Intraperitoneal pyrophosphate treatment reduces renal calcifications in Npt2a null mice.

Mutations in the proximal tubular sodium-dependent phosphate co-transporters NPT2a and NPT2c have been reported in patients with renal stone disease and nephrocalcinosis, however the relative contribution of genotype, dietary calcium and phosphate, and modifiers of mineralization such as pyrophosphate (PPi) to the formation of renal mineral deposits is unclear. In the present study, we used Npt2a-/- mice to model the renal calcifications observed in these disorders. We observed elevated urinary excretion of PPi in Npt2a-/- mice when compared to WT mice. Presence of two hypomorphic Extracellular nucleotide pyrophosphatase phosphodiesterase 1 (Enpp1asj/asj) alleles decreased urine PPi and worsened renal calcifications in Npt2a-/- mice. These studies suggest that PPi is a thus far unrecognized factor protecting Npt2a-/- mice from the development of renal mineral deposits. Consistent with this conclusion, we next showed that renal calcifications in these mice can be reduced by intraperitoneal administration of sodium pyrophosphate. If confirmed in humans, urine PPi could therefore be of interest for developing new strategies to prevent the nephrocalcinosis and nephrolithiasis seen in phosphaturic disorders.

One-pot synthesis of highly greenish-yellow fluorescent nitrogen-doped graphene quantum dots for pyrophosphate sensing via competitive coordination with Eu(3+) ions.

Highly fluorescent nitrogen-doped graphene quantum dots (N-GQDs) with greenish-yellow emission and quantum yield of 13.2% have been synthesized via a one-pot hydrothermal method. The obtained N-GQDs displayed excellent optical properties, high photostability and resistance to strong ion strength. Based on the higher affinity of pyrophosphate (PPi) than carboxyl and amido groups on the surface of the N-GQDs to Eu(3+), a Eu(3+)-modulated N-GQD off-on fluorescent probe for PPi detection was constructed with a detection limit of 0.074 µM. The detection process was simple in design, easy to operate, and showed a highly selective response to PPi in the presence of co-existing anions. This work widens the applications of N-GQDs with versatile functionality and reactivity in clinical diagnostics and as biosensors.

An anionic conjugated polymer as a multi-action sensor for the sensitive detection of Cu(2+) and PPi, real-time ALP assaying and cell imaging.

A Cu(2+) ensemble polyfluorene derivative, poly[5,5'-(((9H-fluorene-9,9-diyl)bis(hexane-6,1-diyl))bis(oxy))diisophthalate] sodium salt (PFT), displays unprecedented selectivity for PPi (LOD = 2.26 ppb) in aqueous solution as well as in random urine samples at physiological pH vis-a-vis monitoring ALP activity. Furthermore, intracellular imaging of Cu(2+) and PPi in mouse macrophage (J774A.1) and human breast cancer cells (MDA-MB231) was achieved to confirm the viability of PFT in biological systems.

Label-free fluorescence polarization detection of pyrophosphate based on 0D/1D fast transformation of CdTe nanostructures.

A novel and label-free fluorescence polarization (FP) method for the determination of pyrophosphate (PPi) is developed based on the change in FP signals during fast reversible transformation between CdTe zero-dimensional (0D) nanocrystals (NCs) and one-dimensional (1D) nanorods (NRs) induced by addition of PPi. Under optimum conditions, the FP ratio was linearly proportional to the logarithm of the concentration of PPi between 2.0 × 10(-5) and 1.0 × 10(-9) M with a detection limit of 8.0 × 10(-10) M. The developed method, with high signal selectivity and stability, was successfully applied to the detection of PPi in human urine samples.

Cysteamine CdS quantum dots decorated with Fe3+ as a fluorescence sensor for the detection of PPi.

A new sensitive and selective fluorescence sensor for the detection of pyrophosphate (PPi) in aqueous media based on the Fe(3+) decorated cysteamine CdS QDs ([Cys-CdS QDs]-Fe(3+)) was proposed. The presence of PPi can induce the fluorescence quenching of [Cys-CdS QDs]-Fe(3+) due to the high formation constants between the phosphate group and Fe(3+). Because the complex between Fe(3+) and PPi acts as an efficient quencher, the concentration of PPi can be evaluated by tracking the degree of fluorescence quenching. The fabricated sensor was optimized to obtain the best sensor selectivity and sensitivity. Under optimal conditions, a linear relationship between the fluorescence response and the concentration of PPi was established in the range of 0.5-10 µM. The limits of detection and quantitation for PPi were found to be 0.11 and 2.78 µM, respectively. Furthermore, the proposed sensor exhibited high selectivity toward PPi relative to other common anions. The proposed sensor was successfully applied to the detection of PPi in urine samples with satisfactory results.

