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The Interaction between Dietary Protein and Bone Health: Dr. David Jesudason Commonwealth Scientific and Industrial Research Organisation1 Department of Medicine, University of Adelaide2 Food and Nutritional Sciences CSIRO PO Box 10041, Adelaide, BC, South Australia 5000. + 618 83631155 (T) + 618 83632665 (F) [email protected] Professor Peter Clifton Commonwealth Scientific and Industrial Research Organisation 1 Department of Medicine, University of Adelaide2 Introduction: Bone consists of calcium and phosphorus as part of hydroxyapatite crystals which 00accounts for approximately 70% of its weight and approximately 20% protein such as type 1 collagen. Traditionally increased dietary protein has been thought to be harmful to bone health predominantly due to its association with increased acid load and subsequent hypercalciuria. However, over the last 40 years researchers have approached this topic by studying animals as well as humans and performed cross sectional as well as longitudinal studies. Some studies have focused directly on fracture risk whilst others have used indirect measures such as bone density or increase in bone formation markers such osteocalcin or decrease in bone resorption markers such as deoxypyridinoline or pyridinoline. IGF1 which may promote bone formation has also been measured. This review considers what role dietary protein intake has in increasing or decreasing bone strength and reducing fracture risk. The review will also address any separate effect of vegetable protein from animal protein such as meat or milk protein. It is also clear that the effect of dietary protein per se must be considered in the context of the effect on acid base balance and interaction with dietary calcium and phosphorus. Method Of Article Selection: We did a search of PubMed looking at all articles generated by searching dietary protein, soy protein and milk protein respectively and either bone, bone density or fractures. All papers generally dealing with human subjects and the interaction between dietary protein and bone density, bone markers or fractures whether crosssectional, longitudinal were deemed suitable for discussion in this article. There were also a few studies not generated by this search but which were frequently cited by the articles generated and these were also included when relevant. Effects of High Protein Diet on Bone Mass and Bone Density: There is evidence from studies that amino acids from dietary protein have an anabolic effect on bone. As early as 1985, essential amino acids were shown by Clemmons et al. to augment the IGF1 response to refeeding after fasting[1]. Cross-sectional Studies: Several cross –sectional studies obtained from Pubmed comparing the effect of dietary protein (usually derived from food diaries or validated questionnaires) have looked for an association between dietary protein and bone mineral density (BMD) or bone mineral content (BMC) at the traditionally measured sites. These are summarised in Table 1. The studies are not directly comparable as the subjects vary according to age, sex, mean daily protein intake, pre and post-menopausal status of women, site for BMD measurement, statistical method to look for a relationship between the two parameters and rigour of the study. Even mean daily protein intake has to be interpreted with caution as it does not tell us mean intake per kilogram body weight nor protein intake as percentage of total energy which is not always published. In young and pre-menopausal women increased dietary protein seems to be positively correlated with higher bone density at least at some sites particularly the radius[2-6] In Teegarden’s study a complex relationship between calcium, protein and phosphorus was obtained so that women with higher protein intakes were better off with higher calcium supplementation. In post-menopausal women, increased protein intake was associated with increased bone density in most not all studies [3, 4, 7-9]. The sites where dietary protein was correlated included radius, hip, spine and total body BMD. This included the study by Chiu et al. of predominantly vegetarian women. Usually some but not all sites would show an association in any given study. Moreover when statistical significance was shown it was not always apparent the degree to which a higher dietary protein might produce a clinical benefit. In the three studies of men, the association between dietary protein and BMD is less convincing [10-12]. In the one positive study, the mean protein intake was much higher at 97.0 g protein daily or 1.17 g/kg body weight. Longitudinal Studies: Table 2 summarises longitudinal studies looking at dietary protein intake and future BMD or bone loss at commonly measured sites. These studies usually adjusted for osteoporotic confounders such as age, weight, height, weight change, total energy intake, smoking, alcohol intake, caffeine, physical activity, calcium intake, and, for women, current estrogen use. Most studies are either positive or neutral for bone when increased protein consumption is noted. There are several interesting observations. Firstly the importance of dietary protein intake during adolescence when peak bone mass is being accrued is suggested by the Saskatchewan Paediatric Bone Mineral Accrual Study[13]. An effect of increased dietary protein was not shown in the study by Beasley JM et al with a slightly older but still pre-menopausal population who may have already largely attained peak bone mass [2]. In older cohorts of men and particularly pre-menopausal women such as the Rancho Bernardo study, the Framingham Osteoporosis Study and the hospital study by Geinoz et al. a favourable effect was seen for increased dietary and particularly animal protein [14-17]. The only neutral study in an older