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Sweat facilitated losses of amino acids in Standardbred horses and the application of supplementation strategies to maintain condition during training

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Little is known about the amino acid composition of horse sweat, but significant fluid losses can occur during exercise with the potential to facilitate substantial nutrient losses. Sweat and plasma amino acid compositions for Standardbred horses were assessed to determine losses during a standardised training regime. Two cohorts of horses 2013 (n=5) and 2014 (n=6) were assessed to determine baseline levels of plasma and sweat amino acids. An amino acid supplement designed to counter losses in sweat during exercise was provided after morning exercise daily for 5 weeks (2013, n=5; 2014, n=4). After the supplementation period, blood and sweat samples were collected to assess amino acid composition changes. From baseline assessments of sweat in both cohorts, it was found that serine, glutamic acid, histidine and phenylalanine were present at up to 9 times the corresponding plasma concentrations and aspartic acid at 0-2.2 μmol/l in plasma was measured at 154-262 μmol/l in sweat. In contrast, glutamine, asparagine, methionine and cystine were conserved in the plasma by having lower concentrations in the sweat. The predominant plasma amino acids were glycine, glutamine, alanine, valine, serine, lysine and leucine. As the sweat amino acid profile did not simply reflect plasma composition, it was proposed that mechanisms exist to generate high concentrations of certain amino acids in sweat whilst selectively preventing the loss of others. The estimated amino acid load in 16 l of circulating plasma was 3.8-4.3 g and the calculated loss via sweat during high intensity exercise was 1.6-3.0 g. Following supplementation, total plasma amino acid levels from both cohorts increased from initial levels of 2,293 and 2,044 µmol/l to post-supplementation levels of 2,674 and 2,663 µmol/l respectively (P<0.05). It was concluded that the strategy of providing free amino acids immediately after exercise resulted in raising resting plasma amino acid levels.
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COMPARATIVE
EXERCISE
PHYSIOLOGY
Wagen in gen Acade mic
Publishe r s
ISSN 1755-2540
Editors-in-chief
David Marlin, David Marlin Consulting Ltd., Newmarket, United Kingdom
Kenneth H. McKeever, Rutgers – e State University of New Jersey, Department of Animal Sciences,
New Brunswick, NJ, USA
Editors
Tat ia na Ar t, University of Liege, Belgium; Er ic Barrey, INRA , France; Warw ick M. Bayly, Washington State University,
USA; Hilary M. Clayton, Michigan State University, USA; Manfred Coenen, University Leipzig, Germany; G. Robert
Colborne, Massey University, New Zealand; Michael S. Davis, Oklahoma State University, USA; Howard H. Erickson,
Kansas State University, USA; Jonathan H. Foreman, University of Illinois, USA; Raymond Geor, Michigan State
University, USA; Allen Goodship, University of London, United Kingdom; Pat Harris, WALTHAM Centre For Pet
Nutrition, United Kingdom; Kenneth William Hinchcliff, University of Melbourne, Australia; David Hodgson,
Virginia Polytechnic and State University, USA; James H. Jones, University of California, USA; Michael I. Lindinger,
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Australia; Jeff omason, University of Guelph, Canada; Stephanie Valberg, University of Minnesota Equine Center,
USA; Micheal Weishaupt, University of Zurich, Switzerland; James Wood, University of Cambridge, United Kingdom
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Comparative Exercise Physiology, 2015; 11 (4): 201-212 Wageningen Academic
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ISSN 1755-2540 print, ISSN 1755-2559 online, DOI 10.3920/CEP150027 201
1. Introduction
Free amino acids can act as a readily oxidisable source of
energy and some amino acids have relevance as indicators of
tissue catabolism (Adibi, 1980; Mortimore and Poso, 1987).
The measurement of plasma amino acid levels therefore
represents a potentially valuable avenue of insight into
muscle condition, protein turnover and energy metabolism
of human and animal athletes in response to exercise. In
healthy resting adults, plasma amino acid levels reflect a
tightly regulated homeostasis between nutritional intake
and release from tissues, versus tissue uptake and excretion
from the body (Cynober, 2002). The onset of exercise
represents a disturbance to the resting homeostasis which
is required to support the metabolic requirements of the
muscles. Adjustments in rates of muscle tissue uptake and
release of specific amino acids occur to support demand
which results in alterations in the post-exercise plasma
profile of amino acids relative to the profile seen pre-
exercise (Assenza et al., 2004; Ohtani et al., 2006).
