Detection of specific forms of plasminogen activator inhibitor type 1-by monoclonal antibodies

Monoclonal antibodies to plasminogen activator inhibitor type-1 (PAI-1) were generated and characterised for their ability to inhibit PAI-1 interaction with tissue plasminogen activator (t-PA) and urokinase (u-PA) and detection of the various forms of PAI-1 (native, complexed, and degraded) by immunoblotting. Mab17 inhibited both complex formation between PAI-1 and t-PA/u-PA and PAI-1 activity in a dose dependent manner by 90%. Mab 25 was much less effective, blocking complex formation less than 30% and PAI-1 activity less than 20%. The Kds of Mab 17 and Mab25 were 2.8×10⁻¹¹ M and 2.6×10⁻¹⁰ M, respectively. Following SDS-PAGE and immunoblotting, Mab 17 detected native PAI-1 only; PAI-1 in complex and the t-PA/u-PA degraded form of PAI-1(M_r=42000) did not react with this antibody. In contrast, Mab25 detected all three forms of PAI-I although the affinity for the native form appeared to be greater than Mab 17 or the PAI-1 polyclonal employed. Despite these differences, both monoclonal antibodies immunoprecipitated native and degraded PAI-1 equally as well. These results suggest that the epitope of Mab 17 is associated with the reactive site of PAI-1 and that this region is either missing or not accessible in the cleaved form or in complex.     

The muscarinic agonist pilocarpine inhibits DNA and interferon-γ synthesis in peripheral blood mononuclear cells

Lymphocyte basal DNA synthesis and proliferative responses to phytohemagglutinin (PHA) showed a dose-dependent (5 × 10⁻⁵ − 5 × 10⁻³ M) inhibition by the muscarinic agonist pilocarpine, in contrast to the basal enhancing effect produced by the M₂ muscarinic-nicotinic agonist carbacol. The effect of pilocarpine was reversed by both atropine (1 × 10⁻⁶ M) and pirenzepine (1 × 10⁻⁷ − 1 × 10⁻⁸ M), M₁ − M₂ and M₁ muscarinic antagonists, respectively. The effect of pilocarpine may thus be specific for the M₁ muscarinic receptor. Pilocarpine also inhibited interferon-gg (IFN-γ)-PHA induced production, but was unable to reverse the pokeweed mitogen (PWM)-induced DNA synthesis. Distinct immunoregulatory activities are suggested for cholinergic muscarinic receptors M₁ and M₂.     

Synthesis and ring-opening polymerization of 2-acetoxymethyl-2-alkyltrimethylene carbonates and of 2-methoxycarbonyl-2-methyltrimethylene carbonate; a comparison with the polymerization of 2,2-dimethyltrimethylene carbonate

The polymerization of 2-acetoxymethyl-2-alkyltrimethylene carbonates (alkyl = methyl: AMTC, alkyl = ethyl: AETC) and of 2-methoxycarbonyl-2-methyltrimethylene carbonate (MMTC) in toluene with sec-butyllithium as initiator results in the respective polymer with yields between 78% and 88%. The analysis of the polymer microstructure by means of NMR spectroscopy reveals linear chains without branching due to an attack of the active chain end at the ester moiety of the repeating units. Poly(MMTC) and poly(-AETC) afford crystalline materials upon precipitation from solution while poly-(AMTC) is amorphous; after quenching from the melt all materials are amorphous. Copolymerization of AMTC, AETC and MMTC with 2,2-dimethyltrimethylene carbonate (DTC) results in random copolymers. The kinetics of the polymerization of AETC and MMTC revealed that the ester side chain enhances the rate of propagation compared to the polymerization of DTC. The apparent rate constants of propagation in toluene with lithium alcoholate as active sites at 23°C were determined to be k_app^DTC = 4 ċ 10⁻³ s⁻¹, k_app^AETC = 4,28 ċ 10⁻² s⁻¹, and k_app^MMTC = 2,57 ċ 10⁻² s⁻¹. Studies of ring-chain equilibria in solution of tetrahydrofuran revealed that on the basis of the theory of Jacobson-Stockmayer the characteristic ratios of the polymers are C_∞^PDTC = 7,1, C_∞^PMMTC = 8,6, C_∞^PAETC = 9,9, and C_∞^PAMTC = 13,4.     

