Detection Of Hydroxyl Radicals Research Articles

The detection of hydroxyl radicals in vivo

Several indirect methods have been developed for the detection and quantification of highly reactive oxygen species (hROS), which may exist either as free hydroxyl radicals, bound "crypto" radicals or Fe(IV)-oxo species, in vivo. This review discusses the strengths and weaknesses associated with those most commonly used, which determine the hydroxylation of salicylate or phenylalanine. Chemical as well as biological arguments indicate that neither the hydroxylation of salicylate nor that of phenylalanine can guarantee an accurate hydroxyl radical quantitation in vivo. This is because not all hydroxylated product-species can be used for detection and the ratio of these species strongly depends on the chemical environment and on the reaction time. Furthermore, at least in the case of salicylate, the high concentrations of the chemical trap required (mM) are known to influence biological processes associated with oxidative stress. Two, newer, alternative methods described, the 4-hydroxy benzoic acid (4-HBA) and the terephthalate (TA) assays, do not have these drawbacks. In each case reaction with hROS leads to only one hydroxylated product. Thus, from a chemical viewpoint, they should provide a better hROS quantitation. Further work is needed to assess any possible biological effects of the required millimolar (4-HBA) and micromolar (TA) concentrations of the chemical traps.

Validation of a robust and sensitive method for detecting hydroxyl radical formation together with evoked neurotransmitter release in brain microdialysis

Sodium terephthalate was shown to be a new robust and sensitive chemical trap for highly reactive oxygen species (hROS), which lacks the drawbacks of the salicylic acid method. Reaction of the almost non-fluorescent terephthalate (TA2-) with hydroxyl radicals or ferryl-oxo species resulted in the stoichiometric formation of the brilliant fluorophor, 2-hydroxyterephthalate (OH-TA). Neither hydrogen peroxide nor superoxide reacts in this system. This procedure was validated for determining hROS formation during microdialysis under in vivo conditions as well as by in vitro studies. The detection limit of OH-TA in microdialysis samples was 0.5 fmol/muL. Derivatization of samples with o-phthalaldehyde, for amino acid detection, had no effect on OH-TA fluorescence, which could easily be resolved from the amino acid derivatives by HPLC, allowing determination in a single chromatogram. Use of terephthalate in microdialysis experiments showed the neurotoxin kainate to evoke hROS formation in a dose-dependent manner. The presence of TA2- in the perfusion fluid did not affect basal or evoked release of aspartate, glutamate, taurine and GABA. Assessment of cell death 'ex vivo' showed TA2- to be non-toxic at concentrations up to 1 mM. The in vitro results in the Fenton system (Fe2+ + H2O2) indicate a mechanism whereby TA2- forms a primary complex with Fe2+ followed by an intramolecular hydroxylation accompanied by intramolecular electron transfer.

Synthesis of Coumarin–Polyamine-Based Molecular Probe for the Detection of Hydroxyl Radicals Generated by Gamma Radiation

To develop a molecular probe for detection of hydroxyl radicals in the vicinity of DNA, the coumarin-polyamine complexes, N(1),N(12)-bis[2-oxo-2H-chromene-3-carbonyl]-1,12-diamine-4,9-diazadodecane (5) and tris[2-(2-oxo-2H-chromene-3-carboxamido)ethyl]amine (7), and their hydroxylated derivatives, N(1),N(12)-bis[7-hydroxy-2-oxo-2H-chromene-3-carbonyl]-1,12-diamine-4,9-diazadodecane (6) and tris[2-(7-hydroxy-2-oxo-2H-chromene-3-carboxamido)ethyl]amine (8), have been synthesized. Using computer-generated molecular modeling, the derivatives have been docked onto DNA dodecamer d(CGCGAATTCGCG)(2), the ligand-DNA complexes have been minimized, and the free binding energies (DeltaG(binding)) and inhibition constants (K(i)) have been calculated. Compound 7 is not water-soluble at the concentrations required for the project. When aqueous solutions of 5 are irradiated with gamma rays, the relationship between induced fluorescence and dose is linear in the range of 0 to 10 Gy. The fluorescence emission spectrum of irradiated 5 is similar to that of its dihydroxy derivative 6, indicating conversion of 5 to 6, and induction of fluorescence records formation of hydroxyl radicals in aqueous solution. The dicoumarin-polyamine 5, a novel compound for the detection of hydroxyl radicals close to DNA, is a sensitive and quantitative probe with potential for applications in biological systems.

