- Email: [email protected]
Influence of organic salts of potassium on bone health: Possible mechanisms of action for the role of fruit and vegetables
International Congress Series 1297 (2007) 268 – 281
www.ics-elsevier.com
Influence of organic salts of potassium on bone health: Possible mechanisms of action for the role of fruit and vegetables H.M. Macdonald ⁎ Osteoporosis Research Unit/Department of Medicine and Therapeutics, Health Science Building, University of Aberdeen, AB25 2ZD, UK
Abstract. There is evidence that fruit and vegetables may be beneficial for bone health. Proteincontaining foods such as meats, fish and cereals are acid-generating because of the sulphur and phosphorous they contain. It has been suggested that fruit and vegetables may be important for bone health because of the alkaline salts they provide (for example potassium citrate or potassium malate), which can balance the acidity produced by eating a Westernized diet. There have been a number of intervention studies investigating the effect of potassium bicarbonate or potassium citrate on markers of bone turnover. This chapter reviews the evidence from these studies and concludes that although shortterm studies show that alkaline salts of potassium reduce calcium excretion and affect bone turnover, the body may adapt so that the benefit on bone metabolism is not continued long-term. Fruit and vegetable consumption may be advantageous to bone health, not because of balancing dietary acidity but because of the nutrients they contain (e.g. certain minerals, vitamins, or bioactive compounds such as flavonoids and phytoestrogens). © 2006 Elsevier B.V. All rights reserved. Keywords: Acid–base balance; Fruit and vegetables Bone health
1. Introduction A conundrum that hampers the setting of nutritional guidelines to help prevent osteoporosis is that countries with highest fracture incidence are those with the highest dietary calcium intakes. Sometimes referred to as the ‘Calcium paradox’, it may partly ⁎ Tel.: +44 1224 555515; fax: +44 1224 555474. E-mail address: [email protected]. 0531-5131/ © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2006.08.019
H.M. Macdonald / International Congress Series 1297 (2007) 268–281
269
be explained by genetic differences; the aging population in industrial countries (although there is evidence that fracture rates are still increasing after age has been taken into account [1]); and other factors such as sunlight exposure and diet (Table 1). This has led the Food and Agriculture Organization of the United Nations and the World Health Organization to suggest that dietary calcium recommendations should differ between countries [2]. They suggested that countries with high fracture incidence should have a minimum daily intake of 400–500 mg calcium. Countries with lower fracture incidence should consider other factors, for example sunlight exposure (which would influence vitamin D status) or dietary salt intake, before increasing calcium recommendations. One factor in particular that is continually under scrutiny with regard to chronic diseases in the Western world is our diet: being high in fat, energy, salt and protein; and low in fruit and vegetables. When comparing different countries, it can be seen that those with the highest dietary calcium intakes consume the most protein and conversely, protein intake is low in countries that have low levels of calcium in the diet (Table 2). The high protein diet is relevant to bone health, because it is known that high protein intakes, which generate excess acidity, are associated with increased calcium excretion. Table 1 Possible reasons for the ‘Calcium paradox’ Factor
Reason in relation to fracture incidence
Genetics and ethnicity
Family history is a risk factor for osteoporosis. Black Americans have more bone mass compared to Caucasians Age demographics are changing with greater representation from older age groups: an older population is more likely to suffer osteoporotic fractures Under-estimation in regions where calcium intake is low (e.g. consumption of animal bones is often overlooked) Medication to help reduce heart disease and stroke have resulted in ‘unhealthy’ people living longer and they may be more prone to fracture Westernized diet is high in fat, protein and salt but low in fruit and vegetables Sedentary lifestyles can result in bone loss, in spite of a sedentary lifestyle leading to weight gain, which in itself can increase bone mineral density Sunlight is the main source of vitamin D for most individuals. At latitudes away from the equator, there is less exposure to the wavelengths required for the cutaneous synthesis of vitamin D so that for many months of the year, vitamin D requirements are met from the store made during the previous season's exposure
Aging population
Calcium intakes
Medication available
Diet Exercise
Sunlight exposure
270
H.M. Macdonald / International Congress Series 1297 (2007) 268–281
Table 2 Daily calcium and protein intakes per person from different parts of the world Region
USA and Canada Europe USSR Australia/New Zealand Africa Latin America Middle East Far East
Calcium (mg/day)
Protein (g/day)
Total
Animal
Vegetable
Total
Animal
Vegetable
1031 896 751 836 368 476 484 305
717 684 567 603 108 305 223 109
314 212 184 233 260 171 261 196
109 102 106 98 54 67 79 58
72 60 56 66 11 29 18 11
37 42 50 32 43 38 61 47
Adapted from FAO {Food and Agriculture Organization of the United Nations 1991 #24210} [2].
This was demonstrated in a study of women aged 38 to 62 years in which daily intakes of calcium (600 mg), magnesium (300 mg) and phosphorous (900 mg) were kept constant. When daily protein intake was increased from 40 g to 100 g, urinary calcium excretion increased [3]. This study also showed that sodium bicarbonate (alkali generating) could reduce the excess calcium excretion. In contrast, a more recent study involving healthy postmenopausal women, which used meat rather than purified proteins showed no change in calcium excretion at daily intakes of 600 mg when comparing a low meat diet (12% of dietary energy) with a high meat diet (20% of dietary energy) [4]. Although net acid excretion was higher to begin with on the high meat diet, this was found to decrease with time. 2. What is acid–base balance? 2.1. General principles Acid–base balance is a well-recognised critical aspect of human health. In order to function properly, the pH of arterial blood must be kept to pH 7.4 and the body is equipped with homeostatic mechanisms to keep it strictly within the limits of 7.35 and 7.45. As the Western diet can generate up to 50 to 100 mEq acid/day, it is essential that we have the means for removing excess acid. Excess hydrogen ions are initially buffered. Buffers include haemoglobin in the red blood cells, and proteins and phosphate buffer systems in the plasma and most importantly, bicarbonate. The unique property of the bicarbonate buffer system is that it is driven in the direction of buffering because carbon dioxide can be removed via the lungs. In the case of respiratory disease this may not always be achieved effectively and it may be a reason for the increased fracture risk associated with chronic pulmonary disease. For non-carbonic or fixed acids, excess acid cannot be removed this way. Although the hydrogen ions may have been buffered allowing pH levels to normalize, the hydrogen ions have not disappeared and must ultimately be removed from the body. The only way this can be done is via the kidneys (Fig. 1). Another important organ thought to be involved in acid–base homeostasis is bone, as discussed in the following section.
H.M. Macdonald / International Congress Series 1297 (2007) 268–281
271
2.2. Bone mineral as a reservoir of buffer Early studies performed in dogs by Jaffe and co-workers showed that bone could be demineralised by giving a continuous high acid load using ammonium chloride ingestion [5]. Many fractures and deformities were observed but the authors noted that the changes were ‘less striking’ if calcium intake was adequate compared to the experiments where calcium intake was low. In the 1960s, the research groups of Wilson, Lemann and Barzel separately carried out studies in obese women, patients with kidney disease, and rats [6–8] that provided further evidence that bone was being used to help buffer excess acidity. Based on these findings a hypothesis was suggested in 1968 by Wachman and Bernstein that osteoporosis may be caused by the lifelong use of bone to buffer acid [9]. The skeleton has been described as a giant ion-exchange column which is loaded with alkali buffer [10]. Barzel explained that the carbonate and citrate, which are stored in a thin hydration cell surrounding the surface of the bone, are released for buffering excess hydrogen ions. In the case of chronic acid overload, the bone itself is broken down to release alkali [11]. Although some disagree with Barzel's interpretation that there is an outer hydration shell that surrounds bone since bone cells are found throughout bone including the surface, there is a general consensus that bone can be used to a greater or lesser extent to keep circulating pH constant. 2.3. Acidosis and treatment For the treatment of metabolic acidosis associated with kidney disease, alkaline salts (such as sodium or potassium bicarbonate) are recommended to balance the acidity that
Fig. 1. Major organs involved in the removal of excess acid from the body.
272
H.M. Macdonald / International Congress Series 1297 (2007) 268–281
ensues because the kidney is incapable of (i) removing all the acid that is generated by the body (Type 1 or distal renal acid tubular acidosis, RTA) or (ii) it is unable to resorb bicarbonate (Type 2 RTA). Studies have been performed in individuals with severe kidney disease to determine whether correcting metabolic acidosis had any influence on calcium balance. Although overall net calcium retention was not achieved, calcium losses were reduced with sodium bicarbonate treatment in patients with chronic end-stage renal disease [7]. Also, improvements in bone histology and bone mineral density were reported as a result of potassium citrate therapy in patients with type 1 RTA [12], helping to support the concept that bone is used as a buffer source when kidneys are not functioning properly. 2.4. Healthy individuals Lemann carried out a number of studies in healthy men in the late 1980s and early 1990s [13,14]. He summarized that increasing dietary potassium reduces calcium excretion and makes calcium balance more positive [15], However he pointed out that because potassium also causes phosphate retention by the kidneys, this would inhibit renal synthesis of calcitriol (the active form of vitamin D) and inhibit intestinal calcium absorption. Over time this might result in dampening the continuously positive calcium balance. Although he concluded that diets containing more potassium would help protect skeletal mass, the long-term effects may not be as important as the short-term studies would perhaps predict. There are currently two schools of thought with regard to acid base balance and bone health for the general population: (1) it is irrelevant if kidneys are functioning properly [16]: (2) it is important because of increasing dietary acidity and the slow decline in kidney function with age, leading to an accumulation of acid albeit within normal limits [17]. This can be referred to as latent acidosis. It has been determined that our Westernised diet is far more acid-generating compared to preagricultural and Neolithic man [18–20]. 3. Dietary factors and latent acidosis Even in the early 1900s there was much interest in how much acid could be generated by different foodstuffs. Protein-containing foods produced acid ash whereas fruits and vegetables produced alkaline ash. Although later work has highlighted some discrepancies, most of the foods would be in the correct category using today's criteria [21]. Remer and Manz calculated the acid-generating potential of different foods using a formula based on potassium, calcium and sodium (base-forming), protein, phosphate and chloride (acid-forming), taking into account the absorption of these nutrients [22]. They also included an allowance for non-carbonic acids based on body size. Using this formula they found that the potential renal acid load (PRAL) of adults varied from a low of 3.7 mEq/day for a lacto-ovo-vegetarian diet to 62.2 and 102.2 mEq/day for two moderate protein diets and 117 mEq/day for a high protein diet [23]. This reflected the urinary net acid excretion measured after consuming the different diets. Since sodium chloride provides these two ions in equimolar quantities they suggest that for most diets, these two ions can be removed from the formula.