cohort was the study by Rapuri et al. [9]. This aspect of the study may have been underpowered. In fact the major negative relationship of increased protein intake and decreased BMD was seen for vegetable protein in the Rancho Bernardo Study. This study also suggested that subjects with lower calcium intake might benefit more from higher protein intake. This is at odds with other studies suggesting calcium adequacy is synergistic with higher protein intakes such as the Saskatchewan Paediatric Bone Mineral Accrual Study and as discussed in the calcium subsection below. Protein Supplementation: Dietary protein has also been shown to be effective later in life. This was shown when an oral protein supplement consisting of 90% milk proteins or isocaloric placebo containing maltodextrins was given to 82 women with a mean age of 80.7 +/- 7.4 years with recent osteoporotic hip fracture [18]. Treatment was for 6 months and calcium and vitamin D were also supplied. The protein supplemented subjects had significantly greater increases in serum levels of IGF 1 (85.6% +/- 14.8% vs. 34.1% +/- 7.2% at 6 months; p = 0.003) and an attenuation of the decrease in proximal femur bone mineral density (-2.29% +/- 0.75% vs. control -4.71% +/- 0.77% at 12 months; p= 0.029). Overall based on these studies there seems to be a small beneficial effect of dietary protein on BMC and BMD particularly in women in adolescence and early adulthood when bone mass is being attained as well as after the menopause when bone loss is accelerated. This was recently tested by Darling et al. who performed a systematic review and meta-analysis of dietary protein and bone health studying sixty-one crosssectional surveys, cohort and ecological studies [19]. In the meta-analysis most studies showed a positive influence or no influence of dietary protein on BMD and BMC with only a minority showing a negative relationship. The 18 cross-sectional surveys with correlation co-efficients were pooled, by population subtype and by outcome. The pooled values for population subgroup (eg men, pre and postmenopausal women) and for almost all bone sites were positive and were significant at most sites including radius BMC and BMD, hip BMD, lumbar spine BMC and BMD and total body BMD. Only 6 protein supplementation trials could be used for the meta-analysis and only then for lumbar spine BMD. For all protein (total protein and milk basic protein), a significant effect of protein supplementation on lumbar spine BMD was seen (weighted mean difference + 0.02; 95% CI: 0.00, 0.04; p = 0.04). No effect was seen on lumbar spine BMD of MBP or soy protein alone. Overall the effect of protein on bone was modest with the authors estimating that protein intake could account for 12% of BMD. With regards to the elderly specifically, the beneficial effects of higher protein diets are not just an attempt to correct protein deficient diets. Gaffney- Stomberg et al. have shown that the anabolic effect of the body to dietary protein is reduced in the elderly, so that the amount of protein needed for anabolism is greater and would exceed the 0.8g/kg/day which is the recommended daily intake. In fact they suggest that the Recommended Dietary Allowance of dietary protein to be increased to 1.0-1.2g/kg day in the elderly to maximise its anabolic effect on muscle and bone[20]. This would be consistent with the findings of previously quoted studies such as by Geinoz et al. where a beneficial effect was shown for total protein intakes exceeding >1g/kg/ day[14]. This is analogous to the study by Wengreen discussed in detail below where the lowest odds ratio for fracture were in the quartile of 50-69 year olds with protein intakes as a percentage of energy intake of 17.4-30.8 % as opposed to the quartile with percentage protein intake of 5.6-13.9%[21]. Relationship between Dietary Protein and Fractures Epidemiological studies do not provide a clear answer about the fracture risk with higher protein diets as the results are contradictory and not easy to reconcile. These studies are displayed in Table 3. For example the Nurses Health Study suggested increased dietary protein consumption may increase fracture risk [22]. In this large cohort study for 12 years, dietary protein was associated with an increased risk of forearm fracture (relative risk (RR) = 1.22, 95% confidence interval (Cl) 1.04-1.43, p for trend = 0.01) for women who consumed more than 95 g per day compared with those who consumed less than 68 g per day. This association was linked to animal but not vegetable protein consumption. The increased rate of forearm fracture was slightly reduced in those consuming more calcium but there was no reduction in fracture risk when women with a high ratio of dietary calcium to total protein (≥ 11) were compared to women with a low ratio (<5.5) (RR = 0.91, 95 percent CI 0.78-1.06). In a group of 19,752 women and 20,035 men from Norway followed for an average of 11.4 years, a semi quantitative dietary questionnaire was completed at baseline[23]. There were 213 hip fractures identified during follow up with no overall association between calcium intake or non-dairy animal protein intake and hip fracture in this cohort. In this instance there was a calcium interaction in that there was an elevated risk of fracture in women with a high intake of protein from non-dairy animal sources in the presence of low calcium intake (relative risk = 1.96 (95% confidence interval 1.09-3.56) for the highest quartile of non-dairy protein intake and the lowest quartile of calcium intake vs. the three lower quartiles of protein intake and the three higher quarters of calcium intake). These two studies involved pre-menopausal women but the numbers were very small in the high protein/low calcium group In older women some of the studies are more convincing for the benefit of a high