A simple electrochemical method for the determination of hydroxyl free radicals without separation process

Generally speaking, measurement of hydroxylated radical products of salicylic acid requires a fussy separation process. In this study, we describe a simple method to electrochemically detect hydroxyl radicals ( OH) using 4-hydroxybenzoic acid (4-HBA) as the OH trap. The OH is generated by the Fenton reaction from iron (II) sulfate and hydrogen peroxide in a phosphate buffer solution. Experimental results show that our method can detect the OH with high sensitivity without any separation process. The differential pulse voltammetric responses show a linear dependence on the concentration of OH in a range of 2.0 × 10 −6 and 1.0 × 10 −3 M with a determination limit down to 5.0 × 10 −7 M. As a demonstration, the kinetics of the Fenton reaction was mapped by measuring the reaction product of hydroxyl radical trapped by 4-HBA. The result is in good agreement with that reported previously. All the results show that the present approach could provide a simple, inexpensive and promising method for biomedicine and iatrology.

Near infrared multiphoton-induced generation and detection of hydroxyl radicals in a biochemical system

Solutions of tryptophan and N-hydroxypyridine-2-thione (mercaptopyridine-N-oxide, MPNO) were irradiated at 335nm. Formation of 5-hydroxytryptophan was inferred from increased fluorescence at 334nm on excitation at 315nm, conditions chosen for selective detection of 5-hydroxytryptophan. Such experiments are complicated by overlapping absorption spectra in the region of 300–350nm. Similar solutions were exposed to multiphoton excitation at 750nm using 180fs pulses from a titanium:sapphire laser. In solutions containing both tryptophan and MPNO strong emission at 500nm was observed that was absent in solutions containing either MPNO or tryptophan only. This emission is ascribed to the characteristic fluorescence (‘hyperluminescence’) from 5-hydroxyindoles resulting from multiphoton photochemistry. The conclusion that MPNO generates hydroxyl radicals by 2-photon activation at 750nm is confirmed by the scavenging effects of ethanol and kinetic analysis of the results. This method has potential applications in intracellular induction of oxidative stress using multiphoton near-infrared illumination, a technology that is gaining momentum as a research tool.

Methylene Blue Dye Test for Rapid Qualitative Detection of Hydroxyl Radicals Formed in a Fenton's Reaction Aqueous Solution

A new procedure, the methylene blue dye test, qualitatively indicates the presence of hydroxyl radicals through the immediate, distinct bleaching of methylene blue dye on a paper test strip. This method employs a simple procedure requiring inexpensive materials, without the addition of competitive probe chemicals that potentially can interfere with the reaction. A Fenton's reaction with an Fe2+:H2O2 molar ratio of 1:20 generated hydroxyl radicals in Milli-Q water. The presence and absence of hydroxyl radicals were determined prior to and following quenching of the Fenton's reaction with 10% sodium sulfite, respectively. Bleaching of methylene blue dye, due to the presence of hydroxyl radicals in a sample,was indicated by a discoloration from a dark blue color to an almost white color, concentrated at the point of application, with a dark blue outline. A lack of bleaching indicated the absence of hydroxyl radicals in the sample. The presence of hydroxyl radicals was verified by benzoic acid chemical probe experiments with thin-layer chromatography (TLC) and spectrophotometric wavelength scans. The presence of hydroxyl radicals was indirectly determined by detection of hydroxylated benzoic acids on TLC plates and a violet solution color with a peak absorbance at a wavelength close to 520 nm.

