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Pseudoxanthoma elasticum (PXE) is a rare inherited systemic disease responsible for a juvenile peripheral arterial calcification disease. The clinical diagnosis of PXE is only based on a complex multi-organ phenotypic score and/or genetical analysis. Reduced plasma inorganic pyrophosphate concentration [PPi]p has been linked to PXE. In this study, we used a novel and accurate method to measure [PPi]p in one of the largest cohorts of PXE patients, and we reported the valuable contribution of a cutoff value to PXE diagnosis. Plasma samples and clinical records from two French reference centers for PXE (PXE Consultation Center, Angers, and FAVA-MULTI South Competent Center, Nice) were assessed. Plasma PPi were measured in 153 PXE and 46 non-PXE patients. The PPi concentrations in the plasma samples were determined by a new method combining enzymatic and ion chromatography approaches. The best match between the sensitivity and specificity (Youden index) for diagnosing PXE was determined by ROC analysis. [PPi]p were lower in PXE patients (0.92 ± 0.30 µmol/L) than in non-PXE patients (1.61 ± 0.33 µmol/L, p < 0.0001), corresponding to a mean reduction of 43 ± 19% (SD). The PPi cutoff value for diagnosing PXE in all patients was 1.2 µmol/L, with a sensitivity of 83.3% and a specificity of 91.1% (AUC = 0.93), without sex differences. In patients aged <50 years (i.e., the age period for PXE diagnosis), the cutoff PPi was 1.2 µmol/L (sensitivity, specificity, and AUC of 93%, 96%, and 0.97, respectively). The [PPi]p shows high accuracy for diagnosing PXE; thus, quantifying plasma PPi represents the first blood assay for diagnosing PXE.

Objective: The pathogenesis of calcinosis cutis, a disabling complication of SSc, is poorly understood and effective treatments are lacking. Inorganic pyrophosphate (PPi) is a key regulator of ectopic mineralization, and its deficiency has been implicated in ectopic mineralization disorders. We therefore sought to test the hypothesis that SSc may be associated with reduced circulating PPi, which might play a pathogenic role in calcinosis cutis., Methods: Subjects with SSc and age-matched controls without SSc were recruited from the outpatient rheumatology clinics at Rutgers and Northwestern Universities (US cohort), and from the Universities of Szeged and Debrecen (Hungarian cohort). Calcinosis cutis was confirmed by direct palpation, by imaging or both. Plasma PPi levels were determined in platelet-free plasma using ATP sulfurylase to convert PPi into ATP in the presence of excess adenosine 5' phosphosulfate., Results: Eighty-one patients with SSc (40 diffuse cutaneous, and 41 limited cutaneous SSc) in the US cohort and 45 patients with SSc (19 diffuse cutaneous and 26 limited cutaneous SSc) in the Hungarian cohort were enrolled. Calcinosis was frequently detected (40% of US and 46% of the Hungarian cohort). Plasma PPi levels were significantly reduced in both SSc cohorts with and without calcinosis (US: P = 0.003; Hungarian: P < 0.001)., Conclusions: Circulating PPi are significantly reduced in SSc patients with or without calcinosis. Reduced PPi may be important in the pathophysiology of calcinosis and contribute to tissue damage with chronic SSc. Administering PPi may be a therapeutic strategy and larger clinical studies are planned to confirm our findings., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Trauma-induced calcification is the pathological consequence of complex injuries which often affect the central nervous system and other parts of the body simultaneously. We demonstrated by an animal model recapitulating the calcification of the above condition that adrenaline transmits the stress signal of brain injury to the calcifying tissues. We have also found that although the level of plasma pyrophosphate, the endogenous inhibitor of calcification, was normal in calcifying animals, it could not counteract the acute calcification. However, externally added pyrophosphate inhibited calcification even when it was administered after the complex injuries. Our finding suggests a potentially powerful clinical intervention of calcification triggered by polytrauma injuries which has no effective treatment., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

The present work constructs a sequentially triggered nanoswitch (STN) for sequential detection of Cr 3+ , P 2 O 7 4- (PPi) and alkaline phosphatase (ALP) depending on polythymine (T40) templated fluorescent Cu nanoparticles (Cu NPs). A significant phenomenon is that Cr 3+ can only causing 5% QE of fluorescent Cu NPs synthesized by lower than 500 μM Cu 2+ , but the fluorescence of the Cu NPs synthesized by more than 500 μM Cu 2+ can be quenched up to 90% QE by the same concentration of Cr 3+ . Then the quenched fluorescence of CuNP-Cr 3+ complex provides a sensing platform for PPi due to the strong binding between Cr 3+ and PPi, resulting in dissociation of Cr 3+ from the surface of Cu NPs and the recovery of fluorescence emission. Further ALP hydrolysis of PPi disrupts Cr 3+ -PPi assemble and Cr 3+ is released to interact with Cu NPs, which induces fluorescence quenching again. Thus, sequentially detection of Cr 3+ (LOD, 0.03 μM), PPi (LOD, 0.005 μM) and ALP (LOD, 0.125 mU/mL) was successfully implemented with high sensitivity and selectivity. The sensor is also successfully used for Cr 3+ , PPi and ALP assays in the human serum. Additionally, the sensitive "on-off-on-off" sensing behavior of the Cu NPs allow three chemical inputs (Cr 3+ , PPi and ALP) to construct a logic gate., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Herein, a zinc ion (Zn 2+ )-triggered aggregation induced emission enhancement (AIEE) fluorescence "on-off-on" nanoswitch was fabricated for inorganic pyrophosphate (PPi) and inorganic pyrophosphatase (PPase) activity detection. Dual ligand functionalized Au NCs were utilized as the substrate of the AIEE nanoswitch. The introduction of Zn 2+ can cause Au NCs aggregated along with the enhanced fluorescence. After the addition of PPi, aggregated Au NCs disaggregated along with decreased fluorescence due to the competitive combination between PPi and Zn 2+ (on-off). When PPase was introduced, PPi was hydrolyzed and release Zn 2+ , resulting in aggregated Au NCs along with enhanced fluorescence again (off-on). On the basis of this, highly selective and sensitive detection PPi (liner range from 0.1 to 300 μM) and PPase activity (liner range from 0.1 to 10 mU) can be achieved. The detection limits are 0.04 μM for PPi and 0.03 mU for PPase, respectively. Furthermore, the as-prepared Zn 2+ -triggered AIEE nanoswitch was successfully used for quantitative analysis of PPase activity in human serum with satisfactory spiked recoveries, and applied for the inhibitors screening., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A novel "on-off-on" super-sensitive conjugated polymer fluorescence sensor (PPE-DPA) was developed and it was applied to realize the continuous recognition of Cu 2+ and pyrophosphate (PPi). The fluorescence intensity decreased linearly with the change of Cu 2+ from 0.05 to 5.0 μmol L -1 and the limit of detection was 24 nmol L -1 . The fluorescence intensity was linearly enhanced with the increase of PPi from 0.5 to 12.0 μmol L -1 and the limit of detection was 230 nmol L -1 . In addition, this method was applied to detect PPi in the blood serum and synovial fluid of patients with arthritis and satisfactory results were obtained. Thus, the PPE-DPA is not only an effective tool for detecting Cu 2+ and PPi in samples, but also presents a potential way to diagnose arthritis.

