All four of the urine calcium meta-analyses demonstrated that when dietary phosphate was increased, urine calcium decreased, whether the subjects had low or high calcium intakes and whether the phosphate supplement was neutral/alkaline or acidic. Three of the four calcium balance analyses revealed that as phosphate supplements are increased, calcium balance increased. However, when the phosphate salt was acidic, there was no important change in calcium balance. The effect of phosphate supplementation on the bone metabolism markers did not provide any clear information. The increased net acid excretion from phosphate supplements in three studies was associated with lowered urinary calcium excretion.
All of the findings of this study contradict the acid ash hypothesis since the hypothesis posits that an "acidic" ion such as phosphate causes increased urinary excretion of bone calcium, and therefore calcium balance would be decreased by phosphate. None of the study findings support the hypothesis since phosphate supplements did not increase urine calcium or decrease calcium balance. Dietary phosphate is considered to be the major source of excreted acid, followed by sulfate derived from protein . However, this current study contradicts the assertion that dietary phosphate is detrimental to bone, and our previous work contradicts the assertion that protein is detrimental to bone . When the evidence regarding the acid-ash hypothesis is critically examined, there is more contradiction than support for the hypothesis.
Most of the evidence in favor of the acid-ash hypothesis is based on the outcomes of changes of urinary calcium and changes of indicators of mineral metabolism or bone turnover markers [2, 6, 8, 9, 15, 31, 60–64]. However, urinary calcium and bone/mineral markers, are not direct measures of osteoporosis. Changes in urine calcium excretion are poor surrogate measures of bone health, as intestinal calcium absorption  and/or secretion  may also be altered by an intervention. Calcium balance changes are much better measures of changes of calcium status than changes of urinary calcium, and calcium balance studies can provide useful information about whole body calcium metabolism when the studies are well designed .
What better bone health outcomes have studies used as to test the association of the acid-ash hypothesis? The National Institutes of Health Consensus Panel defined Osteoporosis as ". a skeletal disorder characterized by compromised bone strength predisposing a person to an increased risk of fracture" . The only currently available measures that address bone strength are the occurrence of low trauma fractures and/or biomechanical testing of bone biopsy material [68, 69]. In the assessment of pharmaceutical interventions for osteoporosis, fracture prevention is considered the appropriate outcome measure for clinical trials, and not change in bone density. In clinical practice, however, bone density is an objective measure often used for following an individual's response to an intervention. Although low bone density is used in the clinical diagnosis of osteoporosis, it is simply a risk factor for bone fragility.
Two randomized controlled studies of the acid ash hypothesis have used changes of BMD as the outcome measure [70, 71]. Both of these studies tested the BMD response to potassium citrate [70, 71] and one of these studies also increased fruit and vegetable intakes in an additional study arm . The results of these studies were opposite, one observed a decreased loss of spine BMD among the potassium citrate arm , while in the other study BMD change did not differ significantly between study arms . This discrepancy may be explained by the use of allocation concealment, an indicator of study rigor . The latter study that saw no differences in BMD used adequate allocation concealment  while the former study that saw a difference did not conceal their allocation . Inadequate allocation concealment is associated with an overestimation of effect , therefore it is possible that the results from the former trial  overestimated the effect of potassium citrate on bone.
Three prospective observational cohort studies found some associations that appear to support the acid-ash hypothesis, using changes in bone mineral density as the outcome [73–75]. However, the three prospective studies with positive associations had inconsistent findings, since for each group in which a positive finding was identified there were other subject groups and/or bone sites for which the finding did not apply. The Framingham Osteoporosis study reported that in men potassium intake was associated with loss of bone mineral density at the femoral neck and trochanter, but not at the radius, and there were no associations found for women . Fruit and vegetable intakes were not significantly associated with the changes of bone mineral density at any site for men or women in this study . Of 12 associations assessed in this study regarding the acid-ash hypothesis, two were significantly associated while 10 were not .
In the EPIC-Norfolk study, vitamin C was associated with less loss of bone mineral density at the total hip among elderly women but not among elderly men, and only after the vitamin C intakes were divided into tertiles . In the Aberdeen Prospective Osteoporosis Screening study, intakes of calcium and phosphorus (positively) and fat (negatively) were associated more strongly with the loss of bone mineral density over time than was potassium .