The first application of terephthalate fluorescence for highly selective detection of hydroxyl radicals in thylakoid membranes.

Possibilities and limitations of the detection of hydroxyl radicals via the conversion of terephthalate (TPA) into the strongly fluorescent hydroxyterephthalate were investigated in order to adapt this method for chlorophyll-containing samples. Using model chemical sources of various reactive oxygen species, we confirmed that TPA detects hydroxyl radicals very sensitively, but is not reactive to either hydrogen peroxide or superoxide radicals. As a new result, we showed that the conversion of TPA to hydroxyterephthalate cannot be induced by singlet oxygen, which may be produced in photosynthetic systems under stress. Until now, the TPA method has not been used in photosynthesis research, so necessary adaptations to minimise the effects of chlorophyll and buffering sugars on hydroxyl radical detection were also explored and optimal conditions for using the method in thylakoid preparations are suggested. Anticipating further plant physiology applications, usefulness of the TPA method was tested in a wider range of pH than reported earlier. To demonstrate that this simple and highly specific method can be used as an alternative approach for the detection of hydroxyl radicals in plant samples, we measured these radicals in isolated thylakoid membranes exposed to 312 nm ultraviolet radiation.

Evidence of ROS generation by mitochondria in cells with impaired electron transport chain and mitochondrial DNA damage

Mitochondrial damage is a well known cause of mitochondria-related diseases. A major mechanism underlying the development of mitochondria-related diseases is thought to be an increase in intracellular oxidative stress produced by impairment of the mitochondrial electron transport chain (ETC). However, clear evidence of intracellular free radical generation has not been clearly provided for mitochondrial DNA (mtDNA)-damaged cells. In this study, using the novel fluorescence dye, 2-[6-(4′-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF), which was designed to detect hydroxyl radicals ( ·OH), intracellular free radical formation was examined in 143B cells (parental cells), 143B-ρ 0 cells (mtDNA-lacking cells), 87 wt (cybrid), and cybrids of 4977-bp mtDNA deletion (common deletion) cells containing the deletion with 0%, 5%, 50% and >99% frequency (HeLacot, BH5, BH50 and BH3.12, respectively), using a laser confocal microscope detection method. ETC inhibitors (rotenone, 3-nitropropionic acid, thenoyltrifluoroacetone, antimycin A and sodium cyanide) were also tested to determine whether inhibitor treatment increased intracellular reactive oxygen species (ROS) generation. A significant increase in ROS for 143B-ρ 0 cells was observed compared with 143B cells. However, for the 87 wt cybrid, no increase was observed. An increase was also observed in the mtDNA-deleted cells BH50 and BH3.12. The ETC inhibitors increased intracellular ROS in both 143B and 143B-ρ 0 cells. Furthermore, in every fluorescence image, the fluorescence dye appeared localized around the nuclei. To clarify the localization, we double-stained cells with the dye and MitoTracker Red. The resulting fluorescence was consistently located in mitochondria. Furthermore, manganese superoxide dismutase (MnSOD) cDNA-transfected cells had decreased ROS. These results suggest that more ROS are generated from mitochondria in ETC-inhibited and mtDNA-damaged cells, which have impaired ETC.

Comparison of chemiluminescence methods for analysis of hydrogen peroxide and hydroxyl radicals

Assessment of alpha radiolysis influence on the chemistry of geologically disposed spent fuel demands analytical methods for radiolytic product determination at trace levels. Several chemiluminescence methods for the detection of radiolytic oxidants hydrogen peroxide and hydroxyl radicals are tested. Two of hydrogen peroxide methods use luminol, catalyzed by either μ-peroxidase or hemin, one uses 10-methyl-9-(p-formylphenyl)-acridinium carboxylate trifluoromethanesulfonate and one potassium periodate. All recipes are tested as batch systems in basic conditions. For hydroxyl radical detection luminophores selected are 3-hydroxyphthalic hydrazide and rutin. Both methods are tested as batch systems.

