Nasser E. Bastani

Vitamin C and E supplementation hampers cellular adaptation to endurance training in humans: a double-blind, randomised, controlled trial

Recent studies have indicated that antioxidant supplementation may blunt adaptations to exercise, such as mitochondrial biogenesis induced by endurance training. However, studies in humans are sparse and results are conflicting. Isolated vitamin C and E supplements are widely used, and unravelling the interference of these vitamins in cellular and physiological adaptations to exercise is of interest to those who exercise for health purposes and to athletes. Our results show that vitamin C and E supplements blunted the endurance training‐induced increase of mitochondrial proteins (COX4), which is important for improving muscular endurance. Training‐induced increases in V̇O2 max and running performance were not detectably affected by the supplementation. The present study contributes to understanding of how antioxidants may interfere with adaptations to exercise in humans, and the results indicate that high dosages of vitamins C and E should be used with caution.

Determination of 8‐epi PGF2α concentrations as a biomarker of oxidative stress using triple‐stage liquid chromatography/tandem mass spectrometry

F2-isoprostanes are a family of prostaglandin F2-like compounds that are formed by free-radical-catalyzed peroxidation of arachidonic acid. Several F2-isoprostanes, but in particular 8-epi PGF(2alpha), are widely used as oxidative stress biomarkers. An analytical method based on liquid chromatography with negative electrospray ionization (ESI) coupled to tandem mass spectrometric detection (LC/MS/MS) was developed for the determination of 8-epi PGF(2alpha) concentrations in human plasma, whole blood, erythrocytes and urine. 8-epi PGF(2alpha)-d4, a stable isotope derivative of 8-epi PGF(2alpha), was used as an internal standard (IS). A 50 microL sample was focused on-column and separated on two 3 microm particle size SUPELCOSIL ABZ+Plus HPLC columns (15 cm x 4.6 mm and 7.5 cm x 4.6 mm) connected in series. An Applied Biosystems 4000 Q TRAP LC/MS/MS system with ESI was operated in multiple reaction monitoring (MRM) mode with the precursor-to-product ion transitions m/z 353.4 --> 193.1 (8-epi PGF(2alpha)), 357.4 --> 197.1 (8-epi PGF(2alpha)-d4), used for quantification. The assay was fully validated and found to have adequate accuracy, precision, linearity, sensitivity and selectivity. The mass limit of detection (mLOD) was 1 pg of analyte eluting from the column. The assay has been successfully applied to the analysis of human plasma, whole blood, erythrocytes and urine samples.

Retinoic acid signalling is activated in the postischemic heart and may influence remodelling

Background All-trans retinoic acid (atRA), an active derivative of vitamin A, regulates cell differentiation, proliferation and cardiac morphogenesis via transcriptional activation of retinoic acid receptors (RARs) acting on retinoic acid response elements (RARE).We hypothesized that the retinoic acid (RA) signalling pathway is activated in myocardial ischemia and postischemic remodelling. Methods and Findings Myocardial infarction was induced through ligating the left coronary artery in mice. In vivo cardiac activation of the RARs was measured by imaging RARE-luciferase reporter mice, and analysing expression of RAR target genes and proteins by real time RT-PCR and western blot. Endogenous retinoids in postinfarcted hearts were analysed by triple-stage liquid chromatography/tandem mass spectrometry. Cardiomyocytes (CM) and cardiofibroblasts (CF) were isolated from infarcted and sham operated RARE luciferase reporter hearts and monitored for RAR activity and expression of target genes. The effect of atRA on CF proliferation was evaluated by EdU incorporation. Myocardial infarction increased thoracic RAR activity in vivo (p<0.001), which was ascribed to the heart through ex vivo imaging (p = 0.002) with the largest signal 1 week postinfarct. This was accompanied by increased cardiac gene and protein expression of the RAR target genes retinol binding protein 1 (p = 0.01 for RNA, p = 0,006 for protein) and aldehyde dehydrogenase 1A2 (p = 0.04 for RNA, p = 0,014 for protein), while gene expression of cytochrome P450 26B1 was downregulated (p = 0.007). Concomitantly, retinol accumulated in the infarcted zone (p = 0.02). CM and CF isolated from infarcted hearts had higher luminescence than those from sham operated hearts (p = 0.02 and p = 0.008). AtRA inhibited CF proliferation in vitro (p = 0.02). Conclusion The RA signalling pathway is activated in postischemic hearts and may play a role in regulation of damage and repair during remodelling.

