Nicholas D. Gillitt

Influence of a Polyphenol-Enriched Protein Powder on Exercise-Induced Inflammation and Oxidative Stress in Athletes: A Randomized Trial Using a Metabolomics Approach

Objectives Polyphenol supplementation was tested as a countermeasure to inflammation and oxidative stress induced by 3-d intensified training. Methods Water soluble polyphenols from blueberry and green tea extracts were captured onto a polyphenol soy protein complex (PSPC). Subjects were recruited, and included 38 long-distance runners ages 19–45 years who regularly competed in road races. Runners successfully completing orientation and baseline testing (N = 35) were randomized to 40 g/d PSPC (N = 17) (2,136 mg/d gallic acid equivalents) or placebo (N = 18) for 17 d using double-blinded methods and a parallel group design, with a 3-d running period inserted at day 14 (2.5 h/d, 70% VO2max). Blood samples were collected pre- and post-14 d supplementation, and immediately and 14 h after the third day of running in subjects completing all aspects of the study (N = 16 PSPC, N = 15 placebo), and analyzed using a metabolomics platform with GC-MS and LC-MS. Results Metabolites characteristic of gut bacteria metabolism of polyphenols were increased with PSPC and 3 d running (e.g., hippurate, 4-hydroxyhippurate, 4-methylcatechol sulfate, 1.8-, 1.9-, 2.5-fold, respectively, P<0.05), an effect which persisted for 14-h post-exercise. Fatty acid oxidation and ketogenesis were induced by exercise in both groups, with more ketones at 14-h post-exercise in PSPC (3-hydroxybutyrate, 1.8-fold, P<0.05). Established biomarkers for inflammation (CRP, cytokines) and oxidative stress (protein carbonyls) did not differ between groups. Conclusions PSPC supplementation over a 17-d period did not alter established biomarkers for inflammation and oxidative stress but was linked to an enhanced gut-derived phenolic signature and ketogenesis in runners during recovery from 3-d heavy exertion. Trial Registration ClinicalTrials.gov, U.S. National Institutes of Health, identifier: NCT01775384

Chia seed supplementation and disease risk factors in overweight women: a metabolomics investigation.

OBJECTIVE/SETTING This study assessed the effectiveness of milled and whole chia seed in altering disease risk factors in overweight, postmenopausal women using a metabolomics approach. DESIGN/INTERVENTION Subjects were randomized to chia seed (whole or milled) and placebo (poppy seed) groups, and under double-blinded procedures ingested 25 g chia seed or placebo supplements each day for 10 weeks. SUBJECTS Subjects included 62 overweight (body-mass index 25 kg/m(2) and higher), nondiseased, nonsmoking, postmenopausal women, ages 49-75 years, with analysis based on the 56 subjects who completed all phases of the study. OUTCOME MEASURES Pre- and poststudy measures included body mass and composition, blood pressure and augmentation index, serum lipid profile, inflammation markers from fasting blood samples, plasma fatty acids, and metabolic profiling using gas chromatography-mass spectrometry with multivariate statistical methods including principal component analysis and partial least-square discriminant analysis (PLS-DA). RESULTS Plasma α-linolenic acid (N=ALA) increased 58% (interaction effect, p=0.002) and eicosapentaenoic acid (EPA) 39% (p=0.016) in the milled chia seed group (N=14) compared to nonsignificant changes in the whole chia seed (N=16) and placebo (N=26) groups. Pre-to-post measures of body composition, inflammation, blood pressure, augmentation index, and lipoproteins did not differ between chia seed (whole or milled) and placebo groups (all interaction effects, p>0.05). Global metabolic difference scores for each group calculated through PLS-DA models were nonsignificant (Q(2)Y<0.40), and fold-changes for 28 targeted metabolites associated with inflammation and disease risk factors did not differ between groups. CONCLUSIONS Ingestion of 25 g/day milled chia seed compared to whole chia seed or placebo for 10 weeks by overweight women increased plasma ALA and EPA, but had no influence on inflammation or disease risk factors using both traditional and metabolomics-based measures.

