Jens Lykkesfeldt

Antioxidant Supplementation Does Not Alter Endurance Training Adaptation

BACKGROUND There is a considerable commercial market, especially within the sports community, claiming the need for antioxidant supplementation. One argument for antioxidant supplementation in sports is that physical exercise is associated with increased reactive oxygen and nitrogen species (RONS) production, which may cause cell damage. However, RONS production may also activate redox-sensitive signaling pathways and transcription factors, which subsequently, may promote training adaptation. PURPOSE Our aim was to investigate the effects of combined vitamin C and E supplementation to healthy individuals on different measures of exercise performance after endurance training. METHODS Using a double-blinded placebo-controlled design, moderately trained young men received either oral supplementation with vitamins C and E (n = 11) or placebo (n = 10) before and during 12 wk of supervised, strenuous bicycle exercise training of a frequency of 5 d x wk(-1). Muscle biopsies were obtained before and after training. RESULTS After the training period, maximal oxygen consumption, maximal power output, and workload at lactate threshold increased markedly (P < 0.01) in both groups. Also, glycogen concentration, citrate synthase, and beta-hydroxyacyl-CoA dehydrogenase activity in the muscle were significantly higher in response to training (P < 0.01) in both groups. However, there were no differences between the two groups concerning any of the physiological and metabolic variables measured. CONCLUSIONS Our results suggest that administration of vitamins C and E to individuals with no previous vitamin deficiencies has no effect on physical adaptations to strenuous endurance training.

Oxidative DNA damage in vivo: Relationship to age, plasma antioxidants, drug metabolism, glutathione-S-transferase activity and urinary creatinine excretion

Oxidative DNA modification has been implicated in development of certain cancers and 8-oxodG, the most abundant and mutagenic DNA modification, has for some time been considered a biomarker of this activity. Urinary excretion of 8-oxodG over 24h has been used to estimate the rate of damage to DNA, and animal studies have supported this rationale. Reported determinants include tobacco smoking, heavy exercise, environmental pollution and individual oxygen consumption. Samples from three published studies were used to determine the association of urinary 8-oxodG excretion with age, plasma antioxidants, the glutathione-S-transferase phenotype and the activity of the xenobiotic metabolising enzyme CYP1A2. In the age range 35-65 years, age was not related to urinary 8-oxodG excretion, and there were no relations to either the glutathione-S-transferase phenotype or to the plasma antioxidants: vitamin C, alpha-tocopherol, beta-carotene, lycopene or coenzyme Q10. The activity of CYP1A2 showed a significant correlation in two of the three studies, as well as a significant correlation of 0.26 (p < 0.05) in the pooled data set. Regression analysis of CYP1A2 activity on 8-oxodG indicated that 33% increase in CYP1A2 activity would correspond to a doubling of 8-oxodG excretion. This finding needs to be confirmed in independent experiments. Spot morning urine samples can under certain circumstances be used to estimate 8-oxodG excretion rate provided that creatinine excretion is unchanged (in paired experiments) or comparable (in un-paired experiments), as evaluated from the correlation between 8-oxodG excretion in 24 h urine samples and in morning spot urine samples corrected for creatinine excretion (r = 0.50, p < 0.05). We conclude that 8-oxodG excretion is determined by factors like oxygen consumption and CYP1A2 activity rather than by factors like plasma antioxidant concentrations.

In vivo toxicity of cationic micelles and liposomes

UNLABELLED This study investigated toxicity of nanocarriers comprised of cationic polymer and lipid components often used in gene and drug delivery, formulated as cationic micelles and liposomes. Rats were injected intravenously with 10, 25 or 100 mg/kg and sacrificed after 24 or 48 h, or 24 h after the last of three intravenous injections of 100 mg/kg every other day. Histological evaluation of liver, lung and spleen, clinical chemistry parameters, and hematology indicated little effect of treatment. DNA strand breaks were increased in the lung and spleen. Further, in the dose response study we found unaltered expression levels of genes in the antioxidant response (HMOX1) and repair of oxidized nucleobases (OGG1), whereas expression levels of cytokines (IL6, CXCL2 and CCL2) were elevated in lung, spleen or liver. The results indicate that assessment of genotoxicity and gene expression add information on toxicity of nanocarriers, which is not obtained by histology and hematology. FROM THE CLINICAL EDITOR This study investigates the toxicity of cationic micelles and liposomes utilized as nanocarriers in gene and drug delivery, demonstrating its effects on the lungs, spleen and liver.

Authors' Perspective: What is the Optimum Intake of Vitamin C in Humans?