Background: It is challenging to find an iron compound that combines good bioavailability with minimal sensory changes when added to seasonings or condiments. Ferric pyrophosphate (FePP) is currently used to fortify bouillon cubes, but its bioavailability is generally low. Previously, the addition of a stabilizer, sodium pyrophosphate (NaPP), improved iron bioavailability from a bouillon drink., Objective: We assessed whether there is a dose-response effect of added NaPP on iron bioavailability from local meals prepared with intrinsically labeled FePP-fortified bouillon cubes in young Nigerian women using iron stable isotope techniques., Methods: In a double-blind, randomized, cross-over trial, women (n = 24; aged 18-40 y; mean BMI 20.5 kg/m2) consumed a Nigerian breakfast and lunch for 5 d prepared with bouillon cubes containing 2.5 mg 57Fe (as FePP) and 3 different molar ratios of NaPP: 57Fe (0:1, 3:1, and 6:1). Iron bioavailability was assessed by measuring 57Fe incorporation into erythrocytes 16 d after each 5 d NaPP: 57Fe feeding period. Data were analyzed using a linear regression model of log iron absorption on NaPP ratio, with body weight and baseline body iron stores as covariates and subject as a random intercept., Results: Of the women included, 46% were anemic and 26% were iron deficient. Iron bioavailability was 10.8, 9.8, and 11.0% for the 0:1, 3:1, and 6:1 NaPP:57Fe treatments, respectively. There was no dose-response effect of an increasing NaPP:57Fe ratio (β ± SE: 0.003 ± 0.028, P = 0.45)., Conclusions: In this study, the addition of NaPP did not increase iron bioavailability from FePP-fortified bouillon cubes. However, iron bioavailability from the Nigerian meals prepared with FePP-fortified bouillon cubes was higher than expected. These results are encouraging for the potential of bouillon cubes as a fortification vehicle. Further studies are needed to assess the effect of FePP-fortified bouillon cubes on improving iron status in low-income populations. This trial was registered at clinicaltrials.gov as NCT02815449., (Copyright © American Society for Nutrition 2019.)

Ectopic mineralization is a global problem and leading cause of morbidity and mortality. The pathomechanisms of ectopic mineralization are poorly understood. Recent studies on heritable ectopic mineralization disorders with defined gene defects have been helpful in elucidation of the mechanisms of ectopic mineralization in general. The prototype of such disorders is pseudoxanthoma elasticum (PXE), a late-onset, slowly progressing disorder with multisystem clinical manifestations. Other conditions include generalized arterial calcification of infancy (GACI), characterized by severe, early-onset mineralization of the cardiovascular system, often with early postnatal demise. In addition, arterial calcification due to CD73 deficiency (ACDC) occurs late in life, mostly affecting arteries in the lower extremities in elderly individuals. These three conditions, PXE, GACI, and ACDC, caused by mutations in ABCC6, ENPP1, and NT5E, respectively, are characterized by reduced levels of inorganic pyrophosphate (PPi) in plasma. Because PPi is a powerful antimineralization factor, it has been postulated that reduced PPi is a major determinant for ectopic mineralization in these conditions. These and related observations on complementary mechanisms of ectopic mineralization have resulted in development of potential treatment modalities for PXE, including administration of bisphosphonates, stable PPi analogs with antimineralization activity. It is conceivable that efficient treatments may soon become available for heritable ectopic mineralization disorders with application to common calcification disorders., (Copyright © 2019 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Pseudoxanthoma elasticum (PXE), a prototype of heritable ectopic mineralization disorders, is caused by mutations in the ABCC6 gene encoding a putative efflux transporter ABCC6. It was recently shown that the absence of ABCC6-mediated adenosine triphosphate release from the liver and, consequently, reduced inorganic pyrophosphate levels underlie the pathogenesis of PXE. Given that tissue-nonspecific alkaline phosphatase (TNAP), encoded by ALPL, is the enzyme responsible for degrading inorganic pyrophosphate, we hypothesized that reducing TNAP levels either by genetic or pharmacological means would lead to amelioration of the ectopic mineralization phenotype in the Abcc6 -/- mouse model of PXE. Thus, we bred Abcc6 -/- mice to heterozygous Alpl +/- mice that display approximately 50% plasma TNAP activity. The Abcc6 -/- Alpl +/- double-mutant mice showed 52% reduction of mineralization in the muzzle skin compared with the Abcc6 -/- Alpl +/+ mice. Subsequently, oral administration of SBI-425, a small molecule inhibitor of TNAP, resulted in 61% reduction of plasma TNAP activity and 58% reduction of mineralization in the muzzle skin of Abcc6 -/- mice. By contrast, SBI-425 treatment of Enpp1 mutant mice, another model of ectopic mineralization associated with reduced inorganic pyrophosphate, failed to reduce muzzle skin mineralization. These results suggest that inhibition of TNAP might provide a promising treatment strategy for PXE, a currently intractable disease., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Sensing of pyrophosphate (PPi) is helpful to better understand many life processes and diagnose various early-stage diseases. However, many traditional reported methods based on artificial receptors for sensing of PPi exhibit some disadvantages including difficulties in designing appropriate binding sites and complicated multi-step assembly/functionalization. Thus, it is significantly important and a big challenge to know how to use a simple molecular self-assembly or an interaction system to solve the above-mentioned limits to achieve the quantitative analysis of specific substances in the system. Based on the natural connection and similarity (such as stimulus responsiveness) between sensing and logic computing, in this study, the Boolean logic tree of molecular self-assembly system based on the cobalt oxyhydroxide (CoOOH) nanoplatform is constructed and applied to organize and connect "plug and play" molecular events (fluorescent dye, acridine orange and anion, PPi). By using molecules as inputs and the corresponding fluorescence signal as the output, the CoOOH-based molecular self-assembly system can be programmed for three-input fluorescent Boolean logic computation, fluorescent three-state logic computation, detection of PPi (linear range from 50 to 6400 nM with a detection limit of 20 nM) and even for imaging in living cancer cells and in vivo (in systems such as Zebrafish and Carassius auratus). Our approach adds a new dimension for expanding molecular logic computing and sensing systems, which will not only provide more opportunities for developing novel logic computing paradigms, but also be helpful in promoting the development and applications of intelligent molecular computing and sensing systems.