H.M. Macdonald / International Congress Series 1297 (2007) 268–281
273
Frassetto found that a formula based only on the ratio of dietary protein to potassium was a good approximation of net endogenous acid production (NEAP) [24]. This has been applied to a number of population studies as detailed below. 4. Evidence for diet influencing bone health through acid–base balance 4.1. Population-based studies There is evidence from both sides of the Atlantic that nutrients associated with eating a diet rich in fruit and vegetables are associated with increased markers of bone health (bone mineral density and bone markers) in late pre-menopausal women [25,26] and in older women and men [27]. Also for men only, alkali-producing nutrients were found to be associated with reduced bone loss [27]. The Framingham group also reported that dietary protein was essential for bone health [28] and that although alkali-inducing components of the diet were beneficial, the role of protein was more complex depending on other components in the diet [29]. In our Aberdeen cohort, we found that although the acid-generating potential of the diet was associated with increasing levels of bone resorption, women in the lowest quartile of protein intake also had higher levels of bone resorption markers, although this association was not significant after adjustment for confounders (Fig. 2) [30]. Alkali-generating diets may be associated with denser bones in children and adolescents although the evidence is not definitive with some studies reporting a beneficial effect [31], some observing that the association is there in girls but not boys [32] and other investigators pointing out that a diet low in acid-generating potential is rich in other nutrients that are beneficial for bone health [33]. 4.2. Dietary interventions The DASH (dietary approaches to stop hypertension) diet involves eating increased amounts of fruit and vegetables (around 13 portions a day) and low fat dairy products [34]. An intervention trial showed that the DASH diet was successful in reducing blood pressure and it was thought that it could also be effective in preserving bone, since it would produce alkaline metabolites. A study of 186 adults aged between 23 and 76 years, found that compared to the control diet, the DASH diet caused a reduction in bone turnover with the bone resorption marker CTX decreasing by 16–18% and the bone formation marker osteocalcin decreasing by 8–11% [35]. There was no change in calcium excretion. Buclin et al. showed in a study of 8 men that compared to an alkaline-producing diet over 4 days, an acidic diet over the same duration caused greater calcium excretion (74% increase) and bone resorption as measured by 19% increase in CTX [36]. 4.3. Cellular studies As detailed by Dr Arnett in a previous chapter in this book, a reduction in pH in the vicinity of bone cells is critical for osteoclast function and bone breakdown. The rapid
274
H.M. Macdonald / International Congress Series 1297 (2007) 268–281
increase in bone resorption occurs in the region of pH 7.1, which is lower than the circulating pH of 7.4. Although interstitial pH has not been measured directly in bone, it was suggested that it could be in this region since skin interstitial pH has been found to be as low as 7.1 [37]. Also, probably more convincing is that although the mean interstitial pH of resting muscle was measured as 7.38, the pH was found to gradually decrease with exercise as follows: with 30 W exercise pH decreased to 7.27, with 50 W exercise the pH was 7.16, and with 50 W exercise the pH was 7.04 [38]. 4.4. Animal studies It was reported in Nature in 1999 that feeding rats extracts of vegetables and herbs reduced bone resorption whereas extracts of milk and meat showed no change [39]. The levels of herb extracts used were compatible with human diets. Using sheep as a model which is sensitive to dietary induced metabolic acidosis [40] bone mineral density was shown to decrease with increased dietary acidity [41]. There appeared to be an additive effect of ovariectomy and dietary acidity on bone loss. However, later experiments by Mulbauer suggested that the benefits of the vegetable extracts in rats were above that
Fig. 2. Mean (±2 S.E.M.) for bone resorption marker-free deoxypyridinoline expressed relative to creatinine (fDPD/Cr) with increasing quartile (Q) of energy-adjusted potassium intake, estimates of dietary acidity [net endogenous acid production (NEAP) and potential renal acid load (PRAL)] and energy adjusted protein intake, for all women who had bone resorption markers measured (n = 2929). Means have been adjusted for age, weight, height, socio-economic status, physical activity level, and menopausal status/HRT use ANCOVA: potassium P b 0.01, NEAP P b 0.01, PRAL, P b 0.01, Protein P = 0.02. Quartiles labelled ‘a’ were significantly different from quartiles labelled ‘b’ within each group of quartiles (P b 0.05 ANOVA with Tukey's test). Reproduced with permission.
H.M. Macdonald / International Congress Series 1297 (2007) 268–281
275
caused by the provision of alkali from the diet, suggesting that there were other components in the diet that may influence bone turnover [42]. 5. Intervention studies in healthy individuals using organic salts of potassium 5.1. Evidence from recent studies The early studies using potassium bicarbonate have already been cited in Sections 2.2 and 2.4. A number of new studies have been carried out since the mid 1990s using organic salts of potassium over different periods of time. Although potassium citrate provides slightly less potassium and alkali compared to potassium bicarbonate (Table 3), the former is thought to produce less severe gastrointestinal side effects, and tends to have been favoured in the most recent studies. In this section a few studies are briefly reviewed in order of study duration. Whiting et al. carried out acute (3 h) studies using 10 women (aged 19–31 years) and 10 men (aged 20–28 years). They compared calcium excretion in diets that contained moderate levels of protein (17 g) and high levels of protein (50 g). They also added extra phosphate to the moderate protein diet to bring the phosphate level up to the same amount found in the high protein diet. They found no evidence of hypercalciuria in the high protein diet compared to moderate protein diet, but more calcium was excreted with the high protein diet when compared to the moderate protein diet with additional phosphate. By giving potassium bicarbonate (70 mmol or 50 mmol) the level of calcium excreted in the high protein diet was reduced [43]. These studies show that body quickly responds to changes in dietary acidity. There are 3 intervention studies lasting 2 to 4 weeks. The first of these, using potassium bicarbonate and involving 18 postmenopausal women, was carried out in 1994 [44]. The women first underwent a 2-week period on a control diet when the baseline measurements were made. Compared to the baseline diet the potassium bicarbonate intervention (60–120 mmol/day/60 kg body weight) resulted in improved calcium balance. In addition, the bone formation marker osteocalcin had increased from 5.5 ± 2.8 to 6.1 ± 2.8 ng/ml (P = 0.001) and hydroxyproline (on older marker of bone resorption) had decreased 220 ± 94 to 204 ± 82 μmol. Wood made some critical observations regarding the study: calcium balance was negative in the control period and although it became less negative, with less calcium excreted in the urine, the women were still losing 124 mg Ca/ 60 kg body weight/day. He also commented that the protein intake was high (96 g) and that although dietary calcium intake was low (652 mg/day) the apparent absorption was only 7%, which is unusually low [45].
Table 3 Comparison of potassium citrate and potassium bicarbonate
Formula Molecular weight (g) Potassium content
Potassium bicarbonate
(tri)Potassium citrate
KHCO3 100 1 g gives 0.39 g K
K3C6H5O7·H2O 324 1 g gives 0.36 g K
276
H.M. Macdonald / International Congress Series 1297 (2007) 268–281
In 2005, Sakhaee et al. investigated 18 women in 2 weeks phases with washout periods of 2 weeks in between [46]. For the intervention phases, potassium citrate (40 mmol/day elemental potassium), calcium citrate (20 mmol Ca/day), a combined treatment, or placebo were given. They noted that potassium citrate reduced urinary calcium excretion and increased urinary pH, as expected. With regard to bone markers the potassium citrate intervention reduced serum CTX but there was no change in urinary NTX. The calcium citrate and combined treatments also showed benefits on bone turnover. Sellmeyer et al. examined the effects of potassium citrate on higher levels of calcium excretion induced by a high salt diet [47]. In total, 52 postmenopausal women were first given a low salt (87 mmol/day) for 3 weeks, then half were subjected to a high salt diet (225 mmol/day) with potassium citrate (90 mmol) and the other half were given a high salt diet (225 mmol/day) and placebo. They found that urinary calcium increased for the high salt and placebo group but maintained at the same level for the high salt and potassium citrate group. Although the bone resorption marker NTX had increased for the high salt and placebo group there was no change in bone resorption for the high salt and potassium citrate group. As expected, net acid excretion had decreased in the potassium citrate-supplemented group. The 3-month potassium citrate intervention carried out by Marangella in 2004 initially recruited 30 women for the intervention and 22 women completed the study [48]. Potassium citrate was given at a daily dose of 1 mEq/kg body weight (between 37 and 74 mmol potassium or base a day). The study was not placebo controlled. Instead, measurements from the intervention were then compared to those from 24 age-matched controls. Compared to the untreated group, the treated group showed an increase in potassium and citrate excretion, and a decrease in net endogenous acid excretion. Although there was a significant decrease in the bone resorption marker DPD/Cr (deoxypyrdinoline/creatinine) for the treated group from 9.08 to 7.01 nmol/mmol P b 0.01 and a non significant change for the untreated group from 6.3 to 7.3 nmol/mmol, it can be seen that the treated group had higher bone resorption at the start. Until the Aberdeen study described in the next section, the longest study examining potassium citrate and bone markers is that of 1-year duration involving 161 postmenopausal women. It was presented at the American Society of Bone and Mineral Research in 2005 but it has not yet been published in full [49]. Jehle et al. found that daily doses amounting to 30 mM potassium chloride caused lumbar spine bone loss (− 1%) but 10 mM tri-potassium citrate resulted in an increase in BMD of + 0.9% after 1 year. Although compared to potassium chloride, potassium citrate caused a slight gain in total hip BMD, there was a slight loss in femoral neck BMD. The bone marker data were mixed. The final study described in this section was of 3 years duration but did not include any bone data. Frassetto et al. calculated that the reduced excretion of calcium seen with longterm potassium citrate treatment could result in an overall gain in bone of 1.7% a year [50]. However, Heaney pointed out that at similar levels of potassium intake, his studies showed that although calcium excretion was reduced, there was a reduction in the amount of calcium that was absorbed, indicating that overall there would be no net gain in calcium [51].
