EDTA Therapy for Vascular 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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Chelation therapy

Adult Chelation Therapy Encephalopathic BAL IM first, followed by EDTA IV. BPb 100 mcg dL Same. BPb 70-100 mcg dL Succimer orally only. BPb 70 mcg dL Reduce exposure, no treatment indicated chelation contraindicated secondary to increased BPb levels from mobilized bone stores. FIGURE 21.6 Metaphysical lead lines. Chelating Agents BAL (British anti-Lewisite, dimercaprol) IM only, developed in World War II as antidote for Lewisite (arsine) and mustard gases sulfur donor and nonspecific chelator of Pb, As, Cu. Side effects hemolysis in G-6-PD deficiency, nephrotoxic in acid urine, peanut allergy give prior to EDTA for lead encephalopathy. CaNa2EDTA (calcium disodium ethylenedi-amine tetraacetic acid) IV IM, water-soluble nonspecific chelator of Pb, Hg, Cu, Zn. Side effects nephrotoxic with oliguria, IM subcutaneous calcinosis, transient increased ALT AST, redistribution of Pb from stomach to brain in encephalopathy. Pediatric Chelation Therapy Encephalopathic BAL IM first, followed by...

Extraction of Brain CSPGs with PBS Solution

B. 0.2 M neutralized ethylenediaminetetraacetic acid (EDTA) as a metalloprotease inhibitor dissolve EDTA with deionized water, neutralize with NaOH, and add deionized water to make 0.2 M EDTA. Store at room temperature. 3. Mix 5 ml of 10 x PBS, 5 ml of 0.2 M neutralized EDTA, and 0.5 ml of 0.2 M PMSF-1 M NEM to make 50 ml of PBS-20 mM EDTA-10 mM NEM-2 mM PMSF (PBS-inhibitors).

Extraction of Cartilage CSPGs with 4 M GuanidineHCl

B. 4 M guanidine-HCl-50 mM Tris-HCl, pH 7.5-20 mM EDTA (Guanidine-HCl-EDTA) Dissolve solid guanidine-HCl in ca.1 2 vol. of deionized water. Add 1 20 vol. of 1 M Tris-HCl, pH 7.5, 1 10 vol. of 0.2 M neutralized EDTA (Section IV.A), and then add deionized water to a guanidine HCl-concentration of 4 M. Store at 4 C. 3. Mix 4 ml of Guanidine-HCl-EDTA and 40 ml of inhibitor mixture (4 M guanidine-HCl solution). The mixed solution should be used immediately since most of the protease inhibitors are unstable in an aqueous solution.

Polyacrylamide Gel Electrophoresis PAGE of Hyaluronan Oligosaccharides and Fragments

Early electrophoretic analyses of hyaluronan and other glycosaminoglycans were designed to separate polysaccharides primarily according to charge density (33-35). Size separation had not been established by analysis of well-characterized samples differing solely in molecular weight. In 1984, three separate groups (36-38) reported high-resolution separation of glycosamino-glycan oligosaccharides according to size. In each case, a polyacrylamide gel was used to separate oligosaccharides into discrete bands, with adjacent bands in the final pattern differing in size by one disaccharide repeat. The gel composition varied from 10 to 25 acrylamide and the continuous buffer systems used were 50-100 mM Tris-borate, pH 8.3, with 1-2.4mM EDTA or 100 mM Tris-glycine, pH 8.9, with 1.25 mM EDTA. If the glycosaminoglycan fragments were radiolabeled, the separation patterns were visualized by fluorography of the gel after incorporation of a fluor and subsequent drying of the gel. Separation patterns...

Materials 21 TRF Analysis

Proteinase K solution 20 mg mL proteinase K (Sigma-Aldrich, St. Louis, MO) in a 10 mM Tris-HCl buffer (pH 8.0) containing 1 mM EDTA, 0.3 M sodium acetate, and 0.2 (w v) sodium dodecyl sulfate (SDS). Aliquots of this stock solution are stored at -20 C. Proteinase K working solutions are prepared freshly by diluting the proteinase K stock in the same buffer 10-fold to 2 mg mL. 5. TE 1X buffer 10 mM Tris-HCl (pH 8.0) containing 1 mM EDTA. 9. TBE 1X buffer 89 mM Tris base, 89 mM boric acid, and 2 mM EDTA in H2O. 17. HIGH buffer 1 (w v) bovine serum albumin (BSA), 0.2 M sodium phosphate, 15 (w v) formamide, 1 mM EDTA, and 7 (w v) SDS in H2O. 20. Solution 1 15 mM Tris-HCl (pH 7.5) supplemented with 60 mM KCl, 15 mM NaCl, 0.5 mM spermine, 0.15 mM spermidine, 14 mM -mercaptoethanol, 2 mM EDTA, and 0.3 M sucrose (store at 4 C). 21. Solution 2 15 mM Tris-HCl (pH 7.5) supplemented with 60 mM KCl, 15 mM NaCl, 0.5 mM spermine, 0.15 mM spermidine, 14 mM -mercaptoethanol, 1 mM EDTA, and 1.37 M...

Agarose Gel Electrophoresis of High Molecular Weight Hyaluronan

Lee and Cowman (61) adapted methods used in the electrophoretic separation of high molecular weight nucleic acids for the separation of high molecular weight hyaluronan. They proposed the use of agarose gel at 0.5 in a continuous Tris-acetate-EDTA buffer for the separation of hyaluronan (Fig. 6). Sample loads of approximately 4-7 mg were required for polydisperse samples and the separated pattern was visualized by staining with the dye Stains-All For hyaluronan standards of known average molecular weight, the electrophoretic mobility was found to be approximately linearly related to the logarithm of the hyaluronan molecular weight over the range of 0.2 X 106 - 6 X 106. Larger molecules may be separable by this method, but no suitable standards have been available. The method was shown to be useable preparatively, but yields are low and some degradation occurs during extraction of the hyaluronan from the gel. Impure hyaluronan samples containing high levels of contaminating protein...

Mechanisms Of Toxic Injury

The antidotes for heavy metals are called chelating agents, a picturesque term invoking an image of lobster claws (chelae) grabbing hold of the metal. Such drugs are rich sources of the ligands to which metals readily bind, and these drugs are able to compete effectively for the metal against the endogenous tissue ligands.

