Amino AcidsFatigue and Immunodepression in Exercise

Chapter 11
Amino Acids, Fatigue and Immunodepression
in Exercise
ERIC A. NEWSHOLME AND LINDA M. CASTELL
Introduction
Amino acids and the athlete
This chapter discusses the importance of some
amino acids in relation to exercise, in particular to prolonged, exhaustive exercise. Protein
metabolism and the protein requirements of
the athlete have already been discussed in
Chapter 10. Nevertheless, it is worth mentioning
here that consideration of daily protein requirements is complicated by the fact that not all
proteins in the diet have the same nutritional
value, since they contain different amounts of
essential amino acids. First-class proteins (e.g. in
eggs, milk and meat) contain enough of each
essential amino acid to allow protein synthesis to
occur without the need to eat extra protein (Fig.
11.1).
By contrast, proteins of plant origin are termed
‘second class’ because they are deficient in the
same amino acids. Adequate amounts of each of
the essential amino acids can be obtained from a
vegetarian diet by eating a wide range of plant
foods, e.g. cereals and legumes. This does mean,
however, that the extra protein consumed takes
the place in the diet of the all-important carbohydrate. The athlete must therefore seek a balance:
too much protein and the diet is distorted; too
little and recovery after intensive training might
be slowed. This problem leads to a consideration
of supplementation of the diet with essential
amino acids, especially during peak training.
Furthermore, from studies on individual cells
in vitro, knowledge is becoming available of the
individual nutritional requirements of particular
cells and how these change under different conditions. Transfer of this knowledge to clinical
situations has occurred over the past 10 years
with considerable success. Consequently, it is
now possible to provide nutrients designed to
deliver fuels to particular cells, tissues and
organs that are involved in the response to injury
or illness, and this will enhance the natural
healing process.
This information can be extended to include
the athlete. Furthermore, a specific response to
exercise, and to physical and mental fatigue, can
provide information that could be applied in the
clinic. In addition, it is possible to suggest some
amino acids with which athletes might consider
supplementing their diet (Table 11.1). Some of
the acquired non-dispensable amino acids that
might be of benefit to athletes if taken as a
supplement have been discussed elsewhere
(Newsholme et al. 1994). In this chapter discussion will centre on glutamine and the branched
chain amino acids, for which evidence of benefit
in the athletic field is available.
Fatigue in physical activity
Fatigue in physical activity can be considered at
physiological or biochemical levels. Potential
mechanisms for fatigue at a physiological level
are as follows (see Fitts 1994).
1 Central fatigue:
(a) excitatory input to higher motor centres;
153
154
nutrition and exercise
Non-essential
Alanine
Glutamine and
asparagine
Arginine
Creatine
Nitric oxide
Cysteine
Aspartate
Pyrimidines
Proline
Cysteine
Taurine
Glycine
Glutamate
γ-Aminobutyrate
Tryrosine
Glutamine
Amino sugars
Purines
Pyrimidines
Glycine
Creatine
Purines
Tetrapyrroles
Hisitidine
Histamine
Lysine
Carnitine
Methionine
Creatine
Methyl derivatives
Serine
Choline
Ethanolamine
Sphingosine
Proline
Ornithine
Putrescine
Tryrosine
Catecholamines
Melanin
Thyroxine
Tryptophan
5-Hydroxytryptamine
Nicotinic acid
Valine
Pantothenic acid
Serine
Arginine
Aspartic acid
Glutamic acid
Valine
Tryptophan
Histidine
Methionine
Phenylalanine
Essential
Threonine
Isoleucine
Leucine
Lysine
+Peptides (e.g. carnosine, glutathione)
Fig. 11.1 The amino acid composition of myosin, one of the two major proteins in muscle — and, therefore, in lean
meat — and the biosynthetic role of some of these amino acids. From Newsholme et al. (1994), with permission.
(b) excitatory drive to lower motor neurones;
(c) motor neurone excitability.
In these three mechanisms there is a decrease in
neural drive. This can be detected by showing
that fatiguing muscle can still maintain power
output if the nerve is stimulated artificially or the
brain is stimulated.
2 Peripheral fatigue:
(a) neuromuscular transmission;
(b) sarcolemma excitability;
(c) excitation–contraction coupling.
Biochemical mechanisms for fatigue can also
be put forward:
• depletion of phosphocreatine in muscle;
• accumulation of protons in muscle;
• accumulation of phosphate in muscle;
• depletion of glycogen in muscle;
• hypoglycaemia;
• changes in the concentrations of key amino
acids in the blood leading to changes in the concentrations of neurotransmitters in the brain.
Some of these mechanisms have been discussed elsewhere (Newsholme et al. 1994). The
first four are explanations for peripheral fatigue
and the last two are explanations of central
fatigue. The advantage of proposing a biochemical mechanism is that, from this knowledge,
ideas can be put forward for manipulations to
delay fatigue. A hypothesis is suggested that
links all of the latter three biochemical mecha-
amino acids, fatigue and immunodepression
155
Table 11.1 A ‘contemporary’ view of dispensable and non-dispensable amino acids.
Category
Amino acid
Totally non-dispensable
Lysine, threonine
Oxoacid non-dispensable*
Branched-chain amino acids†,
methionine, phenylalanine,
tryptophan
Conditionally non-dispensable‡
Cysteine, tyrosine
Acquired non-dispensable§
Arginine, cysteine, glutamine, glycine,
histidine, serine
Dispensable¶
Alanine, asparagine, aspartate,
glutamate
* The carbon skeleton of these amino acids cannot be synthesized by the body. However, if the oxo(keto) acids are
provided, the amino acids can be synthesized from the oxoacids via the process of transamination. The oxoacids
can be provided artificially.