Prion protein does not redox-silence Cu 2+, but is a sacrificial quencher of hydroxyl radicals

Oxidative stress is believed to play a central role in the pathogenesis of prion diseases, a group of fatal neurodegenerative disorders associated with a conformational change in the prion protein (PrP C). The precise physiological function of PrP C remains uncertain; however, Cu 2+ binds to PrP C in vivo, suggesting a role for PrP C in copper homeostasis. Here we examine the oxidative processes associated with PrP C and Cu 2+. 1H NMR was used to monitor chemical modifications of PrP fragments. Incubation of PrP fragments with ascorbate and CuCl 2 showed specific metal-catalyzed oxidation of histidine residues, His 96/111, and the methionine residues, Met 109/112. The octarepeat region protects His 96/111 and Met 109/112 from oxidation, suggesting that PrP(90–231) might be more prone to chemical modification. We show that Cu 2+/+ redox cycling is not ‘silenced’ by Cu 2+ binding to PrP, as indicated by H 2O 2 production for full-length PrP. Surprisingly, although detection of Cu + indicates that the octarepeat region of PrP is capable of reducing Cu 2+ even in the absence of ascorbate, H 2O 2 is not generated unless ascorbate is present. Full-length PrP and fragments cause a dramatic reduction in detectable hydroxyl radicals in an ascorbate/Cu 2+/O 2 system; however, levels of H 2O 2 production are unaffected. This suggests that PrP does not affect levels of hydroxyl radical production via Fentons cycling, but the radicals cause highly localized chemical modification of PrP C.

Novel hydroxyl radical scavenging antioxidant activity assay for water-soluble antioxidants using a modified CUPRAC method

Reactive oxygen species (ROS) such as superoxide anion, hydroxyl ( OH), peroxyl, and alkoxyl radicals may attack biological macromolecules giving rise to oxidative stress-originated diseases. Since OH is very short-lived, secondary products resulting from OH attack to various probes are measured. Although the measurement of aromatic hydroxylation with HPLC/electrochemical detection is more specific than the low-yield TBARS test, it requires sophisticated instrumentation. As a more convenient and less costly alternative, we used p-aminobenzoate, 2,4- and 3,5-dimethoxybenzoate probes for detecting hydroxyl radicals generated from an equivalent mixture of Fe(II) + EDTA with hydrogen peroxide. The produced hydroxyl radicals attacked both the probe and the water-soluble antioxidants in 37 °C-incubated solutions for 2 h. The CUPRAC (i.e., our original method for total antioxidant capacity assay) absorbance of the ethylacetate extract due to the reduction of Cu(II)-neocuproine reagent by the hydroxylated probe decreased in the presence of OH scavengers, the difference being proportional to the scavenging ability of the tested compound. A rate constant for the reaction of the scavenger with hydroxyl radical can be deduced from the inhibition of color formation. The second-order rate constants of the scavengers were determined with competition kinetics by means of a linear plot of A 0/ A as a function of C scavenger/ C probe, where A 0 and A are the CUPRAC absorbances of the system in the absence and presence of scavenger, respectively, and C is the molar concentration of relevant species. The 2,4- and 3,5-dimethoxybenzoates were the best probes in terms of linearity and sensitivity. Iodide, metabisulfite, hexacyanoferrate(II), thiourea, formate, and dimethyl sulfoxide were shown by the modified CUPRAC assay to be more effective scavengers than mannitol, glucose, lysine, and simple alcohols, as in the TBARS assay. The developed method is less lengthy, more specific, and of a higher yield than the classical TBARS assay. The hydroxyl radical scavenging rate constants of ascorbic acid, formate, and hexacyanoferrate(II) that caused interference in other assays could be easily found with the proposed procedure.

