Methodological Considerations on the Detection of 3-Nitrotyrosine in the Cardiovascular System
To the Editor:
Reactive nitrogen species (RNS; eg, •NO, •NO2, ONOO−, NO2Cl) react preferably with tyrosine (Tyr) and protein-associated tyrosine (TyrProt) to form 3-nitrotyrosine, ie NO2Tyr and NO2TyrProt, respectively.1 Therefore, detection of NO2Tyr and/or NO2TyrProt provides evidence for generation of RNS rather than specifically peroxynitrite (ONOO−).1 Besides this difficulty, Tarpey and Fridovich2 have recently discussed, in an article published in Circulation Research, the problematic measurement of NO2Tyr and NO2TyrProt, giving special attention to artifactual formation of NO2Tyr and NO2TyrProt 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 Fridovich2 restricted their discussion exclusively to artifactual formation of 3-nitrotyrosine referring to Yi et al3 and Frost et al,4 whose methods, however, are either not sufficiently sensitive to detect basal NO2Tyr plasma levels3 or yield overestimated values for NO2Tyr and NO2TyrProt.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 techniques3–10⇓⇓⇓⇓⇓⇓⇓ that are presently available to determine levels of NO2Tyr and NO2TyrProt, 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 NO2Tyr and NO2TyrProt.2–9⇓⇓⇓⇓⇓⇓⇓ Avoidance of acidic conditions prevents artifactual formation of 3-nitrotyrosine.3–6,9⇓⇓⇓⇓ Regarding NO2TyrProt, this means that plasma proteins must be hydrolyzed enzymatically under nonacidic conditions.6,10⇓ Especially in MS-based methods, in which artifactual formation of NO2Tyr from the necessary derivatization may occur,3,4,9⇓⇓ separation of NO2Tyr from Tyr is absolutely required and can be easily achieved by HPLC.9
Protein denaturation. When measuring plasma NO2Tyr, its release from denaturing NO2TyrProt 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 −80°C should be thawed once only.9
Interferences in GC/MS. Unlike GC/MS/MS, we9 have convincingly demonstrated that GC/MS does not possess the necessary specificity to selectively measure plasma NO2Tyr and have identified coeluting, NO2Tyr-unrelated interfering compounds as a further source for overestimated levels of NO2Tyr. Insufficient specificity of GC/MS was confirmed by measuring NO2Tyr in 12 older subjects (51±10 years) whose mean plasma NO2Tyr 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), we9 determined a mean basal NO2Tyr 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 NO2Tyr by LC/MS/MS (LOQ, 4.4 nmol/L), are highly supportive of our results.9 Kamisaki et al5 have measured plasma NO2Tyr 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. We10 determined by GC/MS/MS a mean 3-nitrotyrosine:tyrosine ratio of ≈1×1:106 for circulating 3-nitrotyrosinated albumin. By contrast, Frost et al4 have measured by GC/MS a NO2Tyr concentration of 64 nmol/L and a mean 3-nitrotyrosine:tyrosine ratio of ≈35×1:106 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 ELISA7,8⇓ are frequently used to detect 3-nitrotyrosine. However, the highly divergent values reported for plasma 3-nitrotyrosine ranging between not detectable7 and 120 nmol/L,8 strongly suggest that these semiquantitative methods are suffering from serious methodological problems.
We fully agree with Tarpey and Fridovich2 on the necessity to use reliable analytical methods to assess quantitatively the effects of RNS on biomolecules in the cardiovascular system. We are convinced that presently only GC/MS/MS provides the required specificity for NO2Tyr and NO2TyrProt, and LC/MS and LC/MS/MS are best suited to investigate the selectivity of RNS for TyrProt.12 MS-based analytical methods are highly recommended in the l-arginine/•NO pathway.11
- ↵Tarpey MM, Fridovich I. Methods of detection of vascular reactive species: nitric oxide, superoxide, hydrogen peroxide, and peroxynitrite. Circ Res. 2001; 89: 224–236.
- ↵Frost MT, Halliwell B, Moore KP. Analysis of free and protein-bound nitrotyrosine in human plasma by a gas chromatography/mass spectrometry method that avoids nitration artifacts. Biochem J. 2000; 345: 453–458.
- ↵Shigenaga MK, Lee HH, Blount BC, Christen S, Shinego ET, Yip H, Ames BN. Inflammation and NOx-induced nitration: assay for 3-nitrotyrosine by HPLC with electrochemical detection. Proc Natl Acad Sci U S A. 1997; 94: 3211–3216.
- ↵Khan J, Brennand DM, Bradley N, Gao B, Bruckdorfer R, Jacobs M. 3-Nitrotyrosine in the proteins of human plasma determined by an ELISA method. Biochem J. 1998; 330: 795–801.
- ↵Tsikas D, Gutzki FM, Frölich JC. Quantitation of members of the l-arginine/nitric oxide (•NO) pathway by gas chromatography-mass spectrometry. Recent Res Dev Anal Biochem. In press.
- ↵Viner RI, Ferrington DA, Williams TD, Bigelow DJ, Schöneich C. Protein modification during biological aging: selective tyrosine nitration of the SERCA2a isoform of the sarcoplasmic Ca2+-ATPase in skeletal muscle. Biochem J. 1999; 340: 657–669.