Generalized arterial calcification of infancy (GACI) is a rare, life-threatening disorder caused by loss-of-function mutations in the gene encoding ectonucleotide pyrophosphatase phosphodiesterase 1 ( ENPP1 ), which normally hydrolyzes extracellular ATP into AMP and pyrophosphate (PP i ). The disease is characterized by extensive arterial calcification and stenosis of large- and medium-sized vessels, leading to vascular-related complications of hypertension and heart failure. There is currently no effective treatment available, but bisphosphonates - nonhydrolyzable PP i analogs - are being used off-label to reduce arterial calcification, although this has no reported impact on the hypertension and cardiac dysfunction features of GACI. In this study, the efficacy of a recombinant human ENPP1 protein therapeutic (rhENPP1) was tested in Enpp1 asj-2J homozygous mice ( Asj-2J or Asj-2J hom), a model previously described to show extensive mineralization in the arterial vasculature, similar to GACI patients. In a disease prevention study, Asj-2J mice treated with rhENPP1 for 3 weeks showed >95% reduction in aorta calcification. Terminal hemodynamics and echocardiography imaging of Asj-2J mice also revealed that a 6-week rhENPP1 treatment normalized elevated arterial and left ventricular pressure, which translated into significant improvements in myocardial compliance, contractility, heart workload and global cardiovascular efficiency. This study suggests that ENPP1 enzyme replacement therapy could be a more effective GACI therapeutic than bisphosphonates, treating not just the vascular calcification, but also the hypertension that eventually leads to cardiac failure in GACI patients., Competing Interests: Competing interestsS.V., A.A. and H.M. are employees of Alexion Pharmaceuticals. T.K., K.W.S., M.C.L., A.M. and K.L.A. are former employees of Alexion Pharmaceuticals. All authors have equity ownership in Alexion., (© 2018. Published by The Company of Biologists Ltd.)

Intrinsically fluorescent polymer nanoparticles (F-PNPs) were synthetized from 2-hydroxy-5-methylisophthalaldehyde and melamine by solvothermal method. F-PNPs can emit strong yellow green fluorescence at 542 nm without the conjugation to any external fluorescent agent and surface modification. Owing to the abundant amino and hydroxyl groups on their surface, the F-PNPs possess multiple binding sites, good biocompatibility and excellent water-solubility. Addition of Zn 2+ to the F-PNPs solution resulted in a blue shift (Δλ=40 nm) with obvious enhancement in the fluorescence intensity at 502 nm; while there was negligible change in the presence of other metal ions. The subsequent treatment with pyrophosphate (PPi) can cause fluorescence recovery of F-PNPs by pulling the Zn 2+ out of the coordination cavity of F-PNPs-Zn 2+ nanocomposites. No interference was observed from other anions and nucleotides, making the F-PNPs-Zn 2+ ensembles highly sensitive and selective nanoprobes for PPi. The detection limit is 2.75 × 10 -8 M/L and 7.63 × 10 -8 M/L for Zn 2+ and PPi, respectively. The proposed nanoprobes were then used for detecting the recovery of Zn 2+ and PPi in rabbit serum samples, which were found to be 99.4-104.2% and 98.6-104.7%, respectively. The present strategy for the fabrication of nanoparticles may offer a new sight for the preparation of polymer nanostructures. The F-FNPs based probes can provide an accurate method for the detection of Zn 2+ and PPi in serum samples., (Copyright © 2018. Published by Elsevier B.V.)

Vascular calcification (VC) is associated with significant morbidity and mortality of dialysis patients. Previous studies showed an association between loss of plasma pyrophosphate and VC. Moreover, loss of pyrophosphate occurs during dialysis in this population, suggesting that therapeutic approaches that prevent reduction of plasma pyrophosphate levels during dialysis could improve the quality of life of dialysis patients. This study found that pyrophosphate hydrolysis was 51% higher in post- than pre-dialysis plasma. Dialysis sessions modified the kinetic behavior of alkaline phosphatase, increasing its V max and reducing its K m , probably due to the elimination of uremic toxins during dialysis. At least 75% of alkaline phosphatase activity in human plasma was found to depend on a levamisole-sensitive enzyme probably corresponding to tissue non-specific alkaline phosphatase (TNAP). Dialysis increased total plasma protein concentration by 14% and reduced TNAP enzyme by 20%, resulting in an underestimation of pyrophosphate hydrolysis in post-dialysis plasma. Levamisole inhibited TNAP activity (IC 50 , 7.2 µmol/L), reducing pyrophosphate hydrolysis in plasma and increasing plasma pyrophosphate availability. Alkaline phosphatase is also found in many tissues and cells types; therefore, our results in plasma may be indicative of changes in phosphatase activity in other locations that collectively could contribute significantly to pyrophosphate hydrolysis in vivo. In conclusion, these findings demonstrate that dialysis increases pyrophosphate hydrolysis, which, taken together with previously reported increases in alkalization and calcium ion levels in post-dialysis plasma, causes VC and could be prevented by adding calcification inhibitors during dialysis.

The lack of an effective drug therapy against ossification of spinal ligament (OSL) warrants investigation into the therapeutic target of this disease. An endogenous inhibitor of biomineralization, pyrophosphate (PPi) is a potential therapy for ectopic ossification; however, exogenous PPi is rapidly hydrolyzed by tissue non-specific alkaline phosphatase (TNAP) present in body fluids. In this study, we examined whether a drug therapy targeting PPi is efficacious for the treatment of OSL using the Enpp1 ttw/ttw (twy) mouse model. Twenty male twy mice were randomized into four groups: (i) vehicle (Control); (ii) alkaline phosphatase inhibitor levamisole (5 mg/kg/day sc continuously); (iii) levamisole + exogenous PPi (160 µmol/kg/day sc continuously); and (iv) nuclear retinoic acid receptor-γ (RARγ) agonist (6 µg/kg sc daily). The RARγ agonist, which is a proven inhibitor of ectopic endochondral ossification, was used as a positive control. Treatments commenced when the mice were 5 weeks of age and continued for 4 weeks. Longitudinal micro-computed tomography and postmortem histological analysis were performed. Administration of levamisole alone and in combination with PPi increased serum PPi concentration by 17% and 52%, respectively, compared to that in vehicle-treated mice. The development of OSL in twy mice was suppressed by levamisole + PPi and RARγ agonist treatments, but not by levamisole alone. The levamisole + PPi therapy did not cause osteoporosis, whereas RARγ agonist-treated mice developed osteoporosis. Treatment of twy mice with levamisole in combination with exogenous PPi increased serum PPi level, which slowed the progression of OSL without producing adverse effect on bone. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1256-1261, 2018., (© 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Various disorders including pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI), which are caused by inactivating mutations in ABCC6 and ENPP1 , respectively, present with extensive tissue calcification due to reduced plasma pyrophosphate (PPi). However, it has always been assumed that the bioavailability of orally administered PPi is negligible. Here, we demonstrate increased PPi concentration in the circulation of humans after oral PPi administration. Furthermore, in mouse models of PXE and GACI, oral PPi provided via drinking water attenuated their ectopic calcification phenotype. Noticeably, provision of drinking water with 0.3 mM PPi to mice heterozygous for inactivating mutations in Enpp1 during pregnancy robustly inhibited ectopic calcification in their Enpp1 -/- offspring. Our work shows that orally administered PPi is readily absorbed in humans and mice and inhibits connective tissue calcification in mouse models of PXE and GACI PPi, which is recognized as safe by the FDA, therefore not only has great potential as an effective and extremely low-cost treatment for these currently intractable genetic disorders, but also in other conditions involving connective tissue calcification., (© 2017 Institute of Enzymology, RCNS, Hungarian Academy of Sciences Published under the terms of the CC BY 4.0 license.)

