Age-related peculiarities of change in content of free radical oxidation products in muscle during stress

The age-dependent peculiarities of stimulation of free radical processes in subcellular fractions of skeletal muscle of rats subjected to long-term immobilization stress were studied in order to improve knowledge about changes of muscular tissue during ontogenesis. It is found that adult animals do not show accumulation of proteins carbonyls, TBA-reactive substances, and Schiff bases in subcellular fractions of the thigh muscle when immobUized. Long-term immobilization causes apparent manifestation of oxidative stress only in mitochondrial fraction in pubertal rats. Mitochondrial oxidative stress defense systems are sufficiently effective, however, direction of pathways of free radical oxidation carbonyl products catabolism alters in the cytoplasm of myocytes in old rats under long-term immobilization conditions.     

Comparison of serum free thyroxine measurements by chemiluminescence and equilibrium dialysis

A study of 239 patients compared free thyroxine (FT4) measurements made by equilibrium dialysis (ED) with measurements made using the Magic Lite FT4 chemiluminescence (Cl) immunoassay (Ciba Corning Immunodiagnostics). Patient groups: 41 normals; 27 hyperthyroid; 29 hypothyroid; 37 sick euthyroid; 10 chronic renal failure (CRF) and 25 pregnant patients; 13 oestrogen; 10 heparin; 12 salicylate; and 9 dilantin-treated patients; 3 lipaemic; 5 haemolysed; 6 hyperbilirubinaemic patients; 6 low thyroid binding protein (TBP) and 6 high TBP level patients. The two assays gave comparable results in most groups. Both assays tended to give elevated values in heparinized patients but FT4–ED results were more obviously affected. Pregnant patients and women on oral oestrogen had higher mean values with FT4–ED. In both assays the sick euthyroid and CRF patients had mean FT4 values similar to healthy euthyroid patients; the range of values in sick euthyroid and CRF patients was similar in both assays but wider than in healthy euthyroid patients. A supplemental study of 81 unselected acutely ill patients using FT4–Cl alone confirmed the wider range of values to be anticipated in sick euthyroid patients.     

NMDA excitotoxicity and free radical generation in rat brain homogenates: Application of a chemiluminescence assay

NMDA, the specific agonist of glutamate gated ion channels permeable to calcium, is implicated as a causal factor in the pathogenesis of several neurobiological disorders such as stroke, seizures, ischemia, and chronic neurodegenerative disease. On the other hand, evidence on the roles of oxidative mechanisms involved in NMDA-induced neurotoxicity is accumulating. In this study, we have used chemiluminescence measurements as an easy, rapid and sensitive assay to investigate the effects of NMDA and oxidative stress on brain cell vulnerability. Rat brain homogenates were incubated with increasing concentrations of glutamate and NMDA. Production of reactive oxygen species was followed by single photon emission measurements using the specific enhancers luminol and lucigenin. Increases in emission were observed at excitotoxic concentrations of glutamate and NMDA. Other parameters of oxidative stress such as diene conjugates, TBARS and carbonyl groups were also investigated. Our results indicated that chemiluminescence measurements may be used to study involvement of oxidative stress in neurotoxicity.     

Direct and indirect oxidations by peroxynitrite, neither involving the hydroxyl radical

A new mechanism (Mechanism III) that combines features of mechanisms suggested earlier (Goldstein and Czapski, Inorg. Chem. 34:4041–4048; 1995; Pryor, Jin, and Squadrito Proc. Natl. Acad. Sci. USA 91:11173–11177; 1994) is proposed for oxidations by peroxynitrite. In Mechanism III, oxidations by peroxynitrite can take place either directly by ground-state peroxynitrous acid, ONOOH, or indirectly by ONOOH*, where ONOOH* is an activated form of peroxynitrous acid. In the direct oxidation pathway the reaction is first order in peroxynitrite and first order in substrate, and the oxidation yield approaches 100%. In the indirect oxidation pathway the reaction is first order in peroxynitrite and zero order in substrate. In the presence of sufficient concentrations of a substrate that reacts by the indirect oxidation pathway, about 50–60% of the ONOOH directly isomerizes to nitric acid, and about 40–50% of the ONOOH is converted into ONOOH*. Thus, the oxidation yields by the indirect pathway will not exceed 40–50%, and there will always be a residual yield of nitrate even in the presence of very high concentrations of the substrate. Competitive inhibition studies with various free radical scavengers showed that in some cases these scavengers have no effect on oxidation yields. In others, only partial inhibition was observed, far less than that predicted from to the known rate constants for the reactions of these scavengers with the hydroxyl radical. There are some cases where the extent of inhibition correlates well with the known rate constants of the reactions of these scavengers with hydroxyl radical; nevertheless, even in these cases, the involvement of hydroxyl radicals in indirect oxidations by peroxynitrite is ruled out on the basis of kinetics and oxidation yields. Thus, direct oxidations by peroxynitrite are explained in terms of ONOOH, and indirect oxidations in terms of ONOOH*, and substrates can react by one or both of these pathways.     

