Neutrophil function in leukemia patients--the correlation between bactericidal capacity and generation of oxygen radicals, especially hydroxyl radical.
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Blood Bactericidal Activity↗
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Detection of hydroxyl radicals by D-phenylalanine hydroxylation: a specific assay for hydroxyl radical generation in biological systems.
Hydroxylation of l-phenylalanine (Phe) by hydroxyl radical (*OH) yields 4-, 3-, and 2-hydroxyl-Phe (para-, meta-, and ortho-tyrosine, respectively). Phe derivative measurements have been employed to detect *OH formation in cells and tissues, however, the specificity of this assay is limited since Phe derivatives also arise from intracellular Phe hydroxylase. d-Phe, the d-type enantiomer, is not hydroxylated by Phe hydroxylase. We evaluate whether d-Phe reacts with *OH as well as l-Phe, providing a more reliable probe for *OH generation in biological systems. With *OH generated by a Fenton reaction or xanthine oxidase, d- and l-Phe equally gave rise to p, m, o-tyr and this could be prevented by *OH scavengers. Resting human neutrophils (PMNs) markedly converted l-Phe to p-tyr, through non-oxidant-mediated reactions, whereas d-Phe was unaffected. In contrast, when PMNs were stimulated in the presence of redox cycling iron the *OH formed resulted in more significant rise of p-tyr from d-Phe (9.4-fold) than l-Phe (3.6-fold) due to the significant background formation of p-tyr from l-Phe. Together, these data indicated that d- and l-Phe were equally hydroxylated by *OH. Using d-Phe instead of l-Phe can eliminate the formation of Phe derivatives from Phe hydroxylase and achieve more specific, sensitive measurement of *OH in biological systems.
Cell Survival↗
Effect of plant phenolics on the formation of the spin-adduct of hydroxyl radical and the DNA strand breaking by hydroxyl radical.
The effect of plant phenolics, including flavonoids and green tea polyphenolics, on hydroxyl radical was examined by a common method using an electron spin resonance (ESR) technique with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trapping agent. The intensity of the ESR signals of DMPO-OH adduct formed by the interaction of DMPO with Fenton reagent was reduced in the presence of each phenolic in a dose-dependent manner. However, the decrease in the intensity of the signals was due partly to the enhanced disappearance of the spin adduct by the phenolics, as has been previously shown. This spin trapping method was unreliable for evaluation of the effect of the phenolics against hydroxyl radical. Hydroxyl radical induced-DNA single-strand breaks may be a better index for evaluation of the activity of the phenolics regarding hydroxyl radical. The effect of the phenolics on DNA single-strand breaks induced by Fenton reagent was examined. While sesamol and esculetin were inhibitory, most polyphenolics, especially (-)-epigallocatechin (EGC) and (-)-epigallocatechin gallate (EGCG), were rather stimulatory. The results indicate that sesamol and esculetin scavenged hydroxyl radical, and EGC and EGCG generated hydroxyl radical under the conditions where hydroxyl radical was generating.
Catechin↗
An HPLC assay of hydroxyl radicals by the hydroxylation reaction of terephthalic acid.
An HPLC assay for hydroxyl radicals is described. The hydroxyl radical was trapped by terephthalic acid (non-fluorescent), and 2-hydroxyl terephthalic acid (fluorescent) was quantitated by HPLC-fluorescence detection. At a terephthalic acid concentration of 4.25 mmol/L, the hydroxyl radical formed in the Fenton reaction was successfully assayed in the concentration range of hydrogen peroxide of 2.5-50 micro mol/L, where the concentration of Fe(II) was 50 micro mol/L. The fluorescence of 2-hydroxy terephthalate was stable at 24 h, and its detection limit by this method was 5 nmol/L (100 fmol).
Calibration↗
Determination of hydroxyl radical by capillary electrophoresis and studies on hydroxyl radical scavenging activities of Chinese herbs.
High-performance capillary electrophoresis (CE) with electrochemical detection (ED) was employed to determine hydroxyl radicals in the Fenton reaction. Hydroxyl radicals can react with salicylic acid to produce 2,3-dihydroxy benzoic acid and 2,5-dihydroxy benzoic acid, which can be analyzed by CE-ED. Based on this principle, hydroxyl radicals were determined indirectly. In a 20 mmol/L phosphate running buffer (pH 7.4), 2,3-dihydroxy benzoic acid and 2,5-dihydroxy benzoic acid would elute simultaneously from the capillary within 6 min. As the working electrode, a 300 microm diameter carbon-disk electrode exhibits good responses at +0.60 V (vs. SCE) for the two analytes. Peak currents of the two analytes are additive. Excellent linearity was obtained in the concentration range from 1.0x10(-3) mol/L to 5.0x10(-6) mol/L for 2,3-dihydroxy benzoic acid. The detection limit (S/N=3) was 2.0x10(-6) mol/L. This method was successfully applied for studying hydroxyl radical scavenging activities of Chinese herbs. It is testified that Apocynum Venetum L., Jinkgo bibola L., Morus alba L. and Rhododendron dauricum L. have strong hydroxyl radical scavenging activities.
