Scott W. Leonard

Oxidative stress in athletes during extreme endurance exercise

Despite the many known health benefits of exercise, there is a body of evidence suggesting that endurance exercise is associated with oxidative stress. To determine whether extreme endurance exercise induces lipid peroxidation, 11 athletes (3 females, 8 males) were studied during a 50 km ultramarathon (trial 1) and during a sedentary protocol (trial 2) 1 month later. The evening before each trial, with dinner, subjects consumed 75 mg each d(3)-RRR and d(6)-all rac-alpha-tocopheryl acetates. Blood was obtained at baseline, 30 min pre-race, mid-race, post-race, 1 h post-race, 24 h post-race, and at corresponding times during trial 2. All 11 subjects completed the race; average run time was 391 +/- 23 min. Plasma F(2)-isoprostanes increased from 75 +/- 7 pg/ml at pre-race to 131 +/- 17 (p <.02) at post-race, then returned to baseline at 24 h post-race; F(2)-isoprostanes were unchanged during trial 2. Deuterated alpha-tocopherol disappearance rates were faster (2.8 x 10(-4) +/- 0.2 x 10(-4)) during the race compared to the sedentary trial (2.3 x 10(-4) +/- 0.2 x 10(-4); p <.03). These data suggest that extreme endurance exercise results in the generation of lipid peroxidation with a concomitant increase in vitamin E disappearance.

Gamma-tocopherol supplementation alone and in combination with alpha-tocopherol alters biomarkers of oxidative stress and inflammation in subjects with metabolic syndrome

Metabolic syndrome (MetS) is associated with increased incidence of diabetes and cardiovascular disease (CVD). Prospective clinical trials with alpha-tocopherol (AT) have not yielded positive results. Because AT supplementation decreases circulating gamma-tocopherol (GT), we evaluated supplementation with GT (800 mg/day), AT (800 mg/day), the combination or placebo for 6 weeks alone AT and GT concentrations, biomarkers of oxidative stress, and inflammation in subjects with MetS (n=20/group). Plasma AT and GT levels increased following supplementation with AT alone or GT alone or in combination. AT supplementation significantly decreased GT levels. Urinary alpha- and gamma-CEHC, metabolites of the respective Ts, also increased correspondingly, i.e., alpha-CEHC with AT and gamma-CEHC with GT supplementation, compared to placebo. HsCRP levels significantly decreased in the combined AT+GT group. LPS-activated whole blood release of IL-1 and IL-6 did not change. There was a significant decrease in TNF with AT alone or in combination with GT. Plasma MDA/HNE and lipid peroxides were significantly decreased with AT, GT, or in combination. Nitrotyrosine levels were significantly decreased only with GT or GT+AT but not with AT compared to placebo. Thus, the combination of AT and GT supplementation appears to be superior to either supplementation alone on biomarkers of oxidative stress and inflammation and needs to be tested in prospective clinical trials to elucidate its utility in CVD prevention.

Zinc Deficiency Affects DNA Damage, Oxidative Stress, Antioxidant Defenses, and DNA Repair in Rats

Approximately 12% of Americans do not consume the Estimated Average Requirement for zinc and could be at risk for marginal zinc deficiency. Zinc is an essential component of numerous proteins involved in the defense against oxidative stress and DNA damage repair. Studies in vitro have shown that zinc depletion causes DNA damage. We hypothesized that zinc deficiency in vivo causes DNA damage through increases in oxidative stress and impairments in DNA repair. Sprague-Dawley rats were fed zinc-adequate (ZA; 30 mg Zn/kg) or severely zinc-deficient (ZD; <1 mg Zn/kg) diets or were pair-fed zinc-adequate diet to match the mean feed intake of ZD rats for 3 wk. After zinc depletion, rats were repleted with a ZA diet for 10 d. In addition, zinc-adequate (MZA 30 mg Zn/kg) or marginally zinc-deficient (MZD; 6 mg Zn/kg) diets were given to different groups of rats for 6 wk. Severe zinc depletion caused more DNA damage in peripheral blood cells than in the ZA group and this was normalized by zinc repletion. We also detected impairments in DNA repair, such as compromised p53 DNA binding and differential activation of the base excision repair proteins 8-oxoguanine glycosylase and poly ADP ribose polymerase. Importantly, MZD rats also had more DNA damage and higher plasma F(2)-isoprostane concentrations than MZA rats and had impairments in DNA repair functions. However, plasma antioxidant concentrations and erythrocyte superoxide dismutase activity were not affected by zinc depletion. These results suggest interactions among zinc deficiency, DNA integrity, oxidative stress, and DNA repair and suggested a role for zinc in maintaining DNA integrity.

