<i>In Vitro </i>and <i>In Vivo</i> Measurement of pH and Thiols by EPR-Based Techniques

Khramtsov, Valery V.; Grigor'ev, I. A.; Foster, Margaret A.; Lurie, David John

doi:10.1089/152308604773934431

Cited by 57 publications

(63 citation statements)

References 63 publications

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“…The total XO and xanthine dehydrogenase activity, however, is 10-fold above this value. During ischemia, xanthine levels rise from near zero to values on the order of 10 -100 M. Tissue or blood sulfhydryl compound concentrations are typically in the range of 0.5-2 mM under normal physiological conditions (54,55). Thus, pharmacological levels of GTN (10 -25 M) or ISDN (100 -250 M) in the heart could generate about 1-2 nM/s NO from xanthine oxidoreductase-mediated reduction under anaerobic conditions, which is comparable to maximal NO production from tissue NO synthases.…”

Section: Inorganic Nitrite Generation From Xo-mediated Organicmentioning

confidence: 99%

Xanthine Oxidase Catalyzes Anaerobic Transformation of Organic Nitrates to Nitric Oxide and Nitrosothiols

Cui

Li³

et al. 2005

Journal of Biological Chemistry

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Organic nitrates have been used clinically in the treatment of ischemic heart disease for more than a century. Recently, xanthine oxidase (XO) has been reported to catalyze organic nitrate reduction under anaerobic conditions, but questions remain regarding the initial precursor of nitric oxide (NO) and the link of organic nitrate to the activation of soluble guanylyl cyclase (sGC). To characterize the mechanism of XO-mediated biotransformation of organic nitrate, studies using electron paramagnetic resonance spectroscopy, chemiluminescence NO analyzer, NO electrode, and immunoassay were performed. The XO reducing substrates xanthine, NADH, and 2,3-dihydroxybenz-aldehyde triggered the reduction of organic nitrate to nitrite anion (NO 2 ؊ ). Studies of the pH dependence of nitrite formation indicated that XO-mediated organic nitrate reduction occurred via an acid-catalyzed mechanism. In the absence of thiols or ascorbate, no NO generation was detected from XO-mediated organic nitrate reduction; however, addition of L-cysteine or ascorbate triggered prominent NO generation. Studies suggested that organic nitrite (R-O-NO) is produced from XO-mediated organic nitrate reduction. Further reaction of organic nitrite with thiols or ascorbate leads to the generation of NO or nitrosothiols and thus stimulates the activation of sGC. Only flavin site XO inhibitors such as diphenyleneiodonium inhibited XO-mediated organic nitrate reduction and sGC activation, indicating that organic nitrate reduction occurs at the flavin site. Thus, organic nitrite is the initial product in the process of XO-mediated organic nitrate biotransformation and is the precursor of NO and nitrosothiols, serving as the link between organic nitrate and sGC activation. Organic nitrates such as glyceryl trinitrate (GTN)1 and isosorbide dinitrate (ISDN) have been used clinically in the treatment of cardiovascular disease for more than a century.The beneficial effects of organic nitrates result primarily from their vasodilator properties, which have been attributed to their metabolic product, nitric oxide (NO) (1-4). In 1998, the Nobel Prize was awarded for the discovery of "nitric oxide as a signaling molecule in the cardiovascular system." It is well known that organic nitrates require enzymatic metabolism to generate bioactive NO; however, the molecular mechanisms of their biotransformation have not been fully elucidated.It has been reported that flavoenzymes play important roles in the bioactivation of organic nitrates (5-9), and the flavin inhibitor diphenyleneiodonium chloride (DPI) has shown strong inhibition of the bioactivation of organic nitrate (6, 10). Xanthine oxidase (XO) is a ubiquitous flavoenzyme in mammalian cells. XO plays a variety of important roles in normal physiology and disease, and it is the only identified enzyme that can mediate organic nitrate and inorganic nitrate/nitrite reduction (11)(12)(13)(14). It has been reported that XO can catalyze organic nitrate reduction to nitrite anion (NO 2 Ϫ ) under anaerobic conditions (13), ...

