Aromatase deficiency in transplanted bone marrow cells improves vertebral trabecular bone quantity, connectivity, and mineralization and decreases cortical porosity in murine bone marrow transplant recipients.

Estradiol is an important regulator of bone accumulation and maintenance. Circulating estrogens are primarily produced by the gonads. Aromatase, the enzyme responsible for the conversion of androgens to estrogen, is expressed by bone marrow cells (BMCs) of both hematopoietic and nonhematopoietic origin. While the significance of gonad-derived estradiol to bone health has been investigated, there is limited understanding regarding the relative contribution of BMC derived estrogens to bone metabolism. To elucidate the role of BMC derived estrogens in male bone, irradiated wild-type C57BL/6J mice received bone marrow cells transplanted from either WT (WT(WT)) or aromatase-deficient (WT(ArKO)) mice. MicroCT was acquired on lumbar vertebra to assess bone quantity and quality. WT(ArKO) animals had greater trabecular bone volume (BV/TV p = 0.002), with a higher trabecular number (p = 0.008), connectivity density (p = 0.017), and bone mineral content (p = 0.004). In cortical bone, WT(ArKO) animals exhibited smaller cortical pores and lower cortical porosity (p = 0.02). Static histomorphometry revealed fewer osteoclasts per bone surface (Oc.S/BS%), osteoclasts on the erosion surface (ES(Oc+)/BS, p = 0.04) and low number of osteoclasts per bone perimeter (N.Oc/B.Pm, p = 0.01) in WT(ArKO). Osteoblast-associated parameters in WT(ArKO) were lower but not statistically different from WT(WT). Dynamic histomorphometry suggested similar bone formation indices' patterns with lower mean values in mineral apposition rate, label separation, and BFR/BS in WT(ArKO) animals. Ex vivo bone cell differentiation assays demonstrated relative decreased osteoblast differentiation and ability to form mineralized nodules. This study demonstrates a role of local 17ß-estradiol production by BMCs for regulating the quantity and quality of bone in male mice. Underlying in vivo cellular and molecular mechanisms require further study.

Defining hypercalciuria in nephrolithiasis.

The classic definition of hypercalciuria, an upper normal limit of 200 mg/day, is based on a constant diet restricted in calcium, sodium, and animal protein; however, random diet data challenge this. Here our retrospective study determined the validity of the classic definition of hypercalciuria by comparing data from 39 publications analyzing urinary calcium excretion on a constant restricted diet and testing whether hypercalciuria could be defined when extraneous dietary influences were controlled. These papers encompassed 300 non-stone-forming patients, 208 patients with absorptive hypercalciuria type I (presumed due to high intestinal calcium absorption), and 234 stone formers without absorptive hypercalciuria; all evaluated on a constant restricted diet. In non-stone formers, the mean urinary calcium was well below 200 mg/day, and the mean for all patients was 127±46 mg/day with an upper limit of 219 mg/day. In absorptive hypercalciuria type I, the mean urinary calcium significantly exceeded 200 mg/day in all studies with a combined mean of 259±55 mg/day. Receiver operating characteristic curve analysis showed the optimal cutoff point for urinary calcium excretion was 172 mg/day on a restricted diet, a value that approximates the traditional limit of 200 mg/day. Thus, on a restricted diet, a clear demarcation was seen between urinary calcium excretion of kidney stone formers with absorptive hypercalciuria type I and normal individuals. When dietary variables are controlled, the classic definition of hypercalciuria of nephrolithiasis appears valid.

Variability in urinary oxalate measurements between six international laboratories.

