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PubMed2001

The effects of physiologically important nonmetallic ligands in the reactivity of metallothionein towards 5,5'-dithiobis(2-nitrobenzoic acid). A new method for the determination of ligand interactions with metallothionein.

The reaction of Cd5Zn2-metallothionein (MT) with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) has been studied at different reagent stoichiometries, pH and temperature conditions and in the presence of several ligands. At stoichiometries of Nbs2 to MT from 0.5 to 5, the reaction followed first order kinetics. The first order rate constants obtained were independent from the concentration of Nbs2 but were linearly dependent on the concentration of MT. At higher Nbs2/MT stoichiometries, the reaction deviates from first order kinetics and the observed rate constant increases. The reactivity of MT towards Nbs2 has been probed at 4 microM concentration of both reagents where the reaction is monophasic and is characterized by a linear Arrhenius plot (Ea = 45.8 +/- 2.7 kJ.mol-1). It has been demonstrated that metal release at low pH or subtraction from MT by EDTA substantially increases the reactivity of MT towards Nbs2. At the same time, a number of nonmetallic ligands moderately accelerate the reaction of MT with Nbs2 and hyperbolic dose-response curves were obtained. The data have been interpreted with the binding of ligands to MT and following MT. Ligand binding constants were calculated as follows: ATP, K = 0.31 +/- 0.06 mM; ADP, K = 0.26 +/- 0.07 mM. Several compounds such as AMP, S-methylglutathione, and phosphate had no effect on the reaction, but Zn2+ ions showed an inhibitory effect at micromolar concentrations.

Adenosine Diphosphate↗
PubMed1996

Standardization of a flow cytometric assay for phagocyte respiratory burst activity.

The authors optimized the flow cytometric dichlorofluorescin (DCFH)-oxidation assay for buffy coat neutrophil and monocyte respiratory burst activity. Sample handlings were minimized, monocytes identified with a CD14 antibody, and viability evaluated with propidium iodide. Sodium citrate was a better anticoagulant than heparin, with a more intense Yersinia enterocolitica (YER)-induced dichlorofluorescein (DCF)-fluorescence intensity and a higher proportion of DCF-positive cells. EDTA was unsuitable as an anticoagulant with reduced cell viability and poor DCF response. Exposure of cells to YER, phorbol myristate acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanine (FMLP) elicited two neutrophil subpopulations, one with low and the other with high forward light scattering properties. FMLP induced only a marginal DCF response, but after YER or PMA, virtually all neutrophils responded with an increased DCF production. During optimal conditions, the resulting DCF- fluorescence histogram was two-peaked, and the subset of cells with increased forward light scattering properties corresponded to the cells with intense DCF-fluorescence. A similar heterogeneity was frequently but not always observed amongst monocytes. The results indicate that in the peripheral blood there are at least two neutrophil and monocyte populations. One is an effective responder to stimuli, the other exhibiting a moderate response only. Properly optimized, the DCFH-oxidation assay may be used for evaluating neutrophil and monocyte subsets in a clinical setting.

Adult↗
PubMedDate not supplied

The role of carboxylic acids in EDTA-dependent panagglutination.

The investigation of a patient blood sample showing a discrepancy between cell grouping and serum confirmation demonstrated a serum agglutinin which reacted with all red blood cells tested when exposed to EDTA. This reaction was 4+ macroscopic at room temperature, 2+ macroscopic with hemolysis at 37 degrees C in albumin, and 1+ macroscopic in the anti-human globulin phase. Agglutination was abolished following dithiothreitol treatment of the patient's serum or following saline washing of the EDTA-exposed test cells. The agglutination reaction was not limited to EDTA, but could be produced with polycarboxylic acids (citrate, L-tartrate, succinate) and monocarboxylic acids (acetate, lactate, propionate, valerate, butyrate). Non-carboxylic acids and low molecular weight ketones or alcohols failed in the agglutination reaction. This study reports an additional example of an IgM "EDTA dependent agglutinin" and demonstrates the dependence on carboxyl groups for its agglutinating activity.