DNA Damage by Free Radical Production by Aminoguanidine

Aminoguanidine (AG), a prototype therapeutic dicarbonyl scavenger, is the most potent drug available today to inhibit the formation of advanced glycation endproducts (AGEs) and to reverse glycation-mediated damage in normal aging. This paper examines the ability of AG to cause damage to supercoiled plasmid DNA in the presence of the transition metal, Fe(+3). Damage to DNA was dependent on the concentrations of both the transition metal and AG. We could detect hydroxyl radical as well as hydrogen peroxide during the incubation of AG with Fe(+3). Thus this finding further cautions against the indiscriminate use of AG in clinical prophylaxis in diabetes and questions its use as a therapeutic agent.

Hydroxylation of d-phenylalanine as a novel approach to detect hydroxyl radicals: Application to cardiac pathophysiology

Research in the pathophysiology of ischemia/reperfusion or redox signaling is hindered by lack of simple methodology to measure short-lived oxygen radicals. In the presence of hydroxyl radical ((*)OH), d-phenylalanine (d-Phe) yields para-, meta- and ortho-tyrosine. We have previously demonstrated that d-Phe can accurately detect (*)OH formation in chemical, enzymatic and cellular systems by simple HPLC methodology [Anal Biochem 290:138;2001]. In the present study, we tested whether d-Phe hydroxylation can be used to detect (*)OH formation in intact organs. Rat hearts were perfused with buffer containing 5 mM d-Phe and subjected to 30 min of total global ischemia at 37 degrees C followed by 45 min of reperfusion. Quantitative analysis of the three hydroxytyrosine isomers was achieved by HPLC-based electrochemical detection of cardiac venous effluent, with the analytical cells operating in the oxidative mode. The detection limit of this assay was <10 fmol. Under baseline conditions, hydroxytyrosine release from the heart was very low ( congruent with0.8 nmol/min/g). However, a prominent tyrosine burst occurred immediately upon post-ischemic reflow. In cardiac effluent collected 40 s into reperfusion, the hydroxytyrosine concentration was more than 40 times greater than at baseline; hydroxytyrosine concentration then progressively declined, to return to pre-ischemic values by 5 min of reperfusion. In parallel experiments, formation of hydroxytyrosines was markedly reduced in hearts reperfused in the presence of the (*)OH scavenger mannitol. Inclusion of 5 mm d-Phe in the perfusion medium altered neither basal cardiac function nor coronary vascular tone, but it enhanced recovery of myocardial function during post-ischemic reperfusion, consistent with direct reaction with (*)OH. Our results demonstrate that d-Phe is a sensitive method for detection of (*)OH generation in the heart. Since d-Phe is not a substrate for endogenous enzymes, it can be exploited as a reliable method to measure (*)OH formation under a variety of pathophysiological conditions.

Pro-oxidant and cytotoxic activities of atractylenolide I in human promyeloleukemic HL-60 cells

The dried rhizome of Bai Zhu ( Atractylodes ovata) is widely used as a Chinese herbal medicine. Two sesquiterpenolides of similar structures (atractylenolide I, AT-I; atractylenolide III, AT-III) were isolated from dried rhizome of Atractylodes ovata. Incubation of AT-I with recombinant human Cu,Zn-superoxide dismutase (rhCu,Zn-SOD) resulted in rhCu,Zn-SOD fragmentations and Zn releases. However, these were not observed in the AT-III reaction. The AT-1 showed dose-dependent cytotoxic activities (7.5, 15, and 30 μg/ml) on the human promyeloleukemic HL-60 cells while AT-III did not, and the IC 50 of the former being 10.6 μg/ml (corresponding to 46 μM) on 12 h-treated cells. The results of DNA ladder and DNA contents in sub-G1 type revealed that AT-I induced apoptosis in human promyeloleukemic HL-60 cells. The cytotoxic and pharmacological mechanisms of AT-I against human promyeloleukemic HL-60 cells was investigated. The AT-I appeared to exhibit both pro-oxidant and antioxidant properties after an ESR spectrometer was used to detect hydroxyl radical productions in vitro and flow cytometry to detect intracellular ROS productions in AT-I treated cells. The AT-1 also showed dose-dependent Cu,Zn-SOD inhibitory activity in HL-60 cells treated for 12 h, confirmed by activity and immune stainings. However, catalase, Mn-SOD, and glutathione peroxidase did not apparently change activities under the same treatments. The addition of commercial rhCu,Zn-SOD (25–100 U/mL) to the AT-I-treated HL-60 cells (15 μg/ml) resulted in significant differences ( p < 0.01) and could reduce the AT-I cytotoxicity from 78% to 28% on HL-60 cells. It was proposed that the AT-I might work via Cu,Zn-SOD inhibition in HL-60 cells to induce apoptosis and bring about cytotoxicity.