BACKGROUND: Hyperoxaluria is a major risk factor for kidney stone formation. Although urinary oxalate measurement is part of all basic stone risk assessment, there is no standardized method for this measurement. METHODS: Urine samples from 24-h urine collection covering a broad range of oxalate concentrations were aliquoted and sent, in duplicates, to six blinded international laboratories for oxalate, sodium and creatinine measurement. In a second set of experiments, ten pairs of native urine and urine spiked with 10 mg/L of oxalate were sent for oxalate measurement. Three laboratories used a commercially available oxalate oxidase kit, two laboratories used a high-performance liquid chromatography (HPLC)-based method and one laboratory used both methods. RESULTS: Intra-laboratory reliability for oxalate measurement expressed as intraclass correlation coefficient (ICC) varied between 0.808 [95% confidence interval (CI): 0.427-0.948] and 0.998 (95% CI: 0.994-1.000), with lower values for HPLC-based methods. Acidification of urine samples prior to analysis led to significantly higher oxalate concentrations. ICC for inter-laboratory reliability varied between 0.745 (95% CI: 0.468-0.890) and 0.986 (95% CI: 0.967-0.995). Recovery of the 10 mg/L oxalate-spiked samples varied between 8.7 ± 2.3 and 10.7 ± 0.5 mg/L. Overall, HPLC-based methods showed more variability compared to the oxalate oxidase kit-based methods. CONCLUSIONS: Significant variability was noted in the quantification of urinary oxalate concentration by different laboratories, which may partially explain the differences of hyperoxaluria prevalence reported in the literature. Our data stress the need for a standardization of the method of oxalate measurement.

Unusual mid-shaft fractures during long-term bisphosphonate therapy.

BACKGROUND: Bisphosphonates are the most commonly prescribed medications for the treatment of osteoporosis. Although existing evidence supports a good safety profile, there is concern that chronic administration of these agents could result in severe suppression of bone turnover with increased risk of nonvertebral fractures. OBJECTIVE: The objective of this study was to report the clinical presentation, selected bone histomorphometry and X-ray images of patients who developed mid-shaft long bone fractures during bisphosphonate therapy, six of whom had bone biopsy for histomorphometery. RESULTS: Of the 13 patients who sustained atraumatic mid-shaft fractures, 10 were on alendronate and three were on risedronate therapy before the fractures. In addition to bisphosphonates, three patients were on oestrogen and two on tamoxifen concomitantly. Four patients with glucocorticoid-induced osteoporosis were on alendronate for 3-11 years along with glucocorticoid therapy. Bone histomorphometry showed severe suppression of bone turnover in five patients and low bone turnover in one patient. CONCLUSION: Long-term bisphosphonate therapy may increase the risk of unusual long bone mid-shaft fractures. This is probably due to prolonged suppression of bone turnover, which could lead to accumulation of microdamage and development of hypermineralized bone. At present, the scope of this complication in the larger context of patients receiving bisphosphonate therapy remains unknown, but appears to be small.

Bone disease and hypercalciuria in children.

There have been relatively few studies of bone mass in children with idiopathic hypercalciuria (IH). When performed, bone mineral density (BMD) measurements have consistently disclosed decreased Z-scores for children with IH at the lumbar spine and, to a lesser extent, at the femoral neck. Few investigations have delineated the nature of the mechanism(s) that may contribute to the bone loss in these children. Some studies have been consistent, showing increased bone resorption as the probable mechanism of bone loss. To date, there have been no reports regarding the assessment of biochemical markers specific for bone formation in children with IH. However, since most of the children with IH in these reports had demonstrated normal longitudinal growth, it seems less likely that there is an alteration in bone formation. The causes for increased bone resorption also are not firmly established, but genetics, dietary indiscretions, and altered cytokine production have been proposed as being contributory to the decreased BMD observed in these children with IH. Optimal bone mineral accretion during childhood and adolescence is important in attaining peak bone mass and may serve to prevent the development of osteoporosis in adulthood. Thus, a better understanding of bone loss in children with IH is warranted.

Inhibition of osteoclast formation and function by bicarbonate: role of soluble adenylyl cyclase.