Analysis of Core Proteins

0.2 M NEM in ethanol) 0.2 M neutralized EDTA (Section IV.A) 0.5 ml Stock solutions (the 10 x buffer and the 100 x inhibitor mixture) are stored at -20 C. The final reaction mixture contains 100 mM Tris-HCl, pH 7.5, 30 mM NaOAc, 0.2 mM PMSF, 0.072 mM pepstatin, 2 mM NEM, and 2 mM EDTA in addition to CSPGs and chondroitinase ABC. After the reaction mixture is incubated for 1 h at 37 C, the core protein is precipitated by adding 3 vol. (150 ml) of 95 ethanol-1.3 KOAc.

Detection Of Dna Fragmentation

Are extracted from nuclei in buffers containing magnesium chelators.3233 To extract these fragments, cells undergoing apoptosis are lysed in buffer consisting of 20 mM Tris (pH 7 to 8), ethylenedi-aminetetraacetic acid (EDTA), and a neutral detergent. After a 10- to 30-minute incubation at 4 C, the intact chromatin is pelleted, which leaves the nucleosomal fragments in the supernatant. DNA in the two fractions can then be assayed using colorimetric or fluorimetric techniques. Alternatively, if DNA in the cells is uniformly radiolabeled (i.e., greater than one cell cycle) before application of the apoptotic stimulus, DNA in the various fractions can be quantified by scintillation counting. Solubilization of oligonucleosomal fragments in buffers lacking divalent cations also plays a role in the detection of apoptotic cells by flow cytometry after staining with propidium iodide.34 When this approach is applied, cells treated with an apoptotic stimulus are fixed with ethanol, washed with...

Uncorrectedsusceptibility corrected T2imsi

Figure 7 (A) Example of a Gadolinium complex demonstrating the chelation of the otherwise toxic Gd3+ metal. The free water interacts with the surrounding protons thereby locally changing the T1 and thus increasing the MR signal strength on T1 weighted images. (B) Example of a dextran coated iron oxide particle. Source From Ref. 4. Figure 7 (A) Example of a Gadolinium complex demonstrating the chelation of the otherwise toxic Gd3+ metal. The free water interacts with the surrounding protons thereby locally changing the T1 and thus increasing the MR signal strength on T1 weighted images. (B) Example of a dextran coated iron oxide particle. Source From Ref. 4.

Biochemical Characterization of Human Collagenase3

The inclusion of human collagenase-3 in the collagenase subclass of MMPs was initially based on structural comparisons, since its deduced amino acid sequence contained a number of features characteristic of these enzymes. Subsequent studies performed with recombinant protein produced in different eukaryotic expression systems provided definitive support for the proposal that this novel human enzyme was a bona fide collagenase. In this regard, the first studies were performed with a recombinant protein produced in a vaccinia virus expression system.1 This protein was active against type I collagen as well as against synthetic peptides used for assaying vertebrate collagenases. In addition, its pro-teolytic activity was fully abolished by EDTA, a typical inhibitor of metalloproteinases.

Linking the Radionuclide to Protein

Research has sought to balance the conditions required to achieve a radiolabeled product with adequate stability of the resulting complex. However, such scales must be balanced within the constraints imposed by isotope chemistry and half-life. As such, the options of realistic isotopes and chelating agents have become narrowed and focused as this field has matured. However, refinement of bifunctional chelating agents (BCAs) remains an active area of endeavor (Packard et al. 1999). All metallic radionuclides require chelation chemistry for attachment to a mAb (Fig. 6.4). BCAs are chelates possessing specific functional groups that permit the conjugation of proteins to stable metallic radionu-clide complexes. Although 67Cu copper isotopes have been investigated, the choice of BCA remains an open and unresolved topic (Novak-Hofer and Schubiger 2002). Several different macrocyclic chelating agents have been reported as stable and inert with 67Cu, despite reports of its transchelation to...

Impact Of Serum Vs Plasma Specimen On cTn ASSAYS

The mechanism by which plasma gives falsely lower results is not fully understood. Katrukha et al. (43) have suggested that troponin immunoreactivity may be modified either via protein conformational changes or steric hindrance of epitopes upon hepa-rin-cTnl complex formation. In addition, the various types of troponin complexes may create or conceal various epitopes (42,44). These complexes are influenced by the availability of calcium (45) and, thus, anticoagulants that bind calcium (e.g., ethylene diamine-tetraacetic acid EDTA ) may have a marked impact on the assay. Whereas heparin does not appear to have a complex-dissociating effect, addition of EDTA can dissociate the troponin complexes into individual subunits (41).

Effect Of Calcium On Calpain3 Autolysis

The presence of a domain IV containing EF-hand motifs, of a C2-like domain and of the two non EF-hand calcium binding sites in domain IIa and IIb (Moldoveanu et al., 2002), suggests that calpain-3 does bind calcium. Nonetheless, early reports had indicated that it was rapidly degraded in the presence of EDTA (Sorimachi et al., 1993). The paradox was resolved by another study, that directly demonstrated calcium binding to calpain-3 using a gel overlay method, and showed that calpain-3 was stable in the presence of EGTA, autolytic cleavage already occurring at 500nM Ca2+(Branca et al., 1999). Therefore, calpain-3 has a Ca2+-affinity which is far higher than that of conventional calpains. Very recently, the calcium requirement for calpain-3 activity has been determined for two truncated isoforms p94 I-II, comprising domain I and II and the two inserts NS and IS1, and p94I-IIANS IS1 including domain I and II but lacking NS and IS1. The data have suggested cooperative binding of calcium at...

Rapid Whole Blood Analytical System Suitable for POCT or the Emergency Laboratory

This is the most innovative system as it is a robust true POCT device. It uses K-EDTA anticoagulated whole blood as the sample matrix for measurement of a cardiac panel of CK-MM, CK-MB, myoglobin, and cTnl, and incorporates QC into the analytical process. The instrument is a desktop, portable, integrated immunoassay analyzer. The consumable components are a fluid cassette, containing a stabilized, buffered detergent solution used for all reaction and wash steps and reagents, supplied in the form of test-disks. Each test disk is a complete immunoassay system that contains all the required

Applications For The Clinical Laboratory

The LOD of 0.2 mol L using indirect detection and phthalic acid is close to the reference limit of 0.4 mol L, the upper limit seen in normal individuals. Because methylmalonic acid rises dramatically (10-100 times normal levels) in vitamin B12 deficiency, the assay is adequate for clinical purposes. Indirect detection, however, depends entirely on migration time for identification. There is always the possibility that a coeluting compound could be hidden in the methylmalonic acid peak, falsely elevating the value. When methylmalonic acid is elevated, dilution should be used to reduce the possibility of interference by coeluting compounds. Also plasma specimens collected with citric acid as the anticoagulant cannot be analyzed with this assay. The massive peak due to citric acid will overwhelm all other peaks in the vicinity, including the one for methylmalonic acid (see Fig. 1). EDTA plasma does not cause a similar problem. Specimen preparation is based on an organic phase extraction,...