H.M. Macdonald / International Congress Series 1297 (2007) 268–281
277
5.2. Aberdeen potassium citrate, fruit and vegetable intervention study In a 2-year double-blind randomised placebo-controlled trial, two doses of potassium citrate (high dose, 55.6 mmol/day as potassium; low dose, 18.5 mmol/day as potassium) were compared with placebo. Initially 65 postmenopausal women were recruited into each group. For the intention to treat analysis, the numbers in each group were 56, 54 and 55, respectively, for the high dose, low dose and placebo; and for those who continued the treatment the final numbers were 43, 48 and 49, respectively. There was no difference in markers of bone turnover between the different treatment groups, although calcium excretion had decreased for the high dose potassium citrate group at 3 months and 6 months (Macdonald et al., submitted). The study groups were well-matched, including key genotypes that could influence bone metabolism such as VDR polymorphism and Apolipoprotein E polymorphism that has been shown to be associated with increased fracture risk [52,53]. 5.3. Summary of recent studies using organic salts of potassium A summary of recent studies using organic salts of potassium is given in Table 4. It can be seen that although there appear to be changes in bone resorption, the findings are not consistent. It has been suggested that this could be because of short study durations and insufficient washout times [54]. 5.4. Other reasons for the benefits of fruit and vegetables Fruit and vegetables contain a number of other nutrients that may influence bone health (Table 5). For example, vitamin C is involved in the cross-linking of collagen, which is the major structural protein of bone. Vitamin K is needed to modify osteocalcin a Table 4 Summary of recent intervention studies using organic salts of potassium Study (n in each group)
Dose K (mmol/ day)
Study duration
Urinary Bone Bone Comment calcium formation resorption
Whiting et al. [43] (10) Sebastian et al. [44] (18)
50/70 60–120
3h 18 day
Down Down⁎
– Up
– Down
Sakhaee et al. [46] (18) Sellmeyer et al. [47] (26)
40 90
2 weeks 4 weeks
Down No⁎
No No⁎
Down⁎ No
Marangella et al. [48] (22)
37–74
3 months –
–
Down⁎
Jehle et al. [49] (80) 30 Macdonald et al., submitted 56/18.5/0 for publication (56/54/55)
1 years 2 years
Down ⁎Down
Unclear No
Down⁎ No
Frassetto et al. [50] (35/63/ 31 and 72 placebo)
3 years
Down
–
–
30/60/90
No bone data ⁎ Ca balance still negative ⁎ For CTX but not NTX ⁎ With NaCl. Up with NaCl and placebo ⁎ High baseline DPD for treated group ⁎ 6 months only ⁎ For high-dose only ≤6 months, NS other groups No bone data
278
H.M. Macdonald / International Congress Series 1297 (2007) 268–281
Table 5 Nutrients in fruit and vegetables that could influence bone health Vitamins Vitamin C Vitamin K Folate (B vitamin) Carotene Minerals Calcium Magnesium Boron Silicon Anti-oxidants Vitamin Ca Folate Flavonoids Other nutrients Flavonoids Phytoestrogens a
But it can act as pro-oxidant at high concentrations.
protein associated with osteoblasts, and is required for mineralization of the bones. Folate is known to reduce circulating homocysteine levels, which has been shown to be a risk factor for fractures in the elderly [55,56] although other B vitamins may be involved [57,58]. Phytoestrogens, which are found in some vegetables can act as weak agonists and antagonists of oestrogen but further long-term studies are required before we know how important these are for bone health in the Western world [59]. There is evidence that flavonoids may influence bone health [60]. Fruit and vegetables contain a number of compounds that act as antioxidants. These could mop up free radicals that may be promoting bone breakdown. Free radicals have been shown to be involved in osteoclastogenesis and bone resorption, with reactive oxygen species mediating RANK signalling in osteoclasts [61] and oxidative stress inhibiting osteoblast differentiation [62]. In a population study, a urinary biomarker of oxidative stress was found to be negatively associated with BMD and QUS in a study of 48 females and 53 males [63]. A recent publication found that higher fruit intakes were associated with size adjusted bone mineral content in children and older women but previously the investigators had not detected any associations with renal net acid excretion estimated from the diet [64]. They suggest vitamin C and fruit-specific antioxidants may play a role in the positive effects of fruit and vegetables on bone mineral status. 6. Conclusions Organic salts of potassium appear to decrease calcium excretion and lower urinary pH. There appears to be evidence of reduced bone turnover in the short term although this is not unequivocal. It appears that although theoretically, less calcium could be lost in the urine, the negative calcium balance does not appear to continue in the long term. It is likely that more calcium is absorbed in the gut to compensate for the greater urinary losses.
H.M. Macdonald / International Congress Series 1297 (2007) 268–281
279
The role of bone in acid base balance appears to have detrimental effects only in those whose kidney function is severely impaired but for the normal population it does not appear to be a risk factor in osteoporosis. Fruit and vegetable intake may still be important for maintaining healthy bones. Further work is required to establish what nutrients are important and whether some fruit and vegetable groups are more important than others. References [1] I.M. Giversen, Time trends of age-adjusted incidence rates of first hip fractures: a register-based study among older people in Viborg County, Denmark, 1987–1997, Osteoporos. Int. 17 (2006) 552–564. [2] Food and Agriculture Organization of the United Nations and the World Health Organization, Human Vitamin and Mineral Requirements. Report of a Joint FAO/WHO Expert Consultation, WHO/FAO, Bangkok, Thailand, 2002. [3] J. Lutz, Calcium balance and acid–base status of women as affected by increased protein intake and by sodium bicarbonate ingestion, Am. J. Clin. Nutr. 39 (1984) 281–288. [4] Z.K. Roughead, et al., Controlled high meat diets do not affect calcium retention or indices of bone status in healthy postmenopausal women, J. Nutr. 133 (2003) 1020–1026. [5] H. Jaffe, A. Bodansky, J.P. Chandler, Ammonium chloride decalcification, as modified by calcium intake: the relation between generalized osteoporosis and ostitis fibrosa, J. Exp. Med. 56 (1932) 823–834. [6] M.M. Reidenberg, et al., The response of bone to metabolic acidosis in man, Metabolism 15 (1966) 236–241. [7] J.R. Litzow, J. Lemann Jr., E.J. Lennon, The effect of treatment of acidosis on calcium balance in patients with chronic azotemic renal disease, J. Clin. Invest. 46 (1967) 280–286. [8] U.S. Barzel, J. Jowsey, The effects of chronic acid and alkali administration on bone turnover in adult rats, Clin. Sci. 36 (1969) 517–524. [9] A. Wachman, D.S. Bernstein, Diet and osteoporosis, Lancet i (1968) 958–959. [10] J. Green, C.R. Kleeman, Role of bone in regulation of systemic acid–base balance, Kidney Int. 39 (1991) 9–26. [11] U.S. Barzel, The skeleton as an ion-exchange system: implications for the role of acid–base imbalance in the genesis of osteoporosis, J. Bone Miner. Res. 10 (1995) 1431–1436. [12] S. Domrongkitchaiporn, et al., Bone histology and bone mineral density after correction of acidosis in distal renal tubular acidosis, Kidney Int. 62 (2002) 2160–2166. [13] J. Lemann, R.W. Gray, J.A. Pleuss, Potassium bicarbonate, but not sodium bicarbonate, reduces urinary calcium excretion and improves calcium balance in healthy men, Kidney Int. 35 (1989) 688–695. [14] J. Lemann, et al., Potassium administration increases and potassium deprivation reduces urinary calcium excretion in healthy adults, Kidney Int. 39 (1991) 973–983. [15] J. Lemann, J.A. Pleuss, R.W. Gray, Potassium causes calcium retention in healthy adults, J. Nutr. 123 (1993) 1623–1626. [16] M.S. Oh, Irrelevance of bone buffering to acid–base homeostasis in chronic metabolic acidosis, Nephron 59 (1991) 7–10. [17] R.C. Morris, et al., Expression of osteoporosis as determined by diet-disordered electrolyte and acid–base metabolism, in: P. Burckhardt, B. Dawson-Hughes, R.P. Heaney (Eds.), Nutritional Aspects of Osteoporosis. 4th International Symposium on Nutritional Aspects of Osteoporosis, Switzerland, 2000, Academic Press, 2001, pp. 357–378. [18] A. Sebastian, et al., Estimation of the net acid load of the diet of ancestral preagricultural Homo sapiens and their hominid ancestors, Am. J. Clin. Nutr. 76 (2002) 1308–1316. [19] L. Frassetto, et al., Diet, evolution and aging – the pathophysiologic effects of the post-agricultural inversion of the potassium-to-sodium and base-to-chloride ratios in the human diet, Eur. J. Nutr. 40 (2001) 200–213. [20] L. Cordain, et al., Plant–animal subsistence erations and macronutrient energy estimations in worldwide hunter–gatherer diets, Am. J. Clin. Nutr. 71 (2000) 682–692. [21] H.C. Sherman, A.O. Gettler, The balance of acid-forming and base-forming elements in foods, and its relation to ammonia metabolism, J. Biol. Chem. 11 (1912) 323–328.