High [HCO(3)(-)] inhibits and low [HCO(3)(-)] stimulates bone resorption, which mediates part of the effect of chronic acidosis or acid feeding on bone. Soluble adenylyl cyclase (sAC) is a bicarbonate sensor that can potentially mediate the effect of bicarbonate on osteoclasts. Osteoclasts were incubated in 0, 12, and 24 mM HCO(3)(-) at pH 7.4 for 7-8 days and assayed for tartrate-resistant acid phosphatase (TRAP) and vacuolar-ATPase expression, and H+ accumulation. Total number and area of TRAP (+) multinucleated osteoclasts was decreased by HCO(3)(-) in a dose-dependent manner. V-ATPase expression and H+ accumulation normalized to cell cross-sectional area or protein were not significantly changed. The HCO(3)(-) -induced inhibition of osteoclast growth and differentiation was blocked by either 2-hydroxyestradiol, an inhibitor of sAC or sAC knockdown by sAC specific siRNA. The model of HCO(3)(-) inhibiting osteoclast via sAC was further supported by the fact that the HCO(3)(-) dose-response on osteoclasts is flat when cells were saturated with 8-bromo-cAMP, a permeant cAMP analog downstream from sAC thus simulating sAC activation. To confirm our in vitro findings in intact bone, we developed a 1-week mouse calvaria culture system where osteoclasts were shown to be viable. Bone volume density (BV/TV) determined by micro-computed tomography (microCT), was higher in 24 mM HCO(3)(-) compared to 12 mM HCO(3)(-) treated calvaria. This HCO(3)(-) effect on BV/TV was blocked by 2-hydroxyestradiol. In summary, sAC mediates the inhibition of osteoclast function by HCO(3)(-), by acting as a HCO(3)(-) sensor.

Comparison of semi-empirical and computer derived methods for estimating urinary saturation of brushite.

PURPOSE: The Equil 2 computer program has been questioned by the new Joint Expert Speciation System program (Mayhem Unit Trust and Council for Scientific and Industrial Research, Pretoria, South Africa) for estimating the urinary saturation of stone forming salts to gauge the propensity for stone formation. To attempt resolution the supersaturation index according to the Joint Expert Speciation System and the relative saturation ratio according to Equil 2 were compared with the semi-empirically derived concentration-to-product ratio. MATERIALS AND METHODS: Data were obtained from a recent article in The Journal of Urology(R), in which pH, calcium and citrate were varied over a wide range in 72 urine samples. We calculated the relative saturation ratio and the supersaturation index of brushite, and compared them with the available concentration-to-product ratio derived from the growth or dissolution of synthetic brushite. RESULTS: The mean concentration-to-product ratio did not differ from the supersaturation index but the concentration-to-product ratio and the supersaturation index were significantly lower than the relative saturation ratio (p <0.004). On the saturation value and urinary variable plot the relative saturation ratio could be readily distinguished from the concentration-to-product ratio because it was consistently and significantly higher. While the supersaturation index pattern was similar to the concentration-to-product ratio, the supersaturation index was slightly lower at high urinary pH and calcium, and slightly higher at lower urinary pH and calcium (p <0.001). When the Ca(2)H(2)(PO(4))(2) complex was deleted from the Joint Expert Speciation System, the corrected supersaturation index was not significantly different from the relative saturation ratio determined by Equil 2. CONCLUSIONS: The relative saturation ratio overestimates brushite saturation by about 80%. The supersaturation index yields a good approximation of brushite saturation at modest degrees of saturation but it overestimates saturation at low pH or calcium (low saturation) and underestimates it at high pH or calcium (high saturation).

Bone disease and idiopathic hypercalciuria.

Observational and epidemiologic studies alike have shown that idiopathic hypercalciuric (IH) stone-forming patients typically show bone mineral density scores that are significantly lower than those observed for age- and sex-matched normal subjects or those for nonhypercalciuric stone-forming patients. Most of these studies have relied on changes in bone mineral density and have not explored the mechanism(s) involved. There have been a small number of studies that have relied on dynamic bone histomorphometry to ascertain the nature of the bone defect in IH patients. When performed, these studies clearly have shown increased bone resorption and high bone turnover in patients with fasting hypercalciuria whereas suppressed bone formation indices are the most consistent finding in patients with the absorptive variant of IH. The causes of this apparent difference in bone remodeling between the 2 variants of IH still is uncertain. Available evidence suggests that potential mechanisms may be dependent in large part to genetic, metabolic, and nutritional causes of hypercalciuria and bone loss in patients with IH.

