1H-NMR Based Metabolomics Identifies New Predictive Urinary Biomarkers and Highlights the Pathobiology of Ketosis in Periparturient Dairy Cows

The objective of this study was to investigate metabolic fingerprints in the urine of pre-ketotic cows as well as during and after the occurrence of disease and to identify newly predictive and diagnostic urine biomarkers that can be used to distinguish cows with ketosis from healthy controls (CON). In this study, proton nuclear magnetic resonance based metabolomics was performed to analyze urine samples from 6 cows with ketosis and 20 CON cows, at -8 wks, -4 wks, disease diagnosis, +4 wks, and +8 wks relative to parturition. Univariate (t-test or Wilcoxon Mann Whitney test: significance P<0.05) and multivariate analyses (Permutation test: P < 0.05) and biomarker analysis (Empirical P<0.05) were used to select metabolite-sets for the noninvasive prediction and diagnosis of ketosis. All data analyses were performed using MetaboAnalyst 3.0. A total of 14, 21, 14, 2, and 2 differential metabolites between the two groups were identified at -8 wks, -4 wks, disease diagnosis, +4 wks, and +8 wks, respectively. VIP plots ranked the most significant differential metabolites, which contributed to the onset and progression of ketosis. Specifically, concentrations of pantothenic acid (i.e., 3.42 vs 1.44 at -8wks, P = 0.04; 2.9 vs 1.26 at -4wks, P = 0.01; 3.04 vs 1.54 at the disease wk, P = 0.03; unit: μM/mM Creatinine) and myo-inositol (i.e., 31.89 vs 10.73 at -8wks, P < 0.01; 26.35 vs 9.17 at -4 wks, P < 0.01; 21.85 vs 13.26 at the disease wk, P = 0.04) were persistently greater in the urine of both preketotic and ketotic cows when compared with CON cows. Urinary concentrations of urea were lower in preketotic and ketotic cows versus the CON group at -8 wks (41.79 vs 146.55, P < 0.01), -4 wks (44.51 vs 144.3, P =0.04), and the disease wk (43.77 vs 194.57, P < 0.01). Moreover, two promising biomarker models were identified for prediction of ketosis with the excellent level of sensitivity and specificity. Overall, multiple urine metabolite alterations were identified in preketotic, ketotic, and postketotic cows which could be used as potential screening biomarkers as well as to better understand the pathobiology of disease, and to develop new preventive treatments in the future.