Abstract
Trimethylamine (TMA) is a gut microbial metabolite—rendered by the enzymatic cleavage of nutrients containing a TMA moiety in their chemical structure. TMA can be oxidized as trimethylamine N-oxide (TMAO) catalyzed by hepatic flavin monooxygenases. Circulating TMAO has been demonstrated to portend a pro-inflammatory state, contributing to chronic diseases such as cardiovascular disease and chronic kidney disease. Consequently, TMAO serves as an excellent candidate biomarker for a variety of chronic inflammatory disorders. The highly positive correlation between plasma TMAO and urine TMAO suggests that urine TMAO has the potential to serve as a less invasive biomarker for chronic disease compared to plasma TMAO. In this study, we validated a method to simultaneously measure urine TMA and TMAO concentrations by liquid chromatography–mass spectrometry (LC/MS). Urine TMA and TMAO can be extracted by hexane/butanol under alkaline pH and transferred to the aqueous phase following acidification for LC/MS quantitation. Importantly, during sample processing, none of the nutrients with a chemical structure containing a TMA moiety were spontaneously cleaved to yield TMA. Moreover, we demonstrated that the acidification of urine prevents an increase of TMA after prolonged storage as was observed in non-acidified urine. Finally, here we demonstrated that TMAO can spontaneously degrade to TMA at a very slow rate.
Highlights
Trimethylamine N-oxide (TMAO), the oxidative product of trimethylamine (TMA), which is dependent on gut microbiota, has gained wide interest due to its pro-atherogenic and pro-thrombotic properties [1,2,3,4]
Plasma/serum and urine trimethylamine N-oxide (TMAO) can be simultaneously measured with other TMA-related metabolites such as choline, carnitine, γ-butyrobetaine and crotonobetaine by liquid chromatography–mass spectrometry (LC/mass spectrometry (MS))/MS after precipitation of protein with methanol [10]
An increased injection volume can lead to TMAO signal saturation, which is not conducive to linearity throughout the detectable range
Summary
Trimethylamine N-oxide (TMAO), the oxidative product of trimethylamine (TMA), which is dependent on gut microbiota, has gained wide interest due to its pro-atherogenic and pro-thrombotic properties [1,2,3,4]. Apart from the clinical relevance to cardiovascular disease, TMAO has been reported to contribute to chronic kidney disease progression and has been linked to type II diabetes mellitus, a key feature of the human metabolic syndrome [6,7,8,9]. As evidenced by these studies, plasma TMAO has become an important biomarker for determining the human health status in a variety of disease states. When compared to serum or plasma, urine is much easier to collect as it does not require venipuncture
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