Sweat facilitated losses of amino acids in Standardbred horses and the application of
supplementation strategies to maintain condition during training
R.H. Dunstan
1*
, D.L. Sparkes
1
, B.J. Dascombe
2
, C.A. Evans
1
, M.M. Macdonald
1
, M. Crompton
1
, J. Franks
1
, G. Murphy
1
, J. Gottfries
3
,
B. Carlton1 and T.K. Roberts1
1
University of Newcastle, School of Environmental and Life Sciences, University Dr, Callaghan, NSW 2308, Australia;
2
University of Newcastle,
School of Environmental and Life Sciences, 10 Chittaway Road, Ourimbah, NSW 2258, Australia; 3Department of Chemistry, University of
Gothenburg, P.O. Box 100, 405 30 Gothenburg, Sweden; hugh.dunstan@newcastle.edu.au
Received: 4 August 2015 / Accepted: 10 November 2015
RESEARCH ARTICLE
© 2015 Wageningen Academic Publishers
Abstract
Little is known about the amino acid composition of horse sweat, but significant fluid losses can occur during
exercise with the potential to facilitate substantial nutrient losses. Sweat and plasma amino acid compositions for
Standardbred horses were assessed to determine losses during a standardised training regime. Two cohorts of horses
2013 (n=5) and 2014 (n=6) were assessed to determine baseline levels of plasma and sweat amino acids. An amino
acid supplement designed to counter losses in sweat during exercise was provided after morning exercise daily for
5 weeks (2013, n=5; 2014, n=4). After the supplementation period, blood and sweat samples were collected to assess
amino acid composition changes. From baseline assessments of sweat in both cohorts, it was found that serine,
glutamic acid, histidine and phenylalanine were present at up to 9 times the corresponding plasma concentrations
and aspartic acid at 0-2.2 μmol/l in plasma was measured at 154-262 μmol/l in sweat. In contrast, glutamine,
asparagine, methionine and cystine were conserved in the plasma by having lower concentrations in the sweat. The
predominant plasma amino acids were glycine, glutamine, alanine, valine, serine, lysine and leucine. As the sweat
amino acid profile did not simply reflect plasma composition, it was proposed that mechanisms exist to generate
high concentrations of certain amino acids in sweat whilst selectively preventing the loss of others. The estimated
amino acid load in 16 l of circulating plasma was 3.8-4.3 g and the calculated loss via sweat during high intensity
exercise was 1.6-3.0 g. Following supplementation, total plasma amino acid levels from both cohorts increased
from initial levels of 2,293 and 2,044 µmol/l to post-supplementation levels of 2,674 and 2,663 µmol/l respectively
(P<0.05). It was concluded that the strategy of providing free amino acids immediately after exercise resulted in
raising resting plasma amino acid levels.
Keywords: exercise recovery, catabolism, muscle, sweat, amino acids.
R.H. Dunstan et al.
202 Comparative Exercise Physiology 11 (4)
Free amino acids released from tissue into the plasma at the
cessation of exercise are theoretically available for re-uptake
or excretion and restoration of homeostasis. The amino
acid levels excreted in urine and sweat represent a net loss
of amino acids which must ultimately be replenished via
dietary intake. Little is known about equine nutrient loss via
sweat but significant volumes of sweat can be lost during
exercise which could contribute to altering nitrogen balance
during periods of high performance training (Marlin et al.,
1999). It was proposed that comparing the free amino acid
profile of the plasma relative to the excretion profiles of
sweat would provide further insight into sweat facilitated
losses of amino acids (SFLAA) and the potential impact
on effective recovery from exercise.
Literature pertaining to plasma free amino acid levels
in relation to exercise in equine athletes is currently
limited, with many of the published studies confined to
measurement of tryptophan (Trp) and/or the branched-
chain amino acids (BCAA) leucine, isoleucine and valine
(Assenza et al., 2004; Casini et al., 2000; Farris et al.,
1998; Stefanon et al., 2000). These studies have largely
been driven by the apparent significance of the plasma
Trp/BCAA ratio to the synthesis of 5-hydroxytryptamine
and its role in fatigue associated with exercise in humans
comprising the ‘central fatigue hypothesis’ (Blomstrand,
2001). Among these studies, those involving clearly defined
exercise regimes have revealed decreased serum BCAA
levels and increased Trp/BCAA following prolonged
exercise (Assenza et al., 2004); reduced endurance but no
effect on plasma Trp/BCAA ratio following infusion with
Trp (Farris et al., 1998); and no change in performance
of Standardbred trotters following supplementation with
BCAA alone (Casini et al., 2000). Decreased serum BCAA
and increased alanine were observed after prolonged
exercise in one study (Trottier et al., 2002) while another
reported similar results following short term exercise
(Essen-Gustavsson et al., 2010). The latter further reported
that plasma BCAA levels remained higher during exercise
recovery in horses fed a high protein diet than in those
on a diet based on ‘recommended’ crude protein content.
The findings of two further studies involving analysis of a
comprehensive array of amino acids were less comparable.
In a recent study, plasma amino acid levels were measured
in Standardbred trotters before and after intensive short
term exercise, reporting increases in the levels of seven
of 22 amino acids measured and decreases in a further
nine, with indications of muscle catabolism (Hackl et
al., 2009). In contrast, Bergero et al. (2005) observed a
more general increase in serum amino acids levels after
short term exercise and a general decrease in amino acid
concentrations after prolonged exercise, concluding that
the observed short term increases reflected mobilisation of
amino acids, while longer term decreases were indicative
of amino acid catabolism.
The relationship between the amino acid composition
of sweat and plasma has been investigated in regards to
managing temperature with associated losses of electrolytes
and fluid. It is known that the superficial dermal layers of
the horse are highly vascularised, providing the sweat gland
fundus with a rich blood supply (McEwan Jenkinson et al.,
2006) and that equine sweating rates are closely correlated
with blood flow to the dermis (Johnson and Creed, 1982;
Johnson and Hales, 1983; Lovatt Evans and Smith, 1956).
