Calcium EDTA Research Articles

Efficacy of Calcium-EDTA as an Inhibitor for Metallo-β-Lactamase in a Mouse Model of Pseudomonas aeruginosa Pneumonia

In this study, we have evaluated the efficacy of calcium-EDTA (Ca-EDTA) as an inhibitor of bacterial metalloenzymes, such as metallo-β-lactamase (MBL) and other proteases, in a mouse model of Pseudomonas aeruginosa pneumonia. The simultaneous presence of Ca-EDTA (32 μg/ml) reduced the MICs of imipenem (IPM) in all MBL-producing P. aeruginosa isolates (IMP-1, -2, -7, and -10 and VIM-2) but not non-MBL-producing strains. In the pneumonia model, mice were intranasally infected with MBL-producing P. aeruginosa and then kept under conditions of hyperoxia to mimic ventilator-associated pneumonia. With both intranasal and subcutaneous administrations, Ca-EDTA significantly potentiated survival benefits of IPM compared to those of IPM alone. Ca-EDTA combination therapy induced a significant reduction of the bacterial burden in the lungs (P < 0.05). Furthermore, the inhibition activity of Ca-EDTA against MBL activity was confirmed by using the purified IMP-1 enzyme, which was characterized by a 50% inhibitory concentration (IC(50)) of 55 ± 8.2 μM. Finally, the protective effects of Ca-EDTA were demonstrated by culture supernatant-induced epithelial cell damage and acute lung injury in mice. These data suggest the therapeutic potential of Ca-EDTA not only by the blocking of MBLs but also by neutralizing tissue-damaging metalloproteases in P. aeruginosa infections.

The effect of calcium antagonists on the growth of Entamoeba histolytica in vitro

he E. histolytica parasite was maintained in vitro using Locke-egg medium (LEM) and Liver infusion agar medium (LIAM). The effect of two calcium antagonists (Nifedipine and Ethylene-diaminetetraacetic acid EDTA) on the growth and activity of the parasite in the two culture media was investigated. The calcium antagonists Nifedipine and EDTA inhibited the reproduction rate of E. histolytica in a concentration-dependent manner. For Nifedipine, a concentration of 41.6 mg/ml inhibited the reproduction rate to 99.7% in both media. The EDTA had an approximate effect (98.2 and 95.8)% at a concentration of 0.83 mg/ml in LEM and LIAM media, respectively. Additionally, some cases of a parasite encystment were observed in LEM medium that was treated with Nifedipine.

PH statvs. single extraction tests to evaluate heavy metals and arsenic leachability in environmental samples

Here we compared the pH stat test, which examines the leachability of major elements (Ca, Mg, Al, Fe, and Mn), dissolved organic carbon, and trace elements (Cd, Zn, Cu, Pb, and As) in a wide pH range, with single extraction tests based on the use of mild extractants (calcium chloride, acetic acid or EDTA). For this purpose, we examined samples from a variety of environmental conditions (sludges, mineral soils, organic soils, and soils with particulate and/or soluble contamination). Extraction yields obtained with CaCl 2 (0.01 mol L −1) and CH 3COOH (0.43 mol L −1) correlated well with those from the pH stat at the same pH ( r = 0.98 and 0.95, respectively), while the use of EDTA (0.05 mol L −1) led to systematically higher extraction yields than those quantified with the pH stat at the same pH. However, the pH stat test had three distinct advantages: (1) it revealed the relationship between the solubility of the main soil phases and pH; (2) it showed the variation in pollutant leachability due to changes in pH; and (3) it better predicted the maximum contaminant availability. Thus we propose that the pH stat is the best laboratory tests to evaluate the contaminant leachability over a wide range of sample types (soil, sludge, and sediment).

