Journal of Applied Physiology, 87:4, 1999, 1381-1385.
Excerpt with permission of the American Physiological Society
L. C. LANDS, V. L. GREY, AND A. A. SMOUNTAS
Lands, L. C., V. L. Grey, and A. A. Smountas. Effect of supplementation with a cysteine donor on muscular performance. J. Appl. Physiol. 87(4): 1381-1385, 1999.- Oxidative stress contributes to muscular fatigue. GSH is the major intracellular antioxidant, the biosynthesis of which is dependent on cysteine availability. We hypothesized that supplementation with a whey-based cysteine donor [Immunocal (HMS90)] designed to augment intracellular GSH would enhance performance. Twenty healthy young adults (10 men, 10 women) were studied presupplementation and 3 mo postsupplementation with either Immunocal (20 g/day) or casein placebo. Muscular performance was assessed by whole leg isokinetic cycle testing, measuring peak power and 30-s work capacity. Lymphocyte GSH was used as a marker of tissue GSH. There were no baseline differences (age, ht, wt, % ideal wt, peak power, 30-s work capacity). Follow-up data on 18 subjects (9 Immunocal, 9 placebo) were analyzed. Both peak power [13 ± 3.5 (SE) %, P < 0.02] and 30-s work capacity (13 ± 3.7%, P < 0.03) increased significantly in the Immunocal group, with no change (2 ± 9.0 and 1 ± 9.3%) in the placebo group. Lymphocyte GSH also increased significantly in the Immunocal group (35.5 ± 11.04%, P < 0.02), with no change in the placebo group (-0.9 ± 9.6%). This is the first study to demonstrate that prolonged supplementation with a product designed to augment antioxidant defenses resulted in improved volitional performance.
OXIDATIVE STRESS contributes to the development of muscular fatigue (27). GSH is a major intracellular antioxidant, the biosynthesis of which depends on the intracellular availability of cysteine (1). Previous work has shown that supplementation with N-acetylcysteine can slow the onset of muscular fatigue (26). However, there are significant adverse effects with such treatment (18, 26), possibly related to elevations in extracellular cysteine (1, 24). Cysteine, in the form of glutamylcystine moieties, more readily enters into cells. Immunocal, a whey-based oral supplement with a relative abundance of glutamylcystine, has been shown to augment intracellular GSH concen-trations in vitro (5). We hypothesized that if this would occur in vivo, then supplementation with Immunocal would improve muscular performance.
We demonstrated that ingestion of Immunocal, at a dose of 20 g/day, resulted in a 35.5% increase in circulating lymphocyte GSH concentrations. At the same time, supplemented subjects were able to generate more power to perform more work during a 30-s maximal effort. Glutamyl amino acids can be transported into cells. In the case of glutamylcystine, this can effectively increase cellular GSH concentrations (2).
Immunocal is a bovine whey protein concentrate produced by a proprietary lenient technique involving microfiltration and low-temperature pasteurization of milk. Whey protein consists of several compounds, including albumin, lactoferrin, and a-lactalbumin, which are rich in cystine (the oxidized form of cysteine) residues. Albumin and lactoferrin are also rich in glutamylcystine, which is easily transported into cells, making it a more readily available substrate for GSH biosynthesis (4, 5). Immunocal contains 2.5% cystine, compared with 0.3% for casein.
We utilized lymphocyte GSH concentrations as a marker of tissue GSH levels, as evidence from animal studies has suggested that this could track tissue levels, in response to both L-2-oxothiazolidine-4-carboxylic acid, a cysteine precursor, and buthionine sulfoximine, an inhibitor of the first, rate-limiting step in GSH synthesis, -glutamylcysteine synthase. Although a small trial of L-2-oxothiazolidine-4-carboxylic acid raised lymphocyte GSH levels, it also resulted in elevations in plasma cysteine and adverse effects (24). Although some patients did complain of bloating and occasional queasiness while on Immunocal, no other complaints were noted. This study, then, is the first demonstration of a well-tolerated oral supplement that could effectively raise tissue GSH concentrations.