Amino acids present in sweat clearly represent a net loss
of nutrients, yet despite the fact that horses exhibit the
highest known sweating rates of any animal (Marlin et
al., 1999) the overall extent of such losses has not been
well established. It is noted that the authors of the current
paper were unable to locate in the published literature, any
peer-reviewed data on free amino acid levels in the sweat
of equine athletes.
The primary aims of the current study were to (1)
characterise the amino acid composition of equine sweat
to assess the potential for SFLAA resulting from exercise
and (2) assess the potential for amino acid replacement
via supplementation to alter resting plasma composition
of amino acids. Two separate studies were undertaken in
consecutive years at the same Standardbred horse training
facility to assess whether the measures in sweat were
consistent across years with variable weather conditions.
2. Methods and materials
Experimental design
The study involved two cohorts of five and six Standardbred
harness racing geldings, aged from 3 to 5 years, with no
history of significant disease or suffering from any significant
ailments or injuries at the beginning of the project. Cohort
1 (n=5) was studied in 2013, where all horses followed
an identical training schedule under supervision of one
trainer, and were actively engaged in competitive racing
throughout the course of the study. The baseline sampling
was integrated into the regular training regime of the horses
and was scheduled to coincide with sessions involving a
high intensity track workout. As a result, samples were
taken at regular intervals for a period of three weeks prior
to commencing supplementation where four-five samples
were collected per horse. Prior to each training session the
horses were fitted with a GPS/heart rate monitoring device
to allow for the measurement of speed, distance, effort
and recovery. The horses underwent daily training every
morning from Monday to Saturday (except on race days)
prior to receiving supplementation and feed. The horses
received 1 kg Hygain Powatorque
®
(crude protein 17%,
crude fat 10%, maximum crude fibre 10%, added salt 1.5%
calcium 1.5%, phosphorous 0.6%, lysine 11 g/kg, vitamin E
1000 IU/kg, selenium 1.5 mg/kg; Hy Gain Feeds Pty Ltd.,
Victoria, Australia) after the morning workout and again
Sweat amino acid losses in Standardbred horses
Comparative Exercise Physiology 11 (4) 203
in the evening. The horses also received 2.5-4.5 kg Hygain
Micrbarley® (crude protein 11%, crude fat 2%, maximum
crude fibre 9%) for each animal depending on size and
condition. All horses received liberal quantities of wheaten
chaff and lucerne chaff and this feeding regime remained
constant throughout the baseline, supplementation and final
assessment periods. No other vitamins or supplements were
provided during the experimental period. On Sundays, the
horses were allowed to forage on grass in the open paddock.
With the exception of race day restrictions when it was
not possible for the horses to receive the supplement,
the horses were provided with a complex amino acid
supplement daily for 34 days before beginning two weeks
of post-supplementation samplings and evaluations. The
horses were provided with an amino acid formulation
commercially available as Fatigue Reviva™ (Top Nutrition
Pty Ltd., Newcastle, Australia) which was a complex mix
of 20 L-amino acids (glycine, proline, glutamine, carnitine,
threonine, lysine, alanine, valine, taurine, serine, cysteine,
arginine, histidine, isoleucine, phenylalanine, leucine,
methionine, glutamic acid, aspartic acid, and tyrosine),
fructo-oligosaccharide, malic acid, citric acid, succinic
acid, ribose, and 13 minerals and 13 vitamins. The Fatigue
Reviva™ product was developed for human consumption
and has been shown to be efficacious in reducing fatigue
(Dunstan et al., 2013, 2014). The formulation was provided
by the company in a large resealable plastic container and
was mixed daily with mid-chain triglycerides oil (1:1) to
form a paste before oral delivery via 60 ml plastic syringes.
The human dosages were adjusted appropriately for the
horses with 30 g of the Fatigue Reviva™ being provided to
each horse daily, which delivered 14.1 g amino acids (except
as precluded by racing). After 34 days of supplementation,
blood and sweat samples were taken before and after
hard work training sessions on three separate occasions
over a two week period whilst supplementation was
continued. Approval was received from the University of
Newcastle Animal Care and Ethics Committee (approval
number A-2012-257).
The second cohort of horses was studied in 2014 with a
view to replicating the analyses of sweat composition using
a different set of animals. Again all horses followed the same
training and feeding schedules under the supervision of
the same trainer, and were actively engaged in competitive
racing throughout the period of the study. Cohort 2 was
provided with a supplement available as Hygain Omina R3
(Hy Gain Feeds Pty Ltd.) which was formulated to contain
only the 14 amino acids identified as the major amino acid
components lost in sweat (serine, glutamic acid, histidine,
leucine, lysine, aspartic acid, alanine, glycine, phenylalanine,
valine, isoleucine, proline, threonine, and tyrosine). The
proportions were also adjusted to reflect the relative losses
observed for the amino acids in the sweat for these animals.
The amino acids in Hygain Omina R3 were pre-mixed in the
appropriate proportions with the Aqua Gel base product
(1:1) (Hy Gain Feeds Pty Ltd.) to form a paste comprising
30 g amino acids with 500 mg glucose in 30 ml Aqua Gel
per serve for delivery via 60 ml syringes. This volume was
easy to administer and the formulation was well received
by the animals. Five horses were sampled four times
each during an initial two week period to obtain baseline
measures. Two horses were withdrawn after baseline
testing and prior to supplementation for Cohort 2 due to
the development of injury concerns, and were replaced with
another Standardbred horse. This provided six animals for
baseline evaluations and four which were then provided
with the supplement during training and racing for 40 days.
