Chelation for Coronary Heart Disease

Chelation Natural Miracle For Protecting Your Heart

Chelation therapy has been conclusively shown to be up to 82 Percent Effective at dissolving the plaque that blocks arteries! In the ebook Chelation Natural Miracle For Protecting Your Heart you'll discover: The frustrating reason many doctors are ignoring Edta chelationor even openly rejecting it. The deadly heart surgeries even the American Heart Association admits are unnecessary. The hidden signs and symptoms of heart attacks and strokes? Are you in danger right now?. The average number of years stripped away by heart and vessel disease. Can you get them back?. The newest set of risk factors for heart disease (they'll likely surprise you!). Shady government practices that protect Big Pharma and keep Edta chelation out of the public eye. How the Roman Empire could have been savedif only they'd known about Edta chelation. Why Edta chelation is guaranteed to be safeeven in extremely high doses. (It puts aspirin to shame!). The shocking truth about plaque in young childrenand how to keep your little ones safer. Why dentists, artists and welders need Edta chelationand whether your workplace is dangerous too. The differences between IV and oral chelationand which kind of Edta is right for you. Other forms of chelationand how these little-known treatments can dramatically boost your health.

Chelation Natural Miracle For Protecting Your Heart Summary


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Author: Michael Cutler, M.D.
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Iron and Iron Chelation Post Light Activation of Photosensitizer

Sequestering of iron through iron chelation may block this HO* radical generating pathway and therefore reduce the amount of cellular damage caused following PDT. Iron is highly regulated within the cell, ensuring that no free intracellular iron is available. Under oxidative stress, however, the normal iron homeostasis is

Chelation Therapy For Arsenic Poisoning

Chelation therapy often is begun with dimercaprol (3-4 mg kg intramuscularly every 4 12 hours) until abdominal symptoms subside and charcoal (if given initially) is passed in the feces. Oral treatment with penicillamine then may be substituted for dimercaprol and continued for 4 days. Penicillamine is given in 4 divided doses to a maximum of 2 g day. If symptoms recur after cessation of chelation therapy, a second course of penicillamine may be instituted. Succimer (2,3-dimercaptosuccinic acid), a derivative of dimercaprol, is efficacious in the treatment of arsenic poisoning but is FDA-approved only for lead chelation in children. After long-term exposure to arsenic, treatment with dimercaprol and penicillamine also may be used, but oral penicillamine alone usually is sufficient. The duration of therapy is determined by the clinical condition of the patient, and the decision is aided by periodic determinations of urinary arsenic concentrations. Adverse effects of the chelating agents...

Treatment Of Cadmium Poisoning By Chelation Therapy

Effective therapy for cadmium poisoning is difficult to achieve. Although there is no proven benefit, some clinicians recommend chelation therapy with CaNa2EDTA. The dose of CaNa2EDTA is 75 mg kg day in 3-6 divided doses for 5 days. After a minimum of 2 days without treatment, a second 5-day course is given. The total dose of CaNa2EDTA per 5-day course should not exceed 500 mg kg. Animal studies suggest that chelation therapy should be instituted as soon as possible after cadmium exposure because a rapid decrease in effectiveness of chelation therapy occurs in parallel with distribution to sites inaccessible to the chelators. The use of dimercaprol and substituted dithiocarbamates appears promising for individuals chronically exposed to cadmium.


The formation of two or more bonds between a single central atom (often a metal ion) and a chemical species, termed a multidentate ligand. The complex formed is referred to as a chelate. One naturally occurring example is the MgATP2 complex. The terms bidentate, tridentate, etc., refer to the number of sites on the chelating ligand that are bond to the central atom. EDTA and EGTA have four carboxylic acid groups to form a tetradentate ligand, and many crown ethers are polydentate in terms of their ion binding character. See also Sequestration Cryptand

Preanalytical Considerations

Commercially available to an investigator. For instance, if one were to specify EDTA plasma as a biomarker sample, there are at least 25 different collection vacutainers, which yield EDTA plasma from whole blood. These collection tubes vary by EDTA concentration (15 or 7.5 K3EDTA, yielding sample dilution of 2.4 or 1.1 , respectively, or spray-dried K3EDTA, with no sample dilution), draw volume from 2.0mL to 10.0mL, size from 13 X 75mm to 16 X 100mm, glass or plastic, and stopper of the conventional and Hemogard design. With the opportunity for such diverse collection containers for an EDTA plasma specimen, the best opportunity for consistent sample collection is provided by constructing the collection materials into a well-designed protocol visit-customized package. This ensures that the required collection containers appropriate for the planned analysis are utilized for the collection process and that the institutional review board (IRB)-approved volume of sample is obtained. In the...

Sequestration of iron

Silicic acid, a ubiquitously distributed component of cereals and other foods, forms complexes with inorganic iron, which enable the safe sequestration of iron in tissues, decreasing its ability to generate ROS, initiate membrane lipid peroxidation, and to mobilize leukocytes (Birchall, 1993). Iron, per se, as oxygen-bridged ferrous-ferric complexes may initiate lipid peroxidation (Minotti and Aust, 1989). Dietary supplementation of iron-enhanced dimethylhydrazine-induced colorectal cancer in rats, but this was reversed by phytic acid, a component of dietary fibre, probably due to the chelation of the iron by phytic acid, with the consequent decreased ability of the iron to generage ROS (Nelson et al, 1989).

Overview Of Antibody Therapeutics

Another class of Ab variants, immunoconjugates, may be derived through chemical conjugation of Abs to several different types of natural or synthetic compounds. Both radioimmunoconjugates and ADCs fall into this broad class of Ab-based agents. Arming Abs with drugs, toxins, or therapeutic radionuclides can significantly improve their potential therapeutic benefits. Several chemo-therapeutics drugs including doxorubicin, calicheamicin, maytansine, metho-trexate, chlorambucil, and many others have been linked to Abs using various chemical conjugation methods (31,32). The successful use of an ADC depends on a number of factors such as its in vivo stability, its ability to localize and persist at the tumor site with little localization in normal tissues, and its intratumoral and intracellular penetration and metabolism. The sensitivity of the tumor cells themselves to the conjugated drug also influences the overall therapeutic efficacy of this approach (15). In addition, radionuclides...

Postanalytical Considerations

Stability are a combination of the laboratory processes and investigator site. Tests lost due to whole-blood microdots is an evaluation of specimen transport conditions. Inappropriate cooling or heating during transport, in addition to inadequate sample mixing at collection, can generate microdots in EDTA whole-blood specimens. The most common reason for lost data is specimen received beyond stability. The next two most common reasons for loss of data are specimen hemolysis and use of an expired collection container, which are directly linked to site performance. The typical data yield will be greater than 98.5 and direct laboratory causes for lost data are less than 0.1 of the total data. Regardless of the cause, this approach allows the drug development team to track such losses and identify the need for corrective measures should these losses prove unacceptable.

Characteristics of Serum and Plasma Specimens

In contrast to serum, citrate and EDTA inhibit coagulation and other enzymatic processes by chelate formation with ions, thereby inhibiting ion-dependent enzymes. This is in contrast to heparin, which acts through the activation of antithrombin III. The main concern associated with heparinized plasma for proteomic studies is that it is a poly-disperse charged molecule that binds many proteins non-specifically (15,16), and may also influence separation procedures and mass spectrometric detection of peptides and small proteins due to its similar molecular weight (17).

Studies on the Interaction of Vitamin C and Iron Under Physiologically Relevant Conditions

Synovial fluid Iron-EDTA H Human plasma Iron H2O2 Iron-EDTA Iron H2O2 to the presence of metal binding proteins in these fluids, rather than vitamin C, because enzymatic removal of endogenous vitamin C did not alter the results. However, when sufficient exogenous iron (as ferrous ammonium sulfate) is added to plasma to saturate transferrin, resulting in the appearance of non-protein bound, bleomycin-detectable iron, endogenous and exogenous vitamin C strongly inhibits, rather than promotes lipid peroxidation (66) (Table 3.2). This finding is supported by an earlier study in which vitamin C acted as an antioxidant in serum to which excess copper had been added (67). Two other studies carried out with plasma, lymph, and synovial fluid showed that vitamin C can catalyze the formation of OH, but only when the catalytically active form of iron, iron-EDTA, was added (68,69), not ferrous ammonium sulfate (69). Recently, we found that even when H2O2 is added to plasma, in addition to...

Modes Of Regulation

There have been very few reports on the effect of oxidative stress on mRNA stabilization. In vascular smooth muscle cells, heme oxygenase mRNA levels are increased by a combination of decreased mRNA turnover and increased gene transcription (Hartsfield et al., 1997). We have assessed the contribution of mRNA stability to the induction of adaptlS, adapt33, c-fos, c-jun, and gadd45 mRNA following exposure of HA-1 cells to hydrogen peroxide. A significant increase in the stability of adapt33 and gadd45 but not adaptl5, c-fos, nor c-jun mRNAs was observed (Wang et al., 1996). Intracellular chelation of calcium dramatically inhibited the induction of adapt33 mRNA by hydrogen peroxide, indicating a role for calcium in this stabilization. We also observe a significant acceleration of the processing of adaptl5 precursor RNA following the exposure of HA-1 cells to hydrogen peroxide. This apparently accounts for some of the observed increase in the steady-state levels of mature adaptlS mRNA...