protein diet. Dietary nutrient intake by food frequency questionnaire was assessed in a cohort of Iowa women aged 55-69 y at baseline and followed for 104338 person-years [24]. There were 44 cases of incident hip fracture during follow up. In this case fracture risk was negatively associated with total protein intake and animal protein. In a multivariate model with inclusion of age, body size, parity, smoking, alcohol intake, estrogens use, and physical activity, the relative risks of hip fracture decreased across increasing quartiles of intake of animal protein as follows: 1.00 (reference), 0.59 (95% CI: 0.26, 1.34), 0.63 (0.28, 1.42), and 0.31 (0.10, 0.93); P for trend = 0.037. There was a non-significant trend for higher vegetable protein intake to increase fracture risk. In the Adventist Health Study 2, the incidence of wrist fractures were determined in 1865 peri and post-menopausal women who had completed two lifestyle questionnaires 25 years apart [25]. Amongst the vegetarians, those who consumed the least vegetable protein intake were at highest risk for fracture. As levels of plantbased high-protein foods increased the wrist fracture decreased with a 68% reduction in risk (hazard ratio (HR) = 0.32, 95% confidence interval (CI) 0.13-0.79) in the highest intake group. In meat eaters, amongst those with lowest vegetable protein consumption, increasing meat intake decreased the risk of wrist fracture with the highest consumption decreasing risk by 80% (HR = 0.20, 95% CI 0.06-0.66). In a subset of the Shanghai Women’s Health Study, 24 403 post-menopausal women aged 40-70 were followed for a mean of 4.5 years[26] . Their dietary intake including of soy protein was determined on several occasions by food frequency questionnaire and a link with fracture risk was sought. After adjusting for confounders the relative risks (95% confidence intervals) of fracture were 1.00, 0.72 (0.62-0.83), 0.69 (0.590.80), 0.64 (0.55-0.76), and 0.63 (0.53-0.76) across quintiles of increasing soy protein intake (P<.001 for trend). This trend was particularly marked for women soon after the menopause. In men such as in the study by Mussolino et al. the effect of dietary protein on hip fracture seems less convincing [27]. In the study above by Schurch, there were seven vs thirteen new vertebral deformities respectively amongst patients who received protein supplements vs controls, (p > 0.2) [18]. Dietary protein is also anabolic later in life. A study from Utah also supported the role for a higher protein diet for bone health [21]. Wengreen et al. recruited subjects aged 50-89 with previous hip fractures and matched them with controls of the same age and sex in a case-control study. The 2501 subjects’ diets were assessed using a picturesort food frequency questionnaire. Protein intake was stratified into quartiles and logistic regression was applied controlling for gender, smoking, oestrogen, calcium and vitamin D use as well as body mass index and level of physical activity. There was no association between protein intake and hip fractures for the 70-89 year old age group. However, for participants aged 50-69, the odds ratio of a hip fracture decreased from an odds ratios of 1.0 [reference] for the lowest protein intake quartile to 0.35 [0.21-0.59]; (p < 0.001) for the highest protein consuming quartile. In this study, stratifying by high and low calcium did not alter results. Despite the promising results from some of these studies , in the meta-analysis discussed above, there was no overall effect of protein intake on hip fracture risk [19]. Disappointingly however only 4 studies were able to be used (the Nurses’ study, the Norwegian Study, the Iowa study and NHANES 1 study) and then only hip fracture examined. Hence it is hard to draw definitive conclusions from the meta-analysis about fracture risk and it is possible that a small beneficial effect of dietary protein may have been missed. It is possible that whilst higher protein diets may be beneficial in improving bone mass, the effect is modest and dependant on interactions with dietary acid and calcium as discussed below and easier to demonstrate at times of increased bone formation and resorption and in women. With regard to fracture risk, as bone mass is only one factor in the aetiology of fractures, it is harder to demonstrate a beneficial effect of high protein diets when there is interplay with other factors including falls risk, muscle strength, and other nutrients such as Vitamin D. Nevertheless a higher protein diet seems to show a more convincing effect on reducing fractures in older rather than younger subjects, perhaps when its anabolic effect may be more important. Relationship between Dietary Protein, Acid and Bone: Traditionally high dietary protein was thought harmful to bone health. Wachman and Bernstein hypothesised in 1968 that the increased incidence of osteoporosis with ageing was due to “the life-long utilisation of the buffering capacity of the basic salts of bone for the constant assault against pH homeostasis” due to the western meat diets containing “acid ash”[28]. It has long been hypothesised that diets high in acid, cause a low grade metabolic acidosis and mobilise base from the skeleton and thereby contributing to bone loss with ageing. As early as 1994, potassium bicarbonate was administered to post-menopausal women for 18 days whose diet was otherwise controlled for calcium and protein [29]. There was an improvement in calcium and phosphorus balance (due to reduction in urinary excretion of these elements) and reductions in urinary hydroxyproline and increases in osteocalcin suggesting a beneficial effect on bone turnover. When potassium citrate supplementation was given to post-menopausal women for three months, a significant reduction in urinary deoxypyridinolines and hydroxyproline-tocreatinine