ABSTRACT
BACKGROUND: Due to the close correlation between choline, L-carnitine, betaine and their intestinal microbial metabolites, including trimethylamine (TMA) and trimethylamine N-oxide (TMAO), and creatinine, there has been an increasing interest in the study of these compounds in vivo. METHODS: In this study, a rapid stable isotope dilution (SID)-UHPLC-MS/MS method was developed for the simultaneous determination of choline, L-carnitine, betaine, TMA, TMAO and creatinine in plasma, liver and feces of rats. The method was validated using quality control (QC) samples spiked at low, medium and high levels. Second, we applied the method to quantify the effects of Rosa Roxburghii Tratt juice (RRTJ) on plasma, liver, and fecal levels of choline, L-carnitine, betaine, TMA, TMAO, and creatinine in high-fat diet-induced hyperlipidemic rats, demonstrating the utility of the method. RESULTS: The limits of detection (LOD) were 0.04-0.027 µM and the limits of quantification (LOQ) were 0.009-0.094 µM. The linear ranges for each metabolite in plasma were choline1.50-96 µM; L-carnitine: 2-128 µM; betaine: 3-192 µM; TMA: 0.01-40.96 µM; TMAO: 0.06-61.44 µM and creatinine: 1-64 µM (R2 ≥ 0.9954). The linear ranges for each metabolite in liver were Choline: 12-768 µM; L-carnitine: 1.5-96 µM; betaine: 10-640 µM; TMA: 0.5-32 µM; TMAO: 0.02-81.92 µM and creatinine: 0.2-204.8 µM (R2 ≥ 0.9938). The linear ranges for each metabolite in feces were choline: 1.5-96 µM; L-carnitine: 0.01-40.96 µM; Betaine: 1.5-96 µM; TMA: 1-64 µM; TMAO: 0.02-81.92 µM and Creatinine: 0.02-81.92 µM (R2 ≥ 0.998). The intra-day and inter-day coefficients of variation were < 8 % for all analytes. The samples were stabilized after multiple freeze-thaw cycles (3 freeze-thaw cycles), 24 h at room temperature, 24 h at 4 °C and 20 days at -80 °C. The samples were stable. The average recovery was 89 %-99 %. This method was used to quantify TMAO and its related metabolites and creatinine levels in hyperlipidemic rats. The results showed that high-fat diet led to the disorder of TMAO and its related metabolites and creatinine in rats, which was effectively improved after the intervention of Rosa Roxburghii Tratt juiceï¼RRTJï¼. CONCLUSIONS: A method for the determination of choline, L-carnitine, betaine, TMA, TMAO and creatinine in plasma, liver and feces samples was established, which is simple, time-saving, high precision, accuracy and recovery.
ABSTRACT
Hyperlipidemia endangers human health and has become a significant public health problem. This study aimed to investigate the mechanism of the hypolipidemic effects of Fermented Rosa roxburghii Tratt juice (FRRT) on hyperlipidemic rats and a new hypolipidemic intervention strategy was disclosed. The study revealed 12 weeks FRRT treatment significantly decreased the body weight, total cholesterol (TC), triacylglycerol (TG), low-density lipoprotein cholesterol (LDL-c), while high-density lipoprotein cholesterol (HDL-c) increased. We integrated the 16S rDNA sequencing and metabolomic profiling to evaluate the changes in the gut microbiota and metabolites. Significant changes in microbial composition accompanied marked changes in 56 feces metabolites. The results showed that FRRT could decrease the ratio of Firmicutes to Bacteroidetes, while increase the abundance of some bacterial genera (Prevotella, Paraprevotellaceae_Prevotella, Ruminococcus, Oscillospira). Metabolomics analysis displayed that the metabolisms of bile acid, amino acid and lipid were significantly affected by FRRT. Correlation analysis suggest that the reductions in serum lipids by FRRT are associated with the gut microbial community and their associated metabolites (amino acid metabolites, bile acid metabolites, and lipid metabolites). This study confirmed FRRT could be used as a new dietary and therapeutic strategy to dyslipidemia by improving the gut microbiota dysbiosis, metabolomic disorders and regulating the dyslipidemia. Our study also extended the understanding of the relationship between gut microbiota, metabolites, and lipid-lowering functions.