The Microbiota Is Essential for the Generation of Black Tea Theaflavins-Derived Metabolites

Background Theaflavins including theaflavin (TF), theaflavin-3-gallate (TF3G), theaflavin-3′-gallate (TF3′G), and theaflavin-3,3′-digallate (TFDG), are the most important bioactive polyphenols in black tea. Because of their poor systemic bioavailability, it is still unclear how these compounds can exert their biological functions. The objective of this study is to identify the microbial metabolites of theaflavins in mice and in humans. Methods and Findings In the present study, we gavaged specific pathogen free (SPF) mice and germ free (GF) mice with 200 mg/kg TFDG and identified TF, TF3G, TF3′G, and gallic acid as the major fecal metabolites of TFDG in SPF mice. These metabolites were absent in TFDG- gavaged GF mice. The microbial bioconversion of TFDG, TF3G, and TF3′G was also investigated in vitro using fecal slurries collected from three healthy human subjects. Our results indicate that TFDG is metabolized to TF, TF3G, TF3′G, gallic acid, and pyrogallol by human microbiota. Moreover, both TF3G and TF3′G are metabolized to TF, gallic acid, and pyrogallol by human microbiota. Importantly, we observed interindividual differences on the metabolism rate of gallic acid to pyrogallol among the three human subjects. In addition, we demonstrated that Lactobacillus plantarum 299v and Bacillus subtilis have the capacity to metabolize TFDG. Conclusions The microbiota is important for the metabolism of theaflavins in both mice and humans. The in vivo functional impact of microbiota-generated theaflavins-derived metabolites is worthwhile of further study.

Bananas as an Energy Source during Exercise: A Metabolomics Approach

This study compared the acute effect of ingesting bananas (BAN) versus a 6% carbohydrate drink (CHO) on 75-km cycling performance and post-exercise inflammation, oxidative stress, and innate immune function using traditional and metabolomics-based profiling. Trained cyclists (N = 14) completed two 75-km cycling time trials (randomized, crossover) while ingesting BAN or CHO (0.2 g/kg carbohydrate every 15 min). Pre-, post-, and 1-h-post-exercise blood samples were analyzed for glucose, granulocyte (GR) and monocyte (MO) phagocytosis (PHAG) and oxidative burst activity, nine cytokines, F2-isoprostanes, ferric reducing ability of plasma (FRAP), and metabolic profiles using gas chromatography-mass spectrometry. Blood glucose levels and performance did not differ between BAN and CHO (2.41±0.22, 2.36±0.19 h, P = 0.258). F2-isoprostanes, FRAP, IL-10, IL-2, IL-6, IL-8, TNFα, GR-PHAG, and MO-PHAG increased with exercise, with no trial differences except for higher levels during BAN for IL-10, IL-8, and FRAP (interaction effects, P = 0.003, 0.004, and 0.012). Of 103 metabolites detected, 56 had exercise time effects, and only one (dopamine) had a pattern of change that differed between BAN and CHO. Plots from the PLS-DA model visualized a distinct separation in global metabolic scores between time points [R2Y(cum) = 0.869, Q2(cum) = 0.766]. Of the top 15 metabolites, five were related to liver glutathione production, eight to carbohydrate, lipid, and amino acid metabolism, and two were tricarboxylic acid cycle intermediates. BAN and CHO ingestion during 75-km cycling resulted in similar performance, blood glucose, inflammation, oxidative stress, and innate immune levels. Aside from higher dopamine in BAN, shifts in metabolites following BAN and CHO 75-km cycling time trials indicated a similar pattern of heightened production of glutathione and utilization of fuel substrates in several pathways.

Serum Metabolic Signatures Induced By a Three-Day Intensified Exercise Period Persist After 14 h of Recovery in Runners

