Free radical detection methods

Comments on Contemporary Methods of Oxygen and Nitrogen Free Radical Detection... 

Free radicals may be reaction intermediates in biological systems in more situations than are presently recognized. However, progress in detecting such species by ESR has been relatively slow. ESR is a very sensitive technique for free radical detection and characterization. It can be used to investigate very low concentrations of radicals provided that they are stable enough for their presence to be detected. For unstable radicals special techniques have to be employed [901], One of these methods is... 

Laser photolysis of a precursor may also be used to generate a reagent. In a crossed-beam study of the D + FI2 reaction [24], a hypertliennal beam of deuterium atoms (0.5 to 1 eV translational energy) was prepared by 248 mn photolysis of DI. This preparation method has been widely used for the preparation of molecular free radicals, both in beams and in experiments in a cell, with laser detection of the products. Laser photolysis as a method to prepare reagents in experiments in which the products are optically detected is fiirtlier discussed below. 

Detection of an Intermediate. In many cases, an intermediate cannot be isolated but can be detected by IR, NMR, or other spectra. The detection by Raman spectra of NOj was regarded as strong evidence that this is an intermediate in the nitration of benzene (see 11-2). Free radical and triplet intermediates can often be detected by ESR and by CIDNP (see Chapter 5). Free radicals [as well as radical ions and EDA complexes] can also be detected by a method that does not rely on spectra. In this method, a doublebond compound is added to the reaction mixture, and its fate traced. One possible result is cis-trans conversion. For example, cis-stilbene is isomerized to the trans isomer in the presence of RS- radicals, by this mechanism ... 

A number of the techniques that have been employed have the ability to directly monitor free-radical species either in vitro or in vivo [predominantly those involving electron spin resonance (e.s.r.) spectroscopy]. However, since many physiologically relevant free radicals have extremely short half-lives (e.g. 10 s for OH), the majority of the methods utilized detect products arising from their reactions with chemical components present (i.e. indirect methods). These indirect methods for... 

Up to date, several experimental techniques have been developed which are capable of detecting some of these particles under ordinary thermodynamic conditions. One can use these methods to keep track of transformations of the particles. For instance, it is relevant to mention here the method of electron paramagnetic resonance (EPR) with sensitivity of about 10 particles per cm [IJ. However, the above sensitivity is not sufficient to study physical and chemical processes developing in gaseous and liquid media (especially at the interface with solids). Moreover, this approach is not suitable if one is faced with detection of particles possessing the highest chemical activity, namely, free radicals and atoms. As for the detection of excited molecular or atom particles... 

In the following, an overview of the experimental approaches is presented, including the production and detection methods of free radicals and the techniques for studying free radical photodissociation in the molecular beam. The photochemistry of the free radical systems investigated recently will then be discussed in detail. 

REMPI provides high detection sensitivity for free radicals similar to that of LIF.4 In the REMPI method, one or more photons typically from a focused laser radiation initially excite the free radicals to an intermediate excited electronic state. The radicals are further excited and ionized by another photon in the same laser pulse (one-color REMPI) or by a photon of different wavelength from another laser beam overlapping in space and time... 

The spectroscopy methods such as LIF and REMPI are utilized not only to detect the free radicals as discussed above, but also to directly measure the internal state distributions of the photoproducts in the photodissociation of free radicals. In this approach, the photochemistry is carried out in the free radical beam under single-collision conditions with well-defined... 

Since its discovery in the late 1960s [41,42], the method of spin trapping has been extensively used for the detection and identification of short-lived free radicals in chemistry, biology, and medicine studies [41-50]. The method is based on the scavenging of radicals, P by a spin trap, leading to the formation of a spin adduct with higher stability, typically a nitroxide radical. Nitroso and... 

In earlier studies the in vitro transition metal-catalyzed oxidation of proteins and the interaction of proteins with free radicals have been studied. In 1983, Levine [1] showed that the oxidative inactivation of enzymes and the oxidative modification of proteins resulted in the formation of protein carbonyl derivatives. These derivatives easily react with dinitrophenyl-hydrazine (DNPH) to form protein hydrazones, which were used for the detection of protein carbonyl content. Using this method and spin-trapping with PBN, it has been demonstrated [2,3] that protein oxidation and inactivation of glutamine synthetase (a key enzyme in the regulation of amino acid metabolism and the brain L-glutamate and y-aminobutyric acid levels) were sharply enhanced during ischemia- and reperfusion-induced injury in gerbil brain. 

Another important ESR method of NO detection is based on the interaction of NO with the stable radical phenyl-4,4,5,5-tetramethylimidazoline-l-oxyl (PTIO) and its derivatives carboxy-PTIO or trimethylammonio-PTIO [100]. It is interesting that in this assay NO reduces stable free radical PTIO to another stable free radical PTI and the N02 radical, and the reaction can be monitored by both a decrease in the ESR PTIO spectrum and an increase in the ESR PTI spectrum [101]. 

Hermann (2000) described a rapid automated method involving generation of a known amount of free radicals and the detection of the excess by photochemiluminescence. Test kits are available for determination of total water-soluble antioxidants, fat-soluble antioxidants and ascorbic acid. A luminometric method was developed for the determination of antioxidative activity and was subsequently applied to anthocyanin and betalaine colour concentrates (Kuchta et al., 1999). The method involved quantification of the interruption in luminescence from the hydrogen peroxide-horse radish peroxidase-luminol system in the presence of antioxidants. 

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