Antispasmogenic and spasmolytic effects of verapamil and salbutamol,alone and in combination,on histamine-induced guinea pig tracheal contractionin vitro

The antispasmogenic and spasmolytic effects of the calcium entry blocker verapamil were examined on histamine-induced guinea pig tracheal contractionin vitro in comparison with the beta₂-adrenergic agonist salbutamol. The effects of verapamil were found to be about 1000 times weaker than those of salbutamol. The antispasmogenic IC₅₀ for verapamil was 1.99×10⁻⁴ M compared to 7.94×10⁻⁸ M for salbutamol. The spasmolytic EC₅₀ for verapamil was 3.37×10⁻⁵ M and for salbutamol was 1.95×10⁻⁸ M. The combined application of verapamil and salbutamol resulted in additive antispasmogenic and spasmolytic effects.

     

Interaction of IgE with rat basophilic leukemia cells—v. Binding properties of cell free particles

Particulate material from sonicated rat basophilic leukemia cells was isolated and examined for its ¹²⁵I-IgE binding properties by a rapid filter assay. The binding was saturable and was stoichiometrically inhibited with unlabeled IgE. Binding parameters (at 37°C) were similar to those observed with intact cells though the forward rate constant was up to 2 × higher (2 × 10⁵ M⁻¹sec⁻¹ while the backward rate constant was 2–3 × smaller (∼5 × 10⁻⁶ sec⁻¹) yielding an estimated K_a of ∼4 × 10¹⁰M⁻¹. The binding activity was irreversibly lost after brief exposure to temperatures above 50° and at pH's above 9 or below 5. The activity was largely resistant to proteolytic enzymes and neuraminidase, but not to phospholipase C. Non-ionic detergents led to apparent disruption of the particles.     

Bacterial expression and characterization of an anti-desipramine single-chain antibody fragment

Tricyclic antidepressant toxicity is a leading cause of death from intentional drug overdose. Monoclonal antibody Fab' fragments specific for the tricyclic antidepressant, desipramine, reverse acute drug toxicity but may themselves have adverse effects at therapeutic doses. To evaluate the characteristics of smaller antibody fragments, we cloned, expressed and characterized a 26 kD single chain Fv fragment (G5-sFv). A DNA sequence encoding V_H-linker-V_L was constructed from hybridoma mRNA encoding a high affinity monoclonal desipramine-specific IgG₁ and expressed in E. coli. G5-sFv was produced at high levels as insoluble inclusion bodies. Single chain Fv was solubilized, folded in a redox buffer and affinity purified on desipramine-Sepharose. The affinity of G5-sFv for desipramine was similar to that of the corresponding monoclonal Fab' as measured by surface plasmon resonance (Fab′ 5.5±0.5 × 10⁸M⁻¹, sFv 2.3±0.5 × 10⁸ M⁻¹). G5-sFv administered to rats after a tracer dose of ³H-desipramine produced rapid and marked redistribution of drug from tissues into serum. G5-sFv was stable at 4°C for greater than 6 months but lost activity at higher temperatures.We conclude that desipramine-specific-single chain Fv expressed in E. coli retains the affinity of the parent antibody for desipramine. The pharmacokinetic effect of G5-sFv on desipramine distribution suggests that it may be useful as an antidote for desipramine overdose.     