Post-supplementation blood samples were taken before and
after hard work training sessions, while sweat samples were
taken following training, on four separate occasions over
a two week period whilst supplementation was continued.
The sample collection periods were extended due to delays
resulting from inclement weather.
Resting plasma samples were evaluated from another seven
horses at the trainer’s property and assessed as ‘horses not
in work’ (time not in work: 4 months-7 years; age range:
3-14 years old; six geldings and one brood mare). These
horses had not been provided with any of the Hygain high
protein content feeds for at least four months prior to
assessment and were foraging on grass in the paddocks.
Because these horses had not received high protein dietary
support, they were used as a reference group for comparison
of their amino acid levels in plasma.
The exercise sessions for both Cohorts of horses involved
two hard work sessions per week when the horses were not
racing, or one hard work session and a race. The horses
were raced on average once every three weeks. The hard
work sessions involved pacing around a 700 m track in
full racing harness while pulling a sulky and driver. The
session comprised approximately 2.5 ‘warm up’ laps of
the track at a moderate pace, accelerating to racing speed
for 3-4 laps, then decelerating gradually for 2.5 laps as
a final ‘warm down’. All samples were taken before and
after an early morning hard work session and prior to
provision of feed or supplement. The light training sessions
involved approximately 2.5 ‘warm-up’ laps of the track at a
moderate pace and then a light jog at around 19 km/h for
9-12 km. To provide as much consistency as possible in
training tempo and demand between horses and between
sampling events, the same driver was used for all horses in
all sessions. The various aspects of the training, including
duration, session times, distances, speeds and heart rate
parameters were monitored regularly in Cohort 1 during
the hard work sessions to assess consistency between horses
and between pre- and post-supplementation stages of the
project. The data have been summarised in Table 1 where it
was apparent that the majority of the measured parameters
did not show significant differences between the two stages
R.H. Dunstan et al.
204 Comparative Exercise Physiology 11 (4)
of assessment for Cohort 1. The training mean speeds and
session distances varied to some degree based upon the
prevailing season, weather and track conditions. However,
it was concluded that the training regimes were sufficiently
consistent between the pre- and post-supplementation arms
of the study for Cohort 1 to allow comparisons to be made
and a similar regime was applied to Cohort 2.
Biospecimen collection
Each horse had replicate samples taken for assessment of
pre-exercise blood and plasma as well as post-exercise blood,
plasma and sweat at both baseline and post-supplementation
stages. The replicate samples for each phase of testing
were averaged to represent a single representative blood,
plasma or sweat sample for each animal at both the baseline
and subsequent post-supplementation stages. The data
sets of both cohorts were analysed separately to assess
the consistency of amino acid composition in sweat and
responses to amino acid supplementation in two different
cohorts of animals measured across two years. Before
commencement of supplementation, baseline levels of
plasma amino acids for each horse were established by
taking blood samples before and immediately following the
exercise training regimes on four or five separate occasions.
After 34 days of supplementation in Cohort 1, a second set
of samples were taken on three separate occasions whilst
the horses remained on the supplement to establish the
post-supplementation compositions of the plasma and
sweat. The same approach was taken for Cohort 2, where
baseline levels were established for each horse during four
separate sampling events prior to supplementation. After
40 days of supplementation for Cohort 2 a second set of
samples were taken on four separate occasions whilst the
horses remained on the supplement to establish the post-
supplementation compositions of the plasma and sweat.
Blood was collected from the jugular vein of each horse in
a 20 ml syringe and transferred directly to a labelled 9 mL
lithium heparin Vacutainer
®
(BD, Franklin Lakes, NJ, USA)
prior to the hard work session representing the ‘resting’
sample. An equivalent blood sample was also collected
immediately after the training session (within 15-20 min)
and sweat was collected by scraping a sterile 70 ml sample
jar in an upward motion over the surface of the horse’s coat
to allow the fluid to run into the container. In Cohort 1,
a combined sweat sample was collected from three areas
of the body: (1) the chest between and immediately above
the forelegs; (2) the sides and underbelly of the torso;
and (3) the insides of the upper portion of the hind legs
and analysed as a pooled sample. In Cohort 2, sweat was
collected and analysed separately from the same three
regions to determine whether any differences in sweat
composition of amino acids occurred at different locations
on the body. Once collected, each sweat sample was
transferred to a sterile Monovette
®
tube (Sarstedt Australia
Pty Ltd., Mawson Lakes, Australia) and stored immediately
following collection in a chilled container for transport to
the laboratory. All blood and sweat samples were collected
prior to supplementation and feeding.
Biospecimen analysis
Following the blood draw for the horses in Cohort 2, a 100
µl sample of whole blood was drawn into a heparinised
capillary tube (Bacto Laboratories, Liverpool, Australia).