† The branched-chain amino acids may play a role in fatigue but, to place them in context, it is important to outline
various explanations for fatigue (see text).
‡ These are produced from other amino acids — cysteine from methionine and tyrosine from phenylalanine —
provided that these amino acids are present in excess.
§ The demand for these amino acids can increase markedly under some conditions, e.g. infection, severe trauma,
burns and in some premature babies.
¶ It is assumed that all these amino acids can be synthesized at sufficient rates in the body to satisfy all requirements. It is now beginning to be appreciated that this may not always be the case for glutamine.
nisms into one mechanism involving the central
serotoninergic pathway in central fatigue.
There is considerable evidence that depletion
of muscle glycogen results in fatigue. In middledistance events, aerobic and anaerobic metabolism both contribute to adenosine triphosphate
(ATP) generation. The athlete can use the aerobic
system towards its maximum capacity, i.e. that
which is limited by oxygen supply to the muscle
but, in addition, further ATP can be produced
from the conversion of glycogen to lactate. So
what causes fatigue in this situation? It is
suggested that it is either depletion of glycogen
or the accumulation of protons in the muscle;
whichever occurs will depend upon the distance
of the event, the class of the athlete and his/her
fitness. If the rate of conversion of glycogen to
lactate is greater than the capacity to lose protons
from the muscle, protons will eventually accumulate sufficiently to cause fatigue (Newsholme
et al. 1994). However, it is also possible that
depletion of glycogen before the end of the event
can result in fatigue. As glycogen levels fall, fatty
acid mobilization will occur and increase the
plasma fatty acid level. In a prolonged event such
as the marathon, fatty acids must be mobilized
since there is not enough glycogen to provide the
energy required for the whole event. For an
optimum performance, the marathon runner
must oxidize both glycogen and fat simultaneously, but the rate of utilization of the latter
should be such as to allow glycogen to be used
for the whole of the distance — and for depletion
to occur at the finishing post. Consequently,
precision in control of the rates of utilization of
the two fuels, fat and glycogen, is extremely
important.
An interesting question is why glycogen
depletion should result in fatigue. If, as is already
known, it were possible to switch to the enormous store of fat as a fuel, this would delay
fatigue dramatically: theoretically, the runner
should then be able to maintain a good pace for a
considerable period of time — possibly several
days. At least two explanations have been put
forward to account for the fact that this does not
156
nutrition and exercise
occur. First, there is a limitation in the rate of fatty
acid oxidation so that a high rate of ATP generation cannot be supported by fatty acid oxidation
alone so that, once fat becomes the dominant
fuel, the pace must slow. Secondly, and these are
not mutually exclusive, fatty acids must be mobilized from adipose tissue to be oxidized and
mobilization of fatty acids can result in central
fatigue. A mechanism for this is described below.
Plasma levels of tryptophan, branched-chain
amino acids and the 5-HT hypothesis for
central fatigue
The branched-chain amino acids (leucine,
isoleucine and valine), unlike other amino acids,
are taken up largely by muscle and adipose
tissue. Like most amino acids, tryptophan is
taken up and metabolized by the liver. However,
a small amount of tryptophan is taken up by the
brain, where it is converted to the neurotransmitter 5-hydroxytryptamine (5-HT). Once this neurotransmitter is released in the synapses of some
neurones, it can influence a variety of behaviours, including tiredness, sleep, mood and possibly mental fatigue. It is suggested that an
increase in 5-HT level in these neurones makes it
harder mentally to maintain the same pace of
running, cycling, etc.
The basic tenets of the hypothesis are as
follows.
1 Both branched-chain amino acids and tryptophan (and other aromatic amino acids) enter the
brain upon the same amino acid carrier so that
there is competition between the two groups of
amino acids for entry (for review, see Fernstrom
1990).
2 Tryptophan is converted via two enzymes in
the brain to 5-HT. However, an increased level of
brain tryptophan can increase the rate of formation and hence the level of 5-HT in some areas of
the brain (Blomstrand et al. 1989).
3 A high 5-HT level could result in increased
amount of this neurotransmitter being released
into the synaptic cleft during neuronal firing,
therefore leading to a greater postsynaptic stimulation in some 5-HT neurones.
4 It is proposed that some of these neurones are
involved in fatigue.
5 Tryptophan is unique amongst the amino
acids in that it is bound to albumin, so that
it exists in the plasma and interstitial space
in bound and free forms, which are in equilibrium. This equilibrium changes in favour
of free tryptophan as the plasma fatty acid
level increases, since the latter also binds to
albumin and this decreases the affinity for
tryptophan.
6 It is considered that the plasma concentration
of free tryptophan governs, in competition with
branched chain amino acids, the rate of entry of
tryptophan into the brain, the level of tryptophan
in the brain and hence that of 5-HT (Fernstrom
1990).
As a consequence of these basic tenets, it is
proposed that either an increase in the plasma
fatty acid level and/or a decrease in that of
branched-chain amino acids would increase the
plasma concentration ratio of free tryptophan to
branched chain amino acids. This would then
favour the entry of tryptophan into the brain,
and increase the level of 5-HT which would lead
to a decrease in motor drive and a fall in power
output. Hence a marked increase in the plasma
fatty acid level could lead, via changes in the
plasma level of free tryptophan, to fatigue.
This could occur in both the middle-distance or
marathon runner as the muscle and liver glycogen stores are depleted and the fatty acid is mobilized from adipose tissue.
