Identification of methylated avenanthramides in human plasma

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Avenanthramides (AVs) are phytochemicals unique to oats consisting of an anthranilic acid and hydroxycinnamic acid linked by a pseudo‐peptide bond. We have demonstrated AV bioavailability to be low in hamsters and humans, suggesting they may be metabolized by phase II enzymes to facilitate their rapid excretion. Thus, we sought to identify the presence of phase II metabolites of 6 dominant AV isomers (A, B, C, O, P, and Q). AV‐A, ‐B, and ‐C differ in the moiety at position 3 (A: ‐H; B: ‐CH3O; and C: ‐OH) of the cinnamic acid ring, and AV‐O, ‐P, and ‐Q correspond to A, B, and C in terms of position 3 but differ in the length of the pseudo‐peptide linkage. We conducted a placebo‐controlled, crossover study in 10 healthy adults (60.4 y, BMI = 25.3). Blood was collected at 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, and 24 h after consumption of a muffin made with either 20 g AV‐enriched oat bran (containing 46.2 mg total AVs) or regular oat bran. After removal of lipids with hexane, AV phase II metabolites in the defatted plasma were extracted twice with ethyl acetate, dried under N2, and reconstituted in 55% methanol for analysis on an Agilent UPLC‐QToF‐MS equipped with an Agilent Zorbax Extend C‐18 RR HT column (2.1 × 50 mm, 1.8 μm). Utilizing the MassHunter Qualitative Analysis software, we identified with a confidence score ≥80 for aglycones AV‐O and ‐A, and ≥90 for methylated AV‐O. Their detection is consistent with plasma AV results obtained after the removal of the glucuronide and sulfate conjugates that AV‐O and ‐A were the most abundant isomers. AV‐A was detected at fewer time points than AV‐O probably due to its much lower circulating concentrations. AV‐A had a maximum concentration (Cmax) of 2.2 ± 1.6 ng/mL with the time to reach Cmax (Tmax) at 1.9 ± 0.8 h. The Cmax for AV‐O was 6.4 ± 3.9 ng/mL and the Tmax was 2.6 ± 0.8 h, as well as 14.0 ± 8.2 ng/mL AV‐O equivalents and 1.9 ± 0.7 h for methylated AV‐O. Though AVs are extensively transformed to phase II metabolites, our analyses reveal the presence of AV‐O and ‐A aglycones in the circulation. Ours is the first report of a methylated AV metabolite following acute AV consumption. Future studies are warranted to explore the potential bioactivity of methylated AVs.Support or Funding InformationSupported by USDA and PepsiCo