Background: Ready-to-use-therapeutic foods (RUTFs) high in lipid, protein, and iron are used to treat malnutrition. Lipids increase gastric residence time, which could increase iron absorption, particularly from poorly soluble iron compounds and in combination with phytase. Objectives: The objectives were to 1 ) assess the effect on iron absorption of a lipid emulsion given 20 min before or together with an iron-fortified maize meal and 2 ) assess iron absorption from a micronutrient powder (MNP) given with a nutrient-dense RUTF and/or a microbial phytase. Design: A total of 41 women participated in 3 studies. They consumed a maize meal fortified with isotopically labeled ferrous sulfate (FeSO 4 ; study 1) or ferric pyrophosphate (FePP; study 2). In studies 1 and 2, a lipid emulsion was given with or 20 min before the meal. In study 3, with the use of a 2 × 2 factorial design, subjects consumed a maize meal fortified with an MNP containing labeled FeSO 4 (MNP) given with an RUTF (MNP+RUTF), with a phytase (MNP+phytase), or both (MNP+RUTF+phytase). Iron absorption was assessed by isotope incorporation in erythrocytes 14 d after the test meals. Results: The lipid emulsion given either before or with the meal significantly increased iron absorption from FePP by 2.55-fold (95% CI: 1.48-, 4.37-fold; P = 0.001) but not from FeSO 4 There was a trend to increase iron absorption with the MNP+RUTF meal, which did not reach significance (1.21-fold; 95% CI: 0.92-, 1.61-fold; P = 0.060). The addition of phytase to MNP and MNP+RUTF significantly increased iron absorption by 1.85-fold (95% CI: 1.49-, 2.29-fold; P < 0.001), with no interaction between phytase and RUTF. Conclusions: In iron-fortified maize-based meals, the addition of lipids more than doubles iron absorption from FePP. Our results suggest the possibility of an enhancing effect on iron absorption of lipid-rich RUTFs, but more research is needed to determine this. This trial was registered at clinicaltrials.gov as NCT01991626., (© 2017 American Society for Nutrition.)

We present a new copper-mediated on-off switch for detecting either pyrophosphate (PPi) or alkaline phosphatase (ALP) based on DNA-scaffolded silver nanoclusters (DNA/AgNCs) templated by a single-stranded sequence containing a 15-nt polythymine spacer between two different emitters. The switch is based on three favorable properties: the quenching ability of Cu(2+) for DNA/AgNCs with excitation at 550 nm; the strong binding capacity of Cu(2+) and PPi; and the ability of ALP to transform PPi into orthophosphate (Pi). The change in fluorescence of DNA/AgNCs depends on the concentrations of Cu(2+), PPi, and ALP. Copper(II) acts as a mediator to interact specifically with the Probe, while PPi and ALP convert the signal of the Probe by removing and recovering Cu(2+), operating as an on-off switch. In the presence of Cu(2+) only, DNA/AgNCs exhibit low fluorescence because the combination of Cu(2+) and DNA template disturbs the precise formation of DNA/AgNCs. When PPi is added to the system containing Cu(2+), free DNA template is obtained due to the stronger interaction of PPi and Cu(2+), leading to a significant fluorescence increase (ON state) which depends on the concentration of PPi. Further addition of ALP results in the release of free Cu(2+) via ALP-catalysis of hydrolysis of PPi into Pi, thereby returning the system to the low fluorescence OFF state. The switch allows the analysis of either PPi or ALP by observation of the fluorescence status, with the detection limit of 112.69 nM and 0.005 U/mL for PPi and ALP, respectively. The AgNCs on-off switch provides the advantages of simple design, convenient operation, and low experimental cost without need of chemical modification, organic dyes, or separation procedures.

Summary: Calcium supplements have been associated with increased cardiovascular risk, but the mechanism is unknown. We investigated the effects of calcium supplements on the propensity of serum to calcify, based on the transition time of primary to secondary calciprotein particles (T50). Changes in serum calcium were related to changes in T50., Introduction: Calcium supplements have been associated with increased cardiovascular risk; however, it is unknown whether this is related to an increase in vascular calcification., Methods: We investigated the acute and 3-month effects of calcium supplements on the propensity of serum to calcify, based on the transition time of primary to secondary calciprotein particles (T50), and on three possible regulators of calcification: fetuin-A, pyrophosphate and fibroblast growth factor-23 (FGF23). We randomized 41 postmenopausal women to 1 g/day of calcium as carbonate, or to a placebo containing no calcium. Measurements were performed at baseline and then 4 and 8 h after their first dose, and after 3 months of supplementation. Fetuin-A, pyrophosphate and FGF23 were measured in the first 10 participants allocated to calcium carbonate and placebo who completed the study., Results: T50 declined in both groups, the changes tending to be greater in the calcium group. Pyrophosphate declined from baseline in the placebo group at 4 h and was different from the calcium group at this time point (p = 0.04). There were no other significant between-groups differences. The changes in serum total calcium from baseline were significantly related to changes in T50 at 4 h (r = -0.32, p = 0.05) and 8 h (r = -0.39, p = 0.01), to fetuin-A at 3 months (r = 0.57, p = 0.01) and to pyrophosphate at 4 h (r = 0.61, p = 0.02)., Conclusions: These correlative findings suggest that serum calcium concentrations modulate the propensity of serum to calcify (T50), and possibly produce counter-regulatory changes in pyrophosphate and fetuin-A. This provides a possible mechanism by which calcium supplements might influence vascular calcification.