Efficient Synthesis of Macromolecular DO3A@Gn Derivatives for Potential Application in MRI Diagnostics: From Polymer Conjugates to Polymer Nanoparticles

Herein, the synthesis of three different macromolecular DO3A@Gn conjugates based on poly(2-oxazoline)s is presented. Therefore, poly(2-methyl-2-oxazoline) is synthesized by a ring-opening, cationic polymerization and the polymerization is terminated with DO3A(tBu)₃ . The best results are obtained after 48 h at 120 °C with degree of termination of 86%. After deprotection of the DO3A ligand and complexation with Gn³⁺, relaxivity as measured with a magnetic field strength of 9.4 T (400 MHz) reveals values for r₁ of up to 2.32 mm ⁻¹ s⁻¹. The concept is extended to a block copolymer based on 2-heptyl-2-oxazoline and 2-methyl-2-oxazoline that is again terminated with DO3A(tBu)₃ to form micelles with a size of 12.6 ± 0.7 nm after DO3A(tBu)₃ termination and deprotection of the 1,4,7,10-tetraazacyclododecane-N,N,N,N-tetraacetic acid ligand. After complexation with Gn³⁺, relaxivity r₁ is 10.1 mm ⁻¹ s⁻¹ as determined from the slope of the plot of 1/T₁ against the gadolinium(III) concentration at 9.4 T. Finally, crosslinked nanoparticles are prepared from amphiphilic macro-monomers that form micelles in water and are crosslinked throughout the core in the presence of azoisobutyronitrile (AIBN). The nanoparticle is 32.9 ± 7.8 nm in size after Gn³⁺ complexation and reveals a relaxivity r₁ of 6.77 mm ⁻¹ s⁻¹.     

Monoclonal Antibodies Against Human Chorionic Gonadotropin (hCG): II. Affinity and Ability to Neutralize the Biological Activity of hCG

ABSTRACT: Monoclonal antibodies (MCA) against hCG have been characterized with regard to their affinity and their ability to neutralize the biological activity of hCG in vivo. The production and specificities of these reagents were described in the preceeding paper of this series. Equilibrium association constants (K_a) of the MCA, determined by radioimmunological saturation assays, ranged from less than 1 × 10⁸M⁻¹ up to 3.7 × 10⁹M⁻¹ whereas values for conventional polyclonal antisera against hCG ranged from 8.9 × 10⁹M to 1.8 × 10¹⁰M⁻¹. The ability of MCA to neutralize the biological activity of hCG was tested in a rat bioassay in vivo; 9 of 13 different MCA preparations tested could neutralize hCG. Surprisingly, this property did not correlate with affinity or specificity, and was not restricted to those MCA recognizing the hormone specific β-subunit. It could be demonstrated that determinants on each individual subunit as well as epitopes formed by both subunits are involved in the expression of the biological activity of hCG.     

Dye Diffusion in Polymer Films Subjected to Supercritical CO2: Confocal Raman Microscopy and Modelling

Summary: Poly(ethylene terephthalate) films impregnated with Disperse Red 1 from a supercritical CO₂ solution were studied using confocal Raman microscopy. Depth profiles of films dyed for different periods of time were measured using the oil immersion approach and concentration‐depth plots of the relative dye concentration were generated. A one‐dimensional Fickian diffusion model was used to evaluate the diffusion coefficient of the dye from the Raman microscopy data using a non‐linear least squares regression. The calculated diffusion coefficient at 200 bar and 80 °C was 6.75 ± 1.01 × 10⁻¹⁴ m² · s⁻¹.

Typical example of Raman spectra acquired during depth profiling.     

The genome of equine arteritis virus.