ratios was achieved [30]. In terms of dietary protein, it is the effect of the sulphate acid load derived from the amino acids methionine and cysteine that have been postulated to have a demineralising effect. The relationship between meat intake and acid load and also urine pH has been examined in several studies. In a cross-sectional study, 161 post-menopausal women had DEXA measurement of bone density at the lumbar spine and total hip[31]. The dietary intakes of sulphur containing amino acids, protein and minerals were also assessed. Although dietary protein did not predict BMD at lumbar spine, a positive relationship was demonstrated after adjusting for the negative effect of sulphate, suggesting that non sulphur containing amino acids are beneficial to bone. The hip BMD was positively associated with dietary protein; the relationship did not change after adjustment for sulphate. Several groups have tried to reduce dietary protein and acid load and study the effect. Ince et al. studied the effect of lowering protein content in the diet in 39 healthy, premenopausal women consuming ad libitum diets[32]. Mean daily protein was decreased from 1.1 g/kg to the US recommended dietary allowance of 0.8g/kg daily on an isocaloric diet. There was no change to concentration of other minerals such as 819 mg (20.5 mmol) Ca, 1152 mg (37 mmol) P, 129 mmol Na. This required the substitution of carbohydrates and fats for protein as well as using phosphate supplements. Bone markers were measured before and one week after the switch. As the dietary protein was reduced, mean urine pH increased from 6.3 to 6.8 (P < 0.001), and net renal acid excretion (NRAE = urine ammonium plus titratable acids minus bicarbonate) decreased 68% (21.4 mEq/d; P < 0.001. Mean urinary calcium decreased 32% [42 mg (1 mmol)/d; P < 0.001], and bone resorption urine N-telopeptides decreased 17% (74 micromol bovine collagen equivalents/d; P < 0.001). Although in this study, reduced dietary protein had a beneficial effect on bone markers and reduced calciuria due to decreasing acid load, the authors acknowledged that dietary modifications, such as increasing vegetable and fruit intake, could result in sustained reductions in NRAE without reducing protein intake. This is a valid insight as other studies have shown a beneficial effect of dietary protein when these factors are considered. In the Framingham Osteoporosis Study discussed above, the potentially catabolic effect of dietary acid on bone was considered [15, 17]. As expected the dietary intake of basic foods were positively associated with BMD at baseline and with reduced bone loss in men. However higher rather than lower protein intake was associated with less bone loss reflecting the interaction of protein with other components in a mixed diet. Alexy et al. conducted a prospective study of 229 children and adolescents aged 618[33]. Every year for four years, dietary intake was measured from 3 day weighed dietary records followed by bone measurement on one occasion by quantitative computed tomography at the proximal radius. Using an algorithm utilising dietary phosphorus, protein, potassium and magnesium, the potential renal acid load (PRAL) was determined as a marker of dietary acid load. The authors adjusted for confounders such as sex, age, energy intake, body mass index, growth velocity and stage of puberty. Children with low intake of alkalinizing minerals (i.e. with a higher dietary PRAL)had significantly less bone mineral content (p < 0.01) and had significantly less cortical area (p <0.05). In addition the long term protein intake was significantly associated with markers of bone modelling and remodelling such as periosteal circumference (p < 0.01), bone mineral content (p < 0.01), cortical area (p < 0.001) and polar strength strain index (p < 0.0001). Sebastian speculates that this trade off between the potential anabolic effect of a high protein diet and the catabolic effects of a high acid diet interact to determine the overall net effect of any given diet[34]. Thus according to him the ideal diet from a bone viewpoint in order to achieve peak bone mass in early adulthood would be more akin to that of prehistoric hunter gathers. This would combine a high protein diet with a high alkaline load such as fruit and green leafy vegetables, stalks and roots rather than acid producing cereal grains. Not only are fruits and vegetables a good source of dietary potassium which has an alkalinizing effect but cereals are also a major source of phosphorus – an excess of which can increase parathyroid hormone secretion and bone resorption. The importance of fruit and vegetables was also recently emphasised by Heaney and Layman[35]. Protein and Calcium Adequacy: There is evidence that dietary protein has a more favourable effect in the presence of calcium adequacy [13]. Besides the issue of acid-base status, one concern about high protein diets are that they cause calciuria. Theoretically, this could lead to negative calcium balance in the absence of supplementation and cause secondary hyperparathyroidism and calcium loss from bone. In a well quoted study, Dawson-Hughes et al. reviewed the protein intake of 342 healthy subjects aged over 64 who had completed a 3 year study of calcium citrate malate and Vitamin D supplementation or placebo[36]. Overall the mean (+/-SD) protein intake of all subjects was 79.1 +/- 25.6 g/d whilst the mean total calcium intakes of the supplemented and placebo groups were 1346 +/- 358 and 871 +/- 413 mg/d, respectively. In this study only in the group supplemented with calcium was a higher protein intake significantly associated with a favourable 3-y change in totalbody and femoral neck BMD. It should be noted however that New raised the possibility that some of the beneficial effect of calcium may be related to the alkalizing