Spectrofluorometric Determinations of Tetrapyrroles at Room Temperature

For isolated plastids, 1 g of tissue was homogenized by hand in a chilled mortar in 5 ml of homogen-ization buffer consisting of 0.5 M sucrose, 15 mM Hepes, 10 mM Tes, 1 mM MgCl2, and 1 mM EDTA adjusted to pH 7.7 at room temperature. The hom-ogenate was filtered through one layer of Miracloth into cooled 40 ml centrifuge tubes. The homogenate was centrifuged at 200g for 3 min in a Beckman JA-20 fixed angle rotor at 1 C. The supernatant was decanted and centrifuged at 1500g for 10min at 1 C. The pelleted plastids were gently resuspended in 2 ml of cold homogenization buffer glycerol (1 2 v v). Excitation spectra were recorded as described above for crude homogenates 9 .

Materials and Methods

0.05 trypsin ethylenediamine tetracetic acid (EDTA) store at -20'C. 3. Add 2 mls of 0.05 Trypsin-EDTA solution to the dish and incubate at 37'C for 2-5 minutes, then observe cells under an inverted microscope until cell layer is dispersed. (The serum in the medium deactivates the Trypsin-EDTA so this is a crucial step that should not be left out). The dish can be gently tapped to help the cells detach. Cells that are difficult to detach can be placed back in the incubator for an additional 1-2 minutes. 3. Add 2 mls of 0.05 Trypsin-EDTA solution to the dish and incubate at 37'C for 2-5 minutes, then observe cells under an inverted microscope until cell layer is dispersed. 4. Add 2 mls of 0.05 Trypsin-EDTA solution to the dish and incubate at 37'C for 2-5 minutes, then observe cells under an inverted microscope until cell layer is dispersed. Cells that are difficult to detach can be placed back in the incubator for an additional 1-2 minutes. 3. Add 1 ml of0.05 Trypsin-EDTA solution into...

EpoxyalkaneCoM Transferase A Zinc Dependent Epoxidase

The importance of zinc in EaCoMT was established by removing zinc from the enzyme by chelation, which resulted in loss of catalytic activity that was partially restored by adding zinc back to the enzyme (38). Due to the harshness of the chelation conditions, an apoenzyme for biochemical studies was instead prepared by expressing the enzyme in zinc-deficient medium and purifying the enzyme in the presence of ethylenediaminetetraacetic acid (EDTA) (38). Even under these conditions the purified enzyme contained 0.2 mol zinc mol subunit. The activity of this semiapoenzyme was 25 of the holoenzyme, which demonstrated a direct correlation between zinc content and activity.

Protocol 16 Monitoring NFkB Activity by Electrophoretic Mobility Shift Assay EMSA

Suspend cells in 400 l of buffer A (10 mM HEPES acid pH 7.8 , 10 mM KCl, 0.1 mM EDTA, 1 5. Suspend the nuclei in buffer C (50 mM HEPES pH 7.8 , 420 mM KCl, 0.1 mM EDTA, 5 mM MgCl2, 10 glycerol, 1 mM DTT, 2 g ml aprotinin, and 0.5 mM PMSF). 10. Incubate the nuclear extract (3 g) with 300 femtomole (fmol) of the probe in a total of 25 l of binding buffer (10 mM HEPES pH 7.8 , 50 mM KCl, 1 mM EDTA, 5 mM MgCl2, 10 glycerol, and 2 g of poly-dIdC) for 20 minutes at room temperature. Poly-dIdC is used to minimize the binding of nonspecific proteins to the labeled target DNA. These repetitive polymers provide an excess of nonspecific sites to adsorb proteins in crude lysates that will bind to any general DNA sequence. 11. Fractionate the samples on a 4 polyacrylamide gel in 25 mM Tris-HCl (pH 8.5), 190 mM glycine, and 1 mM EDTA.

Preparation of Human Plasma Samples

Blood collection tubes BD Plus Plastic K2EDTA (BD, 367525 10 mL), BD Glass Serum with silica clot activator (367820, 10 mL). 2. Protease inhibitor (Complete Protease Inhibitor Cocktail, Roche, 11 697 498 001, 20 tablets) One tablet contains protease inhibitors (antipain, bestatin, chymostatin, leupeptin, pepstatin, aprotinin, phosphoramidon, and EDTA) sufficient for the processing of 100 mL plasma samples. Prepare 25x stock solutions in 2 mL distilled water.

Targeting Multiple Functional and Pathological Deficits

Five different mechanisms of actions for the treatment of AD are discussed separately in the following chapters. In the opening chapter Drs. Jeffrey Kao and George Grossberg review the use of cholinesterase inhibitors currently on the market as symptomatic treatments for AD. The next four chapters discuss potential disease modifying therapeutic opportunnities that have entered clinical studies. In Chapter 2 Drs. Holly Soares and Larry Sparks review the pleiotropic effects of HMG-CoA reductase inhibitors (statins) that may affect AD pathogenesis. In Chapter 3 Drs. Qingguang Jiang, Shweta Mandrekar and Gary Landreth discuss how PPAR-y agonists can be used to regulate metabolism of lipids and glucose and inflammatory responses to ameliorate deficits in AD patients. Both mechanisms discussed in Chapters 2 and 3 have benefited from the availability of marketed drugs for a different indication and have been fast tracked to Phase III clinical trials for the treatment of AD. In Chapter 4 Drs....

Protocol 17 Monitoring IRF3 Nuclear Translocation by western Blotting

Pellet down the cells, and resuspend them in 150 l of buffer A (10 mM HEPES-KOH pH 7.8 , 10mM KCl, 0.1 mM EDTA pH 8.0 , 1 g ml apro-tonin, 1 ag ml pepstatin, and 0.5 mM PMSF). 5. Resuspend the nuclei in 40-80 jl of buffer C (50 mM HEPES-KOH pH 7.8 , 420 mM KCl, 5 mM MgCI2, 0.1 mM EDTA pH 8.0 , and 2 glycerol) and incubate them at 40 C for 30 minutes to allow nuclear protein extraction.

Synthetic Antioxidants

Synthetic antioxidants which contain phenolic groups, such as gallic acid esters, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tertiary butyl hydroxyquinone (BHQ), are the most widely used food synthetic antioxidants (Fig. 4.1). The effectiveness of phenolic antioxidants depends on the resonance stabilization of the phenoxy radicals determined by the substitution at the ortho and para positions on the aromatic ring and by the size of the substituting group (Shahidi et al., 1992). The presence of carbonylic and carboxylic groups in numerous phenolic compounds can also result in the inhibition of oxidative rancidity by metal chelation (Hudson and Lewis, 1983).

Systemic Delivery of Peptides and Proteins

The addition of absorption enhancers, like bile salts (glycocholate), fatty acids (linoleic acid), surfactants (lecithins, polyoxyethylene-9-lauryl ether or N-lauryl- -D-maltopyra-noside) and chelators (EDTA) can significantly increase the absorption of various proteins. However, the application of enhancers is limited by their toxicity. For example polyoxyethyl-ene-9-lauryl ether and sodium glycocholate caused serious oedema, haemorrhage and inflammation of the lung after intratracheal instillation 39 .

Bad Effects Of Copper On Prion Protein

Other experiments have studied the effects of copper on the neurotoxicity of PrPSc or the peptide mimic PrP106-126. Chelation of copper in culture models of PrP106-126 neurotoxicity abolish the toxicity of the peptide, suggesting that copper is necessary for PrP106-126 toxicity (74). Neurones grown in low concentrations of copper are also more resistant to the toxicity of PrP106-126. Transition metals have been shown to have a critical effect on the ability of the p-amyloid protein to induce fibril formation of that protein (75). Another study has investigated whether this property is common to other prion protein peptides, by studying the effect of metals on PrP106-126 aggregation (76). In that article, the authors show that fibrillization of PrP106-126 was completely inhibited in a transition-metal-depleted environment. Cu2+ and, to a lesser extent, Zn2+ could restore PrP106-126 aggregation. The metal-binding site was localized and found to comprise the N-terminal amino group...

LDL receptor binding assay

Harvested HepG2 cells were assayed for LDL receptor binding activity as described for mononuclear cells (Roach et al., 1993a). Human LDL, 1.025> d> 1.050 g ml-1, was isolated from 2-4 day old blood (Red Cross, Adelaide, Australia) and conjugated to colloidal gold (LDL-gold) as previously described (Roach et al., 1987, 1993a). The cells, 100 pg of protein (Lowry et al., 1951), were incubated for 1 h at room temperature with LDL-gold (20 g protein ml-1) and buffer (60 mM Tris-HCl, pH 8.0, and 20 mg ml-1 bovine serum albumin) in a total volume of 300 l either in the presence of 2 mM Ca(NO3 to measure total binding or 20 mM ethylene diamine tetraacetic acid (EDTA) to measure calcium-independent binding. The cells were then centrifuged at 400 X g for 10 min, resuspended and washed in 300 ml of 2 mM Ca(NO3)2 for total binding or 300 ml of 20 mM EDTA (pH 8.0) for calcium-independent binding and centrifuged at 400 X g for 10 min. The HepG2 cells were finally resuspended in 120 l of 4 (w...

Molecular Size Of Native Aspnat

Whether or not the large molecular size of native Asp-NAT was an artifact of the enzyme preparation methods was tested by using different homogenization and solubilization conditions. A buffer that is commonly used in mitochondrial protein purification (Tris-sucrose medium Tris-HCl, 50 mM 1 mM EDTA 0.32 M sucrose 1 mM DTT protease inhibitor cock-tail added according to the quantity of processed tissue pH adjusted to 7.4) was employed to obtain a crude mitochondrial pellet from rat brains. Various detergents were tested to solubilize the pellet deoxycholate (DC) (negatively charged and one of the most commonly used detergents in mitochondrial studies), hexadecyl trimethyl ammonium bromide (positively charged, commonly called CTAB), Triton X-100 (non-ionic), laurylmaltoside (LM) (non-ionic, commonly used in mitochondrial protein reconstitution experiments) and CHAPS.