ABO Blood-Group System↗
PubMedDate not supplied

Assessment of the optimal anticoagulant solution for storage of whole blood samples prior to measurement of platelet-associated IgG.

The complexity of assays used to measure platelet-associated IgG (PAIgG) often requires blood samples to be sent to a central laboratory, In order to determine the optimal storage conditions, we measured PAIgG, platelet recovery, and platelet size serially over several days in whole blood samples obtained from healthy individuals. Blood was collected into EDTA, acid-citrate-dextrose (ACD) and citrate-phosphate-dextrose-adenine (CPD-A) with and without the fixative paraformaldehyde (P). The level of PAIgG remained with normal limits in all samples stored for up to six days in the ACD, the CPD-A and the CPD-A-P samples. The least drop in platelet count and change in platelet size occurred in the ACD and CPD-A samples. Blood collected into EDTA plus paraformaldehyde was less satisfactory, with PAIgG rising by day three in some samples, and a decline in the platelet count occurring by day two. Specimens collected into EDTA alone were least satisfactory, with a rise in PAIgG by 24 hours after collection. Collection of whole blood into either ACD or CPD-A and storage at 22 degrees C allows a good recovery of platelets whose level of PAIgG is unchanged for up to six days.

Adenine↗
PubMed2001

Statistical process monitoring of WBC-reduced blood components assessed by two types of software.

BACKGROUND: Statistical process control is required for monitoring of the WBC-reduction process. This study focused on some factors that may influence the outcomes of statistical process monitoring, such as WBC-reduction technologies, the anticoagulant used, and WBC-counting technologies, by using two types of software. STUDY DESIGN AND METHODS: Data were collected from January to September 1999, before the implementation of universal WBC reduction. The effects of three major factors were investigated: methods of preparation, the addition of EDTA to the sample, and the WBC-counting technologies used (microvolume fluorimetry, flow cytometry, and Nageotte chamber). The WBC-reduction process capability was assessed by two types of software, EZQC (Gambro BCT) and NWA (Northwest Analytical). In addition, the differences between various sets of results were compared by the t test or ANOVA. RESULTS: There was no statistical difference (at the 0.05 level of significance) in WBC content when the three types of platelets in citrate samples were compared with EDTA samples. In general, the Nageotte chamber appeared to count the lowest, and microvolume fluorimetry appeared to count lower than flow cytometry. There were minor but significant methodologic differences between the software packages. However, these differences had negligible effects on the percentage of conforming components at both <1 x 10(6) and <5 x 10(6) WBCs per unit. CONCLUSION: Only the counting technologies were sufficiently different to warrant consideration. This difference may make unacceptable the interchange of results obtained from various counting methods.

Anticoagulants↗
PubMed2003

Factors that influence platelet recovery after transfusion: resolving donor quality from ABO compatibility.

BACKGROUND: A system was established to examine the extent to which the apheresis donor determines platelet recovery after transfusion, to measure the impact of ABO identity, and to predict outcome by evaluating the donor. STUDY DESIGN AND METHODS: The percentage of platelet recovery was measured after prophylactic transfusion of apheresis units divided from single donors to paired recipients with uncomplicated thrombocytopenia secondary to leukemia chemotherapy. Platelet microaggregation induced by citrate was measured at the time of apheresis. RESULTS: Platelet recoveries in paired recipients correlated strongly when both transfusions were ABO- identical. When one recipient was ABO-identical and the other was ABO-nonidentical, nonidentical transfusions yielded one-third the recovery of ABO-identical transfusions. In ABO-identical transfusions, platelet recovery in donors having microaggregates in the before-apheresis ACD sample was one-third that in donors without microaggregates. This difference was observed at 1 and 24 hours. Expression of P-selectin in the apheresis units at the time of transfusion correlated well with ACD microaggregates in the before-apheresis sample. CONCLUSION: When transfusions of platelets are ABO-identical, donor quality dominates recovery in circulation. Donor quality is predicted by a rapid and simple assay of citrate-induced microaggregation performed at the time of apheresis. When donor quality is factored out, ABO identity prevails.