Vitamin e kinetics and the function of tocopherol regulatory proteins

Plasma and tissue alpha-tocopherol concentrations are remarkably stable, which suggests that they are regulated. alpha-Tocopherol transfer protein, tocopherol-associated protein, and tocopherol-binding protein bind alpha-tocopherol. These proteins might function as tocopherol regulatory proteins, although only tocopherol transfer protein has been shown to influence plasma and tissue alpha-tocopherol concentrations. Tissue alpha-tocopherol concentrations likely depend on tocopherol regulatory protein function and tissue lipid content, vitamin E uptake and efflux, oxidative stress, and interactions between vitamin E and other antioxidants. Pharmacokinetic models often divide tissues into rapidly perfused, slowly perfused, and very slowly perfused compartments. Tissue vitamin E concentrations might equilibrate more rapidly in tissues with greater perfusion, greater vitamin E uptake, increased amounts or activities of tocopherol regulatory protein, and lower lipid contents. The rate at which tissue concentrations approach equilibrium, however, does not predict the final equilibrium concentrations because of redistribution among tissues. Redistribution of vitamin E to adipose tissue from other tissues may be significant. Intracellular trafficking of vitamin E might occur in conjunction with membrane recycling because membrane constituents rapidly recycle between the plasma membrane and intracellular endocytic compartments. Thus, tocopherol regulatory proteins may modulate rather than directly regulate vitamin E tissue distribution and intracellular trafficking.

α- and γ-tocotrienols are metabolized to carboxyethyl-hydroxychroman derivatives and excreted in human urine

Limited information is available regarding metabolism of vitamin E forms, especially the tocotrienols. Carboxyethyl-hydroxychromans (α-and γ-CEHC) are human urinary metabolites of α- and γ-tocopherols, respectively. To evaluate whether tocotrienols are also metabolized and excreted as urinary CEHC, urine was monitored following tocotrienol supplementation. Complete (24 h) urine collections were obtained for 2 d prior to (baseline), the day of, and 2 d after human subjects (n=6) ingested tocotrienol supplements. The subjects consumed 125 mg γ-tocotrienyl acetate the first week, then the next week 500 mg; then 125 mg α-tocotrienyl acetate was administered the third week, followed by 500 mg the fourth week. Urinary α- and γ-CEHC were measured by high-performance liquid chromatography with electrochemical detection. Urinary γ-CEHC levels rose about four- to sixfold in response to the two doses of γ-tocotrienol and then returned to baseline the following day. Significant (P<0.0001) increases in urinary α-CEHC were observed only following ingestion of 500 mg α-tocotrienyl acetate. Typically, 1–2% of α-tocotrienyl acetates or 4–6% of γ-tocotrienyl acetates were recovered as their respective urinary CEHC metabolites. A γ-CEHC excretion time course showed an increase in urinary γ-CEHC at 6 h and a peak at 9 h following ingestion of 125 mg γ-tocotrienyl acetate. In summary, tocotrienols, like tocopherols, are metabolized to CEHC; however, the quantities excreted in human urine are small in relation to dose size.

Vitamin E supplementation increases circulating vitamin E metabolites tenfold in end-stage renal disease patients.

