances-as would be expected in a species that evolved in a protein-rich dietary environment. On the other hand, excessive protein intakes over a prolonged period may be harmful. In fact, an adequate but limited intake of protein (without caloric restriction) may prolong life (Morley et al. 1988). Moreover, concern has been expressed that high protein intakes contribute to a gradual reduction in renal function with age, and high protein intakes have been postulated to be related to the occurrence of renal stones. colon cancer, and atherosclerosis (Pellett 1990). At present, the evidence that high protein intakes are associated with any of these adverse outcomes has not been sufficient to lead to specific recommendations to limit protein consumption. However, it has been “deemed prudent” to maintain an upper bound of no more than twice the recommended daily allowance for protein (Subcommittee on the Tenth Edition of the RDAs 1989). In summary, human metabolism has evolved in the context of considerable nutritional heterogeneity, and has the capacity to adapt to a wide range of dietary protein intakes. In spite of this versatility there are many areas of the world where protein undernutrition is an obvious problem, and in glaring contrast, there are other populations in which protein intakes are very much above those necessary for the maintenance of good health. It is apparently at these extremes that metabolic bone disease may result. Protein Undernutrition

and Metabolic Bone Disease

As in other tissues, protein metabolism in the skeleton can be expected to be altered in states of dietary protein deprivation. In addition, there may be alterations the systemic metabolism induced by low protein intakes that adversely affect bone. Because of the demands of growth on protein requirements and on the skeleton, the effects of dietary protein restriction will be considered independently in children and adults. Children As might be expected in view of the higher requirements of growth, the effects of protein-energy malnutrition on the skeleton are severe in children. In addition to the well known retardation in bone growth, there have also been a variety of reports of reductions in bone integrity in nutritionally deprived children. Endochrondral bone growth is clearly abnormal, with reductions in the zone of cartilage cell proliferation (Higginson 1954). Interestingly, bone length is retarded only in proportion to body weight (Adams & Berridge 1969), and radiographic progression of ossification centers is not particularly impaired in proteindeprived young children (Freudenheim et al. 1986). However, osteopenia is clearly present in those severely affected (kwashiorkor) (Adams & Berridge 1969), and Garn et al. (1964) have demonstrated that cortical osteopenia is not so much a result of impaired periosteal growth, but rather of dramatically increased medullary expansion-suggesting increased endosteal remodeling and bone loss.

The effects of protein restriction on bone modeling/ remodeling are not entirely clear. Kwashiorkor rarely exists as a disorder of isolated protein deficiency, and inadequacies of other nutrients (energy, minerals, vitamins) are usually also present. In animals (rats), experimental semistarvation induced by global reductions in nutrient intake has recently been associated with the development of osteoporosis (Care et al. 1990) in the context of a reduction in bone remodeling and failure to assume a tetracycline label (Shires et al. 1980). Bone mineral density is more reduced in cancellous than in cortical areas (Care et al. 1990). Although this implies that the causation of bone disease in protein-energy malnutrition may be multifactorial, there is good evidence that protein deficiency itself plays a major role. In primates (rhesus macaques), severe dietary protein restriction during the first year of life (without caloric or vitamin restriction) results in osteopenia later in life without a reduction in bone width (Leutenegger et al. 1973), a finding consistent with clinical studies of deprived children (Garn et al. 1969). Moreover, Jha et al. (1968) have demonstrated that severe protein (but not calorie) deprivation in the young rhesus results in histomorphometric abnormalities similar to those described in children with kwashiorkordisruptions of endochondral bone formation (reduced chondrocyte numbers, trabecular stunting), reduced remodeling rates, and clear reductions in appositional bone growth. Similar findings have been described in proteimcalorierestricted pigs, which develop transverse “growth arrest lines” as are found in malnourished children (Platt & Stewart 1962). Finally, in rats protein/calorie deprivation results in a reduction in bone collagen and mineral content (Pucciarelli et al. 1983, 1984). These effects in turn apparently contribute to a reduction in mechanical strength and stiffness, and an increase in commnuted fracture risk with mechanical stress (Ferretti et al. 1988). A resumption in bone growth and mineralization occurs with refeeding (Garn et al. 1964; Platt & Stewart 1962; El-Maraghi et al. 1965). but it is unclear whether a residual deficit in bone mass is present at maturity. Osteomalacia is not a part of the skeletal lesion of protein restriction unless other deficiencies are present, and in fact protein deficiency may preclude the classical histomorphometric manifestations of osteomalacia (hyperosteoidosis). The causation of modeling/remodeling disturbances in severe protein deficiency is probably multifactorial. Abright et al. (1941) suggested protein malnutrition directly impairs the ability of chondrocytes and osteocytes to synthesis matrix, a suggestion that may be valid. Also, intestinal protein content is low in deficient animals, and intestinal inadequacy has been implicated as a cause of calcium malabsorption under those conditions (Shenolikar & Narasinga 1968; McCance et al. 1942). In proteindeficient animals intestinal calcium binding protein levels are increased by exogenous vitamin D administration, but concentrations are low to begin with and fail to increase to the same extent as in control animals (Kalk & Pimstone 1974). In addition, there may be humoral concomitants of deficiency states that contribute to disturbances in skeletal metabolism. For instance, insulin and insulin-like growth factor 1 (IGFI) both have osteoblast and chondrocyte-stimulating properties, and their concentrations are depressed in protein deprivation (Hahn et al. 1971; Anthony & Endozian 1975; Peck & Messinger 1970; Wettenhall et al. 1969; Moats-Staats et al. 1989). Another potentially important factor is adrenocortical function. In states of nutritional stress such as malnutrition or anorexia nervosa, cortisol concentrations are elevated and lack normal diurnal rhythmicity (Gold et al. 1986). It has been suggested that an excess of glucocorticoid action increases the risk of osteopenia in these patients, who are already at risk for bone loss for other reasons. In some animal studies (domestic fowl), isolated protein restriction similarly re-