The recommended dietary allowance (RDA) of vitamin C has traditionally been based on the prevention of the vitamin C deficiency disease, scurvy. While higher intakes of vitamin C may exert additional health benefits, the limited Phase III randomized placebo-controlled trials (RCTs) of vitamin C supplementation have not found consistent benefit with respect to chronic disease prevention. To date, this has precluded upward adjustments of the current RDA. Here we argue that Phase III RCTs—designed principally to test the safety and efficacy of pharmaceutical drugs—are ill suited to assess the health benefits of essential nutrients; and the currently available scientific evidence is sufficient to determine the optimum intake of vitamin C in humans. This evidence establishes biological plausibility and mechanisms of action for vitamin C in the primary prevention of coronary heart disease, stroke, and cancer; and is buttressed by consistent data from prospective cohort studies based on blood analysis or dietary intake and well-designed Phase II RCTs. These RCTs show that vitamin C supplementation lowers hypertension, endothelial dysfunction, chronic inflammation, and Helicobacter pylori infection, which are independent risk factors of cardiovascular diseases and certain cancers. Furthermore, vitamin C acts as a biological antioxidant that can lower elevated levels of oxidative stress, which also may contribute to chronic disease prevention. Based on the combined evidence from human metabolic, pharmacokinetic, and observational studies and Phase II RCTs, we conclude that 200 mg per day is the optimum dietary intake of vitamin C for the majority of the adult population to maximize the vitamins potential health benefits with the least risk of inadequacy or adverse health effects.

Effect of smoking cessation on plasma ascorbic acid concentration.

Numerous studies have shown that cigarette smokers have a lower plasma concentration of ascorbic acid than non-smokers, but only a few have considered the antioxidant status of ex-smokers.1 We report the first controlled study monitoring the early effect of smoking cessation on the concentration of ascorbic acid in plasma. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the local ethics committee. Two hundred volunteers were recruited through local advertisement and gave signed informed consent. Eligible subjects of both sexes (aged 35 to 65 years) had smoked at least 15 cigarettes a day for one year and declared motivation to stop. Exclusion criteria were known presence of disease; daily intake of drugs, including hormonal contraceptives; antioxidant supplements within the last month; …

Effect of antioxidant supplementation on insulin sensitivity in response to endurance exercise training.

While production of reactive oxygen and nitrogen species (RONS) is associated with some of the beneficial adaptations to regular physical exercise, it is not established whether RONS play a role in the improved insulin-stimulated glucose uptake in skeletal muscle obtained by endurance training. To assess the effect of antioxidant supplementation during endurance training on insulin-stimulated glucose uptake, 21 young healthy (age 29 ± 1 y, BMI 25 ± 3 kg/m(2)) men were randomly assigned to either an antioxidant [AO; 500 mg vitamin C and 400 IU vitamin E (α-tocopherol) daily] or a placebo (PL) group that both underwent a supervised intense endurance-training program 5 times/wk for 12 wk. A 3-h euglycemic-hyperinsulinemic clamp, a maximal oxygen consumption (Vo(2max)) and maximal power output (P(max)) test, and body composition measurements (fat mass, fat-free mass) were performed before and after the training. Muscle biopsies were obtained for determination of the concentration and activity of proteins regulating glucose metabolism. Although plasma levels of vitamin C (P < 0.05) and α-tocopherol (P < 0.05) increased markedly in the AO group, insulin-stimulated glucose uptake increased similarly in both the AO (17.2%, P < 0.05) and the PL (18.9%, P < 0.05) group in response to training. Vo(2max) and P(max) also increased similarly in both groups (time effect, P < 0.0001 for both) as well as protein content of GLUT4, hexokinase II, and total Akt (time effect, P ≤ 0.05 for all). Our results indicate that administration of antioxidants during strenuous endurance training has no effect on the training-induced increase in insulin sensitivity in healthy individuals.

Effect of vitamin C and iron chelation on diesel exhaust particle and carbon black induced oxidative damage and cell adhesion molecule expression in human endothelial cells

Exposure to particulate matter is associated with oxidative stress and risk of cardiovascular diseases. We investigated if vitamin C and desferrioxamine (iron chelator) altered the levels of oxidative stress and expression of cell adhesion molecules upon exposure to diesel exhaust particles (DEP) and carbon black in cultured human umbilical vein endothelial cells (HUVECs). We found that the particles were only slightly cytotoxic in the high concentration ranges. Particle-induced intracellular reactive oxygen species (ROS) production was attenuated by vitamin C administration or iron chelation and particularly when combined (p<0.001). Only desferrioxamine protected the DNA from oxidative damage in terms of strand breaks and formamidopyrimidine DNA glycosylase sensitive sites induced by carbon black (p<0.01). Carbon black and small sized DEP generated from an Euro4 engine increased the surface expression of VCAM-1 and ICAM-1, whereas DEP from an engine representing an old combustion type engine (SRM2975) with larger particles did not affect the expression of cell adhesion molecules. These effects were also attenuated by desferrioxamine but not vitamin C. The study shows that exposure to carbon black and DEP in HUVECs can generate both oxidative stress and expression of cell surface adhesion molecules and that these effects can in part be attenuated by vitamin C and desferrioxamine.