Activation of Procollagenase by Trypsin

Trypsin-induced activation of procollagenase involves an initial cleavage between Arg36 and Asn,37 immediately after the triplet of basic amino acids, K34RR (Fig. 6.1), generating a major intermediate form of 46 kDa.28 This intermediate form is then converted to a stable 42 kDa active enzyme by cleavage between Phe81 and Val.82 While formation of the 46 kDa intermediate form is not affected by metalloproteinase inhibitors, EDTA or o-phenanthroline, subsequent conversion to the 42 kDa active form is completely inhibited by these reagents.28 Moreover, the conversion of the intermediate form to the 42 kDa species does not require trypsin activity, cannot be blocked by serine protease inhibitors, and is independent of the initial procollagenase concentration.14,28 These lines of evidence suggest that the proteolytic cleavage responsible for production of the 42 kDa stable active enzyme species is an auto-catalytic reaction requiring initial activation by trypsin.

Detection Methods For Viralload Assessment

Including tumor biopsies and whole blood can be obtained by standard procedures. Alternatively, commercially available system such as Ultraspec II RNA isolation system (Biotecx, Houston, TX) may be used. This system isolates total RNA by disruption and homogenization of samples with 14 M guanidine salts and urea followed by chloroform extraction. The sample is centrifuged and the upper aqueous phase containing the RNA is isolated followed by isopropanol precipitation. A proprietary RNATack resin that specifically binds RNA and then eluted with TE (Tris-EDTA, pH 7.4) buffer purifies the RNA. The RNA concentration is quantitated spectrophotometri-cally. The entire isolation can be completed in approx 1 h, which is a significant advantage over standard methods. RNA can also be extracted directly from lymphocytes obtained from whole blood. Concentrations of 10-15 ng of RNA are routinely obtained from 10 mL of whole blood comparing favorably to standard methods (55).

Exploiting Inherent Material Properties

5.4.2.6 Polymeric Micelles as Nanoreactors Polymeric micelles have been investigated for the entrapment of metallic nanoparticles for use as contrast agents in MRI and other imaging techniques. In some instances, the polymers may entrap a metal salt and interact with it in such a way as to cause metal chelation to occur. The micelle thus provides a microenvironment that serves as a reactor for the production of metallic particles 74 . The microenvironment provided by the core of polymeric micelles may in the future be modified in such a way that it is capable of acting as a microreactor for the synthesis or activation of a biologically active compound like a drug or growth factor.

Enrichment and Isolation Procedures

The culture medium and incubation conditions are similar to those described for Chromatium species. Not more than 1.5 mM sulfide and 3 mM acetate should be used for growth of Thiopedia, and the trace element solution must not contain a chelating agent (e.g., EDTA). Growth is enhanced by the addition of 100 M dithionite to the medium. Incubation is carried out at a temperature of 20-25 C and a light intensity of 100-200 lux. Thiopedia species grow in the form of individual cells, or platelets with 4-128 cells. Colonies and cell suspensions of Thi-opedia rosea exhibit a unique bright purple-red color and can, therefore, be differentiated readily from other purple sulfur bacteria.

High Throughput Screening

Grow HeLa cells in 150-mm tissue-culture dishes so that they are 70-80 confluent the day before screening. Approximately 2 o 109 cells (10-12 150-mm plates) are needed for a 50-plate assay using 384-well plates. Aspirate media and wash cells once with 15 mL PBS. Add 2 mL trypsin-EDTA per plate and incubate plates at 37 C for 2-5 min. Resuspend cells in DMEM to a final concentration of 2 o 105 cells per mL. Add appropriate volume of adenovirus to cell suspension. 1. Grow B-SC-1 cells in 150-mm plates to 70-80 confluence the day before screening. Aspirate media and wash cells once with 15 mL PBS. Add 2 mL trypsin-EDTA per plate and incubate plates at 37 C for 2-5 min. Resuspend cells in DMEM to a final concentration of 2.6 o 105 cells per mL.

Construction of Hammerhead Ribozyme Expression Plasmids

TE buffer 10 mM Tris-HCl, pH 8.0 1 mM ethylenediaminetetraacetic acid (EDTA). 3. Solution of trypsin (0.25 ) and ethylenediamine tetraacetic acid (EDTA) (1 mM) from Life Technologies. 3. 10X RNA loading buffer 50 glycerol, 10 mM EDTA, 0.25 (w v) bromo-phenol blue, 0.25 (w v) xylene cyanol. Use DEPC water and keep at 4 C. 7. 20X MOPS buffer 0.4 M MOPS, pH 7.0, 100 mM sodium acetate, 20 mM EDTA. 8. 2X SSPE buffer with 0.1 sodium dodecyl sulfate (SDS) 1.5 MNaCl, 17.3 mM NaH2PO4, 2.5 mM EDTA, 0.1 SDS.

Cytotoxic Chemotherapy

Change, but the peak concentration of these agents is increased and the risk of toxicity enhanced as a results of volume of distribution (Vd) alterations. The effects of Vd changes may be ameliorated by correction of anaemia, when this is present. Several studies have shown that anaemia is an independent risk factor for chemotherapy-induced myelodepression 28 , as many agents bind to red blood cells consequently, anaemia is associated with increased concentration of circulating free drug. A decline in the glomerular filtration rate (GFR) is almost universal with aging, and may lead to an increased half-life of cytotoxic compounds, such as carboplatin, methotrexate, and bleomycin, whose parent compounds are excreted through the kidneys, and drugs that give origin to active or toxic metabolites excreted from the kidneys. For example, 80 of the activities of idarubicin and daunorubicin are metabolised in the liver and excreted from the biliary tract, but is due to renally excreted...

Nonmechanical Disruption Techniques

An enzyme is used to lyse bacterial cell walls and recover the desired product. Although this technique is expensive, it consumes less energy, damages the product less, and is very specific. Lysozyme is an enzyme that hydrolyzes the murein present in bacterial cell walls, and cellulase can lyse plant cells. Temperature, pH, and cofactor can influence enzymatic lysis. Additives such as ethylene diamine tetra-acetic acid (EDTA), a chelator of divalent ions (divalent ions are common in cell walls) can also disrupt cell walls.