Nitrate Reduction and Nitrate Reductase Activity in Photosynthesizing Leaves

The catalytic activity of NR is rapidly modulated by reversible phosphorylation on a serine residue (Ser 543 in spinach). P-NR binds a 14-3-3 dimer and becomes totally inactive in the presence of divalent cations. Dephosphorylation, or chelation of divalent cations, releases 14-3-3 and activates NR. Partial inactivation occurs in conditions such as darkness or after removal of (for a recent review, see Kaiser et al., 1999). In leaves from spinach grown with good nitrate fertilization, maximum NR activity (+ EDTA, NRmax) was about 20 moles g_l FW fir1 (Fig. 1). NR activity measured in the presence of free NRact) varied between 50 and 80 of NRmax under good photosynthetic conditions (for further experimental details, see Kaiser et al., 2000). If NRact reflects the NR activity in situ, these values suggest that in the leaf, nitrate should be reduced at a rate of 10 to

Strategies to Overcome Solubility Issues in Aqueous Media

Table 6 shows examples of assay buffers used for biological screening of ten different discovery programs. The buffer components and percent organic solvents are very different for each project. A generic buffer system would not be able to closely mimic the different conditions used in the assays. The solubility of the compounds will vary in the different buffers containing drastically different amounts of organic solvents, serum proteins, pHs, chelating agents, surfactants and salts. Correction of activity using solubility data measured in generic buffer can introduce additional uncertainty into the activity data. 50 mM Na2HPO4,pH 6.6 200 mM NaCl 1 mM EDTA 50 mM Tris, pH 7.5 10 mM MgCl2 0.5 mM EDTA

Enzyme Assay Optimization

In its simplest form, building an assay is a matter of adding several reagent solutions to a micro-titer plate (MTP) with a multi-channel pipette, at various incubation times, stopping the reaction if required, and reading the MTP in a plate reader. A typical procedure may involve (1) adding the enzyme solution to a compound-containing microtiter plate and incubating for 15 min (2) adding substrates and incubating for 15 to 60 min (3) adding a stopping reagent such as EDTA (for kinases) or sodium orthovanadate (for phosphatases) (4) adding sensor or detector reagents such as labeled antibodies or coupling enzymes and (5) measuring in a plate reader. The specific detector reagents, assay reagent volumes, concentrations of reagents, incubation times, buffer conditions, MTP types, and assay stopping reagents are all important parameters that must be tested.

Quantitative Determination ofFactin Depolymerization

Toxin-treated cells are rinsed with ice-cold PBS. Cold Triton solution (2 (v v) Triton X-100, 160 mM KCI, 20 mM EDTA, 8mM sodium azide, 40 mM imidazole-HCI pH 7.0 300 1 per 50 mm culture dish) is added, the cells are mechanically removed, transferred into an Eppendorf vial, vortexed and incubated for 15min on ice, followed by centrifugation for 15min at 3000 g.

Measuring Reaction Rates With Isotopes

Because most enzyme reactions are not attended by quantifiable changes in their UV-visible absorption and or fluorescence spectra, stable and radioactive isotopes to measure reaction rate must be employed frequently (Allison and Purich, 1979 1980 Highes, 2002). In most cases, kineti-cists apply the stopped-time assay method, wherein an isotopically labeled substrate is incubated with enzyme and reaction components for a defined period, whereupon further reaction is prevented by addition of strong acid, a chelating agent like EDTA, EGTA or o-phenanthroline, a potent inhibitor, etc., or elevated temperature to denature an enzyme. After applying some chromatographic separation techniques to separate substrates and products, the experimenter then quantifies the amount of isotope in the substrate and product. In a few cases, NMR spectroscopy can detect structural rearrangements of substrates that have been suitably labeled with an isotope substituted at or near the reaction center (i.e.,...

Protocol 33 Detection of Complex Formation by Coimmunoprecipitation

Terminate this stimulation by removing the medium and washing the cells with 15 ml of cold PBS. Add 1 ml of ice-cold lysis buffer (20 mM HEPES pH 7.4 , 150 mM NaCl, 1.5 mM MgCl2, 2 mM EDTA, 2 mM DTT, and 1 Triton X-100) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM sodium fluoride (NaF), 1 mM sodium orthovanadate (Na3VO4), and protease inhibitors (Complete, from Roche Diagnostics, Basel, Switzerland). Detach the cells with a cell lifter (Fisher Scientific, Waltham, MA), and transfer cell suspension into a 1.5 ml tube.

Protocol 36 NEMO Ubiquitination

Lyse cells in RIPA buffer (50 mM Tris pH 7.5 , 150 mM NaCl, 0.25 DOC, 1 NP-40, 1 SDS, 1mM EDTA, 1mM EGTA, 1 mM P-glycerophosphate, 1 mM PSMF, 1 mM sodium orthovanadate, 1 g mL leupeptin, 1 g mL pepstatin, 10 nM calyculin A, 5 mM iodoacetimide, and 5 mM N-ethylma-leimide). Note omit DTT or P-mercaptoethanol from the buffer.

Protocol 38 determination of IKBa phosphorylation

Wash cells with PBS containing 10 nM calyculin (Millipore), and lyse with lysis buffer (20 mM Tris-HCl pH 7.5 , 150 nM NaCl, 1 mM EDTA, 1 mM EGTA, 1 Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM P-glycero-phosphate, 1 mM sodium orthovanadate, 1 pg mL leupeptin, 1 mM PMSF, and 10 pM calyculin A). Incubate on ice for 20 minutes, and then spin at 13,000 rpm for 10 minutes. Discard the pellet.

Thiols sulphides and other sulphur groups

Thiol and sulphide groups are not usually introduced into leads in SAR studies because they are readily metabolised by oxidation (see Table 12.1). However, thiols are sometimes introduced into a lead structure when improved metal chelation is the objective of the SAR study. For example, the antihypertensive captopril was developed from the weakly active carboxyacylprolines by replacement of their terminal carboxylic acid group with a thiol group, which is a far better group for forming complexes with metals than carboxylic acids (see section 9.12.2).

Protective effect of HfPS1 against ethanolinduced oxidative stress

FIGURE 11.6 Effects of Hf-PS-1 on ethanol-induced gastric damage by DNA fragmentation. Animals were treated and gastric tissues were collected. DNA of tissues was extracted with 10 mM Tris-HCl (pH 7.4), 10 mM EDTA, and 1 Triton X-100 and electrophoresis in 1 agarose gels and visualized by ethidium bromide staining with a UV transilluminator. Each lane represents a sample from an individual rat.


EDTA Buffer Dissolve 37.2 g of edetate disodium in 800 mL of water, adjust with ammonium hydroxide to a pH of 7.8, dilute with water to 1000 mL, and mix. Support Phase Add 5 mL of EDTA Buffer to 10 g of acid-washed chromatographic siliceous earth for column chromatography, and mix until the siliceous earth is uniformly moistened.

How Calcium And Ros Can Interact

Through voltage or ligand-gated calcium ion channels and calcium efflux by active transport and through Ca2+ Na+ exchange, through sequestration of calcium in the endoplasmic reticulum (ER), mitochondria, and nucleus, and its chelation by cytoplasmic calcium-binding proteins (Fig. 1) (1,2). In addition to influx through receptor-linked calcium ion channels, stimulation of receptors not directly linked to such channels (e.g., G protein-linked metabotropic glutamate receptors) may also act to raise intracellular calcium levels via activation of IP3 or ryanodine receptors on the ER via second messengers that elicit release of calcium from this intracellular store. Figure 1 Control of intracellular Ca2+ levels. Intracellular Ca2+ levels are maintained due to a balance between calcium influx through agonist and voltage-gated channels or metabotropic receptors via activation of ionositol triphosphate (IP3) and calcium efflux by active transport or ion exchange, through sequestration of...

Cell Culture and Harvest

500 mM EDTA, pH 8 9. 2 mM EDTA in sterile PBS add 80 L of 500 mM EDTA, pH 8, in 20 mL of PBS 1. Lysis buffer 50 mM Tris-HCl, pH 7.2, 1 Triton X-100, 10 mM sodium fluoride (NaF), 1 mM sodium orthovanadate (Na3VO4), and 1 mM EDTA 4. Add 3 mL of 2 mM EDTA-PBS per flask, put flask into the 37 C incubator. Monitor the detachment of cells carefully. Cells usually detach within 5 min. For the celecoxib-treated cells, it takes less than 5 min (see Note 1). 1. Dislodging cells using a low concentration of EDTA preserves the integrity of cell surface proteins, which is critical in quantitative proteomic analysis.

Tissue Specimen and Sample Preparation by Nonenzymatic Method NESP

Glutamine-free RPMI 1640 medium glutamine-free, 5 fetal calf serum, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM ethylenediaminetetraacetic acid tetrasodium salt dehydrate (EDTA), and antibiotics oxacillin 25 g ml, gentamycin 50 g ml, penicillin 100 U ml, streptomycin 100 g ml, amphotericin B 0.25 g ml, nistatin 50 U ml. Store at 4 C.

Procedure 2 Purification of Human Hemoglobin

Frozen cells (150 g) were resuspended in 300 mL 15 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 1 mM dithiothreitol (DTT), 1 mM MgCl2, 0.1 mM MnCl2, 40 mg RNase A, 5 mg RNase B, 10 000 U of DNase. The cells were lysed by sonication and centrifuged (20 min at 17300x g). The supernatant was further clarified at 50 000 rpm for 30 minutes. cm Amersham Pharmacia Biotech) equilibrated with 50 mM Tris-HCl (pH 8.0) containing 0.1 mM EDTA and 1 mM DTT (start buffer). The eluate was loaded onto a DE52 column (4 x 8 cm Whatman, Clifton, NJ, USA) equilibrated with start buffer and washed with a further 500 mL of the same buffer. The hemoglobin fraction was eluted with a gradient of 500 mL each of start buffer and 15 mM Bis-Tris (pH 6.2) containing 0.1 mM EDTA and 1 mM DTT. All of the above buffers were equilibrated with carbon monoxide. The hemoglobin fraction was concentrated through PM30 Amicon membranes (Millipore, Bedford, MA, USA).