effect of the citrate rather than the calcium alone[37]. There is also some reassuring evidence that the hypercalciuria associated with a high protein diet is accompanied by and due to increased gut calcium absorption rather than due to release from bone in a buffering response to the acid load from the protein. In an elegant experiment, Kerstetter JE et al. studied 13 women who received 10 days of an experimental diet after a two week run in period. These women received either a moderate (1.0 g/kg) or high (2.1 g/kg) protein diet in random order[38]. Calcium kinetics was studied using dual stable calcium isotopes. Urinary calcium increased during the high-protein diet in comparison with the moderate (5.23 +/- 0.37 vs. 3.57 +/- 0.35 mmol/d, P < 0.0001, mean +/-sem) but this was due to increased intestinal calcium absorption (26.2 +/- 1.9% vs. 18.5 +/- 1.6%, P < 0.0001, mean +/sem). Hence the high protein diet did not affect net bone balance and in fact caused a significant reduction in the fraction of urinary calcium of bone origin and a non significant trend toward a reduction in the rate of bone turnover. Type of Protein: Much research has also gone toward investigating what might be the best source of protein for bone health in both animals and humans. Soy Protein: Soy protein is notable for its low fat content and for containing phytochemicals such as isoflavones, saponins and phytic acid. Phytates are naturally occurring compounds particularly found in whole grains. Despite concern that they may reduce calcium absorption, there is some evidence that they are beneficial for bone health [39]. Soy protein studies are of three types; firstly studies aiming to see if a higher soy protein intake per se is beneficial usually by cross-sectional analysis, secondly longitudinal studies comparing isoflavone rich versus isoflavone poor soy protein and lastly comparing soy with another protein such as milk protein [40-53]. In the cross-sectional studies by Ho and Horiuchi respectively in post-menopausal women, increased dietary soy protein was associated with increased BMD/BMC at hip, total body and spine respectively. However when high versus low isoflavone concentration was compared in longitudinal studies varying from 3-12 months, a beneficial effect of isoflavones was mainly seen on markers of bone formation and resorption. In a few studies a beneficial study on spine BMD was shown for the higher isoflavones [40, 50, 51]. In the two studies involving men, no effect was seen for BMD. In comparison with milk derived proteins no beneficial effect was seen on bone density. In the subset of the previously mentioned Shanghai Women’s Health Study, women in the lowest quintile of soy intake actually had a relative risk of 0.63 for hip fracture compared to the highest quintile which is at odds with all the other studies suggesting no effect or a beneficial effect of soy protein. This was the only major fracture study looking at soy protein so it is hard to know how to interpret this result. Overall based on these very mixed results there is little to definitely suggest that soy protein itself has any intrinsic properties making it superior to other proteins. Once again dietary interactions may be important. Roughead suggests soy protein may be more favourable at doses above 40g/day and in the presence of calcium supplementation (eg 650-1400mg in previous studies) such as in some of the studies above[40, 41, 51, 52]. Milk Protein and Bone: The effect of milk whey protein supplementation (particularly its basic protein) fraction on bone health has also been studied. The Copenhagen Cohort Study was a cross-sectional study performed on 63 seventeen year old girls and 46 boys[54]. A seven day diet analysis was performed and BMC and serum markers of bone turnover as well as IGF1 were measured. Both total and milk (0.3g/kg) protein intake, but not meat protein intake (0.4g/kg) was positively associated with size-adjusted BMC (P≤ 0.05). After correcting for calcium intake, physical activity and energy, the positive association between milk protein intake and size adjusted BMC remained significant (P≤ 0.01) and did not seem to be mediated via current IGF-1. A series of studies have been performed in Japan some of whose authors are from Snow Brand Milk Products Co Limited[55-59]. They have variably studied healthy women, post-menopausal women, and healthy men and looked at the effect of doses of 40-300mg of milk basic protein supplementation for e.g. periods of 6 months. They have found suppression of bone resorptive markers such as urinary cross-linked Ntelopeptides of type-I collagen/creatinine and deoxypyridinoline/ creatinine and variably increased markers of bone formation like ostocalcin. They have also demonstrated modest effects on bone density compared to control at sites including calcaneus, lumbar spine and radius. These results are somewhat surprising. For example even modest doses of 40mg increased the mean (+/- SD) rate of left calcaneus BMD gain of women in the MBP group by 3.42 +/- 2.05% compared to the BMD of women in the placebo group of 2.01 +/- 1.75%, p = 0.042. However not all studies have shown a convincing a benefit of MBP. In one study 84 healthy young women were divided into 3 groups receiving placebo, whole milk or milk containing 40mg MBP for eight months[60]. BMD was measured at lumbar spine and forearm at baseline and 8 months as well as indices of bone turnover. The total BMD actually significantly increased compared with the baseline values in all groups. However, no significant difference on the mean rate of gain of total BMD was observed among the MBP group (2.19%), the whole milk group (2.63%) and the control group (1.61%). There was no significant increase in markers of bone formation like bone – specific alkaline phosphatase in either milk