Establishing Stable Transfected Cell Line

Prepare 24-well dishes with 1 mL of selective medium. Rinse the 100-mm dish with PBS and then add warm PBS containing 5 trypsin (1 mL standard trypsin-EDTA plus 19 mL PBS). 8. In a day or two, when the cells are confluent, rinse in PBS and trypsinize with 100 L of trypsin EDTA. Split into a 6-well plate for passaging.

Homologous Recombination of a Mutant Transgene

Time course of cleavage at the helical and aminotelopeptide sites in mouse type I collagen by purified rat interstitial collagenase and human fibroblast (MMP-1) collagenase. (A) SDS-PAGE (5 acrylamide) showing reaction products after incubation with + + (Lanes 1-5 and 11-15) or r r collagens (Lanes 6-10 and 16-20) and sufficient purified human fibroblast (MMP-1) collagenase (Lanes 1-10) or rat (lanes 11-20) interstitial collagenase to digest 50 of the pepsinized + + collagen after 24 hr at 20oC. The substrates were collagen extracted from skin of + + mice with pepsin and from skin of r r (Col1a1tm1Jae) mice in 0.5M acetic acid without pepsin as described. 6 Parent solutions containing enzyme and substrate were prepared at 0oC and incubated at 20oC to start the reaction. Aliquots calculated to contain 25 ig of collagen were withdrawn and the reaction stopped by the addition of 1 10 vol of 500mM EDTA at the indicated intervals. After the reactions were stopped by the addition...

Characterization of Collagenase Cleavage

We have expressed in E. coli human MMP-1 collagenase, (HCL1 called CLH in ref. 56 ) and mouse interstitial collagenase (MMP-13 MCL3 or CLM ) cDNAs and chimeric constructs in pET-3d, juxtaposing mouse collagenase (MMP-13) sequences N-termi-nal to the Zn-binding site in the catalytic domain and MMP-1 sequences C-terminal (MCL3 HCL1 or CLMH) and vice versa (HCL1 MCL3 or CLHM).56 MMP-13 (MCL3 or CLM) and chimeric molecules that contained the MMP-13 sequences N-terminal to the Zn-binding site (MCL3 HCL1 or CLMH) cleaved (+ +) collagen at the helical locus and cleaved crosslinked (r r) collagen in the N-telopeptide (p components converted to a chains). Human MMP-1 (HCL1 or CLH) and chimeric MMP-1 MMP-13 with MMP-1 sequences N-terminal to the active site (HCL1 MCL3 or CLHM) cleaved collagen at the helical locus but not in the N-telopeptide. All activities were inhibited by TIMP-1, 1,10-phenanthroline and EDTA. Thus, sequences in the distal 2 3 of the catalytic domain appear to determine the...

The Biosynthesis and Catabolism of Galactosaminoglycans

Rotary shadowing electron microscopy studies have revealed that C6S is capable of forming aggregates of two or more chains (Scott et al., 1992). The charge distribution of the twofold helix of C6S is near the periphery, creating regions of hydrophobicity. Such hydrophobic zones from separate chains can interact with each other. Chondroitin-4-sulfate is incapable of such network formation, as sulfate groups are clustered close to the helical axis, minimizing aggregation behavior. Although C4S cannot self-aggregate, it is capable of association with DS. Alternating IdoA and GlcA in DS can self-associate in the absence of divalent cations under physiological conditions (Fransson and Coster, 1979). Chemical modification of the sugar backbone, hydrogen bonding, and cation chelation are critical determinants of self-aggregation potential and solid-state structural conformation, necessary for conferring the biological information inherent in GAG biomolecules.

CAAs Types Design Strategies Structureactivity Relationships And Isozyme Specificity

Silverman's laboratory reported hCA II activation by histidine (Silverman et al. 1978), but subsequently considered their discovery an artefact owing to the ethyl-enediaminotetraacetic acid (EDTA) added in the buffer, which might form complexes with the adventitious Cu2+ possibly present in the protein preparation, thus restoring the activity of the enzyme (heavy metal ions act as micromolar inhibitors of most CAs Tu et al. 1981). In the light of this study (Supuran et al. 1991), the initial report by Silverman's group regarding hCA II activation with histidine (Silverman et al. 1978) was proposed to be authentic. It was explained that the controversial results of Tu et al. (1981) were because of the experimental protocols used, EDTA probably possessing a competitive suppressing effect for the histidine activating effect (Supuran and Puscas 1994).

Detachment of Bound Particles by Elastic Deformation

No bound microgel particles were eluted with running buffer or with specific eluents, imidazole (up to 0.3 M) or EDTA (up to 50 mM). However, when cryogel with bound microgel particles was compressed in the presence of either eluent, up to 60 of microgel particles was released from the cryogel. As the pulse of the eluent at 20-fold higher flow rate did not succeed in re

Preparation of Yeast for Replicative Life Span Analysis

1X TE buffer 10 mM Tris HCl, pH 8.0 and 1 mM EDTA, pH 8.0. 2. Sorbitol solution 0.9 M sorbitol (Fisher), 0.1 M Tris HCl, pH 8.0, 0.1 M EDTA are prepared with water and filter sterilized using a 0.22-pm filter. 5. 10X TBE electrophoresis stock buffer 0.89 M Tris base 0.89 M boric acid 20 mM EDTA, pH 8.0. A 1X working solution is made by diluting 1 10 with water.

Purification and preparation of spin labelled vimentin

Dissolve IBs (from 500 ml of bacterial culture) in 8 ml of 8 M urea (20 mM Tris pH 8, 1 mM EDTA, 8 M urea) and filter through a 0.2 micron filter (Pall Serum Acrodisc, Fisher Scientific). 2. Chromatograph 4 ml of inclusion body solution on a Hi Load 16 60 Superdex 200 column. The column is run with 20 buffer B, giving conditions of 20 mM Tris, pH 8, 1 mM EDTA, 0.2 M NaCl. Electrophorese column fractions on an SDS-PAG and visualize the proteins by Coomassie blue staining. Pool peak fractions. 7. After spin labelling, add 8 ml of buffer A (8 M urea, 20 mM tris, 1 mM EDTA, pH 8.0) and chromatograph the spin labelled protein over the Source 15S as before. Collect the purified peak and store at -800C. 2. If EPR spectra are to be recorded at multiple steps during vimentin assembly, dialysis can be performed in a stepwise fashion 8 . Starting conditions are 20 mM Tris pH 8.0, 1 mM EDTA, 8 M urea (buffer A + 8 M urea). Vimentin dimers can be produced by dialysis against buffer A + 4 M urea....