Serum Albumin is as Efficient as Paraxonase in the Detoxication of Paraoxon at Toxicologically Relevant Concentrations

Human serum albumin was able to hydrolyze the organophosphorus compounds paraoxon, chlorpyrifos-oxon, and diazoxon at toxicologically relevant concentrations. Human serum displayed two paraoxon hydrolyzing activities: the so-called paraoxonase, which is associated with the lipoprotein fraction and is calcium dependent and EDTA sensitive, and the activity associated with albumin, which is EDTA resistant and sensitive to fatty acids. Human serum albumin hydrolyzed these compounds with the same relative efficacy as lipoproteins (chlorpyrifos-oxon > diazoxon > paraoxon). The capability of detoxication of activity associated with human serum albumin was similar or even higher than paraoxonase associated with lipoproteins in the case of paraoxon at concentrations as low as those noted in an acute in vivo intoxication. However, paraoxonase activity associated with lipoprotein was more effective than paraoxonase activity associated with albumin at toxicologically relevant chlorpyrifos-oxon concentrations. These results explain why mice deficient in paraoxonase associated with lipoprotein are not more sensitive to paraoxon than wild animals.

Chelation of neurotoxic zinc levels does not improve neurobehavioral outcome after traumatic brain injury

Increases of synaptically released zinc and intracellular accumulation of zinc in hippocampal neurons after traumatic or ischemic brain injury is neurotoxic and chelation of zinc has been shown to reduce neurodegeneration. Although our previous studies showed that zinc chelation in traumatically brain-injured rats correlated with an increase in whole-brain expression of several neuroprotective genes and reduced numbers of apoptotic neurons, the effect on functional outcome has not been determined, and the question of whether this treatment may actually be clinically relevant has not been answered. In the present study, we show that treatment of TBI rats with the zinc chelator calcium EDTA reduces the numbers of injured, Fluoro-Jade-positive neurons in the rat hippocampus 24 h after injury but does not improve neurobehavioral outcome (spatial memory deficits) 2 weeks post-injury. Our data suggest that zinc chelation, despite providing short-term histological neuroprotection, fails to improve long-term functional outcome, perhaps because long-term disruptions in homeostatic levels of zinc adversely influence hippocampus-dependent spatial memory.

Effect of pH on the Carbonate Incorporation into the Hydroxyapatite Prepared by an Oxidative Decomposition of Calcium–EDTA Chelate

In this study, the carbonate incorporation into the hydroxyapatite (HAp) lattice under various pH conditions was investigated. Crystalline‐sodium and carbonate‐containing calcium HAp (NaCO3HAp) powders were prepared using an oxidative decomposition of calcium–EDTA chelates in a sodium phosphate solution with hydrogen peroxide. The powders obtained were characterized by X‐ray diffraction, infrared spectroscopy, thermal gravimetric analysis, scanning electron microscopy, energy‐dispersive X‐ray spectroscopy, and elemental analysis. Depending on pH, spherical particles approximately 3.5 μm in diameter or hexagonal prismatic particles measuring 3–9 μm in length were obtained. Various characterization techniques showed that the precipitates were a single‐phase NaCO3HAp. The carbonate content and the lattice parameters of the HAp were a function of solution pH. Maximum carbonate incorporated into the HAp lattice was at pH=10, corresponding to lattice parameters of a=0.93880 nm and c=0.69070 nm. Furthermore, spectroscopic analyses indicate that the as‐prepared samples are B‐type carbonated HAp, in which carbonate ions occupy the phosphate sites. After heat treatment at 965°C, most of the carbonate is removed from the HAp lattice.

Frontotemporal Dementia: Recommendations for Therapeutic Studies, Designs, and Approaches

