Demonstrating a link between diet, gut microbiota and brain: 14C radioactivity identified in the brain following gut microbial fermentation of 14C-radiolabeled tyrosine in a pig model.

Background: There is a need to better understand the relationship between the diet, the gut microbiota and mental health. Metabolites produced when the human gut microbiota metabolize amino acids may enter the bloodstream and have systemic effects. We hypothesize that fermentation of amino acids by a resistant protein-primed gut microbiota could yield potentially toxic metabolites and disturb the availability of neurotransmitter precursors to the brain. However, these mechanisms are challenging to investigate via typical in vitro and clinical methods. Methods: We developed a novel workflow using 14C radiolabeling to investigate complex nutrient-disease relationships. The first three steps of the workflow are reported here. α-Linolenic acid (ALA) was used as a model nutrient to confirm the efficacy of the workflow, and tyrosine (Tyr) was the test nutrient. 14C-Tyr was administered to male weanling pigs fed a high resistant protein diet, which primed the gut microbiota for fermenting protein. The hypotheses were; (1) that expected biodistribution of 14C-ALA would be observed, and (2) that radioactivity from 14C-Tyr, representing Tyr and other amino acids released from resistant protein following gut microbial fermentation, would be bioavailable to the brain. Results: Radioactivity from the 14C-ALA was detected in tissues reflecting normal utilization of this essential fatty acid. Radioactivity from the 14C-Tyr was detected in the brain (0.15% of original dose). Conclusion: Metabolites of gut-fermented protein and specifically amino acid precursors to neurotransmitters such as tyrosine, are potentially able to affect brain function. By extension, resistant proteins in the diet reaching the gut microbiota, also have potential to release metabolites that can potentially affect brain function. The high specificity of detection of 14C radioactivity demonstrates that the proposed workflow can similarly be applied to understand other key diet and health paradigms.

Reduced Growth, Altered Gut Microbiome and Metabolite Profile, and Increased Chronic Kidney Disease Risk in Young Pigs Consuming a Diet Containing Highly Resistant Protein.

High-heat processed foods contain proteins that are partially resistant to enzymatic digestion and pass through to the colon. The fermentation of resistant proteins by gut microbes produces products that may contribute to chronic disease risk. This pilot study examined the effects of a resistant protein diet on growth, fecal microbiome, protein fermentation metabolites, and the biomarkers of health status in pigs as a model of human digestion and metabolism. Weanling pigs were fed with standard or resistant protein diets for 4 weeks. The resistant protein, approximately half as digestible as the standard protein, was designed to enter the colon for microbial fermentation. Fecal and blood samples were collected to assess the microbiome and circulating metabolites and biomarkers. The resistant protein diet group consumed less feed and grew to ~50% of the body mass of the standard diet group. The diets had unique effects on the fecal microbiome, as demonstrated by clustering in the principal coordinate analysis. There were 121 taxa that were significantly different between groups (adjusted-p < 0.05). Compared with control, plasma tri-methylamine-N-oxide, homocysteine, neopterin, and tyrosine were increased and plasma acetic acid was lowered following the resistant protein diet (all p < 0.05). Compared with control, estimated glomerular filtration rate (p < 0.01) and liver function marker aspartate aminotransferase (p < 0.05) were also lower following the resistant protein diet. A resistant protein diet shifted the composition of the fecal microbiome. The microbial fermentation of resistant protein affected the levels of circulating metabolites and the biomarkers of health status toward a profile indicative of increased inflammation and the risk of chronic kidney disease.

Does timing of phytonutrient intake influence the suppression of postprandial oxidative stress? A systematic literature review.

BACKGROUND: Postprandial oxidative stress markers in blood are generated transiently from various tissues and cells following high-fat and/or high-carbohydrate (HFHC) meals, and may be suppressed by certain phytonutrients, such as polyphenols and carotenoids. However, the transient presence of phytonutrients in circulation suggests that timing of consumption, relative to the meal, could be important. This systematic review investigates the effect of timing of phytonutrient intake on blood markers of postprandial oxidative processes. METHOD: EMBASE, Medline, Scopus and Web of Science were searched up to December 2020. Eligible studies met the criteria: 1) healthy human adults; 2) phytonutrient(s) consumed in solid form within 24 h of a HFHC meal; 3) postprandial measurements of oxidative stress or antioxidants in blood; and 4) controlled study design. Cohen's d effect sizes were calculated to compare studies. RESULTS: Nine studies, involving 256 participants, were included. Phytonutrients were consumed either at the same time, 1 h before, or the day (>12 h) before a HFHC meal. Significant decreases in blood markers - plasma lipid hydroperoxides, plasma malondialdehyde, serum sNox2-dp, serum 8-iso-PGF2α, platelet p47phox phosphorylation, and Keap-1 and p47phox protein levels in mononuclear cells (MNCs) - were observed where the phytonutrient was consumed together with the challenge meal (n = 4). Lack of any effect on oxidative stress markers was observed where phytonutrients were consumed with (n = 1), 1 h before (n = 1), and the day before (n = 2) the HFHC meal. CONCLUSION: Phytonutrients consumed with a HFHC meal significantly suppressed some markers of oxidative stress in blood. Although there were only a limited number of studies, it appears that suppression appeared effective at the time of peak phytonutrient concentration in plasma. However, further studies are required to confirm the observations and systematically optimise the effect of timing.