group. Serum (NTx) in MBP group at 8 months and in whole milk group at 6 months were significantly decreased from baseline. Although there was no significant difference between the whole milk group and MBP group, after combining the milk groups, NTx had significantly decreased from baseline. Animal vs Vegetable Protein: Attempts to demonstrate the superiority or inferiority of animal versus vegetable protein to bone health has been confounded by the fact that communities consuming more vegetable protein tend to also consume more fruit, vegetables and potassium which tends be more alkaline with beneficial effects as described above. The effect of varying amounts of dietary calcium is also an issue. Hence epidemiological studies can tend to favour vegetable protein For example a global analysis was performed by recording hip fracture rates per 100 000 person years in women aged over 50, from 33 different countries. [61]. Those 11 countries in the lowest tertile of hip fracture rates had the lowest animal protein consumption with vegetable protein intake exceeding animal protein. Of the 11 countries in the highest risk tertile for hip fractures, animal protein consumption exceeded vegetable protein intake in 10 countries. Overall hip fracture rates varied directly with total (r = +.67, p < .001) and animal (r = +.82, p < .001) protein intake and inversely with vegetable protein intake (r = .37, p < .04). The vegetable/animal protein intake accounted for 70% of the total variation in hip fracture rates. The authors concluded that the critical determinant of hip fracture risk in relation to the acid-base effects of diet is the net load of acid in the diet both from dietary protein as well as from other sources such as fruit and vegetables. There are several weaknesses with cross-sectional between country studies such as these. Firstly the links were associational and confounding factors such as physical activity, and cultural variations were not controlled for. Additionally the study required using dietary data for the whole country and applying it to one subset of the population i.e. post-menopausal women. In the EPIC Potsdam Study, the relationship between bone health and diet, particularly animal and vegetable protein and their interaction with calcium was studied[62]. The 8178 females had bone measurements performed on their right os calcis by broadband ultrasound attenuation (BUA) and the relationship between BUA and dietary protein was assessed by a linear regression model after adjusting for confounding factors. A high vegetable protein intake correlated with an increased BUA (beta = 0.11; p = 0.007) whilst a high intake of animal protein was associated with decreased BUA values (beta = -0.03; p = 0.010). The authors admitted however that they could not exclude the possibility that some other factor such as the alkalinizing effect of the diet (e.g. from vegetables) rather than the amount of protein per se was responsible. Moreover, the apparent detrimental effect of animal protein on bone was attenuated in those subjects with a high calcium intake. There was a significant interaction between calcium and animal protein but not calcium and vegetable protein. Sellmeyer DE et al. followed 1035 women aged over 65 and followed them prospectively for a mean (+/-SD) of 7.0+/-1.5 years[63]. Protein intake whether of vegetable or animal origin was noted from a food frequency questionnaire whilst BMD was measured by dual-energy X-ray absorptiometry. They found that women with a higher ratio of animal to vegetable protein had greater bone loss at the femoral neck (p=0.02) and greater risk of validated hip fracture (relative risk = 3.7, P = 0.04) after adjustment for confounding factors including calcium and total calcium intake. In a subsequent clarification, they explain that they were not suggesting that animal protein per se is harmful to bones. However the animal to vegetable protein ratio of a community is a surrogate for a higher consumption of animal foods with less accompanying foods such as vegetables which are base rich. [64]. Conversely many of the longitudinal studies in table 2 showed a beneficial effect of animal protein and with the Rancho Bernardo study vegetable protein actually had a deleterious effect. [16]. In the Adventist Health Study 2 discussed above, both increasing vegetable and meat protein appeared to attenuate wrist fracture risk [25]. One concern with meat protein is that it may have a negative effect on calcium balance as discussed above compared to vegetable protein or compared to a low meat diet. Two studies by Roughead et al. are reassuring. Roughead et al. compared the effects of a high or low meat diet (20 vs. 12% of energy as protein) on calcium balance in 15 post-menopausal women [65]. Women received 600mg calcium supplementation and either diet for 4 weeks. Calcium retention was measured by extrinsically labelling the 2-d menu with (47) Ca, followed by whole body scintillation counting for 28 Days. Calcium retention was not different during the high and low meat dietary periods (d 28, mean +/- pooled SD: 17.1 and 15.6%, +/-0.6%, respectively; P = 0.09). The diets did not affect urinary calcium loss nor indicators of bone metabolism. An initially higher renal acid excretion in subjects consuming the high meat compared with the low meat diet decreased significantly with time. Another carefully designed feeding study followed 13 post-menopausal women [52] . These women consumed one of two similar diets except that 25g of high isoflavone soy protein was compared with a similar amount of meat protein for 7 weeks in a randomized cross-over trial. No beneficial or harmful effect was noted on calcium homeostasis as determined after a 47Ca retention study followed by whole body counting for 28 days. No difference was noted in bone specific alkaline