Recovery from skeletal fluorosis (an enigmatic, American case).

UNLABELLED: A 52-year-old man presented with severe neck immobility and radiographic osteosclerosis. Elevated fluoride levels in serum, urine, and iliac crest bone revealed skeletal fluorosis. Nearly a decade of detailed follow-up documented considerable correction of the disorder after removal of the putative source of fluoride (toothpaste). INTRODUCTION: Skeletal fluorosis, a crippling bone disorder, is rare in the United States, but affects millions worldwide. There are no data regarding its reversibility. MATERIALS AND METHODS: A white man presented in 1996 with neck immobility and worsening joint pains of 7-year duration. Radiographs revealed axial osteosclerosis. Bone markers were distinctly elevated. DXA of lumbar spine (LS), femoral neck (FN), and distal one-third radius showed Z scores of +14.3, +6.6, and -0.6, respectively. Transiliac crest biopsy revealed cancellous volume 4.5 times the reference mean, cortical width 3.2 times the reference mean, osteoid thickness 25 times the reference mean, and wide and diffuse tetracycline uptake documenting osteomalacia. Fluoride (F) was elevated in serum (0.34 and 0.29 mg/liter [reference range: <0.20]), urine (26 mg/liter [reference range: 0.2-1.1 mg/liter]), and iliac crest (1.8% [reference range: <0.1%]). Tap and bottled water were negative for F. Surreptitious ingestion of toothpaste was the most plausible F source. RESULTS: Monitoring for a decade showed that within 3 months of removal of F toothpaste, urine F dropped from 26 to 16 mg/liter (reference range: 0.2-1.1 mg/liter), to 3.9 at 14 months, and was normal (1.2 mg/liter) after 9 years. Serum F normalized within 8 months. Markers corrected by 14 months. Serum creatinine increased gradually from 1.0 (1997) to 1.3 mg/dl (2006; reference range: 0.5-1.4 mg/dl). Radiographs, after 9 years, showed decreased sclerosis of trabeculae and some decrease of sacrospinous ligament ossification. DXA, after 9 years, revealed 23.6% and 15.1% reduction in LS and FN BMD with Z scores of +9.3 and +4.8, respectively. Iliac crest, after 8.5 years, had normal osteoid surface and thickness with distinct double labels. Bone F, after 8.5 years, was 1.15% (reference range, <0.1), which was a 36% reduction (still 10 times the reference value). All arthralgias resolved within 2 years, and he never fractured, but new-onset nephrolithiasis occurred within 9 months and became a chronic problem. CONCLUSIONS: With removal of F exposure, skeletal fluorosis is reversible, but likely impacts for decades. Patients should be monitored for impending nephrolithiasis.

Aromatase deficiency causes altered expression of molecules critical for calcium reabsorption in the kidneys of female mice *.