Oxidative stress in brain during experimental bacterial meningitis: differential effects of α-phenyl-tert-butyl nitrone and N-acetylcysteine treatment

Antioxidant treatment has previously been shown to be neuroprotective in experimental bacterial meningitis. To obtain quantitative evidence for oxidative stress in this disease, we measured the major brain antioxidants ascorbate and reduced glutathione, and the lipid peroxidation endproduct malondialdehyde in the cortex of infant rats infected with Streptococcus pneumoniae. Cortical levels of the two antioxidants were markedly decreased 22 h after infection, when animals were severely ill. Total pyridine nucleotide levels in the cortex were unaltered, suggesting that the loss of the two antioxidants was not due to cell necrosis. Bacterial meningitis was accompanied by a moderate, significant increase in cortical malondialdehyde. While treatment with either of the antioxidants alpha-phenyl-tert-butyl nitrone or N-acetylcysteine significantly inhibited this increase, only the former attenuated the loss of endogenous antioxidants. Cerebrospinal fluid bacterial titer, nitrite and nitrate levels, and myeloperoxidase activity at 18 h after infection were unaffected by antioxidant treatment, suggesting that they acted by mechanisms other than modulation of inflammation. The results demonstrate that bacterial meningitis is accompanied by oxidative stress in the brain parenchyma. Furthermore, increased cortical lipid peroxidation does not appear to be the result of parenchymal oxidative stress, because it was prevented by NAC, which had no effect on the loss of brain antioxidants.

Simultaneous determination of urinary free cortisol and 6β-hydroxycortisol by high-performance liquid chromatography to measure human CYP3A activity

The ratio of the hydrophilic metabolite 6 beta-hydroxycortisol to its parent compound cortisol has recently been demonstrated to be a specific marker for human CYP3A oxygenase activity. We have developed a sensitive and simple single-run high-performance liquid chromatographic method for the quantification of urinary free cortisol and 6 beta-hydroxycortisol using dexamethasone as internal standard. The urine samples (1 ml) are applied to Sep-Pak cartridges, which are washed with water and eluted with ethyl acetate-diethyl ether (4:1, v/v). The organic extracts are washed sequentially with alkaline and acidic solutions saturated with sodium sulfate and subsequently concentrated to dryness. After reconstitution in ethanolic water, the samples are analyzed on a reversed-phase gradient system using ultraviolet absorbance detection at 254 nm. The within- and between-day coefficients of variation (C.V.) for the assay where both in the range of 5-10%. The reference interval for the 6 beta-hydroxycortisol/cortisol ratio of eleven healthy non-smoking subjects was 2.77-26.88 with an average of 10.09 +/- 6.89 (S.D.). The method constitutes an improvement over previous methods and is suitable for routine assessment of the 6 beta-hydroxycortisol/cortisol ratio requiring only 1 ml of urine or less.

Putative biomarkers for evaluating antibiotic treatment: an experimental model of porcine Actinobacillus pleuropneumoniae infection

Biomarkers of infection were screened for their possible role as evaluators of antibiotic treatment in an aerosol infection model of porcine pneumonia caused by Actinobacillus pleuropneumoniae (Ap). Following infection of 12 pigs, clinical signs of pneumonia developed within 20 h, whereafter the animals received a single dose of either danofloxacin (2.5mg/kg) or tiamulin (10 mg/kg). To test the discriminative properties of the biomarkers, the dosage regimens were designed with an expected difference in therapeutic efficacy in favour of danofloxacin. Accordingly, the danofloxacin-treated pigs recovered clinically within 24h after treatment, whereas tiamulin-treated animals remained clinically ill until the end of the study, 48 h after treatment. A similar picture was seen for the biomarkers of infection. During the infection period, plasma C-reactive protein (CRP), interleukin-6 and haptoglobin increased, whereas plasma zinc, ascorbic acid and alpha-tocopherol decreased. In the danofloxacin-treated animals, CRP, interleukin-6, zinc, ascorbic acid and alpha-tocopherol reverted significantly towards normalisation within 24h of treatment. In contrast, signs of normalisation were absent (CRP, zinc and ascorbic acid) or less marked (interleukin-6 and alpha-tocopherol) in the tiamulin-treated animals. Plasma haptoglobin remained elevated throughout the study in both groups. This indicates that CRP, zinc, ascorbic acid and to a lesser extent interleukin-6 and alpha-tocopherol might be used to evaluate antibiotic treatment of acute Ap-infection in pigs. The present model provides a valuable tool in the evaluation of antibiotic treatments, offering the advantage of clinical and pathological examinations combined with the use of biochemical infection markers.