Background Glutathione is an endogenous antioxidant found in oxidized (GSSG) and reduced (GSH) forms. Glutathione depletion is indicative of oxidative stress and occurs in various pathological conditions and following extreme exercise activity. Raising blood glutathione concentration has potential to attenuate and prevent chronic disease and also to improve recovery from exercise. There are a number of challenges to achieving this through traditional dietary supplements, and thus there is a need to develop optimized delivery methods to improve blood glutathione status. This study evaluated the effect of a novel glutathione formulation on blood glutathione parameters in healthy individuals. Methods 15 (8 male) healthy individuals (25±5y old, 78.0±14.6kg) participated in a single-blinded randomized placebo-controlled crossover study, with a minimum one-week washout period between treatments. Participants were overnight fasted and administered 1mL of a non-liposomal nano-size glutathione solution (NLNG) containing 200mg of glutathione or 1mL of placebo lacking glutathione. The solution was held in the mouth for 90 seconds before the remainder was swallowed. Blood was collected at baseline, 5, 10, 30, 60 and 120 minutes post-treatment. Protein-bound plasma and erythrocyte lysate concentrations of GSH and GSSG were measured at all time points using previously validated procedures. Linear mixed effects models were used to compare differences between baseline and post-treatment glutathione concentrations between NLNG and placebo for each parameter. Results There was a significant main effect for treatment type, such that increases in GSH concentration in erythrocyte lysate were greater following NLNG than placebo (p = 0.001). Similar significant main effects for treatment were also found for total (protein bound + erythrocyte lysate) GSH (p = 0.015) and GSSG (p = 0.037) concentration, as well as total blood glutathione pool (GSH+GSSG, p = 0.006). Discussion NLNG increased multiple blood glutathione parameters compared to placebo. Future research should examine whether NLNG can attenuate oxidative stress.

Introduction: Nitric oxide (NO) is a vasodilator that increases blood flow by promoting relaxation of endothelium. Dietary nitrate supplementation increases plasma nitrite, a marker of overall NO bioavailability. Previously, acute dietary nitrate supplementation has been shown to reduce oxygen consumption and improve tolerance during submaximal exercise in healthy populations. Less is known about the effect of dietary nitrate on oxygen consumption at rest. Hypothesis: We hypothesized that dietary nitrate supplementation would reduce resting metabolic rate (RMR) and oxidative stress (8-isoprostane) at rest, via enhanced NO bioavailability via the oxygen independent Nitrate-Nitrite-Nitric Oxide pathway in healthy, young males. Methods: In a randomized, double-blind, cross-over study, ten healthy, young males (21 ± 2 years) visited the laboratory on 5 separate occasions. Participants completed informed consent paperwork and underwent protocol familiarization during visit 1. Prior to visits 2 and 4, participants fasted for 12 hours and adhered to an NIH-approved low-nitrate diet for 48 hours. During visits 2 and 4, an initial blood draw was performed to analyze baseline plasma nitrite and 8-isoprostane. Participants then completed a 30-minute resting metabolic rate (RMR) test. Two hours prior to visits 3 and 5, participants consumed either a placebo or dietary nitrate supplement (negligible and 6.2 mmol nitrate, respectively). During visits 3 and 5, participants also had blood drawn for analysis of the previously stated measurements, and completed an RMR test. Visits 2 and 3 were on consecutive days, followed by a week-long washout period between visit 3 and visit 4, while visit 4 and 5 also occurred on consecutive days. Results: Plasma nitrite significantly increased following dietary nitrate consumption compared to baseline values (27.56 ± 7.58 and 1.25 ± 1.51 arbitrary units, respectively). No difference was present between nitrate and baseline measurements for 8-isoprostane (155.75 ± 57.01 and 198.42 ± 66.44 pg/mL, respectively; p=0.55) and RMR (2086.60 ± 202.23 and 2050.00 ± 209.23 kcal/day, respectively; p=0.13). Conclusion: Dietary nitrate supplementation increases plasma nitrite, but does not change resting metabolic rate following an acute dose of dietary nitrate in healthy males. Individuals consuming dietary nitrate as an ergogenic aid during exercise may not, however, experience similar changes in their resting metabolism. The lack of change in oxidative stress may have been associated with the overall health of the cohort examined. Future research should investigate the clinical implications of dietary nitrate in populations with decreased NO bioavailability and associated endothelial disfunction (and elevated oxidative stress). In such populations, dietary nitrate may provide benefit. However, in a healthy cohort, dietary nitrate exerts minimal effects.