Vitamin C and E supplementation blunts increases in total lean body mass in elderly men after strength training

The aim of this study was to investigate the effects of vitamin C and E supplementation on changes in muscle mass (lean mass and muscle thickness) and strength during 12 weeks of strength training in elderly men. Thirty‐four elderly males (60–81 years) were randomized to either an antioxidant group (500 mg of vitamin C and 117.5 mg vitamin E before and after training) or a placebo group following the same strength training program (three sessions per week). Body composition was assessed with dual‐energy X‐ray absorptiometry and muscle thickness by ultrasound imaging. Muscle strength was measured as one‐repetition maximum (1RM). Total lean mass increased by 3.9% (95% confidence intervals: 3.0, 5.2) and 1.4% (0, 5.4) in the placebo and antioxidant groups, respectively, revealing larger gains in the placebo group (P = 0.04). Similarly, the thickness of m. rectus femoris increased more in the placebo group [16.2% (12.8, 24.1)] than in the antioxidant group [10.9% (9.8, 13.5); P = 0.01]. Increases of lean mass in trunk and arms, and muscle thickness of elbow flexors, did not differ significantly between groups. With no group differences, 1RM improved in the range of 15–21% (P < 0.001). In conclusion, high‐dosage vitamin C and E supplementation blunted certain muscular adaptations to strength training in elderly men.

Effects of vitamin C and E supplementation on endogenous antioxidant systems and heat shock proteins in response to endurance training.

Reactive oxygen and nitrogen species are important signal molecules for adaptations to training. Due to the antioxidant properties of vitamin C and E, supplementation has been shown to blunt adaptations to endurance training. In this study, we investigated the effects of vitamin C and E supplementation and endurance training on adaptations in endogenous antioxidants and heat shock proteins (HSP). Thirty seven males and females were randomly assigned to receive Vitamin C and E (C + E; C: 1000 mg, E: 235 mg daily) or placebo (PLA), and underwent endurance training for 11 weeks. After 5 weeks, a subgroup conducted a high intensity interval session to investigate acute stress responses. Muscle and blood samples were obtained to investigate changes in proteins and mRNA related to the antioxidant and HSP system. The acute response to the interval session revealed no effects of C + E supplementation on NFκB activation. However, higher stress responses to exercise in C + E group was indicated by larger translocation of HSPs and a more pronounced gene expression compared to PLA. Eleven weeks of endurance training decreased muscle GPx1, HSP27 and αB‐crystallin, while mnSOD, HSP70 and GSH remained unchanged, with no influence of supplementation. Plasma GSH increased in both groups, while uric acid decreased in the C + E group only. Our results showed that C + E did not affect long‐term training adaptations in the antioxidant‐ and HSP systems. However, the greater stress responses to exercise in the C + E group might indicate that long‐term adaptations occurs through different mechanisms in the two groups.

Dried blood spot (DBS) sample collection for determination of the oxidative stress biomarker 8‐epi‐PGF2α in humans using liquid chromatography/tandem mass spectrometry

RATIONALE F2-isoprostanes are a series of prostaglandin F2-like compounds that are formed by free-radical-catalyzed peroxidation of arachidonic acid (ARA). Several F2-isoprostanes, but in particular 8-epi-PGF(2α), are widely used as oxidative stress biomarkers. In this study we have developed an analytical tool for finger-tip blood sampling and analysis of 8-epi-PGF(2α) from dried blood spots (DBS). METHODS We have applied solid-phase extraction (SPE) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the extraction, separation and detection of 8-epi-PGF(2α) in DBS and have studied the stability of this marker using the DBS collection platform. RESULTS The mass limit of detection (mLOD) for 8-epi-PGF(2α) extracted from DBS samples was 1.5 pg while the concentration limit of detection (cLOD) and concentration limit of quantitation (cLOQ) were 6 pg/mL and 18 pg/mL, respectively. All values based on triplicate analysis. Sufficient stability of 8-epi-PGF(2α) in DBS was achieved by preconditioning DBS paper with vitamin E and BHT. CONCLUSIONS The developed method is sensitive, specific, robust, efficient, and can accurately measure endogenous concentrations of 8-epi-PGF(2α) in DBS. Thus, it offers an analytical approach to measure 8-epi-PGF(2α) by a novel sample collection technique that is less invasive and costly than conventional techniques.

Compliance, tolerability and safety of two antioxidant-rich diets: a randomised controlled trial in male smokers.

It has been suggested that antioxidants attenuate oxidative stress and prevent oxidative stress-related diseases. Paradoxically, randomised controlled trials (RCT) using pharmacological doses of antioxidant supplements have demonstrated harmful effects in smokers. The aim of the present study was to test the compliance, tolerability and safety of two food-based antioxidant-rich diets in smokers. One of the diets provided antioxidants at levels similar to that used in RCT using supplements which previously have generated harmful effects. The present study followed a randomised, parallel-arm dietary intervention for 8 weeks (n 102) in male smokers (age ≥ 45 years). Participants were randomised to either antioxidant-rich diet, kiwi fruit or control groups. The antioxidant-rich foods provided about 300 mmol antioxidants/week from a wide range of plant-based food items. The kiwi fruit group consumed three kiwi fruits/d. Compliance to both diets was good. Only mild, undesirable events were reported by a minority of the participants. The safety of both diets was demonstrated as no potentially harmful or pro-oxidative effects were observed. In the antioxidant-rich diet group, the mean intake of antioxidants increased from 30 mmol/d at baseline to 62 mmol/d during the intervention. In conclusion, we have demonstrated that male smokers can comply with two food-based antioxidant-rich diets. Furthermore, the present study is the first to demonstrate the tolerability and safety of dietary antioxidants at levels similar to dosages provided in RCT using supplements. Such diets may be useful in future studies investigating whether dietary antioxidants may reduce oxidative stress and related diseases.