ABO Blood-Group System↗
PubMed1974

Calcium-induced extrusion of secretory granules (exocytosis) in mast cells exposed to 48-80 or the ionophores A-23187 and X-537A.

Isolated peritoneal mast cells from rats were observed, by phase contrast microscopy, to extrude secretory granules when exposed to 48/80 or the ionophores A-23187 and X-537A, which are known to facilitate transmembrane fluxes of calcium. These effects were abolished when the cells were treated with EDTA and suspended in a Ca-free environment. Ca-deprived cells exposed to any one of the three drugs promptly extruded granules when calcium, but not magnesium, was added to the incubation medium. Such Ca-evoked or Ca-dependent responses persisted when Na was omitted from the incubation medium and replaced with sucrose, choline, or K. The responses thus seem independent of possible shifts in the alkali metal ions. The results are considered support for the view that Ca influx mediates stimulus-secretion coupling and does so by initiating exocytosis.

Animals↗
PubMed1987

Oxygen transfer involving nonheme iron: the influence of leaving group ability on the rate constant for oxygen transfer to (EDTA)Fe(III) from peroxycarboxylic acids and hydroperoxides.

Ethylenediaminetetraacetato iron(III) [(EDTA)Fe(III)] has been shown to react with a series of four peroxycarboxylic acids and four alkyl hydroperoxides (YOOH; dry methanol solvent, 30 degrees C) by heterolytic O-O bond scission that accompanies the transfer of an oxygen atom to the iron(III) moiety [(EDTA)Fe(III) + YOOH----[(EDTA)(FeO)]+ + YOH]. A single linear free-energy relationship exists for both peroxycarboxylic acids and alkyl hydroperoxides when the logarithm of the second-order rate constant (kYOOH) for reaction of YOOH species with (EDTA)Fe(III) is plotted vs. the pKa of the YOH leaving group.

Carboxylic Acids↗
PubMed1990

Antibodies to a histidine-rich protein (PfHRP1) disrupt spontaneously formed Plasmodium falciparum erythrocyte rosettes.

Cerebral involvement in Plasmodium falciparum malaria is associated with sequestration of infected red blood cells and occlusion of cerebral vessels. Adhesion of infected erythrocytes along the vascular endothelium as well as binding of uninfected erythrocytes to cells infected with late-stage asexual parasites (rosetting) may be important in erythrocyte sequestration. We report that the recently discovered rosetting phenomenon shares characteristics with other human cell-cell interactions (heparin sensitivity, temperature independence, Ca2+/Mg2+ and pH dependence). Mono- and polyclonal antibodies specific for PfHRP1, a histidine-rich protein present in the membrane of P. falciparum-infected erythrocytes, disrupt rosettes but do not affect attachment of infected erythrocytes to endothelial cells. The inhibitory anti-PfHRP1 antibodies reacted with rosetting parasites in indirect immunofluorescence and with P. falciparum polypeptides of Mr 28,000 and Mr 90,000 in immunoprecipitation and immunoblotting, respectively. No inhibitory effects on erythrocyte rosetting were obtained with antibodies to related histidine-rich or other antigens of P. lophurae or P. falciparum. Whether the epitope that mediates rosetting, and is recognized by the anti-PfHRP1 antibodies, is located on PfHRP1 or on a crossreactive antigen remains to be established. The results suggest that endothelial cytoadherence and erythrocyte rosetting involve different molecular mechanisms.

Animals↗
PubMed1996

Modulation of superoxide-dependent oxidation and hydroxylation reactions by nitric oxide.