Characterising absorption and health-related properties of phytochemicals extracted from Malaysian palm fruit biomass after oil extraction.

After oil extraction, palm fruit biomass contains abundant water-soluble phytochemicals (PCs) with proven bioactivity in regulating oxidative stress and inflammation (OSI). For optimal bioefficacy following oral consumption, the pharmacokinetic plasma peak (Tmax) should be bio-matched with the onset of OSI, which can be predicted from the Phytochemical Absorption Prediction (PCAP) model and methodology. Predicted absorption and potential for regulation of OSI by measures of total phenolic content, antioxidant capacity and hydrogen peroxide production capacity, were applied to characterise eight extracts from mesocarp fibre and kernel shells of oil-depleted palm fruits. Results indicated post-consumption absorption Tmax ranges of 0.5-12 h and 2-6 h for intake in liquid and solid forms, respectively, and generally high antioxidant activity of the extracts. The research supports that PC extracts of palm fruit biomass have broad potential uses for human health as dietary antioxidants in foods, supplements or functional beverages.

Pharmacokinetic properties of phytochemicals in Hypericum perforatum influence efficacy of regulating oxidative stress.

BACKGROUND: Hypericum perforatum is used in ethnopharmacology and has recently become popular in conventional medicine for treatment of mild to moderate depression. The abundance of potentially functional phytochemicals and their broader utilizations in traditional medicine suggests that ingestion of H. perforatum may impart additional secondary health benefits. HYPOTHESIS/PURPOSE: Considering that many phytochemicals are known to display antioxidant activity, it was hypothesized that H. perforatum ingestion may inhibit oxidative stress and inflammation (OSI) which occurs in transient cycles following exercise and consumption of meals. The aim of this study was to explore the pharmacokinetics of H. perforatum phytochemicals after ingestion to predict the absorption timing of putative medicinal phytochemicals. STUDY DESIGN/METHODS: In silico analyses of previously published plant extract phytochemical profiles were performed, wherein the Phytochemical Absorption Prediction (PCAP) model was used to predict the pharmacokinetics of phytochemicals. The predicted times for phytochemicals to reach maximum plasma concentration (Tmax), and associated antioxidant activities, were compared to prior clinical in vivo studies to assess the accuracy and applicability of predictions. RESULTS: The PCAP model identified that phytochemicals with antioxidant activity concurrently accumulate in plasma with Tmax in the range of 1.6-2.3 h after ingestion. Comparison with previously published results identified that attenuation of OSI following H. perforatum ingestion aligns with the predicted Tmax of antioxidant phytochemicals. CONCLUSION: Based on these results it is therefore recommended that H. perforatum administration occurs 2 h before meals to provide optimal secondary health benefits associated with inhibition of postprandial stress. Additionally, these results highlight the use of in silico analyses to inform ingestion time and optimize the health benefits from ingestion of plant-based foods and medicines.

Statistical modelling coupled with LC-MS analysis to predict human upper intestinal absorption of phytochemical mixtures.

A diet rich in phytochemicals confers benefits for health by reducing the risk of chronic diseases via regulation of oxidative stress and inflammation (OSI). For optimal protective bio-efficacy, the time required for phytochemicals and their metabolites to reach maximal plasma concentrations (Tmax) should be synchronised with the time of increased OSI. A statistical model has been reported to predict Tmax of individual phytochemicals based on molecular mass and lipophilicity. We report the application of the model for predicting the absorption profile of an uncharacterised phytochemical mixture, herein referred to as the 'functional fingerprint'. First, chemical profiles of phytochemical extracts were acquired using liquid chromatography mass spectrometry (LC-MS), then the molecular features for respective components were used to predict their plasma absorption maximum, based on molecular mass and lipophilicity. This method of 'functional fingerprinting' of plant extracts represents a novel tool for understanding and optimising the health efficacy of plant extracts.

Dietary Phytochemicals Promote Health by Enhancing Antioxidant Defence in a Pig Model.