Importance of precision in the mobilization
and oxidation of fatty acids
The precise balance between the use of the
two fuels, glycogen and fatty acids, may be
extremely important for the athlete, since too
high or too low a rate of fatty acid mobilization/oxidation could cause problems
(Newsholme et al. 1994). After 20–30 min of exercise, mobilization of fatty acids from adipose
tissue increases, probably as a result of sympathetic stimulation. However, despite increased
rates of mobilization, the plasma concentration
amino acids, fatigue and immunodepression
of fatty acids may be only slightly increased: this
is because the rates of fatty acid uptake and oxidation by the active muscle are increased
(Winder 1996).
It is possible that the plasma fatty acid concentration and therefore the free tryptophan level
increases markedly in exercise only in some
conditions:
• when the muscle (and liver) glycogen store are
totally depleted;
• in unfit subjects, when control of fatty acid
mobilization may not be precisely regulated in
relation to demand and control of oxidation
within the muscle;
• when the rate of fatty acid oxidation by muscle
is somewhat restricted by the intermittent nature
of the exercise that occurs, in games such as
soccer, rugby, tennis, or squash;
• in obese individuals, in whom precision of
release may be restricted by the amount of
adipose tissue.
If the rate of fatty acid mobilization from
adipose tissue is higher than that of oxidation by
muscle, the plasma concentration of fatty acids
will increase, increasing the free tryptophan level
in the plasma, which will result in central fatigue
as described above. If the rate of fatty acid oxidation is too low (e.g. if the rate of mobilization
is too low), the rate of glycogen oxidation will
be high, and the athlete may deplete glycogen
stores before the end of the race, resulting in a
very poor performance. Thus, the endurance
athlete appears to have to run on a metabolic
tightrope of fatty acid mobilization/oxidation
during the race, and the precise rate of mobilization/oxidation for each athlete must be learnt by
training.
A summary of experimental findings which
support the hypothesis is as follows.
1 The plasma concentration ratio of free tryptophan/branched-chain amino acids is increased
in humans after prolonged exhaustive exercise
and, in the rat, the brain levels of tryptophan and
5-HT are increased (Blomstrand et al. 1989,
1991a).
2 Administration of a 5-HT agonist impairs
running performance, whereas a 5-HT antago-
157
nist improved running performance in rats
(Bailey et al. 1992).
3 Administration of a 5-HT re-uptake blocker
to human subjects decreased physical performance — exercise time to exhaustion during standardized exercise was decreased in comparison
with a control condition (Wilson & Maughan
1992).
4 The secretion of prolactin from the hypothalamus is controlled, in part, by 5-HT neurones, and
5-HT stimulates the rate of secretion. During
exercise, there is a correlation between the
plasma levels of prolactin and free tryptophan,
supporting the view that increased free tryptophan level in the blood can influence the 5-HT
level in the hypothalamus (Fischer et al. 1991).
5 The blood prolactin level increased to a much
smaller extent in well-trained endurance athletes, compared with controls, in response to an
agent that increased 5-HT levels in the hypothalamus (e.g. fenfluramine). This could be caused
by down-regulation of 5-HT receptors as a result
of chronic elevation of the 5-HT level in this part
of the brain (Jakeman et al. 1994).
In the past few years, supplementary feeding
with branched-chain amino acids has produced
some results supporting the hypothesis and
some which show no effect. The latter are
described in more detail by Davis in Chapter 12.
Table 11.2 gives a brief comparison of the results
from supplementation studies in exercise of
which the authors are aware. In one of the most
recent studies, a laboratory-based, cross-over
study, seven endurance cyclists were monitored
for perceived effort and mental fatigue (using the
Borg scale), with and without branched-chain
amino acid supplementation. When subjects
received the branched-chain amino acids, compared with the placebo, there was a lower perception of effort required to sustain the level
of exercise required (Blomstrand et al. 1997).
Mittleman et al. (1998) have reported a positive
effect of branched-chain amino acids on performance in moderate exercise during heat stress in
men and women.
In rats, injection of branched-chain amino
acids not only increased the time to fatigue of
158
No. of
subjects
13
10
193c
25c
5
10
7
6
52
10
7
9
10
Exercise
Cycling
Cycling
Cycling
Marathond
30-km rund
Cycling
Cycling
Cycling
Soccerd
30-km rund
Cycling
Cycling
Cyclingi
Cycling
·
Vo2max.
(%)
40
70–75
70–75
N/M
75
72.7
65–75
N/M
N/M
70
70
63.1
N/M
Duration (min)
c. 137–153
c. 122
c. >210
60–80
50–60
c. 30
145
c. 230
60
c. 159
c. 125–212
Amount
of BCAA
ingested
12.8 g
23.4 g
7.8 g
16 g
6.3 g·l-1
16 g·day-1g
30 g
10 gf
5.3 gh
0.74 g·l-1f
6–9 g
18 gj
21 g
p[BCAA]
Effect
on
NH3
Effect on performance
Mental
Physical
Reference
1250 mm
2400 mm
950 mm
1250 mm
None
Rise
Rise
N/M
None
N/M
N/M
Improved
Improveda
Noneb
Noneb
Improvede
Mittleman et al. (1998)
van Hall et al. (1995)
van Hall et al. (1995)
Blomstrand et al. (1991a)
1000 mm
N/M
3000 mm
1200 mm
650 mm
420 mm
1050 mm
1026 mm
c.1250 mm
N/M
N/M
Rise
N/M
N/M
N/M
None
Rise
Rise
N/M
N/M
N/M
Improved
Improved
N/M
Improved
N/M
Improved
None
Improved
None
N/M
N/M
None
Improved
None
None
Blomstrand et al. (1995)
Hefler et al. (1995)
Wagenmakers (1992)
Blomstrand et al. (1991b)
Hassmen et al. (1994)
Galiano et al. (1991)
Blomstrand et al. (1997)
Madsen et al. (1996)
Struder et al. (1998)
N/M, not measured; p[BCAA], the peak plasma concentration of branched-chain amino acids observed in each study.
