**Background:** There is growing interest in the relationship between diet, gut microbiota, and mental health, but the mechanisms linking dietary components to brain function remain poorly understood. Metabolites produced when gut microbes ferment amino acids—including neurotransmitter precursors such as tyrosine—may enter the bloodstream and exert systemic effects. The authors hypothesized that fermentation of amino acids from resistant proteins (produced by high-heat food processing) could yield potentially toxic metabolites and disturb the availability of neurotransmitter precursors to the brain. To investigate this, they developed a novel workflow using ¹⁴C radiolabeling to trace the metabolic fate of nutrients in a clinically relevant pig model.
**Methods:** Male weanling Landrace cross Large White pigs were fed either a standard diet (n=4) or a high resistant protein diet (n=4) for 4 weeks prior to ¹⁴C-nutrient administration. The resistant protein diet contained autoclaved skim milk powder (15 h at 70°C, 20 min at 121°C) to model high-heat/low-moisture processing, resulting in approximately 50% in vitro protein digestibility. α-Linolenic acid (ALA, 20 MBq [1-¹⁴C]-ALA) was used as a model nutrient to validate the workflow, given its well-characterized biodistribution. Tyrosine (Tyr, 20 MBq [¹⁴C(U)]-L-tyrosine) was the test nutrient, encapsulated in acid-resistant capsules to prevent gastric digestion and promote colonic release, mimicking amino acids from resistant proteins. Within each cohort, one animal received the ¹⁴C-labeled nutrient and three received equivalent non-labeled capsules. After a 3-day period for metabolism and distribution, blood, urine, feces, organs, and tissues were collected. Radioactivity was measured by liquid scintillation counting (LSC). Additionally, tryptophan (Trp), kynurenine (Kyn), and tryptamine (TA) were analyzed in plasma and feces from both diet groups by LC-MS to assess differences in aromatic amino acid metabolism.
**Key Results:** For ¹⁴C-ALA, 52.4% of radioactivity was excreted in urine and feces (peaking on day 1 at 46.8% of dose). Only 1.45% of the original dose was recovered in organs, with the highest amounts in liver (0.62%) and small intestine (0.21%). Radioactivity in the brain was very low (<0.01% of dose). The highest concentrations were in kidney (0.002% dose/g), liver (0.001% dose/g), and fat (0.001% dose/g). Total mass recovery was approximately 61%, with the remaining 39% presumed eliminated as CO₂. For ¹⁴C-Tyr, 48.6% of radioactivity was excreted in urine and feces (peaking on day 1 at 43.12% of dose). Higher radioactivity in colon digesta (1.46 kBq/g) compared to stomach (0.10 kBq/g) and small intestine (0.64 kBq/g) confirmed colonic delivery. Of the original dose, 6.9% was recovered in all organs sampled. The highest amounts were in small intestine (3.08%) and liver (2.24%). Critically, 0.15% of the original 20 MBq dose was detected in the brain: brain lobes (0.088% of dose, 0.52 kBq/g), cerebellum (0.03% of dose, 0.62 kBq/g), and mid-brain (0.03% of dose, 0.37 kBq/g). The highest concentrations in organs were in liver (0.008% dose/g) and pancreas (0.006% dose/g). For tryptophan metabolites, fecal Trp concentration was significantly greater in the standard diet group compared to the resistant protein diet group (p<0.05). Fecal Kyn and TA showed non-significant trends toward lower levels on the standard diet. No differences in plasma concentrations of Trp, Kyn, or TA were detected between diet groups.
**Clinical Implications:** This study provides the first direct demonstration that metabolites of gut-microbially fermented tyrosine are bioavailable to the brain, supporting a mechanistic pathway linking dietary resistant protein intake to brain function via the gut-brain axis. The findings suggest that high-heat-processed foods containing resistant proteins may influence mental health through two potential mechanisms: (1) reduced availability of neurotransmitter precursors (tyrosine, tryptophan) to the brain due to microbial fermentation, and (2) production of potentially neuroactive or toxic microbial metabolites (e.g., tryptamine, indoles, phenols) that may affect brain function. The ¹⁴C radiolabeling workflow validated here offers a powerful approach for tracing nutrient metabolism to specific tissues, which could be applied to study other diet-disease relationships where target tissues are not easily sampled (e.g., brain). However, the authors caution that the small sample size (n=1 per ¹⁴C-nutrient), lack of a standard-diet control for ¹⁴C-Tyr, and absence of chemical identification of specific brain metabolites mean these findings require confirmation and extension. Further research is needed to identify which specific tyrosine-derived compounds reach the brain, characterize their neuroactivity in cell models, and validate associations between resistant protein intake and mental health outcomes in clinical studies.