Effect of sulphite on the oxidative metabolism of human neutrophils: Studies with lucigenin- and luminol-dependent chemiluminescence

To assess the effect of sulphite on the oxidative metabolism of human neutrophils, chemiluminescence (CL) measurements were performed using lucigenin and luminol as chemiluminigenic probes. Lucigenin-dependent CL was used for measuring superoxide anion (O) production, and luminol-dependent CL was used for determination of myeloperoxidase (MPO)-connected processes. With sulphite concentrations of 0.01 to 1 mmol/L, resting neutrophils showed an up to sixfold increase of lucigenin-dependent CL, but only a 1.9-fold increase of luminol-dependent CL. Subsequent stimulation of sulphite-treated neutrophils with phorbol myristate acetate (PMA) (soluble stimulant) or zymosan (particulate stimulant) resulted in an additional significant increase of lucigenin-dependent CL compared to stimulated control cells, whereas luminol-dependent CL increased slightly by 0.01 mmol/L sulphite and decreased then continuously. Sulphite concentrations above 1 mmol/L decreased both lucigenin- and luminol-dependent CL of resting and PMA- or zymosan-stimulated neutrophils. Lucigenin-dependent CL of sulphite-treated and subsequently stimulated neutrophils was strongly inhibited by extracellularly added superoxide dismutase, whereas luminol-dependent CL was markedly reduced by the MPO inhibitor azide. The intracellular activity of MPO in neutrophils stimulated with PMA in the presence of sulphite (2 mmol/L) was reduced by 55%. Sulphite (0.1 mmol/L) also inhibited strongly the activity of MPO in a cell-free system. These results indicate that micromolar concentrations of sulphite exert a stimulating effect on the O production of neutrophils extracellularly, but have an inhibitory effect on MPO-catalysed reactions intracellularly.     

Experimental evidence of the reciprocal oxidation of Bovine Serum Albumin and Linoleate in aqueous solution,initiated by HO free radicals

An investigation of radiation-induced oxidation of aqueous bovine serum albumin (BSA) in the presence of linoleate (LH) at pH 10.5 has been carried out in order to better understand the respective oxidative processes involved in both lipid and protein phases. Solutions containing BSA (15 μmol L⁻¹) and linoleate (15–600 μmol L⁻¹) below the critical micellar concentration (cmc = 2000 μmol L⁻¹), have been irradiated by γ-rays (¹³⁷Cs) at radiation doses ranging from 10 to 400 Gy (dose rate 9.5 Gy min⁻¹). It can be noticed that, in the absence of BSA, the main hydroperoxides formed from HO^•-induced linoleate oxidation below the cmc, do not exhibit a conjugated dienic structure. This was also verified in the presence of BSA. Selected chemical markers of oxidation have been monitored: non-conjugated dienic hydroperoxides and conjugated dienes (without hydroperoxide function) for linoleate oxidation, and carbonyl groups for BSA oxidation. We have shown that for the lowest linoleate concentration (15 μmol L⁻¹) in the presence of BSA (15 μmol L⁻¹), the formation of conjugated dienes was not observed, meaning that LH was not exposed to HO^• radicals attack. However, non-conjugated dienic lipid hydroperoxides were simultaneously detected, indicating that LH was secondarily oxidised by BSA oxidised species. Moreover, the oxidation of linoleate was found to be enhanced by the presence of BSA. For the highest linoleate concentration (600 μmol L⁻¹), the expected protection of BSA by LH was not observed, even if LH monomers were responsible for the total scavenging of HO^• radicals. In this latter case, the formation of non-conjugated dienic lipid hydroperoxides was lower than expected. Those results showed that BSA was not oxidised by the direct action of HO^• radicals but was undergoing a secondary oxidation by non-dienic lipid hydroperoxides and/or lipid radical intermediates, coming from the HO^•-induced linoleate oxidation.     