The sample was immediately expelled from the capillary
tubes into the sample well of an i-STAT
®
CG8+ cartridge
(i-STAT Corporation, East Windsor, NJ, USA). All air
bubbles were removed from the samples prior to the
cartridges being closed. The cartridge was analysed by
i-STAT® clinical analyser for measures of haematocrit (Hct)
and haemoglobin (Hb). Prior to each testing session, the
i-STAT analyser was calibrated according to manufacturer’s
specifications by an electronic stimulation and Level 2
i-STAT control solution. Cartridges were stored prior to
use as per the manufacturer’s instructions (2-8°C), and
were removed to room temperature approximately 5 min
prior to use.
The plasma fraction was isolated from the blood samples
via centrifugation (3,000 rpm, 10 min) and the plasma
supernatant was subsequently transferred to sterile 2 ml
Eppendorf tubes. Aliquots of sweat samples were removed
from the Monovette tubes and centrifuged at 2,000 rpm
for 5 min. The clear supernatant was transferred to a clean
tube for extraction. The amino acid compositions of both
plasma and sweat samples were determined via EZ:Faast™
derivatisation (Phenomenex Inc., Torrance, CA, USA)
Table 1. Comparison of baseline and post-supplement training
parameters for the Cohort 1 horses.1
Parameter
Pre-supplement
Post-supplement2
Training duration (s) 301±13 259±43
Training mean heart rate (bpm) 208±8 214±6
Training peak heart rate (bpm) 224±6 230±4
Training peak speed (km/h) 51±0.6 50±2
Session time (s) 1049±24 1062±145
Session mean heart rate (bpm) 158±3 156±9
Session peak heart rate (bpm) 224±6 229±5
Session peak speed (km/h) 54±0.9 53±2
Training distance (km) 3.8±0.2 3.1±0.5*
Training mean speed (km/h) 44±1 41±2*
Session mean speed (km/h) 27±0.5 22±1*
Session distance (km) 7.0±0.3 6.3±0.6*
1 Values are means ± standard error (n=5).
2 Significant difference from values obtained at baseline at *P<0.05.
Sweat amino acid losses in Standardbred horses
Comparative Exercise Physiology 11 (4) 205
followed by gas chromatography-flame ionisation detector
(GC-FID) analysis. The EZ:Faast procedure consists of a
solid phase extraction step, followed by derivatisation and
a liquid/liquid extraction. All samples were derivatised
according to the manufacturer’s protocol, with the following
modifications: (1) addition of 200 µl of sterile de-ionised
water to the initial reaction mixture for all plasma samples;
(2) addition of 200 µl 0.1M HCl to the initial reaction
mixture for all sweat samples. Analysis of the EZ:Faast
derivatised samples was performed on a Hewlett Packard
HP 6890 series GC system (Agilent, Santa Clara, CA, USA)
fitted with a flame ionisation detector and ZB-PAAC-MS
column (10 m × 0.25 mm i.d.), supplied by Phenomenex
Inc. The instrument method comprised split injection
(ratio 15:1) with injector temperature 250°C and column
flow rate 0.5 ml/min. Injection volume was set at 2.5 μl
for all samples. The oven programme comprised an initial
temperature of 110°C, increasing 32°C/min to 320°C
(run time = 8.56 min). Target compounds were identified
according to pre-established retention times of analytical
standards, with quantification calibrated against the signal
response of an internal standard.
Statistical analysis
Comparisons of blood parameters, exercise regimes and
amino acid concentrations between sample sets were
completed by one-way ANOVA using Statistica™ 12
(Statsoft, Tulsa, OK, USA). Effect sizes were calculated and
interpreted accordingly to assess the magnitude of difference
between the means for haematocrit and haemoglobin
assessed at baseline and after supplementation (Cohen’s
d; small = 0.2-0.49, moderate = 0.5-0.79; large ≥0.8). Samples
from both cohorts were pooled for comparison of pre- and
post-supplementation levels of total amino acids in resting
plasma using paired-samples t-test in Statistica. Correlation
analyses were performed on the plasma resting plasma
levels of amino acids and the corresponding haematocrit
and haemoglobin levels in the horses at baseline and again
following the supplementation period using Statistica. The
wellbeing of the horses was assessed daily by the trainer
throughout the study and notes recorded.
3. Results
Baseline sweat amino acid composition relative to post-
exercise plasma
The sweat collected from the three collection sites on
each animal in Cohort 1 was pooled for each horse and
displayed high variability for the amino acid concentrations
(Table 2). The sweat collected from each sample site in the
Cohort 2 samples were analysed separately revealing that
the chest samples had a total amino acid concentration
of 3,214±411 µmol/l compared with the underbelly at
2,777±428 µmol/l and the hind legs at 1,876±315 µmol/l
(P<0.05). The chest area sweat also had the highest levels
of serine, histidine and threonine and was deemed the
easiest and safest collection site for the animal handlers.
This sampling site was therefore used for the reporting
of sweat data for Cohort 2. At baseline, the mean loss in
body weight which occurred during exercise for Cohort 2
was 5.1±0.7 kg.
Twenty amino acids were detected and quantified in post-
exercise plasma taken from the horses. Comparisons
between the plasma compositions from Cohort 1 and
Cohort 2 revealed that the average plasma profile patterns
were similar across the two studies (Table 2), with glycine
as the major plasma amino acid, followed by alanine,
glutamine, valine and serine, comprising 61-64% of the
plasma amino acids. The total levels of amino acids in the
plasma from the two cohorts were similar in both individual
amino acid magnitude and distribution.