E. S. Orwell: Dietary protein and the skeleton sults in increased glucocorticoid levels (Weber et al. 1990), but in rats exposed to more general food deprivation, corticosterone levels were found to be reduced (Shires et al. 1980). The reasons for this discrepancy are unclear. The level of protein deficiency at which detrimental skeletal effects are observed in children is not clear. Severe protein deficiency (kwashiorkor) causes an obvious disruption in skeletal metabolism. Graded increases in protein intake result in greater skeletal growth potential, but protein intake (even in well nourished groups) may be related to bone density as well. For instance, in apparently adequately nourished United States children Chan et al. (1987) reported radial bone mineral density was correlated with protein as well as calcium intake. Hence one of the adverse effects of protein insufficiency may be to blunt the attainment of optimal peak bone mass in early adulthood. On the other hand, moderate degrees of protein deprivation may have less disastrous effects on overall skeletal health. Whereas even small reductions in protein intake affect growth potential, the strength of these smaller skeletons may be preserved. In animal studies, we have shown that there may be adaptations inherent in mineral metabolism which may serve to protect bone mass in the face of nutritional restriction. Rats fed a diet restricted in protein to the point of reducing growth but avoiding clinical kwashiorkor were compared to pair-fed rats consuming protein-replete but otherwise identical diets. In the protein-deprived rats, urinary calcium excretion was dramatically reduced (a well described concomitant of protein restriction) and bone density was preserved (Orwoll 1989). Although calcium absorption has been reported to be impaired by protein restriction (Shenolikar & Narasinga 1968; McCance et al. 1942) and in fact was reduced in these studies, we believe this to be in part secondary to the reduction in urinary calcium excretion. Essentially, the lower protein intake reduces urinary calcium loss, induces a more positive calcium balance, and results in a reduction in calcium absorption, apparently via a reduction in 1,25(OH)*D levels (Orwoll 1989; Raghuramulu 1989). Others have also reported a bone-sparing effect of moderate protein restriction (Shenolikar 1968; Saville & Lieber 1969; Le Roith & Pimstone 1973). Another mechanism by which protein restriction may protect bone density is by reducing the deleterious long-term effects of protein consumption on renal function, and hence avoiding the secondary hyperparathyroidism that results (Kalu 1988). These protective mechanisms apparently are overwhelmed with more severe protein deprivation (kwashiorkor). Adults

In populations with limited nutrition the mean adult body mass is considerably smaller than in nutritionally replete people, reflecting the effects of deprivation during growth (Garn & Kangas 1981). Hence after growth requirements cease the adaptational capacities inherent in protein nutrition (above) allow these small individuals to function on very limited intakes. In some populations, however, even these requirements are not met, and in some segments of affluent populations (those with low income, chronic illness, “dieting” lifestyles) similarly deficient diets are encountered. Some chronic diseases may double the requirements for protein intake (Munro & Crim 1980). thus resulting in relative protein deficiency even in the presence of an otherwise sufficient dietary intake. In adults there is virtually no information concerning dietary protein deficiency and its effects on the skeleton. There are some descriptions of bone mass in subjects previously interred in concentration or prisoner-of-war camps, in whom cortical area is reduced (Gam & Kangas 1981). However, the role of protein

nutrition per se is not separable from the effects of other adverse aspects of these situations. In rats studied after rapid growth had ceased (Care et al. 1990; Shires et al. 1980), protein-calorie restriction resulted in osteopenia and impaired bone formation. Therefore, there is the potential that detrimental effects of protein deprivation contribute to bone loss in some individuals, but little data exist. The group perhaps at most risk is the elderly, in whom bone competence is already compromised by the effects of aging. In fact, in his prescient 1941 publication on postmenopausal osteoporosis, Fuller Albright speculated that protein deprivation may contribute to postmenopausal bone loss (Albright 1941). In fact, a decline in caloric intake is a recognized concomitant of increasing age (McClellan & DuBois 1930). Chronic disease may further impair intake. Moreover, whereas most findings support the adequacy of the recommended daily allowance of 0.8 g protein/kg/d for the maintenance of protein balance in the healthy elderly (Munro & Crim 1980; Morley 1986; Surgeon General’s Report on Nutrition and Health 1988), some suggest that with even minor illness that level of intake is inadequate (Gersovitz et al. 1982; Morley 1986). Particularly in women, or those consuming little energy, protein requirements may also be higher (Young 1984). Serum albumin levels fall with aging in many reports (Gersovitz et al. 1982; Greenblatt 1979; Morley 1986; Morley et al. 1988), and albumin levels correlate with bone mineral density in men (Nordin & Polley 1987). In crosssectional studies, Tylavsky and Anderson (1988) reported that in elderly white women consuming a moderate amount of protein, bone mineral density was positively related to the level of protein intake, and Lacey & Anderson (1991) found a similar relationship in Asian women. No such relationship was found in other cross-sectional studies (Angus et al. 1988; Freudenheim et al. 1986; Mazees & Barden 1991; Nordin 1976; Yano & Heilbrun 1985). In a longitudinal study, Freudenheim et al. (1986) found that a higher protein intake was associated with lower rates of appendicular bone loss in pre- and postmenopausal women, but an attempt to isolate the effects of protein intake from those of other nutrients was not attempted. In a short-duration (but large) longitudinal study of postmenopausal women, Nordin and Polley (1987) found no relationship between protein intake and rate of forearm bone loss. Finally, in Matkovic’s studies of fracture rates in Yugoslavia, protein (as well as calcium and phosphorus) intakes were higher in those with lower fracture rates (Matkovic 1979). It is very difficult to identify the specific effects of protein nutrition in these studies because all populations studied were basically nutritionally replete, and there are a myriad of confounding variables that cannot be controlled (Heaney 1989; Holbrook & Barrett-Connor 1991). A related issue is the possible effect of dietary protein insufficiency on recovery after fracture in the elderly. Delmi et al. (1990) pointed out that epidemiologic evidence suggests that elderly patients who suffer hip fractures are more likely to be malnourished, and hence they studied the clinical outcome of such patients if treated with an oral nutrition supplement after surgical reduction of femoral neck fracture. Compared to a control (placebo-treated) group, the supplemented subjects had improved serum albumin levels, fewer hospital complications, and lower mortality. Others have reported similar results (Bastow et al. 1983; Jensen et al. 1982). Although the supplements used in these studies provided additional energy as well as a variety of nutrients, the protein component may have been instrumental for the beneficial effect observed. In summary, although the available data addressing the issue are controversial, Fuller Albright’s hypothesis that dietary protein insufficiency adversely effects skeletal health remains viable.

