Profiling of Polar Metabolites in Mouse Feces Using Four Analytical Platforms to Study the Effects Of Cathelicidin-Related Antimicrobial Peptide in Alcoholic Liver Disease

Abstract

Alterations in gut bacterial homeostasis result in changes in intestinal metabolites. To investigate the effects of alcohol on fecal metabolites and the role of cathelicidin-related antimicrobial peptide (CRAMP) in alcoholic liver disease (ALD), CRAMP knockout (KO) and their control wild type (WT) mice were fed a Lieber-DeCarli liquid diet with or without alcohol. Polar metabolites in mouse feces were analyzed by GC × GC-MS and 2DLC-MS, and the concentrations of short chain fatty acids (SCFAs) were measured by GC-MS. A total of 95 and 190 metabolites were detected by GC × GC-MS and 2DLC-MS, respectively. Among the significantly changed metabolites, taurine and nicotinic acid were decreased in WT mice fed alcohol, which were also down-regulated in KO mice fed without alcohol. Interestingly, these two metabolites were increased in KO mice fed alcohol compared to them in WT controls. Additionally, SCFAs were significantly decreased in WT mice fed alcohol and in KO mice fed without alcohol, whereas two branched-chain SCFAs were increased by alcohol treatment in KO mice. In summary, the analytical platforms employed in this study successfully dissected the alterations of polar metabolites and SCFAs in fecal samples, which helped understand the effects of alcohol consumption and CRAMP in intestinal metabolism and alcohol-induced liver injury.

Integrating Two-Dimensional Gas and Liquid Chromatography-Mass Spectrometry for Untargeted Colorectal Cancer Metabolomics: A Proof-of-Principle Study

“…However, its abundance change has the same direction, i.e., all up-regulated in the cases. Manual data analysis shows that such discrepancy was mainly caused by the low instrument response ( Figure 3 ), as we reported before [ 7 , 11 ].…”

“…The mzXML files were then subjected to MetSign software for spectrum deconvolution and cross-sample peak list alignment [ 71 , 72 , 73 ]. Metabolite identification was achieved using the 2DLC-MS/MS data of the pooled samples, as described in our previous work [ 7 , 11 , 12 ]. Briefly, MS/MS spectra of pool samples were first matched with the information of authentic standards of metabolites recorded in our in-house database, including parent ion m/z , retention time, and MS/MS spectrum similarity with thresholds of parent ion m/z variation < 5 ppm, retention time variation < 0.15 min, and MS/MS spectral similarity > 0.4.…”

“…To further increase metabolite coverage and achieve high accuracy of metabolite quantification, we have developed a method by analyzing the same metabolite sample on comprehensive two-dimensional gas chromatography-mass spectrometry (GC × GC-MS) and two-dimensional liquid chromatography-mass spectrometry (2DLC-MS). We then integrate their data for metabolite identification and quantification [ 7 , 11 , 12 ]. Such analysis does not only dramatically increase the metabolite coverage but also provide accurate metabolite quantification and reliable metabolic pathway assignment.…”

Role of Gut Microbiota in Neuroendocrine Regulation of Carbohydrate and Lipid Metabolism via the Microbiota-Gut-Brain-Liver Axis

“…In addition, several kinds of liver diseases are associated with alterations in SCFA levels. For example, mice with alcohol-induced liver injury have shown decreased concentrations of straight-chain SCFAs as compared to normal mice [175]. Further, higher fecal acetate and propionate together with a higher abundance of SCFA-producing gut microbiota were observed in NAFLD patients [176].…”

Essential Role of IFN-γ in Regulating Gut Antimicrobial Peptides and Microbiota to Protect Against Alcohol-Induced Bacterial Translocation and Hepatic Inflammation in Mice

“…AMPs are produced and secreted by intestinal epithelial cells and Paneth cells and serve as important innate immune regulators in maintaining gut microbial growth and composition ( Bevins and Salzman, 2011 ; Muniz et al, 2012 ). Chronic alcohol feeding reduces intestinal AMPs, including C-type lectins (Reg3β and Reg3γ; Yan et al, 2011 ; Wang et al, 2016 ), α-defensins ( Zhong et al, 2019 ), and cathelicidin-related antimicrobial peptide (CRAMP; He et al, 2019 ). Moreover, overexpression of Reg3γ in mice restricted bacterial colonization and ameliorated alcohol-induced steatohepatitis ( Wang et al, 2016 ).…”