Of more import are the functional findings. Subjects on Immunocal were able to generate greater power and increased the amount of work they could achieve during an all-out 30-s sprint. We have previously demonstrated that leg muscle 30-s isokinetic work output is a significant factor contributing to progressive exercise performance in patients with cystic fibrosis (20) and patients after lung transplant (22), independent of the effect of pulmonary impairment. Similar results have also been demonstrated in healthy adults (23). The ability to perform exercise has a significant impact on quality of life (10). Our results suggest then that the improvements in volitional performance that we measured in the laboratory can have a direct impact on functional ability. In this regard, it is intriguing that the subjects on Immunocal increased the percentage of time they spent being active.
Oxidative stress is associated with strenuous muscular contraction, leading to fatigue (8, 13, 15, 16, 28, 31). However, as pointed out by both Reid and colleagues (26) and Sen (27), although biochemical parameters of oxidative stress can be altered by supplementation, it has been difficult to demonstrate improvement in muscular performance. Animal models of muscular fatigue have demonstrated a beneficial effect of pretreatment with N-acetylcysteine (29, 32). Reid and co-workers were the first to demonstrate that pretreatment with intravenous N-acetylcysteine could increase force output of the tibilais anterior in humans when electrically stimulated to fatigue at low frequencies. A recent study reported that the time to voluntary task failure (inability to maintain 80% of maximal transdiaphragmatic pressure while breathing against a resistive load) in healthy humans could be increased by the use of intravenous N-acetylcysteine (34).
N-acetylcysteine can serve to maintain adequate stores of GSH through several mechanisms, including supplying cysteine for GSH biosynthesis and directly scavenging reactive oxygen species. Because N-acetylcysteine does not cross the sarcolemma or increase blood total GSH concentrations but does reduce blood GSH oxidation after exercise (28), it is likely that the results of Reid and co-workers (26) were due to N-acetyleysteine's free radical scavenging effects. The prevention of free radical-induced muscular dysfunction by free radical scavenging most likely explains the results of recent animal studies of diaphragm fatigue (32). However, other potential effects, such as improved blood flow or increased central nervous system respiratory drive, could also contribute (12). Unfortunately, N-acetlycysteine is associated with a number of adverse effects that detract from its utility as an ergogenic aid. These include blurred vision, dysphoria, and gastrointestinal discomfort. In the study by Travaline and co-workers (34), four subjects were premedicated with diphenhydramine and ranitidine to prevent the development of adverse effects.
The exact mechanism(s) of how Immunocal improved muscular performance is unclear. The most obvious mechanism would be an increase in intracellular glutathione levels, leading to a decrease in oxidant-induced muscular dysfunction. Our patients increased the percentage of time spent in moderate-to-vigorous activity, so that a central effect leading to increased activity and improved neural regulation of muscular function cannot be excluded. Many of the subjects reported a sense of feeling more energetic. This feeling could relate to central mechansims but could also relate to a decrease in muscular damage from antioxidant protection, as muscle soreness and sarcolemma permeability have been linked to oxidative stress (30). Our activity questionnaire provides us with time spent in activity but does not describe how that time was spent. However, this enhanced activity could have led to a training effect.
Subjects on Immunocal had a decrease in their percentage of body fat while maintaining their weight. Although this result sounds almost too good to be true, in healthy subjects plasma concentrations of cysteine and glutamine have been prognostic of subsequent changes in lean body mass (17). In patients with wasting disorders, such as cancer and human immunodeficiency virus infections, these values are reduced early on, preceding overt cachexia (7, 11).
The biochemical changes seen in wasting disorders have led to the concept of a low cyst(e)ine-glutamine syndrome. In this model, hepatic catabolism of cyst(e)ine to sulfate leads to the generation of hydrogen ions, which remove bicarbonate through buffering. Bicarbonate is required for the first rate-limiting step in the conversion of ammonium to urea. Removal of bicarbonate promotes ammonia's conversion to glutamine, thus conserving nitrogen in the amino acid pool. Our results are consistent with this model of cysteine metabolism. The change in redox state resulting from augmentation of glutathione stores could also alter gene expression to promote muscle growth (6). This suggests that supplementation with a cysteine donor may favorably influence body composition toward increased muscle mass. We do not believe that the changes we saw in body composition and muscle function were simply due to augmented protein intake, as the casein-supplemented group did not demonstrate these changes.
In conclusion, supplementation of healthy young adults with a whey-based oral supplement augmented lymphocyte GSH concentrations, while increasing muscular performance in these subjects. Aside from its potential as an ergogenic aid, such supplementation may have particular benefit in patients with persistent inflammatory conditions.
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