E. S. Orwell: Dietary protein and the skeleton Protein Overnutrition

and Metabolism

Bone Disease

Wachman and Bernstein ( 1968) proposed that one explanation for age-related bone loss was “bone dissolution” related to the need to buffer the acid load imposed by the consumption of protein. Since that time, a variety of reports have drawn an association between high intakes of dietary protein and bone mineral density or fracture rates. For instance, Hegstead (I 986) and others (Chalmers 1970; U .S. Department of Health and Human Services 1984) have pointed out that there is an apparent relationship between the protein consumption of a population and the incidence of hip fracture. Mazees and Mather (1974) reported that Eskimos have lower bone mineral densities than do whites, and speculated that a higher dietary protein intake might be responsible (although there were also differences in dietary calcium and phosphorus intakes). In some cross-sectional studies of omnivorous versus vegetarian women (Ellis et al. 1972; Marsh et al. 1988), bone mineral density has been higher in vegetarian women, who consume less protein. However, other evaluations have not found similar results (Hunt et al. 1989; Tylavsky 1988). These cross-sectional investigations are particularly hard to interpret in view of the difficulties in assessing an issue (bone mass) with multiple effecters acting over a lifetime (Heaney 1989), particularly when the assessment of the effects of protein nutrition is confounded by a variety of other covariates (Holbrook & Barrett-Connor I99 I ). Nevertheless, the reported epidemiologic associations between protein intake and bone mass have raised considerable debate concerning the utility of recommending lower intakes of protein in affluent societies. The danger described by Wachman and Bernstein that the skeleton is necessary to directly buffer the acid load of dietary protein is probably not a large one, as the amount of acid is not great relative to the ability of the kidney to excrete protons (Brosnan & Brosnan 1982). The primary basis for the concern that excess dietary protein intake leads to bone loss now centers on the calciuric effect of protein. Early reports of an increase in urinary calcium excretion in response to protein feeding (Hegsted et al. 1952; McClellan & DuBois 1930; Pittman & Kunerth 1939; Sherman 1920) have been abundantly verified (Allen, Bartlett, & Black 1979; Allen, Oddoye, & Margen 1979; Anand & Linkswiler 1974; Chu et al. 1978; Hegsted & Linkswiler I98 I ; Johnson et al, 1970; Linkswiler et al. 1974; Margen et al. 1974: Schwartz et al. 1973; Walker & Linkswiler 1972). It is clear that protein supplementation results in calciuria, that the effect is observed from very low to very high levels of protein intake, and that the effect of protein intake on urinary calcium excretion is actually more pronounced than that of calcium intake (Hegsted 1981). All else being equal, doubling of dietary protein intake induces a 50% increase in urinary calcium excretion (Heaney & Reeker 1982). These increases in urinary calcium excretion are sustained over prolonged periods of high-protein feeding and are not accompanied by an increase in gastrointestinal calcium absorption, so calcium balance becomes negative (Allen, Bartlett, & Block 1979; Allen, Oddoye, & Margen 1979; Licata et al. 1981; Mahalko & Sandstead 1983; Margen et al. 1974). Urinary calcium excretion is increased virtually immediately following a dietary protein stimulus, and that increase is somewhat unusual in that there is a dissociation between the normally tightly coupled relationship between calcium and sodium excrction (Licata et al. 1981). In fact, after a protein-induced increase in urinary calcium excretion, the addition of more dietary sodium further increases calcium excretion, suggesting the two effects are independent and additive (Kok et al. 1990). The causation of the hypercalciuria that occurs with protein feeding is unclear, but is almost certainly multifactorial. The fact

