Investigation Of Tryptophan Metabolites Relevant To Aryl Hydrocarbon Receptor And Their Detection Methods

Tryptophan(Trp)is one of the 8 essential amino acids in human and is an important indicator for the evaluation of protein nutritional value.Tryptophan metabolites have important regulatory effects on the human’s mood,metabolism,cell growth and development,biological rhythms and immune function.6-Formylindole [3,2-b]carbazole(FICZ)is a photolysis product of tryptophan and can also be produced by indole-3-pyruvate inoculation without light.FICZ can induce high expression of aryl hydrocarbon receptor(AHR)and its downstream genes at the nanomolar level,induce the differentiation of Th17 cells,and positively regulate the immune response.This indicates that FICZ is of special significance for improving immunity and preventing,even controlling tumors.However,there is no direct evidence that FICZ can be synthesized in vivo.Therefore,it is important to find the key enzyme in vivo that can metabolize tryptophan to endogenous FICZ.L-amino acid oxidase(LAAO)is a key enzyme for the metabolism of indole-3-pyruvate by tryptophan,and its human-expressed gene is IL4i1.Therefore,in order to study whether the human LAAO enzyme is the key enzyme in catalyzing the production of FICZ by tryptophan,293 t cells over-expressing IL4i1 has been investigated by non-targeted and targeted metabolomic techniques.The main experiments and results showed as follow:1.The 293 t cells were transiently and stably transfected with IL4i1 gene,and the expression of LAAO enzyme in cell supernatant and cell lysate has been detected by Western-Blot,and the enzyme activity has been evaluated according to luminol luminescence reaction.The results showed that LAAO expressed by IL4i1 gene is expressed intracellularly and could secret into the extracellular,which is a secreted protein;LAAO can efficiently catalyze tryptophan at 37°C and pH 5.5,and the conversion rate is approximately 2.2%.2.The expression of luciferase in the AHR reporter cell line has been detected to evaluate how many AHR ligands could be produced by the LAAO enzyme.After catalyzed by LAAO,the ability of tryptophan to induce luciferase expression was significantly increased,indicating that at least one of the catalytic products of the LAAO is a strong AHR agonist.3.Liquid chromatography tandem mass spectrometry detection method was established for FICZ,indole-3-aldehyde,indole acetic acid,tryptophan,kynurenine and kynuric acid analysis.Linear ranges were wide,and the linear correlation coefficients were all above 0.999.Quantification limits ranged from 0.05 to 10 ng/mL,detection limits ranged from 0.02 to 5 ng/mL,and recoveries ranged from 85% to 102%.These works laid the root for the determination of tryptophan and its metabolites.4.The untargeted metabolomics combined with targeted metabolite detection were performed to quantify the tryptophan products produced by the LAAO.The results showed that under the experimental conditions,FICZ is not the product of the LAAO,but LAAO can metabolize tryptophan to indole-3-aldehyde,indole-3-acetic acid,and their contents are positively correlated with the concentration of tryptophan in the cell culture environment.Both indole-3-aldehyde and indole-3-acetic acid are AHR ligands and important immunomodulatory substances.For the first time in our study,it was confirmed that indole-3-acetic acid is the decomposition product of tryptophan catalyzed by LAAO,and it is also the first time that the indole-3-carboxaldehyde can be produced by a non-bacterial synthesis pathway,which laid a theoretical foundation for the in-depth study of indole-3-carboxaldehyde synthesis and regulation in vivo.In addition to this,our study provided an unprecedented orientation for evaluating the nutritional value of high tryptophan diet.