In this work, chemiluminescence (CL) reagent and catalyst metal ion complexes bifunctionalized gold nanoparticles (BF-AuNPs) with high CL efficiency were synthesized via an improved synthesis strategy. Biothiols, such as cysteine (Cys), cysteinyl-glycine (Cys-Gly), homocysteine (Hcy), and glutathione (GSH), instead of 2-[bis[2-[carboxymethyl-[2-oxo-2-(2-sulfanylethylamino)ethyl]amino]ethyl]amino]acetic acid (DTDTPA), were used as new chelators. N-(aminobutyl)-N-(ethylisoluminol) (ABEI) was used as a model of CL reagents and Cu(2+) as a model of metal ion. In this strategy, biothiols were first grafted on the surface of ABEI-AuNPs by Au-S bond. Then, Cu(2+) was captured onto the surface of ABEI-AuNPs by the coordination reaction to form BF-AuNPs. The CL intensity of Cu(2+)-Cys/ABEI-AuNPs was 1 order of magnitude higher than that of DTDTPA/Cu(2+)-ABEI-AuNPs synthesized by the previous work. Moreover, strong CL emission of Cu(2+)-Cys/ABEI-AuNPs was also observed in neutral pH conditions. In addition, the present BF-AuNPs synthesis method exhibited advantages over the previous method in CL efficiency, simplicity, and synthetic rate. Finally, by virtue of Cu(2+)-Cys/ABEI-AuNPs as a platform, a simple CL chemosensor for the sensitive and selective detection of pyrophosphate ion (PPi) was established based on the competitive coordination interactions of Cu(2+) between Cys and PPi. The method exhibited a wide detection range from 10 nM to 100 μM, with a low detection limit of 3.6 nM. The chemosensor was successfully applied to the detection of PPi in human plasma samples. It is of great application potential in clinical analysis. This work reveals that BF-AuNPs could be used as ideal nanointerface for the development of novel analytical methods.

Objective: Mutations in ABCC6 underlie the ectopic mineralization disorder pseudoxanthoma elasticum (PXE) and some forms of generalized arterial calcification of infancy, both of which affect the cardiovascular system. Using cultured cells, we recently showed that ATP-binding cassette subfamily C member 6 (ABCC6) mediates the cellular release of ATP, which is extracellularly rapidly converted into AMP and the mineralization inhibitor inorganic pyrophosphate (PPi). The current study was performed to determine which tissues release ATP in an ABCC6-dependent manner in vivo, where released ATP is converted into AMP and PPi, and whether human PXE ptients have low plasma PPi concentrations., Approach and Results: Using cultured primary hepatocytes and in vivo liver perfusion experiments, we found that ABCC6 mediates the direct, sinusoidal, release of ATP from the liver. Outside hepatocytes, but still within the liver vasculature, released ATP is converted into AMP and PPi. The absence of functional ABCC6 in patients with PXE leads to strongly reduced plasma PPi concentrations., Conclusions: Hepatic ABCC6-mediated ATP release is the main source of circulating PPi, revealing an unanticipated role of the liver in systemic PPi homeostasis. Patients with PXE have a strongly reduced plasma PPi level, explaining their mineralization disorder. Our results indicate that systemic PPi is relatively stable and that PXE, generalized arterial calcification of infancy, and other ectopic mineralization disorders could be treated with PPi supplementation therapy., (© 2014 American Heart Association, Inc.)

Plasma levels of pyrophosphate, an endogenous inhibitor of vascular calcification, are reduced in end-stage renal disease and correlate inversely with arterial calcification. However, it is not known whether the low plasma levels are directly pathogenic or are merely a marker of reduced tissue levels. This was tested in an animal model in which aortas were transplanted between normal mice and Enpp1(-/-) mice lacking ectonucleotide pyrophosphatase phosphodiesterase, the enzyme that synthesizes extracellular pyrophosphate. Enpp1(-/-) mice had very low plasma pyrophosphate and developed aortic calcification by 2 months that was greatly accelerated with a high-phosphate diet. Aortas of Enpp1(-/-) mice showed no further calcification after transplantation into wild-type mice fed a high-phosphate diet. Aorta allografts of wild-type mice calcified in Enpp1(-/-) mice but less so than the adjacent recipient Enpp1(-/-) aorta. Donor and recipient aortic calcium contents did not differ in transplants between wild-type and Enpp1(-/-) mice, demonstrating that transplantation per se did not affect calcification. Histology revealed medial calcification with no signs of rejection. Thus, normal levels of extracellular pyrophosphate are sufficient to prevent vascular calcification, and systemic Enpp1 deficiency is sufficient to produce vascular calcification despite normal vascular extracellular pyrophosphate production. This establishes an important role for circulating extracellular pyrophosphate in preventing vascular calcification.

Medial arterial calcification is accelerated in patients with CKD and strongly associated with increased arterial rigidity and cardiovascular mortality. Recently, a novel in vitro blood test that provides an overall measure of calcification propensity by monitoring the maturation time (T50) of calciprotein particles in serum was described. We used this test to measure serum T50 in a prospective cohort of 184 patients with stages 3 and 4 CKD, with a median of 5.3 years of follow-up. At baseline, the major determinants of serum calcification propensity included higher serum phosphate, ionized calcium, increased bone osteoclastic activity, and lower free fetuin-A, plasma pyrophosphate, and albumin concentrations, which accounted for 49% of the variation in this parameter. Increased serum calcification propensity at baseline independently associated with aortic pulse wave velocity in the complete cohort and progressive aortic stiffening over 30 months in a subgroup of 93 patients. After adjustment for demographic, renal, cardiovascular, and biochemical covariates, including serum phosphate, risk of death among patients in the lowest T50 tertile was more than two times the risk among patients in the highest T50 tertile (adjusted hazard ratio, 2.2; 95% confidence interval, 1.1 to 5.4; P=0.04). This effect was lost, however, after additional adjustment for aortic stiffness, suggesting a shared causal pathway. Longitudinally, serum calcification propensity measurements remained temporally stable (intraclass correlation=0.81). These results suggest that serum T50 may be helpful as a biomarker in designing methods to improve defenses against vascular calcification.