Characterization of PEG Reagents

Dissolve PEG-SH (or any SH-carrying compound) in oxygen-free phosphate-EDTA, pH 7.0, at a final concentration of 2 mM (if PEG-SH has a MW of 5000, then dissolve it at 10 mg mL). This solution should be used immediately after preparation. 2. In separate test tubes, mix 30 L of PEG-SH solution (or the solution containing SH) or 30 L of phosphate EDTA, pH 7.0 (as blank), and 970 L of phosphate-EDTA, pH 7.0. 1. Dissolve the reactive PEG in phosphate-EDTA, pH 6.0, at a final concentration of 2 mM (e.g., if the PEG molecular weight is 5000 Daltons, then dissolve it at 10 mg mL). 2. In separate test tubes, mix 500 L of reactive PEG solution or phosphate-EDTA, pH 6.0 (as a blank), and 500 L of cysteine solution.

Light Scattering and Size Exclusion Chromatography

Figure 7 Agarose gel electrophoresis of nearly monodisperse hyaluronan standards and commercial hyaluronan. Gel was 0.7 agarose in Tris-acetate-EDTA (minigel format), stained with Stains-All by the method of Lee and Cowman (61). S a mixture of 5 different monodisperse SelectHA preparations with indicated Mw determined by SEC-MALS C and C0 commercial hyaluronan samples D DNA standards, Bioline Hyperladder 1, containing DNA of 10, 8, 6, 5, 4, 3, 2.5, 2, 1.5 kb D0 DNA standards, BioRad 1 Kilobase Ruler, containing DNA of 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 kb. Figure kindly provided by P DeAngelis and W Jing. Figure 7 Agarose gel electrophoresis of nearly monodisperse hyaluronan standards and commercial hyaluronan. Gel was 0.7 agarose in Tris-acetate-EDTA (minigel format), stained with Stains-All by the method of Lee and Cowman (61). S a mixture of 5 different monodisperse SelectHA preparations with indicated Mw determined by SEC-MALS C and C0 commercial hyaluronan samples D DNA...

Conjugation of radioisotopes to antibodies

The identification of suitable chelating agents for radiometals has been challenging. Studies have shown that the use of the macrocyclic ligand 1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA) can result in stable 90Y immunoconjugates and significantly reduce bone uptake of radioyttrium (11). Because DOTA has been shown to be immunogenic (12), several diethylenetri-amine pentaacetic acid (DTPA)-derived chelates have been evaluated. Although none have held yttrium as well as DOTA, cyclohexyl-A (CHX-A)-DTPA was found to be a suitable chelate for 90Y (13). This chelate has also been used to generate bismuth-containing radioimmunoconjugates for clinical use (14). Another radiometal, 188Re, has been directly labeled to the anti-CD66c antibody BW250 183 using tris-(2-carboxyethyl) phosphine as a reducing agent (15). Radiolabeling of MAbs can cause loss of biologic function, especially when they are labeled with 131I at high specific activities (16). This decrease in immunoreactivity...

Rapid HIV diagnostic tests

The Determine assay (Arai et al., 1999) is based on the sandwich immunoassay technique with HIV-1 and HIV-2 antigens conjugated to selenium colloid and a capture site containing HIV-1 and HIV-2 antigens. If a sample contains anti-HIV-1 or anti-HIV-2 antibodies, the antibodies first react with the antigen-selenium colloid conjugates. As the antibody-antigen selenium colloid complex flow past the capture site, the antibodies react with the antigens at the site with the formation of a visible red line within 15 min. For serum or plasma, 50 ml is placed on the sample application pad. For an EDTA coagulated whole blood sample, 50 ml is placed on the pad, followed by the addition of one drop of buffer. The test also contains a procedural control site that confirms the validity of the assay by the formation of a visible red line. Test devices stored at room temperature (30 C) for 12 months had sensitivity equivalent to that of test devices stored at 2-8 C. No difference in sensitivity was...

Routine Passaging of Zebrafish Cell Cultures

Detach the cells from the culture flask substrate by a 5-min treatment with 1X trypsin-EDTA at room temperature. Use a volume of trypsin just sufficient to cover the cell layer. Dilute the suspension of detached cells with L15 complete culture medium (see Note 6) and pipet the cell suspension vigorously up and down through a 5-mL or 10-mL pipet to break up cell clumps.

Interfering Particles

The presence of fibrin strands in serum that is processed for analysis prior to complete clot formation is known to cause errors in many immunoassay systems. The susceptibility of the different automated immunoassay analyzers to such interference varies. Fibrin strands are only of concern for NT-proBNP measurement when conducted using serum samples. Blood for BNP determination must be collected in EDTA-coated plastic tubes. EDTA is important for in vitro stabilization of BNP because it inhibits degradation by metalloproteases (36). NT-proBNP may also be measured using fresh plasma samples. If serum is used, it is important to ensure that the clotting process is complete prior to cen-trifuging the sample in order to eliminate the presence of microfibrin strands. Icteric and hemolyzed samples may also cause inaccuracy in immunoassays based on fluorimetric detection of the signal.

Other CAM Therapies

Multiple CAM therapies are unstudied or unlikely to be beneficial at the same time, they are possibly unsafe, expensive, or labor-intensive. Because of these concerns, these therapies should be fully investigated and well understood before use calcium EAP, Candida (yeast) therapy, chelation therapy, dental amalgam removal, DHEA, hyperbaric oxygen, Prokarin, and toxin avoidance.

Optimization of Expression Level for Assays

Grow cells in a T-75 flask until they are 70-80 confluent. Aspirate media and wash cells once with 15 mL PBS. Add 2 mL trypsin-EDTA and incubate plates at 37 C for 2-5 min. Resuspend cells in DMEM to a final concentration of 3.3 o 105 cells per mL (HeLa cells) or 2.64 o 105 per mL (BS-C-1 cells). Dispense 30 L of cell suspension into each well (providing 10,000 cells per well for HeLa cells, or 8000 cells per well for BS-C-1). For luciferase assays, dispense cells into 384-well white plates for imaging assays, use 384-well black plates with clear bottoms.