that calcium excretion is rapidly and proportionately affected when amino acid infusion rates are increased during total parenteral nutrition (Bengoa & Sitrin 1983) argues that the effect is not dependent on gastrointestinal function, and is much more likely to be the direct result of a change in renal calcium handling. In fact, daily urinary calcium excretion during total parenteral nutrition can greatly exceed the amount of infused calcium (Berkelhammeret al. 1988; Wood et al. 1985; Sloan et al. 1983). The fixed acid load imposed by the ingestion or infusion of protein plays a major role in the induction of hypercalciuria. Acid loading results in a decline in renal tubular calcium reabsorption, which is apparently independent of changes in parathyroid hormone secretion or sodium excretion (Adams & Berridge 1979; Heaney 1989; Hunt et al. 1989; Nordin & Polley 1987). That acid handling is intimately involved in the calciuric effect of protein feeding is illustrated by the ability of simultaneous bicarbonate ingestion or infusion (or the infusion of acetate, which is metabolized to bicarbonate in the liver) to greatly reduce the increase in calcium excretion induced by dietary or parenteral protein supplements (Lutz 1984; Berkelhammer et al. 1988). Although the addition of bicarbonate in these studies neutralized all the urinary acid associated with the protein load, the increase in calcium excretion was not completely blocked, implicating other factors in the genesis of the hypercalciuria. The dietary content of sulfur has been postulated to be a major contributor to net acid excretion, since there is a strong correlation between urinary sulfate and calcium, and the addition of sulfur-containing amino acids to the diet increases calcium excretion (Trilok & Draper 1989; Tschpoe & Ritz 1985; Whiting SJ, 1980; Whiting SJ, 1986; Zemel MB, 1981). However, these effects are apparently secondary to the fixed acid load imposed by sulfur-containing amino acids, rather than a direct effect of sulfur itself. This was recently demonstrated to be the case in chronic studies of rats in which supplementation with ammonium sulfate (the ammonium is metabolized to urea and hence avoids imposing a cationic load) was no more calciuric than was supplementation with ammonium chloride (Whiting & Cole 1987). In addition to the effects of acid loading, increases in protein intake clearly elevate the glomerular filtration rate, and calcium excretion is correspondingly increased (Nordin 1976). Finally, endocrine influences have been invoked to explain proteininduced hypercalciuria. Insulin (Allen et al. 1981) and glucagon (Anderson & Thomsen 1987) have been suggested as potential mediators of this effect. IGF-I (insulin-like growth factor-l) also may play a role, as it is mediated by protein intake, and in turn influences calcium excretion (Care et al. 1990; Moats-Staats et al. 1989). Which, if any. of these endocrine factors play important roles, or whether there are other unappreciated issues involved, is at present unclear. The source of dietary protein has been postulated to be an important determinant of the degree of hypercalciuria induced. Specifically, soy (vegetable) protein has been suggested to be less potent than is protein from animal sources (Anderson & Thomsen 1987; Calvo et al. 1982). When examined under carefully controlled conditions, however, soy protein is also hypercalciuric. In carefully controlled experiments in rats, in which the amounts of phosphorus and protein (purified from various sources) were fixed, soy protein was clearly hypercalciuric (Whiting & McNally 1989). Although mean rates of calcium excretion were lower with supplementation with soy protein than with lactalbumin. the differences were not significant. Lactalbumin contains more sulfate, and resulted in greater sulfate and net acid excretion, than soy. Beef protein supplementation resulted in effects similar to those of lactalbumin. In previous experiments, the same group had shown the propensity of proteins

E. S. Orwell:

Dietary protein and the skeleton

from various sources to cause calciuria was in rough proportion to their effects on net acid and sulfate excretion (Whiting & Druper 1980). Hence, soy protein is capable of promoting calcium excretion, but in light of its somewhat lower sulphate content and lesser effect on net acid excretion, may be somewhat less potent. The potential advantages of soy protein intake on mineral metabolism were also apparent in studies of the effects of soy versus casein feeding in rats (Kalu et al. 1988). In long-term experiments, protein (casein) feeding resulted in well reported deleterious effects on renal function, which were accompanied by increases in parathyroid hormone levels and reductions in femoral mineral density. On the other hand, soy feeding was associated with much more modest changes in renal function, parathyroid hormone levels, and bone density. The effects of these manipulations on renal calcium handling were not evaluated, and the adverse effects of casein feeding were attributed to the renal insufficiency and hyperparathyroidism that presumably resulted. Excess protein intake has been implicated in similar effects on renal function in humans, but the skeletal consequences (or whether the use of soy protein is similarly protective) have not been directly examined in humans. Despite the unequivocal impact of dietary protein on renal calcium handling and subsequent calcium balance, the importance of these effects on mineral and bone metabolism remains quite controversial. First, Spencer (Spencer et al. 1978) has pointed out that the effects of protein intake on urinary calcium excretion have been noted primarily in short-term studies performed with the addition of protein supplements (rather than in the form of meat) to the diets of study subjects. In studies of only nine subjects divided into four groups on the basis of calcium intake (mean daily calcium intakes in the groups: 217 mg, 807 mg, 1113 mg, and 2020 mg) , Spencer et al. ( 1978) found no change in any group in urinary or fecal calcium excretion, or calcium balance, after increasing the average intake of meat protein from 50 to 300 gm/day. Urinary calcium excretion was not affected in long-term studies, and in fact was found to gradually decline in one subject on a high-protein intake (Spencer et al. 1978). They point out that the intake of phosphorus increases considerably when protein is provided in the form of meat, thereby possibly blunting the effect of protein per se, and they argue that these results indicate there is little if any danger of bone loss posed by highprotein (meat) intakes. Although these studies raise legitimate issues (the duration of the hypercalciuric effect and the magnitude of the effect under normal dietary situations), they are very limited in scope (and power), and utilized some subjects with conditions that may have influenced mineral metabolism (obesity, osteoporosis, hypothyroidism, hypercalciuria). Heaney & Reeker (1982) pointed out that the hypocalciuric effect of dietary phosphorus is apparently the result of increased fecal calcium excretion without any change in calcium balance, an effect probably independent of the predominantly renal alterations induced by protein feeding. In fact, the addition of phosphorus to the diet blunts but does not obliterate the effects of protein on calcium balance (Hegsted & Linkswiler 1981), and other studies have reported that meat feeding can result in an increase in calcium excretion (Cummings et al. 1979; Hegsted et al. 1979; Licata 1981; Schwartz et al. 1973). The duration of the effect has also been addressed by other investigators, and it would appear that the hypercalciuric effect can be prolonged (Allen 1979; Hegsted & Linkswiler 1981). Finally, Heaney and Reeker (1982) have reported that the level of dietary protein intake in free living subjects is negatively correlated with calcium balance. Hence there remains concern that high levels of protein may be detri-