The rapid and spontaneous interaction between superoxide (O2-.) and nitric oxide (NO) to yield the potent oxidants peroxynitrite (ONOO-) and peroxynitrous acid (ONOOH), has been suggested to represent an important pathway by which tissue may be injured during inflammation. Although several groups of investigators have demonstrated substantial oxidizing and cytotoxic activities of chemically synthesized ONOO-, there has been little information available quantifying the interaction between O2-. and NO in the absence or the presence of redox-active iron. Using the hypoxanthine (HX)/xanthine oxidase system to generate various fluxes of O2-. and H2O2 and the spontaneous decomposition of the spermine/NO adduct to produce various fluxes of NO, we found that in the absence of redox-active iron, the simultaneous production of equimolar fluxes of O2-. and NO increased the oxidation of dihydrorhodamine (DHR) from normally undetectable levels to approximately 15 microM, suggesting the formation of a potent oxidant. Superoxide dismutase, but not catalase, inhibited this oxidative reaction, suggesting that O2-. and not hydrogen peroxide (H2O2) interacts with NO to generate a potent oxidizing agent. Excess production of either radical virtually eliminated the oxidation of DHR. In the presence of 5 microM Fe+3-EDTA to insure optimum O2-.-driven Fenton chemistry, NO enhanced modestly HX/xanthine oxidase-induced oxidation of DHR. As expected, both superoxide dismutase and catalase inhibited this Fe-catalyzed oxidation reaction. Excess NO production with respect to O2-. flux produced only modest inhibition (33%) of DHR oxidation. In a separate series of studies, we found that equimolar fluxes of O2-. and NO in the absence of iron only modestly enhanced hydroxylation of benzoic acid from undetectable levels to 0.6 microM 2-hydroxybenzoate. In the presence of 5 microM Fe+3-EDTA, HX/xanthine oxidase-mediated hydroxylation of benzoic acid increased dramatically from undetectable levels to 4.5 microM of the hydroxylated product. Superoxide dismutase and catalase were both effective at inhibiting this classic O2-.-driven Fenton reaction. Interestingly, NO inhibited this iron-catalyzed hydroxylation reaction in a concentration-dependent manner such that fluxes of NO approximating those of O2-. and H2O2 virtually abolished the hydroxylation of benzoic acid. We conclude that in the absence of iron, equimolar fluxes of NO and O2-. interact to yield potent oxidants such as ONOO-/ONOOH, which oxidize organic compounds. Excess production of either radical remarkably inhibits these oxidative reactions. In the presence of low molecular weight redox-active iron complexes, NO may enhance or inhibit O2-.-dependent oxidation and hydroxylation reactions depending upon their relative fluxes.

Benzoates↗
PubMed2003

Iron absorption from ferrous fumarate in adult women is influenced by ascorbic acid but not by Na2EDTA.

Ascorbic acid and Na2EDTA enhance Fe absorption from the water-soluble Fe compound FeSO4 but their effect on poorly water-soluble Fe compounds such as ferrous fumarate is less well established. In the present study, the effects of ascorbic acid and Na2EDTA on Fe absorption from ferrous fumarate were evaluated in adult women (ten women/study) from the erythrocyte incorporation of Fe stable isotopes ((57)Fe or (58)Fe) 14 d after administration. Two separate studies were made with test meals of Fe-fortified infant cereal (5 mg Fe/meal). Data were evaluated by paired t tests and the results are presented as geometric means. In study 1a, the comparison between Fe absorption from ferrous fumarate- and FeSO4-fortified cereal showed that adult women absorb Fe as well from ferrous fumarate as from FeSO4 (3.0 and 3.1 % respectively, P=0.85). After addition of Na2EDTA (Na2EDTA:fortification Fe molar ratio of 1:1), Fe absorption from FeSO4 was significantly higher than from ferrous fumarate (5.3 v. 3.3 % respectively, P<0.01; study 1b). In study 2, Fe absorption was compared from ferrous fumarate-fortified meals with and without ascorbic acid added at a 4:1 molar ratio (relative to fortification Fe) and the results showed that ascorbic acid increased Fe absorption from ferrous fumarate significantly (6.3 v. 10.4 %, P=0.02). The results of the present studies show that Fe absorption from ferrous fumarate is enhanced by ascorbic acid but not by Na2EDTA, thus emphasising that not all findings from Fe absorption studies made with FeSO4 can be extrapolated to Fe compounds with different solubility properties.