Drugs, Chinese Herbal↗
Cimetidine and other H2 receptor antagonists as powerful hydroxyl radical scavengers.
Hydroxyl radical scavengers are able to compete with deoxyribose for the hydroxyl radicals generated in a reaction mixture. We found that the H2 receptor antagonists like cimetidine, burimamide, ranitidine, famotidine and tiotidine except for being good inhibitors in histamine-stimulated gastric acid secretion, were also very powerful hydroxyl radical scavengers. Rate constants for reaction of these drugs with hydroxyl radicals ranged from 7.7 x 10(9) Ms-1 to 14.8 x 10(9) M-1 s-1. These rate constants are much higher than for the well-known hydroxyl radical scavenger mannitol (1.7 x 10(9) M-1 s-1). In this study we investigated which part of the cimetidine molecule might be responsible for its potent hydroxyl radical scavenging activity. Testing fragments of the cimetidine molecule revealed that the guanidine moiety of cimetidine had little hydroxyl radical scavenging activity. However the other part of the molecule, the methylated imidazole with a sulfur and amino group containing side chain appeared to be a powerful hydroxyl radical scavenger.
Cimetidine↗
A spectroscopic study of some of the peptidyl radicals formed following hydroxyl radical attack on beta-amyloid and alpha-synuclein.
There is clear evidence implicating oxidative stress in the pathology of many neurodegenerative diseases. Reactive oxygen species (ROS) are the primary mediators of oxidative stress, and hydrogen peroxide, a key ROS, is generated during aggregation of the amyloid proteins associated with some of these diseases. Hydrogen peroxide is catalytically converted to the aggressive hydroxyl radical in the presence of Fe(II) and Cu(I), which renders amyloidogenic proteins such as beta-amyloid and alpha-synuclein (implicated in Alzheimer's disease (AD) and Parkinson's disease (PD), respectively) vulnerable to self-inflicted hydroxyl radical attack. Here, we report some of the peptide-derived radicals, detected by electron spin resonance spectroscopy employing sodium 3,5-dibromo-4-nitrosobenzenesulfonate as a spin-trap, following hydroxyl radical attack on Abeta(1-40), alpha-synuclein and some other related peptides. Significantly, we found that sufficient hydrogen peroxide was self-generated during the early stages of aggregation of Abeta(1-40) to produce detectable peptidyl radicals, on addition of Fe(II). Our results support the hypothesis that oxidative damage to Abeta (and surrounding molecules) in the brain in AD could be due, at least in part, to the self-generation of ROS. A similar mechanism could operate in PD and some other "protein conformational" disorders.
Alzheimer Disease↗
Scavenging capacity of berry crops on superoxide radicals, hydrogen peroxide, hydroxyl radicals, and singlet oxygen.
The antioxidant activities against superoxide radicals (O(2)(*)(-)), hydrogen peroxide (H(2)O(2)), hydroxyl radicals (OH(*)), and singlet oxygen ('O(2)) was evaluated in fruit juice from different cultivars of thornless blackberries (Rubus sp.), blueberries (Vaccinium spp.), cranberries (Vaccinium macrocarpon Aiton), raspberries (Rubus idaeus L. and Rubus occidentalis L.), and strawberries (Fragaria x ananassa Duch.). Among the different cultivars, juice of 'Hull Thornless' blackberry, 'Earliglow' strawberry, 'Early Black' cranberry, 'Jewel' raspberry, and 'Elliot' blueberry had the highest antioxidant capacity against superoxide radicals (O(2)(*)(-)), hydrogen peroxide (H(2)O(2)), hydroxyl radicals (OH(*)), and singlet oxygen ('O(2)). In general, blackberries had the highest antioxidant capacity inhibition of O(2)(*)(-), H(2)O(2), and OH(*). Strawberry was second best in the antioxidant capacity assay for these same free radicals. With regard to 'O(2) scavenging activity, strawberry had the highest value, while blackberry was second. Cranberries had the lowest inhibition of H(2)O(2) activity. Meanwhile, blueberries had the lowest antioxidant capacity against OH(*) and 'O(2). There were interesting and marked differences among the different antioxidants in their abilities to scavenge different reactive oxygen species. beta-Carotene had by far the highest scavenging activity against 'O(2) but had absolutely no effect on H(2)O(2). Ascorbic acid was the best at inhibiting H(2)O(2) free radical activity. For OH(*), there was a wide range of scavenging capacities from a high of 15.3% with alpha-tocopherol to a low of 0.88% with ascorbic acid. Glutathione had higher O(2)(*)(-) scavenging capacity compared to the other antioxidants.