341

mental even when consumed as a part of a normally mixed diet. Secondly, if high protein intakes are associated with a negative calcium balance, it would be predicted that alterations in biochemical indices of mineral metabolism should occur. In fact, protein-induced alterations in urinary calcium excretion or calcium balance have not consistently resulted in increased levels of parathyroid hormone, urinary cyclic AMP, 1,25(OH),D, or hydroxyproline excretion (Allen, Bartlett, & Block 1979; Lutz & Linkswiler 1981; Kim & Linkswiler 1979). Current studies are needed to examine the issue with more recent, sensitive measures of mineral and bone metabolism (for instance, serum intact parathyroid hormone and osteocalcin levels, urinary pyridinium excretion). Similarly, it has been difficult to demonstrate that protein-rich diets actually lead to osteopenia in experimental situations (Yano & Heilbrun 1985). Finally, the interactions of dietary calcium, phosphorus, and protein intakes is unclear. It has been pointed out that the clearly negative calcium balance induced by the consumption of high protein, low calcium, low phosphorus experimental diets might be considerably ameliorated by diets with higher calcium:protein ratios, and that the slightly negative calcium balance induced by high protein, high phosphorus, low calcium diets (Schuette & Linkswiler 1982) can be improved by increasing calcium intakes (Reinhard et al. 1976; Schuette & Linkswiler 1982). It has been proposed that diets relatively high in all these nutrients (protein, phosphorus, and calcium) are most beneficial for skeletal health (Hegsted 1981; van Geresteijn et al. 1990). This is, in fact, the situation in the classic studies of Matkovic et al. (1979) in which the calcium, phosphorus, and protein intakes of the population with greater bone mass and lower fracture rates were all higher than in the population with lower mass and more fractures. In addition, there are other factors, particularly carbohydrate intake (Shahkhalili & Mettraux 1991), that also influence calcium absorption (and potentially skeletal metabolism) and add to the complexity of the dietary relationships involved. The sum of all these interactions needs to be further evaluated to design specific dietary recommendations more confidently. In summary, the issue of whether a relatively high intake of protein influences mineral and bone metabolism, and contributes to the risk of osteopenia, remains controversial. At present there is sufficient evidence of an effect to stimulate the pursuit of more knowledge. Specifically, carefully performed metabolic studies utilizing modem, sensitive biochemical markers of bone and mineral metabolism are needed to assess better the metabolic effects of dietary manipulations. Already there exists some information that commonly consumed diets, replete in phosphate but low in calcium, may be associated with potentially harmful metabolic changes (Calvo et al. 1990). In this dietary setting (high phosphorus, low calcium) the addition of excess dietary protein may be of particular concern.

The author would like to acknowledge by Carol Albertson.

Acknowledgment:

preparation

the manuscript

References Adams, P.; Benidge, F. R. Effects of kwashiorkor on cortical and trabecular bone. Arch. Dis. Childh. 44~70~09; 1969. Albright, F.; Smith, P. H.; Richardson, A. M. Postmenopausal osteoporosis. JAMA 116~2465-2474; 1941. Allen, L. H.; Bartlett, R. S.; Block, G. D. Reduction of renal calcium reabsorption in man by consumption of dietary protein. J. Nurr. 109~1343-50; 1979.

E. S. Otwoll:

348 Allen. L. H.: Oddoye. E. A.: Margen. S. Protein-Induced hypercalciurw A longer term study. Am. J C/in. Nufr. 32:741-9: 1979. Allen. L. H.; Block. G. D.: Wood, R. J ; Bryce. G. F The role of insulin and pamthyroid hormone in the protein-induced calciuria of man Nutr. Re.r I:3 II: 1981. Anand. D. R.: Linkswiler. H. M. Effect of protein intake on calcium balance of young men given 500 mg calcium daily J. Nutr. 104:695-700: 1974 Anderson, J. J. B.: Thomsen. K. High protein meals. insular hormones and urmar) calcium excretion in human SubJects. ChristIansen. C.; Johansen. J S eds International Symposium on Osteoporosis, Viborg. Denmark. 1987. Angus. R. M.: Samhrwk, P. N : Pocock. N. A.: Eisman. J. A. Dietary intake and bone mineral density. Bone Min. 4:265-77: 1988. Anthony, L. E.; Edozien, J. C. Experimental protein and energy defnencies m the rat. J. Nurr. 631-48; 1975. Bastow, M. D.: Rawlings. I.; Allison, S. P. Benefits of supplementary tube feeding after fractured neck of femur: A randomlsed controlled trial Rr Med. .I 287:1589-92; 1983. Bengoa. J. M.; Sitrin, M. D. Amino acid-induced hypercalciuria in patients on total parenteral nutrition. Am. J. Clin. Nutr. 38:26&269: 1983 Berkelhammer. C. H.: Wood. R. J.: Sitrin, M. D. Acetate and hypercalciuria during total parenteral nutrition Am. J C/in. Nutr. 48:1482-9: 1988. Brosnan, J. T.: Brosnan. M. E. Dietary protein, metabolic acidosis, and calcium balance. Adv. Nutr. Res. 4~77-105; 1982. Calve. M. S.: Bell. R. R., Forbes. R M. Effect of protenmduced calciuria on calcium metahohsm and bone status in adult rats. J. Nutr 112: 140 I: 1982 Calve. M. S.; Kumar. R.: Heath, H., III. Persistently elevated pamthyroid hermane secretion and action in young women after four weeks of ingesting high phosphorus, low calcium diets J C/in. Endocrinol. Metub. 70:133&40, 1990. Care. M. M.; Pacheco. C. R.: Coble. T.. Kimmel, D. B. Effect 01 chrome festnction of food intake on bone mass and dynamics in aged rats. J. Bow Min. Re.\ [Abst.] 5:179:Sl18: 1990. Caverzasio, I.: Montessuit, C.: Bonjour, J. P. Stimulatory effect of msubn-llke growth factor-l on renal Pi transport and plasma 1.25.dihydroxyvitamin D, Endocrinology 127:453-9; 1990 Chalmers. 1. Geographical 52B:667-75: 1970.

variations

in senile osteoporoats. J Bone Jo~nr SW-~.