phosphatase, osteocalcin, serum IGF1, serum IGF-IBP3 nor tartrate resistant acid phosphatase. Calcium was similarly retained during the control and soy diets (d 28, percent dose, mean +/- pooled sd: 14.1 and 14.0 +/- 1.6, respectively). Although during the soy diet, there was a 15-20% lower renal acid excretion, urinary calcium loss was unaffected by diet. Conclusion: On balance, dietary protein by itself appears to have an anabolic effect on bone and has variably shown favourable effects on bone markers including IGF1, bone density and much less consistently on fracture risk where other non-dietary factors such as falls risk are important. Dietary protein may be more important at key times in the life cycle such as adolescence when bone mass is being acquired, after the menopause when increased bone loss occurs and in late life when dietary protein intake might be otherwise low. The benefit of increased dietary protein may be harder to show in men than women because their baseline osteoporosis and fracture risk is lower. Early fears about protein induced hypercalciuria have been somewhat allayed by studies showing increased gut absorption of calcium. However several studies have suggested that dietary protein works best in the presence of calcium sufficiency. Also essential is the presence of adequate fruit and vegetables which will reduce the acidity of the diet as a whole. Protein from vegetable, milk and meat sources has each been (inconsistently) demonstrated to aid bone health. The relative superiority of one source over another particularly in epidemiological studies may relate more to other components of the diet such as calcium and fruit and vegetables rather than to characteristics of the protein itself. There is not enough evidence to suggest that one protein source (e.g. soy, milk protein) should be favoured over another source. Because the overall benefit of dietary protein on bone is modest, recommendations about its use should be targeted to higher risk groups such as the elderly and only in the presence of adequate calcium and fruit and vegetables. Moreover further trials aimed at longitudinally studying the effect of high protein diets using hard end-points like fracture reduction are indicated. Ideally these should be performed in high risk groups like the elderly in the presence of calcium and acid base sufficiency. References: 1. 2. 3. Clemmons DR, Seek MM, Underwood LE (1985) Supplemental essential amino acids augment the somatomedin-C/insulin-like growth factor I response to refeeding after fasting. Metabolism 34:391-395 Beasley JM, Ichikawa LE, Ange BA, Spangler L, LaCroix AZ, Ott SM, Scholes D Is protein intake associated with bone mineral density in young women? 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J Nutr 133:1020-1026 Table 1: Cross-sectional Studies linking protein with BMD/BMC Study Name Age Number Diet Endpoint Results Composition Teegarden D 18-31 215 women et al. [6] Usual diet BMD FN, Protein, (mean protein spine, hip intakes positively 76.0g/day) radius, TB correlated with and BMC radius, spine, spine BMD and radius, TB spine BMC Protein calcium interaction. Beasley JM Women 14-40 560 women et al. [2] Baseline AP and LS, HIP No relationship VP intake from and TB between TP, AP questionnaire as BMD. and adjusted BMD. % of energy Low VP associated intake. lower BMD hip Mean TP was (0.03) and TB (p= 15.5% total 0.04). energy. Michaelsson K. Et al. [5] (Sweden) 28-74 175 Usual diet TB, L2- Protein associated women (mean protein L4, TB and FN BMD 59g/day) proximal not spine with femur linear regression BMD Lacey JM et 89 35-40 178 Usual diet Mid-radial Protein intake was al. [4] years, and 89 Japanese (mean protein bone a positive correlate 55-60 years pre and post 70.9 and mineral of MBMC for menopause 72.6g/day in pre content pre and post women and post- (MBMC) menopause menopausal) and density (MBMD) Cooper C et Premenopausal 72 pre and Usual diet BMD LS, Protein intake al. [3] mean age 39 218 post ( pre- prox correlated with Postmenopaus menopausal menopausal femur, BMD distal radius, al mean age 68 women mean protein distal and proximal femur in 75g/day, mid radius premenopausal. Chiu et al. Mean 60.8 [7] Ilich et al. [8] Mean 68.7 postmenopausal No relationship in 72 g/day) post-menopausal 258 pm Usual diet LS and FN Dietary protein Buddhist (vegetarian) BMD women (mean protein BMD in regression 61.0g/day) analysis associated LS 136 PM Usual diet TB BMD, Protein associated women (mean protein TB BMC, with TBBMD, 70.7g/day) Hip , TBBMC, Ward’s, forearm, hand in regression hand analysis BMD Rapuri P.B. 68-77 [9] Whiting et Quartile 1 mean BMD Quartile 4 had Mean daily protein 53.7 spine, higher BMD spine, protein g/day cf Quartile radius, mid-radius total 71.5/day 4 71.2 g/day femur, body cf quartile 1 total body &2 Usual diet TB, hip Protein predicted (mean protein and spine TB, spine not hip 97.0g/day) BMD BMD Usual diet Femoral No association with (mean protein neck BMD dietary protein 39-42 489 57 men al. [12] Jaime et al. Mean age 62.3 277 men [10] 86.5g/day) Kyriazopoul 18-30 300 men Usual diet BMC and No association with os et. al. [11] (mean protein BMD protein from meat (Greece) 196±63 g/week) radius and fish FN – Femoral Neck, LS - Lumbar Spine, TB – Total body, AP- Animal Protein, VP= Vegetable Protein Table 2: Longitudinal Studies linking protein with BMD/BMC Study Name Age Number Diet Endpoint Results Duration Saskatchewan 8-15 at start 133 young Usual Diet TB BMC, Adult protein Paediatric Mean age 23 adults (74 Annual 24 hour TB BMC intake predicted Study [13] at end females and record net gain TB-BMC net gain 59 males) including from peak β= protein height 0.11; P = 0.015). velocity TB BMD annually Rancho 55-92 Bernardo [16] 388 men Usual diet from BMD HIP, BMD increased at and 572 questionnaire