Phytochemical-rich diets are protective against chronic diseases and mediate their protective effect by regulation of oxidative stress (OS). However, it is proposed that under some circumstances, phytochemicals can promote production of reactive oxygen species (ROS) in vitro, which might drive OS-mediated signalling. Here, we investigated the effects of administering single doses of extracts of red cabbage and grape skin to pigs. Blood samples taken at baseline and 30 min intervals for 4 hours following intake were analyzed by measures of antioxidant status in plasma, including Trolox equivalent antioxidant capacity (TEAC) and glutathione peroxidase (GPx) activity. In addition, dose-dependent production of hydrogen peroxide (H2O2) by the same extracts was measured in untreated commercial pig plasma in vitro. Plasma from treated pigs showed extract dose-dependent increases in non-enzymatic (plasma TEAC) and enzymatic (GPx) antioxidant capacities. Similarly, extract dose-dependent increases in H2O2 were observed in commercial pig plasma in vitro. The antioxidant responses to extracts by treated pigs were highly correlated with their respective yields of H2O2 production in vitro. These results support that dietary phytochemicals regulate OS via direct and indirect antioxidant mechanisms. The latter may be attributed to the ability to produce H2O2 and to thereby stimulate cellular antioxidant defence systems.

Physicochemical properties of dietary phytochemicals can predict their passive absorption in the human small intestine.

A diet high in phytochemical-rich plant foods is associated with reducing the risk of chronic diseases such as cardiovascular and neurodegenerative diseases, obesity, diabetes and cancer. Oxidative stress and inflammation (OSI) is the common component underlying these chronic diseases. Whilst the positive health effects of phytochemicals and their metabolites have been demonstrated to regulate OSI, the timing and absorption for best effect is not well understood. We developed a model to predict the time to achieve maximal plasma concentration (Tmax) of phytochemicals in fruits and vegetables. We used a training dataset containing 67 dietary phytochemicals from 31 clinical studies to develop the model and validated the model using three independent datasets comprising a total of 108 dietary phytochemicals and 98 pharmaceutical compounds. The developed model based on dietary intake forms and the physicochemical properties lipophilicity and molecular mass accurately predicts Tmax of dietary phytochemicals and pharmaceutical compounds over a broad range of chemical classes. This is the first direct model to predict Tmax of dietary phytochemicals in the human body. The model informs the clinical dosing frequency for optimising uptake and sustained presence of dietary phytochemicals in circulation, to maximise their bio-efficacy for positively affect human health and managing OSI in chronic diseases.

In vitro heme and non-heme iron capture from hemoglobin, myoglobin and ferritin by bovine lactoferrin and implications for suppression of reactive oxygen species in vivo.

Lactoferrin (Lf), present in colostrum and milk is a member of the transferrin family of iron-binding glyco-proteins, with stronger binding capacity to ferric iron than hemoglobin, myoglobin or transferrin. Unlike hemoglobin and myoglobin, iron-bound Lf is reasonably stable to gastric and duodenal digestive conditions. Unlike ferrous iron, ferric iron is not directly reactive with oxygen supporting the capacity of Lf capture of heme iron to suppress reactive oxygen species (ROS) production. We therefore hypothesized that bovine Lf could capture and thereby terminate the cycle of ROS production by heme iron. The transfer of heme iron from either intact or digested forms of hemoglobin and myoglobin and from intact ferritin was demonstrated by in vitro methods, monitoring Fe-saturation status of Lf by changes in absorptivity at 465 nm. The results are discussed in the context of new proposed opportunities for orally administered Lf to regulate oxidative damage associated with heme iron. In addition to potentially suppressing oxidative heme-iron-mediated tissue damage in the lumen, Lf is expected to also reverse the overload of ferritin-bound iron, that accompanies chronic inflammation and aging. These new proposed uses of Lf are additional to known host defense functions that include anti-microbial, anti-viral properties, immune and cancer cell growth regulation. The findings and interpretations presented require clinical substantiation and may support important additional protective and therapeutic uses for Lf in the future.

Regulation of milk protein solubility by a whey-derived proline-rich peptide product.

The effects of a bovine whey peptide product enriched in proline (wPRP) on the solubility of milk proteins were tested under ambient conditions or following heat treatment at 75 and 100 °C, for 1 and 15 min, followed by post-incubation storage at either ambient temperature or 4 °C for up to 7 d. wPRP promoted solubilisation of milk proteins in a concentration-dependent manner without heat treatment and also after heat treatment at 75 and 100 °C, and the effect was enhanced after storage under either ambient or refrigerated storage conditions. Interactions of wPRP and milk proteins were monitored by particle size analysis and tryptic digestion and specifically linked with solubilisation of αS1 casein (αS1-Cn), which supported observed changes in milk protein solubility. The results suggested that wPRP preferably prevented or reversed physical versus covalent protein aggregation, with the relaxation of hydrophobic interactions at 4 °C providing an additive effect. This application of wPRP represents a novel approach to stabilisation of dairy proteins following thermal processing with industrial usefulness yet to be explored.