A novel pharmacodynamic assay to evaluate the effects of crystallization inhibitors on calcium phosphate crystallization in human plasma.

Cardiovascular calcification (CVC) is a progressive complication of chronic kidney disease and a predictor of CV events and mortality. The use of biomarkers to predict CV risk and activities of potential or current treatment drugs in these patients could have a crucial impact on therapeutic approaches. Our aim was to develop a novel assay for measurement of the rate of calcium phosphate crystallization in human plasma and provide a tool to evaluate the effects of crystallization inhibitors. The efficacy of inhibitors was determined by adding inhibitory compounds (polyphosphates, fetuin-A, sodium thiosulfate or citrate) to control samples. The assay was additionally validated for SNF472, an experimental formulation of phytate being developed for the treatment of calciphylaxis and CVC in patients with end-stage renal disease (ESRD) undergoing hemodialysis (HD). The method was repeatable and reproducible. The plasma crystallization rate was reduced up to 80% in a concentration-dependent manner following treatment with inhibitors in vitro, among which SNF472 was the most potent. This method appears beneficial in evaluating and discriminating between inhibitory activities of compounds such as polyphosphates on calcium phosphate crystallization, which present a novel therapeutic approach to treat CVC in ESRD patients.

Dietary interventions for mineral and bone disorder in people with chronic kidney disease.

BACKGROUND: Chronic kidney disease-mineral and bone disorder (CKD-MBD) is a systemic dysfunction of mineral and bone metabolism in people with CKD. Recent research shows that phosphate retention plays a significant role in the development of CKD-MBD. Compared with drug therapies, dietary interventions may be simple, inexpensive and feasible for phosphate retention. However, there is little evidence to support these interventions. OBJECTIVES: Our objective was to assess the benefits and harms of any dietary intervention for preventing and treating CKD-MBD. SEARCH METHODS: We searched Cochrane Kidney and Transplant's Specialised Register to 27 August 2015 through contact with the Trials' Search Co-ordinator using search terms relevant to this review. We also searched the Chinese Biomedicine Database (CBM) (1976 to August 2015), China Knowledge Resource Integrated Database (CNKI) (1979 to August 2015), and VIP (1989 to August 2015). SELECTION CRITERIA: Randomised controlled trials (RCTs) and quasi-RCTs looking at dietary interventions for prevention or treatment of CKD-MBD were eligible for inclusion. DATA COLLECTION AND ANALYSIS: Two authors independently assessed the eligibility, methodological quality, and extracted data. Continuous outcomes (serum calcium level, serum phosphorus level, calcium × phosphate product, parathyroid hormone (PTH), fibroblast growth factor 23 (FGF-23) and alkaline phosphatase) were expressed as mean difference (MD) with 95% confidence interval (CI). Dichotomous outcomes (mortality) were expressed as risk ratio (RR) with 95% CI. We used a random-effects model to meta-analyse studies. MAIN RESULTS: Nine studies were included in this review which analysed 634 participants. Study duration ranged from 4 to 24 weeks. The interventions included calcium-enriched bread, low phosphorus intake, low protein intake, very low protein intake, post haemodialysis supplements and hypolipaemic diet. Only one study reported death; none of the included studies reported cardiovascular events or fractures. There was insufficient reporting of design and methodological aspects among the included studies to enable robust assessment of risk of bias.There was limited and low-quality evidence to indicate that calcium-enriched bread increased serum calcium (1 study, 53 participants: MD -0.16 mmol/L, 95% CI -0.51 to -0.31), decreased serum phosphorus (53 participants: MD -0.41 mmol/L, 95% CI -0.51 to -0.31) and decreased the calcium × phosphate product (53 participants: MD -0.62 mmol²/L², 95% CI -0.77 to -0.47).Very low protein intake was not superior to conventional low protein intake in terms of effect on serum phosphorus (2 studies, 41 participants: MD -0.12 mmol/L, 95% CI -0.50 to 0.25), serum calcium (MD 0.00 mmol/L, 95% CI -0.17 to 0.17), or alkaline phosphatase (MD -22.00 U/L, 95% CI -78.25 to 34.25). PTH was significantly lower in the very low protein intake group (2 studies, 41 participants: MD -69.64 pmol/L, 95% CI -139.83 to 0.54).One study reported no significant difference in the number of deaths between low phosphorus intake and normal diet (279 participants: RR 0.18, 95% CI 0.01 to 3.82). Low phosphorus intake decreased serum phosphorus (2 studies, 359 participants: MD -0.18 mmol/L, 95% CI -0.29 to -0.07; I(2) = 0%).One study reported post-haemodialysis supplements did not increase serum phosphorus compared to normal diet (40 participants: MD 0.12 mmol/L, 95% CI -0.24 to 0.49).One study reported low phosphorus intake plus lanthanum carbonate significantly decreased FGF-23 (19 participants: MD -333.80 RU/mL, 95% CI -526.60 to -141.00), but did not decrease serum phosphorus (19 participants: MD -0.10 mg/dL, 95% CI -0.38 to 0.58) or PTH (19 participants: MD 31.60 pg/mL, 95% CI -29.82 to 93.02). AUTHORS' CONCLUSIONS: There was limited low quality evidence to indicate that dietary interventions (calcium-enriched bread or low phosphorus/protein intake) may positively affect CKD-MBD by increasing serum calcium, decreasing serum phosphorus, the calcium × phosphate product and FGF-23. Large and well-designed RCTs are needed to evaluate the effects of various interventions for people with CKD-MBD.

