Quantitative UPLC-MS/MS analysis of the gut microbial co-metabolites phenylacetylglutamine, 4-cresyl sulphate and hippurate in human urine: INTERMAP Study

Abstract

The role of the gut microbiome in human health, and non-invasive measurement of gut dysbiosis are of increasing clinical interest. New high-throughput methods are required for the rapid measurement of gut microbial metabolites and to establish reference ranges in human populations. We used ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) -- positive and negative electrospray ionization modes, multiple reaction monitoring transitions -- to simultaneously measure three urinary metabolites (phenylacetylglutamine, 4-cresyl sulphate and hippurate) that are potential biomarkers of gut function, among multi-ethnic US men and women aged 40–59 from the INTERMAP epidemiologic study (n = 2000, two timed 24-hr urine collections/person). Metabolite concentrations were quantified via stable isotope labeled internal standards. The assay was linear in the ranges 1ng/mL (lower limit of quantification) to 1000ng/mL (phenylacetylglutamine and 4-cresyl sulfate) and 3ng/mL to 3000ng/mL (hippurate). These quantitative data provide new urinary reference ranges for population-based human samples: mean (standard deviation) 24-hr urinary excretion for phenylacetylglutamine was: 1283.0 (751.7) μmol/24-hr (men), 1145.9 (635.5) μmol/24-hr (women); for 4-cresyl sulphate, 1002.5 (737.1) μmol/24-hr (men), 1031.8 (687.9) μmol/24-hr (women); for hippurate, 6284.6 (4008.1) μmol/24-hr (men), 4793.0 (3293.3) μmol/24-hr (women). Metabolic profiling by UPLC-MS/MS in a large sample of free-living individuals has provided new data on urinary reference ranges for three urinary microbial co-metabolites, and demonstrates the applicability of this approach to epidemiological investigations.

NMR-Based Metabolomic Profiling of Overweight Adolescents: An Elucidation of the Effects of Inter-/Intraindividual Differences, Gender, and Pubertal Development

“…In addition, females may utilize less protein as an energy source owing to a greater part of exercise energy from fat [44]. In addition, Table 1 shows that girls have a relatively higher urinary excretion of hippurate and phenylacetylglutamine than boys, which is different from results obtained for adults aged 40–59 years [45]. They found a higher hippurate excretion in men compared to women, but no gender difference in phenylacetylglutamine excretion.…”

Urinary Metabolomics Profiles Associated to Bovine Meat Ingestion in Humans

“…The urinary concentrations of PAG in healthy people show an important intra individual variability, with pronounced differences depending on sex and age and may be influenced by medications or contaminants and/or ingestion of foods rich in phenylalanine. Phenylalanine is highly concentrated in wheat grain and high protein foods, such as meat or cottage cheese.…”

Microbial Determinants of Biochemical Individuality and Their Impact on Toxicology and Pharmacology

“…This guild theory, although still plausible, has been weakened by metabolomic analyses of human samples, which demonstrate that the concentration of microbial end products also varies between individuals, especially in those of different age, gender, race, or ethnicity (Wijeyesekera et al, 2012). Quantification of the gut microbial co-metabolites phenylacetylglutamine, 4-cresol sulfate, and hippuric acid in individuals from multiple ethnic groups revealed marked variations both within and between populations.…”