a Subjects experienced heat stress.
b Very high day-to-day intraindividual variation in time to fatigue for some subjects.
c Not all subjects gave blood samples.
d Field study.
e In subset of slower runners.
f 6% carbohydrate added.
g 14-day study.
h 7% carbohydrate added.
i 100-km trials.
j 5% carbohydrate added.
nutrition and exercise
Table 11.2 A comparison of studies on branched-chain amino acid (BCAA) supplementation in humans during endurance exercise.
amino acids, fatigue and immunodepression
exercising rats but also prevented the normal
increase in brain tryptophan level caused by
exhaustive exercise (T. Yamamoto, personal
communication). Calders et al. (1997) observed
an increase in the time to fatigue, as well as
an increase in plasma ammonia, in fasting rats
injected with 30 mg of branched-chain amino
acids 5 min before exercise, compared to those
injected with placebo.
The data in Table 11.2 indicate that administration of branched-chain amino acids alone
appears to have a more beneficial effect than
when added to carbohydrate. It also seems that
the higher the dose of branched-chain amino
acids, the more likely it is that plasma ammonia
levels will be elevated. This suggests that lower
doses are more likely to be beneficial. In the
majority of studies, the branched-chain amino
acids have been administered before exercise. It
may be that administration during exercise was
the reason that Blomstrand et al. (1997) and
Mittleman et al. (1998) failed to observe a change
in plasma ammonia. Whether a bolus dose is
given or whether separate doses are given
during exercise could be important for the
release of ammonia from muscle.
In conclusion, beneficial effects of branchedchain amino acids have been seen on aspects of
both mental and physical fatigue in exhaustive
exercise. Most studies have not investigated
effects on mental fatigue. However, the mental
exertion necessary to maintain a given power
output is an integral feature of central fatigue.
Cellular nutrition in the
immune system
For many years, it was thought that both
lymphocytes and macrophages obtained most
of their energy from the oxidation of glucose.
However, it has now been shown that these cells
also use glutamine and that its rate of utilization
is either similar to or greater than that of glucose.
There are clear lines of evidence which support
the view that glutamine is used at a very high
rate by lymphocytes and by macrophages in vivo.
1 The maximal catalytic activity of glutaminase,
159
the key enzyme in the glutamine utilization
pathway, is high in freshly isolated resting
lymphocytes and macrophages (Ardawi &
Newsholme 1983, 1985).
2 The rates of utilization of glutamine are
high: (i) in freshly isolated lymphocytes and
macrophages (Ardawi & Newsholme 1983,
1985) and (ii) in cultured lymphocytes and
macrophages, and in T- and B-lymphocytederived cell lines (Ardawi & Newsholme 1983,
1985; Newsholme et al. 1988).
3 A high rate of glutamine utilization by lymphocytes in vitro maintains unusually high intracellular concentrations of glutamine, glutamate,
aspartate and lactate. Very similar levels of these
intermediates are seen in intact lymph nodes
removed from anaesthetized rats and frozen
rapidly prior to extraction of the tissues (T. Piva
and E.A. Newsholme, unpublished data).
In addition, although various lymphocyte
subsets have not been studied, the available
evidence suggests that B- and T-lymphocytes
utilize glutamine at similar rates (Ardawi &
Newsholme 1983, 1985; Newsholme et al. 1988).
Surprisingly, little of the carbon of glucose
(< 10%) and only some of that of glutamine
(10–30%) is oxidized completely by these cells:
glucose is converted almost totally into lactate,
glutamine into glutamate, aspartate, alanine
and CO2. The partial oxidation of these fuels is
known as glycolysis and glutaminolysis, respectively. From these simple metabolic characteristics several questions arise.
1 What is the significance of these high rates?
2 Why is the oxidation only partial?
3 What are the consequences for the whole
organism?
4 Does the plasma glutamine level ever decrease
sufficiently to decrease the rate of such utilization by these cells, and hence decrease their
ability to respond to an immune challenge?
High rates of glycolysis and glutaminolysis
will provide energy for these cells. In addition,
glutamine provides nitrogen for synthesis of
several important compounds, e.g. purine and
pyrimidine nucleotides, which are needed for
the synthesis of new DNA and RNA during pro-
160
nutrition and exercise
liferation of lymphocytes and for mRNA synthesis and DNA repair in macrophages. This will
also be important for the production of these
and other cells in the bone marrow, especially
when stimulated to increase their production
during trauma, infection, burns and if white cells
are damaged, for example, during exercise.
However, when it has been quantitatively
studied — so far, only in lymphocytes — the rate of
glutaminolysis is very markedly in excess of the
rates of synthesis of these compounds. For
example, the rate of utilization of glutamine by
lymphocytes is very much greater than the
measured rate of synthesis of uridine nucleotides
and much higher than the maximum activity of
the rate limiting enzyme, carbamoyl phosphate
synthase II (Newsholme & Leech 1999).