UNLABELLED: Kidney stones increase after menopause, suggesting a role for estrogen deficiency. ArKO mice have hypercalciuria and lower levels of calcium transport proteins, whereas levels of the klotho protein are elevated. Thus, estrogen deficiency is sufficient to cause altered renal calcium handling. INTRODUCTION: The incidence of renal stones increases in women after menopause, implicating a possible role for estrogen deficiency. We used the aromatase deficient (ArKO) mouse, a model of estrogen deficiency, to test the hypothesis that estrogen deficiency would increase urinary calcium excretion and alter the expression of molecular regulators of renal calcium reabsorption. MATERIALS AND METHODS: Adult female wildtype (WT), ArKO, and estradiol-treated ArKO mice (n = 5-12/group) were used to measure urinary calcium in the fed and fasting states, relative expression level of some genes involved in calcium reabsorption in the distal convoluted tubule by real-time PCR, and protein expression by Western blotting or immunohistochemistry. Plasma membrane calcium ATPase (PMCA) activity was measured in kidney membrane preparations. ANOVA was used to test for differences between groups followed by posthoc analysis with Dunnett's test. RESULTS: Compared with WT, urinary Ca:Cr ratios were elevated in ArKO mice, renal mRNA levels of transient receptor potential cation channel vallinoid subfamily member 5 (TRPV5), TRPV6, calbindin-D28k, the Na+/Ca+ exchanger (NCX1), and the PMCA1b were significantly decreased, and klotho mRNA and protein levels were elevated. Estradiol treatment of ArKO mice normalized urinary calcium excretion, renal mRNA levels of TRPV5, calbindin-D(28k), PMCA1b, and klotho, as well as protein levels of calbindin-D28k and Klotho. ArKO mice treated with estradiol had significantly greater PMCA activity than either untreated ArKO mice or WT mice. CONCLUSIONS: Estrogen deficiency caused by aromatase inactivation is sufficient for renal calcium loss. Changes in estradiol levels are associated with coordinated changes in expression of many proteins involved in distal tubule calcium reabsorption. Estradiol seems to act at the genomic level by increasing or decreasing (klotho) protein expression and nongenomically by increasing PMCA activity. PMCA, not NCX1, is likely responsible for extruding calcium in response to in vivo estradiol hormonal challenge. These data provide potential mechanisms for regulation of renal calcium handling in response to changes in serum estrogen levels.

Severely suppressed bone turnover: a potential complication of alendronate therapy.

Alendronate, an inhibitor of bone resorption, is widely used in osteoporosis treatment. However, concerns have been raised about potential oversuppression of bone turnover during long-term use. We report on nine patients who sustained spontaneous nonspinal fractures while on alendronate therapy, six of whom displayed either delayed or absent fracture healing for 3 months to 2 yr during therapy. Histomorphometric analysis of the cancellous bone showed markedly suppressed bone formation, with reduced or absent osteoblastic surface in most patients. Osteoclastic surface was low or low-normal in eight patients, and eroded surface was decreased in four. Matrix synthesis was markedly diminished, with absence of double-tetracycline label and absent or reduced single-tetracycline label in all patients. The same trend was seen in the intracortical and endocortical surfaces. Our findings raise the possibility that severe suppression of bone turnover may develop during long-term alendronate therapy, resulting in increased susceptibility to, and delayed healing of, nonspinal fractures. Although coadministration of estrogen or glucocorticoids appears to be a predisposing factor, this apparent complication can also occur with monotherapy. Our observations emphasize the need for increased awareness and monitoring for the potential development of excessive suppression of bone turnover during long-term alendronate therapy.

Effect of subcutaneous leptin replacement therapy on bone metabolism in patients with generalized lipodystrophy.

The adipocyte-derived hormone leptin, which plays an important role in energy homeostasis, has been suggested to have an influence on bone development and remodeling. However, it is not clear from animal studies whether leptin is a stimulator or an inhibitor of bone growth. Cross-sectional studies in humans suggest that serum leptin levels are positively associated with bone mineral density (BMD), but these observations are not consistent, and whether this relationship is independent of obesity remains unclear. We therefore examined the effect of sc leptin administration on BMD and markers of bone turnover in two women, one with congenital generalized lipodystrophy and the other with acquired generalized lipodystrophy. Both patients had regular menstrual cycles. At baseline, the BMD for both patients, measured at the lumbar spine and total hip, was within 1 SD of the peak bone mass. There was no significant change in BMD in both patients after 16-18 months of leptin therapy. Similarly, concentrations of serum osteocalcin and bone-specific alkaline phosphatase or urinary excretion of deoxypyridinoline and N-telopeptides remained unchanged after 6-8 months of leptin therapy, suggesting no effects of leptin on osteoblastic or osteoclastic activity. Our preliminary data suggest that sc leptin replacement in hypoleptinemic patients with generalized lipodystrophy has no effect on the mature adult skeleton.