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.     

Protection of membrane cholesterol by sphingomyelin against free radical-mediated oxidation

Although the free radical-mediated oxidation of free cholesterol (FC) is critical in the generation of regulatory sterols and in atherogenesis, the physiological regulation of this process is poorly understood. We tested the hypothesis that sphingomyelin (SM), a major phospholipid of cell membranes, which is closely associated with FC, protects FC against oxidation, because of its unique structure, and affinity to the sterol. We employed phosphatidylcholine (PC) liposomes containing varying amounts of SM, and either radioactive FC or a fluorescent analog, dehydroergosterol (DHE), and determined the oxidative decay of the sterol in presence of 2,2′-azo-bis(2-amidinopropane hydrochloride) (AAPH). Incorporation of 25 mol% of SM in the liposomes inhibited the oxidation of FC or DHE by up to 50%. This inhibition was specific for SM among phospholipids, and was abolished by sphingomyelinase treatment. SM was not degraded during the oxidation reaction, and its effect was not dependent on the nature of the oxidizing agent, because it also inhibited sterol oxidation by FeSO₄/ascorbate, and by cholesterol oxidase. These studies show that SM plays a physiological role in the regulation of cholesterol oxidation by free radicals.     

On the chemiluminescence in the oxidation of tetravalent uranium to the uranyl ion by dimethyldioxirane

The reaction of the tetravalent uranium [U(IV)] with dimethyldioxirane (DMD) in strongly acidic water–acetone solutions is accompanied by chemiluminescence (CL) in the visible (Vis) and infra‐red (IR) regions. At least three independent reaction pathways are involved in the U(IV)–DMD oxidation: the first entails the non‐chemiluminescent oxidation of U(IV) to the uranyl ion (UO₂²⁺); the second involves the catalytic decomposition of DMD by U(IV) to afford singlet oxygen, as manifested by its characteristic IR‐CL; and in the third process, slow Vis‐CL (510–540 nm) is emitted, following DMD consumption. Copyright © 2002 John Wiley & Sons, Ltd.     

Enhanced chemiluminescence in the oxidation of luminol and an isoluminol cortisol conjugate by hydrogen peroxide in reversed micelles

The chemiluminescent oxidation of luminol and an isoluminol cortisol conjugate (ABICOR) by hydrogen peroxide has been studied in cetyltrimethylammonium bromide (CTAB) reversed micelles in octane-chloroform (1 : 1). The maximum chemiluminescence intensity of both compounds is dependent on the initial concentrations of the H₂O₂ and substrates, the pH value of the micelle polar phase and the H₂O/CTAB ratio. The optimum pH ranged from 8.5 to 9.5. Under comparable conditions, the chemiluminescence intensity for luminol was 15-fold higher than for the ABI-COR conjugate. A mechanism of oxidation of the substrates in reversed micelles is proposed and the possible mechanisms of inhibition by the substrate and oxidant is discussed.     

Inhibition and enhancement by organic compounds of luminol-KIO4-H2O2 chemiluminescence.

The effects of 36 organic compounds on luminol-KIO(4)-H(2)O(2) chemiluminescence (CL) were studied. It was found that most of the tested compounds could inhibit or enhance the CL intensity. The activities of such inhibitors or enhancers were related to the pH of the CL system and the number and position of functional groups such as -OH and -NH(2) on aromatic rings. The mechanism of the CL inhibition and enhancement was considered. Based on the CL inhibition or enhancement, the possibility of analytical applications was explored. The results demonstrated that numerous compounds were detectable at the ng/mL level using the CL system.     

