Metabolic Phenotyping Using Mass Spectrometry-Based Metabolomics: A Cross-Sectional Pilot Study of Lean and Overweight Domestic Cats
Overnutrition and sedentary behavior increases adiposity in domestic cats, a consequence of positive energy balance that can be detrimental to health. Adiposity is a risk factor for feline diabetes mellitus, characterized by insulin resistance. Chromatography and mass spectrometry (MS) technologies in the field termed "Metabolomics" may be a means to identify biomarkers for predicting pre-onset insulin resistance. The objective was to evaluate MS, coupled with gas chromatography (GC) or liquid chromatography (LC), techniques to perform metabolic phenotyping in lean and overweight domestic cats in preparation for a large-scale study aimed at identifying predictive biomarkers for feline insulin resistance. To evaluate methods, lean (n=6; ≤4.5 kg) and overweight (n=6; ≥6.5 kg), adult (2-10 y of age), neutered, client-owned male domestic shorthair cats were enrolled in a cross-sectional pilot study. Cats were not diagnosed with diabetes. Plasma samples were collected by a veterinarian following an overnight fast. For GC/MS, plasma metabolites were methanol extracted, derivatized, and analyzed by a GC tandem MS system. For LC/MS, plasma metabolites were chloroform-methanol extracted and analyzed by high pressure LC coupled to an electrospray ionization source of a triple quadrupole tandem MS system. Normalized data were analyzed using Student's t-test. For metabolomics data, statistics were performed on the log of the normalized, median-scaled data. To validate analytical methods, total free fatty acids (FA) and glucose were determined by colorimetry. Mean weights and ages for lean and overweight cats were 3.9 ±0.5 and 7.1 ±1.0 kg (P <0.05), and 4.2 ±2.6 and 6.8 ±2.4 y of age (P >0.10), respectively. Untargeted GC/MS analysis detected 40 metabolites including lactate, urea, glycerol, amino acids, citrate, saccharides, saturated and unsaturated free FA, uric acid, vitamin E, and cholesterol. Overweight cats had a nearly two-fold increase in palmitic acid, stearic acid, and oleic acid (P <0.05), results validated by an 83% increase in total free FA (P <0.05). In overweight cats, glucose levels were increased by 38% (MS; P <0.05) and a similar non-significant numerical increase when determined by colorimetry. Cholesterol, vitamin E, and glycerol were elevated in overweight cats (P <0.05). Targeted LC/MS analysis detected 28 sphingolipids including ceramides, monohexylceramides, and dihexylceramides. Of interest, palmitoyl-linked ceramide level was increased 80% in overweight cats (P <0.05); elevated plasma ceramides are associated with insulin resistance progression in humans. In overweight cats, GC/MS and LC/MS methodologies revealed metabolite phenotypes similar to patterns observed in diabetic humans.
Feline diabetes mellitus