Glomerular function Glomerular filtration

Filtration of radioactively labelled compounds, such as 51Cr-EDTA, 131I-iothalamate and 99Tc-DPTA, gives a GFR value generally comparable to that of inulin clearance. The complex safety procedures and legislation governing the handling of such compounds has led to a decline in their use. The contrast agents iohexol (Krutzen et al. 1984) and isothalamate (Gaspari et al. 1992 Isaka et al. 1992) are eliminated from plasma mainly by glomerular filtration and have an excellent correlation with the plasma clearance of 51Cr-EDTA and inulin (Gaspari et al. 1995). Iohexol is more commonly used as it reportedly has lower allergenic potential. The most accurate estimate of GFR is obtained by sampling up to 600 min after injection, particularly in individuals with GFR 40 ml min 1.73 m2, sampling may be limited to only at 120, 150, 180, 210 and 240 min after injection.

Decreasing Catheter Risk

Mix adjustment, 0.48 baseline covariates adjustment, RR 0.41 baseline covariates and follow-up adjustment) 19 . One recent report demonstrated that a systematic program of education and mapping followed by AV fistula creation successfully converted 57.9 (70 121, intention to treat) of all catheter patients and 81.4 (70 81) of patients who agreed to venous mapping to a functioning alternative access 18 . An alternative approach is to make catheters safer by decreasing the bacteremia risk. In one report, mortality decreased by 76 and hospitalization for catheter-related bacteremia decreased by 63 with the routine application of an antiseptic antimicrobial solution (polysporin) at the catheter exit site 20 . Similar reductions in bacteremia have been reported with the use of muperacin ointment. Antimicrobial catheter lock solutions which decrease or prevent the formation of biofilm are also effective. Studies using citrate, taurolidine, gentamycin heparin, gentamycin EDTA have reported...

Mass Spectrometric Analysis of Heparin and Heparan Sulfate

(MALDI) and electrospray ionization (ESI). Despite its current importance in HP and HS structural analysis, only recently have there been significant advances in the development of MS as an analytical tool for structural characterization of these complex polysaccharides. Whereas historically, MS procedures for biological samples have largely been investigated and optimized using peptides and proteins, recent efforts have been aimed at identifying experimental conditions amenable to the analysis of acidic GAGs. Such an endeavor has proven critical since early on it was discovered that application of traditional mass spectrometric approaches to the analysis of GAG structure were generally ineffective. GAGs are difficult to detect as molecular ions due to differential chelation of positively charged metal ions (such as sodium) and because they possess the tendency to fragment extensively due to the loss of sulfate prior to detection. A number of techniques have been developed to analyze...

Isolation and purification of inclusion bodies IBs [35

Thaw bacterial pellets in centrifuge bottles by the addition of a GET buffer (50 mM glucose, 25 mM Tris pH 8, 10 mM EDTA) containing 10 mg ml egg white lysozyme (Sigma L-6876), 10 ml per centrifuge bottle (20 ml per 500 ml culture). The bottle is vigorously vortexed to resuspend the pellet and create a homogeneous suspension. The cell suspension is transferred to a disposable 50 ml screw-cap tube using a Pasteur pipette. Any remaining clumps are broken up by rapid pipetting. 3. Produce complete lysis by the addition of an equal volume of 20 mM Tris pH 7.5, 0.2 M NaCl, 1 mM EDTA 1 deoxycholic acid, 1 NP-40. The bacterial solution and the lysis buffer should be gently rocked back and forth several times over several minutes to mix the solutions the solution should rapidly turn viscous (more of a gelatinous blob than a suspension). This indicates lysis and release of bacterial genomic DNA. 6. Add 10 ml of 0.5 TritonX-100 (TX-100), 1 mM EDTA to the tube and resuspend the IBs by pipetting....

Cis Trans Isomerism of Aryl Compounds

Cis-trans isomerism around single bonds has been widely used in supramolecular chemistry, in particular with bulky biaryl compounds such as the metal-chelating agents bipyridines and sterically hindered binaphthyl motifs. In these compounds, a moderate barrier to rotation exists due to the resonance of the two con

Measurement Of Bnp In Clinical Specimens

A wealth of scientific literature exists that establishes the relevance of BNP measurements in assisting the diagnosis and management of patients with congestive heart failure (CHF) (for reviews see 28-31). Furthermore, there is evidence that BNP measurements in patients with acute coronary syndromes (ACS) within the first few days after onset are of prognostic significance (32). Early analysis of BNP in clinical specimens was performed using an immunoradiometric assay (33). Shionogi & Co., Ltd. (Osaka, Japan) produces the Shionoria BNP test, an immunoradiometric assay that quantifies the amount of BNP in ethylenediaminetetraacetic acid (EDTA)-anticoagulated plasma, that is available in the United States not for diagnostic purposes, but for research use only (34). The Shionoria BNP test procedure is completed in approx 24 h. The test measures BNP molecules sandwiched between a bead-coated mouse monoclonal anti-BNP antibody specific The Triage BNP Test is a fluorescence immunoassay...

Freezing

Dissociate monolayers with trypsin EDTA (see Note 1) and resuspend cells in complete medium. 1. Most cell types require a 0.25 (w v) trypsin 0.2 (w v) EDTA solution prepared in sterile Hank's Balanced Salt Solution (HBSS) or 0.9 (w v) NaCl to detach cells and chelate Ca2+ and Mg2+ ions that could hinder the action of trypsin.

Gelatinase B

Collier et al59 expressed gelatinase B residues 93-708. IBs were solubilized in 8 M urea 25 mM Tris-HCl (pH 7.5) 5 mM CaCl2 1 mM PMSF 0.2 M NaCl 10 mM EDTA. Soluble protein was obtained after dialysis against buffers containing successively, 6, 4, 2 and 0 M urea. The active recombinant protein was purified by gelatin-agarose chromatography.

Homogenization

For most purposes, an isoosmotic medium containing 0.25 M sucrose, 1 mM EDTA, 10 mM Tris-HCl, pH 7.4 may be used for any soft tissue or cultured animal cell. There are a number of minor variations such as adding EGTA instead of, or in addition to, EDTA and substituting Tris by an alternative organic buffer (Hepes or Tricine). Such modifications do not materially affect the result of the fractionation procedure they are normally introduced to be more compatible with some subsequent add-on procedure or analysis. Others, summarized in Table 4.1, are more critical to the isolation of a particular organelle or use of a particular tissue or cell type. The presence of divalent cations is detrimental to the functioning of mitochondria hence EDTA is regularly included in media for the isolation of these organelles (and also lysosomes and peroxisomes). Mannitol-containing media (sometimes also supplemented with K+) are also suited to the preservation of oxidative phosphorylation in...