Equine arteritis virus (EAV) contains an infectious RNA. [³H]uridine-labeled RNA was released from purified virus (density, 1.155 g/ml in sucrose; s_20,w, 224 ± 8 S) with sodium dodecyl sulfate and 2-mercaptoethanol. An s_20,w value of 48 S was found in isokinetic sucrose gradients in 0.1 M saline. In 1 mM saline, sedimentation was slower (33 S), in 0.1 M saline plus 1 mM MgCl₂, a value of 56 S was measured. A molecular weight of 4.0 × 10⁶ was determined by polyacrylamide-agarose-gel electrophoresis. Heating of purified RNA in the presence of 1.1 M formaldehyde and subsequent centrifugation in gradients containing formaldehyde did not result in degradation to smaller RNA's. From this procedure a molecular weight of 4.1 × 10⁶ was calculated. Buoyant density in Cs₂SO₄ of the RNA was 1.65 g/ml. In most experiments Semliki forest virus RNA was taken as a reference. It behaved almost indistinguishably from the RNA of EAV. In conclusion EAV contains an infectious, colinear molecule of single-stranded RNA with a molecular weight of about 4 million. These data justify a definite inclusion of this virus in the family Togaviridae.     

Plasminogen activator inhibitor-1 aids nerve growth factor-induced differentiation and survival of pheochromocytoma cells by activating both the extracellular signal-regulated kinase and c-Jun pathways

Astrocytes are thought to be critical to neurons' surviving damage caused by ischemic stroke or other injury. Plasminogen activator inhibitor-1 is one of the active soluble factors released by astrocytes and regulates plasminogen activator-plasmin proteolytic sequence in the CNS as a serpin. In this study, we show that plasminogen activator inhibitor-1 can promote neurite outgrowth and survival of rat pheochromocytoma cells in serum-deprived conditions, and that this neuroprotective activity is correlated with enhanced activation of both extracellular signal-regulated kinases following a direct phosphorylation of nerve growth factor receptor, Trk A, and of c-Jun. Our results suggest that plasminogen activator inhibitor-1 can act as a neurotrophic factor, protecting neurons from serum deprivation-induced neuron death not only by compensating for nerve growth factor functions, but also by activating the c-Jun/activating protein-1 pathway.     

Specificity of training velocity and training load on gains in isokinetic knee joint strength

The present study investigated the effects of three different strength training regimes on the isokinetic strength profile of the knee extensors (quadriceps, Q) and flexors (hamstrings, H) and if increases in isokinetic strength were accompanied by an enhanced performance during a more complex leg movement, the soccer kick. Twenty-two elite soccer players performed 12 weeks of strength training (three times per week) at either high resistance (HR group: 4 sets, 8 reps, 8RM loading), low resistance (LR group: 4 sets, 24 reps, 24RM loading), loaded kicking movements (LK group: 4 sets, 16 reps, 16RM loading) while one group served as controls (CO group). Isokinetic concentric and eccentric moment of force was obtained (KinCom) as peak moment (M_peak) and moment at 50° knee flexion (M₅₀) at angular velocities of 30, 120, 240° s^-1. Isokinetic knee joint strength was unchanged in groups LR, LK, CO. However, after the HR strength training, concentric M_peak (±SD) increased (P<0.01) at 30° s^-1 (Q, 258±37 to 297±57 Nm; H, 122±22 to 140±21 Nm). Furthermore, eccentric M_peak increased at 30, 120 and 240° s^-1 (Q, 274±60 to 345±57 Nm (P<0.01), 291±56 to 309±49 Nm and 275±43 to 293±36 Nm (P<0.05), respectively; H, 143±32 to 158±25 Nm, 152±39 to 169±31 Nm and 148±27 to 163±19 Nm (P<0.05)). Corresponding increases (P<0.05) were observed for M₅₀. The H/Q ratio calculated as eccentric hamstring strength divided by concentric quadriceps strength (H_ecc/Q_con, representative for knee extension) at 240° s^-1 increased (P<0.05) from 107 to 118% (based on M_peak) and from 90 to 105% (M₅₀). Kicking performance estimated by maximal ball flight velocity was unaffected by any of the strength training regimes investigated. In conclusion, only heavy-resistance strength training induced increases in isokinetic muscle strength in the absence of learning effects. Concentric strength gains were observed at the actual velocity of training, while eccentric strength gains were found over the entire range of velocities examined. The capacity of the hamstring muscles for providing stability to the knee joint during fast extension was augmented as a result of the heavy-resistance strength training. Strength training should be integrated with other types of training involving the actual movement pattern in order to increase the performance within more complex movement patterns.     