Pathogenesis of hypercalciuric nephrolithiasis.

The major contribution of hypercalciuria in raising urinary state of saturation with respect to calcium salts and subsequent risk of nephrolithiasis is appreciated. Derangements in the physiological mechanisms that regulate calcium homeostasis and contribute to hypercalciuria have also been identified. New avenues of research are beginning to explore the specific defects that may contribute to hypercalciuria. From such studies, an understanding of the role of certain dietary excesses as contributors to the development of hypercalciuria and, in some cases, attendant bone loss, is beginning. The contribution of genetics to hypercalciuria has provided a powerful means of identifying genes that contribute to the hypercalciuric phenotype in a number of hypercalciuric conditions. Such studies have disclosed that hypercalciuria is probably polygenic in nature and will require a concerted effort to better understand the defects while attempting to develop gene-specific countermeasures.

Nutrition and renal stone disease in space.

There is a growing body of evidence from the National Aeronautics and Space Administration and the Russian space program showing that humans exposed to the microgravity environment of space have a greater risk for developing renal stones. Increased bone resorption and the attendant hypercalciuria and hyperphosphaturia contribute significantly to raising the urinary state of saturation with respect to the calcium salts, namely calcium oxalate and calcium phosphate. In addition, other environmental and dietary factors may adversely affect urine composition and increase stone formation risk during space flight. For example, reductions in urinary volume, pH, and citrate contribute to raising stone formation risk. In addition to raising the risk for calcium stone formation, this metabolic profile is conducive to the formation of uric acid stones. Although observations to date have suggested that there may actually be a reduced food intake during the early phase of flight, crew members on longer-duration flights may increase food intake and be at increased risk for stone formation. Taken together, these findings support the use of nutritional recommendations for crew members that would serve to reduce the stone-forming propensity of the urinary environment. Pharmacologic intervention should be directed at raising urinary volumes, diminishing bone losses, and preventing reductions in urinary pH and citrate. Success in reducing the risk for stone formation in astronauts would also be of potential major benefit to the estimated 20 million Americans with nephrolithiasis.

The measurement of vitamin D: analytical aspects.

In the past quarter of a century, our understanding of the metabolism and mechanism of action of vitamin D has been elucidated. During this period, many metabolites of vitamin D have been identified and a small proportion of these assayed in blood. The ability to assay these vitamin D metabolites has led to a better appreciation of the pathological role that altered vitamin D metabolism plays in the development of diseases of calcium homeostasis. However, for many physicians it is not clear which vitamin D metabolites should be quantitated and what the information gained tells us. Of the four major circulating vitamin D metabolites in blood, only two, namely 25-hydroxyvitamin D (25OHD) and 1,25-dihydroxyvitamin D [1,25(OH)(2)D], have warranted measurement. Of these, the need for assessing serum 1,25(OH)(2)D is actually quite limited and should therefore not be considered as part of the standard vitamin D testing regimen. 25OHD, on the other hand, provides us with the single best assessment of vitamin D nutritional status and should be the only vitamin D assay typically ordered for this reason. Which of the many methods that are available should a laboratory use for quantitating either of these vitamin D metabolites? Early methods required large volumes of blood, organic solvent extractions, and extensive purification of the vitamin D metabolites prior to assay. Today, these time-consuming and costly methods have given way to a range of radioimmunoassays and enzyme-linked immunosorbent assays that can accurately and conveniently provide important information concerning an individual's vitamin D status. This review will consider when vitamin D measurements should be undertaken and how best to perform such assays.

Biphasic and dosage-dependent regulation of osteoclastogenesis by ß-catenin.