Fluorescence detection of hydroxyl radicals

The hydroxyl radical ( OH), a product of water radiolysis, reacts to hydroxylate aromatic organic compounds. In some cases, these hydroxylated products are fluorescent. Examples include the benzoate, coumarin, and phenoxazinone systems. For representative members of these systems, we have determined both the rate constants for reaction with OH and the yields of the fluorescent products. The rate constants all fall in the range 2×10 9 to 2×10 10 L mol −1 s −1, and the yields 5–11% per OH. These results suggest that it may prove feasible to construct a probe consisting of two groups both of which must react with OH to become fluorescent. The efficient process of fluorescence resonance energy transfer implies that such a probe might be able to detect OH clusters, which are generally assumed to be a characteristic feature of energy deposition by ionizing radiation.

Antioxidant Activities of Mucilages from Different Taiwanese Yam Cultivars

The antioxident effects of crude mucilages (CM) and partially purified mucilages (PPM) from three different Taiwanese yam cultivars-including Dioscorea alata L. cv. Tainong 1 (Th1), Dioscorea alata L. cv. Tainong 2 (TN2), and D. alata L. var. purpurea (Roxb.) Ming-Jen (MJ)-were evaluated, including 1,1-diphenyl-2-picrylhy-drazyl (DPPH) radical, hydroxyl radical, and superoxide radical scavenging activities Electron spin resonance (ESR) spectrometry was used to measure hydroxyl radical scavenging activities. The IC50 stands for the concentration required for 50% scavenging activity. The IC50 of CM and PPM against DPPH radical was 0.329, 0.279; 0.547, 0.653; and 0.847, 0.631 mg/ml, respectively, for TN1, TN2 and MJ. The IC50 of CM and PPM against hydroxyl radical by spectrophotometry was 0.668, 1.146; 1.461, 1.096; and 0.946, 1.554 mg/ml, respectively, for TN1, 1N2 and MJ. The IC50 of CM and PPM against superoxide radical was 0.802, 0.368; 0.681, 0.258; and 0.086, 0.148 mg/ml, respectively, for TN1, TN2 and MJ. Using ESR to detect hydroxyl radicals, the IC50 of PPM against hydroxyl radical was 0.083, 0.47, and 0.004 mg/ml, respectively, for TN1, TN2 and MJ. The results demonstrated that different cultivars of yams exhibited different antioxidant ability, and the purification process was able to partially increase the antioxidant activity of the mucilage polysaccharide. Taken together, these results suggest that mucilage polysaccharides of the yam tuber might play an important role on antiradicals and antioxidants.

Probing the Hydroxyl Radical-Mediated Reactivity of Peroxynitrite by a Spin-Labeling Fluorophore

The decomposition of peroxynitrite at physiological pH yielded a hydroxyl radical, which reacted rapidly with dimethyl sulfoxide (DMSO) to produce a methyl radical (*CH3), which was then trapped by a spin-label fluorophore nitroxide-linked naphthalene (NTEMPO), a carbon-centered radical probe with a low fluorescence intensity, and transformed to a stable diamagnetic O-alkoxyamine, a high-fluorescence compound. The fluorescence increment was proportional to the concentration of the hydroxyl radical, and then to the concentration of peroxynitrite. NTEMPO therefore was demonstrated to be capable of detecting hydroxyl radicals generated from peroxynitrite, and the method was proved to be simple and sensitive. The hydroxyl radical-mediated reactivities of peroxynitrite to several amino acids, such as tyrosine, phenylalanine and histidine, were then evaluated by the spin-labeling fluorophore NTEMPO at pH 7.4 and, 37 degrees C. The obtained data were in good agreement with the reference values, respectively.