Ghan, G. M.: Mclnnes. R.: &II, G. Nutrmonal factors affectmg bone mmeral status in children. Clinrcal Research [Ahst.] 35:227A: 1987 Chu, J. Y.: Costa. F. M.: Margen. S. Effects of low calcium and variable protrm intake on human radio-calcium kinetics. Nutr. Rapt. Intern. 17:503X+: 1978. Chu. J. Y.; Margen. S.; Costa, F. M. Studies in calcium metabolism. II. Effects of low calcium and variable protein intake on human calcium metabolism. Am. J. C/in. Nutr. 28:1028%35: 1975 Cummings, J. H ; Hill. M. J.; Jlvrai. T : Houston, H.; Branch, W J.: Jenkn. D. I. A. The effect of meat protein and dietary fiber on colomc function and metabolism. 1. Changes in bowel habit bile acid excretion and calcium absorption. Am. J. C/in. Nutr. 32:208&93: 1979. Delmi, M.: Rapin. C. H.; Bengoa, J M.. Delmas. P D.; Vasey. H.. BWIJWX. J. P Clinical practice: Dietary supplementation in elderly patxnts with fractured neck of the femur. Lancer 335:101_~16; 1990.

Dietary protein and the skeleton

C&m. S. M.; Kangas. J. Protein intake. bone mass, and bone loss. DeLuca, H. F.; Frost, H. M.; Jee, W. S. S.; Johnston. C. C.: Pariitt, A. M., eds. Osteoporosis: Recent advances in pathogenesls and treatmenl. Baltimore. MD: University Park Press: 1981: 257. Gersovitr. M.: Motil. K.: Munro. H. N.; Scrimshaw, N. S.; Young, V. R. Human protem requirements: Assessment of the adequacy of the current recommended dietary allowance for dietary protein in elderly men and women. Am. J. Ckn Nutr 35:&14: 1982. Gold, P W.; Gwirtsman. H., Avgerinos, P. C.: Nieman. L. K.; Gallucci, W. T.. Kaye. W.: Jimerson. D.. Ebert, M.; Rittmaster, R.; Loriaux, D. L.; Chmusos. G. P Abnormal hypothalamic-pituitary-adrenal function in anorexia nervosa. N i?n~/. J. Med. 314:1335: 1986. Greenhlatt, D. 1. Reduced semm albumin concentration in the elderly: A report from the Boston collaborative drug surveillance program. J. Am. Geri. SoNutr loo: 1425-30: 1970 Kalk. W J.; Pimstone, B. L Calcium-bmding protein and vitamin D metabolism in experimental protein malnutrition. Br. J. Nutr. 32:56%78; 1974.

of its nature and

Kalu. D N.: Masoro, E. J.: Yu, B P.: Hardin. R. R.: Hollis. B. W. Modulation of age-related hyperparathyroidism and senile bone loss in fischer rats by joy protein and food restriction. Endocrinology 122: 1X47-54: 1988.

El-Maraghi N. R. H.. Platt, 8. S.; Stewart. R. J. C. The effect of the rnteract~on of dietary protem and calcium on the growth and mamtenance of the bones of young adult and aged rats. Br. J. Nutr. 19:491-508: 1965

Kim, Y Lmkswiler, H. M. Effect of level of protein intake on calcium metabolism and on pamthyroid and renal function in the adult human male. J. Nutr. 109:1399-1404: 1979.

Ellis. F. R.; Holesch. S ; Ellis, J. W. Incidence of osteoporosxs m vegetarians and omnivores. Am. J Clin. Nutr. 25:555-58: 1972.

Kok. D. J : lestra,J. A.; Doorenbos, C. J.: Papapoulos, S. E. The effecta of dietary excesses in ammal protein and in sodium on the composition and the crystallization kinetics of calcium oxalate monohydrate m urines of healthy men. J. C/in. Endo. Merab. 71:861-67; 1990.

Eaton. S B.; Konner, M. Paleohthic nutrition: A consideration current implications. N. Eng J. Med. 312:283-89; 1985

Ferretti, J. L.; Tessam, R. D.: Delgado. C. J.: Bouini, C. E.: Ahppi, R M.. Barcelo. A. C. Biomechanical performance of diaphyseal shafts and bone tissue of femurs from protein-restricted rats. Bone Min. 4~329-39; 198X. Freudenheim. J. L.: Johnson. N. E.; Smith, E. L. Relationships between usual nutrient intake and bone-mineral content of women 35-65 years of age: Longltudinal and cross-sectional analysis. Am. J. C&z. Nurr. 44~863~76; 1986. Garlick, P. 1.; McNurlan, M. A., Ballmer. P. E. Influence of dietary protein intake on whole-body protein tumove~ in humans. Diabetes Care 14:118P-98: 1991. Cam. S. M.; Rohmann, C. G., Behar, M., Vital, F.; Gusman, M. A. Compact bone deficiency in pmtem-calorie malnutrition. Science 145: 144& 1445; 1964 Cam. S. M.; Guzman. M. A.; Wagner, B. Subperiosteal gain and endosteal 10% in protein-calorie malnutrition. Am. J. Phys. Anrhrop. 30:153-6: 1969

Lacey. J. M.: Anderson, I. J. B. Correlates of comcal bone mass among premenopauaal and postmenopausal Japanese women. J. Bone Min. Res. 6:651-659: 1991 Lx Roith, D.; Pimstone, B. L. Bone densitometry protein-deprived rat. S. Afr. Med. J. 47:72-4;

of the femoral midshaft in the 1973.

Latenegger. W.; Larsen, R. M.; Bravo, S. The effects of temporary protein deprivation on bone width and hone mineral content in rhesus macaques. Growth 37:36%72:

1973.

A A. Acute effects of Increased meat protein on unnary electrolytes and cychc adenosme monophosphate and serum pamthyroid hormone. Am. J. Clin. Nutr. 34:1779-84: 1981.

Lmta,

E. S. Orwell:

Dietary protein and the skeleton

349

Licata, A. A.; Bou, E.; Bartter, F. C.; West, F. Acute effects of dietary protein on calcium metabolism in patients with osteoporosis. J. Geronrol. 3&l&19; 1981.

Reinhard, M. K.; Mahan, D. C.; Workman, B. L.; Cline, J. H.; Fetter, A. W.; Grifo, A. P. Effect of increasing dietary protein level, calcium, and phosphorus on feedlot performance, bone mineralization and serum mineral values with growing swine. J. Anim. Sci. 43:77&80; 1976.