Adult↗
PubMed2005

Endoscopic biopsies in Ussing chambers evaluated for studies of macromolecular permeability in the human colon.

OBJECTIVE: Studies of mucosal permeability to protein antigens in humans are limited to in vitro techniques. The use of surgical specimens for such studies has major shortcomings. Endoscopic biopsies in Ussing chambers have been introduced as a means of studying secretion and transepithelial permeability, but have not been evaluated for studies of protein antigen uptake in human intestine. MATERIAL AND METHODS: Standard forceps biopsies from the sigmoid colon of 24 healthy volunteers were mounted in Ussing chambers with an exposed tissue area of 1.76 mm2. 51Cr-EDTA (paracellular probe) and horseradish peroxidase (HRP; 45 kDa protein antigen) were used as permeability markers. Mucosal permeability, electrophysiology, histology and energy contents of the biopsies were studied over time. To evaluate the ability of the technique to detect permeability changes, the mucosa was modulated with capric acid, a medium-chain fatty acid, known to affect tight junctions. RESULTS: In the Ussing chamber the mucosal biopsies were viable for 160 min with stable levels of ATP and lactate, and only minor changes in morphology. Steady-state permeability with low variability was seen for both markers during the 30-90 min period. Exposure to capric acid induced a rapid decrease in short-circuit current (Isc) and a slower reversible decrease in transepithelial resistance (TER), as well as an increased permeability to 51Cr-EDTA and HRP. CONCLUSIONS: Endoscopic biopsies of human colon are viable in Ussing chambers and are reliable tools for studies of mucosal permeability to protein antigens. The technique offers a broad potential for studies of mucosal function in the pathophysiology of human gastrointestinal diseases.

Adenosine Triphosphate↗
PubMed1996

Comparison of 5-hydroxy-2, 3-dihydrophthalazine-1, 4-dione and luminol as co-substrates for detection of horseradish peroxidase in enhanced chemiluminescent reactions.

The utility of 5-hydroxy-2, 3-dihydrophthalazine-1, 4-dione (HDP) as a co-substrate for the chemiluminescent detection of horseradish peroxidase was assessed. Several substituted aryl boronic acid derivatives (4-phenyl, 4-iodo) acted as potent enhancers of the peroxidase catalyzed reaction. Addition of chelating agents (EDTA) and surfactants (Tween-20 and [poly (vinylbenzyl)tributylphosphonium chloride-poly (vinylbenzyl) trioctylphosphonium chloride copolymer]) modulated background light emission and the intensity and duration of the signal from both HDP and luminol. However, HDP was found to be inferior to luminol in the peroxidase assay. Comparative studies revealed that at 500 amol of peroxidase the S/B was ten-fold higher using a commercial luminol-based signal reagent as compared with an HDP-EDTA-Tween-20 reagent (S/B t = 0 min 21.8 vs 1.7, S/B t = 10 min 17.8 vs 2.0).

Boronic Acids↗
PubMed1996

Influence of anticoagulants on the level of soluble HLA class I and class II antigens measured in blood samples.