Intro: Caffeine intake may elevate blood pressure (BP), while habitual consumption leads to developed tolerance, providing conflicting evidence for incurred changes in BP. Dietary nitrate consumption decreases BP and reduces arterial stiffness in patients with atherosclerosis and hypertension due to increased plasma nitrite and enhanced bioavailability of nitric oxide. However, a decline in BP for a healthy cohort could be an unwanted side effect for normotensive individuals. Less is known about the combined effects of dietary nitrate and caffeine on resting hemodynamics in a healthy population. Hypothesis: We hypothesized that caffeine supplementation would increase systolic blood pressure (SBP) and pulse wave velocity (PWV) while combined dietary nitrate consumption would attenuate this rise. Methods: In a randomized, double-blind, counterbalanced study, eight healthy, young adults who typically consumed some but not more than 500 mg caffeine/day completed 4 separate visits to the laboratory, each visit separated by one week. Prior to each visit, participants consumed a 4-day supplementation regimen of either dietary nitrate (12.4 mmol, NIT) or a placebo nitrate (PLN) combined with either caffeine (3 mg/kg, CAF) or placebo caffeine (PLC); this comprised 1 of 4 treatments. The final dose of NIT or PLN was consumed 2.5 hours prior to assessments while CAF or PLC was consumed 1 hour prior. Participants minimized consumption of dietary nitrate rich foods and caffeine during the 4 days leading up to their scheduled visit. Each visit consisted of measurements of peripheral SBP and diastolic blood pressure (DBP) with aortic central systolic and diastolic blood pressure (cSBP and cDBP, respectively) assessed noninvasively using pulse wave analysis. Measurement of arterial stiffness via applanation tonometry determined PWV. A linear mixed effects model analysis was performed to determine if supplementation regimen influenced hemodynamic parameters. Results: SBP of NIT+CAF (115±6 mmHg), PLN+CAF (117±9 mmHg), NIT+PLC (115±6 mmHg) and PLN+PLC (115±6 mmHg)(p=0.591) and PWV of NIT+CAF (5.5±1.1 m/s), PLN+CAF (5.8±0.7 m/s), NIT+PLC (5.8±0.6 m/s) and PLN+PLC (5.6±1.0 m/s) (p=0.353) were not statistically different between treatments. In addition, DBP (p=0.496), cSBP (p=0.656), cDBP (p=0.548), aortic augmentation index (p=0.054), and resting heart rate (p=0.646) were not different between treatments. Conclusion: Following 4 days of caffeine supplementation, habitual caffeine users did not experience any changes in resting hemodynamics when compared to a placebo supplement. In addition, dietary nitrate did not incur any changes in resting metrics whether or not it was consumed alongside caffeine. In this regard, habitual caffeine users are not affected by 3mg/kg doses of caffeine, and dietary nitrate (12.4 mmol/day) does not further reduce the BP of healthy normotensive adults.

Introduction: Dietary nitrate has been shown to reduce submaximal oxygen consumption (VO 2 ), but less is known about the changes to excess post-exercise oxygen consumption (EPOC). In contrast, caffeine intake increases both exercise VO 2 and EPOC. Minimal research has reported on the combined effects of dietary nitrate/caffeine on exercise and post-exercise metabolism. Hypothesis: Caffeine will elevate exercise VO 2 and EPOC while dietary nitrate will attenuate the change in exercise VO 2 and EPOC. Methods: Seven healthy individuals participated in a double-blind, placebo controlled, crossover experiment. The first of five visits consisted of a maximal volume of oxygen consumption (VO 2max ) treadmill test. Prior to visit 2 - 5, participants consumed either a dietary nitrate (~12.4 mmol, NIT) or placebo nitrate supplement (PLN) combined with either a caffeine (3 mg/kg, CAF) or placebo caffeine (PLC) dose. Supplements were consumed on each of 4 days and the final doses of NIT or PLN and CAF or PLC were consumed 2.5 and 1-hr pre-exercise, respectively. Visits 2 - 5 consisted of a 30-min treadmill run at ~65% VO 2max followed by a 60-min seated recovery. During exercise, VO 2 and heart rate (HR) were measured continuously. During recovery, EPOC, HR, and peripheral (SBP/DBP) and aortic (cSBP/cDBP) blood pressure (via pulse wave analysis) were measured every 20 min. A linear mixed effects model analysis was performed