This study investigated changes in the human serum metabolome elicited by a 3-day period of intensified training. Runners (N = 15, mean ± SD age, 35.2 ± 8.7 years) ran for 2.5 h/day on treadmills at ∼70% VO2max for 3 days in a row, with blood samples collected pre-exercise, and immediately and 14 h post-exercise. Samples were analyzed using gas and liquid chromatography/mass spectrometry (GC-MS, LC-MS), with compounds identified based on comparison to more than 2800 purified standards. Repeated measures ANOVA was used to identify metabolites that differed significantly across time, with multiple testing corrected by the false discovery rate (FDR) (q-value). Immediately following the 3-day exercise period, significant 2-fold or higher increases in 75 metabolites were measured, with all but 22 of these metabolites related to lipid/carnitine metabolism, 13 to amino acid/peptide metabolism, 4 to hemoglobin/porphyrin metabolism, and 3 to Krebs cycle intermediates (q-values < 0.001). After a 14 h overnight recovery period, 50 of the 75 metabolites remained elevated, with 8 decreased (primarily amino acid-related metabolites) (q-values < 0.05). Among the top 20 metabolites, the mean fold changes were 12.4 ± 5.3 and 2.9 ± 1.3 immediately and 14-h post-exercise, respectively. Significant decreases (40-70%, q < 0.01) in 22 metabolites (primarily related to lysolipid and bile acid metabolism) were measured post-exercise, with all but 4 of these still decreased after 14 h rest recovery (q < 0.025). Runners experienced a profound systemic shift in blood metabolites related to energy production especially from the lipid super pathway following 3 days of heavy exertion that was not fully restored to pre-exercise levels after 14 h recovery.

The Protective Effects of a Polyphenol-Enriched Protein Powder on Exercise-Induced Susceptibility to Virus Infection

Prolonged and intensive exercise induces transient immunosuppression and is associated with an increased risk and severity of infections. The goal of this study was to characterize the antiviral and antibacterial properties of the bioactive metabolites of a blueberry–green tea‐polyphenol soy protein complex (PSPC) in the serum of supplemented subjects during a 3‐day intensified training period. Long‐distance runners, randomly divided into two groups, ingested 40 g/day PSPC or placebo (soy protein and colorings) for 17 days, with a 3‐day running period inserted at day 14. Blood serum samples were collected pre‐14 days and post‐14 days supplementation, and immediately and 14 h after the third day of running. The post‐exercise serum from both groups significantly promoted the growth of Escherichia coli and Staphylococcus aureus in culture by 20–70%, but returned to normal levels following recovery. Furthermore, the serum from subjects ingesting PSPC did not display antibacterial properties at any time point. In contrast, there was a significant difference in the ability of serum from PSPC‐supplemented versus placebo‐supplemented athletes to protect cells in culture from killing by vesicular stomatitis virus following strenuous exercise. In addition, the serum of subjects who ingested PSPC significantly delayed an exercise‐induced increase in virus replication. These results indicate that polyphenol complexes containing blueberry and green tea have the potential to protect athletes from virus infections following rigorous exercise. Copyright

Metabolomics-Based Analysis of Banana and Pear Ingestion on Exercise Performance and Recovery

Bananas and pears vary in sugar and phenolic profiles, and metabolomics was utilized to measure their influence on exercise performance and recovery. Male athletes (N = 20) cycled for 75 km while consuming water (WATER), bananas (BAN), or pears (PEAR) (0.6 g carbohydrate/kg each hour) in randomized order. UPLC-MS/MS and the library of purified standards maintained by Metabolon (Durham, NC) were used to analyze metabolite shifts in pre- and postexercise (0-h, 1.5-h, 21-h) blood samples. Performance times were 5.0% and 3.3% faster during BAN and PEAR versus WATER (P = 0.018 and P = 0.091, respectively), with reductions in cortisol, IL-10, and total leukocytes, and increases in blood glucose, insulin, and FRAP. Partial Least Square Discriminant Analysis (PLS-DA) showed a distinct separation between trials immediately (R(2)Y = 0.877, Q(2)Y = 0.457) and 1.5-h postexercise (R(2)Y = 0.773, Q(2)Y = 0.441). A total of 107 metabolites (primarily lipid-related) increased more than 2-fold during WATER, with a 48% and 52% reduction in magnitude during BAN and PEAR recovery (P < 0.001). Increases in metabolites unique to BAN and PEAR included fructose and fruit constituents, and sulfated phenolics that were related to elevated FRAP. These data indicate that BAN and PEAR ingestion improves 75-km cycling performance, attenuates fatty acid utilization and oxidation, and contributes unique phenolics that augment antioxidant capacity.