280
H.M. Macdonald / International Congress Series 1297 (2007) 268–281
[22] T. Remer, F. Manz, Potential renal acid load of foods and its influence on urine pH, J. Am. Diet. Assoc. 95 (1995) 791–797. [23] T. Remer, F. Manz, Estimation of the renal net acid excretion by adults consuming diets containing variable amounts of protein, Am. J. Clin. Nutr. 59 (1994) 1356–1361. [24] L.A. Frassetto, et al., Estimation of net endogenous noncarbonic acid production in humans from diet potassium and protein contents, Am. J. Clin. Nutr. 68 (1998) 576–583. [25] S.A. New, et al., Nutritional influences on bone mineral density: a cross-sectional study in menopausal women, Am. J. Clin. Nutr. 65 (1997) 1831–1839. [26] H.M. Macdonald, et al., Low dietary potassium intakes and high dietary estimates of net endogenous acid production are associated with low bone mineral density in premenopausal women and increased markers of bone resorption in postmenopausal women, Am. J. Clin. Nutr. 81 (2005) 923–933. [27] K.L. Tucker, et al., Potassium, magnesium, and fruit and vegetable intakes are associated with greater bone mineral density in elderly men and women, Am. J. Clin. Nutr. 69 (1999) 727–736. [28] M.T. Hannan, et al., Effect of dietary protein on bone loss in elderly men and women: the Framingham Osteoporosis Study, J. Bone Miner. Res. 15 (2000) 2504–2512. [29] K.L. Tucker, M.T. Hannan, D.P. Kiel, The acid–base hypothesis: diet and bone in the Framingham Osteoporosis Study, Eur. J. Nutr. 40 (2001) 231–237. [30] H.M. Macdonald, et al., Low dietary potassium intakes and high dietary estimates of net endogenous acid production are associated with low bone mineral density in premenopausal women and increased markers of bone resorption in postmenopausal women, Am. J. Clin. Nutr. 81 (2005) 923–933. [31] C.P. McGartland, et al., Fruit and vegetable consumption and bone mineral density: the Northern Ireland Young Hearts Project, Am. J. Clin. Nutr. 80 (2004) 1019–1023. [32] S.J. Whiting, et al., Factors that affect bone mineral accrual in the adolescent growth spurt, J. Nutr. 134 (2004) 696S–700S. [33] C.J. Prynne, et al., Dietary acid–base balance and intake of bone-related nutrients in Cambridge teenagers, Eur. J. Clin. Nutr. 58 (2004) 1462–1471. [34] L.J. Appel, et al., A clinical trial of the effects of dietary patterns on blood pressure, New Engl. J. Med. 336 (1997) 1117–1124. [35] P.H. Lin, et al., The DASH diet and sodium reduction improve markers of bone turnover and calcium metabolism in adults, J. Nutr. 133 (2003) 3130–3136. [36] T. Buclin, et al., Diet acids and alkalis influence calcium retention in bone, Osteoporos. Int. 12 (2001) 493–499. [37] G.R. Martin, R.K. Jain, Noninvasive measurement of interstitial pH profiles in normal and neoplastic tissue using fluorescence ratio imaging microscopy, Cancer Res. 54 (1994) 5670–5674. [38] D. Street, J. Bangsbo, C. Juel, Interstitial pH in human skeletal muscle during and after dynamic graded exercise, J. Physiol. 537 (2001) 993–998. [39] R.C. Muhlbauer, F. Li, Effect of vegetables on bone metabolism, Nature 401 (1999) 343–344. [40] J.M. Macleay, D.L. Wheeler, A.S. Turner, The ovine model for the study of dietary acid base, estrogen depletion and bone health, in: P. Burckhardt, B. Dawson-Hughes, R.P. Heaney (Eds.), Nutritional Aspects of Osteoporosis. 4th International Symposium on Nutritional Aspects of Osteoporosis, Switzerland, 2000, Academic Press, 2001, pp. 331–348. [41] J.M. MacLeay, et al., Dietary-induced metabolic acidosis decreases bone mineral density in mature ovariectomized ewes, Calcif. Tissue Int. 75 (2004) 431–437. [42] R.C. Muhlbauer, A. Lozano, A. Reinli, Onion and a mixture of vegetables, salads, and herbs affect bone resorption in the rat by a mechanism independent of their base excess, J. Bone Miner. Res. 17 (2002) 1230–1236. [43] S.J. Whiting, D.J. Anderson, S.J. Weeks, Calciuric effects of protein and potassium bicarbonate but not of sodium chloride or phosphate can be detected acutely in adult women and men, Am. J. Clin. Nutr. 65 (1997) 1645–1672. [44] A. Sebastian, et al., Improved mineral balance and skeletal metabolism in postmenopausal women treated with potassium bicarbonate, New Engl. J. Med. 330 (1994) 1776–1781. [45] R.J. Wood, Potassium bicarbonate supplementation and calcium metabolism in postmenopausal women: are we barking up the wrong tree? Nutr. Rev. 52 (1919) 278–280. [46] K. Sakhaee, et al., Contrasting effects of various potassium salts on renal citrate excretion, J. Clin. Endocrinol. Metab. 72 (1991) 396–400.
H.M. Macdonald / International Congress Series 1297 (2007) 268–281
281
[47] D.E. Sellmeyer, M. Schloetter, A. Sebastian, Potassium citrate prevents increased urine calcium excretion and bone resorption induced by a high sodium chloride diet, J. Clin. Endocrinol. Metab. 87 (2002) 2008–2012. [48] M. Marangella, et al., Effects of potassium citrate supplementation on bone metabolism, Calcif. Tissue Int. 74 (2004) 330–335. [49] S. Jehle, et al., Neutralization of the acidogenic Western diet with K citrate increases DXA BMD in postmenopausal osteopenic women: results of DBRCT, J. Bone Miner. Res. 20 (2005) s289. [50] L. Frassetto, R.C. Morris Jr., A. Sebastian, Long-term persistence of the urine calcium-lowering effect of potassium bicarbonate in postmenopausal women, J. Clin. Endocrinol. Metab. 90 (2005) 831–834. [51] K. Rafferty, K.M. Davies, R.P. Heaney, Potassium intake and the calcium economy, J. Am. Coll. Nutr. 24 (2005) 99–106. [52] M. Kohlmeier, et al., Bone fracture history and prospective bone fracture risk of hemodialysis patients are related to apolipoprotein E genotype, Calcif. Tissue Int. 62 (1998) 278–281. [53] J.A. Cauley, et al., Apolipoprotein E polymorphism: a new genetic marker of hip fracture risk – the study of osteoporotic fractures, J. Bone Miner. Res. 14 (1999) 1175–1181. [54] Z.K. Roughead, Is the interaction between dietary protein and calcium destructive or constructive for bone? J. Nutr. 133 (2003) 866S–869S. [55] R.R. McLean, et al., Homocysteine as a predictive factor for hip fracture in older persons, N. Engl. J. Med. 350 (2004) 2042–2049. [56] J.B. van Meurs, et al., Homocysteine levels and the risk of osteoporotic fracture, N. Engl. J. Med. 350 (2004) 2033–2041. [57] H.M. Macdonald, D.M. Reid, Vitamin B-complex, methylenetetrahydrofolate reductase polymorphism and bone: potential for gene–nutrient interaction, in: P. Burckhardt, B. Dawson-Hughes, R.P. Heaney (Eds.), Nutritional Aspects of Osteoporosis. 4th International Symposium on Nutritional Aspects of Osteoporosis, Switzerland, 2000, Academic Press, 2001, pp. 127–138. [58] H.M. Macdonald, et al., Methylenetetrahydrofolate reductase polymorphism interacts with riboflavin intake to influence bone mineral density, Bone 35 (2004) 957–964. [59] A. Cassidy, et al., Critical review of health effects of soyabean phyto-oestrogens in post-menopausal women, Proc. Nutr. Soc. 65 (2006) 76–92. [60] H. Chiba, et al., Hesperidin, a citrus flavonoid, inhibits bone loss and decreases serum and hepatic lipids in ovariectomized mice, J. Nutr. 133 (2003) 1892–1897. [61] H. Ha, et al., Reactive oxygen species mediate RANK signaling in osteoclasts, Exp. Cell Res. 301 (2004) 119–127. [62] X.C. Bai, et al., Oxidative stress inhibits osteoblastic differentiation of bone cells by ERK and NF-kappaB, Biochem. Biophys. Res. Commun. 314 (2004) 197–207. [63] S. Basu, et al., Association between oxidative stress and bone mineral density, Biochem. Biophys. Res. Commun. 288 (2001) 275–279. [64] C.J. Prynne, et al., Fruit and vegetable intakes and bone mineral status: a cross-sectional study in 5 age and sex cohorts, Am. J. Clin. Nutr. 83 (2006) 1420–1428.
