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.

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.

Measurement of inorganic pyrophosphate in biological fluids. Elevated levels in some patients with osteoarthritis, pseudogout, acromegaly, and uremia.

A rapid and relatively simple method for measurement of inorganic pyrophosphate (PPi) in biological samples has been described. The mean +/-SEM of plasma samples from 94 normal subjects was 1.8+/-0.06 muM, giving a normal range (99% confidence limits) of 0.16 - 3.40 mumol/liter. Analysis of 17 plasma samples in duplicate showed a standard deviation of 0.18, giving a 99% probability that a single determination of plasma PPi would be +/-0.68 muM of the true value. The mean PPi levels in plasma from subjects with osteoarthritis, pseudogout, acromegaly, and uremia were significantly greater than the normal mean (P < 0.01). Samples from rheumatoid arthritis showed PPi levels distributed about a mean identical to the normal mean. Plasma inorganic orthophosphate levels correlated positively with PPi levels in samples from normal subjects and in samples from patients with osteoarthritis, pseudogout, and uremia, but not with acromegaly. This correlation was statistically significant only in the normal samples and in those from patients with osteoarthritis.

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.

Time-dependent image quality using 99mTc-pyrophosphate.

Technetium-99m-labeled pyrophosphate has proved to be a useful skeletal-imaging agent. In this study, specific areas of the skeleton were imaged at times ranging from 1/2 to 6 1/2 hr after injection of 99mTc-pyrophosphate. Count ratios between abnormal and normal bone with respect to adjacent soft tissue were obtained for selected regions of interest on computer-stored scintillation camera images. The results show that image quality improves most rapidly from 1/2 to 2 hr, but further modest gain in quality does occur on views recorded between 2 and 6 hr. All lesions detected on the later images were also observed on the early ones and the ratios of uptake between abnormal and normal bone from computer-processed scintillation camera images did not change appreciably with time after the 1/2-hr images. Our results confirm the clinical impression that overall image quality is better on views obtained at least 3 hr after injection. Further delays in imaging beyond 3-4 hr after injection probably will not result in any appreciable gain in diagnostic accuracy.

Kinetics of 99mTc-labeled pyrophosphate and polyphosphate in man.

Thekinetic of 99mTc-labeled pyrophosphate were compared with those of polyphosphate in ten patients in a combined study. Both agents cleared from the blood in a biexpoential fashion. The clearance half-time of Exponent I was the same for both and was shorter than the clearance half-time of Exponent ii. Urinary excretion of both agents was the same during the first hour but during the next 3 hr Tc-pyrophosphate cleared at a slightly more rapid rate, resulting in lower blood background radioactivity. Both agents were bound loosely to plasma proteins, mainly to globulin fractions. The sensitivity of lesion detection was similar for both. Excellent bone images were obtained with both agents. The images with Tc-pyrophosphatewere consistently superior owing to the low blood background and they took less time to accumulate an identical number of counts from identical regions. With the amount of 99mTc-complex used, no hyocalcemia or tetany was noted, nor was there any significant effect on 1-hr serum levels of inorganic phosphours and alkaline phosphatase. Four hours after injection, 9.5% of the dose of Tc-pyrophosphate was circulating in blood, 31.7% was excreted in urine, and the remaining 58.8% was taken up by bone and other tissues. The corresponding values with Tc-polyphosphate were 12.5% in blood, 29.0% in urine, and 58.5% in bone and other tissues. Among the soft tissues, the genitourinary system is most consistently visualized. It is concluded that both Tc-pyroposphate and Tc-polyphosphate are excellent skeletal-imaging agents and that Tc-pyrophosphate appears slightly superior to Tc-polyphosphate.

The estimation of pyrophosphate in urine with uridine-5'-diphosphoglucose pyrophosphorylase.

The concentration of pyrophosphate in urine has been determined using the enzyme uridine-5'-diphosphoglucose pyrophosphorylase. The technique is relatively quick and easy to perform. Reproducibility of results was good, and results from recovery experiments were excellent. The mean concentration of pyrophosphate found in early morning urine samples from 14 normal adults was identical with that reported by other workers using an ion-exchange method to measure pyrophosphate. The pyrophosphate concentration was related approximately to the phosphate concentration.

An assay method for the measurement of urinary inhibitors of calcium phosphate formation.

An assay method has been developed for studying quantitatively the formation of calcium phosphate. The method will detect both substances which affect the solubility of calcium phosphate and substances which affect the crystal growth of the material. It is sensitive as or more sensitive than other methods described in the literature for detecting such compounds.

