Reactive oxygen species methods

Degli Esposti M. Measuring mitochondrial reactive oxygen species. Methods 2002 26 335-340. 

Spin trapping methods were also used to show that when carotenoid-P-cyclodextrin 1 1 inclusion complex is formed (Polyakov et al. 2004), cyclodextrin does not prevent the reaction of carotenoids with Fe3+ ions but does reduce their scavenging rate toward OOH radicals. This implies that different sites of the carotenoid interact with free radicals and the Fe3+ ions. Presumably, the OOH radical attacks only the cyclohexene ring of the carotenoid. This indicates that the torus-shaped cyclodextrins, Scheme 9.6, protects the incorporated carotenoids from reactive oxygen species. Since cyclodextrins are widely used as carriers and stabilizers of dietary carotenoids, this demonstrates a mechanism for their safe delivery to the cell membrane before reaction with oxygen species occurs. 

In this section we will illustrate the application of ESR methods in order to detect and identify polymer fragments, reactive intermediates, as well as reactive oxygen species that attack the polymer structure. Some examples are selected from studies of polymer degradation performed at the University of Detroit Mercy laboratory. 

Gomes, A., Fernandes, E. and Lima, J. L. F. C. (2005). Fluorescence probes used for detection of reactive oxygen species. J. Biochem. Biophys. Methods 65, 45-80. 

A possible pro-/antioxidant effect of C60 was tested in the presence of fluorescence probe 2, 7 -dichlorofluorescein. This compound is oxidized by reactive oxygen species (ROS), primarily by hydrogen peroxide (H202) with the transformation into the fluorescent oxidized form (A = 385 nm A = 535 nm). The method is supposed to be one of the most direct techniques of ROS indication (Bass et al., 1983 Clothier et al., 2002 Lautraite et al., 2003). 

Selected entries from Methods in Enzymology [vol, page(s)] Assay with 2,2-dithiobisnitrobenzoic acid method, 233, 381-382 pH effects, 233, 384-385 formed by incubation with dithiothrei-tol, labeling, 233, 409-410 protein thiol assay, 234, 273-274 labeling, 233, 414 reactivity with free radicals and reactive oxygen species, 233, 405 reaction with ferrylmyoglobin, 233, 196-197. 

Multireference character, complete active space calculation, 12, 29 Multivariate analysis analytical methods, 624 SIMPLISMA program, 624, 702 Mutations, DNA oxidative damage, 616 Myeloperoxidase, tyrosyl free radicals, 610 Myocardial ischemia, reactive oxygen species in blood, 612... 

Hanson, K. M., and Clegg, R. M. 2005. Two-photon fluorescence imaging and reactive oxygen species detection within the epidermis. Methods Mol. Biol. 289 413-22. 

M. Fontecave and C. Gerez, Methods Enzymol, 2002, 348 (Protein Sensors and Reactive Oxygen Species, Part B), 21. 

A method based on inhibition of ABAP-induced oxidation of a fluorogenic probe, 2, 7-dichlorodihydrofluorescein diacetate (H2-DCF-DA), commonly used for detection of production of reactive oxygen species, has been proposed (V2). Oxidation of the probe can be monitored either spectrophotometrically or fluori-metrically. A drawback of the method is the necessity of hydrolysis of the ester because only the deesterified from can be oxidized. The rate of hydrolysis depends on the hydrolytic activity of the samples measured. A modification consisting of chemical hydrolysis of H2-DCF-DA before measurement is impractical because 2,7 -dichlorodihydrofluorescein oxidizes rapidly. 

Another method of neurotoxicant entry into the CNS is by exploitation of a compromised blood-brain barrier. Aluminum itself compromises the blood-brain barrier and reactive species, both directly and indirectly, can open the barrier. Blood-borne inflammation, possibly through the release of reactive oxygen species, is widely known to create a leaky blood-brain barrier. This allows inflammatory cells, endotoxins, and neurotoxicants to enter the CNS. Inflammation within the brain itself also affects blood-brain barrier permeability, an aspect that potentially links psychological stress to increased susceptibility to neurotoxicants. It has been shown that inflammatory responses are induced in animals under stressful conditions, such as electrical shock, restraint, or adverse social interactions. 

Cash TP, Pan Y, Simon MC. Reactive oxygen species and cellular oxygen sensing. Free Radic. Biol. Med. 2007 43 1219-1225. Gorlach A, Kietzmann T. Superoxide and derived reactive oxygen species in the regulation of hypoxia-inducible factors. Methods Enzymol. 2007 435 421-446. 

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