www.ics-elsevier.com
Influence of organic salts of potassium on bone health: Possible mechanisms of action for the role of fruit and vegetables H.M. Macdonald ⁎ Osteoporosis Research Unit/Department of Medicine and Therapeutics, Health Science Building, University of Aberdeen, AB25 2ZD, UK
Abstract. There is evidence that fruit and vegetables may be beneficial for bone health. Proteincontaining foods such as meats, fish and cereals are acid-generating because of the sulphur and phosphorous they contain. It has been suggested that fruit and vegetables may be important for bone health because of the alkaline salts they provide (for example potassium citrate or potassium malate), which can balance the acidity produced by eating a Westernized diet. There have been a number of intervention studies investigating the effect of potassium bicarbonate or potassium citrate on markers of bone turnover. This chapter reviews the evidence from these studies and concludes that although shortterm studies show that alkaline salts of potassium reduce calcium excretion and affect bone turnover, the body may adapt so that the benefit on bone metabolism is not continued long-term. Fruit and vegetable consumption may be advantageous to bone health, not because of balancing dietary acidity but because of the nutrients they contain (e.g. certain minerals, vitamins, or bioactive compounds such as flavonoids and phytoestrogens). © 2006 Elsevier B.V. All rights reserved. Keywords: Acid–base balance; Fruit and vegetables Bone health
1. Introduction A conundrum that hampers the setting of nutritional guidelines to help prevent osteoporosis is that countries with highest fracture incidence are those with the highest dietary calcium intakes. Sometimes referred to as the ‘Calcium paradox’, it may partly ⁎ Tel.: +44 1224 555515; fax: +44 1224 555474. E-mail address: [email protected]. 0531-5131/ © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2006.08.019
H.M. Macdonald / International Congress Series 1297 (2007) 268–281
269
be explained by genetic differences; the aging population in industrial countries (although there is evidence that fracture rates are still increasing after age has been taken into account [1]); and other factors such as sunlight exposure and diet (Table 1). This has led the Food and Agriculture Organization of the United Nations and the World Health Organization to suggest that dietary calcium recommendations should differ between countries [2]. They suggested that countries with high fracture incidence should have a minimum daily intake of 400–500 mg calcium. Countries with lower fracture incidence should consider other factors, for example sunlight exposure (which would influence vitamin D status) or dietary salt intake, before increasing calcium recommendations. One factor in particular that is continually under scrutiny with regard to chronic diseases in the Western world is our diet: being high in fat, energy, salt and protein; and low in fruit and vegetables. When comparing different countries, it can be seen that those with the highest dietary calcium intakes consume the most protein and conversely, protein intake is low in countries that have low levels of calcium in the diet (Table 2). The high protein diet is relevant to bone health, because it is known that high protein intakes, which generate excess acidity, are associated with increased calcium excretion. Table 1 Possible reasons for the ‘Calcium paradox’ Factor
Reason in relation to fracture incidence
Genetics and ethnicity
Family history is a risk factor for osteoporosis. Black Americans have more bone mass compared to Caucasians Age demographics are changing with greater representation from older age groups: an older population is more likely to suffer osteoporotic fractures Under-estimation in regions where calcium intake is low (e.g. consumption of animal bones is often overlooked) Medication to help reduce heart disease and stroke have resulted in ‘unhealthy’ people living longer and they may be more prone to fracture Westernized diet is high in fat, protein and salt but low in fruit and vegetables Sedentary lifestyles can result in bone loss, in spite of a sedentary lifestyle leading to weight gain, which in itself can increase bone mineral density Sunlight is the main source of vitamin D for most individuals. At latitudes away from the equator, there is less exposure to the wavelengths required for the cutaneous synthesis of vitamin D so that for many months of the year, vitamin D requirements are met from the store made during the previous season's exposure
Aging population
Calcium intakes
Medication available
Diet Exercise
Sunlight exposure
270
H.M. Macdonald / International Congress Series 1297 (2007) 268–281
Table 2 Daily calcium and protein intakes per person from different parts of the world Region
USA and Canada Europe USSR Australia/New Zealand Africa Latin America Middle East Far East
Calcium (mg/day)
Protein (g/day)
Total
Animal
Vegetable
Total
Animal
Vegetable
1031 896 751 836 368 476 484 305
717 684 567 603 108 305 223 109
314 212 184 233 260 171 261 196
109 102 106 98 54 67 79 58
72 60 56 66 11 29 18 11
37 42 50 32 43 38 61 47
Adapted from FAO {Food and Agriculture Organization of the United Nations 1991 #24210} [2].
This was demonstrated in a study of women aged 38 to 62 years in which daily intakes of calcium (600 mg), magnesium (300 mg) and phosphorous (900 mg) were kept constant. When daily protein intake was increased from 40 g to 100 g, urinary calcium excretion increased [3]. This study also showed that sodium bicarbonate (alkali generating) could reduce the excess calcium excretion. In contrast, a more recent study involving healthy postmenopausal women, which used meat rather than purified proteins showed no change in calcium excretion at daily intakes of 600 mg when comparing a low meat diet (12% of dietary energy) with a high meat diet (20% of dietary energy) [4]. Although net acid excretion was higher to begin with on the high meat diet, this was found to decrease with time. 2. What is acid–base balance? 2.1. General principles Acid–base balance is a well-recognised critical aspect of human health. In order to function properly, the pH of arterial blood must be kept to pH 7.4 and the body is equipped with homeostatic mechanisms to keep it strictly within the limits of 7.35 and 7.45. As the Western diet can generate up to 50 to 100 mEq acid/day, it is essential that we have the means for removing excess acid. Excess hydrogen ions are initially buffered. Buffers include haemoglobin in the red blood cells, and proteins and phosphate buffer systems in the plasma and most importantly, bicarbonate. The unique property of the bicarbonate buffer system is that it is driven in the direction of buffering because carbon dioxide can be removed via the lungs. In the case of respiratory disease this may not always be achieved effectively and it may be a reason for the increased fracture risk associated with chronic pulmonary disease. For non-carbonic or fixed acids, excess acid cannot be removed this way. Although the hydrogen ions may have been buffered allowing pH levels to normalize, the hydrogen ions have not disappeared and must ultimately be removed from the body. The only way this can be done is via the kidneys (Fig. 1). Another important organ thought to be involved in acid–base homeostasis is bone, as discussed in the following section.
H.M. Macdonald / International Congress Series 1297 (2007) 268–281
271
2.2. Bone mineral as a reservoir of buffer Early studies performed in dogs by Jaffe and co-workers showed that bone could be demineralised by giving a continuous high acid load using ammonium chloride ingestion [5]. Many fractures and deformities were observed but the authors noted that the changes were ‘less striking’ if calcium intake was adequate compared to the experiments where calcium intake was low. In the 1960s, the research groups of Wilson, Lemann and Barzel separately carried out studies in obese women, patients with kidney disease, and rats [6–8] that provided further evidence that bone was being used to help buffer excess acidity. Based on these findings a hypothesis was suggested in 1968 by Wachman and Bernstein that osteoporosis may be caused by the lifelong use of bone to buffer acid [9]. The skeleton has been described as a giant ion-exchange column which is loaded with alkali buffer [10]. Barzel explained that the carbonate and citrate, which are stored in a thin hydration cell surrounding the surface of the bone, are released for buffering excess hydrogen ions. In the case of chronic acid overload, the bone itself is broken down to release alkali [11]. Although some disagree with Barzel's interpretation that there is an outer hydration shell that surrounds bone since bone cells are found throughout bone including the surface, there is a general consensus that bone can be used to a greater or lesser extent to keep circulating pH constant. 2.3. Acidosis and treatment For the treatment of metabolic acidosis associated with kidney disease, alkaline salts (such as sodium or potassium bicarbonate) are recommended to balance the acidity that
Fig. 1. Major organs involved in the removal of excess acid from the body.
272
H.M. Macdonald / International Congress Series 1297 (2007) 268–281
ensues because the kidney is incapable of (i) removing all the acid that is generated by the body (Type 1 or distal renal acid tubular acidosis, RTA) or (ii) it is unable to resorb bicarbonate (Type 2 RTA). Studies have been performed in individuals with severe kidney disease to determine whether correcting metabolic acidosis had any influence on calcium balance. Although overall net calcium retention was not achieved, calcium losses were reduced with sodium bicarbonate treatment in patients with chronic end-stage renal disease [7]. Also, improvements in bone histology and bone mineral density were reported as a result of potassium citrate therapy in patients with type 1 RTA [12], helping to support the concept that bone is used as a buffer source when kidneys are not functioning properly. 2.4. Healthy individuals Lemann carried out a number of studies in healthy men in the late 1980s and early 1990s [13,14]. He summarized that increasing dietary potassium reduces calcium excretion and makes calcium balance more positive [15], However he pointed out that because potassium also causes phosphate retention by the kidneys, this would inhibit renal synthesis of calcitriol (the active form of vitamin D) and inhibit intestinal calcium absorption. Over time this might result in dampening the continuously positive calcium balance. Although he concluded that diets containing more potassium would help protect skeletal mass, the long-term effects may not be as important as the short-term studies would perhaps predict. There are currently two schools of thought with regard to acid base balance and bone health for the general population: (1) it is irrelevant if kidneys are functioning properly [16]: (2) it is important because of increasing dietary acidity and the slow decline in kidney function with age, leading to an accumulation of acid albeit within normal limits [17]. This can be referred to as latent acidosis. It has been determined that our Westernised diet is far more acid-generating compared to preagricultural and Neolithic man [18–20]. 3. Dietary factors and latent acidosis Even in the early 1900s there was much interest in how much acid could be generated by different foodstuffs. Protein-containing foods produced acid ash whereas fruits and vegetables produced alkaline ash. Although later work has highlighted some discrepancies, most of the foods would be in the correct category using today's criteria [21]. Remer and Manz calculated the acid-generating potential of different foods using a formula based on potassium, calcium and sodium (base-forming), protein, phosphate and chloride (acid-forming), taking into account the absorption of these nutrients [22]. They also included an allowance for non-carbonic acids based on body size. Using this formula they found that the potential renal acid load (PRAL) of adults varied from a low of 3.7 mEq/day for a lacto-ovo-vegetarian diet to 62.2 and 102.2 mEq/day for two moderate protein diets and 117 mEq/day for a high protein diet [23]. This reflected the urinary net acid excretion measured after consuming the different diets. Since sodium chloride provides these two ions in equimolar quantities they suggest that for most diets, these two ions can be removed from the formula.