Acute and 3-month effects of microcrystalline hydroxyapatite, calcium citrate and calcium carbonate on serum calcium and markers of bone turnover: a randomised controlled trial in postmenopausal women.

Ca supplements are used for bone health; however, they have been associated with increased cardiovascular risk, which may relate to their acute effects on serum Ca concentrations. Microcrystalline hydroxyapatite (MCH) could affect serum Ca concentrations less than conventional Ca supplements, but its effects on bone turnover are unclear. In the present study, we compared the acute and 3-month effects of MCH with conventional Ca supplements on concentrations of serum Ca, phosphate, parathyroid hormone and bone turnover markers. We randomised 100 women (mean age 71 years) to 1 g/d of Ca as citrate or carbonate (citrate-carbonate), one of two MCH preparations, or a placebo. Blood was sampled for 8 h after the first dose, and after 3 months of daily supplementation. To determine whether the acute effects changed over time, eight participants assigned to the citrate dose repeated 8 h of blood sampling at 3 months. There were no differences between the citrate and carbonate groups, or between the two MCH groups, so their results were pooled. The citrate-carbonate dose increased ionised and total Ca concentrations for up to 8 h, and this was not diminished after 3 months. MCH increased ionised Ca concentrations less than the citrate-carbonate dose; however, it raised the concentrations of phosphate and the Ca-phosphate product. The citrate-carbonate and MCH doses produced comparable decreases in bone resorption (measured as serum C-telopeptide (CTX)) over 8 h and bone turnover (CTX and procollagen type-I N-terminal propeptide) at 3 months. These findings suggest that Ca preparations, in general, produce repeated sustained increases in serum Ca concentrations after ingestion of each dose and that Ca supplements with smaller effects on serum Ca concentrations may have equivalent efficacy in suppressing bone turnover.

Vitamin D deficiency during winter months among an adult, predominantly urban, population in Northern Poland.