Chronic stress and calcium oxalate stone disease: influence on blood cortisol and urine composition.

OBJECTIVE: To evaluate the influence of chronic stress (CS) on urine composition of calcium oxalate (CaOx) stone patients and controls. METHODS: This case-control study enrolled 128 patients during a period of 20 months. The cases were CaOx stone formers with a recent stone episode. Controls were matched by sex and age. Dimensions of CS were evaluated in cases and controls by validated self-report questionnaires measuring stressful life events, perceived stress, anxiety, depression, burnout, and satisfaction with life. Blood and urine samples were collected to determine cortisol levels and urinary composition. RESULTS: More relations between CS dimensions and blood and urine parameters were observed in cases than in controls. In cases, the blood cortisol level was related positively with the number of stressful life events (P = .03), intensity of these events (P = .04), and anxiety (P = .04). In addition, urinary magnesium (P = .03) and pyrophosphate (P = .05) levels were positively related with satisfaction with life and burnout, respectively. In contrast, urinary magnesium levels were negatively related with perceived stress (P = .01), anxiety (P = .016), and depression (P = .03). In controls, the number of stressful life events and the intensity of stressful life events was related positively with magnesium (P = .06, P = .02) levels and negatively with blood cortisol levels (P = .03, P = .004). CONCLUSION: Based on the variation between cases and controls in relations between CS dimensions and biochemical parameters, we hypothesize that CS may trigger a differential biological response in CaOx stone formers and controls, which in turn may promote or protect against CaOx stone formation.

A kinetic method for expeditious detection of pyrophosphate anions at nanomolar concentrations based on a nucleic acid fluorescent sensor.

A kinetic method has been developed for highly sensitive and selective detection of pyrophosphate anions (PPi) by using a fluorophore labelled single-stranded-DNA-Al(III) complex system. The facile and spectrum-tunable fluorescent sensor enables rapid detection of PPi in urine and cell lysate solutions without the need for complex pre-cleanup procedures.

Quantitative determination of the anticancer prodrug combretastatin A1 phosphate (OXi4503, CA1P), the active CA1 and its glucuronide metabolites in human urine and of CA1 in plasma by HPLC with mass spectrometric detection.

Validated methods for the determination of CA1, the active agent derived from the prodrug CA1P, in human plasma and urine, and of CA1P and three glucuronides CA1G1, CA1G2 and CA1DG in human urine were developed using LC-MS. Plasma CA1 was extracted using solid phase extraction and validated over the range 5-1000 nM. Urine samples were analysed without extraction, and the assays validated over the range 50-2000 nM (CA1P), 25-2000 nM (CA1), 50-40,000 nM (CA1G1 and CA1G2) and 25-4000 nM (CA1DG). The mean correlation coefficient (r²) was ≥ 0.997 for all assays. The intra-day and inter-day accuracy and precision were within the generally accepted criteria for bioanalytical methods (<15%). Mean recovery of CA1 from plasma was 101%, and 97% from urine. Mean urine recovery of CA1P was 98%, CA1G1 96%, CA1G2 93% and CA1DG 93%. The method was applied to plasma and urine samples from a recently completed clinical trial of the prodrug. Peak plasma concentrations of up to 470 nM CA1 were seen. The majority of drug-related material measured in urine comprised of the two monoglucuronides; CA1 and the diglucuronide were about 10-fold lower. No CA1P was detectable in urine.

Aggregation of freshly precipitated calcium oxalate crystals in urine of calcium stone patients and controls.

