What do we measure with luminol-, lucigenin- and penicillin-amplified chemiluminescence? 1. Investigations with hydrogen peroxide and sodium hypochlorite

Evidence is provided that the amplifiers luminol and lucigenin react with different reactive oxygen species (ROS), depending on the ROS-generating system used. H₂O₂ is used to produce calibration curves for luminol- and lucigenin-amplified chemiluminescence. With this chemiluminescence generator we characterized the specificity and sensitivity of luminol- and lucigenin-amplified chemiluminescence and also studied penicillin G, a known enhancer of luminol-amplified chemiluminescence. The combination of luminol and lucigenin in reciprocally changing concentrations is effective in an additive manner, but the weak amplifier penicillin increases luminol-amplified chemiluminescence distinctly more than in an additive manner in different combinations. Lucigenin-amplified chemiluminescence is increased by penicillin at about 1% of the optimum concentration of penicillin; increasing concentrations of penicillin are less and less effective. On the other hand, low lucigenin concentrations enhance penicillin-amplified chemiluminescence at optimum penicillin concentrations more than in an additive manner. Fe²⁺ does not alter luminol-, lucigenin- or penicillin-amplified chemiluminescence. Co²⁺ increases luminol-amplified chemiluminescence by a factor of 100. Lucigenin- and penicillin-amplified chemiluminescence are minimally enhanced by Co²⁺. Cu²⁺ enhances luminol-amplified chemiluminescence with increasing concentrations by a factor of 1000. Lucigenin-amplified chemiluminescence increases also by the factor of 1000, but the concentration–reaction curve is not as steep. NaOCl enhances H₂O₂/Fe²⁺-driven luminol-amplified chemiluminescence in a concentration-dependent manner by a factor of 10⁴ (in the highest concentration of 10 mmol/L) and lucigenin amplified chemiluminescence only by a factor of about 25. Catalase (CAT) abolishes luminol-, lucigenin- and penicillin-amplified chemiluminescence completely, whereas superoxide dismutase (SOD) has no effect on luminol- or penicillin-amplified chemiluminescence, but enhances lucigenin-amplified chemiluminescence five-fold increasingly with increasing SOD activity. © 1998 John Wiley & Sons, Ltd.     

In vitro screening of Fe2+‐chelating effect by a Fenton's reaction–luminol chemiluminescence system

In vitro screening of a Fe²⁺‐chelating effect using a Fenton's reaction–luminol chemiluminescence (CL) system is described. The luminescence between the reactive oxygen species generated by the Fenton's reaction and luminol was decreased on capturing Fe²⁺ using a chelator. The proposed method can prevent the consumption of expensive seed compounds (drug discovery candidates) owing to the high sensitivity of CL detection. Therefore, the assay could be performed using small volumes of sample solution (150 μL) at micromolar concentrations. After optimization of the screening conditions, the efficacies of conventional chelators such as ethylenediaminetetraacetic acid (EDTA), diethylentriaminepentaacetic acid (DETAPAC), deferoxamine, deferiprone and 1,10‐phenanthroline were examined. EC₅₀ values for these compounds (except 1,10‐phenanthroline) were in the range 3.20 ± 0.87 to 9.57 ± 0.64 μM (n = 3). Rapid measurement of the Fe²⁺‐chelating effect with an assay run time of a few minutes could be achieved using the proposed method. In addition, the specificity of the method was discussed. Copyright © 2014 John Wiley & Sons, Ltd.     

The effect of quenchers on the chemiluminescence of luminol and lucigenin

The chemiluminescence of luminol and lucigenin is often used to detect the production of reactive oxygen derivatives by phagocytic cells. Also, several quenchers and enzyme inhibitors are used to determine which oxygen derivatives are responsible for the observed effects. In the present work we have assessed the reliability of dimethylthiourea and cysteamine (OH^. quenchers), desferrioxamine (iron chelator) and diethyldithiocarbamate (superoxide dismutase inhibitor). They all react with CIO^? and are also strong inhibitors of the luminescence of luminol catalysed by horseradish peroxidase (HRP); cysteamine and diethyldithiocarbamate also react with H₂O₂. NaN₃ is an inhibitor of myeloperoxidase and a quencher of singlet O₂, but we found that under certain conditions it can amplify the the luminescence of luminol triggered by CIO^? or Fenton's reagent. A complex of copper and penicillamine that had been proposed as an $ {\rm O}_{\rm 2} ^{\bar .} $ quencher, quenches all luminescent reactions studied. On the other hand, we were able to confirm the relative specificity of other quenchers: taurine for CIO^?, benzoate for OH^. and mannitol for both OH^. and ‘crypto-OH^.’.     

