Frank McArdle

Effect of Vitamin C Supplements on Antioxidant Defence and Stress Proteins in Human Lymphocytes and Skeletal Muscle

Oxidative stress induces adaptations in the expression of protective enzymes and heat shock proteins (HSPs) in a variety of tissues. We have examined the possibility that supplementation of subjects with the nutritional antioxidant, vitamin C, influences the ability of lymphocytes to express protective enzymes and HSPs following exposure to an exogenous oxidant and the response of skeletal muscle to the physiological oxidative stress that occurs during exercise in vivo. Our hypothesis was that an elevation of tissue vitamin C content would reduce oxidant‐induced expression of protective enzymes and HSP content. Lymphocytes from non‐supplemented subjects responded to hydrogen peroxide with increased activity of superoxide dismutase (SOD) and catalase, and HSP60 and HSP70 content over 48 h. Vitamin C supplementation at a dose of 500 mg day−1 for 8 weeks was found to increase the serum vitamin C concentration by ∼50 %. Lymphocytes from vitamin C‐supplemented subjects had increased baseline SOD and catalase activities and an elevated HSP60 content. The SOD and catalase activities and the HSP60 and HSP70 content of lymphocytes from supplemented subjects did not increase significantly in response to hydrogen peroxide. In non‐supplemented subjects, a single period of cycle ergometry was found to significantly increase the HSP70 content of the vastus lateralis. Following vitamin C supplementation, the HSP70 content of the muscle was increased at baseline with no further increase following exercise. We conclude that, in vitamin C‐supplemented subjects, adaptive responses to oxidants are attenuated, but that this may reflect an increased baseline expression of potential protective systems against oxidative stress (SOD, catalase and HSPs).

Ascorbic acid supplementation does not attenuate post-exercise muscle soreness following muscle-damaging exercise but may delay the recovery process.

Exercise involving lengthening muscle actions, such as downhill running, results in delayed onset muscle soreness (DOMS), which may be attributable to reactive oxygen species (ROS). Although exercise causes oxidative stress, any link between ROS and DOMS remains speculative. There is emerging evidence to suggest that ROS play an important physiological role, assisting in the recovery process and protecting the cell from future damage; however, this has not been fully established. Despite this uncertainty as to the precise role of ROS, attempts to prevent post-exercise ROS production through antioxidant intervention are still common. The study investigated the effects of ascorbic acid supplementation on ROS production and DOMS following downhill running. Subjects were assigned to two groups. The ascorbic acid group (group AA) received 1 g ascorbic acid 2 h pre-, and for 14 d post-downhill running, whilst the placebo group (Pl group) received a placebo. Blood samples were drawn pre-supplement, pre- and post-exercise, and then 1, 2, 3, 4, 7 and 14 d post-exercise for analysis of ascorbate, malonaldehyde and total glutathione. DOMS was assessed using a visual analogue scale and pressure algometry. Muscle function was assessed using isokinetic dynamometry. Plasma ascorbate was elevated throughout in group AA compared with the Pl group. Downhill running resulted in DOMS in both groups. Muscle function was impaired post-exercise in both groups, although a delayed recovery was noted in group AA. Malonaldehyde increased 4 d post-exercise in the Pl group only. Ascorbic acid supplementation attenuates ROS production following downhill running, without affecting DOMS. Furthermore, ascorbic acid supplementation may inhibit the recovery of muscle function.

Aging‐Related Muscle Dysfunction: Failure of Adaptation to Oxidative Stress?

Oxygen-free radicals and oxidant-induced damage to cellular molecules have been implicated as important contributors to the aging process. In aged muscle, there is an increased content of products of oxidative damage to proteins and DNA1,2 together with a marked increase in the number and variety of mitochondrial DNA rearrangements.3 Mammalian cells respond to oxidative stress by variations in the rate of cell growth, changes in cell cycle length, and marked responses in resistance to oxidative stress.4 In muscle there is a transient response to contractile activity by increased expression of several stress or heat shock proteins (HSPs) and by increasing activity of superoxide dismutase and catalase enzymes.5 Evidence also exists that aging results in a reduced ability to express HSPs in response to stress.6 The decline in skeletal muscle function that occurs with age appears to be due primarily to a loss of muscle fibers. Reduction in fiber number may be related to an increased susceptibility of aged muscle to contraction-induced damage and a reduced rate of regeneration following damage.7 Muscles from aged animals take significantly longer to recover from exercise involving lengthening contractions than do muscles from adult animals and do not show the same adaptive response to preventing muscle damage induced by further exercise protocols.8 Oxidative stress therefore plays an important role in the aging process, and studies in nonmammalian systems suggest that a reduction in this stress may reduce agerelated tissue dysfunction. It is also clear that cells respond to episodes of oxidative stress to prevent further tissue damage and that failure of adaptation is a feature of the aging process.

Characterisation of the expression of the Renin-Angiotensin system in primary and immortalised human renal proximal tubular cells.

Background/Aims: Angiotensin II (AngII) is pivotal in the pathogenesis of progressive kidney disease. We have recently shown that AngII induced an increase in markers of oxidative stress, adaptive responses and upregulated stress-related gene expression in immortalised human proximal tubular (HK-2) cells. However, these observed effects of AngII were not mediated solely via AngII type 1 receptor (ATR1). Both HK-2 cells and primary human renal proximal tubular cells (RPTEC) are useful tools to investigate the renin-angiotensin system (RAS), but data on the local expression of the RAS in these cells remain limited. We therefore characterised RAS expression in RPTEC and HK-2 cells. Methods: The mRNA and protein expression of RAS in RPTEC and HK-2 cells was examined by RT-PCR, Western blotting and immunoprecipitation. Results: In both cell lines, mRNA for angiotensin-converting enzyme (ACE) and mRNA and protein expression for angiotensinogen, renin, ACE2, ATR1 and ATR4 were detected. Candesartan, a specific ATR1 blocker, effectively blocked the expression of 80% of the stress-related genes that were upregulated in HK-2 cells following exposure to AngII. Con clusion: These data support a role for AngII in mediating oxidative stress via other receptor types stimulated by AngII and confirm that it is possible to investigate ATR4 pathways of potential injury in RPTEC.

Oxidative Stress in a Novel Model of Chronic Acidosis in LLC-PK1 Cells

Chronic metabolic acidosis occurs commonly in chronic renal failure (CRF). The proximal renal tubular cell is the site in the kidney of high oxidative metabolic activity and in CRF is associated with adaptive hypertrophy and hypermetabolism. We hypothesised that chronic acidosis may lead to increased generation of reactive oxygen species due to increased oxidative activity. We developed a novel model of chronic acidosis in LLC-PK1 cells and measured markers of oxidative stress and metabolism. Acidosis led to a reduction in cellular total glutathione and protein thiol content and an increase in glutathione peroxidase activity and NH3 generation. The expression of constitutively expressed heat stress protein (HSP) HSC70 and HSP60 increased at pH 7.0.

Chapter 8 – Antioxidants and free radicals

Antioxidants are intimately involved in the prevention of cellular damage -the common pathway for cancer, aging, and a variety of diseases. The scientific community has begun to unveil some of the mysteries surrounding this topic, and the media has begun whetting our thirst for knowledge. Athletes have a keen interest because of health concerns and the prospect of enhanced performance and/or recovery from exercise. The purpose of this article is to serve as a beginners guide to what antioxidants are and to briefly review their role in exercise and general health. What follows is only the tip of the iceberg in this dynamic and interesting subject.