Combined Urinary Biomarkers to Assess Coffee Intake Using Untargeted Metabolomics: Discovery in Three Pilot Human Intervention Studies and Validation in Cross-Sectional Studies

Abstract

Coffee is a widely consumed beverage worldwide and has a high content of chlorogenic acids, polyphenols, methylxanthines, and volatile flavor compounds. Scientific evidence to support the beneficial health effects of coffee is limited, and validated urinary biomarkers of coffee intake are therefore needed. We observed 23 common putative biomarkers of coffee intake in three separate parallel intervention studies by ultra-high-performance liquid chromatography-electrospray ionization-quadrupole time-of-flight-mass spectrometry (UHPLC-ESI-QTOF-MS) and multivariate analyses. Baseline samples from the NU-AGE study were used to confirm and validate 16 of these candidate biomarkers, including their robustness, time response, and dose response. These validated candidate biomarkers are N-methylpyridinium cation, 1-methyl-1H-pyrrole-2-carboxaldehyde, 1H-pyrrole-2carboxaldehyde sulfate, 3-piperidinemethanol, furfurylidene-furfurylamine, 2-furoylglycine, N-substituted-5-(aminoethyl) furan-2carbaldehyde derivative, 3′,4′-dihydroxyacetophenone sulfate, caffeine, dihydroxystyrene glucuronide, ferulic acid sulfate, 4ethylcatechol glucuronide, 3-feruloylquinic acid, 3,4-dihydroxystyrene sulfate, one unknown glucuronide, and one unknown sulfate. Combinations of candidate biomarkers gave a better prediction of coffee consumption than individual biomarkers. The robustness of the combined biomarkers requires additional validation in cohort studies covering other populations.

Metabolites of Key Flavor Compound 2,3,5-Trimethylpyrazine in Human Urine

“…In conclusion, we report the application of a quantitative UHPLC–MS/MS-based stable isotope dilution assay and results from urine analysis of metabolites of TMP delivered by real-life doses of roasted coffee brew. The study itself, however, has some limitations, as the biosamples were spot urines and therefore did not allow time-resolved analysis and kinetic interpretations, and calculation of excretion rates. ,− In contrast to untargeted metabolomics utilizing high-resolution MS-screening aiming at biomarker discovery, − the number of participants in the study the biosamples were available from was relatively small with six participants, and substantial interindividual differences in excreted concentrations were detected. Our targeted approach nevertheless succeeded in unambiguous identification and quantification of TMP metabolites as the data show that consumption of roasted coffee brew leads to elevated concentrations of TMP metabolites 3a and 3b .…”

Validation of N-Methylpyridinium as a Feasible Biomarker for Roasted Coffee Intake

“…Reliable biomarkers that indicate coffee consumption are highly interesting for healthrelated nutritional studies, and several metabolomics studies have been dedicated to discovering suitable compounds [26][27][28][29][30]51]. However, whether such proposed candidates are actually applied for biomarker purposes is still being determined.…”

“…Promising candidates, e.g., phase II metabolites of hydroxycinnamic acids [11,28], furane metabolites [51], and diterpenes [26,29], are not readily available as standard substances or have yet to be sufficiently validated. Using a profile of compounds has been suggested to increase accuracy for classifying study participants [11,27]. However, in real-life scenarios, analyzing and processing multiple compounds per individual to verify or substitute dietary records appears cumbersome, making a quick classification of an individual as a coffee drinker or a non-coffee drinker impossible.…”

“…Bresciani et al (2020) reported that the average plasma concentration (Cavg) and 24 h area under the curve (AUC 0-24 h) of NMP in plasma was roughly three times that when three cups of coffee were consumed over the day compared to consumption of a single cup [41]. Xi et al (2021) recently reported that urinary NMP concentrations followed a dose-response dependency based on data from high-and low-coffee consumers [27].…”

“…Discovery of new food-specific biomarker candidates is regularly reported in the literature [21][22][23][24][25][26][27][28][29][30]. Usually, the discovery process involves sample collection, e.g., urine or plasma, of low and high-consuming populations followed by untargeted metabolomics, approaches using sophisticated techniques like nuclear magnetic resonance (NMR) or ultra high-performance liquid chromatography coupled to high-resolution mass spectrometry (UPLC-HR-MS) [31,32].…”