Pseudoxanthoma elasticum (PXE) is an autosomal recessive disease characterized by progressive ectopic mineralization of the skin, eyes, and arteries, for which no effective treatment exists. PXE is caused by inactivating mutations in the gene encoding ATP-binding cassette sub-family C member 6 (ABCC6), an ATP-dependent efflux transporter present mainly in the liver. Abcc6(-/-) mice have been instrumental in demonstrating that PXE is a metabolic disease caused by the absence of an unknown factor in the circulation, the presence of which depends on ABCC6 in the liver. Why absence of this factor results in PXE has remained a mystery. Here we report that medium from HEK293 cells overexpressing either human or rat ABCC6 potently inhibits mineralization in vitro, whereas medium from HEK293 control cells does not. Untargeted metabolomics revealed that cells expressing ABCC6 excrete large amounts of nucleoside triphosphates, even though ABCC6 itself does not transport nucleoside triphosphates. Extracellularly, ectonucleotidases hydrolyze the excreted nucleoside triphosphates to nucleoside monophosphates and inorganic pyrophosphate (PPi), a strong inhibitor of mineralization that plays a pivotal role in several mineralization disorders similar to PXE. The in vivo relevance of our data are demonstrated in Abcc6(-/-) mice, which had plasma PPi levels <40% of those found in WT mice. This study provides insight into how ABCC6 affects PXE. Our data indicate that the factor that normally prevents PXE is PPi, which is provided to the circulation in the form of nucleoside triphosphates via an as-yet unidentified but ABCC6-dependent mechanism.

Generalized arterial calcification of infancy (GACI), an autosomal recessive disorder, is characterized by early mineralization of blood vessels, often diagnosed by prenatal ultrasound and usually resulting in demise during the first year of life. It is caused in most cases by mutations in the ENPP1 gene, encoding an enzyme that hydrolyzes ATP to AMP and inorganic pyrophosphate, the latter being a powerful anti-mineralization factor. Recently, a novel mouse phenotype was recognized as a result of ENU mutagenesis - those mice developed stiffening of the joints, hence the mutant mouse was named 'ages with stiffened joints' (asj). These mice harbor a missense mutation, p.V246D, in the Enpp1 gene. Here we demonstrate that the mutant ENPP1 protein is largely absent in the liver of asj mice, and the lack of enzymatic activity results in reduced inorganic pyrophosphate (PPi) levels in the plasma, accompanied by extensive mineralization of a number of tissues, including arterial blood vessels. The progress of mineralization is highly dependent on the mineral composition of the diet, with significant shortening of the lifespan on a diet enriched in phosphorus and low in magnesium. These results suggest that the asj mouse can serve as an animal model for GACI.

Background: The active metabolites of tenofovir (TFV) and emtricitabine (FTC) in peripheral blood mononuclear cells (PBMCs) have been used as markers of long-term antiretroviral (ARV) adherence. However, the process of isolating PBMCs is expensive, complex, and not feasible in many settings. We compared concentrations of TFV-diphosphate (TFV-DP) and FTC-triphosphate (FTC-TP) in the upper layer packed cells (ULPCs) obtained after whole blood centrifugation to isolated PBMCs as a possible alternative marker of adherence., Methods: Ten HIV+ adults with HIV RNA <50 copies/mL on a TDF/FTC-containing regimen provided 5 paired PBMC and ULPC samples over 6 hours. TFV-DP and FTC-TP concentrations were analyzed by liquid chromatography/mass spectrometry. Partial areas under the curve were calculated using noncompartmental methods and Spearman Rank Correlations (rho) between PBMC and ULPC were determined., Results: The median (25th-75th percentile) concentration of TFV-DP in PBMCs was 143 (103-248) fmol/10(6) cells and in ULPC was 227 (160-394) fmol/10(6) cells (rho = 0.65; P < 0.0001). The concentration of FTC-TP in PBMCs was 6660 (5650-10,000) fmol/10(6) cells and in ULPC was 19.0 (12.0-27.8) fmol/10(6) cells (rho = 0.55; P < 0.0001). Compared to PBMCs, ULPC TFV-DP was 64% higher and FTC-TP was 99.7% lower. ULPC concentrations of TFV-DP and FTC-TP in one additional subject receiving a single dose of TDF/FTC were only 0.05% and 25%, of the other 10 subjects, respectively., Conclusions: ULPC concentrations significantly correlated with PBMC concentrations. Preliminary single-dose data suggest some discrimination between intermittent versus consistent dosing. ULPC concentrations of TFV-DP and FTC-TP should be further investigated as a simply collected surrogate measure of ARV adherence.

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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Vascular calcification (VC) is a strong prognostic marker of mortality from cardiovascular disease. Extracellular inorganic pyrophosphate (PPi) is a critical inhibitor of vascular calcification and it has been reported that hemodialysis patients have reduced plasma PPi levels, suggesting that altered PPi metabolism could contribute to VC in hemodialysis patients. Platelets are rich in PPi and release of PPi from platelets during storage or processing of plasma can lead to falsely elevated plasma PPi levels. To prepare plasma samples that are suitable for measuring PPi levels, ultracentrifugation has been used to remove platelets. Consequently, plasma PPi measurements have been limited to research laboratories since the majority of clinical laboratories do not have access to an ultracentrifuge. The purpose of the present study was to test the validity of an improved method of preparing platelet free plasma that uses filtration with a 300,000 Dalton molecular weight cut-off filter to exclude platelets, while minimizing their release of PPi., Methods: In 20 maintenance hemodialysis patients, PPi levels were measured in plasma samples prepared by the conventional technique of low-speed centrifugation to remove red and white blood cells versus a novel filtration technique., Results: Plasma prepared by filtration had significantly lower platelet counts (0 vs. 3 - 7 10(3)/microL) and PPi levels (1.39 +/- 0.30 microM vs. 2.74 +/- 1.19 microM; mean +/- SD, p < 0.01)., Conclusions: The filtration method appears effective in excluding platelets without causing trauma to platelets and can be used by clinical laboratories to prepare platelet-depleted plasma for PPi measurement.

We present 2 human immunodeficiency virus-infected patients with tenofovir disoproxil fumarate-induced Fanconi syndrome, leading to osteomalacia. Intracellular tenofovir diphosphate levels were measured in 1 patient and were found to be very high, with plasma tenofovir levels just slightly elevated. Fibroblast growth factor-23, a phosphaturic hormone, was decreased in both patients and is therefore unlikely to have a pathophysiological role in this pathology. The different potential factors contributing to the development of tenofovir-related kidney proximal tubular dysfunction are discussed and the data presented may help to further elucidate its pathogenesis.