Frontotemporal dementia (FTD) is one of three neurobehavioural syndromes produced by frontotemporal lobar degeneration. Despite the importance of FTD as a cause of dementia, especially in younger age groups, and a rationale for therapies targeting serotonergic and dopaminergic systems, there have been no large scale treatment trials in FTD. Moreover, there is no consensus on standards to facilitate comparison across therapeutic trials. This paper reviews the literature on therapeutic trials in FTD and outlines general recommendations for standards related to the development of future treatment studies in this disorder. Drugs tested in FTD include trazodone, galantamine, idazoxan, lithium plus fluoxetine, lithium plus paroxetine, SSRIs, 1-deprenyl, moclobemide, methylphenidate, piracetam, rivastigmine, donepezil, olanzapine, risperidone, amantadine, guanfacine, allopurinol, and bromocriptine. Improvement has been reported in FTD for all drugs except piracetam, guanfacine and galantamine, although there was improvement on galantamine in primary progressive aphasia. Whereas improvement has been reported for paroxetine and other SSRIs, as well as idazoxan and methylphenidate, paroxetine and idazoxan have also been reported to cause a decline in function, and a marginally significant decline has been reported for methylphenidate. In addition, patients with Pick's disease, which is part of the spectrum of frontotemporal lobar degeneration, showed improvement on calcium EDTA. Six studies are double-blind placebo-controlled trials: two reports of cases using idazoxan and group trials using trazodone, paroxetine, galantamine and methylphenidate. It is recommended that experts in FTD arrive at a consensus to define standards for all clinical trials in FTD. These should include standards for diagnostic criteria, tests of severity, experimental design, and outcome measures.

IgE-receptor activation induces survival and Bfl-1 expression in human mast cells but not basophils

The contribution of mast cells to the pathology of allergic diseases are facilitated by their long life span in tissue and ability to regranulate. Bcl-2 genes are one of the main regulators of cell death and survival. The aim of this study was to elucidate the mechanisms responsible for mast cell survival in allergy. Bcl-2 family gene expression in human mast cells and basophils was analyzed by ribonuclease protection assay and by reverse-transcriptase polymerase chain reaction. Cell survival was measured by mixing cells with the vital dye, trypan blue, and the number of living cells was enumerated. Apoptotic cells were measured by a Cell Death Detection ELISA. We found that cross-linking of FcepsilonRI on human cord blood cultured mast cells (CBCMCs) promoted cell survival and induced expression of the pro-survival gene Bfl-1. CBCMCs were found to express both Bfl-1 and Bfl-1S, two splicing variants of Bfl-1. Bfl-1 induction was mediated through Syk, PI3-kinase and intracellular calcium mobilization, since piceatannol, wortmannin and EDTA, respectively, significantly reduced Bfl-1 expression levels. In contrast to CBCMCs, no evidence was found for Bfl-1 expression and survival promotion in human basophils. Immunoglobulin E (IgE)-dependent activation-induced mast cell survival was correlated with Bfl-1 gene upregulation, providing a possible explanation for mast cell longevity in allergic reactions.

Heavy metals extraction from contaminated soil: Recovery of the flushing solution

The optimum conditions for the recovery of copper from a contaminated soil by using the soil flushing technique are evaluated. Tests on a soil artificially contaminated with copper chloride were carried out in order to evaluate the influence of the speed of percolation and of the chelating agent concentration (aqueous solution of an ethylendiaminotetraacetic acid di-sodium salt Na 2–EDTA). At pH=7.3 an efficiency up to 93.9% for copper extraction was achieved by flushing 500 ml of Na 2–EDTA 0.05 M solution and 100 ml of pure water at 0.792 cm/h. At these operating conditions the formation of EDTA complexes with other competitive cations (calcium and iron) was negligible. The experimental results were in agreement with the ones obtained using a model describing the chemistry of metal extraction. This model assessed that above pH=6 the formation of calcium and iron EDTA complexes was excluded and only the chelation of copper was allowed. The recovery of 91.6% of EDTA was also achieved by evaporating and acidifying the extracted solution: after filtration, solid EDTA was obtained, through the addition of sodium hydroxide Na 2–EDTA. About 99.5% of the extracted copper was finally precipitated under alkaline conditions from the liquid phase.

