Application of an optimized and validated LC-MS/MS method for the quantification of free 3-nitrotyrosine in plasma, urine and liver tissue of lactating dairy cows

In dairy cows, the high energy demand and oxygen requirements during the transition period could increase oxidative stress (OS), impairing the challenging metabolic adaptation needed during this period. Also, OS and health could be affected by some environmental conditions such as heat stress. In this sense, 3-nytrotyrosine (3 NT) has been described as a biomarker of OS. Therefore, the aim of this study was to develop and validate a fast, reliable method for the accurate determination of free 3-NT in blood, urine and liver samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS). To this end, during the transition period, 3-NT concentrations were analyzed in samples from dairy cows grouped according to different environmental conditions: cows that gave birth during winter (winter group, WG; n = 18) and cows that gave birth during spring (spring group, SG; n = 18). Additionally, other plasma parameters such as beta-hydroxybutyrate acid (BHBA) and glucose concentrations and liver triacylglycerol (TAG) content and aspartate transaminase activity were exhaustively studied. Samples were collected at 21 (+/- 3) days before the expected calving date, and at 7 (+/- 3) and 21 (+/- 3) days after calving. The analysis of the samples involved a purification step by solid-phase extraction. The method developed was validated in terms of linearity, specificity, precision, accuracy, recovery, matrix effect, dilution and analyte stability. The lower limit of quantification of the method was 1.02 ng/mL for plasma, 0.24 mg/L for urine and 0.05 mg/kg for liver tissue. Coefficients of variation for the concentrations tested were < 15% and accuracy was within 85-115%. 3-NT concentration was higher in cows of the SG than in those of the WG in the three matrices evaluated. BHBA and glucose concentrations were higher and lower, respectively, in cows of the SG. BHBA concentration was particularly higher in cows of the SG on day 21 postpartum. Liver TAG content and aspartate transaminase activity were also higher in cows of the SG on the postpartum days. These results suggest that the methodology developed and validated by LC-MS/MS for the quantification of 3-NT in different fluids and liver tissue could be a very useful parameter to evaluate OS in dairy cows during the transition period.

Automated summary

In dairy cows, oxidative stress during the transition period can be influenced by environmental conditions such as heat stress. A study developed and validated a method for accurate determination of 3-NT in blood, urine, and liver samples in dairy cows using LC-MS/MS. Results showed higher 3-NT concentrations in cows that gave birth during spring, along with higher BHBA and lower glucose concentrations. Liver TAG content and aspartate transaminase activity were also higher in cows that gave birth during spring.