A validated method for the determination of Combretastatin A1 phosphate (CA1P, OXi4503), a bisphosphate prodrug of the vascular disrupting agent Combretastatin A1 in human plasma has been developed using fluorescence detection after post-column photolysis. The separation used the ion-pairing agent tetrabutylammonium hydrogen sulphate, and this agent was also required to give consistently high recovery from plasma. Initially, the range was shown to be linear (r(2)>0.995) from the LOQ of 0.025 μM to 5 μM, but as the trial progressed to much higher doses, using a lower injection volume, the assay was subsequently subject to limited revalidation to cover the range from 0.05 to 50 μM. Intra-assay precision and accuracy ranged from 2.2 to 11.8% and 1.8 to 13% respectively, and for inter-assay from 4.4 to 14.9% and 1.7 to 6.5%. Mean recovery of OXi4503 from plasma was 80.2%., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A novel fluorescent sensor composed of a naphthalene functionalized tetraazamacrocycle ligand 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3-methyl naphthalene (1) and Zn(2+) has been designed and prepared, which can be utilized for selective and ratiometric sensing of pyrophosphate (PPi) over other phosphate-containing anions in aqueous solution at physiological pH. Notably, the water soluble 1 itself also exhibits a selective enhanced fluorescent response to Zn(2+), and the complex 1-Zn(2+) thus formed eventually fulfils the synergic Zn(2+) coordination-altered strategy with PPi. Furthermore, the ratiometric sensing of 1-Zn(2+) towards PPi performed well even in blood serum milieu. Finally, the sensor 1-Zn(2+) was successfully employed to monitor a real-time assay of inorganic pyrophosphatase (PPase) by means of ratiometric fluorescent measurements for the first time.

Background: Pyrophosphate (PPi) is a potent inhibitor of vascular calcification and may be deficient in renal failure. We sought to determine whether plasma PPi is affected by dialysis or the mode of dialysis and whether it correlates with vascular calcification., Methods: PPi was measured in plasma samples stored from a recent study of vascular calcification in 54 HD patients, 23 peritoneal dialysis (PD) patients and 38 patients with stage 4 chronic kidney disease (CKD). Calcification was quantified in a standardized section of the superficial femoral artery using computed tomography, and PPi was measured by enzyme assay, at both baseline and 1 year., Results: Baseline plasma PPi was weakly correlated with age and serum phosphate, but not with alkaline phosphatase activity or other biochemical parameters, and did not differ between HD, PD and CKD patients. Both baseline calcification score and change in the calcification score at 1 year decreased with increasing quartiles of plasma PPi. In a multivariate analysis, plasma PPi was independently correlated with baseline calcification (P = 0.039) and the change in calcification (P = 0.029)., Conclusion: Plasma PPi is negatively associated with vascular calcification in end-stage renal disease (ESRD) and CKD but is not affected by dialysis, the mode of dialysis or nutritional or inflammatory status. Although these data are consistent with an inhibitory effect of PPi on vascular calcification, further studies are needed to establish a causal role.

In bone and teeth formation, coordinated calcification is a highly desirable biological process. However, heterotopic calcification at unwanted tissue sites leads to dysfunction, disease and, potentially, to death and therefore requires prevention and treatment. With the recent discovery of calcification inhibitors we now know that biological calcification is not passive but a complex, active and highly regulated process. Calcification at vascular sites is the most threatening localization and manifests as part of atherosclerosis or arteriosclerosis. Atherosclerosis is often accompanied by intimal plaque calcification, whereas arteriosclerosis is characterized by calcification of the media. The severity of calcification of cerebral or coronary atherosclerotic plaques is associated with an increased incidence of events such as stroke or myocardial infarction. Medial calcification is the major cause of arterial stiffness, which contributes to left ventricular dysfunction and heart failure. Patients with chronic kidney disease are at especially increased risk for both intimal and medial calcification. In this context, it is currently thought that calcium-regulatory factors including fetuin-A, matrix Gla protein, osteoprotegerin, and pyrophosphates act in a local or systemic manner to prevent calcifications of the vasculature, and that dys-regulations of such calcification inhibitors may contribute to progressive calcifications. Nephrolithiasis represents another process of unwanted calcification responsible for significant morbidity. More than 80% of renal stones contain calcium. Urinary factors inhibiting calcification are citrate, glycosaminoglycans, Tamm-Horsfall protein, and osteopontin. This review summarizes current experimental and clinical data underlining the biological importance of these calcification inhibitors.

Pyrophosphate (PPi) is a known inhibitor of hydroxyapatite formation and has been shown to inhibit medial vascular calcification in vitamin D-toxic rats. It was demonstrated recently that endogenous production of PPi prevents calcification of rat aorta that are cultured in high concentrations of calcium and phosphate. For determining whether PPi metabolism is altered in hemodialysis patients, plasma levels and dialytic clearance of PPi were measured in stable hemodialysis patients. Predialysis plasma [PPi] was 2.26 +/- 0.19 microM in 38 clinically stable hemodialysis patients compared with 3.26 +/- 0.17 in 36 normal subjects (P < 0.01). Approximately 30% of plasma PPi was protein bound, and this was not altered in dialysis patients. There was a weak inverse correlation with age in normal individuals but not in dialysis patients. Plasma [PPi] in dialysis patients was correlated with plasma [PO4(3-)] (r = 0.56) but not with [Ca2+], parathyroid hormone, or the dose of dialysis, and levels did not vary between interdialytic periods of 2 and 3 d. Plasma [PPi] decreased 32 +/- 5% after standard hemodialysis in 17 patients. In vitro clearance of PPi by a 2.1-m2 cellulose acetate dialyzer was 36%, and the mean PPi removal in five patients was 43 +/- 5 micromol, consistent with a similar in vivo clearance. Cleared PPi was greater than the plasma pool but less than the estimated extracellular fluid pool. Erythrocyte PPi content decreased 24 +/- 4%, indicating that intracellular PPi is removed as well. It is concluded that plasma [PPi] is reduced in hemodialysis patients and that PPi is cleared by dialysis. Plasma levels in some patients were below those that have previously been shown to prevent calcification of vessels in culture, suggesting that altered PPi metabolism could contribute to vascular calcification in hemodialysis patients.

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.

The absorption of ciprofloxacin has been reported to be impaired by concomitant administration of ferrous sulphate. The effects of sodium ferrous citrate and ferric pyrophosphate, which have been used as extensively as ferrous sulphate, on the absorption of ciprofloxacin were compared with that of ferrous sulphate. The effects of ascorbic acid on the interactions between ciprofloxacin and each iron compound were studied in mice. Mice were treated orally with ciprofloxacin (50 mg kg(-1)) alone, the iron compound (ferrous sulphate, sodium ferrous citrate or ferric pyrophosphate; 50 mg elemental iron kg(-1)) alone, ciprofloxacin with each iron compound or ciprofloxacin in combination with each iron compound and ascorbic acid (250 mg kg(-1)). The maximum serum concentration of ciprofloxacin was significantly (P < 0.01) reduced from 1.15+/-0.11 microg mL(-1) (ciprofloxacin alone) to 0.17+/-0.01, 0.27+/-0.01 or 0.28+/-0.02 microg mL(-1), respectively, when ferrous sulphate, sodium ferrous citrate or ferric pyrophosphate was administered along with ciprofloxacin. The addition of ascorbic acid did not affect the inhibitory effects of each iron compound on the absorption of ciprofloxacin. Ciprofloxacin did not affect the variation of serum iron levels after administration of each iron compound. The addition of ascorbic acid significantly (P < 0.01) enhanced the increase in serum iron concentration after administration of sodium ferrous citrate, showing an increase from 270+/-6 microg dL(-1) to 463+/-11 microg dL(-1) compared with an increase from 248+/-8 microg dL(-1) to 394+/-18 microg dL(-1) after administration of sodium ferrous citrate alone. Ascorbic acid also caused a significant (P < 0.01) increase in serum iron concentration from 261+/-16 microg dL(-1) to 360+/-12 microg dL(-1) after administration of ferric pyrophosphate, although it did not affect the levels after ferrous sulphate administration. The results suggest that sodium ferrous citrate and ferric pyrophosphate should not be administered with ciprofloxacin (as for ferrous sulphate) and that sodium ferrous citrate is converted to the ferric form more easily than ferrous sulphate. This difference in convertibility might contribute to a clinical difference between sodium ferrous citrate and ferrous sulphate.