Late Calcium EDTA Rescues Hippocampal CA1 Neurons from Global Ischemia-Induced Death

Transient global ischemia induces a delayed rise in intracellular Zn2+, which may be mediated via glutamate receptor 2 (GluR2)-lacking AMPA receptors (AMPARs), and selective, delayed death of hippocampal CA1 neurons. The molecular mechanisms underlying Zn2+ toxicity in vivo are not well delineated. Here we show the striking finding that intraventricular injection of the high-affinity Zn2+ chelator calcium EDTA (CaEDTA) at 30 min before ischemia (early CaEDTA) or at 48-60 hr (late CaEDTA), but not 3-6 hr, after ischemia, afforded robust protection of CA1 neurons in approximately 50% (late CaEDTA) to 75% (early CaEDTA) of animals. We also show that Zn2+ acts via temporally distinct mechanisms to promote neuronal death. Early CaEDTA attenuated ischemia-induced GluR2 mRNA and protein downregulation (and, by inference, formation of Zn2+-permeable AMPARs), the delayed rise in Zn2+, and neuronal death. These findings suggest that Zn2+ acts at step(s) upstream from GluR2 gene downregulation and implicate Zn2+ in transcriptional regulation and/or GluR2 mRNA stability. Early CaEDTA also blocked mitochondrial release of cytochrome c and Smac/DIABLO (second mitochondria-derived activator of caspases/direct inhibitor of apoptosis protein-binding protein with low pI), caspase-3 activity (but not procaspase-3 cleavage), p75NTR induction, and DNA fragmentation. These findings indicate that CaEDTA preserves the functional integrity of the mitochondrial outer membrane and arrests the caspase death cascade. Late injection of CaEDTA at a time when GluR2 is downregulated and caspase is activated inhibited the delayed rise in Zn2+, p75NTR induction, DNA fragmentation, and cell death. The finding of neuroprotection by late CaEDTA administration has striking implications for intervention in the delayed neuronal death associated with global ischemia.

A Review of Chronic Lead Intoxication: An Unrecognized Cause of Chronic Kidney Disease

Chronic lead nephropathy occurs as a result of years of lead exposure. This disease has been prevalent throughout human history. It is important that primary care providers and internists recognize this disorder because it can contribute to progressive loss of kidney function. Diagnosis is made by a thorough history in combination with physical examination. A history of lead intoxication often requires knowledge of the various sources of lead exposure. Laboratory tests, many previously known and some newly described, are available to further support this diagnosis. Therapy to reduce lead burden may be useful when employed early in the disease; new data sheds more light on which patients should be treated and when such patients should undergo chelation therapy. In particular, treatment with calcium EDTA chelation may benefit certain patients with chronic kidney disease by slowing the progression to end-stage renal disease.

Protective effects of zinc chelation in traumatic brain injury correlate with upregulation of neuroprotective genes in rat brain

Chelation of excessive neuronal zinc ameliorates zinc neurotoxicity and reduces subsequent neuronal injury. To clarify the molecular mechanisms of this neuroprotective effect, we used a focused cDNA array of stress-response genes with zinc chelation (calcium EDTA) in our rat model of fluid percussion brain injury at 2 h, 24 h, and 7 days after injury. In parallel experiments, we compared neuronal cell death in TUNEL-stained brain sections in traumatized rats with and without calcium EDTA treatment. Zinc chelation induced the expression of several neuroprotective genes; neuroprotective gene expression correlated with substantially decreased numbers of TUNEL-positive cells.

Additivity and the physical basis of multivalency effects: a thermodynamic investigation of the calcium EDTA interaction.

To better understand the origin of multivalency effects in ligand binding, the binding of a series of mono-, bi-, tri- and tetravalent carboxylate ligands to Ca(II) was examined by isothermal titration calorimetry (ITC). The data are inconsistent with an entropic origin of enhanced affinity, but rather show that at least in this instance the multivalency effect is enthalpic in origin. Analysis of binding data using the Jencks model shows the addition of incremental carboxylate "ligands" produces an unfavorable interaction entropy that is more than offset by a strongly favorable interaction enthalpy. The most likely source of this interaction enthalpy is the relief of repulsive Coulombic interactions in the unbound state. The conformational entropy penalty arising from the restriction of flexible dihedrals is negligible, within experimental error. On the other hand, an enthalpic contribution from linker restriction contributes strongly to the overall thermodynamics of ligand binding. Together, these data suggest that enthalpic effects dominate ligand binding, and design strategies should seek to optimize these interactions. The incorporation of unfavorable interactions in the unbound ligand that are relieved during binding provides an important mechanism by which to enhance ligand affinities.