Beverages↗
Inhibition of firefly bioluminescence by scavengers of singlet oxygen, superoxide radicals and hydroxyl radicals.
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Adenosine Triphosphate↗
The generation of hydrogen peroxide, superoxide radical, and hydroxyl radical by 6-hydroxydopamine, dialuric acid, and related cytotoxic agents.
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Amines↗
Bupivacaine hydrochloride induces muscle fiber necrosis and hydroxyl radical formation-dimethyl sulphoxide reduces hydroxyl radical formation.
We induced acute skeletal muscle necrosis in rats using bupivacaine hydrochloride and found that both 2,5- and 2,3-dihydroxybenzoic acid significantly increased in skeletal muscle. A single administration of dimethyl sulphoxide, a free radical scavenger, significantly lowered concentrations of 2,5- and 2,3-dihydroxybenzoic acid. These results suggest that dimethyl sulphoxide is an effective hydroxyl radical scavenger and may be useful in the treatment of myopathy.
Anesthetics, Local↗
Ferrous ion-EDTA-stimulated phospholipid peroxidation. A reaction changing from alkoxyl-radical- to hydroxyl-radical-dependent initiation.
The stimulatory effect of ferrous salts on the peroxidation of phospholipids can be enhanced by EDTA when the concentration of Fe2+ in the reaction is greater than that of EDTA. Hydroxyl-radical scavengers do not inhibit peroxidation until the concentrations of Fe2+ and EDTA in the reaction are equal. Lipid peroxidation is then substantially initiated by hydroxyl radicals derived from a Fenton-type reaction requiring hydrogen peroxide. Superoxide radicals appear to play some role in the formation of initiating species.
Chemical Phenomena↗
Colorimetric detection of the hydroxyl radical: comparison of the hydroxyl-radical-generating ability of various iron complexes.
An aromatic substrate for hydroxylation by OH radicals has been synthesized. Neither the substrate nor its hydroxylated product binds either iron(III) or iron(II). A spectrophotometric assay based on the hydroxylation of this substrate, N,N'-(5-nitro-1,3-phenylene)bisglutaramide, is described.
Colorimetry↗
Ascorbic acid radical, superoxide, and hydroxyl radical are detected in reperfusion injury of rat liver using electron spin resonance spectroscopy.
Using electron spin resonance (ESR) spectroscopy, we examined whether reactive oxygen intermediates (ROIs) are produced after reperfusion of isolated, perfused rat livers subjected to global ischemia (10, 30, 60, and 90 min of ischemia). A few drops of effluent (200 microliters) flowed from reperfused liver was mixed immediately with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO, final concentration 1 M), and ESR measurements were then performed. Immediately after reperfusion, a small amount of ascorbic acid radical and DMPO-OOH, which was formed by the reaction of superoxide with DMPO, was detected. The amount of ascorbic acid radical peaked immediately after reperfusion, but became hardly detectable at 6 min after reperfusion, irrespective of the period of ischemia. Six minutes after reperfusion, not only DMPO-OOH but also DMPO-OH, which was formed by the reaction of hydroxyl radical with DMPO, was detected. Thereafter, DMPO-OOH and DMPO-OH gradually declined and became hardly detectable at 40 min after reperfusion. In accordance with the duration of ischemia, the concentrations of DMPO-OOH and DMPO-OH were highest after 60 min of ischemia, and then after 30, 90, and 10 min of ischemia in decreasing order. These results directly demonstrate that the production of ROIs occurs at an early stage of reperfusion in ischemic liver and that the amount of ROI production is closely related to the duration of ischemia.
Animals↗
Formation of hydroxyl radicals in biological systems. Does myoglobin stimulate hydroxyl radical formation from hydrogen peroxide?
Incubation of horse-heart oxymyoglobin or metmyoglobin with excess H2O2 causes formation of myoglobin(IV), followed by haem degradation. At the time when haem degradation is observed, hydroxyl radicals (.OH) can be detected in the reaction mixture by their ability to degrade the sugar deoxyribose. Detection of hydroxyl radicals can be decreased by transferrin or by .OH scavengers (mannitol, arginine, phenylalanine) but not by urea. Neither transferrin nor any of these scavengers inhibit the haem degradation. It is concluded that intact oxymyoglobin or metmyoglobin molecules do not react with H2O2 to form .OH detectable by deoxyribose, but that H2O2 eventually leads to release of iron ions from the proteins. These released iron ions can react to form .OH outside the protein or close to its surface. Salicylate and the iron chelator desferrioxamine stabilize myoglobin and prevent haem degradation. The biological importance of .OH generated using iron ions released from myoglobin by H2O2 is discussed in relation to myocardial reoxygenation injury.