Immunoprecipitation of PKC Isoforms

After treatment, cells were harvested in 1 mL lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 v vl Triton X-100, 1 mM EGTA, 2 mM EDTA, chymostatin, leupeptin, antipain, pepstatin 5 mg L each , 1 mg L E64, and 1 mM PMSF. Cell lysates were forced through a 25-gauge syringe (15 times) and cleared by centrifugation at 15,800 X g for 10 min. Immunoprecipitation was carried out on equal amounts of protein with the indicated anti-PKC antibody (3 ig) incubated for 1-3 h at 4 C followed by adsorption to protein A-Sepharose beads (10 mg) for 1 h at 4 C. Precipitated samples were recovered by centrifugation and proteins were either resolved by SDS-PAGE or used in autophosphorylation and kinase reactions.

Autophosphorylation of PKC Isoforms

Immunoprecipitated PKCs bound to protein A-Sepharose beads were washed three times with lysis buffer and once with the same buffer containing 0.4 M NaCl and without EDTA EGTA. Samples were incubated in 40 mL of a mixture containing 5 lCi of y-32P ATP, 10 mM ATP, 400 (iM MgCl2, 5 mM CaCl2, 400 iM phosphatidylserine, 100 nM phorbol 12,1-dibutyrate, 1 mM sodium orthovanadate, and 20 mM Tris, pH 7.4 at 37 C for 10 min. The reactions were terminated by addition of 10 iL of boiling SDS sample buffer and electrophoresed on a 10 polyacrylamide gel. Gels were stained using the SYPRO protein gel stain kit (Molecular Probes), blotted, and radioactivity in the membranes was detected by using a BioRad GS-250 Molecular Imager. Alternatively, gels were dried down for autoradiography on Kodak X-Omat S films. Quantification was done by using a BioRad GS-700 imaging densitometer.

Basis To Understand Toxic Effects Associated With Chronic Exposure To Copper

Ring copper from the intestinal lumen to the portal circulation (32,45). Our results indicate that in Caco-2 cells grown in a low copper medium, 64 of the cellular-to-basolateral flux corresponds to newly incorporated copper (64Cu). This suggests that in these conditions, copper uptake is strongly associated to efflux pathway(s) (Fig. 2). In contrast, only a minor fraction (4 ) of the 64Cu was delivered to the basolateral domain, when copper concentration was increased in the medium. In these latter conditions, the intracellular copper was elevated and the copper efflux was enhanced (14). When copper offered to the cell is high, copper uptake by the cell may be higher than the concentration of the copper-chelating agents, making it possible that intracellular copper becomes toxic unless the cells can induce protective mechanisms. Induction of MT is one protective mechanism (55). Another mechanism is given by the facilitation of copper efflux during excess, by increasing the...

Assay Of Human Antithrombin

Prepare 2 independent series of 3 or 4 dilutions in the range 1 75 to 1 200 from 1 IU ml, for both the preparation to be examined and the reference preparation, using tris-EDTA BSA buffer solution pH 8.4 R containing 15 IU of heparin per millilitre. Warm 200 pl of each dilution at 37 C for 1-2 min. Add to each dilution 200 pl of a solution of bovine thrombin R containing 2 IU ml in tris-EDTA BSA buffer solution pH 8.4 R. Mix and maintain at 37 C for exactly 1 min. Add 500 pl of a suitable chromogenic substrate (for example, reconstituted in water R to give a solution containing 4 mmol l and further diluted to a concentration suitable for the assay using tris-EDTA BSA buffer solution pH 8.4 R without albumin). Immediately start measurement of the change in absorbance at 405 nm (2.2.25), continuing the measurement for at least 30 s. Calculate the rate of change of absorbance (AA min). (Alternatively, an end-point assay may be used by stopping the reaction with acetic acid and...

In vitro dissolution of amyloid

The process of metal chelation involves the interaction between at least two donor groups on a ligand and an ionic substrate resulting in a 'ring' structure 86 . The potential use of a metal ligand as a therapeutic is affected by many properties such as size, charge, hydrophobicity and density. The latter is a measure of how many available groups can bind to the ionic substrate, i.e., bidentate has two groups whereas hexadentate has six 86 . The selection of optimal metal ligands for therapeutics may depend on a trade-off between several characteristics. For example, a hexadentate ligand may have a higher specificity for a particular metal, however, its increased size can reduce its ability to cross the blood-brain barrier. 1,10 phenanthroline has a high affinity for Cu, Zn and Fe through its nitrogen groups, however, binding of these metals results in a positive charge, which reduces its ability to cross membranes. Alternatively, 8-hydroxyquinolines chelate through both nitrogen and...

Labeling and Hybridization of Serum Samples

Buffers phosphate buffered saline (PBS), pH 7.4 (137 mM NaCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4) carbonate buffer, pH 8.5 (50 mM NaHCO3) PBST, PBS containing 0.5 (v v) Tween-20 0.1 M PBS, pH 7.2 (68.4 ml 1 M Na2HPO4, 31.6 ml 1 M NaH2PO4, 900 ml dH2O) NP40 lysis buffer 50 mM Hepes-OH, EDTA, 50 mM NaCl, 10 mM NaPPi (Tetrasodium Diphosphate Decahydrate), 50 mM NaF, 1 (v v) NP40, 10 mm Sodium- Vanadate, pH 7.5-8.0 saturated NaCl (Sigma) blocking buffer 1 (w v) bovine serum albumin (BSA) in PBST 7-10 mM dye stock in DMSO Dissolve one tube of Cy3 or Cy5 dyes in 30 pl of DMSO. Aliquot and freeze at -80 C.

Treatment of Alzheimers disease patients with CQ

The decrease in plasma Ap is congruent with the lowering of plasma Ap in CQ-treated transgenic mice as the treatment debulks the brain Ap burden (there was a significant correlation between brain Ap and plasma Ap levels) 179 . The increase in plasma Zn normalized the levels from the abnormally low baseline levels, which is typical of levels in older subjects. This effect is compatible with the debulking of cerebral amyloid burden upon treatment with CQ, so preventing Zn from being trapped in plaques and CAA. 15 of plasma Zn is in communication with Zn released from synaptic activity 140 . The elevation (and normalization) of plasma Zn in CQ-treated AD patients underscores that the mechanism of CQ activity in AD does not involve systemic chelation.

Treatment of Alzheimers disease patients with traditional hydrophilic chelators

Desferrioxamine (DFO) is a hexadentate Fe-selective chelator used in a 2-year single-blind trial for treatment of AD (Table 2). The basis for this trial at the time was the potential removal of Aluminium (Al), which once had been considered as possibly linked to AD aetio-pathology 192 . The association between Al and AD has not been supported by the weight of recent studies 193 , however, in the DFO trial 48 patients were treated with either 125 mg DFO twice daily, oral placebo or no treatment. DFO treatment resulted in a significant reduction in rate of decline of daily living activities. These findings are interesting as DFO does not penetrate the BBB and therefore its access to the brain would be limited to sites of putative BBB disruption 182 . Although the target in this case was Al, it is likely that the beneficial effect of DFO may have resulted from chelation of Fe, Cu or Zn. Further clinical research into DFO has diminished due to difficulty of administration (painful...

Plimstex Results for RasGDP Titrated with Mg2B

Fig. 11.4 PLIMSTEX curve for 1.5 iM Ras-GDP titrating with Mg2+. Conditions 90 D2O, 50 mM HEPES buffer, 100 mM KCl, pH 7.4, H D exchange time 3 h. EDTA was used to control Mg2+ in solution. The error bars shown for each data point were based on the deviation from two independent runs. Fig. 11.4 PLIMSTEX curve for 1.5 iM Ras-GDP titrating with Mg2+. Conditions 90 D2O, 50 mM HEPES buffer, 100 mM KCl, pH 7.4, H D exchange time 3 h. EDTA was used to control Mg2+ in solution. The error bars shown for each data point were based on the deviation from two independent runs.

Objective and Rationale

AlphaScreen is an energy transfer method that employs donor and acceptor beads the donor bead absorbs light and energy in the form of a singlet oxygen molecule and is transferred to an acceptor bead to generate a signal. In these assays, the AlphaScreen histidine (nickel chelate) detection kit was used. The kit contained streptavidin-coated beads for binding to biotin-GA and nickel-coated beads for chelation to the His tag on the full length Hsp90 isoforms. Binding of the biotin-GA to the ATP-binding pocket of the His-tagged isoforms (Hsp90a, Hsp90 , and Grp94) brought the strepta-vidin and nickel-coated beads into close enough proximity to produce a signal (Figure 5.3A).

Experimental Issues Regarding Using Metal Chelators

In most PLIMSTEX experiments, the total ligand concentration is used in the curve fitting. The determination of free ligand concentration is not required because the relationship between the free and total ligand concentrations is resolved in the modeling procedure 24 . For the ras titration, however, we had no Mg2+-free ras-GDP stock but only a limited amount of ras-GDP-Mg2+ stock solution (1.5 mM ras, 10 mM MgCl2, in 64 mM Tris-HCl, 10 mM MgCl2, 1 mM sodium azide, 1 mM DTT buffer, pH 7.6). After diluting by 100 times with 50 mM acid (HEPES) and 0.1 M KCl, pH 7.4 buffer, there was still excess of Mg2+. To conduct the Mg2+ titration of free ras-GDP, we added EDTA to control free Mg2+ in solution free Mg2+ was calculated using a WebChelators program (Webmaxc Standard) on the internet 43, 44 .