Pectins as possible source of the copper involved in the green staining alteration of cv. Gordal table olives.

The pectic and pigment compositions and Ca and Cu contents of the alcohol-insoluble solid (AIS) residues were determined in cv. Gordal olives treated with NaOH solution and kept at different constant pH values (3.5-6.5). The same controls were made in table olives presenting green staining alteration. The ratio between the various pectin fractions of the more acid pH experiment samples remained similar in fruits not showing green staining. In altered fruits, the protopectin fraction was lower, and the calcium pectate or EDTA soluble pectins were higher. Regarding the presence of Ca and Cu in the AIS, it was observed that, whereas Ca levels fell at the most acid pH values, those of Cu increased. The concentration of Ca was higher in the AIS of altered olives than in nonaltered ones. The same trend was seen for the zone with or without green staining of an altered fruit. In the case of Cu, the relationship was the opposite: a decrease in the levels of AIS Cu in fruits and zones of fruits with green staining. This result was correlated with the highest concentration of Cu-chlorophyll complexes found in such samples and suggested that pectins might act as a reservoir of Cu involved in the alteration.

Lead, renal disease and hypertension

Lead, renal disease and hypertension

Un cas « historique » de saturnisme hydrique : difficultés diagnostiques et thérapeutiques

Introduction. – It is likely that lead poisoning via drinking water is often overlooked because of its supposed rarity and nonspecific early symptoms, which result in delayed management. Exegesis. – One case of severe lead poisoning via drinking water is reported. The diagnosis was long missed and a particularly long chelating treatment was required. The clinical features included lead colic, a Burton’s lead line, anemia, polyneuritis and arterial hypertension. Eighteen courses of calcium EDTA were required to obtain ‘biological recovery’. The poisoning was linked to a very long water supply lead pipe and potomania secondary to alcohol withdrawal. Conclusion. – This case report illustrates how difficult the early recognition of lead poisoning can be, and underlines the need to inquire about a toxic aetiology, particularly via the environment, of otherwise unexplained pathological conditions.

The Effect of Estrogen on Renal Phosphorus Handling in the Rat

Background/Aims: Phosphorus reabsorption in the kidney is regulated by parathyroid action on the sodium phosphorus cotransporter (Na-Pi cotransporter). There is some evidence that estrogen may also regulate renal phosphorus handling but it is not known if this is an effect of estrogen on filtered phosphorus load. Methods: This study examined the effect of estrogen on renal phosphorus handling during infusion with calcium or EDTA. Six month old Sprague Dawley rats were bilaterally oophorectomized (OOX) or underwent a sham operation under ether anaesthesia 6 weeks before undergoing infusion with calcium chloride or EDTA. A second study examined renal phosphorus handling after estrogen replacement in the OOX rat injected with 20 µg estradiol valerate, or vehicle alone, prior to calcium infusion. Results: A comparison of filtered phosphorus load with renal phosphorus excretion indicated that the estrogen replete rat had a higher renal excretion of phosphorus when infused with both calcium (p = 0.004) and EDTA (p = 0.037) at all filtered phosphorus loads. A similar analysis in calcium infused, estrogen replaced, OOX rats indicated an effect of estrogen to increase renal phosphorus excretion (p = 0.007) at all filtered phosphorus loads. OOX resulted in a mild metabolic alkalosis, an effect not reversed by estrogen administration, that was not related to renal phosphorus excretion. OOX decreased renal sodium excretion and uncoupled the relation between renal phosphorus and sodium excretion, an effect reversed by estrogen replacement. Conclusion: Ovarian hormone deficiency in the rat results in a decrease in renal phosphorus excretion. This finding is comparable to effects of the menopause and hormone replacement therapy in postmenopausal women.

