Sensitive determination of nitrotyrosine in human plasma by isocratic high-performance liquid chromatography

Kamisaki, Yoshínori; Wada, Kouichirou; Nakamoto, Kentaro; Kishimoto, Yosuke; Kitano, Masayuki; Itoh, Tadao

doi:10.1016/s0378-4347(96)00202-2

Cited by 89 publications

(60 citation statements)

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“…By contrast, Frost et al 4 have measured by GC/MS a NO 2 Tyr concentration of 64 nmol/L and a mean 3-nitrotyrosine:tyrosine ratio of Ϸ35ϫ1:10 6 in plasma of healthy humans. These values are 23 and 35 times higher than those measured by us, 9,10 respectively, and even 2 times higher than those found by Kamisaki et al 5 Immunohistochemical methods and ELISA 7,8 are frequently used to detect 3-nitrotyrosine. However, the highly divergent values reported for plasma 3-nitrotyrosine ranging between not detectable 7 and 120 nmol/L, 8 strongly suggest that these semiquantitative methods are suffering from serious methodological problems.…”

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confidence: 49%

“…4 The discussion on this subject is incomplete and leaves out other serious problems and solutions. We would like to discuss critically these issues and to evaluate the most relevant techniques 3-10 that are presently available to determine levels of NO 2 Tyr and NO 2 TyrProt, which include MS-based methods, ie LC/MS/MS, 3 GC/MS, 4 and GC/MS/MS, 9,10 HPLC, 5,6 and ELISA. 7,8 Acidification.…”

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confidence: 99%

“…2-9 Avoidance of acidic conditions prevents artifactual formation of 3-nitrotyrosine. [3][4][5][6]9 Regarding NO 2 TyrProt, this means that plasma proteins must be hydrolyzed enzymatically under nonacidic conditions. 6,10 Especially in MS-based methods, in which artifactual formation of NO 2 Tyr from the necessary derivatization may occur, 3,4,9 separation of NO 2 Tyr from Tyr is absolutely required and can be easily achieved by HPLC.…”

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Methodological Considerations on the Detection of 3-Nitrotyrosine in the Cardiovascular System

Tsikas

Schwedhelm

Frölich

2002

Circulation Research

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Reactive nitrogen species (RNS; eg, • NO,• NO 2 , ONOO Ϫ , NO 2 Cl) react preferably with tyrosine (Tyr) and protein-associated tyrosine (TyrProt) to form 3-nitrotyrosine, ie NO 2 Tyr and NO 2 TyrProt, respectively. 1 Therefore, detection of NO 2 Tyr and/or NO 2 TyrProt provides evidence for generation of RNS rather than specifically peroxynitrite (ONOO Ϫ ). 1 Besides this difficulty, Tarpey and Fridovich 2 have recently discussed, in an article published in Circulation Research, the problematic measurement of NO 2 Tyr and NO 2 TyrProt, giving special attention to artifactual formation of NO 2 Tyr and NO 2 TyrProt by acidification of biological samples. This methodological pitfall is very important and well-recognized, 3-10 but it is not the sole methodological problem in 3-nitrotyrosine detection. Tarpey and Fridovich 2 restricted their discussion exclusively to artifactual formation of 3-nitrotyrosine referring to Yi et al 3 and Frost et al,4 whose methods, however, are either not sufficiently sensitive to detect basal NO 2 Tyr plasma levels 3 or yield overestimated values for NO 2 Tyr and NO 2 TyrProt. 4 The discussion on this subject is incomplete and leaves out other serious problems and solutions. We would like to discuss critically these issues and to evaluate the most relevant techniques 3-10 that are presently available to determine levels of NO 2 Tyr and NO 2 TyrProt, which include MS-based methods, ie LC/MS/MS, 3 GC/MS, 4 and GC/MS/MS, 9,10 HPLC, 5,6 and ELISA. 7,8 Acidification. Acidification of Tyr-and TyrProt-containing biological samples convincingly and inevitably results in the artifactual formation of NO 2 Tyr and NO 2 TyrProt. 2-9 Avoidance of acidic conditions prevents artifactual formation of 3-nitrotyrosine. [3][4][5][6]9 Regarding NO 2 TyrProt, this means that plasma proteins must be hydrolyzed enzymatically under nonacidic conditions. 6,10 Especially in MS-based methods, in which artifactual formation of NO 2 Tyr from the necessary derivatization may occur, 3,4,9 separation of NO 2 Tyr from Tyr is absolutely required and can be easily achieved by HPLC. 9 Protein denaturation. When measuring plasma NO 2 Tyr, its release from denaturing NO 2 TyrProt must be avoided by gentle blood drawing, immediate generation of plasma and plasma ultrafiltrate under mild nonacidic conditions (eg, 2°C to 4°C), 9,10 and immediate analysis of the plasma ultrafiltrate. Plasma samples stored at Ϫ80°C should be thawed once only. 9 Interferences in GC/MS. Unlike GC/MS/MS, we 9 have convincingly demonstrated that GC/MS does not possess the necessary specificity to selectively measure plasma NO 2 Tyr and have identified coeluting, NO 2 Tyr-unrelated interfering compounds as a further source for overestimated levels of NO 2 Tyr. Insufficient specificity of GC/MS was confirmed by measuring NO 2 Tyr in 12 older subjects (51Ϯ10 years) whose mean plasma NO 2 Tyr was determined to be 4.5 nmol/L by GC/MS, but only 1.2 nmol/L by GC/MS/MS.Basal plasma levels of NO2Tyr and NO2TyrProt. By means of GC/MS/MS (limit of quantitat...