H.M. Macdonald / International Congress Series 1297 (2007) 268–281
273
Frassetto found that a formula based only on the ratio of dietary protein to potassium was a good approximation of net endogenous acid production (NEAP) [24]. This has been applied to a number of population studies as detailed below. 4. Evidence for diet influencing bone health through acid–base balance 4.1. Population-based studies There is evidence from both sides of the Atlantic that nutrients associated with eating a diet rich in fruit and vegetables are associated with increased markers of bone health (bone mineral density and bone markers) in late pre-menopausal women [25,26] and in older women and men [27]. Also for men only, alkali-producing nutrients were found to be associated with reduced bone loss [27]. The Framingham group also reported that dietary protein was essential for bone health [28] and that although alkali-inducing components of the diet were beneficial, the role of protein was more complex depending on other components in the diet [29]. In our Aberdeen cohort, we found that although the acid-generating potential of the diet was associated with increasing levels of bone resorption, women in the lowest quartile of protein intake also had higher levels of bone resorption markers, although this association was not significant after adjustment for confounders (Fig. 2) [30]. Alkali-generating diets may be associated with denser bones in children and adolescents although the evidence is not definitive with some studies reporting a beneficial effect [31], some observing that the association is there in girls but not boys [32] and other investigators pointing out that a diet low in acid-generating potential is rich in other nutrients that are beneficial for bone health [33]. 4.2. Dietary interventions The DASH (dietary approaches to stop hypertension) diet involves eating increased amounts of fruit and vegetables (around 13 portions a day) and low fat dairy products [34]. An intervention trial showed that the DASH diet was successful in reducing blood pressure and it was thought that it could also be effective in preserving bone, since it would produce alkaline metabolites. A study of 186 adults aged between 23 and 76 years, found that compared to the control diet, the DASH diet caused a reduction in bone turnover with the bone resorption marker CTX decreasing by 16–18% and the bone formation marker osteocalcin decreasing by 8–11% [35]. There was no change in calcium excretion. Buclin et al. showed in a study of 8 men that compared to an alkaline-producing diet over 4 days, an acidic diet over the same duration caused greater calcium excretion (74% increase) and bone resorption as measured by 19% increase in CTX [36]. 4.3. Cellular studies As detailed by Dr Arnett in a previous chapter in this book, a reduction in pH in the vicinity of bone cells is critical for osteoclast function and bone breakdown. The rapid
274
H.M. Macdonald / International Congress Series 1297 (2007) 268–281
increase in bone resorption occurs in the region of pH 7.1, which is lower than the circulating pH of 7.4. Although interstitial pH has not been measured directly in bone, it was suggested that it could be in this region since skin interstitial pH has been found to be as low as 7.1 [37]. Also, probably more convincing is that although the mean interstitial pH of resting muscle was measured as 7.38, the pH was found to gradually decrease with exercise as follows: with 30 W exercise pH decreased to 7.27, with 50 W exercise the pH was 7.16, and with 50 W exercise the pH was 7.04 [38]. 4.4. Animal studies It was reported in Nature in 1999 that feeding rats extracts of vegetables and herbs reduced bone resorption whereas extracts of milk and meat showed no change [39]. The levels of herb extracts used were compatible with human diets. Using sheep as a model which is sensitive to dietary induced metabolic acidosis [40] bone mineral density was shown to decrease with increased dietary acidity [41]. There appeared to be an additive effect of ovariectomy and dietary acidity on bone loss. However, later experiments by Mulbauer suggested that the benefits of the vegetable extracts in rats were above that
Fig. 2. Mean (±2 S.E.M.) for bone resorption marker-free deoxypyridinoline expressed relative to creatinine (fDPD/Cr) with increasing quartile (Q) of energy-adjusted potassium intake, estimates of dietary acidity [net endogenous acid production (NEAP) and potential renal acid load (PRAL)] and energy adjusted protein intake, for all women who had bone resorption markers measured (n = 2929). Means have been adjusted for age, weight, height, socio-economic status, physical activity level, and menopausal status/HRT use ANCOVA: potassium P b 0.01, NEAP P b 0.01, PRAL, P b 0.01, Protein P = 0.02. Quartiles labelled ‘a’ were significantly different from quartiles labelled ‘b’ within each group of quartiles (P b 0.05 ANOVA with Tukey's test). Reproduced with permission.
H.M. Macdonald / International Congress Series 1297 (2007) 268–281
275
caused by the provision of alkali from the diet, suggesting that there were other components in the diet that may influence bone turnover [42]. 5. Intervention studies in healthy individuals using organic salts of potassium 5.1. Evidence from recent studies The early studies using potassium bicarbonate have already been cited in Sections 2.2 and 2.4. A number of new studies have been carried out since the mid 1990s using organic salts of potassium over different periods of time. Although potassium citrate provides slightly less potassium and alkali compared to potassium bicarbonate (Table 3), the former is thought to produce less severe gastrointestinal side effects, and tends to have been favoured in the most recent studies. In this section a few studies are briefly reviewed in order of study duration. Whiting et al. carried out acute (3 h) studies using 10 women (aged 19–31 years) and 10 men (aged 20–28 years). They compared calcium excretion in diets that contained moderate levels of protein (17 g) and high levels of protein (50 g). They also added extra phosphate to the moderate protein diet to bring the phosphate level up to the same amount found in the high protein diet. They found no evidence of hypercalciuria in the high protein diet compared to moderate protein diet, but more calcium was excreted with the high protein diet when compared to the moderate protein diet with additional phosphate. By giving potassium bicarbonate (70 mmol or 50 mmol) the level of calcium excreted in the high protein diet was reduced [43]. These studies show that body quickly responds to changes in dietary acidity. There are 3 intervention studies lasting 2 to 4 weeks. The first of these, using potassium bicarbonate and involving 18 postmenopausal women, was carried out in 1994 [44]. The women first underwent a 2-week period on a control diet when the baseline measurements were made. Compared to the baseline diet the potassium bicarbonate intervention (60–120 mmol/day/60 kg body weight) resulted in improved calcium balance. In addition, the bone formation marker osteocalcin had increased from 5.5 ± 2.8 to 6.1 ± 2.8 ng/ml (P = 0.001) and hydroxyproline (on older marker of bone resorption) had decreased 220 ± 94 to 204 ± 82 μmol. Wood made some critical observations regarding the study: calcium balance was negative in the control period and although it became less negative, with less calcium excreted in the urine, the women were still losing 124 mg Ca/ 60 kg body weight/day. He also commented that the protein intake was high (96 g) and that although dietary calcium intake was low (652 mg/day) the apparent absorption was only 7%, which is unusually low [45].
Table 3 Comparison of potassium citrate and potassium bicarbonate
Formula Molecular weight (g) Potassium content
Potassium bicarbonate
(tri)Potassium citrate
KHCO3 100 1 g gives 0.39 g K
K3C6H5O7·H2O 324 1 g gives 0.36 g K
276
H.M. Macdonald / International Congress Series 1297 (2007) 268–281
In 2005, Sakhaee et al. investigated 18 women in 2 weeks phases with washout periods of 2 weeks in between [46]. For the intervention phases, potassium citrate (40 mmol/day elemental potassium), calcium citrate (20 mmol Ca/day), a combined treatment, or placebo were given. They noted that potassium citrate reduced urinary calcium excretion and increased urinary pH, as expected. With regard to bone markers the potassium citrate intervention reduced serum CTX but there was no change in urinary NTX. The calcium citrate and combined treatments also showed benefits on bone turnover. Sellmeyer et al. examined the effects of potassium citrate on higher levels of calcium excretion induced by a high salt diet [47]. In total, 52 postmenopausal women were first given a low salt (87 mmol/day) for 3 weeks, then half were subjected to a high salt diet (225 mmol/day) with potassium citrate (90 mmol) and the other half were given a high salt diet (225 mmol/day) and placebo. They found that urinary calcium increased for the high salt and placebo group but maintained at the same level for the high salt and potassium citrate group. Although the bone resorption marker NTX had increased for the high salt and placebo group there was no change in bone resorption for the high salt and potassium citrate group. As expected, net acid excretion had decreased in the potassium citrate-supplemented group. The 3-month potassium citrate intervention carried out by Marangella in 2004 initially recruited 30 women for the intervention and 22 women completed the study [48]. Potassium citrate was given at a daily dose of 1 mEq/kg body weight (between 37 and 74 mmol potassium or base a day). The study was not placebo controlled. Instead, measurements from the intervention were then compared to those from 24 age-matched controls. Compared to the untreated group, the treated group showed an increase in potassium and citrate excretion, and a decrease in net endogenous acid excretion. Although there was a significant decrease in the bone resorption marker DPD/Cr (deoxypyrdinoline/creatinine) for the treated group from 9.08 to 7.01 nmol/mmol P b 0.01 and a non significant change for the untreated group from 6.3 to 7.3 nmol/mmol, it can be seen that the treated group had higher bone resorption at the start. Until the Aberdeen study described in the next section, the longest study examining potassium citrate and bone markers is that of 1-year duration involving 161 postmenopausal women. It was presented at the American Society of Bone and Mineral Research in 2005 but it has not yet been published in full [49]. Jehle et al. found that daily doses amounting to 30 mM potassium chloride caused lumbar spine bone loss (− 1%) but 10 mM tri-potassium citrate resulted in an increase in BMD of + 0.9% after 1 year. Although compared to potassium chloride, potassium citrate caused a slight gain in total hip BMD, there was a slight loss in femoral neck BMD. The bone marker data were mixed. The final study described in this section was of 3 years duration but did not include any bone data. Frassetto et al. calculated that the reduced excretion of calcium seen with longterm potassium citrate treatment could result in an overall gain in bone of 1.7% a year [50]. However, Heaney pointed out that at similar levels of potassium intake, his studies showed that although calcium excretion was reduced, there was a reduction in the amount of calcium that was absorbed, indicating that overall there would be no net gain in calcium [51].
