Journal of Applied
Physiology, 87:4, 1999, 1381-1385.
Excerpt with permission of the American Physiological
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
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 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
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
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
perfor-mance in these subjects. Aside from its potential
as an ergogenic aid, such supplementation may have particular
benefit in patients with persistent inflammatory conditions.
1. Anderson, M. E. Glutathione
and glutathione delivery compounds. Adv. Pharmacol. 38:
2. Anderson, M. E., and AL Meister. Transport and direct
utilization of gamma-glutamylcyst(e)ine for gIutathione
synthesis. Proc. Natl. Acad Sci. USA 80: 707-711, 1983.
3. Boucher, G. P., L. C. Lands, J. A. Hay, and L. Hornby.
Activity levels and the relationship to lung function
and nutritional status in children with cystic fibrosis.
Am. J. Phys. Med. Rehab.76:311-315,1997.
4. Bounous, G., G. Batist, and P. Gold. Immunoenhancing
property of dietary whey protein in mice: role of glutathione.
Clin. Invest. Med. 12: 154-161, 1989.
5. Bounous, G., and P. Gold. The biological activity of
undenatured dietary whey proteins: role of glutathione.
Clin. Invest. Med. 14:296-309,1991.
6. Burdon, R. H. Superoxide and hydrogen peroxide in relation
to mammalian cell proliferation. Free Radic. Biol. Med.
7. Droge, W., and E. Holm. Role of cysteine and glutathione
in HIV infection and other diseases associated with muscle
wasting and immunological dysfunction. FASEB J. 11: 1077-1089,
8. Gohill, YL, C. Viguie, W. C. Stanley, G. A. Brooks,
and Packer. Blood glutathione oxidation during human exercise.
J. Appl. Physiol. 64:115-119,1988.
9. Grey, V. L., V. G. Kramer, and L. C. Lands. The determination
of oxidised and reduced glutathione in circulating lymphocytes
using the Cobas Mira S (Abstract). Clin. Biochem. 31:
10. Guyatt, G. H., L. B. Berman, M. Townsend, S. O. Puggley,
and L. W. Chambers. A measure of quality of life for clinical
trials in chronic lung disease. Thorax 42: 773-778,1987.
11. Hack, V., D. Schmid, R. Breitkreutz, C. Stahl-Henning,
P. Drings, R. Kinscherf, F. Taut, E. Holm, and W. Droge.
Cystine levels, cystine flux, and protein catabolism in
cancer eachexia, HIV/SIV infection, and senescence. FASEB
J. 11: 84-92,1997.
12. Harrison, P. M., J. A. Wendon, A. E. Gimson, G. J.
Alexander, and R. Williams. Improvement by acetyleysteine
of hemodynamics and oxygen transport in fulminant hepatic
failure. N. Engl. J. Med. 324:1852-1857, 1991.
13. Jackson, M. J., R. H. T. Edwards, and M. C. H. Symons.
Electron spin-resonance studies of intact mammalian skeletal
muscle. Biochim. Biophys. Acta 847:185-190, 1985.
14. Jetté, K, S. Hendricks, D. Kroetsch, H. Nielsen, P.
Soucy, and P. Verdier. Guide for Anthropometric Meaurements
of Canadian Adults. Ottawa, ON: Health-Welfare Canada,
15. Ji, L. L., A. Katz, R. Fu, M. Griffiths, and M. Spencer.
Blood glutathione status during exercise: effect of carbohydrate
supplementation. J. Appl. Physiol. 74: 788-792, 1993.
16. Kanter, M. M., L. A. Nolte, and J. O. Holloszy. Effects
of an antioxidant vitamin mixture on lipid peroxidation
at rest and postexercise. J. Appl. Physiol. 74: 965-969,
17. Kinscherf, R., V. Hack, T. Fischbach, B. Friedmann,
C. Weiss, L. Edler, P. Bartsch, and W. Droge. Low plasma
glutamine in combination with high glutamate levels indicate
risk for loss of body cell mass in healthy individuals:
the effect of N-acetyl-cysteine. J. Mol. Med. 74: 393-400,
18. Koch, S. M.9 A. A. Leis, D. S. Stokic, F. A. Khawli,
and M. B. Reid. Side effects of intravenous N-acetylcysteine
(Abstract). Am. J. Respir Crit. Care Med. 149: A321, 1994.