Wnt/ß-catenin signaling is a critical regulator of skeletal physiology. However, previous studies have mainly focused on its roles in osteoblasts, while its specific function in osteoclasts is unknown. This is a clinically important question because neutralizing antibodies against Wnt antagonists are promising new drugs for bone diseases. Here, we show that in osteoclastogenesis, ß-catenin is induced during the macrophage colony-stimulating factor (M-CSF)-mediated quiescence-to-proliferation switch but suppressed during the RANKL-mediated proliferation-to-differentiation switch. Genetically, ß-catenin deletion blocks osteoclast precursor proliferation, while ß-catenin constitutive activation sustains proliferation but prevents osteoclast differentiation, both causing osteopetrosis. In contrast, ß-catenin heterozygosity enhances osteoclast differentiation, causing osteoporosis. Biochemically, Wnt activation attenuates whereas Wnt inhibition stimulates osteoclastogenesis. Mechanistically, ß-catenin activation increases GATA2/Evi1 expression but abolishes RANKL-induced c-Jun phosphorylation. Therefore, ß-catenin exerts a pivotal biphasic and dosage-dependent regulation of osteoclastogenesis. Importantly, these findings suggest that Wnt activation is a more effective treatment for skeletal fragility than previously recognized that confers dual anabolic and anti-catabolic benefits.

Biochemical and histological assessment of alkali therapy during high animal protein intake in the rat.

The Westernized diet is acidogenic due to the high content of sulfur-containing amino acids and relative deficiency of potassium organic anions. Chronic acid loads result in hypercalciuria and negative calcium balance often associated with loss of bone mineral. Alkali therapy tends to reverse the hypercalciuria but little is known regarding its effect on bone as assessed by bone histomorphometry. The present study utilized dynamic bone histomorphometry to evaluate the effects of alkali therapy on acid-induced changes in bone turnover. Serum and urine analyses and bone histomorphometry were assessed in adult rats after 2 months of either a low casein (LC) or high casein (HC) diet supplemented with either potassium chloride (KCl) or potassium citrate (KCit). Compared to animals on LC-KCl diet, HC-KCl diet delivered a substantial acid load as shown by significant increases in urinary sulfate, ammonium, and net acid excretion, and a lower urinary pH and citrate excretion without detectable changes in serum parameters. The acid load also resulted in hypercalciuria. Dynamic and static bone histomorphometry disclosed a significant reduction in cancellous bone volume and trabecular number associated with a 2.5-fold increase in eroded and a 3.5-fold increase in osteoclastic surfaces. There was also a near 2-fold increase in bone formation rate in rats on the HC-KCl diet. When animals on the HC diet were given KCit instead of KCl, all of the aforementioned changes in urine biochemistry and bone turnover were significantly attenuated or entirely prevented. These findings underscore the deleterious effects of high animal protein intake in promoting hypercalciuria and increasing bone turnover. Co-administration of potassium alkali attenuates or prevents these changes. In this animal model of high dietary animal protein intake, the major skeletal effect of alkali therapy is to reduce bone resorption, with little or no effect on bone formation.

Blood biomarkers of vitamin D status.

In the past quarter century, more than 50 metabolites of vitamin D have been described. To date, only a few of these have been quantified in blood, but this has widened our understanding of the pathologic role that altered vitamin D metabolism plays in the development of diseases of calcium homeostasis. Currently, awareness is growing of the prevalence of vitamin D insufficiency in the general population in association with an increased risk of several diseases. However, for many researchers, it is not clear which vitamin D metabolites should be quantified and what the information gained from such an analysis tells us. Only 2 metabolites, namely, 25-hydroxyvitamin D [25(OH)D] and 1,25-dihydroxyvitamin D [1,25(OH)2D], have received the greatest attention. Of these, the need for measuring serum 1,25(OH)2D is limited, and this metabolite should therefore not be considered as part of the standard vitamin D testing regimen. On the other hand, serum 25(OH)D provides the single best assessment of vitamin D status and thus should be the only vitamin D assay typically performed. Currently, numerous formats exist for measuring serum 25(OH)D concentrations, each with its own advantages and disadvantages. This article reviews the currently available methods for serum 25(OH)D quantitation and considers important issues such as whether both the D2 and the D3 forms of the vitamin should be assayed, whether total or free concentrations are most important, and what measures should be taken to ensure the fidelity of the measurements.