Aggregation (AGN) of freshly precipitated calcium oxalate crystals was photometrically studied in urine of 30 calcium stone patients and 30 controls, in solutions containing urinary macromolecules (UMS) and in an inhibitor free control solution (CS). Crystals were produced by oxalate titration and crystallization was monitored measuring optical density (OD). Tests were repeated adding hydroxyapatite (HAP) to urine and UMS and adding citrate and pyrophosphate (PPi) to UMS of the controls. AGN was recognized as a rapid OD decrease being at least three times faster than sedimentation of single crystals (p < 0.001) and used to calculate an extent of AGN (EA%). The time between the end of titration and the beginning of AGN was determined as suspension stability (SS). The main effect of urinary inhibitors was retardation of AGN without changing EA, SS being higher in urine than UMS (p < 0.001) and in UMS than CS (p < 0.001). In urine of 63% of controls but only in 33% of patients, no AGN was recorded (p < 0.05). The high inhibitory activity of urine could not be reproduced in UMS even in combination with 3.5 mM citrate or 0.05 mM PPi. 0.05 mg/mL HAP reduced SS in all urine samples to low values and increased the rate of rapid OD decrease, being a measure for the size of aggregates. Retarding AGN of crystals during their passage through the kidney seems to be an important mechanism to prevent stone formation during crystalluria. The promotion of AGN by HAP reveals a new role of Randall's plaques in nephrolithiasis.

Spiropyran-based fluorescent anion probe and its application for urinary pyrophosphate detection.

In recent decades, numerous spiropyran derivatives have been designed and utilized for optical sensing of metal ions. However, there is still less research on spiropyran-based anion sensors. In this work, a new spiropyran compound (L) appended with a pendant bis(2-pyridylmethyl)amine was synthesized and used in fluorescent sensing of pyrophosphate ion (PP(i)) in aqueous solution. The molecular recognition and signal transduction are based on the cooperative ligation interactions and the ligation-induced structural conversion of the spiropyran, which leads to a significant change in the photophysical property of the spiropyran. In an ethanol/water solution (30:70, v/v) at pH 7.4, ligation of L with Zn(2+) causes an intense fluorescence emission at 620 nm at the expense of the original fluorescence at 560 nm. Once PP(i) was introduced, interaction between PP(i) and the L-Zn(2+) complex leads to full quenching of the 620 nm band emission which was concomitant with recovery of the 560 nm band emission, and the fluorescence intensity ratio, F(560)/F(620), is proportional to the PP(i) concentration. Under the optimum condition, the L-Zn(2+) complex responds to PP(i) over a dynamic range of 1.0 x 10(-6) to 5.0 x 10(-4) M, with a detection limit of 4.0 x 10(-7) M. The fluorescence response is highly selective for PP(i) over other biologically related substrates, especially the structurally similar anions, such as phosphate and adenosine triphosphate. The mechanism of interaction among L, Zn(2+), and PP(i) was primarily studied by (1)H NMR, (31)P NMR, and HRMS. To demonstrate the analytical application of this approach, the PP(i) concentration in human urine was determined. It was on the order of 3.18 x 10(-5) M, and the mean value for urinary PP(i) excretion by three healthy subjects was 62.4 micromol/24 h.

Minimum handling method for the analysis of phosphorous inhibitors of urolithiasis (pyrophosphate and phytic acid) in urine by SPE-ICP techniques.

Pyrophosphate (PPi) and phytic acid (IP6) are natural phosphorous compounds with growing interest in the biomedical field due to their ability as potential inhibitors of urolithiasis among others. Existing methodologies for their evaluation show inconveniences mainly associated with sample treatment, matrix interferences and lack of resolution. The objective of the present work is the validation of a new method to determine both inhibitors in urine samples selectively and its application to the diagnosis of lithiasic patients. After urine purification by an off-line anion exchange solid phase extraction (SPE), based in an appropriate acidic elution gradient, the phosphorous compounds were analyzed by (31)P measurements by inductively coupled plasma mass spectrometry (ICP-MS) in the purified urine extracts. Linear range and limit of detection obtained were adequate for the analysis of the physiological amounts of the compounds in urine. The method was successfully applied to human urine samples, resulting in adequate accuracy and precision and allowing for the analysis of phosphorus inhibitors of urolithiasis in urine. The method simplicity and high sample throughput leads to a clear alternative to current determinations of the mentioned species in urine. Moreover, PPi and IP6 concentrations found in patients suffering from oxalocalcic urolithiasic were significantly lower than those for healthy controls, supporting the fact that the risk for oxalocalcic urolithiasis increases when urinary phosphorus inhibitors decrease. Thus, speciation of phosphorus inhibitors of urolithiasis in urine of stone formers can be performed, which is of unquestionable value in diagnostic, treatment and monitoring of urolithiasis.

