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

Background

Trimethylamine 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).

Methods

Serum 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.

Results

Retention 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.

Conclusions

Overall, 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.