Accurate quantification of basal plasma levels of 3-nitrotyrosine and 3-nitrotyrosinoalbumin by gas chromatography–tandem mass spectrometry

Measurement of 3-nitro-l-tyrosine (NO2Tyr) and protein-related 3-nitro-l-tyrosine in human plasma is associated with numerous methodological problems which result in highly divergent basal plasma levels often ranging within two orders of magnitude. Recently, we have described an interference-free GC–tandem MS-based method for NO2Tyr which yielded the lowest basal plasma NO2Tyr levels reported thus far. This method was extended to quantify protein-associated 3-nitrotyrosine and in particular 3-nitrotyrosinated albumin (NO2TyrALB) in human plasma. NO2TyrALB and albumin (ALB) were extracted from plasma by affinity column extraction and digested enzymatically at neutral pH. 3-Nitro- l-[2H3]tyrosine was used as internal standard. In plasma of 18 healthy young volunteers the molar ratio of NO2TyrALB to albumin-derived tyrosine (TyrALB), i.e. NO2TyrALB/TyrALB, was determined to be 1.55±0.54×1:106 (mean±SD). The plasma concentration of NO2TyrALB was estimated as 24±4 nM. The NO2Tyr plasma levels in these volunteers were determined to be 0.73±0.53 nM. In the same volunteers, NO2TyrALB/TyrALB, NO2TyrALB and NO2Tyr were measured 15 days later and the corresponding values were determined to be 1.25±0.58×1:106, 25±6 nM and 0.69±0.16 nM. For comparison, NO2Tyr and NO2TyrALB were measured in six plasma samples from healthy volunteers by GC–MS and GC–tandem MS. Different values were found for NO2Tyr, i.e. 5.4±2.8 versus 2.7±1.5 nM, and comparable values for NO2TyrALB/TyrALB, i.e. 0.5±0.2×1:106 versus 0.4±0.1×1:106, by these methods. The ratio of the values measured by GC–MS to those measured by GC–tandem MS were 2.9±3.1 for NO2Tyr and 1.2±0.2 for NO2TyrALB/TyrALB. The present GC–tandem MS method provides accurate values of NO2Tyr and NO2TyrALB in human plasma.