The amino acid compositions of the sweat were significantly
different from those of the corresponding plasma samples
for both cohorts (Table 2). The average total concentrations
of amino acids in the sweat samples were double that of the
plasma for Cohort 1 (P<0.05) and although 1.2 times higher
in Cohort 2, this latter difference did not reach levels of
statistical significance. The sweat contained five amino acids
which were consistently present in higher concentrations
in the sweat compared with the corresponding plasma
levels for both study Cohorts and included serine (3.9-5.4
times higher), glutamic acid (7.0-9.5 times higher) histidine
(4.3-4.5 times higher), phenylalanine (1.9-3.4 times higher),
and aspartic acid. Aspartic acid was not detected in the
plasma from the horses in Cohort 1 but was present at
262±29 µmol/l in the sweat. Similarly, it was measured
at 2±2 µmol/l in plasma from Cohort 2 compared with a
corresponding 154±21 µmol/l in the sweat. Alanine, leucine,
valine, proline and tyrosine were higher in sweat relative
to plasma in Cohort 1, but not Cohort 2. Both valine and
ornithine were more concentrated in the sweat relative to
the plasma in Cohort 1, but were less concentrated in the
sweat in Cohort 2. Glutamine, cystine, methionine and
asparagine were consistently lower in concentration in
the sweat relative to the plasma in both cohorts. Glycine
and tryptophan were lower in the sweat compared with
plasma in Cohort 2 and present at equivalent levels in both
matrices in Cohort 1.
Changes in resting blood, plasma and sweat post-
supplementation
Average amino acid levels were assessed in resting plasma
samples at baseline and after supplementation to assess
potential changes in metabolic homeostasis following
amino acid supplementation (Table 3). Cohort 1 had
a higher average baseline level of total plasma amino
acids at 2,293±68 µmol/l compared with Cohort 2 at
R.H. Dunstan et al.
206 Comparative Exercise Physiology 11 (4)
2,044±135 µmol/l, but the resting levels displayed similar
profile characteristics between the two years. Following
supplementation, average total plasma amino acid
levels increased in both cohorts compared with their
corresponding mean baseline levels to 2,674±41 µmol/l and
2,663±124 µmol/l respectively (P<0.05). The average total
levels of amino acids in plasma were therefore equivalent
for the two cohorts post-supplementation. The two cohorts
were pooled to perform a paired-samples t-test to compare
plasma amino acid concentrations at baseline and post-
supplement with a Bonferroni correction. There were
significant increases observed in the resting plasma after
the supplementation period compared with the baseline
levels for glycine (baseline: 582 µmol/l vs post-supplement:
769 µmol/l), threonine (93 µmol/l vs 118 µmol/l), serine
(175 µmol/l vs 312 µmol/l), and glutamine (213 µmol/l
vs 343 µmol/l) (n=9, P<0.002). The average total amino
acid concentration in plasma after supplementation
(mean = 2,669 µmol/l, standard deviation = 165) was also
assessed using a paired-samples t-test and was found to
be significantly higher compared with the levels observed
prior to supplementation (mean = 2,138 µmol/l, standard
deviation = 313) (t(8) = -4.29, P<0.003). The data indicated
that the process of amino acid supplementation immediately
after exercise resulted in increased circulatory levels of
amino acid levels in resting plasma.
As well as having a lower plasma total amino acid content
at baseline, Cohort 2 also had a lower initial average
total amino acid level in the sweat at 3,213±411 µmol/l
compared with the Cohort 1 level of 5,696±932 µmol/l
(Table 4). Following supplementation, the average total
Table 2. The mean concentrations of amino acids in post-exercise sweat and plasma samples taken from horses prior to
commencement of supplementation.1
Cohort 1 Cohort 2
Amino acid type based on sweat levels higher
than corresponding plasma levels
Post-exercise
amino acids
Sweat Blood plasma2Sweat Blood plasma2
Type A. Sweat amino acid concentrations higher than
or equivalent to plasma levels
serine 893±103 164±8* 791±108 202±14*
glutamic acid 429±53 45±7* 167±25 24±2*
histidine 396±184 88±5* 206±28 48±6*
aspartic acid 262±29 0.0* 154±21 2±2*
phenylalanine 222±48 66±2* 131±18 69±4*
Type B. Amino acids with variable sweat concentrations
relative to the plasma concentrations, depending on
study cohort
Type C. Sweat amino acid concentrations lower than
or equivalent to plasma levels
alanine 594±69 392±12* 489±68 345±12
leucine 489±106 144±2* 128±20 138±7
lysine 448±136 156±7 150±23 96±3*
valine 389±49 266±6* 137±27 207±14*
proline 164±18 96±3* 111±22 110±4
tyrosine 158±31 66±2* 87±15 77±4
ornithine 129±35 83±2 39±5 59±4*
glycine 616±65 607±34 409±55 637±30*
tryptophan 30±8 44±1 13±2 60±7*
cystine 21±3 36±2* 3±1 28±2*
methionine 18±3 29±2* 7±2 22±1*
cystathionine 15±5 14±6 0.2±0.2 9±4
glutamine 14±9 286±15* 9±5 251±19*
hydroxylysine 2±2 30±3* 3±2 0±0
asparagine 1±1 30±2* 8±3 21±1*
Total35,696±932 2,834±18* 3,213±411 2,584±101
1 Values are means ± standard error (µmol/l). Sample size: Cohort 1 (n=5); Cohort 2 (n=6).
2 Plasma values significantly different compared with the corresponding sweat values (* P<0.05).
3 Total includes some minor amino acid derivatives not shown: α-aminopimelic acid, α-aminoadipic acid, glycine-proline and β-aminoisobutyric acid.