INTRODUCTION: Vitamin D is vital in the regulation of the calcium-phosphate metabolism, has a direct impact on the musculoskeletal system, and also affects numerous other systems. Widespread vitamin D deficiency and its detrimental effect on health have been reported globally. Data concerning vitamin D status in Polish adult population is scarce. MATERIAL AND METHODS: Ambulatory patients of an outpatient clinic in Gdansk were included in the study. Serum concentrations of 25(OH) D, parathyroid hormone (PTH), alkaline phosphatase (ALP), calcium and phosphorus were determined. In a questionnaire declared UVB exposure, dietary vitamin D and calcium intake, and health status of the subjects were assessed. Non- and parametric tests, logistic regression and population attributable risk were applied in data analysis. RESULTS: 448 adults were examined from February to mid-April 2012, 305 women and 143 men, aged 19 to 86 (mean 46.3 ± 14.9 years). Mean 25-hydroxyvitamin D concentration was 14.3 ± 6.6 ng/mL. 84.4% of subjects were vitamin D deficient (25(OH)D < 20 ng/mL); 13.2% presented insufficient (20-30 ng/mL), and 2.5% (or 11 subjects) sufficient 25(OH)D concentrations. Significantly higher 25(OH)D concentrations were found in subjects who reported more UVB exposure, supplemented vitamin D orally and those who declared more physical activity. 21% of subjects had elevated serum PTH concentration (i.e. > 62 pg/mL); mean parathormone was 48.6 ± 25.2 pg/mL. A linear correlation was found between the logarithm of PTH and logarithm of 25(OH)D concentrations (r = -0.21, p < 0.001). CONCLUSIONS: Results obtained here demonstrate the necessity of implementing a monitoring and prophylaxis programme of vitamin D deficiency in Poland.

Postprandial effects of calcium phosphate supplementation on plasma concentration-double-blind, placebo-controlled cross-over human study.

BACKGROUND: The aim of the present study was to examine the postprandial calcium and phosphate concentrations after supplementation with pentacalcium hydroxy-triphosphate (CaP). METHODS: Ten men participated in this double-blind, placebo-controlled, cross-over study. The participants were divided into two groups. One group consumed bread enriched with CaP (plus 1 g calcium/d) and the other group a placebo product for three weeks. After a two week wash-out, the intervention was switched between the groups for another three weeks. Blood samples were drawn at the beginning (single administration) and at the end (repeated administration) of the intervention periods at 0, 30, 60, 120, 180 and 240 min. Between 0 and 30 min, a test meal, with or without CaP was consumed. The plasma concentrations of calcium and phosphate were examined. One participant dropped out due to personal reasons. RESULTS: CaP supplementation resulted in a significantly higher plasma calcium concentration after 240 min compared to placebo. After repeated CaP administration, the AUC for the increment in plasma calcium concentration was significantly higher compared to placebo.After single and repeated CaP supplementation, plasma phosphate concentration significantly decreased after 30, 60, 120 and 180 min compared to 0 min. The placebo administration resulted in significant decreases after 30, 60 and 120 min compared to 0 min. CONCLUSION: Our results show that CaP contributes to an adequate calcium supply, but without increasing the plasma concentration of phosphate. TRIAL REGISTRATION: www.clinicaltrials.gov; NCT01296997.

[Secondary hyperparathyroidism and anemia. Effects of a calcimimetic on the control of anemia in chronic hemodialysed patients. Pilot Study]. / Hyperparathyroïdie secondaire et anémie. Effets d'un calcimimétique sur le contrôle de l'anémie des hémodialysés chroniques. Étude pilote.