Uric acid oxidation by peroxynitrite: multiple reactions, free radical formation, and amplification of lipid oxidation

Uric acid has been considered to be an efficient scavenger of peroxynitrite but the reaction between urate and peroxynitrite has been only partially characterized. Also, previous studies have indicated that urate may increase peroxynitrite-mediated oxidation of low density lipoprotein (LDL). Here, we examined the reaction between urate and peroxynitrite by combining kinetic, oxygen consumption, spin trapping, and product identification studies; in parallel, we tested the effect of urate upon peroxynitrite-mediated lipid oxidation. Our results demonstrated that urate reacts with peroxynitrite with an apparent second order rate constant of 4.8 x 10(2) M(-1). s(-1) in a complex process, which is accompanied by oxygen consumption and formation of allantoin, alloxan, and urate-derived radicals. The main radical was identified as the aminocarbonyl radical by the electrospray mass spectra of its 5, 5-dimethyl-l-pyrroline N-oxide adduct. Mechanistic studies suggested that urate reacts with peroxynitrous acid and with the radicals generated from its decomposition to form products that can further react with peroxynitrite anion. These many reactions may explain the reported efficiency of urate in inhibiting some peroxynitrite-mediated processes. Production of the aminocarbonyl radical, however, may propagate oxidative reactions. We demonstrated that this radical is likely to be the species responsible for the effects of urate in amplifying peroxynitrite-mediated oxidation of liposomes and LDL, which was monitored by the formation of lipid peroxides and thiobarbituric acid-reactive substances. The aminocarbonyl radical was not detectable during urate attack by other oxidants and consequently it is unlikely to be responsible for all previously described prooxidant effects of uric acid.     

Oxidative modification of low density lipoprotein (LDL) by activated human monocytes and the cell lines U937 and HL60

Summary Human peripheral blood monocytes, upon activation, have the capacity to oxidize low density lipoprotein (LDL) and render the LDL toxic to cultured cells. Previous studies by our laboratory indicate that this process is mediated by free radicals in that it can be prevented by addition of free radical scavengers and antioxidants during the incubation of monocytes with LDL. Here we report that optimal modification of LDL by monocytes was influenced by media composition. In the absence of added metal ions, oxidation was distinctly dependent on the concentration of monocytes as well as LDL concentration. Exposure of monocytes to lipopolysaccharide or stimulation of phagocytosis by opsonized zymosan resulted in marked enhancement of LDL oxidation compared to other activating agents. After exposure to activated monocytes, lipid oxidation products in the supernatant were found both in a high molecular weight fraction containing LDL (>30 000 Daltons) and in a lipoprotein-free, low molecular weight fraction (<30 000 Daltons), yet only the high molecular weight, LDL-containing fraction was toxic to target cells. In addition, human myelomonocytic cell lines U937 and HL60 were shown to mediate oxidation of LDL. As with monocytes, exposing these cells to opsonized zymosan caused the level of LDL oxidation to be significantly enhanced. These findings offer further insight into the mechanisms of monocyte-mediated oxidation of lipoproteins and will facilitate studies investigating the role of monocyte-modified LDL in tissue injury. This project was funded by grants form the American Heart Association-Northeast Ohio Affiliate and the National Institutes of Health, Bethesda, MD (HL-29582).     

Spectroscopic characterization of a Ni-organic radical intermediate in the aerobic oxidation of methanol catalyzed by a Ni(II)(polyoximate) complex

In the aerobic oxidation of methanol catalyzed by a Ni(II)(TRISOX) complex [H₃TRISOX = tris(1-propan-2-onyl oxime)amine], an intermediate is observed spectroscopically. The intensities of both the UV-Vis absorption and electron paramagnetic resonance (EPR) spectra associated with this intermediate maximize during the time period of maximum formaldehyde production, and decrease as the methanol oxidation activity decreases. The UV-Vis spectrum has prominent features at 350, 420, and 535 nm. The EPR spectrum is centered at g = 2.00 and shows splittings of 28 ± 5 G. Both of these spectra are consistent with characterization of the intermediate as including one or more iminoxyl radicals derived from the oximate groups of the TRISOX ligand. Spectroscopic features very similar to those in the air-oxidized intermediate are observed in electrochemically oxidized samples, suggesting that the electrochemically generated complex will be a useful model for the intermediate observed during catalytic turnover. The crystal structure of a Ni(II) complex with an intermediate protonation state of the ligand, [Ni(II)₂(H₂TRISOX)₂(μ₂:η¹-ONO₂)](NO₃) · (CH₃CN) · 5(H₂O), 4, has been structurally characterized. Comparison to the previously reported [Ni(II)(H₂TRISOX)(CH₃CN)]₂(ClO₄)₂, 3, shows that bis(μ-oximate) dimers can form either with or without an additional bridging ligand. Addition of the nitrato bridge decreases the Ni-Ni distance from 3.5752(13) Å in 3 to 3.2014(4) Å in 4. It is intriguing to note that the reactions catalyzed by the Ni(II)(TRISOX) complex, the net transfer of two hydrogen atoms from an alcohol or amine substrate to O₂, are the same reactions catalyzed by several different metalloenzymes that also incorporate both a redox active metal and a redox active organic component in their active sites.     