Chemiluminescence of lucigenin by electrogenerated superoxide ions in aqueous solutions

The chemiluminescence reaction of lucigenin (Luc²⁺?2NO₃^?, N,N′‐dimethyl‐9,9′‐biacridinium dinitrate) at gold electrodes in dioxygen‐saturated alkaline aqueous solutions (pH 10) was investigated in detail by the use of electrochemical emission spectroscopy. We noted that both O₂ and Luc²⁺ are reduced on a gold electrode in aqueous solution of pH 10 in almost the same potential region. From this fact, we expected chemiluminescence based on a radical–radical coupling reaction of superoxide ion (O₂·^?) and one‐electron reduced form of Luc²⁺ (Luc·⁺, a radical cation). Chemiluminescence was actually observed in the potential range where O₂ and Luc²⁺ were simultaneously reduced at the electrodes. The effects were examined upon addition of enzymes, i.e. superoxide dismutase (SOD) and catalase, into the solution and the substitution of heavy water (D₂O) for light water (H₂O) as a solvent on the chemiluminescence. In the presence of native and active SOD, chemiluminescence was completely absent. On the other hand, chemiluminescence was observed, unchanged in the presence of either denatured and inert SOD or catalase. In addition, the amount of chemiluminescence in D₂O solution was about three times greater than that in H₂O solution. These results, together with cyclic voltammetric results, suggest that O₂·^? participates directly in the chemiluminescence but H₂O₂ does not, and the chemiluminescence results from the coupling reaction between O₂·^? and Luc^·+ under the present experimental conditions. These chemically unstable species, O₂·^? and Luc·⁺, are produced during the simultaneous electroreduction of O₂ and Luc²⁺. The coupling reaction between those radical species would lead to the formation of a dioxetane‐type intermediate and, finally, to chemiluminescence. The chemiluminescence reaction mechanism is discussed. Copyright © 2002 John Wiley & Sons, Ltd.     

The lactate-dependent enhancement of hydroxyl radical generation by the Fenton reaction

The effect of lactic acid (lactate) on Fenton based hydroxyl radical (^·OH) production was studied by spin trapping, ESR, and fluorescence methods using DMPO and coumarin-3-carboxylic acid (3-CCA) as the ^·OH traps respectively. The ^·OH adduct formation was inhibited by lactate up to 0.4mM (lactate/iron stoichiometry = 2) in both experiments, but markedly enhanced with increasing concentrations of lactate above this critical concentration. When the H₂O₂ dependence was examined, the DMPO-OH signal was increased linearly with H₂O₂ concentration up to 1 mM and then saturated in the absence of lactate. In the presence of lactate, however, the DMPO-OH signal was increased further with higher H₂O₂ concentration than 1 mM, and the saturation level was also increased dependent on lactate concentration. Spectroscopic studies revealed that lactate forms a stable colored complex with Fe³⁺ at lactate/Fe³⁺ stoichiometry of 2, and the complex formation was strictly related to the DMPO-OH formation. The complex formation did not promote the H₂O₂ mediated Fe³⁺ reduction. When the Fe³⁺-lactate (1:2) complex was reacted with H₂O₂, the initial rate of hydroxylated 3-CCA formation was linearly increased with H₂O₂ concentrations. All the data obtained in the present experiments suggested that the Fe³⁺-lactate (1:2) complex formed in the Fenton reaction system reacts directly with H₂O₂ to produce additional ^·OH in the Fenton reaction by other mechanisms than lactate or lactate/Fe³⁺ mediated promotion of Fe³⁺/Fe²⁺ redox cycling.     

Luminol Luminescence Induced by 2,2'-Azo-Bis(2-Amidinopropane) Thermolysis

2-2'-Azo-bis(2-amidinopropane) thermolysis induces luminol luminescence. The luminescence intensity is quenched by SOD, catalase, Trolox and human blood serum. However, the time course of the light intensity profile is different for the different additives. In particular, the quenching efficiency of Trolox and human blood serum decreases with time after addition. Double quenching experiments show that SOD and Trolox are not competitive quenchers, while a simple competition can be established between Trolox and human blood serum in trapping a common intermediate. From the kinetic analysis of the data it is concluded that, at least at low additive concentrations, Trolox scavenges a luminol derived radical. Higher concentrations of Trolox or human blood serum produce induction times that are proportional to the additives concentrations. The possibility of employing luminol luminescence in the evaluation of TRAP levels and the capacity of biological samples to scavenge free radicals is discussed.     

