RESUMO
Gut microbiota-host cometabolites are closely related to various diseases. Monitoring dynamic changes of cometabolites can provide a more comprehensive understanding of pathophysiology. Here, a novel liquid chromatography-tandem mass spectrometry method was performed for the analysis of aromatic amino acids and their gut microbiota-host cometabolites in rat serum and urine. In the developed method, seven key gut microbiota-host cometabolites were chromatographically separated on a Kinetex Phenyl-Hexyl column by gradient elution, and the run time was 6 min. Serum and urine were extracted by protein precipitation. This method was linear between 10.20 and 1000.00 ng/mL for phenylalanine and p-cresyl sulfate; 25.60-2500.00 ng/mL for tryptophan; 51.20-5000.00 ng/mL for tyrosine, indole, and indoxyl sulfate; and 75.50-7500.00 ng/mL for p-cresol. The linearity, accuracy, precision, and recovery of seven analytes were all satisfactory. The method was sufficiently sensitive and robust. It was successfully applied to characterize the alterations of gut microbiota-host cometabolites in inflammatory disorders. All of these results suggest that the developed method is able to simultaneously monitor aromatic amino acids and their gut microbiota-host cometabolites. This method will be expected to be a valuable tool for clinical researches and comprehensive studies of the pathophysiological roles.
Assuntos
Aminoácidos Aromáticos/sangue , Aminoácidos Aromáticos/urina , Microbioma Gastrointestinal , Metabolômica , Animais , Cromatografia Líquida , Ratos , Espectrometria de Massas em TandemRESUMO
2-Amino-9H-pyrido[2,3-b]indole (AαC) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are carcinogenic heterocyclic aromatic amines (HAAs) formed during the combustion of tobacco and during the high-temperature cooking of meats. Human enzymes biotransform AαC and PhIP into reactive metabolites, which can bind to DNA and lead to mutations. We sought to understand the relative contribution of smoking and diet to the exposure of AαC and PhIP, by determining levels of AαC, its ring-oxidized conjugate 2-amino-9H-pyrido[2,3-b]indole-3-yl sulfate (AαC-3-OSO3H), and PhIP in urine of smokers on a free-choice diet before and after a six week tobacco smoking cessation study. AαC and AαC-3-OSO3H were detected in more than 90% of the urine samples of all subjects during the smoking phase. The geometric mean levels of urinary AαC during the smoking and cessation phases were 24.3 pg/mg creatinine and 3.2 pg/mg creatinine, and the geometric mean levels of AαC-3-OSO3H were 47.3 pg/mg creatinine and 3.7 pg/mg creatinine. These decreases in the mean levels of AαC and AαC-3-OSO3H were, respectively, 87% and 92%, after the cessation of tobacco (P < 0.0007). However, PhIP was detected in <10% of the urine samples, and the exposure to PhIP was not correlated to smoking. Epidemiological studies have reported that smoking is a risk factor for cancer of the liver and gastrointestinal tract. It is noteworthy that AαC is a hepatocellular carcinogen and induces aberrant crypt foci, early biomarkers of colon cancer, in rodents. Our urinary biomarker data demonstrate that tobacco smoking is a significant source of AαC exposure. Further studies are warranted to examine the potential role of AαC as a risk factor for hepatocellular and gastrointestinal cancer in smokers.
Assuntos
Aminoácidos Aromáticos/química , Aminoácidos Aromáticos/urina , Aminopiridinas/química , Compostos Heterocíclicos/urina , Indóis/química , Fumar , Aminopiridinas/urina , Cromatografia Líquida , Compostos Heterocíclicos/química , Humanos , Indóis/urina , Limite de Detecção , Espectrometria de Massas , Estrutura Molecular , Abandono do Hábito de FumarRESUMO
The gut microbiota-host co-metabolites are good indicators for representing the cross-talk between host and gut microbiota in a bi-direct manner. There is increasing evidence that levels of aromatic amino acids (AAAs) are associated with the alteration of intestinal microbial community though the effects of long-term microbial disturbance remain unclear. Here we monitored the gut microbiota composition and host-microbiota co-metabolites AAA profiles of mice after gentamicin and ceftriaxone treatments for nearly 4 months since their weaning to reveal the relationship between host and microbiome in long- term microbial disturbances. The study was performed employing targeted LC-MS measurement of AAA-related metabolites and 16S RNA sequence of mice cecal contents. The results showed obvious decreased gut microbial diversity and decreased Firmicutes/Bacteroidetes ratio in the cecal contents after long-term antibiotics treatment. The accumulated AAA (tyrosine, phenylalanine and tryptophan) and re-distribution of their downstreaming metabolites that produced under the existence of intestinal flora were found in mice treated with antibiotics for 4 months. Our results suggested that the long-term antibiotic treatment significantly changed the composition of the gut microbiota and destroyed the homeostasis in the intestinal metabolism. And the urinary AAA could be an indicator for exploring interactions between host and gut microbiota.