FN, Spine, hip (p = 0.005), at TB, FN (p = 0.02), at women 4 year spine (p = 0.08), follow up and at TB(p = 0.04) for every 15-g/day increase in AP but decreased at hip (p = 0.03), FN and (p = 0.04), and spine (p = 0.02) for every 15-g/day increase in VP. Framingham 69-97 855 subjects Dietary TP, Hip, Bone loss in Osteoporosis followed for as % energy forearm Quartile 1 (lower Study [15] 4 years BMD TP) greater than intake from questionnaire Quartile 4 at N divided into (P<0.001) and LS quartiles. (P< 0.05). Similar Mean % protein results for AP. intake 15.9%. Beasley JM et Women 14- 560 women Baseline AP and LS, HIP No relationship al. [2] 40 3 year VP intake from and TB between % increase Mean age follow up questionnaire as BMD. in dietary protein 24.3 at start. % of energy and BMD intake. longitudinally Mean TP was 15.5% total energy. Geinoz et al. Men mean 48 women, Diet record after BMD FN, Higher protein [14] age 80 26 men 28 days hospital LS group had higher Women mean Divided into TP BMD at FN age 82 >1g/kg/ day or < (P<0.05). Higher 1g/kg/ day protein men had higher BMD at LS (p<0.05). Rapuri PB. et 68-77 96 women 7 day food BMD No association al. [9] Mean daily 3 years. diaries at start spine, between BMD loss and end. radius, and protein intake. protein 71.5/day femur, total body FN – Femoral Neck, LS- Lumbar Spine, TB – Total body, AP- Animal Protein, VP= Vegetable Protein Table 3: Dietary Protein and Fractures Study Age Number Name Diet Duration Endpoints Results 12 years 234 hip, 1628 AP > 95g/d distal forearm cf 68 g/d fractures RR 1.22 Composition Nurses 35- 89 500 Health 59 women Usual diet Study [22] Forearm # No association with hip # Norweg mean 19,752 Usual diet 47.1 women from baseline 20,035 questionnaire ian [23] 11.4 213 hip RR hip # fractures 1.96 for highest men quartile non-dairy protein, lowest quartile calcium Iowa 55- 32050 Usual diet 104338 44 hip Multivariate [24] 69 Women from person fractures analysis. questionnaire years RR highest quartiles AP 0.31 cf lowest quartile Adventi mean 1865 Usual peri/post baseline diet Health menopa from highest VP Study 2 usal questionnaire cf lowest. [25] women then 25 years 80% RR if later meat > 4x st 52.2 25 years 216 wrist Vegetarians; fractures 68% RR in week Shangh 40- 24 403 Soy protein 4.5 1770 incident RR 0.63 in ai 70 post- from years fractures lowest cf Women menopa questionnaire including highest SP ’s usal wrist (17.6%), quintile Health women vertebrae Study (14.9%), and [26] hip (3.3%). Utah 50- 1167 hip Usual Cross- 1167 hip OR hip Hip 89 fracture baseline diet sectional fractures fracture Fractur cases from 0.35 highest e Study (831f, questionnaire protein [21] 336m) quartile vs matched lowest to 1334 protein controls quartile for subjects 5069 only Nhanes 45- 2879 Dietary Up to 22 71 hip No 1 74 men protein from years fractures association follow questionnaire hip # up study [27] Geneva 80.7 82 men Usual + 550 6 13 vertebral Non- Protein or calcium daily, months deformities in significant Supple women 200000 units treatment vs 7 mentati with D3 once in control on recent + 20g protein Study hip daily or [18] fracture isocaloric control AP – Animal Protein, VP- Vegetable protein, SP- Soy Protein Table 4: Soy Protein intake and bone health Study Subjects Duration Intervention Results Ho et al. 454 Chinese cross- Usual diet No difference (cross- post- sectional Soy protein in BMD/BMC sectional) [46] menopausal quartiles spine. For hip women. Mean Q1: 1.38±0.81 and TB 55.1 y.o. g/day BMD/BMC Q4: 19.41±11.58 significant dose g/day response for increased soy in women. 4years PM Horiuchi T. et 85 post- cross- Usual diet SP associated al. [47] menopausal sectional Mean SP 12.4 ± with L2-4 and (cross- women mean 5.4 g/day UDPYR z sectional) age 66.9 y.o. score on regression analyses Alekel et al. 69 peri- [40] 24 week Isoflavone rich Isoflavone rich menopausal soy (SPI+) vs soy only women. Isoflavone poor attenuated bone Mean age soy (SPI-) loss in LS in 50.2 y.o. Vs whey placebo perimenopausal 40g SP daily No effect on Dalais et al. 106 PM 3 months [43] women (including 118mg urine DPYR (longitudinal) Mean age 60 isoflavones) or /PYR placebo Gallagher JC 65 PM et al. [45] (longitudinal) 9 months SP with 96mg No effect on women isoflavones vs BMD after 15 Mean age 55 52mg vs < 4mg months isoflavones Vupadhyayula 203 PM et al. [53] women 24 months (longitudinal) 25g SP/ 90mg No change in isoflavones vs hip/LS bone 25g of SP/0 mg loss without isoflavones or 25g of milk protein/ casein and whey Kreijkamp- 202 PM Kaspers et al. [49] 12 months 25.6 g of soy No effect on women aged protein/ 99 mg of BMD 60-75 isoflavones (longitudinal) vs placebo Arjmandi BH 71 PM et al. [41] women 3 months 40g SP or MBP Urine DPD decreased more (2003) and IGF1 (longitudinal) increased more for SP vs MP Arjmandi BH 87 PM et al. [42] women (2005) (longitudinal) 12 months 25g SP / 60mg Soy improves isoflavone vs IGFBP3 cf placebo placebo, no other change in BMD or DPD Evans EM et 61PM al. [44] women 9 months (longitudinal) 25.6g SPI/91.2mg No difference isoflavones vs in BMD. 25.6 g MPI SPI improved bALP and decreased Cterminal crosslinked telopeptides Potter SM et 66 PM al. [51] 6 months 40g SP with 1.39 Increased women mg isoflavones vs BMD and Mean age 40g SP with BMC in higher 60.8 2.25mg?? isoflavone isoflavones vs group in LS 40g casein/ nonfat only. milk protein Newton KM et 145 subjects 12 months SP with 83 mg No difference al. [50] (22 women, isoflavones(+ISO) for BMD in 123 men) vs SP with 3mg men nor hip in Aged 50-80 isoflavone (−ISO) women. +ISO women had modest benefit in spine. Khalil DA et Men al. [48] 59.2 +/- 17.6 3 months 40g SP vs MP SP significantly y increased IGF1 cf MP. No effect on other bone markers SPI- Soy Protein Isolate, MBP – Milk Based Protein, MPI- Milk Protein Isolate, PMPost-Menopausal