[The effect of urinary tract calculosis to levels of low molecular inhibitors of crystallization in the urine].

The incidence of urinary tract calculosis continuously progresses. The triggering event in the process of stone formation is decreased urinary level of crystallizing inhibitors. The aim of our study was to investigate whether the existing stone or applied therapeutic procedure - extracorporeal shock waves lithotripsy (ESWL) - has effect to urinary levels of Mg, citrate and pyrophosphate. Study included 128 patients with the upper urinary tract stones. ESWL using the Lithostar (Siemens) device was used as a mode of treatment. Out of all patients, 76 (59%) were free of stone particles before 1 month, while 52 (41%) had residual stone fragments even 3 months after ESWL. Mg, citrate and pyrophosphate were measured in 24h-urine specimens: before, between days 2 and 3, as well as 1 and 3 months after ESWL. The analysis of the results revealed that stone itself had no effect on urinary crystallizing inhibitors. Detected increased urinary levels of Mg, citrate and pyrophosphate after ESWL, compared with pre-treatment values, could be attributed to applied therapeutic procedure.

Enzymatic determination of pyrophosphate in urine by flow methods.

Two flow methods for the enzymatic determination of pyrophosphate are described that are used to diminish the consumption of reagents. One method is based on the use of an open-close circuit with manual injection using a syringe. The other is a sequential injection method. The analytical features of both methods are: a linear range of 0.4 - 20 mg L(-1), an LOD of 0.38 mg L(-1), and a CV of 2.0% for the sequential injection method, and a linear range of 0.3 - 15 mg L(-1), an LOD of 0.29 mg L(-1), and CV of 2.2% for the open-close circuit method. The methods were applied to the determination of pyrophosphate in urine. The pyrophosphate concentration determined in urine samples varied from 1.26 to 6.67 mg L(-1).

Development and validation of a highly sensitive RIA for zoledronic acid, a new potent heterocyclic bisphosphonate, in human serum, plasma and urine.

Zoledronic acid is a new, highly potent bisphosphonate drug under clinical evaluation. A radioimmunoassay has been developed to determine zoledronic acid concentration in human serum, plasma, and urine. The assay utilizes rabbit polyclonal antisera against a zoledronic acid-BSA conjugate and a [125I]zoledronic acid derivative as tracer in a competitive format adapted to microtiter plates. The assay shows a LLOQ 0.4 ng/ml in serum or plasma (interassay%CV=17%, accuracy 97%), 5 ng/ml in urine (21%, 98%). In 23 patients receiving 4, 8 or 16 mg of zoledronic acid, drug concentrations in plasma were dose proportional and showed a multiphasic profile, followed by a prolonged gradual decline to concentrations near the LLOQ. Zoledronic acid disposition in plasma and the recovery of only 40-50% of the dose in urine are consistent with the rapid and extensive uptake by and slow release from bone in parallel with renal clearance, typically shown by bisphosphonates.

Synergism between the brushite and hydroxyapatite urinary crystallization inhibitors.

The aim of this paper is to study possible synergic effects between crystallization-inhibitor molecules of low molecular weight on the hydroxyapatite and brushite crystal nucleation. Kinetic-turbidimetric measurements were performed to follow the nucleation process in synthetic urine at 37 degrees C. Only pyrophosphate + phytate mixture manifested synergic effects on the brushite nucleation, whereas the mixture pyrophosphate + citrate exhibited synergic effects only on the hydroxyapatite nucleation. It seems clear that synergic effects between the crystallization inhibitory capacity of some substances in urine can take place and as a consequence, the high crystallization inhibitory capacity of healthy urine could be assigned not only to the individual inhibitory capacity of each product but also to the synergic effects between different products.

Determination of pyrophosphate in renal calculi and urine by means of an enzymatic method.