Sample

DNA is usually obtained from EDTA or ACD anticoagulated venous blood samples. Heparin interferes with Taq polymerase activity, and should not be used as an anticoagulant. By using the GenoPrep B350 method, which is described in this chapter, DNA extraction can also be performed with heparin-ized samples. If the white blood cell (WBC) counts are very low, the use of buffy coat is suggested. DNA can also be extracted from fresh or frozen blood samples. Storage of samples for less than 5 d at +4 C is recommended. Bone marrow aspiration material is also acceptable

First catch your DNA

Ethylenediamine tetra-acetate (EDTA) sodium dodecyl sulfate (SDS) phenol (carbolic acid) Method 4. Add a drop or two of EDTA and SDS. Between them, these two chemicals break open the bacterial cell walls and release all the cell contents. (EDTA weakens the cell walls by removing their magnesium ions while SDS is a detergent-like chemical that dissolves the fat molecules in the cell walls.) After about 30 minutes you should have a clear, viscous quid the consistency of egg white.

Transfusions

Direct replacement of red blood cells through transfusion is the cornerstone of palliating anemia-related symptoms in MMM. Currently, there are no data to support the routine use of iron chelation therapy for prevention of secondary iron overload in these transfusion-dependent individuals. Indeed, there are no data demonstrating definitive end-organ toxicity from iron overload in MMM patients. Unfortunately, the

Protein Purification

Thaw bacterial pellets in 24-well blocks at room temperature. While waiting, add protease inhibitors, lysozyme, and Benzonase to the chilled lysis buffer 1 mg mL lysozyme, 10 units Benzonase mL, 2 pL mL protease inhibitor cocktail (no ethyl-enediamene tetraacetic acid EDTA ), and 1 mM PMSF. Keep on ice.

Complexing Agents

Inulin can be modified to compounds that display good heavy metal complexing properties similar to ethylene diamine tetra-acetic acid (EDTA) but with better biodegradation properties (Bogaert et al., 1998). Inulin is first oxidized using sodium periodate to the dialdehyde, and then reduced to a polyol using Pt C and hydrogen. The polyol can then be modified with carbon disulfide to form xanthate or with SO3-pyridine to obtain an inulin sulfate. Alternatively, the dialdehyde can be aminated with diaminoethane and sodium cyanoborohydride and the product reacted with monochloroacetic acid sodium salt to form carboxymethylamino inulin. Each of these compounds can be used to precipitate heavy metals.

Chromium

In this method separation of Cr (IV) or Cr (III) requires complexation with either cyclohexanediamine-tetraacetic acid (CDTA) or ethyldiaminetetraacetic acid (EDTA) to to cause the chromium species to migrate towards the cathode. Using CDTA and direct detection at 240 nm gave 19 and 59 ppb (ng mL) detection levels for Cr (VI) and Cr (III), respectively. About 10-fold lower detection limits noted when EDTA used (23). Although the limit of sensitivity for the method using EDTA is good, another one to two orders of magnitude is still required for CE to be clinical useful.

Methods

Modification of the checkerboard DNA-DNA hybridization method 38 . In brief, subgingival plaque samples are obtained with the help of periodontal curettes and placed in individual pre-labeled bar-coded tubes containing 0.1 ml TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) and 0.1 ml of 1 M NaOH. Samples are stored at -80 C until assay. The sample DNA was denatured through boiling for 5 min and neutralized using 0.8 ml 5 M ammonium acetate. The DNA was placed into the slots of a Minislot device and thereby deposited as 'lanes' onto a Roche Diagnostics nylon membrane. The DNA was then fixed to the membrane by exposure to ultraviolet light. Pooled DNA of known bacterial numbers (105 and 106 CFU ml) for each one of the microorganisms included in the analysis were deposited with the patient's samples. Digoxigenin-labeled whole genomic DNA probes were prepared for each of the reference strains using a random primer technique. For these analyses the level of all 8 organisms was summed to...

Abbreviations

The abbreviations used are AAGE6, 250 mM ammonium acetate, 6 M guanidine hydrochloride, 10 mM EDTA (pH 6.0) AAGE9, 250 mM ammonium acetate, 6 M guanidine hydrochloride, 10 mM EDTA (pH 9.0) ABTS, acid) Ado, adenosine BPB, bromophenol blue BSA, bovine serum albumin DBD, DNA binding domain DHBB, dihydroxyboronyl Bio-Rex DTT, dithiothreitol ELISA, enzyme-linked immunosorbent assay HEPES, acid HPLC, high-performance liquid chromatography PES, phenazine ethosulfate PMSF, phenylmethylsulfonyl fluoride MNNG, 1-methyl-3-nitro-1-nitrosoguanidine MTT, phenylmethylsulfo-nylfluoride, bromide NAD,

Transgenic Animals

1.5 X binding buffer 50 mM Tris-HC1 (made from a 1 M stock solution, pH 7.5), 5 mM EDTA, 50 mM MgCl2, 25 glycerol (v v 31.5 g 100 mL). Adjust pH to 6.8 with HCl and filter-sterilize the solution. 4. IPTG-elution buffer 10 mM Tris-HCl (made from a 1 M stock solution, pH 7.5), 1 mM EDTA, 125 mM NaC1. Filter-sterilize and store at room temperature. 23. Lysis buffer 10 mM Tris-HCl, pH 8.0 10 mM EDTA 150 mM NaCl (autoclaved). 26. TE buffer 10 mM Tris, pH 8.0, 1 mM EDTA, pH 8.0 (autoclaved). 33. 10X DIANA buffer (100 mL) 25 mL 1 M Tris-HCl, pH 8.0, 30 mL 5 M NaCl, 2 mL 0.5 M EDTA, 5 mL 1 M MgCl2. Autoclave, allow to cool to room temperature. Add 0.5 mL Tween20 and store at 4 C.

In Vitro Stability

In general terms, the apparent in vitro stability of a given peptide will depend on the type of assay and the characteristics of the antibodies used. Although the plasma halflife of NT-proBNP remains to be determined in humans, the in vitro stability of this pep-tide in plasma and full blood samples stored at room temperature has been investigated (23,55). In one study, using an immunoluminometric assay with antibodies directed at the middle and C-terminal portions of NT-proBNP, EDTA and aprotinin plasma samples showed no significant difference in immunoreactive peptide levels between samples centrifuged immediately and stored at -70 C or kept at room temperature or on ice for 24 or 48 h (55). In contrast, using a competitive radioimmunoassay with a polyclonal antibody directed at the N-terminal fragment (amino acid sequence 1-21) of NT-proBNP, plasma concentrations were stable after storage at room temperature for 3 and 6, but not for 24 or 72 h (23). Two freeze-thaw cycles did not...