Animals↗
Estimation of hydroxyl radical generation by salicylate hydroxylation method in multiple organs of mice exposed to whole-body X-ray irradiation.
Appropriate experimental conditions for the estimation of hydroxyl radical generation by salicylate hydroxylation were determined for multiple organs of X-irradiated mice in vivo. The in vitro experiments showed that there were significant correlations between the salicylic acid (SA) concentration, the amount of 2,3-dihydroxy benzoic acid (2,3-DHBA) and the X-ray exposure dose, and we obtained two linear-regression equations to calculate the amounts of hydroxyl radicals generated by the X-irradiation. The optimum dosage of SA and the appropriate sampling time for in vivo experiments was determined, and significant increases in the ratio of 2,3-DHBA to SA were detected in several organs of mice after X-irradiation. The hydroxyl radical equivalents of the 2,3-DHBA increases were also calculated. Our results clearly demonstrated the usefulness of the salicylate hydroxylation method in estimating hydroxyl radical generation in multiple organs in vivo.
Animals↗
Transformation of barbituric acid into alloxan by hydroxyl radicals: interaction with melatonin and with other hydroxyl radical scavengers.
Barbituric acid (2,4,6-pyrimidinetrione) can be transformed by a non-enzymatic hydroxylation into alloxan (2,4,5,6-pyrimidinetetrone). This transformation can be used as a reaction indicating the formation of hydroxyl radicals (.OH). This conversion was detected using HPLC. Formation of .OH was demonstrated by electron spin resonance (ESR) spectroscopy combined with spin-trapping techniques. It was shown that .OH generated via the Fenton reaction abstracts first a hydrogen atom from barbituric acid (BA) and forms intermediately a paramagnetic derivative of BA. After a second attack by another .OH, the BA radical is transformed into dialuric acid (DA), which autoxidizes via the alloxan radical (.ALX) to ALX. Superoxide radicals (.O2-) are formed during autoxidation of DA and.ALX. They are able to regenerate ferrous ions. As a result, traces of iron salts are capable of catalyzing the conversion of large amounts of BA into ALX. Several scavengers of .OH were tested with regard to their efficiency in preventing the transformation of BA into ALX. Of all the scavengers analyzed, melatonin was shown to be one of the most potent compounds.
Alloxan↗
One-electron reduction of chromium(VI) by alpha-lipoic acid and related hydroxyl radical generation, dG hydroxylation and nuclear transcription factor-kappaB activation.
Reaction of chromium(VI) with alpha-lipoic acid (reduced form, also called 1,2-dithiolane-3-pentanoic acid) generated Cr(V) and hydroxyl radical (*OH) as measured by electron spin resonance and ESR spin trapping. 5,5-Dimethyl-1-pyrroline was used as a spin trapping agent. Catalase inhibited the *OH generation and enhanced the Cr(V) formation. Superoxide dismutase had an opposite effect. H2O2 enhanced the *OH generation and decreased the Cr(V) formation in a dose-dependent manner. Metal chelators, EDTA, diethylenetriaminepentaacetic acid, deferoxamine, and 1, 10-phenanthroline inhibited *OH radical generation in the order of EDTA > 1,10-phenanthroline > DTPA > deferoxamine. Oxygen consumption measurements indicated that molecular oxygen was used to generate *OH radical in the mixture of Cr(VI) and alpha-lipoic acid. H2O2 and superoxide radical (O2-) were involved as reactive intermediates. The *OH radical was generated via Cr(V)-mediated Fenton-like reaction (Cr(V) + H2O2 --> Cr(VI) + OH- + *OH). HPLC measurements show that the *OH radical generated by this reaction is capable of generating 8-hydroxyl-2'-deoxyguanosine from 2-deoxyguanosine. Incubation of Cr(VI) with cultured Jurkat cells resulted in an activation of DNA binding activity of the nuclear factor (NF)-kappaB. Addition of alpha-lipoic acid enhanced the NF-kappaB activation, while the *OH radical scavenger, sodium formate, inhibited it, showing that alpha-lipoic acid enhanced Cr(VI)-induced NF-kappaB activation via free radical reactions. The results indicate that while alpha-lipoic acid is considered to be an antioxidant, it may be a cellular one-electron Cr(VI) reductant and could be involved in the mechanism of Cr(VI)-induced carcinogenesis.
Antioxidants↗