Other Considerations

False positives may be observed with the AlphaScreen histidine detection kit certain inhibitors of Ni chelation to histidine are known. Compounds such as imidazoles can displace histidine from nickel chelates, resulting in a loss of signal. Compounds of interest should be tested for AlphaScreen interference by testing against a biotinylated (His)6 peptide provided in the kit.

Rapid Mixing Quenching Devices Permit Kinetic Analysis of a Wide Range of Chemical Reactions

Although rates are most conveniently assayed when attended by large changes in the UV visible or fluorescence spectrum, the majority of enzyme-catalyzed reactions do not generate any observable change in their electronic spectrum. In such cases, it is necessary to purchase or prepare substrates containing one or more radioactive atoms so that changes in substrate depletion and or product accumulation can be analyzed. The experimenter uses a rapid mix quench apparatus, a device containing two or more syringes and one or more mixers (Fig. 10.6). Two syringes advance and force solutions containing reactants A and B to combine at the start mixer at time t0. The reaction proceeds through an aging loop whose length determines the duration of reaction. Then at time t, the reaction is quenched (i.e., stopped) by a change in pH to denature the enzyme or the addition of sufficient EDTA or EGTA to sequester a metal ion essential for enzymatic activity.

Pmpa And 2mppa Are Potent And Specific Gcp Ii Inhibitors

Research at Guilford has focused on the potential utility of GCP II inhibitors wherein excess glutamate neurotransmission has been implicated. 2-(Phosphonomethyl) pentanedioic acid (2-PMPA) is a potent, competitive GCP II inhibitor with a Ki value of 0.2 nM15 16 (Fig 2). It exhibits a fast association rate and a slow dissociation rate. The high potency of 2-PMPA can be attributed to the strong chelation of the phosphonate group to an active site zinc atom as well as the interaction of the glutamate moiety (pentanedioic) portion of the inhibitor with the glutamate recognition site of GCPII.18 2-PMPA seems to be quite specific for GCP II, i.e., no significant activities were observed at 10 j.M (more than 10,000-fold higher than the Ki for NAALADase inhibition) in over 100 different receptor and enzyme assays, including glutamate receptors and

Concentration Analysis CCA

The authors used synthetic double-stranded poly d(G-C) and poly d(A-T) in 0.1 M NaCI, 10 mM Tris HCL, 1 mM EDTA (pH 7.2) at 24 C. Reaction progress was monitored as the fluorescence of intercalated ethidium at wavelengths greater than 610 nm upon excitation at 545 nm. The DNA concentration was varied from 1 to 45 mM and the ethidium bromide concentration from 0.5 to 25 mM. The data fit a single-step bimolecular association reaction (Macgregor, Clegg and Jovin, 1985).

Procedure 1 Phytochrome Photoassay

The protein sample, either from plant tissue or reconstituted recombinant phytochrome (see later section), is diluted or reconstituted in the desired amount of TEGE buffer 25 mM Tris-HCl, pH 7.8, containing 25 (vol vol) ethylene glycol, 1 mM EDTA, 2 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM dithiothreitol (DTT) , typically 500 pL. During irradiation and the subsequent analysis, the sample is kept at 10 to 16 C.

Solutions for the assay of exocytosis

KG-buffer I with Mg2VATP for permeabilization and incubation before the stimulation (150 mM K+-glutamate, 2mM EGTA, 2mM EDTA, 20mM PIPES, 2mM Na+ ATP, 1 mM free Mg2+) For 500ml dissolve 13.9 g K+-glutamate, 0.38 g EGTA (free acid), 0.2 g EDTA (free acid), 3.02 g PIPES (free acid) and 0.61 g Na ATP in about 300 ml of water, add KOH to give a pH of 7.0 and stir at room temperature. Add 2.4 ml of 1 M Mg(CH3COO)2 to give a final free Mg2+ concentration of 1 mM and adjust pH exactly to 7.0 with KOH. Make up to 500 ml.

Holophytochrome Assembly

The PEB is dissolved in Me2SO, with the final Me2SO concentration in the assay no greater than 2 (vol vol). The assay mixture is rapidly mixed and placed in a fluorescence spectropho-tometer. Typically, measurements are performed with samples maintained at room temperature (22 -25 C).

Potential Strategies For Radical Scavenging

One of the main pathological features of PD is the appearance of abundant deposition of iron at the site of neurodegeneration, but the basis of the increased iron levels remains unclear (8). Iron has the capacity to promote oxidation reactions and the formation of cytotoxic free radicals (9). A marked increase in the concentration of iron in affected brain areas has been confirmed for PD, Huntington's disease, supra nuclear palsy, multiple system atrophy, as well as Alzheimer's disease (10). The late state of PD is characterized by an accumulation of iron in substantia nigra pars compacta (11). This raises the possibility that therapies designed to chelate iron and prevent it from participating in oxidation reactions may protect vulnerable neurons and slow the rate of disease progression in patients suffering from neurodegenerative disorders. Iron chelators have been demonstrated to effectively prevent iron-catalyzed reactions from taking place and to correspondingly limit free...

Helena Mira Vicente Sancenn and Lola Pearrubia

Different ecotypes from plant species can be classified as sensitive or tolerant to a certain metal, depending on the characteristics of their growth response curves (3), thus indicating that metal sensitivity or tolerance in plants are controlled by a relatively low number of genes. Metal tolerance can either be constitutive or inducible by acclimation, as a part of the adaptive response after plant pre-treatment with moderate metal concentrations. In general, tolerance in plants is not conferred by metal-extruding mechanisms through ionic pumps as it occurs in bacteria, instead, it is mainly based on metal chelation and storage processes that prevent its deleterious effects (3,4).

Y and InLabeling of DTPAConjugated Integrin avp3 Receptor Antagonists

Recently, Liu et al. 94 reported the synthesis of DTPA-c(RGDfK) (Fig. 4 SQ169) and DTPA c(RGDfK) 2 (Fig. 4 SQ170), and their 90Y complexes (90Y-SQ169 and 90Y-SQ170). 90Y-SQ169 and 90Y-SQ170 can be prepared in high yield (more than 95 ) by reacting 2 ig SQ169 and SQ170, respectively, with 20 mCi 90YCl3 (SQ169 Y ratio approximately 4 1) in a 0.5 M ammonium acetate buffer (pH 6.5-8.0) at room temperature. In both cases, the 90Y chelation was instan- 111In-SQ169 and 111In-SQ170 can be prepared in high yield by reacting SQ169 and SQ170, respectively, with 111InCl3 in the 0.5 M ammonium acetate buffer (pH 6.0-7.0) at room temperature 95 . The 111In chelation was almost instantaneous for both SQ169 and SQ170.111In-SQ169 and 111In-SQ170 are stable in solution for more than 24 h without significant decomposition. It is surprising to find that 111In-SQ169 and 111In-SQ170 are more hydrophilic than their corresponding 90Y analogs (Fig. 6), suggesting a different coordination sphere in 111In...

Composition and Concentrations of the Exported Carbon and Nitrogen Compounds

1957 Hall and Baker, 1972) or leaf petiole exudation. Chelating agents (EDTA) are added to avoid the sealing of the wounds by callose formation (King and Zeevart, 1974). Interpretation of data obtained by either method is complicated by the presence of stem or petiole metabolites that may contaminate phloem sap samples. Also, normal transport patterns are seriously impaired by the incision. In an excellent survey, Zimmermann and Ziegler (1975) have compiled data on the composition of sugars in the phloem exudates from more than five hundred species. However, since these exudates were collected using the incision method artifacts in the composition can not be excluded and concentrations of metabolites had not been determined.

Y and InLabeling of DOTAConjugated Integrin av3 Receptor Antagonists

SU015 (Fig. 4) is a DOTA-conjugated cyclic peptide dimer 96,97 . 90Y-SU015 can be prepared by reacting SU015 with 90YCl3 in ammonium acetate buffer (pH 5.0-7.5). There are many factors influencing the rate of 90Y chelation and the radiolabeling efficiency of SU015. These include the pH, reaction temperature and heating time, as well as the presence of trace metal contaminants, such as Ca2+, Fe3+, and Zn2+. The chelation of 90Y by SU015 is slow, so heating at elevated temperatures (50-100 C) is needed to complete the 90Y-labeling.

Attaching Apophycobiliproteins to Agarose Beads

The covalent attachment of apo-a-PC to agarose beads greatly facilitated reconstitution studies because it was possible to perform addition reactions in a small microcentrifuge tube, to wash away excess bilin after the reaction was terminated if necessary, and then to measure the fluorescence of the sample after this process (21,22). The apoprotein in 50 mM Na-phosphate, pH 7.0, 5 mM EDTA was mixed with Affi-Gel 15 (Bio-Rad Laboratories) at 1 mg of protein per mL of beads (22). The covalent attachment of the protein to the beads continued for 30 minutes at 4 C until the reaction was stopped by the addition of 0.05 volumes of 1 M glycylglycine, pH 7.0 (incubated for 1 hour at 4 C). To remove excess unbound protein, the beads were washed with 50 mM Na-phosphate, pH 7.0, 5 mM EDTA, 0.5 M NaCl, followed by 50 mM Na-phosphate, pH 7.0, 5 mM EDTA. The air was evacuated out of the flask containing the beads, and the beads were stored at 4 C in the same buffer with the addition of 5 mM DTT.