The existence of soluble forms of HLA class I and class II antigens in human serum is well established and altered concentrations of these serum proteins have been described to be associated with various diseases. Since soluble HLA antigens (sHLA) can be measured both in serum and plasma samples, we investigated whether anticoagulant treatment influences the determined levels of soluble HLA class I (sHLA-I) or soluble HLA-DR (sHLA-DR). Analyzing paired samples of serum and plasma of 40 healthy individuals we found significantly lower serum levels of sHLA-DR (0.31 +/- 0.15 ng/ml) compared to EDTA plasma levels (0.58 +/- 0.20 ng/ml). By contrast, serum levels of sHLA-I (0.89 +/- 0.74 micrograms/ml) were only slightly lower than EDTA plasma values (0.95 +/- 0.86 micrograms/ml), a situation similar to that of sIL-2R and sCD4 levels. Further experiments intended to clarify the reasons of the reduced sHLA-DR serum levels revealed that (i) the blood storage time before centrifugation did not influence the sHLA-DR level, (ii) treatment of serum with anticoagulant did not augment the measured sHLA-DR concentration, and (iii) the recovery of spiked sHLA-DR was significantly lower when added to native blood than to serum or anticoagulant-treated blood. These results suggest that sHLA-DR is partly removed by the process of blood clotting thus resulting in diminished sHLA-DR serum levels.

Anticoagulants↗
PubMed2000

Blood platelet activation evaluated by flow cytometry: optimised methods for clinical studies.

A variety of flow cytometry techniques are in use to evaluate in vivo blood platelet activation. We have in this study further developed and optimised these methods to be suitable for use in clinical studies. By preloading the Monovette EDTA vacuum blood sampling tubes with 1/8 vol 4% (w/v) paraformaldehyde (PFA), we were able to assess platelet CD62P (P-selectin) expression in whole blood with less than 0.2% activated platelets. No washing or neutralising steps were required to remove excess fixative. Both basal and agonist-stimulated CD62P expression were stable for at least 48 h after sampling. The standard curve was linear from 1.9 (basal) to 8.1 x 10(3) (TRAP-stimulated) molecules of equivalent soluble fluorochrome units (MESF) in phycoerythrein-conjugated anti-CD62P labelled whole blood samples. These assay conditions were also well suited for assessment of platelet expression of CD41, CD42a, CD61 and CD63. The preanalytic storage period was extended from 10 min to at least 2 h for platelet PAC-1 and fibrinogen binding analysis by preloading Monovette citrate tubes with 8/10 vol buffer. With PFA preloading, blood sampled into citrated tubes could be analysed for fractions of microparticles and platelet-platelet aggregates as well as for aggregate size.

Adult↗
PubMed2006

Iron humate as a low-cost sorbent for metal ions.

Iron humate produced as a waste by-product during an industrial manufacture of humic substances from low-rank brown coals was tested as a sorbent for the removal of metal cations--Cd (II), Cu (II), Co (II), Ni (II), Zn (II), TI (I), Eu (III), Cr (III)--as well as hexavalent chromium from waters. The Langmuir-type isotherms were used to describe the metal sorption; the respective equations may be derived and interpreted on the basis of a concept of surface-complexation reactions. Parameters of the sorption isotherms were estimated from experimental dependencies measured in a batch arrangement. The sorption capacities ranged from 0.024 mmol g(-1)for Co (II) to 0.324 mmol g(-1)for Tl(I). The metal uptake was affected by the presence of complexing agents (EDTA, salicylate, citrate), but this effect depends strongly on pH and the kind of complexing agent. The sorption of Cu (II) ions was suppressed in the presence of EDTA in an almost whole examined pH range (ca. 1-6.5) and in the presence of citrate at higher pH values (above pH 4). In the presence of salicylate, on the other hand, a slight sorption enhancement was observed. The metal sorption was suppressed also in the presence of anionic surfactant (sodium dodecylsulfate). Both in the presence as well as in the absence of complexing agents, the metal sorption showed a strong dependence on pH--the sorption was reduced considerably at low pH values below ca. 2. A typical working range for iron humate as a sorbent is ca. pH 3-5, where it exhibits a high buffering capacity, a sufficient stability and metal-binding capability.

Adsorption↗