Isolation and identification of some urinary inhibitors of calcium phosphate formation.

Normal urine has been examined for substances which inhibit formation of calcium phosphate. A separation scheme involving ultrafiltration, precipitation, electrophoresis and paper chromatography was devised to isolate these substances. Contrary to what has been suggested in the literature for many years, the urines examined did not contain a potent unidentified inhibitor. The major anionic inhibitors were citric acid, pyrophosphate and isocitric acid. These substances together with a small contribution from the cations appeared to account for most, if not all, of the inhibitory activity of urine.

Raised urinary excretion of inorganic pyrophosphate in asymptomatic members of a hypophosphatasia kindred.

We report the screening of an Anglo-Welsh kindred in which two children were affected by different clinical forms of hypophosphatasia. Among the clinically normal adult members of the kindred, a raised urinary concentration of pyrophosphate was the commonest biochemical abnormality. The concentration of phosphate in serum was elevated in only one adult member of the kindred, the mother of the propositus. Consanguinity in this kindred suggests probable recessive inheritance, and the obligate heterozygotes each exhibited a low serum AP activity plus one other biochemical abnormality indicative of a carrier state.

The effect of urine, pyrophosphate, citrate, magnesium and glycosaminoglycans on the growth and aggregation of calcium oxalate crystals in vitro.

The effects of pyrophosphate, citrate, magnesium, heparin and chondroitin sulphate on the growth and aggregation of calcium oxalate crystals were measured at concentrations likely to be found in 1% urine. The degrees of inhibition of growth and of aggregation caused by these compounds were related to the effects of normal 1% urine on these processes. It was concluded that chondroitin sulphate is responsible for the major portion of the inhibitory effect of urine on crystal aggregation, but that the effect on crystal growth is probably due to the additive or synergistic effects of a number of urinary constituents.

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).

Effects of glycosaminoglycan polysulphate in the treatment of chondrocalcinosis.

The effect of intra-articular injections of glycosaminoglycan polysulphate (Arteparon) on pain, joint mobility, inflammatory reactions, cartilage calcification and the urinary excretion of inorganic pyrophosphate was studied in 12 patients with chondrocalcinosis. All cases were bilateral and the less affected homologous joint served as an untreated control. The patients were followed over a one-year period. The glycosaminoglycan polysulphate treatment brought about a significant reduction of pain (p less than 0.01) and improvement of joint mobility (p less than 0.001). This effect continued for the whole one-year follow-up period, but could not be seen in the control joints. After the treatment period of 2-7 weeks there was a marked decrease in cartilage calcification paralleled with an increase in the excretion of inorganic pyrophosphate.

[Inorganic pyrophosphates and parathormone in hypophosphatasia. Study of a family]. / Pyrophosphates inorganiques et parathormone dans l'hypophosphatasie. Etude d'une famille.

The serum concentration of parathormone is usually normal in hypophosphatasia, a rare disease with a defect of bone mineralisation and low serum alkaline phosphatase activity. Nevertheless there are three cases in the literature presenting a hyperparathyroidism with or without hypercalcemia. No anomaly of parathyroid was found at autopsy. The authors describe the first cases of hypophosphatasia with low serum concentration of parathormone and raise the possibility of a trouble in the calcium-parathormone feed-back. They also emphasize the interest of the urinary pyrophosphate excretion. Its increase seem to be the most constant and the most specific biological disorder.

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.

[The distribution of Sn2+ after application as a complex (author's transl)]. / Das Verhalten von Sn2+ nach seiner Applikation als Komplexsalz

On animal experiments was shown that Sn2+ after i.v. application with 113Sn as radioactiv indicator was deposited in the skeleton. Experiments were done with citrat-, tartrat-, pyrophosphat-, phosphat- and Sn2+/HCl-complexes. The importance of the results for the use of Sn2+-solutions in Nuclear Medicine for men are discussed.

Kinetics of various 99mTc-Sn-pyrophosphate compounds in the rat. I. In vivo studies.

The kinetic data of two different 99mTc-Sn-pyrophosphate compounds (compound A and B) were evaluated in non-adult rats. Only compound A concentrated in bone. Both compounds dispersed rapidly in the intravascular as well as the extravascular space. The plasma protein bond of both compounds increased with time after injection and impaired both the renal clearance of both compounds and the bone clearance of compound A. The renal clearance of both compounds was somewhat above that of 51Cr-EDTA. It is concluded that compound A and B is mainly excreted by glomerular filtration. About one fourth of the glomerular filtrate of compound B is reabsorbed and accumulated by the tubular cells.