Heavymetal Antagonists

The effectiveness of a chelating agent for the treatment of poisoning by a heavy metal depends on numerous factors the relative affinity of the chelator for the heavy metal as compared with essential body metals, the distribution of the chelator in the body as compared with the distribution of the metal, and the capacity of the chelator to remove the metal from the body once chelated. Consider the properties of an ideal chelating agent high solubility in water, resistance to biotransformation, ability to reach sites of metal storage, capacity to form nontoxic complexes with toxic metals, ability to retain chelating activity at the pH of body fluids, and ready excretion of the chelate. A low affinity for Ca2+ also is desirable because Ca2+ in plasma is readily available for chelation, and a drug might produce hypocalcemia despite high affinity for heavy metals. A therapeutic chelating agent must bind the metal more avidly than endogenous ligands bind the metal. The large number of...

Mechanism Of Action

The pharmacological effects of CaNa2EDTA result from formation of chelates with divalent and trivalent metals in the body. Accessible metal ions (both exogenous and endogenous) with an affinity for CaNa2EDTA that is higher than that of Ca2+ will be chelated, mobilized, and usually excreted. Because EDTA is charged at physiological pH, it does not significantly penetrate cells its volume of distribution approximates extracellular fluid space. Experimental studies in mice have shown that administration of CaNa2EDTA mobilizes several endogenous metallic cations, including those of zinc, manganese, and iron. The main therapeutic use of CaNa2EDTA is in the treatment of metal intoxications, especially lead intoxication (see below). CaNa2EDTA is available as edetate calcium disodium (calcium disodium versenate). Intramuscular administration of CaNa2EDTA results in good absorption, but pain occurs at the injection site consequently, the chelator injection often is mixed with a local...

Expression in Bacteria and Purification of Recombinant Proteins

For purification of aFGF-CaaX, the bacterial pellet was suspended in 20 mM Tris-HCI, pH 7.5, 0.5 M NaCI, 1 mM DTT, 1 mM EDTA and sonicated. The clear supernatant, after centrifugation, was applied to a heparin cartridge (Bio-Rad), and the bound material was eluted with a linear NaCI gradient (0.5-2 M) in the above buffer.

Pigment Biosynthesis

The pigments attached to the PBs are linear tetrapyrroles, called phycobilins. They are bound to the protein moiety at conserved positions by cysteinyl thioether linkages through the vinyl substituent of the pyrrole ring A. Occasionally, a second linkage is established through the vinyl substituent of the pyrrole ring D 2 . Phycobilin synthesis follows the same pathway as Chl until the metal chelation step. As

Radioactive Heavy Metals

The widespread production and use of radioactive heavy metals for nuclear generation of electricity, nuclear weapons, laboratory research, manufacturing, and medical diagnosis have generated unique problems in dealing with accidental poisoning by such metals. Because the toxicity of radioactive metals is almost entirely a consequence of ionizing radiation, the therapeutic objective following exposure is chelation of the metals and their removal from the body as rapidly and completely as possible. Treatment of the acute radiation syndrome is largely symptomatic. Attempts have been made to investigate the effectiveness of organic reducing agents, such as mercaptamine (cysteamine), administered to prevent the formation of free radicals success has been limited. Major products of a nuclear accident or the use of nuclear weapons include 239Pu, 137Cs, 144Ce, and 90Sr. Isotopes of Sr and Ra are extremely difficult to remove from the body with chelating agents. Several factors are involved in...

Radiolabeled Nonpeptide Antagonists

TA138 (Fig. 5) is a quinolone-based nonpeptide integrin avfi3 receptor antagonist, and has very high binding affinity and specificity for the integrin avfi3 receptor 124 . DOTA is chosen for 111In- and 90Y-chelation owing its ability to form indium and yttrium complexes with high thermodynamic stability and kinetic inertness. The dicysteic acid linker was selected as a pharmacokinetic modifier to increase the hydrophilicity and to improve the renal clearance of the 111In- and 90Y-labeled DOTA-conjugates 124 . Selected images of

Added Substances Excipients in Parenteral Formulations

To provide efficacious and safe parenteral dosage forms, added substances must frequently be incorporated into the formula to maintain pharmaceutical stability, control product attributes, ensure sterility, or aid in parenteral administration. These substances include antioxidants, antimicrobial agents, buffers, bulking materials, chelating agents, inert gases, solubilizing agents, and protectants. In parenteral product development work, any additive to a formulation must be justified by a clear purpose and function. In addition, every attempt should be made to choose added substances that are accepted by regulatory agencies throughout the world, since most pharmaceutical development is international in scope. Because of the extensive pharmacological and toxicological data required to obtain approval for any new additive, most formulators continue to depend on materials already used in marketed parenteral products.

Protocol 37 Determination of IKK Activity

After stimulation for 0-90 minutes, lyse cells (106) in 1 ml lysis buffer (20 mM Tris pH 7.5 , 150 nM NaCl, 1 mM EDTA, 1 mM EGTA, 1 Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM P-glycerophosphate, 1 mM sodium orthovanadate, 1 g ml leupeptin, 1 mM PMSF, and 10 nM calycu-lin A). After incubation for 10 minutes at 4 C, clear the lysate via centrifu-gation at 13,000 rpm for 10 minutes at 4 C.

Iron and the Enhancement of ALAPDT

One of the most clinically feasible methods listed above is the use of iron-chelating agents. The mechanism of this enhancement technique brings together elements of classical PDT, control of intracellular metal homeostasis and redox control by formation of ROS. It is centered on the heme biosynthesis pathway, where PpIX is the immediate precursor of heme. The PpIX produced in the cell is converted into the nonphotosensitizing compound heme by the enzyme ferrochelatase, which inserts iron into the tetrapyrrole ring of PpIX (Figure 15.3). Sequestering the iron required for this reaction using an iron-chelating agent therefore reduces the amount of PpIX that is converted to heme, resulting in greater levels of PpIX temporarily accumulating in the cell - and thus a greater PDT effect on irradiation. The lower levels of heme will also reduce negative feedback on the heme biosynthesis pathway as described in Figures 15.1 and 15.2. This in turn will increase the production of ALA by ALA...

Physicochemical drug interactions and incompatibilities

10.5 Chelation and other forms of Appendix 425 complexation 405 References 429 Chelation - in which a chelator molecule binds with a metal ion to form a complex Ion-exchange interactions - in which ionised drugs interact with opposites charged resins Adsorption to excipients and containers - causing loss of drug Interactions with plastics - another source of loss of material of this book. There is no reason why the same forces and phenomena that operate in vitro cannot explain many of the observed interactions that occur in vivo, although of course the interplay of physicochemical forces and physiological conditions makes simple interpretations a little hazardous. Interactions such as protein binding, whether as a result of hydrophobic or electrostatic interactions, adsorption of drugs onto solids, or chelation and complexation, all occur in physiological conditions and are predictable to a large degree, provided that certain assumptions are made. We can also observe interactions...

Protocol 35 Determination of IRAK4 and TAK1 Activation

Remove the medium by aspiration, and wash the cells quickly with 10 ml of PBS. Scrap off cells in 1 ml of lysis buffer (50 mM Tris pH 7.5 , 150 mM NaCl, 1 NP-40, 1 mM P-glycerophosphate, 2.5 mM sodium pyrophosphate, 1 mM sodium fluoride, 1 mM EDTA, 1 mM ethylene glycol tetraace- 4. Collect the beads by centrifugation at 4,000 rpm for 1 minute, and carefully remove the supernatant by aspiration. Wash the beads with 1 ml of lysis buffer four times, followed by two more washes with 1 ml of kinase assay buffer (20 mM HEPES pH 7.4 , 20 mM MgCl2, 1 mM DTT, 20mM P-glycerophosphate, 20 mM paranitrophenylphosphate, and 1 mM EDTA) supplemented with 1 mM NaF, 1 mM sodium vanadate, and protease inhibitors (Complete, from Roche Diagnostics, Basel, Switzerland).

Antagonism Or Chemical Inactivation Of An Absorbed Poison

Specific chemical antagonists of a toxicant, such as opioid antagonists (see Chapter 21) and atropine as an antagonist of pesticide-induced acetylcholine excess (see Chapter 7), are rare. A recently approved antagonist is fomepizole, an inhibitor of alcohol dehydrogenase, approved for treatment for poisoning by ethylene glycol and methanol. Chelating agents with high selectivity for certain metal ions are used more commonly (see Chapter 65). Antibodies offer the potential for the production of specific antidotes a notable example is the use of purified digoxin-specific Fab fragments of antibodies in the treatment of potentially fatal cases of poisoning with digoxin (see Chapter 33). The development of human monoclonal antibodies directed against specific toxins has significant potential.

Drugs and Biological Agents Used in Ophthalmic Surgery

Corneal band keratopathy Edetate disodium (disodium EDTA endrate) is a chelating agent that can be used to treat band keratopathy (i.e., a calcium deposit at the level of Bowman's membrane on the cornea). After the overlying corneal epithelium is removed, it is applied topically to chelate the calcium deposits from the cornea.

