Determination of nitrotyrosine concentrations in plasma samples of diabetes mellitus patients by four different immunoassays leads to contradictive results and disqualifies the majority of the tests

BACKGROUND: In the course of type 2 diabetes mellitus, insulin resistance has a severe impact on endothelial function leading to decreased synthesis of nitric oxide (NO). Postprandial hyperglycemia leads to the generation of reactive oxygen species, which counteracts the beneficial NO effects. NO and superoxide combine very fast in solution to form peroxynitrite, which is a potent protein-oxidizing agent. The peroxynitrite concentrations can be indirectly monitored by the detection of nitrotyrosine residues in proteins, reflecting the extent of damage caused by oxidative stress. METHODS: Four commercially available nitrotyrosine-specific immunoassays were evaluated by parallel measurement of nitrotyrosine in 224 serum samples derived from 16 patients with type 2 diabetes and 12 healthy controls (13 male and 15 female, age: 33+/-11 years) following a standardized meal. RESULTS: The available ELISA tests were not applicable for nitrotyrosine determination in human plasma samples due to technical issues and implausible results. However, a competitive luminescence assay was able to provide sufficient sensitivity and lead to clinically meaningful results in our test samples. CONCLUSIONS: All three ELISA methods were disqualified and conclusions previously derived from clinical experiments using these tests should be carefully reconsidered or reconfirmed. In the absence of a liquid tandem chromatography-mass spectrometry reference method, the luminescence test appears to be the method of choice for determination of nitrotyrosine in human plasma.