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

BARCAROLO, D; ANGELI, E; RIBAS, LE; ADDONA, SM; ORTEGA, HH; HEIN, GJ

LIVESTOCK SCIENCE

ELSEVIER SCIENCE BV

Año: 2022 vol. 257

1871-1413

In dairy cows, the high energy demand and oxygenrequirements 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 heatstress. In this sense, 3-nytrotyrosine (3-NT) has been described as a biomarkerof 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 andliver samples by liquid chromatography?tandem mass spectrometry (LC-MS/MS). Tothis end, during the transition period, 3-NT concentrations were analyzed insamples from dairy cows grouped according to different environmental conditions:cows that gave birth during winter (winter group, WG; n=18) and cows that gavebirth during spring (spring group, SG; n=18). Additionally, other plasmaparameters such as β-hydroxybutyrate acid (BHBA) and glucose concentrations andliver triacylglycerol (TAG) content and aspartate transaminase activity wereexhaustively studied. Samples were collected at 21 (±3) days before the expectedcalving date, and at 7 (±3) and 21 (±3) days after calving. The analysis of thesamples involved a purification step by solid-phase extraction. The method developedwas validated in terms of linearity, specificity, precision, accuracy, recovery,matrix effect, dilution and analyte stability. The lower limit ofquantification of the method was 1.02 ng/mL for plasma, 0.24 mg/L for urine and0.05 mg/kg for liver tissue. Coefficients of variation for the concentrations testedwere < 15% and accuracy was within 85?115%. 3-NT concentration was higher incows of the SG than in those of the WG in the three matrices evaluated.  BHBA and glucose concentrations were higherand lower, respectively, in cows of the SG. BHBA concentration was particularlyhigher in cows of the SG on day 21 postpartum. Liver TAG content and aspartatetransaminase 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/MSfor the quantification of 3-NT in different fluids and liver tissue could be avery useful parameter to evaluate OS in dairy cows during the transition period.