UNLABELLED: The main cause of resistance to erythropoiesis-stimulating agents (ESA) used for treatment of anemia in chronic hemodialysed patients (CHP) is the iron deficiency, absolute or functional. Secondary hyperparathyroidism (SHPT) is a secondary factor of resistance. Indeed, it has been reported in the literature an improvement of anemia parameters after surgical parathyroidectomy (PTX). The objective of this study is to assess in CHP, the impact of the correction of SHPT by a calcimimetic, cinacalcet (CI), (which is considered as a pharmacological PTX) on the response to ESA, measured by the erythropoietin resistance index (ERI). Twenty-two CHP with severe SHPT documented by an intact parathyroid hormone (iPTH) above 800pg/mL were included in this prospective pilot study. Mineral bone metabolism, anemia and nutritional parameters were measured baseline and after 6 months of treatment by CI. The effect on anemia was assessed at the end of study by the ERI, the change in Hb concentration, and the proportion of patients with Hb levels above 11g/dL. RESULTS: At the end of study there was a significant decrease (M6 vs M0) in iPTH (1302 vs 674pg/mL or -48%, p=0.006), serum calcium (2.39 vs 2.15mmol/L or -10%), serum phosphate (2 vs 1.7mmol/L or -15%), serum calcium-phosphorus product (CaxP) (4.8 vs 3.8mmol(2)/L(2) or - 20% (p<0.05), and the number of patients with CaxP>4.4mmol(2)/L(2) (64 vs 32%, p<0.05). The level of bone alkaline phosphatase remained stable during the study (28 vs 27 IU/L). The Hb levels increased from 11 to 11.4g/dL, as did the proportion of patients whose Hb concentration reached 11g/dL or higher (50 vs 70%, p<0.05) without important change of the median weekly ESA dosis in the majority of patients, 18 cases (81%) vs four (19%). Two subgroups were identified from the median decreases in iPTH (delta iPTH) between M0 and M6, Group 1 (delta iPTH≥400pg/mL, n=10) and group 2 (delta iPTH<400pg/mL, n=12): in group 1, we found a correlation between the decrease in iPTH by CI and the stability or decrease in ERI (group 1), at comparable dose of dialysis, nutritional and iron intakes and inflammatory profiles; in group 2 without a significant effect of CI on PTH reduction the levels of ERI and ESA dosis were more elevated. CONCLUSION: A treatment by calcimimetic improves the control of anemia by ESA in CHP and interferes positively on a cause of secondary resistance to ESA represented by SHPT. The mechanism of these effects could be linked to the decreased of bone marrow fibrosis and inflammation and to the triptych formed by the reduction in iPTH, CaxP and phosphate.

Left ventricular function and calcium phosphate plasma levels in uraemic patients.

UNLABELLED: BACKGROUND. Recent investigations have focused on the pathogenetic role of disturbances of calcium phosphate metabolism in causing cardiovascular morbidity and mortality in haemodialysis patients. The aim of the present study was to assess left ventricular function and its relationship to phosphate and calcium plasma levels in stable uraemic patients on haemodialysis treatment. METHODS: Twenty uraemic patients (mean age 51+/-13 years) on maintenance haemodialysis and free from overt cardiac dysfunction, and 20 healthy volunteers underwent standard echocardiography, tissue Doppler-derived early (E(m)) and late (A(m)) diastolic velocities, tissue characterization with cyclic variations of integrated backscatter (CV-IBS), and serum biochemistry. RESULTS: With respect to tissue Doppler imaging (TDI), uraemic patients showed a lower E(m) peak, a higher A(m) peak, and a reduced E(m)/A(m) ratio of both interventricular septum and lateral wall (0.01>P<0.001) than controls. CV-IBS of both septum and posterior wall was significantly smaller in uraemic patients than in the control subjects (P<0.001). Moreover, the E(m)/A(m) ratio of septum and lateral wall were negatively related to serum phosphorus and to calcium phosphate product (P<0.001 for all). Accordingly, an inverse relationship was also found between CV-IBS of septum and lateral wall and calcium phosphate product and phosphorus (P<0.05 for all). CONCLUSIONS: These results showed early cardiac impairment of diastolic myocardial function evaluated by TDI and IBS analysis, and a close relationship between these changes and the calcium-phosphate plasma levels. These findings are well in keeping with the important role of hyperphosphataemia as a risk factor for cardiovascular damage, and justify the effort for optimal control of calcium phosphate metabolism in uraemic patients.

An open-label, crossover study of a new phosphate-binding agent in haemodialysis patients: ferric citrate.