Pyrophosphate as substrate for alkaline phosphatase activity: A convenient flow‐injection chemiluminescence assay

A sensitive and convenient flow‐injection chemiluminescence (FI‐CL) turn‐on assay for alkaline phosphatase (ALP) activity without any label and synthesis is developed. Cu²⁺ can catalyze the luminol–H₂O₂ CL reaction. Pyrophosphate (PPi) can chelate Cu²⁺ and therefore the Cu²⁺‐mediated luminol‐H₂O₂ CL reaction is inhibited. The addition of ALP can catalyze the hydrolysis of PPi into phosphate ions, Cu²⁺ is released and the chemiluminescence recovers. A detection limit of 1 mU/mL ALP is obtained.     

Thioctic Acid and Dihydrolipoic Acid are Novel Antioxidants Which Interact With Reactive Oxygen Species

Thioctic acid (TA) and its reduced form dihydrolipoic acid (DHLA) have recently gained somc recognition as useful biological antioxidants. In particular, the ability of DHLA to inhibit lipid peroxidation has been reported. In the present study, the effects of TA and DHLA on reactive oxygen species (ROS) generated in the aqueous phase have been investigated. Xanthine plus xanthine oxidase-generated superoxide radicals (O₂), detected by electron spin resonance spectroscopy (ESR) using DMPO as a spin trap. were eliminated by DHLA but not by TA. The sulhydryl content of DHLA, measured using Ellman's reagent decreased subsequent to the incubation with xanthine plus xanthine oxidase confirming the interaction between DHLA and O₂^-. An increase of hydrogen peroxide concentration accompanied the reaction between DHLA and O₂x, suggesting the reduction of O₂^- by DHLA. Competition of O₂^- with epinephrine allowed us to estimate a second order kinetic constant of the reaction between O₂^- and DHLA, which was found to be a 3.3 × 10⁵ M^-1 s^-1. On the other hand, the DMPO signal of hydroxyl radicals (HO ·) generated by Fenton's reagent were eliminated by both TA and DHLA. Inhibition of the Fenton reaction by TA was confirmed by a chemiluminescence measurement using luminol as a probe for HO ·. There was no electron transfer from Fe²⁺ to TA or from DHLA to Fe^{3 +} detected by measuring the Fe²⁺ -phenanthroline complex. DHLA did not potentiate the DMPO signal of HO · indicating no prooxidant activity of DHLA. These results suggest that both TA and DHLA possess antioxidant properties. In particular. DHLA is very effective as shown by its dual capability by eliminating both O₂^-; and HO ·.     

Optimization of peroxynitrite–luminol chemiluminescence system for detecting peroxynitrite in cell culture solution exposed to carbon disulphide

We established a peroxynitrite–luminol chemiluminescence system for detecting peroxynitrite in cell culture solution exposed to carbon disulphide (CS₂). Three factors, including exposure time to ozone (Factor A), volume of peroxynitrite (ONOO^?) solution (Factor B) and luminol concentrations (Factor C) at three levels were selected and the combinations were in accordance with orthogonal design L₉ (3⁴). Peroxynitrite was generated from the reaction of ozone and 0.01 mol/L sodium azide (NaN₃) dissolved in carbonic acid buffer solution (pH 11), and it was reacted with luminol to yield chemiluminescence. The peak value, peak time and kinetic curve of the light emission were observed. The selected combination conditions were 50 s ozone, 800 µL peroxynitrite and 0.001 mol/L luminol solution. Cell culture solution with CS₂ enhanced the emission intensity of chemiluminescence (F = 8.38, p = 0.018) and shortened the peak time to chemiluminescence (F = 139.00, p = 0.0001). The data demonstrated that this luminol chemiluminescence system is suitable for detecting peroxynitrite in cell culture solutions for evaluating the effect of CS₂ on endothelial cells. Copyright © 2003 John Wiley & Sons, Ltd.     