Tomato Paste Alters NF-κB and Cancer-Related mRNA Expression in Prostate Cancer Cells, Xenografts, and Xenograft Microenvironment

Tomatoes may protect against prostate cancer development, possibly through targeting signaling pathways such as nuclear factor-κB (NF-κB). We investigated whether tomato paste could modulate NF-κB activity and cancer-related gene expression in human derived prostate cancer cells (PC3) and PC3 xenografts. PC3-cells were stably transduced with an NF-κB-luciferase construct, and treated with tomato extracts or vehicle control. Nude mice bearing PC3 xenografts were fed a Western-like diet with or without 10% tomato paste for 6.5 wk. The tomato diet significantly inhibited TNFα stimulated NF-κB activity in cultured PC3 cells, and modulated the expression of genes associated with inflammation, apoptosis, and cancer progression. Accumulation of lycopene occurred in liver, xenografts, and serum of mice fed tomato diet. Tomato paste in the diet did not affect tumor size in mice; however, there was a trend toward inhibition of NF-κB activity in the xenografts. The effect of tomato on gene expression was most prominent in the xenograft microenvironment, where among others NFKB2, STAT3, and STAT6 showed higher expression levels after tomato treatment. Our findings support biological activity of tomatoes in cancer-related inflammation.

Vitamin C in Cultured Human (HeLa) Cells: Lack of Effect on DNA Protection and Repair

Aims: Dietary antioxidants, including vitamin C, may be in part responsible for the cancer-preventive effects of fruits and vegetables. Human intervention trials with clinical endpoints have failed to confirm their protective effects, and mechanistic studies have given inconsistent results. Our aim was to investigate antioxidant/ pro-oxidant effects of vitamin C at the cellular level. Experimental approach: We have used the comet assay to investigate effects of vitamin C on DNA damage, antioxidant status, and DNA repair, in HeLa (human tumor) cells, and HPLC to measure uptake of vitamin C into cells. Results: Even at concentrations in the medium as high as 200 μM, vitamin C did not increase the background level of strand breaks or of oxidized purines in nuclear DNA. Vitamin C is taken up by HeLa cells and accumulates to mM levels. Preincubation of cells with vitamin C did not render them resistant to strand breakage induced by H2O2 or to purine oxidation by photosensitizer plus light. Vitamin C had no effect on the rate of repair of strand breaks or oxidized bases by HeLa cells. However, vitamin C at a concentration of less than 1 μM, or extract from cells preincubated for 6 h with vitamin C, was able to induce damage (strand breaks) in lysed, histone-depleted nuclei (nucleoids). Conclusion: In these cultured human cells, vitamin C displays neither antioxidant nor pro-oxidant properties; nor does it affect DNA strand break or base excision repair.

Inhibitory effects of kiwifruit extract on human platelet aggregation and plasma angiotensin-converting enzyme activity.

Abstract Previous human studies suggest that supplementation with kiwifruits lowers several cardiovascular risk factors such as platelet hyperactivity, blood pressure and plasma lipids. The cardiovascular health benefit of fruit and vegetables is usually attributed to the complex mixture of phytochemicals therein; however, kiwifruit’s cardioprotective factors are not well studied. In this study, we investigated the effects of kiwifruit extract on human blood platelet aggregation and plasma angiotensin-converting enzyme (ACE) activity. A sugar-free, heat-stable aqueous extract with molecular mass less than 1000 Da was prepared from kiwifruits. Typically, 100 g kiwifruits produced 66.3 ± 5.8 mg (1.2 ± 0.1 mg CE) of sugar-free kiwifruit extract (KFE). KFE inhibited both human platelet aggregation and plasma ACE activity in a dose-dependent manner. KFE inhibited platelet aggregation in response to ADP, collagen and arachidonic acid, and inhibitory action was mediated in part by reducing TxA2 synthesis. The IC50 for ADP-induced platelet aggregation was 1.6 ± 0.2 mg/ml (29.0 ± 3.0 μg CE/ml), whereas IC50 for serum ACE was 0.6 ± 0.1 mg/ml (11.0 ± 1.2 μg CE/ml). Consuming 500 mg of KFE (9.0 mg CE) in 10 g margarine inhibited ex vivo platelet aggregation by 12.7%, 2 h after consumption by healthy volunteers (n = 9). All these data indicate that kiwifruit contains very potent antiplatelet and anti-ACE components. Consuming kiwifruits might be beneficial as both preventive and therapeutic regime in cardiovascular disease.