BACKGROUND: Hyperphosphataemia contributes to secondary hyperparathyroidism and renal osteodystrophy in patients with end-stage renal disease (ESRD). Calcium salts are widely employed to bind dietary phosphate (P) but they may promote positive net calcium balance and metastatic calcification. We recently reported that ferric compounds bind intestinal phosphate in studies of normal and azotemic rats. METHODS: To extend this observation, we performed an open-label, random order, crossover comparison study of ferric citrate and calcium carbonate in haemodialysis patients from two teaching hospitals. The study sample consisted of 23 women and 22 men with an average age of 52.5 +/- 11.8 (SD) years and an average weight of 54.5 +/- 10.7 kg. All forms of iron therapy were discontinued. Two weeks before the study, patients were instructed to discontinue all P-binding agents. The patients were randomly assigned to receive either calcium carbonate (3 g/day) or ferric citrate (3 g/day) for 4 weeks followed by a 2 week washout period, and then crossed over to the other P-binding agent for 4 weeks. RESULTS: From a baseline concentration of 5.6 +/- 1.5 mg/dl, the serum P increased during the washout period to 7.2 +/- 1.9 mg/dl prior to calcium carbonate treatment, and to 6.7 +/- 1.9 mg/dl prior to ferric citrate treatment. The serum P concentration fell significantly during treatment with both calcium carbonate (7.2 +/- 1.9 to 5.2 +/- 1.5 mg/dl, P<0.0001) and ferric citrate (6.7 +/- 1.9 to 5.7 +/- 1.6 mg/dl, P<0.0001). The results were not influenced by order of treatment. Under the conditions of the study protocol, ferric citrate was less effective than calcium carbonate at lowering the serum phosphate concentration. The serum Ca concentration increased during treatment with calcium carbonate but not ferric citrate. Ferric citrate treatment did not affect the serum concentration of aluminium. Ferric citrate treatment was associated with mild and generally tolerable gastrointestinal symptoms. CONCLUSION: Ferric citrate shows promise as a means of lowering the serum phosphate concentration in haemodialysis patients. Further studies are needed to find the optimal dose.

A new method for radioiodination of polysaccharides and its use in biodistribution studies of an immunomodulating glycoconjugate (Immunoferon).

A new method for the labeling of polysaccharides by using tyrosine derivatives after CNBr activation is presented, along with its use for pharmacokinetic studies of an orally active immunomodulating noncovalent glycoconjugate, which was administered labeled either in the polysaccharide or the protein moiety. The relationship between the obtained results and the mechanism of action are also discussed.

[Calcium phosphate metabolism in thalassemia]. / Metabolismo calciofosforico nella talassemia.

The AA. performed a screening test on 113 patients affected by beta thalassemia major ranging between 2 and 40 years of age, randomized among those who come to the Microcitemic Center of our Institute, and on a control group. In all of them serum levels of calcium, phosphate, parathyroid hormone (PTH), calcitonin and 25-OH vitamin D were measured. Average serum levels of PTH were significantly (P < 0.001) lower in our patients than in control group and 12.4% of the former were clearly under normal range, especially in the group over 16 years of age. Also serum levels of 25-OH vitamin D were lower in thalassemic patients than in controls, because of the presence of 32 patients with average values under normal limit. Our results are in agreement with current literature and underline the increasing incidence of endocrine complications in thalassemic patients who undergo high transfusion regimens, because of to the increase of hemosiderosis due to the low compliance to iron chelation therapy. Controversial is the pathogenesis of the absence of hypocalcemia in many patients with hypoparathyroidism and the cause of the deficit of vitamin D.

Association of serum phosphorus and calcium x phosphate product with mortality risk in chronic hemodialysis patients: a national study.