H.M. Macdonald / International Congress Series 1297 (2007) 268–281
277
5.2. Aberdeen potassium citrate, fruit and vegetable intervention study In a 2-year double-blind randomised placebo-controlled trial, two doses of potassium citrate (high dose, 55.6 mmol/day as potassium; low dose, 18.5 mmol/day as potassium) were compared with placebo. Initially 65 postmenopausal women were recruited into each group. For the intention to treat analysis, the numbers in each group were 56, 54 and 55, respectively, for the high dose, low dose and placebo; and for those who continued the treatment the final numbers were 43, 48 and 49, respectively. There was no difference in markers of bone turnover between the different treatment groups, although calcium excretion had decreased for the high dose potassium citrate group at 3 months and 6 months (Macdonald et al., submitted). The study groups were well-matched, including key genotypes that could influence bone metabolism such as VDR polymorphism and Apolipoprotein E polymorphism that has been shown to be associated with increased fracture risk [52,53]. 5.3. Summary of recent studies using organic salts of potassium A summary of recent studies using organic salts of potassium is given in Table 4. It can be seen that although there appear to be changes in bone resorption, the findings are not consistent. It has been suggested that this could be because of short study durations and insufficient washout times [54]. 5.4. Other reasons for the benefits of fruit and vegetables Fruit and vegetables contain a number of other nutrients that may influence bone health (Table 5). For example, vitamin C is involved in the cross-linking of collagen, which is the major structural protein of bone. Vitamin K is needed to modify osteocalcin a Table 4 Summary of recent intervention studies using organic salts of potassium Study (n in each group)
Dose K (mmol/ day)
Study duration
Urinary Bone Bone Comment calcium formation resorption
Whiting et al. [43] (10) Sebastian et al. [44] (18)
50/70 60–120
3h 18 day
Down Down⁎
– Up
– Down
Sakhaee et al. [46] (18) Sellmeyer et al. [47] (26)
40 90
2 weeks 4 weeks
Down No⁎
No No⁎
Down⁎ No
Marangella et al. [48] (22)
37–74
3 months –
–
Down⁎
Jehle et al. [49] (80) 30 Macdonald et al., submitted 56/18.5/0 for publication (56/54/55)
1 years 2 years
Down ⁎Down
Unclear No
Down⁎ No
Frassetto et al. [50] (35/63/ 31 and 72 placebo)
3 years
Down
–
–
30/60/90
No bone data ⁎ Ca balance still negative ⁎ For CTX but not NTX ⁎ With NaCl. Up with NaCl and placebo ⁎ High baseline DPD for treated group ⁎ 6 months only ⁎ For high-dose only ≤6 months, NS other groups No bone data
278
H.M. Macdonald / International Congress Series 1297 (2007) 268–281
Table 5 Nutrients in fruit and vegetables that could influence bone health Vitamins Vitamin C Vitamin K Folate (B vitamin) Carotene Minerals Calcium Magnesium Boron Silicon Anti-oxidants Vitamin Ca Folate Flavonoids Other nutrients Flavonoids Phytoestrogens a
But it can act as pro-oxidant at high concentrations.
protein associated with osteoblasts, and is required for mineralization of the bones. Folate is known to reduce circulating homocysteine levels, which has been shown to be a risk factor for fractures in the elderly [55,56] although other B vitamins may be involved [57,58]. Phytoestrogens, which are found in some vegetables can act as weak agonists and antagonists of oestrogen but further long-term studies are required before we know how important these are for bone health in the Western world [59]. There is evidence that flavonoids may influence bone health [60]. Fruit and vegetables contain a number of compounds that act as antioxidants. These could mop up free radicals that may be promoting bone breakdown. Free radicals have been shown to be involved in osteoclastogenesis and bone resorption, with reactive oxygen species mediating RANK signalling in osteoclasts [61] and oxidative stress inhibiting osteoblast differentiation [62]. In a population study, a urinary biomarker of oxidative stress was found to be negatively associated with BMD and QUS in a study of 48 females and 53 males [63]. A recent publication found that higher fruit intakes were associated with size adjusted bone mineral content in children and older women but previously the investigators had not detected any associations with renal net acid excretion estimated from the diet [64]. They suggest vitamin C and fruit-specific antioxidants may play a role in the positive effects of fruit and vegetables on bone mineral status. 6. Conclusions Organic salts of potassium appear to decrease calcium excretion and lower urinary pH. There appears to be evidence of reduced bone turnover in the short term although this is not unequivocal. It appears that although theoretically, less calcium could be lost in the urine, the negative calcium balance does not appear to continue in the long term. It is likely that more calcium is absorbed in the gut to compensate for the greater urinary losses.
H.M. Macdonald / International Congress Series 1297 (2007) 268–281
279
The role of bone in acid base balance appears to have detrimental effects only in those whose kidney function is severely impaired but for the normal population it does not appear to be a risk factor in osteoporosis. Fruit and vegetable intake may still be important for maintaining healthy bones. Further work is required to establish what nutrients are important and whether some fruit and vegetable groups are more important than others. References [1] I.M. Giversen, Time trends of age-adjusted incidence rates of first hip fractures: a register-based study among older people in Viborg County, Denmark, 1987–1997, Osteoporos. Int. 17 (2006) 552–564. [2] Food and Agriculture Organization of the United Nations and the World Health Organization, Human Vitamin and Mineral Requirements. Report of a Joint FAO/WHO Expert Consultation, WHO/FAO, Bangkok, Thailand, 2002. [3] J. Lutz, Calcium balance and acid–base status of women as affected by increased protein intake and by sodium bicarbonate ingestion, Am. J. Clin. Nutr. 39 (1984) 281–288. [4] Z.K. Roughead, et al., Controlled high meat diets do not affect calcium retention or indices of bone status in healthy postmenopausal women, J. Nutr. 133 (2003) 1020–1026. [5] H. Jaffe, A. Bodansky, J.P. Chandler, Ammonium chloride decalcification, as modified by calcium intake: the relation between generalized osteoporosis and ostitis fibrosa, J. Exp. Med. 56 (1932) 823–834. [6] M.M. Reidenberg, et al., The response of bone to metabolic acidosis in man, Metabolism 15 (1966) 236–241. [7] J.R. Litzow, J. Lemann Jr., E.J. Lennon, The effect of treatment of acidosis on calcium balance in patients with chronic azotemic renal disease, J. Clin. Invest. 46 (1967) 280–286. [8] U.S. Barzel, J. Jowsey, The effects of chronic acid and alkali administration on bone turnover in adult rats, Clin. Sci. 36 (1969) 517–524. [9] A. Wachman, D.S. Bernstein, Diet and osteoporosis, Lancet i (1968) 958–959. [10] J. Green, C.R. Kleeman, Role of bone in regulation of systemic acid–base balance, Kidney Int. 39 (1991) 9–26. [11] U.S. Barzel, The skeleton as an ion-exchange system: implications for the role of acid–base imbalance in the genesis of osteoporosis, J. Bone Miner. Res. 10 (1995) 1431–1436. [12] S. Domrongkitchaiporn, et al., Bone histology and bone mineral density after correction of acidosis in distal renal tubular acidosis, Kidney Int. 62 (2002) 2160–2166. [13] J. Lemann, R.W. Gray, J.A. Pleuss, Potassium bicarbonate, but not sodium bicarbonate, reduces urinary calcium excretion and improves calcium balance in healthy men, Kidney Int. 35 (1989) 688–695. [14] J. Lemann, et al., Potassium administration increases and potassium deprivation reduces urinary calcium excretion in healthy adults, Kidney Int. 39 (1991) 973–983. [15] J. Lemann, J.A. Pleuss, R.W. Gray, Potassium causes calcium retention in healthy adults, J. Nutr. 123 (1993) 1623–1626. [16] M.S. Oh, Irrelevance of bone buffering to acid–base homeostasis in chronic metabolic acidosis, Nephron 59 (1991) 7–10. [17] R.C. Morris, et al., Expression of osteoporosis as determined by diet-disordered electrolyte and acid–base metabolism, in: P. Burckhardt, B. Dawson-Hughes, R.P. Heaney (Eds.), Nutritional Aspects of Osteoporosis. 4th International Symposium on Nutritional Aspects of Osteoporosis, Switzerland, 2000, Academic Press, 2001, pp. 357–378. [18] A. Sebastian, et al., Estimation of the net acid load of the diet of ancestral preagricultural Homo sapiens and their hominid ancestors, Am. J. Clin. Nutr. 76 (2002) 1308–1316. [19] L. Frassetto, et al., Diet, evolution and aging – the pathophysiologic effects of the post-agricultural inversion of the potassium-to-sodium and base-to-chloride ratios in the human diet, Eur. J. Nutr. 40 (2001) 200–213. [20] L. Cordain, et al., Plant–animal subsistence erations and macronutrient energy estimations in worldwide hunter–gatherer diets, Am. J. Clin. Nutr. 71 (2000) 682–692. [21] H.C. Sherman, A.O. Gettler, The balance of acid-forming and base-forming elements in foods, and its relation to ammonia metabolism, J. Biol. Chem. 11 (1912) 323–328.