to determine how each supplementation influenced each dependent variable. Treatments (NIT+CAF, CAF+PLN, NIT+PLC, PLN+PLC) and exercise timepoints (10, 20, 30 min) and recovery timepoints (20, 40, and 60 min post-exercise) served as fixed factors. If p<0.05, p ost-hoc pairwise comparisons were performed. Results: Exercise VO 2 (p=0.450) and HR (p=0.622) were not different between treatments at any timepoint. However, EPOC was different between treatments (p<0.001); NIT+PLC (4.0±0.6 ml/kg/min) was significantly lower than NIT+CAF (4.7±0.7 ml/kg/min, p<0.001), CAF+PLN (4.6±0.7 ml/kg/min p=0.001), and PLN+PLC (4.7±0.8 ml/kg/min, p=0.001). Recovery PLN+PLC brachial SBP (117±8 mmHg) was significantly lower than NIT+CAF (122+7 mmHg, p=0.041), CAF+PLN (123+9 mmHg, p=0.003), and NIT+PLC (122+6 mmHg, p=0.013). Recovery treatment differences were found for cSBP (p=0.005); PLN+PLC (104+7 mmHg) was significantly lower than CAF+PLN (109+9 mmHg, p=0.001), and NIT+PLC (108+7 mmHg, p=0.013), but not NIT+CAF (107+7 mmHg, p=0.063). Recovery HR was elevated in PLN+PLC (91+14 bpm) compared to CAF+PLN (86+15 bpm, p=0.029) and NIT+PLC (86+12 bpm, p=0.002). Conclusion: A modest dose of caffeine (3 mg/kg) did not elevate exercise VO 2 or EPOC. Dietary nitrate reduced EPOC and elevated peripheral and aortic SBP in recovery. When caffeine was consumed alongside nitrate, the decrease in EPOC was abolished. Dietary nitrate alone may not be advised to those seeking additional workout caloric expenditure.

Intro: Glutathione is endogenous within human plasma, erythrocyte lysate and is also bound to the protein within plasma. Glutathione mediates redox chemistry and prevents oxidative damage within and around cellular components via reduction of reactive species (e.g. reactive oxygen, nitrogen, or sulfur species). Polyphenols and antioxidants have been shown to improve NO bioavailability which may reduce long term incidence of endothelial dysfunction. Less is known about whether changes in antioxidant capacity augments the risk of developing hypertension. Hypothesis: We hypothesized that acute glutathione supplementation would decrease arterial stiffness and reduce both brachial (bBP) and central blood pressure (cBP) in healthy male and female volunteers. Methods: Six males and six females (25 ± 3 and 22 ± 1 years, respectively) participated in a randomized, double blind, placebo controlled, crossover protocol. On two visits separated by 1 week, following a 12-hour fast, participants consumed either a placebo or glutathione (negligible and 200 mg, respectively) supplement via 90 second sublingual absorption which was then swallowed. Concentrations of oxidized (GSSG) and reduced glutathione (GSH) were spectrophotometrically measured in plasma (protein-bound) and erythrocyte lysate using a kinetic, enzymatic assay. Arterial stiffness was measured via pulse wave velocity (PWV) using applanation tonometry, and cBP was determined non-invasively using pulse wave analysis. All data were recorded before supplementation (baseline) and at 10, 30, 60 and 120 minutes post-consumption. Results: Linear mixed effect models revealed a significant (p<0.01) increase in total glutathione (GSH+GSSG) in the supplement group compared to placebo across all post-supplementation time points with the greatest increase occurring at 120 minutes (mean 99.0; 95%CI: 7.9,190.1). At 120 minutes post-consumption, no difference was present between glutathione and placebo groups for PWV (5.86 ± 1.19 and 6.08 ± 1.25 m/s, respectively; p=0.43), resting heart rate (52.95 ± 3.55 and 55.83 ± 6.36, respectively; p=0.16), systolic bBP (123.05 ± 12.75 and 123.13 ± 14.52 mmHg; p=0.22), diastolic bBP (71.81 ± 7.87 and 74.21 ± 6.53; p=0.48), systolic cBP (108.05 ± 10.45 and 108.68 ± 11.14 mmHg, respectively; p=0.11) and diastolic cBP (72.03 ± 7.82 and 74.94 ± 6.42 mmHg, respectively; p=0.46). Conclusion: Young healthy males and females experienced an increase in circulating humoral antioxidants in response to glutathione supplementation. However, supplementation had minimal effects on resting hemodynamics. Future research should examine glutathione supplementation’s effect in participants with decreased antioxidant capacity and increased oxidative stress including patients with known disease such as hypertension or peripheral artery disease.