Elevated serum phosphorus is a predictable accompaniment of end-stage renal disease (ESRD) in the absence of dietary phosphate restriction or supplemental phosphate binders. The consequences of hyperphosphatemia include the development and progression of secondary hyperparathyroidism and a predisposition to metastatic calcification when the product of serum calcium and phosphorus (Ca x PO4) is elevated. Both of these conditions may contribute to the substantial morbidity and mortality seen in patients with ESRD. We have analyzed the distribution of serum phosphorus in two large national, random, cross-sectional samples of hemodialysis patients who have been receiving dialysis for at least 1 year. Data were obtained from two special studies of the United States Renal Data System, the Case Mix Adequacy Study (1990) and the Dialysis Morbidity and Mortality Study Wave 1 (1993). The relative risk of death by serum phosphorus quintiles is described after adjusting for age at onset of ESRD, race, sex, smoking status, and the presence of diabetes, the acquired immunodeficiency syndrome, and/or neoplasm. Logistic regression analysis is then used to describe the demographic, comorbid, and laboratory parameters associated with high serum phosphorus. Serum phosphorus was similar in these two study populations and averaged 6.2 mg/dL. Ten percent of patients had levels greater than 9 mg/dL and at least 30% of each group had serum phosphorus levels greater than 7 mg/dL. The adjusted relative risk of death by serum phosphorus level was not uniform across all quintiles, being constant below a level of 6.5 mg/dL and increasing significantly above this level. The relative risk of death for those with a serum phosphorus greater than 6.5 mg/dL was 1.27 relative to those with a serum phosphorus of 2.4 to 6.5 mg/dL. This increased risk was not diminished by statistical adjustment for coexisting medical conditions, delivered dose of dialysis, nutritional parameters, or markers of noncompliance. Evaluation of predictors of serum phosphorus greater than 6.5 mg/dL revealed in multivariate analysis that younger age at onset of ESRD, female sex, white race, diabetes, active smoking, and higher serum creatinine levels were all significant predictors. Analysis of serum calcium revealed no correlation with relative risk of death. The Ca x PO4 product, however, showed a mortality risk trend similar to that seen with serum phosphorus alone. Those in the highest quintile of the Ca x PO4 product (>72 mg2/dL2) had a relative mortality risk of 1.34 relative to those with products of 42 to 52 mg2/dL2. The relative mortality risk by log parathyroid hormone (PTH) level was elevated for patients with higher levels, but the mortality risk associated with hyperphosphatemia was independent of PTH. For hemodialysis patients who have been receiving dialysis for at least 1 year, we conclude that a large percentage have a serum phosphorus level above 6.5 mg/dL and that this places them at increased risk of death. This increased risk is independent of PTH. The mechanism(s) responsible for death is unknown, but may be related to an abnormally high Ca x PO4 product. Although mechanisms are not clearly established, this study supports the need for vigorous control of hyperphosphatemia to improve patient survival.

The effects of high-dose glucocorticoid administration on serum bone gamma carboxyglutamic acid-containing protein, serum alkaline phosphatase and vitamin D metabolites in normal subjects.

The effects of 40 mg of prednisone given daily for 5 days to normal individuals on serum levels of bone Gla-protein (BGP), alkaline phosphatase, calcium phosphate, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D and immunoreactive parathyroid hormone (S-iPTH) and on renal excretions of calcium, phosphate and hydroxyproline were evaluated in a double-blind, placebo controlled study. In the prednisone group a 75% decrease (P less than 0.001) was found in serum BGP compared to a 6% decrease (P less than 0.05) in serum alkaline phosphatase. The renal hydroxyproline excretion remained unchanged. Serum calcium was unchanged while the fasting urinary calcium excretion showed a 2-fold increase (P less than 0.001). Serum 1,25-dihydroxyvitamin D increased (P less than 0.01) in spite of unchanged serum 25-hydroxyvitamin D, serum phosphate and parathyroid function (as judged by S-iPTH and the maximal tubular reabsorption capacity for phosphate (TmP/GFR]. The data suggest a direct inhibition of osteoblast number and/or function by short-term glucocorticoid administration with unchanged bone resorption leading to a negative bone mineral balance. The increase in serum 1,25-dihydroxyvitamin D is probably due to a direct stimulation by glucocorticoids of the renal 1 alpha-hydroxylase. The effects of the vitamin D metabolite, however seem to be blunted.

Sudden fatal pulmonary calcification following renal transplantation.

A 42-year-old male was hemodialyzed for 2 years with excellent control of calcium-phosphate metabolism. He received a cadaveric renal transplant but experienced a prolonged episode of acute tubular necrosis during which he could not tolerate phosphate-binding antacids. His calcium X phosphate product became markedly elevated for 20 days. Following a brief period of function, the homograft was removed on the 45th post-transplant day after severe rejection and subsequent infection. Chest X-ray was normal. Six days after graft nephrectomy, he became acutely dyspneic and markedly hypoxemic. Diffuse, flocculent pulmonary infiltrates appeared on the chest film. The patient expired 1 day later. At postmortem examination, there was severe, diffuse pulmonary alveolar calcification demonstrated by chemical and histologic examination. Although unlikely, the prolonged post-transplant period characterized by elevated calcium X phosphate product may have played a pathogenetic role. Calciphylaxis may have occurred, with hyperparathyroidism as the sensitizing agent and any of several drugs acting as challenger.