Effects of calcium source and temperature regime on calcium deficiency during hydroponic forcing of tulip

Tulips ( Tulipa gesneriana L.) forced hydroponically in distilled water developed Ca deficiency symptoms, including topple and flower bud abortion. Prevention of Ca deficiency and uptake of Ca was greater when Ca was supplied in the NO 3 − form rather than as Cl − or SO 4 2−. Calcium EDTA was injurious to plants at a concentration of 2.5 mM, a level too low to correct Ca deficiency. Addition of BO 3 3−, K +, H 2PO 4 −, or Hoagland's all nitrate solution with modified micronutrient levels to the Ca substrate failed to improve growth. The complete nutrient formulation reduced Ca uptake. Ca deficiency could not be prevented at the high temperature regime (22°C day–18°C night), due in part to suppress uptake of Ca. In the low temperature regime (18°C day–14°C night), a 5-mM solution of Ca(NO 3) 2 prevented Ca deficiency during all forcing periods. Similar results were obtained with a 7.5 mM solution of CaCl 2.

Spectroscopic and metabolic effects of MnCl2 and MnDPDP on the isolated and perfused rat heart.

Several works have shown that the hepatobiliary magnetic resonance imaging contrast agent manganese dipyridoxyl diphosphate (MnDPDP) partly releases its metallic ion and exhibits cardiovascular effects that are supposed to arise from the free manganese ions (Mn++). In the current study, the cellular internalization of Mn by the isolated rat heart is monitored through the mechanical function of the organ and the relative broadening of the P-31 nuclear magnetic resonances. Rat hearts were perfused with manganese chloride (MnCl2; 15 and 25 microM) or MnDPDP (25 microM). Variations of the linewidths, heights, and surfaces of phosphocreatine and adenosine triphosphate peaks were monitored. Cardiac function was monitored simultaneously through heart rate, left ventricular pressure, and coronary flow. Influx of Mn++ induces a significant broadening of the P-31 resonances of adenosine triphosphate and phosphocreatine because of a strong scalar paramagnetic interaction between the nuclei and the ion. Compared with MnDPDP administered at the same concentration, MnCl2 induced a more pronounced and dose-dependent line broadening as well as a coronary vasodilation. Calcium channel blockers (nifedipine and verapamil) and EDTA inhibit MnCl2 influx. Similarly, verapamil, EDTA, and DPDP reduce the alterations provoked by MnDPDP. The effects of MnDPDP are smaller but of the same type than those induced by MnCl2. Their inhibition by calcium channel blockers (verapamil and nifedipine) and by an excess of strong chelators such as DPDP or EDTA confirms that they originate from a partial release of Mn++ by the contrast agent.

A Novel Degradation Pathway of Tissue Factor Pathway Inhibitor: Incorporation Into Fibrin Clot and Degradation by Thrombin

AbstractTissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor with three tandem inhibitory domains (K1, K2, and K3) that regulates the initial reactions of the extrinsic blood coagulation pathway through K1 and K2. In the present study, the effect of thrombin on TFPI in a purified system was first examined using recombinant TFPI from Chinese hamster ovary (CHO) cells. TFPI was inactivated by thrombin with cleavage of three peptide bonds, Lys 254-Thr 255 in the C-terminal basic region, Arg 107-Gly 108 (reactive site toward factor Xa in K2), and Lys 86-Thr 87 between K1 and K2. Then, degradation of radiolabeled TFPI by thrombin was examined in two systems: (1) mixed with plasma and then tissue factor (TF ) and calcium ion, and (2) mixed with fibrinogen and then thrombin. TFPI degradation was detected in serum from normal plasma and more extensively from antithrombin (AT)-depleted plasma by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Significant radioactivity was found in the clot after coagulation of the plasma, which decreased after 20 hours' incubation. These changes were more prominent in AT-depleted plasma than in normal plasma. When TFPI lacking the C-terminal basic region was used instead of full-length TFPI, most of the radioactivity was found in serum rather than in fibrin clots. Incorporation of TFPI into the fibrin clot was prevented by a synthetic C-terminal peptide of TFPI. Similar results were obtained after mixing radiolabeled TFPI with fibrinogen and then thrombin in the presence of calcium ion or EDTA. These results demonstrate a novel degradation pathway of TFPI, ie, incorporation into fibrin via the C-terminal basic region and degradation by thrombin (possibly fibrin-bound thrombin).