Sweat amino acid losses in Standardbred horses
Comparative Exercise Physiology 11 (4) 207
sweat amino acid levels did not increase significantly for
Cohort 1 at 6,228±546 µmol/l, but more than doubled in
concentration for Cohort 2 at 8,682±563 µmol/l (P<0.05).
All of the amino acids measured were observed at higher
levels in the sweat post-supplement compared with baseline
levels for Cohort 2 (P<0.05).
The mean resting haematocrit and haemoglobin levels
were 0.41±0.025 and 141±8.6 g/l, respectively, for the
four Cohort 2 horses at baseline who completed the
supplementation program. Following supplementation,
large increases (Cohen’s d>0.8) were observed for both
parameters where the resting haematocrit increased to
0.46±0.015 and haemoglobin increased to 155±5.3 g/l.
The correlation analyses of the resting levels of plasma
amino acids indicated that threonine was the only amino
acid with a strong correlation to the resting blood levels
of haemoglobin. After the supplementation period, a
number of amino acids showed strong associations with
haemoglobin where histidine, valine, asparagine, glutamic
acid, glutamine and lysine displayed R2>0.98.
4. Discussion
The results from this study indicated that free amino acids
were lost in sweat during exercise and that consistently
the concentrations of nine out of 20 amino acids were
not isotonic with the plasma in both studies. Serine,
glutamic acid, histidine, phenylalanine and aspartic acid
were present in the sweat at levels three times greater
Table 3. Comparisons of the amino acid composition (μmol/l) of pre-exercise plasma before and after supplementation for both
Cohort 1 and Cohort 2.1,2
Cohort 1
Plasma concentrations (Mean ± SE)
Cohort 2
Plasma concentrations (Mean ± SE)
Horses not in work
(Mean ± SE)
Literature values3
Mean (range)
Amino acid Baseline (n=5) Post-supplement (n=5) Baseline (n=6) Post-supplement (n=4) (n=7) (n=10)
Glycine 588±42 795±29* 608±49 736±29 423±57 487 (298-641)
Serine 167±5 338±15* 198±26 279±24 287±34 223 (88-332)
Glutamine 256±18 354±16* 172±14 330±17* 393±24 322 (179-440)
Valine 228±7 191±4* 169±5 177±7 291±21 301 (199-457)
Alanine 134±9 189±29 127±9 185±14* 207±26 245 (131-420)
Proline 81±4 90±4 103±9 110±8 121±15 NR
Leucine 98±5 90±4 96±5 106±3 157±16 144 (73-252)
Threonine 96±8 115±5 89±5 121±5* 184±22 171 (73-235)
Tryptophan 51±3 50±2 67±8 67±3 65±8 NR
Lysine 116±7 75±6* 65±6 87±5* 156±27 144 (64-201)
Tyrosine 58±2 53±1* 64±6 67±4 90±10 93 (48-100)
Phenylalanine 56±3 48±3 59±4 60±2 76±8 80 (50-95)
Isoleucine 53±1 58±3 52±3 53±4 83±7 78 (66-130)
Ornithine 74±3 58±3* 51±5 68±2* 77±10 56 (24-81)
Histidine 90±3 54±4* 33±5 61±4* 112±16 76 (60-97)
Cystine 40±3 23±2* 27±3 31±2 50±4 28 (10-51)
Methionine 25±3 22±2 21±2 19±0.3 38±4 33 (23-45)
Asparagine 24±3 32±1* 21±3 32±3* 8±1 NR
Glutamic acid 17±5 31±3 11±1 21±1* 26±3 19 (<10-32)
Cystathionine 5±1 4±2 1±0.4 1±0.7 50±4 NR
Aspartic acid 0.0 0 0±0 14±2* 8±1 <10 (7-11)
Total 2,293±68 2,674±41* 2,044±135 2,663±124* 3,450±61 2,754 4
1 SE = standard error.
2 Significant differences between baseline and post-supplement concentrations within each cohort indicated with * P<0.05.
3 Results reported for non-grazing mixed breed horses, 2-20 years of age (McGorum and Kirk, 2001); NR indicates not reported.
4 There were four amino acids reported in the current study which were not reported in the McGorum and Kirk (2001) study. Thus the values for proline
tryptophan, asparagine and cystathionine from the current study for the horses out of work were substituted into the calculation of totals for the purposes of
reference comparison. The reference data reported additional values for arginine, taurine and citrulline which were not measured by the current technique
(representing an additional combined 195 µmol/l).
R.H. Dunstan et al.
208 Comparative Exercise Physiology 11 (4)
than those in the plasma and represented 39-45% of the
free amino acids measured in sweat. In contrast, the
concentrations of glutamine, cystine, methionine and
asparagine were consistently reduced in sweat compared
with the corresponding plasma suggesting the presence of
mechanisms to protect against the loss of these particular
amino acids from the plasma. These observations provided
evidence that SFLAA was actively regulated, potentially
impacting nitrogen balance, electrolyte and fluid loss, and
thermoregulation.