Linkswiler, H. M.; Joyce, C. L.; Anand, C. R. Calcium retention of young adult males as affected by level of protein and of calcium intake. Trans. N.Y. Acad. Sci. 36:33&40; 1974.

Saville, P. D.; Lieber, C. S. Increases in skeletal calcium and femur cortex thickness produced by undernutrition, J. Nub. 99rl41-4; 1969.

Lutz, 1. Calcium balance and acid-base status of women as affected by increased protein intake and by sodium bicarbonate ingestion Am. J. Clin. Nutr. 39:281-288; 1984.

Schuette, S. A.; Linkswiler, H. M. Effects on Ca and P metabolism in humans by adding meat, meat plus milk, or purified proteins plus Ca and P to a low protein diet. J. Nurr. 112338-49; 1982.

Lutz, J.; Linkswiler, H. M. Calcium metabolism in postmenopausal and osteopo rotic women consuming two levels of dietary protein. Am. J. C/in. NW. 34:2178-86; 1981.

Schwartz, R.; Woodcock, N. A.; Blakely, 1. D.; Mackellar, I. Metabolic responses of adolescent boys to two levels of dietary magnesium and protein. 11. Effect of magnesium and protein level on calcium balance. Am. J. Clin. Nutr. 26:51923; 1973. Shahkhalili, Y.; Mettraux, C. Relative importance of carbohydrate and protein sources in the differential effects of soy-based vs casein-based fortnulas on bone minerals in rats. Am. J. Clin. Nurr. 53:947-53; 1991.

Mahalko, 1. R.; Sandstead, H. H. Effect of a moderate increase in dietary protein on the retention and excretion of Ca, Cu, Fe, Mg, P, and Zn by adult males. Am. J. Clin. Nutr. 37%14; 1983. Margen, S.: Chu, 1. Yu.; Kaufmann, N. S.; Galloway, D. H. Studies in calcium metabolism. 1. The calciuretic effect of dietary protein, Am. J. C/in. NW. 27:584-9: 1974. Marsh, A. G.; Sanchez, T. V.; Michelsen, 0.; Chaffee, F. L.; Fagal, S. M. Vegetarian lifestyle and bone mineral density. Am. J. Clin. Nurr. 48:837-41; 1988.

Shenolikar, 1. S.; Narasinga Rae, B. S. Influence of dietary protein on calcium metabolism in young rats. Ind. Jour. Med. Res. 56:1412-22; 1968. Sherman, H. C. Calcium requirements of maintenance in man. J. Biol. Chem. 44:21-7; 1920.

Matkovic, V.; Kostial, K.; Simonovic, I.; Buzina, R.; Brodarec, A.; Nordin, B. E. C. Bone status and fracture rates in two regions of Yogoslavia. Am. J. Clin. Nufr. 32540-9; 1979.

Shires, R.; Avioli, L. V.; Bergfeld, M. A. ; FaIlon, M. D. ; Slatopolsky, E.; Teitelhaum, S. L. Effects of semistarvation on skeletal homeostasis. Endocrinology 107:153&35; 1980.

Mazees, R. B.; Mather, W. Bone mineral content of North Alaskan eskimos. Am. J. Clin. NW. 27:91625; 1974.

Sloan, G. M.; White, D. E.; Brettnan, M. F. Calcium and phosphorus metabolism during total parenteral nutrition. Ann. Surg. 197:1-6; 1983.

Mazes, R. 8.; Barden, H. S. Bone density in premenopausal women: Effects of age, dietary intake, physical activity, smoking, and birth-control pills. Am. J. Clin. Nun. 53:132-41; 1991.

Spencer, H.; Kramer, L.; Osis, D.; Norris, C. Effect of a high protein (meat) intake on calcium metabolism in man. Am. J. Clin. Nurr., 31:2167-80; 1978.

McCance, R. A.; Widdowson, E. M.; Lehmann, H. The effect of protein intake on the absorption of calcium and magnesium. Biochem. J. 36r686; 1942. McClellan, W. S.; DuBois, E. F. J. Biol. Chem. 87:66%80; 1930. Moats-Staats, B. M.; Brady, J. L.; Undersood, L. E.; D’Ercole, A. J. Dietary protein restriction in artificially reared neonatal rats causes a reduction of insulin-like growth factor-1 gene expression. Endocrinology 125:2363-74; 1989. Morley, J. E. Nutritional

StaNs of the elderly. Am. J. Med. 81:67%95;

1986.

Morley, J. D.; Mooradian, A. D.; Silver, A. J.; Heber, D.; Alfin-Slater, Nutrition in the elderly. Ann. Intern. Med. 109:890-904; 1988.

Spencer, H.; Kramer, L.; DeBartolo, M.; Norris, C.; Osis, D. Further studies of the effect of a high protein diet as meat on calcium metabolism. Am. J. Clin. Nun. 37:92429; 1983. Spencer, J.; Kramer, L.; Osis, D. Do protein and phosphorus cause calcium loss? J. Nutr. llRr657-60; 1988. Subcommittee Allowances.

on the Tenth Edition of the RDAs, et al. Recommended Washington, D.C.: National Academy Press; 1989.

Surgeon General’s Report on Nutrition and Health. Aging, tion No. 88-50201, pp. 595-627; 1988.

Dierary

DHHS (PHS) Publica-

R. B.

Trilok, G.; Draper, H. H. Effect of a high protein intake on acid-base balance in adult rats. C&if. Tissue fnr. 44:33%42; 1989.

Munro. H. N.; Grim, M. C. The proteins and amino acids. Goodhart, R. S.; Shids, M. E.; eds. Modern nutririon in health and disease. Philadelphia; Lea & Febiger; 1980;5 1.

Tschpoe, W.; Ritz, E. Sulfur-containing amino acids are a major determinant of urinary calcium. Mineral Electrolyte M&b. 11:137-9; 1985.