Pathways of Glucose Mediated Oxidative Stress in Diabetic Neuropathy

The intellectual challenge to basic and clinical scientists exploring the pathogenesis of DPN is the identification and characterization of the important initiators, compartments, and mediators common to various pathogenetic hypotheses. These common elements may serve as a linchpins around which to array and perhaps unify otherwise competing pathogenetic mechanisms. Glucose-induced generation of reactive oxygen species (ROS) may subserve this purpose (Fig. 1). Autooxidation of glucose, catalyzed by trace amounts of free transition metals such as iron and copper (26), generate ROS in vitro (27), and metal chelating agents to preserve normal nerve conduction velocity

Potential Excipient Effect on Bioavailability

The Biopharmaceutics Classification System (BCS) (Amidon et al., 1995) allows waivers of in vivo bioequivalence for rapidly dissolving immediate-release (IR) formulations of drugs with high solubility and high permeability. One potential issue in possibly extending BCS biowaivers to low-permeability drugs is the potential for excipients to modulate the intestinal permeability of the drug. Rege et al. (2001) investigated the effects of common excipients on Caco-2 transport of such low-permeability drugs. The effects of nine individual excipients (lactose monohydrate, hydroxypropyl methyl cellulose (HPMC), sodium lauryl sulfate (SLS), EDTA, Tween 80, docusate sodium (dioctyl sodium sulfosuccinate), propylene glycol, poly(ethylene glycol 400), and anhydrous cherry flavor) on the Caco-2 permeability were investigated using seven low-permeability compounds that differ in their physical properties. With the exception of SLS no excipients affected Caco-2 cell monolayer integrity. SLS...

Centrally Acting Adrenergic Drugs

Methyldopa is used as a step 2 agent and is recommended for patients with high blood pressure who are not responsive to diuretic therapy alone. Methyldopa, suitable for oral use, is a zwitterion and is not soluble enough for parenteral use. The problem was solved by making the ester, leaving the amine free to form the water-soluble hydrochloride salt. It is supplied as a stable, buffered solution, protected with antioxidants and chelating agents.

Mixed PN2 X X is P O S Ligands

At noncarrier added level, stable mononuclear complexes, 99mTcO2(P2S2) +, analogous to 74, were synthesized with high radiochemical purity (above 95 ). These compounds, which are stable in vitro over a wide range of pH, were obtained by simply mixing 99mTcO4 - with the ligands or by trans-chelation from 99mTc citrate 76 . The high in vitro and in vivo stability of 99mTcO2(P2S2) + prompted the group of Katti to develop a bifunctional chelating agent (P2S2-COOH) based on the P2S2 framework. Using low concentrations (10-5 M) of the P2S2-COOH ligand,the complex 99mTcO2(P2S2-COOH) + (75a) was prepared by direct reduction of 99mTcO4 - with Sn2+, or by trans-chelation using 99mTc gluconate (Scheme 19). This water-soluble complex (75a) exhibited fast clearance from the bloodstream and its excretion was through the renal and the hepatobiliary pathways, as revealed by biodistribution studies in mice 77 .

Examples Of Infusion Drugs That Photodegrade

Ketoprofen Decarboxylation

An obvious precaution to minimize oxidation is to avoid contact of the drug with ions such as iron, cobalt, or nickel that are initiators of the oxidation process. Similarly, the oxygen above the formulation should be replaced with nitrogen or carbon dioxide. Even traces of oxygen are, however, sufficient to initiate oxidation, and because it is difficult to remove all of the oxygen from a container, it is common practice to add low concentrations of antioxidants and chelating agents to protect drugs against autoxidation. Mechanistically, some antioxidants, such as ascorbic acid, ascorbyl palmitate, sodium bisulfite, sodium metabisulfite, sodium sulfite, acetone sodium bisulfite, sodium formaldehyde sulfoxylate, thioglycerol, and thioglycolic acid, act as reducing agents. They are easily oxidized, preferentially undergo autoxidation, thereby consuming oxygen and protecting the drug or excipient. They are often called oxygen scavengers because their autoxidation reaction consumes...

Potential Excipient Effect on Tight Junctions

Calcium depletion by chelating agent (e.g., EDTA, EGTA) has been reported to increase paracellular permeability. These agents induce general changes in the cell physiology such as disruption of actin filaments and adherence junctions, diminished cell adhesion, and activation of protein kinases (Citi, 1992). It was proposed that EGTA provokes alterations on the tight junctions, being a consequence of its effects on Ca2+ dependent adhesion molecules (which are concentrated in adherence junctions), through a contraction of the junction-associated microfilament cytoskeleton (Citi and Denisenko, 1995). It has been demonstrated that serosal rather than apical Ca2+ levels play a more important role in this process (Collares et al., 1994). Basolateral Ca2+ levels vary and a particular chelation enhancer cannot accomplish full depletion of the calcium ions from the adherence junction to provoke the paracellular widening. Therefore, the approach of using chelating agent as permeation enhancers...

Treatment Of Lead Poisoning

Initial treatment of the acute phase of lead intoxication involves supportive measures. Prevention of further exposure is important. Seizures are treated with diazepam or phenytoin (see Chapter 19), fluid and electrolyte balances must be maintained, and cerebral edema is treated with mannitol and dexamethasone or controlled hyperventilation. The concentration of lead in blood should be determined or at least a blood sample obtained for analysis prior to initiation of chelation therapy. Chelation therapy is indicated in symptomatic patients or in patients with a blood lead concentration in excess of50-60 pg dL (about 2.5 jjM). Four chelators are employed edetate calcium disodium (CaNa2EDTA), dimercaprol, D-penicillamine, and succimer (2,3-dimercaptosuccinic acid DMSA , chemet). CaNa-EDTA and dimercaprol usually are used in combination for lead encephalopathy. CaNa2EDTA is initiated at a dose of30-50 mg kg day in 2 divided doses either by deep intramuscular injection or slow intravenous...

Edetate Calcium Disodium

Ethylenediaminetetraacetic acid (EDTA) is a polycarboxylic acid chelator its sodium salt (edetate disodium, Na2EDTA), and a number of closely related compounds chelate many divalent and triva-lent metals. The cation used to make a water-soluble salt of EDTA has an important role in the toxicity of the chelator. Na2EDTA causes hypocalcemic tetany. However, edetate calcium disodium (CaNa2EDTA) can be used for treatment of poisoning by metals that have higher affinity for the chelating agent than does Ca2+.

HPLC with Electrochemical Detection Hplcecd

In invertebrates, HPLC-ECD is used to measure N-acetyltransferase activity against octopamine, dopamine and 5-hydroxytryptamine as well as for perfoming the bi-substrate kinetics of this enzyme in the cerebral ganglion of the American cockroach Periplaneta Americana 66 . In addition, HPLC-ECD is used to determine octopamine levels in various insect species, including locust Locusta migratoria 66 , cockroach Periplaneta Americana 67-68 , and fruit fly Drosophila melanogaster 69 . Robinson's group has used HPLC-ECD to determine behavior-related changes and age-related changes in the levels of various biogenic amines, such as octopamine, dopamine and serotonin, in the antennal lobes and mushroom bodies of individual honeybee Apis mellifera brains 70-71 . The mobile phase in this analysis contains 15 methanol, 15 acetonitrile (a chemical modifier), 1.5 mmol. l-1 sodium dodecyl sulfate, 75 mmol.l-1, sodium phosphate monobasic, and trace amounts of triethylamine and EDTA 70 . The LOD's for...

Other stabilisers and additives for solution formulations

Proteins can also be stabilised by solutes that interact indirectly with the protein molecules. Compounds, which modify bulk solvent properties or act as a scavenger of certain solutes, can also stabilise proteins from certain chemical degradation pathways. The so-called antioxidants, which are scavengers of radicals, can protect cysteine residues from oxidising to disulphide bonds. Commonly used metal chelators such as EDTA chelate transition metal ions to prevent oxidative degradation.

Combinatorial synthesis and screening of heterocyclic druglike scaffold libraries

Screening Synthesis

Resin-bound a-amino esters 17 react cleanly with aromatic and heteroaromatic aldehydes at room temperature in neat trimethyl orthoformate to afford the corresponding imines 20. Lewis acid mediated ionization of these immobilized imines leads to a-amino aldimines which undergo chelation-controlled regio- and stereoselective 3 + 2 cycloaddition with a wide variety of electron-deficient olefin dipolarophiles to yield the five-membered pyrrolidines. A pool of pyrrolidines prepared combinatorially from four AAs, five aromatic aldehydes, and four olefins was acylated with three different mercaptoacyl chlorides to provide, after appropriate deprotections and final TFA cleavage from resin, a library of > 480 mercaptoacyl proline analogs 21. Such molecules have found well-pre-cedented use as inhibitors of ACE, as exemplified by the antihypertensive drug Captopril. Serial deconvolutive screening of this library for activity against ACE led to the identification of 22 (Ki 160 pM) as an analog...

Small and Large Intestine

Intestinal inflammation may influence opioid pharmacodynamics, and conversely, opioids acting through neural pathways responsive to inflammatory mediators appear to have antiinflammatory activity. Inflammation in peripheral locations increases the analgesic potency of delta opioid agonists after their peripheral or central administration, a phenomenon attributed to an inflammation-induced increase in opioid receptor expression at local sites of inflammation and in the CNS 140 . In acute or chronic inflammation of the murine intestine produced by chemical irritants such as croton oil, delta opioid agonists exhibit increased potency in slowing gut transit and decreasing mucosal permeability to the nonpermeant marker 51Cr-EDTA 101,141 .

Formation of Lipid Hydroperoxides

Hydroxy Acid Decomposition

Mutase, catalase, and reduced glutathione (GSH)-dependent peroxidases (26). Also, endogenous processes such as the sequestration of hydrogen peroxide-generating enzymes or the chelation of free transition metal ions by transferrin, ferritin, and ceru-loplasmin can protect against ROS-mediated damage. However, it is always possible that cellular macromolecules and lipids can be damaged by the ROS that escape from these defense systems. It has been suggested that ROS generation is a major contributor to the degenerative diseases of aging, including cardiovascular disease, cancer, immune-system decline, and brain dysfunction (26). ROS-mediated formation of lipid hydroperoxides is a complex process, which involves initiation by the abstraction of a to-allylic methylene hydrogen atom of PUFA followed by addition of molecular oxygen (13). This results in the formation of 9- and 13-hydroperoxyoctadecadienoic acid (HPODE) isomers from linoleic acid, the major n-6 PUFA present in plasma...

