Orthogonal label and label-free dual pretreatment for targeted profiling of neurotransmitters in enteric nervous system

Highlights

• NT metabolome was summarized by the distribution of functional groups of compounds.

• Ingenious design of orthogonal dual pretreatment in sample preparation for NT metabolome profiling.

• Capturing changes of NT metabolome developed an understanding of neuroimmune-mediated inflammation.

• Divide-and-conquer concept is of great significance in the targeted metabolomics.

Abstract

Neurotransmitter (NT) abnormalities in the enteric nervous system have been reported as crucial roles to regulate the intestinal inflammation and gut immune homeostasis. Capturing quantitative changes at the NT metabolome provides an opportunity to develop an understanding of neuroimmune-mediated inflammation. Given the wide diversity of chemical characterizations in the NTs, only partial coverage of the NT metabolome can be simultaneously quantified in a single-run analysis. Herein, we summarized the distribution of functional groups of compound entries in the NT metabolome. Based on this information, an orthogonal dansyl-labeling and label-free dual pretreatment approach was separately designed to target phenol and amine NTs and tertiary amine and choline NTs. By combining the dansyl-labeled and unlabeled NTs within a single vial, a comprehensive and practical approach was optimized for quantifying high coverage of NT metabolome in a single-run analysis on the reversed-phase C18 column. Method validation indicated good linearity with correlation coefficients (R²) > 0.99, intra- and interday accuracy with relative error < ±20%, and precision with relative standard deviations of ≤15%. With this method, we could simultaneously monitor the alterations of cholines, amines, amino acids, tryptophan and phenylalanine biological pathways in dextran sulphate sodium-induced colitis mice. The measured levels of NT metabolome ranged from 0.0007 to 3.540 μg/mg in intestinal contents and 0.013–154.54 μg/mL in serum samples. The NT metabolism was disrupted by colitis, characterized by the changed NT levels in serum and excessive amino acid NTs accumulation in the intestinal contents. We envisage that the orthogonal approach is of great significance for the comprehensive determination of targeted metabolomics. NTs have the potential to be biomarkers for clinical metabolomics.

Introduction

The immune system in the gastrointestinal tract is continuously exposed to numerous microbiota, pathogens, and food-derived antigens. Unlike other peripheral organs, the gastrointestinal tract has a specific nervous system defined as enteric nervous system (ENS), which has critical roles in regulating the intestinal barrier function and interaction with immune systems [1,2]. Notably, in enteric reflection pathways, the neurons utilize a broad range of chemical neurotransmitters (NTs) and their metabolites that signal through an even more extensive range of receptors [3]. Previous researchers have observed that NTs orchestrate intestinal immune. The principal NT acetylcholine could drive the intestinal cholinergic anti-inflammatory pathway through the function in efferent vagal neurons [4]. Tryptophan and its metabolites, including tryptamine and kynurenine, could activate the aryl hydrocarbon receptor (AHR) in lymphoid tissues and promote Treg cell development [5,6].

Nowadays, targeted metabolic profiling becomes an essential tool in prior studies to predict outcomes and provide insights into the pathophysiology of disease processes in clinical research [7,8]. Given such crucial immunological significance, global profiling of NT metabolome may serve as a clinical indicator of the diagnosis and treatment of immune-inflammation diseases, thereby offering novel insights into the in-depth mechanism by which endogenous metabolites interact with the neuro-immune system. Moreover, NT metabolome may be useful in clinical research for understanding neurodegenerative disease mechanisms through microbiome-gut-brain axis [9].

Compared with validated single biomarkers used in clinical practice, panel-based NT metabolome profiling potentially represents more biochemical pathways and biological disturbance under inflammation [10,11]. The NT metabolome consists of small molecule metabolites, including cholines, amines, amino acids, and their metabolites [9] (Fig. 1). From the angle of chemical structures, amine and phenol NTs could be detected by LC-MS/MS-based chemical labeling approach [12,13]. More recently, some highly polar NTs were successfully determined by LC-MS/MS with a hydrophilic interaction liquid chromatography (HILIC) column [14]. However, only partial coverage of the NT metabolome can be simultaneously quantified by any of these approaches. This is due to the lack of all-purpose labeling reagents for vast chemical group diversity and the ionization efficiency differences in positive and negative ion modes.

Previous studies have shown that sample pretreatment optimization could increase metabolite coverage [15,16]. We recently achieved significant enhancement in microbial metabolite detectability and much higher metabolome coverage by integrating biphasic extractions with chemical derivatizations [17]. Adhering to a divide-and-conquer concept where the NTs could be divided into different chemical groups (Fig. 2), an orthogonal dual pretreatment approach was proposed for the NT metabolome simultaneous determination. First, a high-performance dansyl-labeling method was applied for targeting amine and phenol NTs. Meanwhile, for targeted analyzing tertiary amine and choline NTs, the label-free aliquot was designed to combine with the dansyl-labeling aliquot in a single vial. The orthogonal design will allow us to prioritize the development efforts on labeling chemistry to target specific groups of metabolites and compensate for the deficiency of the rest compounds in a metabolome to guide future method optimization.

With this method, we can monitor the global changes of enteric NTs in serum and intestinal contents samples from a dextran sulphate sodium (DSS)-induced colitis and the intervention effects of a Chinese patent drug. Comprehensive analysis of NTs in the host and microbiome showed that amino acids and cholines shifted in response to inflammation, and highlighted the biological significance of tryptophan and phenylalanine metabolism. Our approach aims at increasing the clinical attention of NT metabolome changes in inflammatory bowel disease and the mechanism of how NT metabolome plays a crucial part in maintaining the neuro-immune system. We hope that combining the divide-and-conquer concept with orthogonal dual pretreatment makes it possible to quantify more metabolites in targeted metabolomics.