Influence of vitamin D mushroom powder supplementation on exercise-induced muscle damage in vitamin D insufficient high school athletes

Abstract Incidence of vitamin D deficiency is increasing worldwide. The purpose of this study was to determine if supplementation with vitamin D2 from Portobello mushroom powder would enhance skeletal muscle function and attenuate exercise-induced muscle damage in low vitamin D status high school athletes. Participants were randomised to Portobello mushroom powder (600 IU/d vitamin D2) or placebo for 6 weeks. Participants then completed a 1.5-h exercise session designed to induce skeletal muscle damage. Blood samples and measures of skeletal muscle function were taken pre-supplementation, post-supplementation/pre-exercise and post-exercise. Six weeks supplementation with vitamin D2 increased serum 25(OH)D2 by 9.9-fold and decreased serum 25(OH)D3 by 28%. Changes in skeletal muscle function and circulating markers of skeletal muscle damage did not differ between groups. In conclusion, 600 IU/d vitamin D2 increased 25(OH)D2 with a concomitant decrease in 25(OD)D3, with no effect on muscular function or exercise-induced muscle damage in high school athletes.

Vitamin D2 supplementation amplifies eccentric exercise-induced muscle damage in NASCAR pit crew athletes.

This study determined if 6-weeks vitamin D2 supplementation (vitD2, 3800 IU/day) had an influence on muscle function, eccentric exercise-induced muscle damage (EIMD), and delayed onset of muscle soreness (DOMS) in National Association for Stock Car Auto Racing (NASCAR) NASCAR pit crew athletes. Subjects were randomized to vitD2 (n = 13) and placebo (n = 15), and ingested supplements (double-blind) for six weeks. Blood samples were collected and muscle function tests conducted pre- and post-study (leg-back and hand grip dynamometer strength tests, body weight bench press to exhaustion, vertical jump, 30-s Wingate test). Post-study, subjects engaged in 90 min eccentric-based exercise, with blood samples and DOMS ratings obtained immediately after and 1- and 2-days post-exercise. Six weeks vitD2 increased serum 25(OH)D2 456% and decreased 25(OH)D3 21% versus placebo (p < 0.001, p = 0.036, respectively), with no influence on muscle function test scores. The post-study eccentric exercise bout induced EIMD and DOMS, with higher muscle damage biomarkers measured in vitD2 compared to placebo (myoglobin 252%, 122% increase, respectively, p = 0.001; creatine phosphokinase 24 h post-exercise, 169%, 32%, p < 0.001), with no differences for DOMS. In summary, 6-weeks vitD2 (3800 IU/day) significantly increased 25(OH)D2 and decreased 25(OH)D3, had no effect on muscle function tests, and amplified muscle damage markers in NASCAR pit crew athletes following eccentric exercise.

Effects of a freeze-dried juice blend powder on exercise-induced inflammation, oxidative stress, and immune function in cyclists

A freeze-dried fruit and vegetable juice powder (JUICE) was investigated as a countermeasure nutritional strategy to exercise-induced inflammation, oxidative stress, and immune perturbations in trained cyclists. Thirty-four cyclists (25 male, 9 female) were randomized to control (nonJUICE) or JUICE for 17 days. JUICE provided 230 mg·day(-1) of flavonoids, doubling the typical adult daily intake. During a 3-d period of intensified exercise (days 15-17), subjects cycled at 70%-75% V̇O2max for 2.25 h per day, followed by a 15-min time trial. Blood samples were collected presupplementation, post supplementation (pre-exercise), and immediately and 14-h post exercise on the third day of exercise. Samples were analyzed for inflammation (interleukin (IL)-6, IL-8; tumor necrosis factor alpha (TNFα); monocyte chemoattractant protein-1 (MCP-1)), oxidative stress (oxygen radical absorbance capacity (ORAC), ferric reducing ability of plasma (FRAP), reduced and oxidized glutathione, protein carbonyls), and innate immune function (granulocyte (G-PHAG) and monocyte (M-PHAG) phagocytosis and oxidative burst activity). A 2 (group) × 4 (time points) repeated measures ANOVA revealed significant time effects due to 3 days of exercise for IL-6 (396% increase), IL-8 (78% increase), TNFα (12% increase), MCP-1 (30% increase), G-PHAG (38% increase), M-PHAG (36% increase), FRAP (12.6% increase), ORAC (11% decrease at 14 h post exercise), and protein carbonyls (82% increase at 14 h post exercise) (p < 0.01). No significant interaction effects were found for any of the physiological measures. Although providing 695 gallic acid equivalents of polyphenols per day, JUICE treatment for 17 days did not change exercise-induced alterations in inflammation and oxidative stress or immune function in trained cyclists after a 3-day period of overreaching.