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confidence: 49%

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confidence: 99%

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Methodological Considerations on the Detection of 3-Nitrotyrosine in the Cardiovascular System

Tsikas

Schwedhelm

Frölich

2002

Circulation Research

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“…The dried extract was dissolved in 100 l ethanol:H 2 O (70:30) by rapid vortexing and then centrifuged at 12,000 ϫ g for 10 minutes. The supernatant was frozen at Ϫ20°C until derivatization and quantitation by HPLC, as previously described (Kamisaki et al, 1996). Derivatization of nitrotyrosine was performed by adding 10 l sodium borate, 0.1 M, pH 8.7 and 10 l 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole (10 mg/ml in ethanol) to 50 l of the ethanol:H 2 O solution containing islet extract and incubating at 60°C for 2 minutes.…”

Section: Nitrotyrosine Assaymentioning

confidence: 99%

Peroxynitrite Is a Mediator of Cytokine-Induced Destruction of Human Pancreatic Islet β Cells

Lakey

Suarez-Pinzon

Strynadka

et al. 2001

Laboratory Investigation

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SUMMARY:The proinflammatory cytokines, interleukin-1␤ (IL-1␤), tumor necrosis factor ␣ (TNF␣), and interferon ␥ (IFN␥), are cytotoxic to pancreatic islet ␤ cells, possibly by inducing nitric oxide and/or oxygen radical production in the ␤ cells. Peroxynitrite, the reaction product of nitric oxide and the superoxide radical, is a strong oxidant and cytotoxic mediator; therefore, we hypothesized that peroxynitrite might be a mediator of cytokine-induced islet ␤-cell destruction. To test this hypothesis we incubated islets isolated from human pancreata with the cytokine combination of IL-1␤, TNF␣, and IFN␥. We found that these cytokines induced significant increases in nitrotyrosine, a marker of peroxynitrite, in islet ␤ cells, and the increase in nitrotyrosine preceded islet-cell destruction. Peroxynitrite mimicked the effects of cytokines on nitrotyrosine formation and islet ␤-cell destruction. L-N G -monomethyl arginine, an inhibitor of nitric oxide synthase, prevented cytokine-induced nitric oxide production but not hydrogen peroxide production, nitrotyrosine formation, or islet ␤-cell destruction. In contrast, guanidinoethyldisulphide, an inhibitor of inducible nitric oxide synthase and scavenger of peroxynitrite, prevented cytokine-induced nitric oxide and hydrogen peroxide production, nitrotyrosine formation, and islet ␤-cell destruction. These results suggest that cytokine-induced peroxynitrite formation is dependent upon increased generation of superoxide (measured as hydrogen peroxide) and that peroxynitrite is a mediator of cytokine-induced destruction of human pancreatic islet ␤ cells. (Lab Invest 2001, 81:1683-1692.

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“…Tissue homogenenates were prepared according to the method described by Kamisaki et al [15]. Briefly, 0.5 g heart tissue was homogenized in 1.5 ml of 50 mM potassium phosphate buffer (pH: 7.2) and then centrifuged for 5 min at 600 g. Following the acid hydrolysation of the precipitate, it was evaporated under nitrogen.…”

Section: Measurement Of Tyrosine Nitrationmentioning

confidence: 99%

Asymmetric Dimethylarginine and 3-Nitrotyrosine Levels of the Heart Tissue aſter Endotoxemia

Kurt¹

2011

Ann Clin Anal Med

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Sensitive determination of nitrotyrosine in human plasma by isocratic high-performance liquid chromatography

Cited by 89 publications

References 13 publications

Methodological Considerations on the Detection of 3-Nitrotyrosine in the Cardiovascular System

Methodological Considerations on the Detection of 3-Nitrotyrosine in the Cardiovascular System

Peroxynitrite Is a Mediator of Cytokine-Induced Destruction of Human Pancreatic Islet β Cells

Asymmetric Dimethylarginine and 3-Nitrotyrosine Levels of the Heart Tissue aſter Endotoxemia

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