280
H.M. Macdonald / International Congress Series 1297 (2007) 268–281
[22] T. Remer, F. Manz, Potential renal acid load of foods and its influence on urine pH, J. Am. Diet. Assoc. 95 (1995) 791–797. [23] T. Remer, F. Manz, Estimation of the renal net acid excretion by adults consuming diets containing variable amounts of protein, Am. J. Clin. Nutr. 59 (1994) 1356–1361. [24] L.A. Frassetto, et al., Estimation of net endogenous noncarbonic acid production in humans from diet potassium and protein contents, Am. J. Clin. Nutr. 68 (1998) 576–583. [25] S.A. New, et al., Nutritional influences on bone mineral density: a cross-sectional study in menopausal women, Am. J. Clin. Nutr. 65 (1997) 1831–1839. [26] H.M. Macdonald, et al., Low dietary potassium intakes and high dietary estimates of net endogenous acid production are associated with low bone mineral density in premenopausal women and increased markers of bone resorption in postmenopausal women, Am. J. Clin. Nutr. 81 (2005) 923–933. [27] K.L. Tucker, et al., Potassium, magnesium, and fruit and vegetable intakes are associated with greater bone mineral density in elderly men and women, Am. J. Clin. Nutr. 69 (1999) 727–736. [28] M.T. Hannan, et al., Effect of dietary protein on bone loss in elderly men and women: the Framingham Osteoporosis Study, J. Bone Miner. Res. 15 (2000) 2504–2512. [29] K.L. Tucker, M.T. Hannan, D.P. Kiel, The acid–base hypothesis: diet and bone in the Framingham Osteoporosis Study, Eur. J. Nutr. 40 (2001) 231–237. [30] H.M. Macdonald, et al., Low dietary potassium intakes and high dietary estimates of net endogenous acid production are associated with low bone mineral density in premenopausal women and increased markers of bone resorption in postmenopausal women, Am. J. Clin. Nutr. 81 (2005) 923–933. [31] C.P. McGartland, et al., Fruit and vegetable consumption and bone mineral density: the Northern Ireland Young Hearts Project, Am. J. Clin. Nutr. 80 (2004) 1019–1023. [32] S.J. Whiting, et al., Factors that affect bone mineral accrual in the adolescent growth spurt, J. Nutr. 134 (2004) 696S–700S. [33] C.J. Prynne, et al., Dietary acid–base balance and intake of bone-related nutrients in Cambridge teenagers, Eur. J. Clin. Nutr. 58 (2004) 1462–1471. [34] L.J. Appel, et al., A clinical trial of the effects of dietary patterns on blood pressure, New Engl. J. Med. 336 (1997) 1117–1124. [35] P.H. Lin, et al., The DASH diet and sodium reduction improve markers of bone turnover and calcium metabolism in adults, J. Nutr. 133 (2003) 3130–3136. [36] T. Buclin, et al., Diet acids and alkalis influence calcium retention in bone, Osteoporos. Int. 12 (2001) 493–499. [37] G.R. Martin, R.K. Jain, Noninvasive measurement of interstitial pH profiles in normal and neoplastic tissue using fluorescence ratio imaging microscopy, Cancer Res. 54 (1994) 5670–5674. [38] D. Street, J. Bangsbo, C. Juel, Interstitial pH in human skeletal muscle during and after dynamic graded exercise, J. Physiol. 537 (2001) 993–998. [39] R.C. Muhlbauer, F. Li, Effect of vegetables on bone metabolism, Nature 401 (1999) 343–344. [40] J.M. Macleay, D.L. Wheeler, A.S. Turner, The ovine model for the study of dietary acid base, estrogen depletion and bone health, in: P. Burckhardt, B. Dawson-Hughes, R.P. Heaney (Eds.), Nutritional Aspects of Osteoporosis. 4th International Symposium on Nutritional Aspects of Osteoporosis, Switzerland, 2000, Academic Press, 2001, pp. 331–348. [41] J.M. MacLeay, et al., Dietary-induced metabolic acidosis decreases bone mineral density in mature ovariectomized ewes, Calcif. Tissue Int. 75 (2004) 431–437. [42] R.C. Muhlbauer, A. Lozano, A. Reinli, Onion and a mixture of vegetables, salads, and herbs affect bone resorption in the rat by a mechanism independent of their base excess, J. Bone Miner. Res. 17 (2002) 1230–1236. [43] S.J. Whiting, D.J. Anderson, S.J. Weeks, Calciuric effects of protein and potassium bicarbonate but not of sodium chloride or phosphate can be detected acutely in adult women and men, Am. J. Clin. Nutr. 65 (1997) 1645–1672. [44] A. Sebastian, et al., Improved mineral balance and skeletal metabolism in postmenopausal women treated with potassium bicarbonate, New Engl. J. Med. 330 (1994) 1776–1781. [45] R.J. Wood, Potassium bicarbonate supplementation and calcium metabolism in postmenopausal women: are we barking up the wrong tree? Nutr. Rev. 52 (1919) 278–280. [46] K. Sakhaee, et al., Contrasting effects of various potassium salts on renal citrate excretion, J. Clin. Endocrinol. Metab. 72 (1991) 396–400.
H.M. Macdonald / International Congress Series 1297 (2007) 268–281
281
[47] D.E. Sellmeyer, M. Schloetter, A. Sebastian, Potassium citrate prevents increased urine calcium excretion and bone resorption induced by a high sodium chloride diet, J. Clin. Endocrinol. Metab. 87 (2002) 2008–2012. [48] M. Marangella, et al., Effects of potassium citrate supplementation on bone metabolism, Calcif. Tissue Int. 74 (2004) 330–335. [49] S. Jehle, et al., Neutralization of the acidogenic Western diet with K citrate increases DXA BMD in postmenopausal osteopenic women: results of DBRCT, J. Bone Miner. Res. 20 (2005) s289. [50] L. Frassetto, R.C. Morris Jr., A. Sebastian, Long-term persistence of the urine calcium-lowering effect of potassium bicarbonate in postmenopausal women, J. Clin. Endocrinol. Metab. 90 (2005) 831–834. [51] K. Rafferty, K.M. Davies, R.P. Heaney, Potassium intake and the calcium economy, J. Am. Coll. Nutr. 24 (2005) 99–106. [52] M. Kohlmeier, et al., Bone fracture history and prospective bone fracture risk of hemodialysis patients are related to apolipoprotein E genotype, Calcif. Tissue Int. 62 (1998) 278–281. [53] J.A. Cauley, et al., Apolipoprotein E polymorphism: a new genetic marker of hip fracture risk – the study of osteoporotic fractures, J. Bone Miner. Res. 14 (1999) 1175–1181. [54] Z.K. Roughead, Is the interaction between dietary protein and calcium destructive or constructive for bone? J. Nutr. 133 (2003) 866S–869S. [55] R.R. McLean, et al., Homocysteine as a predictive factor for hip fracture in older persons, N. Engl. J. Med. 350 (2004) 2042–2049. [56] J.B. van Meurs, et al., Homocysteine levels and the risk of osteoporotic fracture, N. Engl. J. Med. 350 (2004) 2033–2041. [57] H.M. Macdonald, D.M. Reid, Vitamin B-complex, methylenetetrahydrofolate reductase polymorphism and bone: potential for gene–nutrient interaction, in: P. Burckhardt, B. Dawson-Hughes, R.P. Heaney (Eds.), Nutritional Aspects of Osteoporosis. 4th International Symposium on Nutritional Aspects of Osteoporosis, Switzerland, 2000, Academic Press, 2001, pp. 127–138. [58] H.M. Macdonald, et al., Methylenetetrahydrofolate reductase polymorphism interacts with riboflavin intake to influence bone mineral density, Bone 35 (2004) 957–964. [59] A. Cassidy, et al., Critical review of health effects of soyabean phyto-oestrogens in post-menopausal women, Proc. Nutr. Soc. 65 (2006) 76–92. [60] H. Chiba, et al., Hesperidin, a citrus flavonoid, inhibits bone loss and decreases serum and hepatic lipids in ovariectomized mice, J. Nutr. 133 (2003) 1892–1897. [61] H. Ha, et al., Reactive oxygen species mediate RANK signaling in osteoclasts, Exp. Cell Res. 301 (2004) 119–127. [62] X.C. Bai, et al., Oxidative stress inhibits osteoblastic differentiation of bone cells by ERK and NF-kappaB, Biochem. Biophys. Res. Commun. 314 (2004) 197–207. [63] S. Basu, et al., Association between oxidative stress and bone mineral density, Biochem. Biophys. Res. Commun. 288 (2001) 275–279. [64] C.J. Prynne, et al., Fruit and vegetable intakes and bone mineral status: a cross-sectional study in 5 age and sex cohorts, Am. J. Clin. Nutr. 83 (2006) 1420–1428.