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Identifying Biomarkers for Pre-Onset Insulin Resistance Using Mass Spectrometry-Based Metabolomics: Plasma Ceramides are Elevated in Overconditioned Transition Dairy Cows
Metabolomics is a systems biology analytical approach used to study disease phenotypes, an established field in biomedicine that is emerging in the dairy sciences. Parallel with ‘transcriptome’ and ‘proteome’, the comprehensive set of small molecules in biological systems constitutes its ‘metabolome’. Because no single analytical methodology is suited to identify all metabolites, a combination of untargeted and targeted methods (measurement of any molecule that ionizes within a specific mass range, and measurement of specific metabolites, respectively) are employed using gas chromatography (GC) or liquid chromatography (LC), coupled with mass spectrometry (MS). Metabolome screening provides the opportunity to discover molecules (biomarkers) that are associated with disease progression. Similar to overweight monogastrics, overconditioned transition dairy cows experience greater insulin resistance compared with lean cows. Since insulin resistance accelerates NEFA mobilization, overconditioned dairy cattle are at greater risk of developing postpartum disease. Therefore, our objective was to screen the bovine metabolome using GC/MS and LC/MS technologies in search for metabolic phenotypes associated with decreased insulin sensitivity. Our data set included multiparous Holstein cows grouped according to BCS at d-30 prepartum: lean (BCS <3.0; n=10; LEAN) or overconditioned (BCS >4.0, n=11; OVER), with blood samples collected at d-45, -30, -15 and -7, relative to expected calving, and at d4 postpartum. For untargeted detection, derivatized plasma methanol extracts were analyzed using GC/MS. For targeted analysis, plasma chloroform-methanol extracts were analyzed by LC/MS. Following normalization, log transformation, and median-scaling, data were analyzed using ANOVA and cluster analysis. Non-parametric spearman’s correlations were used to evaluate the associations between NEFA and insulin sensitivity to ceramides. Relative to LEAN, OVER had reduced insulin sensitivity and greater NEFA mobilization at d-30, -15 and -7 prepartum (P <0.05). GC/MS and LC/MS analysis detected lactate, urea, glycerol, amino acids, citrate, mono- and disaccharides, saturated and unsaturated free FA, uric acid, vitamin E, nonesterified cholesterol, ceramides, hexylceramides, and others. Of interest, relative to LEAN, OVER had elevated ceramides at d-15, -7, and 4 (e.g. C20:0-ceramide; P <0.05), and mono- and dihexylceramides at d4 (P <0.05). Multiple ceramides were positively associated with NEFA, and negatively associated with insulin sensitivity (e.g. C22:0-, C24:0-, C24:1-, and C26:0-ceramide; P <0.05). Currently used markers (e.g. NEFA and BHBA) have limited predictive power for pre-onset insulin resistance, as they are delayed indications of metabolic stress. Metabolomics may improve our ability to predict prepartum cows at risk of developing greater insulin resistance.
Keywords:
Insulin resistance
Metabolomics
Transition cow