[12 Lysozyme Osmotic Shock Methods for Localization of Periplasmic Redox Proteins in Bacteria

The two procedures described below and summarized in Fig. 1 share the common features of treatment of cells with lysozyme plus EDTA in conjunction with the induction of a mild osmotic shock. The rationale for this method19 is as follows. Lysozyme will digest, at least partially, the murein layer of the cell wall. Divalent cations are believed to play a role in maintaining the integrity of the cell wall and outer membrane. EDTA will modify the outer cell membrane of the cell wall to make it more permeable to lysozyme and agents that are used to adjust the osmotic strength. In this way the combined actions of lysozyme and EDTA make the

Ocular Pharmacokinetics

Some excipients, such as the preservative benzalkonium chloride (BAC), can also act as penetration enhancers. Madhu et al. studied the influence of BAC EDTA (disodium edetate) on ocular bioavailability of ketorolac tromethamine following topical ocular instillation onto normal and de-epithelialized rabbit corneas in vitro and in vivo (28), demonstrating its ability to disrupt epithelial cell tight junctions, and so enhance penetration. As alluded to earlier in this chapter, the rate and extent of systemic absorption via the conjunctiva relative to corneal absorption is dependent on the physicochemical properties of a drug or its formulation. Ahmed and Patton showed that the conjunctival pathway is particularly important for drugs with low corneal permeability and that noncorneal permeation is limited by nonproductive loss to the systemic circulation (30). Hitoshe et al. demonstrated that drugs and prodrugs could be designed to reduce conjunctival absorption selectively and thus...

Experimental Diabetic Neuropathy Oxidative Stress and Antioxidant Therapy

Glucose, by a process of autooxidation in the presence of decompartmenta-lized trace transitional metals, can cause lipid peroxidation (6). We have evaluated the role of hyperglycemia in lipid peroxidation in vitro, using an in vitro lipid peroxidation model, with an ascorbate-iron-EDTA system. The addition of 20 raM glucose to the incubation medium increased lipid peroxidation fourfold, confirming rapid and marked glucose-mediated autooxidative lipid peroxidation (7). Glucose autooxidation results in the production of protein reactive ketoaldehydes, hydrogen peroxide highly reactive oxidants, and the fragmentation of proteins (free radical mechanisms). Glycation and oxidation are simultaneous and inextricably linked (8).

Model Of Parkinsonism

Singlet Oxygen Disease

Metallothionein, despite its high metal binding constant (Kzn 3.2 X 1013 MM at pH 7.4), can transfer zinc to the apoforms of zinc enzymes that have inherently lower stability constants. In order to further clarify this paradox, Jacob et al. (104) studied zinc transfer between zinc enzymes and MT. Zinc can be transferred in both directions, i.e., from the enzymes to thioneine (the apo form of MT) and from MT to the apoenzymes. Agents that mediate or enhance zinc transfer have been identified that provide kinetic pathways in either direction. MT does not transfer all of its seven zinc atoms to an apoenzyme, but apparently contains at least one that is more prone to transfer than the others. Modification of thiol ligands in MT zinc clusters increases the total number of zinc ions released and, hence, the extent of transfer. Aside from disulfide reagents, the authors showed that selenium compounds are potential cellular enhancers of zinc transfer from MT to apoenzymes. Zinc transfer from...

[23 Redox Control of 20S Proteasome

Proteasome can be conducted only after its isolation. Depending on the source and the amount of starting biological material, different purification protocols must be set up in order to obtain both good yields and purification factors. Accordingly, ammonium sulfate precipitation followed by conventional ion-exchange and gelfiltration chromatographic methods is recommended with such organs as human placenta or rat liver. Ultracentrifugation followed by ion-exchange or affinity chromatography is advisable when dealing with smaller organs or tissues such as rat heart, human epidermis biopsies, or cell cultures (e.g., fibroblasts or keratinocytes). Addition of EDTA to the homogenization buffer and working at a low temperature (4 ) are required to prevent inactivation and oxidative modification of the proteasome during the sample preparation procedure. Organs or tissues that have been kept frozen at 70 are thawed and homogenized with either a Potter-Elvehjem glass homogenizer with a Teflon...

Measurement of Dihydroxyphenylacetic Acid DOPAC and DA in Tissue Homogenates

Frozen tissue obtained from one hemisphere was homogenized in HPLC mobile phase (MDTM-70-1332 ESA, Chelmsford, MA) consisting of the following NaH2PO4 75 mM EDTA 25 M octanesulfonic acid sodium salt 1.7 mM triethylamine 100 l L 10 acetonitrile pH 3.0 with phosphoric acid. Homogenates were centrifuged and supernatants were analyzed for DA and DOPAC content using a DECADE Digital Electrochemical Amperometric Detector (Antec Leyden, Netherlands). Mobile phase was delivered to the detector at a rate of 0.2 ml min using an LC1150 Solvent Delivery System (GBC Scientific Equipment, Victoria, Australia). Catecholamines were separated on a ThermoHypersil-Keystone column (150 x 2.1 mm, pore size 5.0 m). WinChrom Chromatography data management software (GBC Separations, Hubbardston, MA) was used for data analysis. Tissue protein content was determined using the Bradford method (Bio-Rad, Hercules, CA). DA and DOPAC (ng mg protein) were determined based on comparison to an external standard curve....

Choice of Radionuclide

Fig. 1 Schematic presentation of the radiopharmaceutical design. The targeting biomole-cule serves as the vehicle to carry a radionuclide to the receptor site on tumor cells. The radionuclide is the radiation source. The bifunctional chelator (BFC) is used for radiometal chelation and attachment of the targeting molecule. The linker is often used for modification of pharmacokinetics. Diethylenetriaminepentaacetic acid (DTPA), acid (DOTA)

Selective Removal of Copper Bound to Metallothionein

Diagram Copper Transport Liver

The concentration of Cu accumulating in the form bound to MT can be decreased either by reducing the intake of Cu or by removing the Cu accumulating in the liver. The former approach is possible by feeding diets and water of low Cu content, by competing the uptake absorption of Cu with Zn (30,31), or by inhibiting the uptake absorption of Cu by complexing with chelating agents, trientine, and D-penicillamine (31-34). The chelating agents may remove Cu not bound to CP in the bloodstream as well. However, the affinity of Cu to MT is greater than those of these chelating agents, and the Cu bound to MT in the liver cannot be removed. The former approach with diets of low Cu content may be possible for experimental animals but not practical for humans. The use of chelating agents is effective in retarding the onset of hepatitis (35). However, chelating agents are not powerful enough to remove Cu accumulating in the form bound to MT in the liver (36).

Clinical Responses and Monitoring of Phase I and Phase II Neuromuscular Blockade by Succinylcholine Infusion

Aminoglycoside antibiotics produce neuromuscular blockade by inhibiting ACh release from the preganglionic terminal (through competition with Ca2+) and to a lesser extent by noncompetitively blocking the receptor. Tetracyclines also can produce neuromuscular blockade, possibly by chelation of Ca2+. Additional antibiotics that have neuromuscular blocking action, through both presynaptic

Dissociation Enhanced Lanthanide Fluoroimmunoassay

The luminescent lanthanide chelates designed for DELFIA are not stable in biological media. To circumvent this limitation, a heterogeneous assay based on a two-step approach was designed. In the first step, biomolecule conjugates bearing nonlumines-cent chelates (derivatives of EDTA or diethylenetriaminepentaacetate (DTPA)) were used to carry the lanthanide ions during the biological reaction. In the second step, the lanthanide ions were dissociated from the nonfluorescent chelates by adding a large excess of a different chelating agent that generates a strongly luminescent complex.36

Ocular Toxicity and Irritation

Some agents commonly used in topical ocular drugs can be used only sparingly or not at all for intraocular use, and pH and buffering capacity must be taken into account. Most preservative agents commonly used in topical ophthalmic preparations are not compatible with the tissues of the anterior segments of the eye (113). The USP recognizes this problem and specifically warns against their use in intraocular solutions (2,114). Drug stabilizers, such as antioxidants and chelating agents, must be used with care and should be used in absolutely minimal quantities only when necessary. Occasionally, it may seem desirable to solubilize an otherwise sparingly soluble ingredient. Only fairly low concentrations of typical cosolvents such as glycerin and propylene glycol can be employed because of their osmotic effect on the surrounding tissues. Hyperosmotic solutions may elicit some transient desiccation of the anterior chamber tissues whereas hypotonic solutions may cause edema that could lead...

Competition Assays for Dopamine D2 Receptors

The method was also applied to the D2 receptor. In this case however, an incubation medium with nonvolatile components frequently used in radioligand binding assays consisting of 50 mM Tris-HCl, 120 mM NaCl, 5 mM MgCl2, 5 mM KCl and 1 mM EDTA was deliberately employed to demonstrate that the incubation medium in MS binding assays quantifying the nonbound marker is not restricted to volatile buffers. As in the D1 receptor binding assay, a crude membrane fraction of pig striatum was used as the source for the D2 receptors and with spiperone (Fig. 7.4) again the native form of a well established radioligand was chosen as a marker. Preliminary experiments showed, as expected, that the signal of the marker was substantially suppressed, when the nonbound marker was analyzed by LC-ESI-MS MS directly out of the matrix of the binding sample. Therefore a solid phase extraction (SPE) method was employed to remove most of the interfering sample matrix and to enhance the spiperone signal, allowing...