A theory has been proposed which accounts
both for these high rates of glutamine utilization
and the fact that its oxidation is partial. The synthetic pathways for de novo nucleotide synthesis
require specific and precise increases in the rate
of synthesis of these nucleotides during the
proliferative process. This theory is known as
branched point sensitivity and has been discussed
in detail elsewhere (Newsholme et al. 1985). The
important point to emerge from this is that glutamine (and glucose) must be used at a high rate by
some of the cells of the immune system even
when they are quiescent, since an immune challenge can occur at any time so that cells must be
‘primed’ to respond whenever there is an invasion by a foreign organism. This requires glutamine to be available in the bloodstream at a fairly
constant level. Furthermore, if pyruvate produced from glutamine were fully oxidized via
the Krebs cycle, the cells might produce too
much ATP, and this could lead to inhibition of the
rates of glutaminolysis and branched-point
sensitivity would be lost. Consistent with the
branched-point sensitivity theory, it has been
shown that a decrease in the glutamine concentration in culture medium below that normally
present in plasma decreases the maximum rate of
proliferation and slows the response to a mitogenic signal in both human and rat lymphocytes,
even though they are provided with all other
nutrients and growth factors in excess (ParryBillings et al. 1990b). In addition, a decrease in
glutamine concentration also decreased phagocytosis and the rate of cytokine production by
macrophages.
Several tissues, including liver, muscle,
adipose and lung, can synthesize and release glutamine into the bloodstream. This is important,
since 50–60% of the glutamine that enters the
body via protein in the diet is utilized by the
intestine. Thus, the glutamine required by other
tissues, including the immune system, must be
synthesized within the body. Quantitatively, the
most important tissue for synthesis, storage and
release of glutamine is thought to be skeletal
muscle. As much glutamine is stored in muscle
as glycogen is stored in liver, and the rate of
release across the plasma membrane, which
occurs via a specific transporter, appears to be
controlled by various hormones (Newsholme &
Parry-Billings 1990). Because of the importance
of glutamine for cells of the immune system, it is
suggested that immune cells may communicate
with skeletal muscle to regulate the rate of glutamine release. This may also involve some
cytokines and glucocorticoids.
The plasma concentration of glutamine is
decreased in conditions such as major surgery
(Powell et al. 1994); burns (Stinnett et al. 1982;
Parry-Billings et al. 1990b); starvation (Marliss
et al. 1971); sepsis (Clowes et al. 1980; Roth et al.
1982). There is also evidence that the immune
system is suppressed in clinical trauma (Baker et
al. 1980; Green & Faist 1988). The requirement for
glutamine, synthesized within muscle and other
cells, will therefore be increased in these conditions, since there will be increased activity of the
immune system, and an increased number of
cells involved in proliferation and repair. Similarly, damage caused to muscle by prolonged,
exhaustive exercise will also lead to a greater
demand for glutamine. Although the plasma
glutamine concentration is increased in athletes
undertaking short-term exercise (Decombaz et al.
1979), it is decreased in prolonged, exhaustive
exercise (Poortmans et al. 1974; Castell et al. 1996)
and in overtraining (Parry-Billings 1989; Parry-
amino acids, fatigue and immunodepression
Billings et al. 1990a; Rowbottom et al. 1996; see
also Budgett et al. 1998).
In a study on athletes with the overtraining
syndrome (Parry-Billings 1989), the plasma concentrations of alanine and branched-chain amino
acids were similar in trained and overtrained
athletes. However, the plasma concentration
of glutamine was lower in overtrained athletes
compared with that in trained athletes and
the concentration in trained subjects was lower
than in recreational runners (Parry-Billings et
al. 1990a). Moreover, after a 6-week recovery
period, despite a significant improvement
in the exercise performance of these subjects,
the plasma glutamine concentration remained
below control values. This suggests that
immunodepression due to overtraining may
persist for longer periods than indicated by the
decrease in physical performance.
Exercise-induced immunodepression has
been demonstrated in a large number of different
types of athletes, including runners, swimmers,
skiers (Noakes 1992) and ballet dancers (Sun et al.
1988). It is therefore suggested that intense, prolonged exercise, particularly if it is undertaken
regularly, can cause a marked decrease in the
plasma glutamine level, and that this might
result in immunodepression. Can muscle, together with other tissues, always respond sufficiently to release enough glutamine to maintain
the normal blood concentration? This may be a
particularly relevant question if muscle is
damaged due to excessive exercise. However, the
reason for the decrease in the plasma glutamine
concentration in longer term, strenuous exercise
is not understood.
Enzymes which are normally localized in
muscle fibres appear in the blood and are
assumed to be evidence of disruption or increased permeability of the muscle cell membranes (Altland & Highman 1961; Newham et al.
1983). The occurrence of muscle damage after
prolonged exercise has been reported by Appell
et al. (1992), who observed increased levels of
circulating complement anaphylotoxin, which
is a likely result of tissue damage. Tiidus and
Ianuzzo (1983) observed that the extent of injury
161
is proportional to the intensity of exercise.
Muscle injuries have been found to be widespread in military personnel during strenuous
training (Greenberg & Arneson 1967; Armstrong
1986).