Bioactive products of phospholipid oxidation: isolation, identification, measurement and activities

There is considerable evidence to suggest that oxidation of LDL plays an important role in atherogenesis. Polyunsaturated fatty acids, a major oxidative target, are present as phospholipids in the outer core of the lipoprotein particle. Studies from several laboratories have shown an increase in the levels of phospholipid oxidation products in atherosclerotic lesions and of antibodies to oxidized phospholipids in mice and humans with lesions. Significantly, phospholipid oxidation products have been demonstrated (in vitro) to selectively activate processes in vascular wall cells that may contribute to atherogenesis. This review discusses activities, methods for isolation, identification and measurement of bioactive phospholipids. Past studies suggest that defined and relatively simple current technologies allow identification of bioactive phospholipid oxidation products and measurement of their levels in tissue.     

Inhibition of oxidative catabolism and oxy free radical production as a possible mode of cytokinin control of foliar senescence

Increment of levels of both lipoxygenase and superoxide-dismutase which typically increase with age or upon light deprivation is significantly lowered by cytokinin treatment. It is suggested that cytokinin on the one hand by means of inhibition of polyunsaturated fatty acid catabolism prevents incipient formation of free radicals while on the other serves as a scavenger of radicals already formed.     

复方中药提取物对大鼠不同状态下脑组织自由基代谢影响的研究

目的:探讨复方中药提取物对大鼠脑组织自由基代谢和抗氧化系统能力的影响机制。方法:选取70只健康Wistar大鼠,随机分为2组(n=35):对照组(N)和服药组(M)。适应性喂养1周,服药组大鼠连续服用8周的复方中药提取物,9周后将2组大鼠分别于安静状态、定量负荷、力竭运动即刻、力竭恢复12 h、力竭恢复24 h状态下处死。分别测定上述2组大鼠在不同功能状态下脑组织中丙二醛(MDA)含量,谷胱甘肽过氧化物酶(GSH-PX)、还原性谷胱甘肽(GSH)、超氧化物歧化酶(SOD)、总抗氧化能力(T-AOC)活性。结果:五种状态下,服药组MDA含量均显著低于对照组,GSH-PX、GSH、SOD、T-AOC活性均不同程度的高于对照组。结论:复方中药提取物可降低不同功能状态下大鼠脑组织中的MDA含量,提高其脑组织GSH-PX、GSH、SOD、T-AOC活性。     

Degradation of alkali lignin by two ascomycetes and free radical scavenging activity of the products

Biodegradation and bioconversion of extracted alkali lignin was performed under varying concentrations of carbon and nitrogen sources, by two potential Ascomycetes ligninolytic fungus isolated from soil. Fungus, F10 was identified as Aspergillus flavus, while APF4 as Emericella nidulans based upon closed similarity with their morphology and high homology in 18S rRNA gene sequences. The alkali lignin degradation was checked in term of disappearance of lignin content and colority. Selected fungus, degraded 19–41.6% of alkali lignin (0.25%, w/v) within 21 days of incubation and reduced the colority up to 14.4–21%. The activity of ligninolytic enzymes was periodically checked. During alkali lignin degradation manganese peroxidase (13.31?U/ml), lignin peroxidase (13.73?U/ml) and laccase (0.05?U/ml) activities were observed (at highest level). The alkali lignin degradation products and functional group changes in degraded lignin were analysed through gas chromatography-mass spectroscopy (GC-MS) and solid state ¹³C-NMR spectroscopy, respectively. The functional group modifications in alkali lignin moiety, alter its biochemical property, thus fungal mediated modified alkali lignin was further tested for reactive free radical scavenging potential with respect to hydroxyl, nitric oxide and superoxide radicals. Results demonstrate that the alkali lignin undergo degradation in studied nutritional conditions (high-carbon low nitrogen) and consequently increase its free radical scavenging activity up to 1–18%.