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Section: Inorganic Nitrite Generation From Xo-mediated Organicmentioning

confidence: 99%

Xanthine Oxidase Catalyzes Anaerobic Transformation of Organic Nitrates to Nitric Oxide and Nitrosothiols

Cui

Li³

et al. 2005

Journal of Biological Chemistry

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“…Thus, thiol-targeted labels R6-R8 (Fig. 2) provide a tool for probing local electrostatics of the biological macromolecules (45,46,68,93). Figure 3A demonstrates the pH dependence of the nitrogen hyperfine splitting of the imidazolidine methanethiosulfonate label R7 (X = SSO 2 CH 3 ).…”

Section: Methods Of Thiol Detectionmentioning

confidence: 99%

“…Figure 3A demonstrates the pH dependence of the nitrogen hyperfine splitting of the imidazolidine methanethiosulfonate label R7 (X = SSO 2 CH 3 ). An attachment of this label to the protein sulfhydryl groups allows the probing of the local electrostatics by EPR (45). The use of highfield EPR with pH-sensitive methanethio-sulfonate labels opens a new possibility for the investigation of site-directed spin-labeled proteins, as was demonstrated for the label R7 (X = SSO 2 CH 3 ) (68,93).…”

Section: Methods Of Thiol Detectionmentioning

confidence: 99%

“…Figure 7 demonstrates the kinetics of the increase of the peak intensity, I m , of the biradical R 2 SSR 2 measured by L-band EPR spectroscopy in rat blood. The initial part of the kinetics in a range of <400 s allows a linear approximation, which can be used to estimate the GSH concentration (45). (R 2 SSR 2 freely penetrates erythrocyte membranes reacting preferably with GSH, while its reaction with the protein sulfhydryls is extremely slow (47)).…”

Section: Noninvasive Measurement Of Thiols Using Disulfide R 2 Ssr 2 mentioning

confidence: 99%

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Use of Electron Paramagnetic Resonance Spectroscopy to Evaluate the Redox StateIn Vivo

Swartz

Khan

Khramtsov

2007

Antioxidants & Redox Signaling

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The aim of this article is to provide an overview of how electron paramagnetic resonance (EPR) can be used to measure redox-related parameters in vivo. The values of this approach include that the measurements are made under fully physiological conditions, and some of the measurements cannot be made by other means. Three complementary approaches are used with in vivo EPR: the rate of reduction or reactions of nitroxides, spin trapping of free radicals, and measurements of thiols. All three approaches already have produced unique and useful information. The measurement of the rate of decrease of nitroxides technically is the simplest, but difficult to interpret because the measured parameter, reduction in the intensity of the nitroxide signal, can occur by several different mechanisms. In vivo spin trapping can provide direct evidence for the occurrence of specific free radicals in vivo and reflect relative changes, but accurate absolute quantification remains challenging. The measurement of thiols in vivo also appears likely to be useful, but its development as an in vivo technique is at an early stage. It seems likely that the use of in vivo EPR to measure redox processes will become an increasingly utilized and valuable tool. OVERVIEWThe goal of this article is to indicate how the unique capabilities of EPR can be used very effectively to follow redox status/events in vivo. This will be done in the context of a general overview and two more detailed complementary mini-reviews of the current status of the use of in vivo EPR to measure free radicals and thiols. The article also seeks to delineate areas where developments are needed and the potential pathways to achieve them.The ability to measure redox status and redox processes in vivo is very attractive for several reasons. It has been clearly established that redox-active species are intrinsically involved in many physiological and pathophysiological processes. The roles of these species include being essential intermediates in key physiological reactions and, in many cases, being directly involved in the causal chain leading to pathology. The redox state is an important parameter for many key events in cell signaling (13). Also, therapeutic approaches for many types of pathophysiology involve redox reactions (84).Whereas many useful insights can be achieved by measurements in model systems and cell cultures, the complexity of redox processes and physiology makes it difficult to understand fully the redox processes that occur in vivo without making direct measurements with the intact NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript system. The dynamics of the vascular system, metabolism in different organs, and the complex web of oxidants and antioxidants, cannot be modeled readily. There are few techniques that are capable of making such measurements in vivo, but fortunately EPR can follow many of the most important aspects of these complex processes.Some of the redox-active species are free radicals, and for these, EPR is t...

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“…The functionally-enhanced NRs for EPR detection of corresponding biologically relevant molecules were developed, namely pH-, SH-, and NO-sensitive spin probes. 35,36 The main types of the nitroxides discussed in this chapter are shown in Scheme 16.1. …”

Section: Nitroxyl Radicalsmentioning

confidence: 99%

Functional in vivo EPR Spectroscopy and Imaging Using Nitroxide and Trityl Radicals

Khramtsov¹,

Zweíer²

2010

Stable Radicals

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In Vitro and In Vivo Measurement of pH and Thiols by EPR-Based Techniques

Cited by 57 publications

References 63 publications

Xanthine Oxidase Catalyzes Anaerobic Transformation of Organic Nitrates to Nitric Oxide and Nitrosothiols

Xanthine Oxidase Catalyzes Anaerobic Transformation of Organic Nitrates to Nitric Oxide and Nitrosothiols

Use of Electron Paramagnetic Resonance Spectroscopy to Evaluate the Redox StateIn Vivo

Functional in vivo EPR Spectroscopy and Imaging Using Nitroxide and Trityl Radicals

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