An enzymatic method for the determination of pyrophosphate which has been applied to renal calculi is described. The method involves the preconcentration of pyrophosphate using anionic exchange resin and development of the enzymatic reactions with the pyrophosphate retained on the resin. The study of calculi treatment according to calculi composition is also reported. The pyrophosphate content was dependent on the calculi composition. The highest amount of pyrophosphate was found in hydroxyapatite calculi (of the order of 10 microg/g), struvite and oxalate calculi showed a lower amount (the order was 2.5 and 4.5 microg/g, respectively) and was not detected in uric acid and cystine stones. The method was also successfully applied to the determination of pyrophosphate in human urine. For urinary pyrophosphate determination, a modification based on a clean-up of urine using activated carbon has been proposed. Pyrophosphate in human urine was of the order of 4 mg l(-1).

Development of a turbidimetric assay to study the effect of urinary components on calcium oxalate precipitation.

The pharmacological treatment of calcium urinary stones, most of which are made of calcium oxalate (CaOx), is only prophylactic. The causes of nephrolithiasis are often unclear, and a number of patients were found to be deficient in physiological inhibitors, e.g. citrate, pyrophosphate, magnesium, and specific proteins. The identification and characterization of these inhibitors can be performed in vitro by a number of methods, most of which are complex and time-consuming. Thus, we developed a simple turbidimetric method based on the precipitation of CaOx from a supersaturated solution. Using this approach, we determined that ionic strengths > 0.2 and pH < 5 inhibited the precipitation of CaOx. The first observation is of interest if one considers that the osmolarity of urine varies in the range of 50-1,400 mmol/kg, while the effect of pH is not usually seen in vivo because of the influence of other phenomena, such as the precipitation of uric acid. The activity of sodium chloride, magnesium and citrate was displayed at concentrations not far from their normal urinary level. Among several mono-, di- and tricarboxylic acids, like acetic, ascorbic, citric, isocitric, formic, fumaric, gluconic, glutaric, alpha-ketoglutaric, maleic, malic, malonic, propionic, pyruvic, succinic, and tartaric acid, only isocitric acid was more potent than citric acid. Pyrophosphate was a potent inhibitor in vitro, but its urinary level may not be sufficient for a significant effect in vivo. Amino acids like Ala, Arg, Asp, Glu, Gly, and Ser which are known to bind calcium, showed little activity. Work is in progress to search for new compounds potentially useful in the treatment of nephrolithiasis.

Pyrophosphate in synovial fluid and urine and its relationship to urinary risk factors for stone disease.

Inorganic pyrophosphate (PPi) measurement in urine and synovial fluid has been established using the PPi-dependent phosphorylation of fructose-6-phosphate and subsequent reduction of dihydroxyacetone phosphate by NADH. The assay is linear up to 200 mumol/L, easy to perform and gives results comparable to more complex methods. Daily urinary output of PPi was independently related to both age (P = 0.0014) and sex (P = 0.0002). Men had higher values than women and older individuals excreted greater amounts. Male stone formers, younger than 45 years, had lower values than age matched male controls (P = 0.012). Younger female stone formers also tended to have lower values. In stone formers' urine significant and independent correlations were found of PPi excretion with urine volume (P = 0.004) and with phosphate excretion (P = 0.008). Oxalate excretion and that of other urine constituents and the degree of supersaturation with common stone-forming salts were not correlated with PPi. PPi excretion was markedly elevated in the urine of two patients with hypophosphatasia. The PPi concentration in synovial fluid from painful, swollen knee joints was elevated, but unrelated to the presence or absence of PPi or urate crystals.

Urinary excretion of inorganic pyrophosphate by normal subjects and patients with renal calculi in north-western India and the effect of diclofenac sodium upon urinary excretion of pyrophosphate in stone formers.

24 h urinary pyrophosphate excretion was studied in 20 normal healthy subjects and 75 idiopathic stone formers from north-western regions of India. The mean 24-hour urinary excretion of pyrophosphate was significantly low in stone formers (50.67 +/- 2.16 mumol/24 h) as compared to that of normal subjects (71.46 +/- 5.46 mumol/24 h) (p less than 0.01). Diclofenac sodium, a non-steroidal anti-inflammatory agent, was administered 50 mg thrice daily for 1 week to 18 stone formers and 24-hour urinary pyrophosphate excretion was studied before and after drug therapy. The 24-hour urinary excretion of pyrophosphate increased from 54.32 +/- 21.40 to 78.31 +/- 28.03 mumol subsequent to diclofenac sodium therapy (p less than 0.01).