Metabolomic Workflow for the Accurate and High-Throughput Exploration of the Pathways of Tryptophan, Tyrosine, Phenylalanine, and Branched-Chain Amino Acids in Human Biofluids

Abstract

The modulation of host and dietary metabolites by gut microbiota (GM) is important for maintaining correct host physiology and in the onset of various pathologies. An ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry method was developed for the targeted quantitation in human plasma, serum, and urine of 89 metabolites resulting from human-GM cometabolism of dietary essential amino acids tryptophan, tyrosine, and phenylalanine as well as branched-chain amino acids. Ninety-six-well plate hybrid-SPE enables fast clean-up of plasma and serum. Urine was diluted and filtered. A 15 min cycle enabled the acquisition of 96 samples per day, with most of the metabolites stable in aqueous solution for up to 72 h. Calibration curves were specifically optimized to cover expected concentrations in biological fluids, and limits of detection were at the order of ppb. Matrix effects were in acceptable ranges, and analytical recoveries were in general greater than 80%. Inter and intraday precision and accuracy were satisfactory. We demonstrated its application in plasma and urine samples obtained from the same individual in the frame of an interventional study, allowing the quantitation of 51 metabolites. The method could be considered the reference for deciphering changes in human-gut microbial cometabolism in health and disease. Data are available via Metabolights with the identifier MTBLS4399.

Feed your microbes to deal with stress: a psychobiotic diet impacts microbial stability and perceived stress in a healthy adult population

“…Blood and urine targeted metabolomics: Blood and urine metabolomics analysis, targeting key metabolites resulting from human-gut-microbiota co-metabolism of dietary essential amino acids tryptophan, tyrosine, phenylalanine and branched chain amino acids were performed at Fondazione Edmund Mach (Trento, Italy) according to established methods in the laboratory using UHPLC-ESI-MS/MS analysis [31].…”

A metabolome-wide case-control study of african american breast cancer patients

“…Each sample was used within ± 15% recovery. This protocols were widely used in metabolomics studies [ 41 – 43 ]. As suggested by the literature, the protocols used in this study offered the greatest extraction performance, though the survivability of certain metabolites cannot be evaluated [ 41 ].…”

Role of the Gut Bacteria-Derived Metabolite Phenylacetylglutamine in Health and Diseases

“…Following the microbial generation, PAA enters the portal circulation and reaches hepatic and renal systems. In the liver, it conjugates with glutamine and glycine through amino acid acetylation mediated by the enzyme phenylacetyltransferase and glycine N -phenylacetyltransferase to complete the meta-organismal transformation of phenylacetylglutamine and phenylacetylglycine . Meta-organismal production of phenylacetylglutamine is predominantly found in humans whereas phenylacetylglycine is more common in rodents ,− (Figure ).…”

“…In the liver, it conjugates with glutamine and glycine through amino acid acetylation mediated by the enzyme phenylacetyltransferase and glycine N-phenylacetyltransferase to complete the meta-organismal transformation of phenylacetylglutamine and phenylacetylglycine. 14 Meta-organismal production of phenylacetylglutamine is predominantly found in humans whereas phenylacetylglycine is more common in rodents 5,15−17 (Figure 1). Bacterial isolates from three major phyla: Bacteroidetes, 1,2 Firmicutes, 3 and Proteobacteria are involved in PAA formation in humans and other vertebrates.…”