In Vitro Effect of H2O2, Some Transition Metals and Hydroxyl Radical Produced Via Fenton and Fenton-Like Reactions,on the Catalytic Activity of AChE and the Hydrolysis of ACh

It is well known that the principal biomolecules involved in Alzheimer’s disease (AD) are acetylcholinesterase (AChE), acetylcholine (ACh) and the amyloid beta peptide of 42 amino acid residues (Aβ42). ACh plays an important role in human memory and learning, but it is susceptible to hydrolysis by AChE, while the aggregation of Aβ42 forms oligomers and fibrils, which form senile plaques in the brain. The Aβ42 oligomers are able to produce hydrogen peroxide (H₂O₂), which reacts with metals (Fe²⁺, Cu²⁺, Cr³⁺, Zn²⁺, and Cd²⁺) present at high concentrations in the brain of AD patients, generating the hydroxyl radical (^·OH) via Fenton (FR) and Fenton-like (FLR) reactions. This mechanism generates high levels of free radicals and, hence, oxidative stress, which has been correlated with the generation and progression of AD. Therefore, we have studied in vitro how AChE catalytic activity and ACh levels are affected by the presence of metals (Fe³⁺, Cu²⁺, Cr³⁺, Zn²⁺, and Cd²⁺), H₂O₂ (without Aβ42), and ^· OH radicals produced from FR and FLR. The results showed that the H₂O₂ and the metals do not modify the AChE catalytic activity, but the ^·OH radical causes a decrease in it. On the other hand, metals, H₂O₂ and ^·OH radicals, increase the ACh hydrolysis. This finding suggests that when H₂O₂, the metals and the ^·OH radicals are present, both, the AChE catalytic activity and ACh levels diminish. Furthermore, in the future it may be interesting to study whether these effects are observed when H₂O₂ is produced directly from Aβ42.     

Control of superoxide and nitric oxide formation during human sperm capacitation

We studied the modulation of superoxide anion (O₂·^?) and nitric oxide (NO·) generation during human sperm capacitation (changes needed for the acquisition of fertility). The production of NO· (diaminofluorescein-2 fluorescence assay), but not that of O₂·^? (luminescence assay), related to sperm capacitation was blocked by inhibitors of protein kinase C, Akt, protein tyrosine kinase, etc., but not by those of protein kinase A. Extracellular calcium (Ca²⁺) controlled O₂·^? synthesis but extra- and intracellular Ca²⁺ regulated NO· formation. Zinc inhibited capacitation and formation of O₂·^? and NO·. Zinc chelators (TPEN and EDTA) and sulfhydryl-targeted compounds (diamide and N-ethylmaleimide) stimulated capacitation and formation of O₂·^? and NO·; superoxide dismutase (SOD) and nitric oxide synthase inhibitor (L-NMMA) prevented these events. Diphenyliodonium (flavoenzyme inhibitor) blocked capacitation and related O₂·^? synthesis but promoted NO· formation, an effect canceled by SOD and L-NMMA. NADPH induced capacitation and NO· (but not O₂·^?) synthesis and these events were blocked by L-NMMA and not by SOD. Integration of these data on O₂·^? and NO· production during capacitation reinforces the concept that a complex, but flexible, network of factors is involved and probably is associated with rescue mechanisms, so that spermatozoa can achieve successful fertilization.     

Degradation of DMPO Adducts from Hydroxyl and 1-Hydroxyethyl Radicals by Rat Liver Microsomes

Hydroxyl and 1-hydroxyethyl radical adducts of 5, 5-dimethylpyrroline N-oxide (DMPO) were prepared by photolysis, and mechanisms for loss of their EPR signals in rat liver microsomal suspensions were evaluated. Rates of NADPH-dependent EPR signal loss were more rapid in phosphate buffer than in Tris buffer. Addition of superoxide dismutase (SOD) partially protected the adducts when Tris was used as a buffer, but was relatively ineffective in the presence of phosphate. The ferrous iron chelator bathophenanthrolene partially protected the spin adducts in the presence and absence of phosphate, but complete protection was observed when SOD was also added. The spin adducts were unstable in the presence of Fe⁺² and K₃Fe(CN)₆, but Fe⁺³ alone had little effect on the EPR signals. The data are consistent with two mechanisms for microsomal degradation of DMPO spin adducts under these conditions. Microsomes form superoxide in the presence of oxygen and NADPH, which attacks these DMPO spin adducts directly. The spin adducts are also degraded in the presence of Fe⁺², and phosphate stimulates this iron-dependent destruction of DMPO spin adducts.     

