Methodological Considerations on the Detection of 3-Nitrotyrosine in the Cardiovascular System
Authors: Dimitrios Tsikas, Edzard Schwedhelm, Jürgen C. Frölich
Journal: Circulation Research (2002)

To the Editor: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. 1Therefore, 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. 4The 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. 7,8cidification.Acidification of Tyr-and TyrProt-containing biological samples convincingly and inevitably results in the artifactual formation of NO 2 Tyr and NO 2 TyrProt. 2-95][6]9 Regarding NO 2 TyrProt, this means that plasma proteins must be hydrolyzed enzymatically under nonacidic conditions. 6,10Especially 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. 9rotein 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. 9nterferences 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 quantitation [LOQ], 0.125 nmol/L), we 9 determined a mean basal NO 2 Tyr level of 2.8 nmol/L in young and 1.2 nmol/L in older subjects.The findings by Yi et al, 3 who could not detect basal NO 2 Tyr by LC/MS/MS (LOQ, 4.4 nmol/L), are highly supportive of our results. 9Kamisaki et al 5 have measured plasma NO 2 Tyr at 31 nmol/L by a less specific and less sensitive (LOQ, 6 nmol/L) HPLC method with fluorescence detection, which is therefore not useful to determine basal plasma levels in humans.We 10 determined by GC/MS/MS a mean 3-nitrotyrosine:tyrosine ratio of Ϸ1ϫ1:10 6 for circulating 3-nitrotyrosinated albumin.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