Analysis of two intestinal bacterial metabolites (trimethylamine N-oxide and phenylacetylglutamine) in human serum samples of patients with T2DM and AMI using a liquid chromatography tandem mass spectrometry method

Abstract

BackgroundTrimethylamine N-oxide (TMAO) and phenylacetylglutamine (PAGln) are associated with acute myocardial infarction (AMI) and type 2 diabetes mellitus (T2DM). This study developed and validated a simple method firstly for simultaneously quantifying serum TMAO and PAGln using liquid chromatography-tandem mass spectrometry (LC–MS/MS). MethodsSerum samples from patients with T2DM, AMI, and healthy subjects were analyzed using a new LC–MS/MS method to evaluate TMAO and PAGln levels. Statistical analyses were performed to evaluate TMAO and PAGln distributions among different groups. ResultsRetention and separation of the two metabolites were achieved within 5 min. For both analytes, the assay was linear in a 0.02–10 µg/mL range, with >0.99 average linear correlation coefficients, and quantification limit values of approximately 0.010 µg/mL. The average recoveries of TMAO and PAGln were 96.3 % and 96.4 %, respectively. The intra-run and total coefficient variations were 3.5–4.8 % and 3.9–5.7 % respectively for TMAO; and 4.0–5.1 % and 4.6–6.3 % respectively for PAGln. TMAO and PAGln showed a moderate correlation (P < 0.001) and their levels in patients with T2DM were significantly higher than those in healthy individuals (P < 0.001). TMAO levels were higher in patients with T2DM than in patients with AMI (P < 0.01). Patients with AMI had higher PAGln levels than healthy individuals (P < 0.05). After adjusting for sex and age, the top tertile of PAGln was positively correlated with T2DM and AMI while that of TMAO was positively correlated with T2DM. ConclusionsOverall, a robust isotope dilution LC–MS/MS method was established, which may be beneficial for assessing the association between two metabolites with AMI and T2DM.