Chemiluminescence intensities and spectra of luminol oxidation by sodium hypochlorite in the presence of hydrogen peroxide

Hydrogen peroxide amplifies the chemiluminescence in the oxidation of luminol by sodium hypochlorite. A linear relationship between concentration of hydrogen peroxide and light intensity was found in the concentration range 5 × 10^?8?7.5 × 10^?6 mol/l. At 7.5 × 10^?6 mol/l H₂O₂ the chemiluminescence is amplified 550—fold. The chemiluminescence spectra of these reactions have a wavelength maximum at 431 nm independent of the concentration of hydrogen peroxide. The results indicate that hydrogen peroxide is a necessary component in the chemiluminescent oxidation of the luminol by sodium hypochlorite.     

The Effects of Different Buffers on the Oxidation of DNA by Thiols and Ferric Iron

Because buffers can act as metal ligands, they can effect several reactions necessary for DNA oxidation by ferric iron and thiols, such as iron reduction. Therefore, these reactions were studied in Hepes and phosphate buffers and unbuffered NaCl. Reduction of Fe³⁺ by dithiothreitol (DTT) and cysteine was observed in either Hepes or NaCl solutions, but not in phosphate buffer. Thiyl radicals were observed in Hepes, but there was much less thiyl radical production in the saline or phosphate solutions. Redox cycling between either DTT or cysteine and Fe³⁺ also resulted in dioxygen consumption in Hepes buffer. Reduction of Fe³⁺ and O₂ resulted in the formation of an oxidant capable of producing 8-hydroxy-2′-deoxyguanosine (8-OHdG) in calf-thymus DNA. The highest levels of 8-OHdG were detected when DTT or cysteine and Fe³⁺ were incubated in Hepes, while much less DNA oxidation was detected when the experiment was done in a saline solution, and almost no DNA oxidation occurred in the phosphate buffer. These results demonstrate that the use of different buffers can greatly affect the ability of thiols to promote iron-dependent oxidations. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 12: 125–132, 1998     

Mechanisms of inhibition of chemiluminescence in the oxidation of luminol by sodium hypochlorite

Two different mechanisms of inhibition of chemiluminescence in the oxidation of luminol by sodium hypochlorite were found. Most substances investigated in these experiments acted by scavenging NaOCI. This mechanism was independent of the concentration of hydrogen peroxide and the incubation time between luminol and inhibitors. The most potent inhibitors were substances containing SH groups. Compounds with amino groups as a target for HOCI/OCI^? to yield chloramines were much less effective inhibitors. Another mechanism of inhibition was found for catalase. It depended on the presence of hydrogen peroxide in the incubation medium and the incubation time between luminol and catalase. The enzyme inhibited the luminescence by removing H₂O₂ at molar concentrations much smaller than those found for all other inhibitors. Our results confirm the present models of the mechanism of generation of luminescence in luminol oxidation.     

A stopped‐flow study of the dual chemiluminescence for the luminol–KIO4–Mn2+ system in strong alkaline solutions

A novel phenomenon of dual chemiluminescence (CL) was observed for the KIO₄–luminol–Mn²⁺ system in strong alkaline solutions using the stopped‐flow technique. Scavenging study of the reactive oxygen species (ROS) suggested that the two CL peaks originated from different CL pathways precipated by distinct ROS (O₂^? and ^?OH for the first peak, mainly ¹O₂ for the second peak). Generation of these ROS at different time intervals from the reactions involving IO₄^?, O₂, and Mn²⁺ and their subsequent reactions with luminol induced the intense CL emission. The relative intensity of the two CL peaks can be tuned over a wide range by varying the concentrations of Mn^2?, luminol and KIO₄. Because of the involvement of different ROS in each pathway, the two CL peaks could respond quite differently to various substances. Moreover, variation of the intensity ratio of the two CL peaks altered the relative proportions of the corresponding ROS, thereby changing their responses to a given substance. The dual CL emission acts like a pair of tunable probes and it is believed that this CL system has great potential in analytical applications. Copyright © 2010 John Wiley & Sons, Ltd.     

Carvedilol inhibition of lipid peroxidation. A new antioxidative mechanism

To define the molecular mechanism(s) of carvedilol inhibition of lipid peroxidation we have utilized model systems that allow us to study the different reactions involved in this complex process.

