Metabolomics analysis of urine from rats given long-term high-protein diet using ultra-high-performance liquid chromatography-mass spectrometry

Previous studies have indicated high-protein diet (HPD) promotes weight loss and improves metabolic parameters, but most of these studies have focused on the impact of short-term, long-term effects remain unclear. In this study, male Wistar rats were fed two diets for 88 weeks: normal control diet (NCD, 20.5% of energy as protein) or HPD (30.5% of energy as protein). At 88 weeks intervention, compared to NCD rats, HPD rats had lower fat tissue and higher skeletal muscle to body weight ratio, but there were no significantly differences in body weight and food intake. To explore the mechanism underlying metabolism and diet, we further collected rat urine samples at 16, 40, 64 and 88 weeks diet treatment and analyzed metabolomics profiles using ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS). Partial least squares-discriminant analysis (PLS-DA) scores plots from ESI- or ESI+ model revealed a perfect separation between two diets at four time points. We identified 11 dramatically different metabolites (with VIP cut-off value > 1) in HPD, including 3 up-regulated and 8 down-regulated. And these 11 metabolites were identified as effective biomarkers, which were significantly related to HPD-induced metabolism related outcomes (fat tissue and skeletal muscle to body weight ratio). Our results provided vital information regarding metabolism in long-term HPD and more importantly, a few potentially promising metabolites were firstly identified which may related to metabolic responses.