Although it might be hypothesized that the
marked decrease in plasma glutamine after prolonged exhaustive exercise could be due to an
inhibition of the glutamine release mechanism, it
seems unlikely, since increased non-specific permeability of muscle cell membranes would be
expected to lead to greater release of glutamine
from muscle. The possibility arises that muscle
damage caused by prolonged exercise presents
an area of tissue which is larger than normal, to
which immune cells might migrate (see Galun
et al. 1987; Pabst & Binns 1989, 1992). As the
numbers of these cells increase, activity increases
and/or proliferation of some cells may result
which, in turn, increases the local demand for
glutamine. It is suggested that failure of muscle
to provide enough glutamine could result in an
impairment of the function of the immune
system via lack of precision for the regulation of,
for example, the rates of purine and pyrimidine
nucleotide synthesis for DNA and RNA formation in lymphocytes (Newsholme 1994). It can be
speculated that excessive damage could produce
resistance to the proposed stimulatory effect of
cytokines and glucocorticoids on glutamine
release, e.g. by a reduction in the number of
cytokine receptors and/or glucocorticoid receptors on muscle.
Glutamine feeding in clinical situations
Over many years, there has been considerable
physiological interest in the phenomenon of
hypoglycaemia, since this can cause abnormal
function of the brain, which is normally dependent upon glucose as a fuel. Similar considerations should be applied to the maintenance of
the plasma glutamine level, which can be considered to be as important a plasma fuel as that of
glucose, but for different cells. Furthermore, the
requirement for glutamine, synthesized within
muscle and other cells, will increase after pro-
162
nutrition and exercise
longed exhaustive exercise, since there will be
increased activity of the immune system, and an
increased number of cells involved in proliferation to carry out the necessary repair. The
question therefore arises as to whether extra glutamine should be provided after exhaustive
exercise.
Evidence that both parenteral and enteral glutamine feeding can have beneficial effects comes
from several clinical studies: of particular relevance to this chapter is the evidence that glutamine has a beneficial effect upon some cells of the
immune system in the patients investigated
(Table 11.3).
Exercise, infections and
immunodepression
Strenuous exercise and
upper respiratory tract infections
Upper respiratory tract infections (URTI) occur
frequently in athletes after prolonged, exhaustive exercise compared with the normal seden-
Table 11.3 Some beneficial effects of glutamine feeding upon the immune system.
Recipients
Clinical situation
Method of feeding
Beneficial effects
Reference
Humans
Bone marrow
transplant
TPN (l-glutamine)
Decreased number of positive
microbial cultures
Ziegler et al. 1992, 1998
Decreased number of clinical
infections
Enhanced recovery of
circulating lymphocytes,
total T-lymphocytes, CD4
helper, CD8 suppressor
Humans
Colorectal cancer
TPN (glycylEnhanced postoperative
glutamine dipeptide) T-lymphocyte DNA synthesis
O’Riordain et al. 1994
Humans
Severe, acute
pancreatitis
TPN (glycylEnhanced T-cell response,
glutamine dipeptide) decreased interleukin 8
production
O’Riordain et al. 1996
Rats
Healthy, suppressed
biliary
immunoglobulin A
TPN (l-glutamine)
Rats
Tumour-bearing
TPN (alanylIncreased phagocytic activity
glutamine dipeptide) of alveolar macrophages
Kweon et al. 1991
Rats
Tumour-bearing
Oral
Shewchuk et al. 1997
Rats
Sepsis
TPN (alanylIncreased rate of lymphocyte
glutamine dipeptide) proliferation and increased
number of lymphocytes
Yoshida et al. 1992
Rats
Chemotherapy
Oral
Klimberg et al. 1992
TPN, total parenteral nutrition.
Increased biliary concentration Burke et al. 1989
of immunoglobulin A
normally suppressed by TPN
Increased mitogenic response
in splenocytes, increased NK
cell numbers in spleen but not
activity
Decreased sepsis defined as
decreased white blood cell
count plus decreased positive
blood cultures
amino acids, fatigue and immunodepression
tary population or with non-competing athletes
(Linde 1987; Fitzgerald 1991; Brenner et al. 1994;
Nieman 1994a; Weidner 1994). For example, in
a study on participants in the Los Angeles
marathon who did not have an infection before
the race, the number of runners who became ill
during the week after the race was almost sixfold
higher than that of the control group. The control
group comprised endurance athletes who had
undergone a similar level of training but who did
not participate in the marathon (Nieman et al.
1990). A high incidence of infections has also
been observed in military personnel undergoing
prolonged and repeated intensive training (Lee
1992; Gray et al. 1994).
It has been suggested that moderate, regular
exercise helps to reduce the level of infection in
sedentary individuals but that, in individuals
who undertake intensive or excessive training,
the incidence of infection can increase sharply.
An overall view of this situation has been graphically described by a ‘J-curve’ (Fig. 11.2) which is
emphasized as being descriptive, rather than
quantitative (Nieman 1994a).
r i s k fact ors for
upper respiratory tract infection
163
demiological studies which have investigated
the incidence of URTI in different sports. The
majority of the studies which showed an
increased incidence of URTI after physical activity have been performed on runners. A longitudinal study on 530 male and female runners
suggested that an URTI was more likely to occur
with higher training mileage (Heath et al. 1991).
Similarly, the risk of illness increased in
endurance runners when training exceeded 97
km · week–1 (Nieman 1994b). Another study, on
marathon runners, demonstrated that the stress
of competition more than doubled the risk of
getting an URTI (O’Connor et al. 1979). A low
body mass may be another risk factor for infections (Heath et al. 1991).
One problem associated with prolonged exercise is that athletes start at some point to breathe
through the mouth rather than through the
nose, thus bypassing the nasal filter mechanism
(Niinima et al. 1980). This dries up bronchial
secretions, thus impeding the protective activity
of the cilia which cover the cell surface with
mucous (Rylander 1968). The high incidence of
infections after prolonged, exhaustive exercise
suggests therefore that immunodepression may
occur in some athletes due to the stress of hard
training and/or competition.