Determination of the antioxidant capacity of different food natural products with a new developed flow injection spectrofluorimetry detecting hydroxyl radicals

This paper presents an automatic spectrofluorimetric method (flow injection analysis spectrofluorimetry) for detecting hydroxyl radicals. Based on H 2O 2 catalyzed by Co 2+ yielding HO , the method utilized sodium terephthalate to trap hydroxyl radicals and obtained sodium 2-hydroxyterephthalate by aromatic hydroxylation, which resulted in an increase in the fluorescence intensity. The relative fluorescence intensity was proportional to the concentration of hydroxyl radicals. Hydroxyl radicals could be determined indirectly, based on the increase of fluorescence. It was a simple, rapid, precise, sensitive and automatic technique for the determination of HO . The relative standard deviation of 11 determinations was 0.57%. The method can be used to sieve antioxidant medicines and will have theoretical and practical guidance on the mechanism of hydroxyl radicals-damaging biology.

N-[3H]Benzoylglycylglycylglycine as a probe for hydroxyl radicals

N-[4-3H]Benzoylglycylglycylglycine ([3H]BzG3) was tested as a probe for detecting hydroxyl radicals (OH). Aerated solutions of l-ascorbate generated OH, which oxidized [3H]BzG3, yielding hydrophilic (probably hydroxylated) derivatives plus tritiated water. The 3H2O was separated from organic products and remaining [3H]BzG3 on Dowex-1. 3H2O production was much greater with OH than with other reactive oxygen species (ROS) (e.g., H2O2, superoxide). The slight 3H2O production in the presence of H2O2 or superoxide was blocked by OH scavengers (e.g., glycerol, mannitol, butan-1-ol) that do not scavenge H2O2 or superoxide. This indicates that 3H2O production was caused by OH and that other ROS only generated any 3H2O by forming traces of OH. Doses of OH that caused detectable nonenzymic polysaccharide scission also caused 3H2O production, indicating that [3H]BzG3 is a sensitive OH probe in studies of polymer scission. The ability of scavengers and chelators to protect against ascorbate-mediated polysaccharide scission paralleled their ability to inhibit concurrent 3H2O production, indicating that both processes were due to OH. Thus, [3H]BzG3 is a simple, specific, sensitive, and robust probe for detecting OH production in vitro. It may have applications for in vivo detection of extracellular OH in arthritic joints and of apoplastic OH in plant cell walls.

Pro-Oxidative Interactions of Cobalt with Human Neutrophils

The primary objectives of this study were to investigate the effects of cobalt(II) chloride (Co, 1.5–25 μ M) on the reactivity of hydrogen peroxide (H2O2, 100 μ M) or oxidants generated by activated human neutrophils. The prooxidative interactions of Co with H2O2 or cells were measured by luminol-enhanced chemiluminescence (LECL), and according to the extent of oxidative inactivation of added alpha-1-proteinase inhibitor (API). Cobalt dramatically potentiated the oxidation of luminol and API by both H2O2 and neutrophils activated with phorbol 12-myristate 13-acetate (5 ng/ml), without affecting the assembly of NADPH oxidase or the magnitude of oxygen consumption by the cells. Using 5,5-dimethyl-pyrolline 1-oxide-based electron spin resonance spectroscopy we were unable to detect hydroxyl radical formation by Co in the presence of either H2O2 or activated neutrophils, while the corresponding LECL responses were unaffected by the hydroxyl radical scavengers benzoate and mannitol (50 mM). These observations indicate that Co potentiates the reactivity of neutrophil-derived oxidants, primarily H2O2, which if operative in vivo during exposure to the heavy metal may pose the risk of oxidant- and protease-mediated tissue injury.