Vitamin E supplementation could elevate circulating vitamin E metabolites while modulating oxidative and inflammatory status in end-stage renal failure patients undergoing hemodialysis. Plasma concentrations of carboxyethyl-hydroxychromanols (α-and γ-CEHC), ascorbic acid, α-and γ-tocopherols, E2-isoprostanes, and inflammatory biomarkers [tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), ferritin, and C-reactive protein (CRP)] were measured in blood samples obtained from patients (n=11) before and after dialysis on two occasions prior to, and at 1 and 2 mon of daily vitamin E supplementation (400 IU RRR-α-tocopherol). Supplementation nearly doubled plasma α-tocopherol concentrations (from 18±0.5 to 31±1.7 μM, P<0.0001), whereas γ-tocopherol concentrations decreased (from 2.8±0.3 to 1.7±0.2 μM, P=0.001). Serum α-CEHC increased 10-fold from 68±3 to 771±175 nM (P<0.0001), and γ-CEHC increased from 837±164 to 1136±230 nM (P=0.008). Vitamin E supplementation also increased postdialysis hematocrits from 38±1% to 41±1% (P<0.001). Dietary antioxidant intakes (vitamins E and C) were low in most subjects; plasma ascorbic acid levels (88±27 μM) decreased significantly with dialysis (33±11 μM, P=0.01). Plasma Il-6, CRP, TNF-α, and free F2-isoprostane concentrations were elevated throughout the study. There is a complex relationship between chronic inflammation and oxidative stress that is not mitigated by short-term vitamin E supplementation. Importantly, serum vitamin E metabolite concentrations that increased 10-fold within 30 d of supplementation did not increase further, suggesting routes other than urine for removal of metabolites.

γ-Tocopherol nebulization by a lipid aerosolization device improves pulmonary function in sheep with burn and smoke inhalation injury

Fire accident victims who sustain both thermal injury to skin and smoke inhalation have gross evidence of systemic and pulmonary oxidant damage and acute lung injury. We hypothesized that gamma-tocopherol (gT), a reactive O(2) and N(2) scavenger, when delivered into the airway, would attenuate lung injury induced by burn and smoke inhalation. Acute lung injury was induced in chronically prepared, anesthetized sheep by 40% total burn surface area, third-degree skin burn and smoke insufflation (48 breaths of cotton smoke, <40 degrees C). The study groups were: (1) Sham (not injured, flaxseed oil (FO)-nebulized, n=6); (2) SA-neb (injured, saline-nebulized, n=6); (3) FO-neb (injured, FO-nebulized, n=6); and (4) gT+FO-neb (injured, gT and FO-nebulized, n=6). Nebulization was started 1 h postinjury, and 24 ml of FO with or without gT (51 mg/ml) was delivered into airways over 47 h using our newly developed lipid aerosolization device (droplet size: 2.5-5 microm). The burn- and smoke inhalation-induced pathological changes seen in the saline group were attenuated by FO nebulization; gT addition further improved pulmonary function. Pulmonary gT delivery along with a FO source may be a novel effective treatment strategy in management of patients with acute lung injury.

Antioxidant Supplements Reduced Oxidative Stress and Stabilized Liver Function Tests but Did Not Reduce Inflammation in a Randomized Controlled Trial in Obese Children and Adolescents