Munro, H. N.; McGandy, R. B.; Hartz, S. C.; Russell, R. M.; Jacob, R. A.; Otradovec, C. L. Protein nutriture of a group of free-living elderly. Am. J. Clin. Nutr. 46:58692; 1987. Nordin, B. E. C. ed. Calcium, phosphate Livingstone; 1976;11%144.

and magnesium

metabolism.

Churchill

Nordin, B. E. C.; Polley, K. J. Metabolic consequences of the menopause. Calcified Tissue International 41;1987. Orwell, E. Protein restriction produces a striking reduction in urinary calcium excretion and enhances bone mineral density. [Ahst.] J. Bone Min. Res. 4:529;S250; 1989. Orwell, E. S.; We&l, R. M.; Oviatt, S. K.; Meier, D. E.; McClung, M. R. Serum protein concentrations and bone mineral content in aging normal men. Am. J. Clin Nutr. 46:614-21; 1987. Peck, W. A.; Messinger, K. Nucleotide and rihonucleic acid metabolism in isolated bone cells. Effects on insulin and cortisol in vitro. J. Eiol. Chem. l&2722; 1970. Pellett, P. L. Protein requirements in humans. Am. J. Clin. NW.

.51:723-37; 1990.

Pittman, M. S.; Kunerth, B. L. A long-time study of nitrogen, calcium and phosphorus metabolism on a medium protein diet. J. NW. 17:17>85; 1939. Platt, B. S.; Stewart, R. J. C. Transverse trabeculae and osteoporosis in bones in experimental protein-calorie deficiency. Br. J. Nuw. l&483-95; 1962. Pucciarelli, H. M.; Oyhenart, E. E.; Mune, M. C. Alterations in protein and mineral contents of rat skull bones, evoked by different protein levels of the diet. Acra Anat. 117:331-38; 1983.

Tylavsky, F. A.; Anderson, 1. J. B. Dietary factors in bone health of elderly lactoovovegetarian and omnivorous women. Am. J. Clin. Nua. 48:842-9; 1988. U.S. Department of Health and Human Services. Natiomd Institutes of Heolrh Consensus Development Conference Sratement. program and abstracts. Bethesda, MD: Osteoporosis. Uauy, R.; Scrimshaw, N. S.; Young, V. R. Human protein requirements: Nitrogen balance response to graded levels of egg protein in elderly men and women. Am. J. Clin. Nutr. 31:77%85; 1978. van Beresteijn, E. C. H.; et al. Relationship between the calcium-to-protein ratio in milk and the urinary calcium excretion in healthy adult-a controlled crossover study. Am. J. Clin. Nutr. 52:142-6; 1990. Wachman, A.; Bernstein, D. S. Hypothesis: Diet and osteoporosis. 1968.

Lancer 958-9;

Walker, R. M.; Linkswiler, H. M. Calcium retention in the adult human male as affected by protein intake. J. Nurr. 102:1297-1302; 1972. Waterlow, J. C. Metabolic adaptation to low intakes of energy and protein. Ann. Rev. Nurr. 6:49%526; 1986. Weber, H.; Kocsis, 1. F.; Lauterio, T. J.; Carsia, R. V. Dietary protein restriction stress and adrenocortical function: Evidence for transient and long-term induction of enhanced cellular function. Endocrinology 127:3138-50; 1990. Wettenhall, R. E. H.; Schwartz, P. L.; Bornstein, I. Actions of insulin and growth hormone on collagen and chondroitin sulfate synthesis in bone organ culNres. Dioberes 18:280; 1969.

Pucciarelli, H. M.; Oyhenart, E. E.; Terreros, M. C. Variations of rat skull bone robusticity evoked by malnutrition. Am. J. Phys. Anthro. 64:119-24; 1984.

Whiting, S. I.; Cole, D. E. The comparative effects of feeding ammonium carbonate, ammonium sulfate, and ammonium chloride on urinary calcium excretion in the rat. Can. J. Physiol. Phormacol. 65:2202-2204; 1987.

Raghurarnulu, N.; Anita, M. C. Influence of protein nutritional status on calcium homeostasis. J. Bone Min. Res. 4:74o,S302; 1989.

Whiting, S. J.; Draper, H. H. Effect of chronic high protein feeding on bone composition in the adult rat. J. N&r. 111:178-83; 1981.

Reeds, P. 3.; C&lick, P. I. Nutrition and protein turnover in man. Draper, H. H., ed. Advances in nutritional research. New York, London: Plenum Press; 1984.

Whiting, S. J.; Draper, H. H. The role of sulfate in the calciuria of high protein diets in adult rats. J. Nutr. 110:212; 1980.

350

Whiting, S. J.; McNally, M. E. Calciuric effect of diets high in soy, beef or l&albumin protein in the rat. Nut. Rep. Inr. 40: 199-205: 1989. Wood, R. I.; Bengoa, J. M.: Sitrin, M. D.; Rosenberg, I. H. Calciuretic effect 01 cyclic versus continuous total parenteral nutrition. Am. J. Clin. Nutr. 41:6149; 1985. Yano, K.; Heilbnm, L. K. The relatmnship between diet and bone mineral content of multiple skeletal sites in elderly Japanese-American men and women living in Hawaii. Am. J. Clin. Nun. 42:877-88; 1985. Young. V. R. Impact of aging on protein metabolism. Armbrecht, H. J.: Prender-

E. S. Otwoll: Dietary protein and the skeleton J. M.; Coe, R. M., eds. Nutritional intervention in the aging process. Berlin, Heidelberg, Tokyo: Springer-Verlag; 1984;27-47. Zemel, M B.; Schuette, S. A.; Hegsted, M.; Linkswiler, H. M. Role of the sulfur-containing amino acids in protein-induced hypemalciuria in men. J. NW. 111:545-52; 1981. gast,

New York,

Date Received:

Date

Revised:

Date Accepted:

October 19, 1991 January 21, 1992 February 28. 1992