Kinetics and molecular weight evolution during controlled radical polymerization

Automatic, continuous online monitoring of polymerization reactions (ACOMP) was applied to the controlled radical polymerization (CRP) of butyl acrylate (BA) using N‐tert‐butyl‐1‐diethylphosphono‐2,2‐dimethylpropyl nitroxide (SG1), to determine monomer conversion, evolution of molecular weight, reduced viscosity, and rate constants. The conversion is roughly first order, but depends only on the initial ratio of free SG1 to initiator; i.e., it is zeroeth order in initiator concentration. While it was found that the weight‐average molecular weight M _w, and viscosity‐average mass increase in approximately linear fashion with conversion, their values are finite at zero conversion. Although ACOMP involves no chromatographic separation columns, a useful measure of polydispersity evolution was found from combining M _w and viscosity‐based masses. CRP is contrasted with monitoring results for conventional free‐radical polymerization. Distinct light‐scattering signatures are expected, and found experimentally, for the two cases. The CRP kinetic findings allowed the determination of the equilibrium constant between active and dormant species at 120 °C (K_eq = 1.53 × 10⁻¹⁰ M ), as well as the corresponding kinetic constant of deactivation (k_deact = 2.8 × 10⁺⁷ L · mol⁻¹ · s⁻¹) and activation (k_act = 4.2 × 10⁻³ s⁻¹). Cross‐checks on the monitoring results were made with conventional Gel Permeation Chromatography (GPC), and kinetic behavior was also analyzed in the light of numerical integration software.

Raw data for PBA CRP reaction #6 in Table 1 vs. detector time, which lags the reaction time by 800 s. Shown are viscosity, RI, UV and 90 degree light‐scattering signals, together with the time at which stabilization, monomer flow, and polymerization began. The temperature curve is offset to the left 800 s with respect to the detector signals.     

Radiation-induced radical formation and crosslinking in aqueous solutions of N-isopropylacrylamide

To investigate chain-initiating and crosslinking mechanisms, radical formation in dilute aqueous solutions of N-isopropylacrylamide (NIPAAm) and poly-NIPAAM was studied using electron pulse radiolysis with optical detection at room temperature. Several transients of NIPAAm generated by reactions with electrons, hydroxyl radicals and hydrogen atoms were observed. Electron attachment to the carboxyl group (k_e = 9.0 x 10⁹ dm³ · mol⁻¹ · s⁻¹) forms the radical anion, which undergoes fast and reversible protonation (pK_a = 7.8) at the carboxyl oxygen. At pH > pK_a, slow and irreversible protonation of the electron adduct at the vinyl group leads to the α-carboxyalkyl radical CH₃(^.CH)CONHCH(CH₃)₂, which is also formed by addition of H atoms to NIPAAm (k_H = 7.3 × 10⁹ dm³ · mol⁻¹ · s⁻¹). Addition of OH radicals (k_OH = 5.4 × 10⁹ dm³ · mol⁻¹ · s⁻¹) forms CH₂(OH)(^.CH)CONHCH(CH₃)₂. Hydrogen abstraction was not observed in the case of NIPAAm monomer, but it was found for the reaction of OH radicals with thermally polymerized NIPAAm. Semi-empirical quantum chemical calculations support the assignment of the observed spectra to the radicals. A reaction mechanism for the formation of crosslinks is discussed.     