Calcium

Prepare 100 l mixture of myosin (10 g with final concentration 0.1 mg ml) and S100A4 (50 g) in a buffer (0.6 M NaCl, 10 mM MES pH 6.2, 1 mM DTT) containing 1 mM CaCl2 or 5 mM EDTA. 2. Dialyse proteins for 5 h at RT against 200 ml of buffer, 10 mM MES pH 6.2, 1 mM DTT, 1 mM CaCl2 or 5 mM EDTA and different NaCl concentrations, 50, 100, 200, 300 and 400 mM.

Inulin Amino Acids

Covalent bonding of an amino acid to inulin (Figure 5.6) allows further chemical modification of the polymers for possible medical use, peptide synthesis, or producing chelating agents for metal ions. The terminal primary amino acid could be more reactive toward acylating agents and potentially allow the attaching of a cross section of molecules of interest. Such compounds are attractive since both amino acids and inulin are expected to be nontoxic, biocompatible, and biodegradable

Treatment Method

In chelation therapy, ethylenediaminetetraacetic acid (EDTA) is given through an intravenous infusion. EDTA binds strongly to (chelates) harmful metals, and the metal-EDTA complexes then are excreted in the urine. Vitamin and mineral supplements are also frequently given. A course of treatment may involve 20 to 30 infusions that are given over the course of a few months. This type of therapy is effective for known situations of heavy-metal toxicity, such as lead poisoning. Some chelation products can be taken orally. These are of no proven value, and the U.S. Food and Drug Administration (FDA) has determined that they should not be sold.

Transferrin

Essentially all circulating plasma iron normally is bound to transferrin. In addition, two-thirds of serum transferrin exists as apotransferrin and will quickly capture the free iron which is released from the cell 42 . This chelation serves three purposes it renders iron soluble under physiologic conditions, it prevents iron-mediated free radical toxicity, and it facilitates transport into cells.

Safety

Although GdCl3 is very toxic, chelating Gd(III) (as with DTPA) largely reduces its toxic profile (32-34). With a high thermodynamic stability constant (KGd-L 1026 for Gd-DTPA), the chelated metal complex is essentially inert and is cleared at a significantly faster rate than the rate of dissociation of the metal ion from the complex. However, the plasma clearance rate of chelated Gd is still sufficiently slow to provide ample contrast for MRI applications. Thus, the chelation process transforms an otherwise highly toxic salt into a useful diagnostic reagent (35).

Absorption

A cornerstone of the treatment of CKD patients is the use of phosphate-binding agents. Aluminum is rarely used any more, but calcium, magnesium, and lanthanum-based agents all pose the possibility of chelation of drugs as well as the intended gastrointestinal phosphorus. Tetracycline chelation to antacids is well known to clinicians, but tetracycline is not used often in the CKD patient population. Of more importance to caregivers of CKD patients is the well-described drug interaction

Johannes Mair MD

B-type natriuretic peptide (BNP) and the N-terminal fragment of its prohormone (NT-proBNP) have emerged as the preferred candidates for diagnosis of heart failure, as well as other clinical applications. Several commercially available assays have been developed for point-of-care determination as well as for high-throughput automated laboratory platforms. Knowledge of preanalytic and analytic issues as well as sources of inter- and intraindividual variability in BNP and NT-proBNP is crucial for clinicians to allow correct interpretation of test results in routine practice. A few practical points for the clinical use of BNP and NT-proBNP include the following Blood sampling may be performed without a standardized period of rest or posture. However, heavy physical exercise should be avoided before blood sampling. Blood for BNP determination must be collected in EDTA-coated plastic tubes, whereas for NT-proBNP serum or plasma collected in glass or plastic tubes is acceptable. Although the...

Supportive Care

Chronic PRBC transfusion therapy can result in iron overload, and prevention of hemochromatosis should be considered in patients with stable disease. Liver biopsy is the gold standard for assessing iron overload, but it may not be feasible in some patients because of thrombocytopenia or other medical concerns. Superconducting quantum interference device imaging can quantitate hepatic iron deposition, but this technology is available at few sites throughout the world. MRI of the liver or heart can be used to document iron overload,6 and despite its limitations, serial serum ferritin levels can also be used to estimate systemic iron burden. Patients with chronically elevated ferritin levels (1500-2500 ng mL or greater), and those who have received 25-50 units of PRBCs, should be considered for iron chelation therapy with subcutaneous desferoxamine.7'8 Unfortunately, because of the cost, discomfort, and time involved in its administration, desferrioxamine therapy is frequently refused or...

Sample Stability

The in vitro stability of both BNP and NT-proBNP is sufficient for routine clinical use. The in vitro stability of BNP is dependent on the assay used for determination (see Issues for Assay Development). BNP is stable in EDTA whole blood at room temperature for 4 h (Biosite and Abbott assays), and at 4 C for 24 h (Bayer assay). EDTA plasma is stable at 4 C for 24 (Abbott assay) and 48 h (Bayer assay). A significant decrease in plasma BNP concentrations was noted when BNP was measured after 24 h of storage at 4 C with the Biosite assay (12). If samples are not to be analyzed within several hours, it is recommended that the plasma be frozen at or below -20 C. NT-proBNP is stable in whole blood or in EDTA or heparinized plasma samples for 2 to 3 d at room temperature (18,19). NT-proBNP is stable in serum samples if serum is separated from cells within 24 h. NT-proBNP is stable in serum or plasma samples for several days at 4oC and for 12 mo at or below -20 C (12,18,19).

BNP Stability

There have been various reports on the stability of BNP in whole blood and plasma. Various studies indicate that BNP is stable in EDTA-anticoagulated whole blood or plasma specimens at room temperature for at least 24 h, and the stability is prolonged through refrigerated storage (45-49). However, it is recommended that BNP measurements using the Triage BNP Test be performed within 4 h of specimen collection (36). The presence of the proteinase inhibitor aprotinin may be useful in prolonging the stability of BNP in specimens frozen at -20 C (49). It has been reported that the stability of BNP is enhanced when the blood specimen is collected in plastic polyethylene ter-ephthalate collection tubes (34,50). Although the selection of blood collection tube type does not significantly affect BNP measurements within the first 4 h after blood sampling, it appears that the stability of BNP in whole blood specimens may be enhanced by collecting the blood specimen in plastic tubes (50).