Weidner (1994) critically evaluated 10 epiImmune response to exhaustive exercise
Incidence
of infection
Excessive
training
Sedentary
Moderate
regular
exercise
Fig. 11.2 The incidence of infection in sedentary
individuals can be decreased with moderate exercise
but increases sharply in individuals who undertake
excessive amounts of exercise, or who suffer from
over-training. From Nieman (1994a), with permission.
There is evidence that numbers of circulating
white blood cells and subsets, together with
cytokine levels, are markedly altered as a result
of prolonged, exhaustive exercise. A substantial
increase in numbers of circulating white blood
cells, mainly due to a large increase in circulating
neutrophils, was first observed by Larrabee
(1902). Despite earlier reports of leucocytosis
and, particularly, an increase in circulating
numbers of neutrophils, relatively little work has
been undertaken on this phenomenon until the
past few years. Recently, however, several publications have reported not only that the total
number of white blood cells in the circulation are
substantially increased during the recovery
period immediately after a marathon or inten-
164
nutrition and exercise
sive training session but that numbers of lymphocytes in the circulation decrease below preexercise levels during the recovery period and
lymphocyte proliferation is impaired (see Fry et
al. 1992; Haq et al. 1993; Nieman 1994a, 1994b;
Castell & Newsholme 1998). There is now also
considerable evidence that prolonged, exhaustive exercise is associated with adverse effects on
immune function (for reviews, see Brenner et al.
1994; Shinkai et al. 1994; Nieman 1997; Pedersen
et al. 1998).
These effects include:
• decreased cytolytic activity of natural killer
cells;
• lower circulating numbers of T-lymphocytes
for 3– 4 h after exercise;
• a decrease in the proliferative ability of
lymphocytes;
• impaired antibody synthesis;
• decreased immunoglobulin levels in blood
and saliva;
• a decreased ratio of CD4 to CD8 cells.
In contrast, it has been suggested that lowintensity exercise is beneficial for the immune
system (Fitzgerald 1988; Nieman 1994a, 1994b).
Nehlsen-Cannarella et al. (1991) reported a
20% increase in serum immunoglobulins and a
decrease in circulating T-cell numbers in mildly
obese women after 6 weeks of brisk walking.
Natural killer cell activity is enhanced by moderate exercise (Pedersen & Ullum 1994).
Hack et al. (1997) reported a correlation
between a decreased plasma glutamine concentration and circulating levels of CD4 cells after 8
weeks of anaerobic training. Rohde et al. (1996),
in in vitro studies on T-cell derived cytokines,
found that glutamine influenced the production
of the cytokines interleukin 2 and g-interferon. In
a study on triathletes, they also found that a time
course of changes in serum glutamine correlated
with changes in lymphokine-activated killer cell
activities.
If, as indicated above, glutamine is important
for the immune system, then provision of glutamine might be beneficial for athletes at particular
times during their training.
glutamine feeding after
prolonged exhaustive exercise
Since the plasma concentration of glutamine is
decreased by approximately 25% in endurance
runners after a marathon as well as in clinical
conditions, a series of studies was undertaken in
which glutamine was administered. The first
study established a suitable glutamine dose and
timing in resting, normal subjects (Castell &
Newsholme 1997). The results showed that
glutamine (at concentrations of 0.1 g · kg–1 body
weight and 5 g per subject), given as a drink, significantly increased the plasma glutamine concentration within 30 min in healthy humans. This
level returned to close to baseline levels after
approximately 2 h.
The effect of giving glutamine or a placebo
after exercise was subsequently investigated in
full and ultramarathon runners. Glutamine (5 g
l-glutamine (GlutaminOx5, Oxford Nutrition
Ltd) in 330 ml mineral water) or placebo (maltodextrin) was given to athletes on a doubleblind basis after prolonged, exhaustive exercise.
Athletes were asked to take two drinks (glutamine/placebo), the first drink immediately after
exercise and the second drink 1 or 2 h after exercise. This timing was chosen as a result of information obtained from the glutamine feeding
studies in normal subjects. Blood samples were
also taken after exercise, before and after a glutamine or placebo drink. In addition to measurement of plasma glutamine and cytokine
concentrations and acute phase markers,
numbers of leucocytes and lymphocytes (for
details, see Castell et al. 1996) were measured
before and after the drinks, as well as CD4
and CD8 cells (Castell & Newsholme 1997).
The plasma concentrations of glutamine and
branched-chain amino acids were decreased
(23% and 26%, respectively) 1 h after the
marathon but had returned to normal the next
morning (Castell et al. 1997). The number of leucocytes tripled in the blood samples taken from
runners immediately after the marathon. This
leucocytosis, due mainly to a substantial increase
amino acids, fatigue and immunodepression
in numbers of neutrophils, was sustained in the
sample taken at 1 h postexercise. A substantial
decrease, to below baseline, was observed in
numbers of circulating T-cells 1 h after exhaustive exercise. However, there was a 30% decrease
in total lymphocytes at the same time point.
There was no significant difference in leucocyte
or lymphocyte numbers between the glutamine
and the placebo group. The provision of glutamine appeared to have a beneficial effect
upon the ratio of CD4 to CD8 T-cells (Castell &
Newsholme 1997). A decrease in this ratio has
been suggested as being a possible cause and
indicator of immunosuppression in athletes
(Nash 1986; Keast et al. 1988; Shepherd et al.
1991).
Questionnaires were given during the studies
to establish the incidence of infection for 7 days
after exercise (for details, see Castell et al. 1996).