19. Lands, L. C., C. Gordon, O. Bar-Or, C. J. Blimkie,
R. M. Hanning, N. L. Jones, L. A. Moss, C. E. Webber,
W. 1VL Wilson, and G. J. Heigenhauser. Comparison of three
techniques for body composition analysis in cystic fibrosis.
J. Appl. Physiol. 75:162-166,1993.
20. Lands, L. C., G. J. Heigenhauser, and N. L. Jones.
Analysis of factors limiting maximal exercise performance
in cystic fibrosis. Clin. Sci. 83:391-397,1992.
21. Lands, L. C., L. Hornby, G. Desrochers, T. Her, and
G. J. Heigenhauser. A simple isokinetic cycle for measurement
of leg muscle function. J. Appl. Physiol. 77: 2506-2510,
22. Lands, L. C., A. A. Smountas, G. Messiano, L. Brosseau,
H. Shennib, 1VL Charbonneau, and R. Gauthier. Maximal
exercise capacity and peripheral skeletal muscle function
following lung transplantation. J. Heart Lung. Transplant.
18: 113-120, 1999.
23. Makrdies, L., G. J. Heigenhauser, N. McCartney, and
N. L. Jones. Maximal short term exercise capacity in healthy
subjects aged 15-70 years. Clin. Sci. Lond. 69: 197-205,
24. Porta, P., S. Aebi, K Summer, and B. H. Lauterburg.
L-2-oxothiazolidine-4-carboxylic acid, a cysteine prodrug:
pharmacokinetics and effects on thiols in plasma and lymphocytes
in humans. J. Pharmacol. Exp. Ther. 257: 331-334, 1991.
25. Putman, C. T., N. L. Jones, L. C. Lands, T. M. Bragg,
M. G. Hollidge-Horvat, and G. J. Heigenhauser. Skeletal
muscle pyruvate dehydrogenase activity during maximal
exercise in humans. Am. J. Physiol. 269 (Endocrinol. Metab.
32): E458-E68, 1995.
26. Reid, M. B.t D. S. Stokic, S. M. Koch, F. A. Ehawli,
and A. AL Leis. N-acetylcysteine inhibits muscle fatigue
in humans. J. Clin, Invest. 94: 2468-2474, 1994.
27. Sen, C. K Oxidants and antioxidants in exercise. J.
Appl. Physiol. 79: 675-686,1995.
28. Sen, C. K., M. Atalay, and O. Hanninen. Exercise-induced
oxidative stress: glutathione supplementation and deficiency.
J. Appl. Physiol. 77: 2177-2187, 1994.
29. Shindoh, C., A. DiMarco, A. Thomas, P. Manubay, and
G. Supinski. Effect of N-acetylcysteine on diaphragm fatigue.
J. Appl. Physiol. 68:2107-2113,1990.
30. Sjödin, B., Y. Hellsten Westing, and F. S. Apple.
Biochemical mechanisms for oxygen free radical formation
during exercise. Sports Med 10: 236-254, 1990.
31. Sumida, S., YL Tanaka, H. Kitao, and F. Nakadomo.
Exerciseinduced lipid peroxidation and leakage of enzymes
before and after vitamin E supplementation. Int. J. Biochem.
21: 835-838, 1989.
32. Supinski, G. S., D. Stofan, R. Ciufo, and AL DiMarco.
N-acetylcysteine administration alters the response to
inspiratory loading in oxygen-supplemented rats. J. Appl.
Physiol. 82: 1119-1125, 1997.
33. Tietze, F. Enzymic method for quantitative determination
of nanograrn amounts of total and oxidized glutathione:
applications to mammalian blood and other tissues. Anal.
Biochem. 27: 502-522,1969.
34. Traveline, J. M., S. Sudarshan, B. G. Roy, F. Cordova,
V. Uyenson, and G. J. Criner. Effect of N-acetyleysteine
on human diaphragm strength and fatigability. Am. J. Respir.
Crit. Care Med. 156:1567-1571,1997.
35. Trump, M. E., G. J. Heigenhauser, C. T. Putman, and
L. L. Spriet. Importance of muscle phosphocreatine during
intermittent maximal cycling. J. Appl. Physiol. 80: 1574-1580,