Age and aerobic training status effects on plasma and skeletal muscle tPA and PAI-1

Introduction

Reductions in fibrinolytic potential occur with both aging and physical inactivity and are associated with an increased cardiovascular disease risk. Plasmin, the enzyme responsible for the enzymatic degradation of fibrin clots, is activated by tissue plasminogen activator (tPA), while plasminogen activator inhibitor-1 (PAI-1) inhibits its activation. Currently, fibrinolysis research focuses almost exclusively on changes within the plasma. However, tPA and PAI-1 are expressed by human skeletal muscle (SM). Currently, no studies have focused on changes in SM fibrinolytic activity with regard to aging and aerobic fitness.

Purpose

The purpose of this study was to cross-sectionally evaluate effects of age and aerobic fitness on tPA and PAI-1 expressions and activity in SM.

Methods

Twenty-six male subjects were categorized into the following groups: (1) young aerobically trained (n = 8); (2) older aerobically trained (n = 6); (3) young aerobically untrained (n = 7); and (4) older aerobically untrained (n = 5). Muscle biopsies were obtained from each subject. SM tPA activity was assessed using gel zymography and SM tPA and PAI-1 expressions were assessed using RT-PCR.

Results

Trained subjects had higher SM tPA activity compared to untrained (25.3 ± 2.4 × 10³ vs. 21.5 ± 5.6 × 10³ pixels, respectively; p = 0.03) with no effect observed for age. VO_{2 max} and SM tPA activity were also significantly correlated (r = 0.42; p < 0.04). SM tPA expression was higher in older participants, but no effect of fitness level was observed. No differences were observed for PAI-1 expression in SM.

Conclusions

Higher levels of aerobic fitness are associated with increased fibrinolytic activity in SM.     

Inhibition of histamine release from human FcεRI+ cells by nimesulide

We have previously demonstrated that pharmacological concentrations of non-steroidal anti-inflammatory drugs (NSAID) such as indomethacin, acetylsalicylic acid, and meclofenamic acid enhance IgE-mediated histamine release (HR) from human basophils. We have now examined the effects of nimesulide (NIM), a new NSAID, on mediator release from human basophils. Preincubation (10 min at 37°C) of basophils with pharmacological concentrations (10⁻⁶−10⁻³ M) of NIM resulted in a concentration-dependent decrease of HR induced by anti-IgE, the Ca²⁺-ionophore A23187 and the formylated tripeptide f-Met-Leu-Phe (FMLP). Maximal inhibition of anti-IgE-induced HR was achieved after preincubation with 10⁻⁴ M NIM and ranged between 14.5% and 44.5% (mean±SEM, 29.7±4.5%). The drug had a marked inhibitory effect on HR from basophils induced by A23187 (80.6±5.5%), FMLP (63.5±10.3%), 12-O-tetradecanoyl-phorbol-13-acetate (57.0±8.7%) and bryostatin 1 (65.7±7.6%). NIM also inhibited the IgE-mediated synthesis of peptide leukotriene C₄ from basophils.

     

EPR Investigations into the Kinetics of Trithiocarbonate‐Mediated RAFT‐Polymerization of Butyl Acrylate

Electron paramagnetic resonance (EPR) spectroscopy is used for measuring rate coefficients of addition, k_ad, and fragmentation, k_β, together with the associated equilibrium constants, K_eq, for butyl acrylate polymerizations mediated by S‐ethyl propan‐2‐ylonate‐S’‐propyl trithiocarbonate (EPPT) and by S‐S’‐bis(methyl‐2‐propionate) trithiocarbonate (BMPT). Experiments at ?40 °C yield k_ad = (3.4 ± 0.3) × 10⁶ L mol^?1 s^?1, k_β = (1.4 ± 0.4) × 10² s^?1, and K_eq = (2.6 ± 0.8) × 10⁴ L mol^?1 for EPPT and k_ad = (4.1 ± 0.9) × 10⁶ L mol^?1 s^?1, k_β = (4.5 ± 0.5) × 10¹ s^?1, and K_eq = (8 ± 4) × 10⁴ L mol^?1 for BMPT. The K_eq values are in satisfactory agreement with data from ab initio calculations.