The two recent prospective cohort studies of the acid-ash hypothesis used fractures as the outcome measure. Fractures are considered a direct and clinically valid measure of osteoporosis [68, 69]. A study among 1865 vegetarian and omnivorous peri- and postmenopausal women found that the risk of wrist fracture decreased significantly as protein intakes increased, whether the protein was from plants or meat . This study provides further support for the concept that dietary protein is supportive of bone health [77, 78]. A French study among 36,217 women examined the association between protein or the diet acid load on fractures (excluded high trauma or metastases fractures) found no overall support for the acid ash hypothesis as neither protein nor the diet acid load were associated with fracture risk . However, in a subgroup analysis, both higher protein intake and diet acid load were associated with higher fracture risk among women in the lowest quartile of calcium intake . In comparison, in this meta-analysis we found no important differences between phosphate intakes and calcium metabolism between those with low or high calcium intakes. In summary, the findings from prospective cohort studies of the acid-ash hypothesis of bone health demonstrated few findings consistent with the hypothesis, and numerous findings that did not support the hypothesis. It must be remembered that observational studies do not have the rigor of randomized control trials, and the findings from observational studies can be confounded or confused by other related variables. An example that might confound observational studies of nutrition and bone health could be due to those subjects who eat more fruit and vegetables may also get more bone protective exercise.
The acid-ash hypothesis posits that meats, grains and dairy products are detrimental to bone health due to their phosphate contents [16, 3, 4, 10], however, this meta-analysis revealed that higher dietary intakes of phosphate do not increase either urinary calcium excretion or whole body calcium loss. Further to question the acid ash hypothesis, the evidence regarding higher net acid excretion from changes in dietary protein type or amount does not support the hypothesis . No studies have evaluated the effect of grain foods on bone health. Dairy products, an important source of dietary phosphate, are also an important dietary calcium source  and an inexpensive source of high quality protein. Protein has been found to be supportive of bone health in a prospective cohort study and a randomized controlled trial [77, 78]. Considering that this study has not upheld the concept that higher intakes of phosphate are detrimental to bone mineral maintenance, the consideration of meats, grains and dairy products as detrimental to bone health on the basis of their phosphate content must be questioned. Additionally, these foods that produce acid on metabolism are important sources of nutrients that are important for bone health, including calcium  (dairy products), protein [82, 83] (dairy products and meats), and vitamin D  (some dairy products).
The recommendation for calcium balance studies by the Institute of Medicine to have study subjects consume the study calcium intake for at least a week prior to the outcome measurement  is designed to allow the subjects to adapt to the experimental calcium intake . This adaption to the study calcium intake is important to lessen the chance that the adaption confuses or biases the effect of the intervention. Randomization of the interventions is very important in calcium balance cross-over studies since the adaptations to the calcium intake would be uneven in the two arms of before-after non-randomized cross-over studies.
Overall, the methodological quality of the studies of phosphate supplementation was poor since only one of the studies used superior methodology : having randomization and using all of the Institute of Medicine's recommendations for calcium balance studies. Therefore, there is a chance that the results of the other studies are biased due to poor methodology. The magnitude of this bias would vary depending on whether the study calcium intakes were greater or less than the subjects' usual intakes. In spite of the questionable methodological quality, all of the other meta-analyses were in congruence with the one study  that used superior methodology, which suggests that the bias may not have been severe.
Concern has been raised about the phosphate, as well as caffeine, contents of cola soft drinks and their potential deleterious effects on bone health. Observational studies that measured BMD  or asked subjects about previous fractures  in relation to carbonated beverage intake documented associations between these beverages and poorer bone health. As well, a non-randomized cross-over intervention study identified higher bone resorption markers from colas compared to milk . The results from this meta-analysis does not support the concept that the phosphate in soda is deleterious, therefore, these potentially deleterious effects could be due to lower milk consumption, and therefore protein, calcium or other nutrient intakes  since those that consume more carbonated beverage drink less milk [88, 89], however generalizations from the studies on soft drinks are limited by the study designs used.
This meta-analysis has three strengths: First, this is the first study to systematically assess the calcium balance literature in response to changes of phosphate supplementation. Second, this meta-analysis includes an evaluation of methodological quality, and therefore an assessment of potential confounding or bias. Third, the stratified analyses allowed assessment of the effect of calcium intakes and the degree of protonation of the phosphate supplement on the findings.
This meta-analysis also has limitations. First, although calcium balance is neither a direct measure of bone health nor of the progression of osteoporosis, but rather it is a surrogate measure of this disease progression. Therefore this study lacks a direct measures of bone health. However, calcium balance is important since prolonged negative calcium balance leads to bone loss. Second, this study is limited by the poor methodological quality of the majority of the studies of phosphate intake on calcium metabolism.
In conclusion, this meta-analysis revealed that raising phosphate intakes decreases urine calcium and slightly increases calcium retention over a broad range of calcium intakes. This work does not support the acid-ash hypothesis concepts that "acidic" ions such as phosphate contribute to demineralization of bone and bone calcium excretion in the urine. A definitive study is needed that follows all of the recommendations for methodological quality for both calcium balance studies  as well as recommendations for intervention studies [25, 26], with measurement of outcomes that are direct measures of bone strength, to determine whether or not there is an association between phosphate intake and osteoporosis.