Objective: To quantify inorganic pyrophosphate (PPi) production from extracellular adenosine triphosphate (ATP) by human serum or plasma., Methods: Serial measurements of ATP hydrolysis (t1/2) were performed by the luciferase method from a starting concentration of 1 microM in serum or platelet-poor plasma incubated under physiologic conditions. ATP was then pumped into another sample of each specimen using the rate constant derived from the ATP t1/2 of that specimen. Trace (32P) gamma ATP was added at the start of the infusion; conversion to (32P) inorganic orthophosphate (Pi) and to (32P) PPi was determined by precipitation of Pi as reduced phosphomolybdate before and after treatment with yeast pyrophosphatase., Results: ATP was hydrolyzed by all serum and plasma specimens; the rate of hydrolysis in serum and plasma from the same blood sample was nearly identical. PPi was the major product, averaging 71%., Conclusion: PPi is the major product of ATP catabolism in serum and platelet-poor plasma.

Pyrophosphate arthropathy (PA) has been reported in association with a number of diseases, usually occurring in the older age group. We report a 40-year-old female with untreated X-linked hypophosphataemic rickets who presented with PA.

A simple, rapid enzymatic assay for the determination of inorganic pyrophosphate in tissue and plasma has been developed using the enzyme pyrophosphate--fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.90) which was purified from extracts of Propionibacterium shermanii. The enzyme phosphorylates fructose-6-phosphate to produce fructose-1,6-bisphosphate using inorganic pyrophosphate as the phosphate donor. The utilization of inorganic pyrophosphate is measured by coupling the production of fructose-1,6-bisphosphate with the oxidation of NADH using fructose-bisphosphate aldolase (EC 4.1.2.13), triosephosphate isomerase (EC 5.3.1.1), and glycerol-3-phosphate dehydrogenase (NAD+)(EC 1.1.1.8). The assay is completed in less than 5 min and is not affected by any of the components of tissue or plasma extracts. The recovery of pyrophosphate added to frozen tissue powder was 97 +/- 1% (n = 4). In this assay the change in absorbance is linearly related to the concentration of inorganic pyrophosphate over the curvette concentration range of 0.1 microM to 0.1 mM.

Soluble pyrophosphate was measured in the plasma and synovial fluid of various groups of patients and in the plasma of two control groups. The two control groups consisted of 13 healthy subjects and 19 patients suffering from benign lumbar back pain. The other group of patients had rheumatoid arthritis (RA) (14 plasma and 19 synovial fluid examinations), osteoarthrosis (OA) (19 plasma and 26 synovial fluids) and articular chondrocalcinosis (ACC) (27 plasma and 43 synovial fluids). The level of soluble pyrophosphate in the plasma was 3.5 mumol/l in healthy subjects, 4.0 mumol/l in patients with lumbar back pain, 4.1 mumol/l in individuals having OA and 3.5 mumol/l in the group suffering from RA as well as for those with ACC. The differences between these values are not significant statistically. In the synovial fluid the values were 4.6 mumol/l for the group with RA, 12.7 mumol/l for those with OA and 34.2 mumol/l in the group having ACC. If a normal distribution of these values is assumed and the average values and standard deviations recalculated for each group after elimination of cases more than 3 standard deviations above the mean, then we obtain 9.8 mumol/l for the group with OA and 23.8 mumol/l for those with ACC. The difference between the group with RA and that with OA is highly significant (p greater than 0.0001). Even more significant is the difference between the group with RA and ACC (p less than 0.0005). The difference between the OA and the ACC is also highly significant (p less than 0.001). On the basis of these observations various mechanisms leading to the pyrophosphage crystal deposition disease are discussed.

A case of adult hypophosphatasia under treatment with a high orthophosphate (P1) intake is described. The patient is a 53-year-old woman. Her symptoms have progressed for seven years, and it has been necessary to perform osteosynthesis of both crura. The diagnosis rests upon a characteristic clinical picture, low serum alkaline phosphatase activity, high urinary excretion of phosphoethanolamine, and an invariably elevated concentration of inorganic pyrophosphate (PP1) in plasma accompanied by a very high excretion of this compound in the urine. An improved technique allowed specific determinations of microquantities of PP1 in biologic materials. The concentrations of PP1 in the plasma and urine remained unchanged when the patient's intake of phosphorus was increased to 1.98 g/day. The PP1/P1 ratio in the urine was 10-20 before treatment. During treatment P1 excretion increased. PP1 excretion did not change, and the ratio decreased to around 7. The renal tubular transport of PP1 probably was saturated, and therefore PP1, which was circulating in abnormally high concentrations in the patient's fluids, could not be removed by loading with P1. Four months of treatment did not benefit the patient.

The platelet content of PPi is 1.90 +/- mumol/10(11) platelets (S.E.M., n = 19) or about 10.5 nmol/mg of protein, several hundred times that found for rat liver. Some 80% of this PPi is secreted by platelets treated with thrombin with a time course and dose-response relationship similar to secretion of ATP, ADP and 5-hydroxytryptamine (serotonin) from the platelet dense granules. During platelet aggregation induced by ADP and adrenaline, substantial amounts of PPi were secreted, but no release of acid hydrolases was observed. Subcellular-fractionation studies showed that the PPi is highly enriched in the same fraction that contains the storage organelles which store ATP, ADP, Ca2+ and 5-hydroxytryptamine. Inorganic pyrophosphatase was present mainly in the soluble fraction and in the mitochondria. Secretion studies done with platelets prelabelled with [32P]Pi showed that the sequestered PPi was relatively metabolically inactive, as is the ATP and ADP in the storage organelles. The possible participation of PPi in the formation of a bivalent-cation-nucleotide complex associated with amine storage is discussed.

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.