Carvedilol inhibits the peroxidation of sonicated phosphatidylcholine liposomes triggered by FeCl₂ addition whereas atenolol, pindolol and labetalol are ineffective. The inhibition proved not to be ascribable (a) to an effect on Fe²⁺ autoxidation and thus on the generation of oxygen derived radical initiators; (b) to the scavenging of the inorganic initiators O^·-₂ and ^·OH; (c) to an effect on the reductive cleavage of organic hydroperoxides by FeCl₂; (d) to the scavenging of organic initiators. The observations that (a) carvedilol effectiveness is inversely proportional to the concentration of FeCl₂ and lipid hydroperoxides in the assay; (b) the drug prevents the onset of lipid peroxidation stimulated by FeCl₃ addition and; (c) it can form a complex with Fe³⁺, suggest a molecular mechanism for carvedilol action. It may inhibit lipid peroxidation by binding the Fe³⁺ generated during the oxidation of Fe²⁺ by lipid hydroperoxides in the substrate. The lag time that carvedilol introduces in the peroxidative process would correspond to the time taken for carvedilol to be titrated by Fe³⁺; when the drug is consumed the Fe³⁺ accumulates to reach the critical parameter that stimulates peroxidation. According to this molecular mechanism the antioxidant potency of carvedilol can be ascribed to its ability to bind a species, Fe³⁺, that is a catalyst of the process and to its lipophilic nature that concentrates it in the membranes where Fe³⁺ is generated by a site specific mechanism.     

The effect of antioxidants on the formation of free radicals and primary products of the peroxidase reaction

Antioxidants suppress the formation of radicals in peroxidase processes. The chemiluminescence kinetic curves in the horseradish peroxidase/luminol/H₂O₂ system have been compared with those obtained from the mathematical model of the reaction. It was shown that the effect of trolox, ascorbate, and mexidol is a result of the reaction of the luminol radical with the inhibitor molecule (rate constants, 1.0·10¹⁰, 9.0·10⁹, and 2.3·10⁵ mol^–1 min^–1, respectively). The antiradical action of quercetin has been described by eight reactions that were based on the assumption of two reaction centers in the molecule, each reacting with two radicals. The hypothesis that the antioxidant molecule captures the enzyme intermediate radicals in peroxidase cycle, rather than the radical of the reaction product was not confirmed because the calculated curves differed from the experimental point positions. Apparently, the formation of radicals in the presence of peroxidases in living cells and the subsequent events, such as apoptosis, may be prevented not only by the inhibition of an enzyme but also by antioxidants that capture free-radical reaction products     

Copper-Ligand Interactions and Physiological free Radical Processes. Part 2. Influence of Cu2+ Ions on Cu+-Driven Oh Generation and Comparison with Their Effects on Fe2+-Driven Oh Production

In our search to establish a reference ·OH production system with respect to which the reactivity of copper(II) complexes could then be tested, the influence of free Cu²⁺ ions on the Cu⁺/H₂O₂ reaction has been investigated.

This influence depends on the C_Cu²⁺/C_Cu⁺ ratio. At low Cu²⁺ concentrations, ·OH damage to various detector molecules decreases with increasing Cu²⁺ concentrations until C_Cu²⁺/C_Cu⁺ reaches unity. Above this value, ·OH damage increases sharply until C_Cu²⁺/C_Cu⁺ becomes equal to 5 with salicylate and 2 with deoxyribose, ratios for which the protective effect of Cu²⁺ cancels. Finally, at higher concentrations, Cu²⁺ ions logically add their own ·OH production to that normally expected from Cu⁺ ions. The possible origin of this unprecedented alternate effect has been discussed. The possible influence of Cu⁺ ions on the generation of ·OH radicals by water gamma radiolysis has also been tested and, as already established for Cu²⁺ in a previous work, shown to be nonexistent. This definitely confirms that either form of ionised copper cannot scavenge ·OH radicals in the absence of a Iigand.     

Semi‐micro flow injection analysis method for evaluation of quenching effect of health foods or food additive antioxidants on peroxynitrite

A semi‐micro flow injection analysis (SMFIA) method for evaluation of quenching effect of food additive antioxidants or health foods on peroxynitrite (ONOO^‐) is described. The injected sample was carried with phosphate buffer containing NaNO₂, mixed with a trigger solution to generate ONOO^‐ and then detected CL generated after mixing with luminol solution. Selective chemiluminescence caused by ONOO^‐ in this generation system was confirmed by catalase treatment. Ascorbic acid (ASA), Trolox and ascorbyl palmitate (ASP) were used as food additive antioxidants. EC₅₀ values of ASA, Trolox and ASP were 2.6, 6.4 and 43 µg/mL, respectively. The amount of reagents required for an assay by this SMFIA system could reduce the time by a third compared with the conventional method previously reported. Furthermore, as an application of the proposed method, the quenching effect of commercially available Noni (Morinda citrifolia) juices was evaluated. Copyright © 2010 John Wiley & Sons, Ltd.