     

Contributions of the Side Groups to the Characteristics of Water Absorption in Cured Epoxy Resins

Novolac resin and the esterified novolac resins with CH₃COO—, CH₃CH₂CH₂COO—, and C₆H₅CH₂COO— side groups are used as curing agents for epoxy resin. The cured resins are referred to as EP, EPA, EPB and EPP for the respective agents. The influence of the side groups on the water absorption behavior of the cured epoxy resins was investigated using a gravimetric method, differential scanning calorimetry (DSC), positron annihilation (PA) and Brunauer–Emmett–Teller (BET) adsorption. The experimental results show that at 20°C the equilibrium water content (M_∞) of the cured resins is in the following order: EP (2.13%) > EPA (0.77%) > EPP (0.65%) > EPB (0.39%); however, the order of the diffusion coefficients of water in the resins (D) is: EPB (1.81×10^–8 cm²·s^–1) > EPA (9.5×10^–9 cm²·s^–1) > EPP (8.4×10^–9 cm²·s^–1) > EP (2.4×10^–9 cm²·s^–1). But at 60 and 80°C, the sequence of D changes into, EPB > EPP > EPA > EP. Furthermore, the M_∞ of EP varies slightly with the temperature, ranging from 20 to 80°C. However, for the modified resins, M_∞ increases significantly with temperature. The results suggest that the higher the polarity, the higher the M_∞ and the lower the D; and the better the flexibility, the lower the M_∞ and the higher the D. There is no evidence for a dependence of M_∞ on the volume of the nanopores, as measured by the BET surface area.     

Electron spin resonance (ESR) estimation of propagation rate constants of radical polymerization of butyl methacrylate

Propagation rate constants (k_p) for the free radical polymerization of butyl methacrylate (BMA) in toluene solution were measured by ESR spectroscopy in the temperature range from –30 to 90°C. The polymerization was photochemically induced at a constant initiator concentration of di‐tert‐butyl peroxide (tBPO). The Arrhenius parameters A and E_a were estimated to be (3.40 ± 0.4)>×10⁶ M^–1·s^–1 and (22.0 ± 1.0) kJ/mol, respectively. The k_p values estimated from ESR analysis were compared with the corresponding data obtained from the pulsed laser polymerization (PLP).     

Analysis of Kinetics of Dihydroethidium Fluorescence with Superoxide Using Xanthine Oxidase and Hypoxanthine Assay

Superoxide (O₂ ^?) is an important reactive oxygen species (ROS), and has an essential role in physiology and pathophysiology. An accurate detection of O₂ ^? is needed to better understand numerous vascular pathologies. In this study, we performed a mechanistic study by using the xanthine oxidase (XOD)/hypoxanthine (HX) assay for O₂ ^? generation and a O₂ ^? sensitive fluorescent dye dihydroethidium (DHE) for O₂ ^? measurement. To quantify O₂ ^? and DHE interactions, we measured fluorescence using a microplate reader. We conducted a detailed reaction kinetic analysis for DHE–O₂ ^? interaction to understand the effect of O₂ ^? self-dismutation and to quantify DHE–O₂ ^? reaction rate. Fluorescence of DHE and 2-hydroethidium (EOH), a product of DHE and O₂ ^? interaction, were dependent on reaction conditions. Kinetic analysis resulted in a reaction rate constant of 2.169 ± 0.059 × 10³ M^?1 s^?1 for DHE–O₂ ^? reaction that is ~100× slower than the reported value of 2.6 ± 0.6 × 10⁵ M^?1 s^?1. In addition, the O₂ ^? self-dismutation has significant effect on DHE–O₂ ^? interaction. A slower reaction rate of DHE with O₂ ^? is more reasonable for O₂ ^? measurements. In this manner, the DHE is not competing with superoxide dismutase and NO for O₂ ^?. Results suggest that an accurate measurement of O₂ ^? production rate may be difficult due to competitive interference for many factors; however O₂ ^? concentration may be quantified.