Oxidative stress and low-grade systemic inflammation may contribute to the pathogenesis of obesity-induced comorbidities, including nonalcoholic fatty liver disease. Increasing intake of dietary antioxidants might be beneficial, but there are few data in obese children. To examine the effect of antioxidant supplementation on biomarkers of oxidative stress, inflammation, and liver function, we randomly assigned overweight or obese children and adolescents (n = 44; mean ± SD age: 12.7 ± 1.5 y) participating in a lifestyle modification program to a 4-mo intervention with daily antioxidants (vitamin E, 400 IU; vitamin C, 500 mg; selenium, 50 μg) or placebo. We measured anthropometrics, antioxidant status, oxidative stress (F(2)-isoprostanes, F(2)-isoprostane metabolites), inflammation, liver enzymes, fasting insulin and glucose, and lipid profile at baseline and endpoint. There was a significant treatment effect of antioxidant supplementation on antioxidant status [α-tocopherol, β = 23.2 (95% CI: 18.0, 28.4); ascorbic acid, β = 70.6 (95% CI: 51.7, 89.4); selenium, β = 0.07 (95% CI: 0.01, 0.12)] and oxidative stress [8-iso-prostaglandin F2α, β = -0.11 (95% CI: -0.19, -0.02)] but not on any of the inflammatory markers measured. There was a significant treatment effect on alanine aminotransferase [β = -0.13 (95% CI: -0.23, -0.03)], a trend toward a significant effect on aspartate aminotransferase [β = -0.04 (95% CI: -0.09, 0.01)], and no significant effect on γ-glutamyltransferase [β = -0.03 (95% CI: -0.11, 0.06)]. In summary, antioxidant supplementation for 4 mo improved antioxidant-oxidant balance and modestly improved liver function tests; however, it did not reduce markers of systemic inflammation despite significant baseline correlations between oxidative stress and inflammation. The study was registered at clinicaltrials.gov as NCT01316081.

Absence of VLDL secretion does not affect α-tocopherol content in peripheral tissues

α-Tocopherol is a lipid-soluble antioxidant that helps to prevent oxidative damage to cellular lipids. α-Tocopherol is absorbed by the intestine and is taken up and retained by the liver; it is widely presumed that α-tocopherol is then delivered to peripheral tissues by the secretion of VLDL. To determine whether VLDL secretion is truly important for the delivery of α-tocopherol to peripheral tissues, we examined α-tocopherol metabolism in mice that lack microsomal triglyceride transfer protein (Mttp) expression in the liver and therefore cannot secrete VLDL (MttpΔ/Δ mice). MttpΔ/Δ mice have low plasma lipid levels and increased stores of lipids in the liver. Similarly, α-tocopherol levels in the plasma were lower in MttpΔ/Δ mice than in controls, whereas hepatic α-tocopherol stores were higher. However, α-tocopherol levels in the peripheral tissues of MttpΔ/Δ mice were nearly identical to those of control mice, suggesting that VLDL secretion is not critical for the delivery of α-tocopherol to peripheral tissues. When fed a diet containing deuterated α-tocopherol, MttpΔ/Δ and control mice had similar incorporation of deuterated α-tocopherol into plasma and various peripheral tissues. We conclude that the absence of VLDL secretion has little effect on the stores of α-tocopherol in peripheral tissues, at least in the mouse.

Vitamin E and C supplementation does not ameliorate muscle dysfunction after anterior cruciate ligament surgery

Muscle atrophy and weakness are predominant impairments after anterior cruciate ligament (ACL) surgical repair. We tested the hypothesis that vitamin E and C supplementation will improve recovery from ACL injury. Men undergoing elective ACL surgery were randomly assigned to twice-daily supplements of either antioxidants (AO; vitamins E and C, n=10) or matching placebos (n=10) from 2 weeks before until 3 months after surgery. Each subject provided several fasting blood draws, two muscle biopsies from the thigh muscle of the injured limb, and strength and thigh circumference measurements of the lower limbs. Muscle atrophy was apparent in both groups before and several days after surgery. Compared with baseline measurements, peak isometric force of the injured limb increased significantly (P<0.05) by 3 months postsurgery in both treatment groups; however, AO supplementation did not augment these strength gains. By contrast, baseline plasma ascorbic acid concentrations correlated (r=0.59, P=0.006) with subsequent improvement in the strength of the injured limb. In summary, vitamin E and C supplementation was ineffective in potentiating the improvement in force production by the injured limb; however, baseline vitamin C status was associated with beneficial outcomes in strength, suggesting that long-term dietary habits are more effective than short-term supplements.