Simultaneous determination of choline, L-carnitine, betaine, trimethylamine, trimethylamine N-oxide, and creatinine in plasma, liver, and feces of hyperlipidemic rats by UHPLC-MS/MS.

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 juice（RRTJ）. 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.

Tribuloside acts on the PDE/cAMP/PKA pathway to enhance melanogenesis, melanocyte dendricity and melanosome transport.

ETHNOPHARMACOLOGICAL RELEVANCE: Tribuloside, a natural flavonoid extracted from Chinese medicine Tribulus terrestris L., has shown potent efficacy in treating various diseases. In China, the fruits of Tribulus terrestris L. have long been utilized for relieving headache, dizziness, itchiness, and vitiligo. Water-based extract derived from Tribulus terrestris L. can enhance melanogenesis in mouse hair follicle melanocytes by elevating the expression of α-melanocyte stimulating hormone (α-MSH) and melanocortin-1 recepter (MC-1R). Nevertheless, there is a lack of information regarding the impact of tribuloside on pigmentation in both laboratory settings and living organisms. AIM OF THE STUDY: The present research aimed to examine the impact of tribuloside on pigmentation, and delve into the underlying mechanism. MATERIALS AND METHODS: Following the administration of tribuloside in human epidermal melanocytes (HEMCs), we utilized microplate reader, Masson-Fontana ammoniacal silver stain, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) to measure melanin contents, dendrite lengths, melanosome counts; L-DOPA oxidation assay to indicate tyrosinase activity, Western blotting to evaluate the expression of melanogenic and associated phosphodiesterase (PDE)/cyclic adenosine monophosphate (cAMP)/cyclic-AMP dependent protein kinase A (PKA) pathway proteins. A PDE-Glo assay to verify the inhibitory effect of tribuloside on PDE was also conducted. Additionally, we examined the impact of tribuloside on the pigmentation in both zebrafish model and human skin samples. RESULTS: Tribuloside had a notable impact on the production of melanin in melanocytes, zebrafish, and human skin samples. These functions might be attributed to the inhibitory effect of tribuloside on PDE, which could increase the intracellular level of cAMP to stimulate the phosphorylation of cAMP-response element binding (CREB). Once activated, it induced microphthalmia-associated transcription factor (MITF) expression and increased the expression of tyrosinase, Rab27a and cell division cycle protein 42 (Cdc42), ultimately facilitating melanogenesis, melanocyte dendricity, and melanin transport. CONCLUSION: Tribuloside acts on the PDE/cAMP/PKA pathway to enhance melanogenesis, melanocyte dendricity, and melanosome transport; meanwhile, tribuloside does not have any toxic effects on cells and may be introduced into clinical prescriptions to promote pigmentation.