Completed questionnaires on the incidence of
infection were received from more than 200
individuals in 14 studies, who participated in
rowing, or endurance or middle-distance
running. The levels of infection were lowest in
middle-distance runners, and were highest in
runners after a full or ultramarathon and in elite
rowers after a period of intensive training (Table
11.4). The majority of the infections reported
were URTIs. Athletes who consumed two drinks,
containing either glutamine or a placebo, immediately after and 2 h after a marathon, also completed 7-day questionnaires (n = 151). Overall,
the level of infections reported by the glutamine
group was considerably less than that reported
Table 11.4 Incidence of infections in athletes during
7 days after different types of exercise (mean
values ± SEM). After Castell et al. (1996a), with
permission.
Event
No. of
studies
No. of
participants
Infections
(%)
Marathon
Ultramarathon
Mid-distance race
Rowing
5
2
3
4
88
40
41
45
46.8 ± 4.8
43.3 ± 4.8
24.7 ± 4.0
54.5 ± 7.8
165
by the placebo group (Table 11.5). A simple explanation for the effects of glutamine observed in
these studies may be the fact that its provision
after prolonged exercise might make more glutamine available for key cells of the immune
system at a critical time for induction of infection.
This series of studies provide more evidence
for the fact that prolonged, exhaustive exercise
such as a marathon produces a response which is
analogous to some aspects of the acute phase
response. Increases were demonstrated in acute
phase response markers, such as C-reactive
protein, interleukin 6 and complement C5a in
blood samples taken after a marathon race
(Castell et al. 1997). An increase in the activation
of complement indicates enhanced macrophage
activity which may be involved in clearance of
fragments from damaged muscle tissue. The
fourfold increase in the plasma concentration of
C-reactive protein, observed 16 h after the race, is
consistent with damage to muscle after prolonged, exhaustive exercise.
The studies also confirm observations made
by others (for reviews, see Brenner et al. 1994;
Nieman 1994a; Shinkai et al. 1994), viz. that, after
marathon running, decreases occurred in the
numbers of some circulating immune cells which
were sustained until at least the next day (Castell
et al. 1997). In one glutamine feeding study in this
series, the numbers of circulating lymphocytes,
Table 11.5 Overall incidence of infections during
7 days for athletes given either glutamine or placebo
after running a marathon (mean values ± SEM). After
Castell et al. (1996a), with permission.
No. of
participants
No. of
participants
with no
reported
infections
Participants
with no
reported
infections
(%)
Glutamine
72
57
80.8 ± 4.2*
Placebo
79
31
48.8 ± 7.4
* Statistical significance between glutamine and
placebo groups (P < 0.001).
166
nutrition and exercise
which decreased 1 h after a marathon, were
restored next morning to baseline levels in the
glutamine group, compared with the placebo
group (Castell et al. 1997). In another of these
studies, white blood cells and neutrophils were
elevated after a marathon, but were closer to
baseline levels (P < 0.05 and < 0.001, respectively)
the next morning in the glutamine group compared with the placebo group. For future studies,
it would be of interest to take samples at daily
intervals after a marathon in order to monitor
immune cell function, since the effect of a viral
attack should be manifest within 2–3 days of
running a marathon.
No increase in the plasma concentration of
glutamine was observed in samples from those
marathon runners who received glutamine
drinks after the race. However, for logistical
reasons, blood samples were not taken until
an hour after glutamine feeding, whereas the
peak concentration of plasma glutamine after a
bolus dose at rest occurred at 30 min (Castell &
Newsholme 1997). Glutamine supplementation in the doses used, and at the times ingested
after this series of marathon studies, appeared
to modify the incidence of URTI and to effect
two or three changes in concentrations of acute
phase response markers or in circulating cell
populations.
An important issue is whether measurements
of the numbers and activities of leucocytes in the
blood properly reflect the performance of the
immune system in the whole body. In human
studies, it is the only measurable link we have
with the much larger number of cells in the
whole immune system but the authors are
aware of the dangers of overinterpretation of
these data. The safety and efficacy of glutamine
feeding have been discussed by Ziegler et al.
(1990).There are now more than 120 published
reports on glutamine feeding studies: no problems of toxicity have been reported. It is
suggested that, in situations where plasma glutamine levels in individuals are low, provision of
exogenous glutamine is a safe and simple
method of restoring physiological levels. This
might enhance the functional ability of cells of
the immune system, as well as improving the
digestive and defence mechanisms of the intestine, both for the patient and the athlete.
Conclusion
In summary, the picture which emerges from
these studies is that infection levels are higher in
athletes undergoing exhaustive exercise of long
duration than in those undertaking shorter or
more moderate exercise. Glutamine concentration in the blood is decreased more by prolonged,
exhaustive exercise than by anaerobic/aerobic or
moderate exercise. More marked leucocytosis
and subsequent decrease in lymphocytes occurs
as a result of prolonged, exhaustive exercise
than anaerobic/aerobic or moderate exercise.
The decreases tend to occur at similar times and
within 3–4 h after prolonged, exhaustive exercise: this creates an opportunity for apparent
immunosuppression to occur, which may coincide with exposure to viral or bacterial agents.
The net result is an increase in the number of
infections, which appears to be modified by
glutamine feeding.
Much more work needs to be done, preferably
in well-controlled field studies, to obtain a more
accurate picture of precisely how glutamine
might be affecting the levels of infection perceived in athletes after prolonged, exhaustive
exercise.
Acknowledgements
The authors are indebted to the subjects for their
willing participation in the studies, and to Professor Jacques Poortmans for his helpful comments on this chapter.
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