Identification and Distribution of the Dietary Antioxidant Ergothioneine in Humans and Animal Models Combining NMR, RANSY, and MS Methods

Ergothioneine (ERG), a unique, naturally occurring antioxidant of dietary origin, is gaining increasing attention due to its crucial roles in human health and diseases. Despite its significance, ERG is rarely detected in biospecimens by mass spectrometry (MS) and, to date, has not been characterized by nuclear magnetic resonance (NMR) spectroscopy, two widely used analytical techniques in metabolomics. In this study, we investigated human plasma, whole blood (WB), and red blood cells (RBC), as well as mouse blood and tissues, combining NMR, LC-MS, and ratio analysis techniques. The results demonstrate the ability of simple 1D ¹H NMR to routinely identify and quantify ERG in various biological samples. The levels of ERG vary widely and depend on the type of biological sample, with human RBC exhibiting remarkably high concentrations, often exceeding 1.5 mM. The average levels of ERG in human plasma, WB, and RBC were in ratios of 1:70:140, respectively. Moreover, ERG levels showed a linear correlation between plasma and WB (<i>R</i>² = 0.59), plasma and RBC (<i>R</i>² = 0.75), and WB and RBC (<i>R</i>² = 0.98). In mice, ERG levels exhibit a distinct whole-body distribution, with average levels in the mouse skeletal muscle, brain, heart, kidney, and liver in ratios of 0:1:10:35:45, respectively. The demonstration of surprisingly high levels of ERG in biological samples using ¹H NMR opens new avenues for its analysis using both NMR and MS methods to explore its roles in human health and diseases, as part of routine global or targeted metabolomics studies.