Amino acid homeostasis in the horse is dependent upon
the daily quantities and quality of ingested protein balanced
against the loads of physical activity, which would influence
rates of protein turnover, energy metabolism and amino
acid excretion. For horses out of work, the maintenance
levels of protein synthesis have been estimated at 15 g/kg
BW
0.75
which would represent 1,400 g per day for a 430 kg
animal. Horses generally synthesise 3-5 times more protein
per day than they consume, which is achieved via processes
of recycling amino acids released from protein degradation
(Milward et al., 1983). The combined daily protein losses
from dead skin cells, sweat, urine and faeces was estimated
at 40-49 g for the 430 kg horse using previously derived
rates of loss/kg BW (McDonald et al., 2009; Meyer, 1983).
When horses engage in regular work, nitrogen requirements
increase, more amino acids are consumed for energy and
excess nitrogen is primarily excreted as urea via the sweat
(Martin-Rosset, 2008). The animals in work in the current
study were receiving 615-835 g per day of crude protein as
well as liberal quantities of wheaten chaff and lucerne chaff
to support the extra activity loads from training.
Based on the assumption that approximately 85% of weight
loss during exercise could be attributed to fluid loss in sweat
(McCutcheon et al., 1999), it was calculated that the loss of
free amino acids was 1.6-3.0 g during the defined exercise
regime. To set this in context, although the plasma volume
in horses is variable between breeds and the levels of fitness
and hydration, an average 450 kg horse would have a plasma
Table 4. Comparisons of the amino acid composition of baseline and post-supplement sweat for Cohort 1 and Cohort 2.1
Amino acid Cohort 1
Sweat concentrations
Cohort 2
Sweat concentrations
Baseline (n=5) Post-supplement (n=5) Baseline (n=6) Post-supplement (n=4)
Serine 893±103 1,588±178* 791±108 1,752±91*
Glycine 616±65 787±61 409±55 1,119±111*
Alanine 594±69 792±87 489±68 1,292±78*
Leucine 489±106 166±11* 128±20 485±64*
Lysine 448±136 291±15 150±23 473±52*
Glutamic acid 429±53 411±31 167±25 571±106*
Valine 389±49 154±14* 137±27 325±33*
Histidine 396±184 924±175 206±28 600±206*
Aspartic acid 262±29 221±21 154±21 372±32*
Phenylalanine 222±48 152±8 131±18 304±32*
Isoleucine 210±36 87±7* 66±10 218±28*
Proline 164±18 153±9 111±22 206±19*
Threonine 165±20 189±22 97±16 302±19*
Tyrosine 158±31 101±5 87±15 202±23*
Ornithine 129±35 85±10 39±5 82±13*
Tryptophan 30±8 19±1 13±2 33±2*
Cystine 21±3 5±2* 3±1 13±1*
Methionine 18±3 8±3* 7±2 30±11*
Cystathionine 15±5 3±2 0.2±0.2 4±2*
Glutamine 14±9 38±4* 9±5 134±32*
Asparagine 1±1 18±14 8±3 58±21*
Total25,696±932 6,228±546 3,213±411 8,682±563*
1 Values are means ± standard error (µmol/l). Significant difference from values obtained pre-supplement at *P<0.05.
2
Total includes some minor amino acid derivatives not shown in the above table: a-aminopimelic acid, a-aminoadipic acid, glycine-proline, and
β-amino-isobutyric acid.
Sweat amino acid losses in Standardbred horses
Comparative Exercise Physiology 11 (4) 209
volume of around 16 l with a corresponding red cell volume
around 20 l (Carlson, 1987). The total amino acid loading
in the 16 L plasma volume was thus calculated as 3.8-4.3 g
across the two cohorts of horses in the present study. Based
upon the above calculations, approximately 40-70% of the
plasma loading of amino acids may be lost through sweat
during exercise that resulted in a 1% reduction of the body
mass via sweating. This indicated that sweating increased
the requirement for amino acids to enter circulation via the
catabolic turnover of muscle storage proteins. It was thus
argued that the loss of amino acids via sweat would place
additional demands on muscle reserves to replenish plasma
levels required to support metabolic processes including
energy production (glucose-alanine cycle), oxygen delivery,
muscle repair and recovery (Boirie, 2009; Castaneda et al.,
1999; Jagoe and Engelen, 2003; Kingsbury et al., 1998).
During exercise the blood supply is diverted from the
digestive tract to the muscles allowing increased delivery
of oxygen and energy substrates to support muscle activity
as well as facilitating the removal of waste products such
as CO
2
(Burton et al., 2004; Jeukendrup, 2004; Rennie and
Tipton, 2000). The digestive tract is not fully functional
during or immediately after exercise and when the process of
digestion does resume, it takes time to break down proteins
to yield amino acids for absorption. The catabolism of
storage proteins thus continues for several hours following
cessation of exercise to provide energy and amino acids for
protein synthesis in recovery and repair (Martin-Rosset,
2008). During this time following high intensity training,
the first phase of recovery may be viewed as critical since
the continued degradation of muscle proteins could extend
to proteolysis of actin and myosin fibrillar proteins leading
to muscle damage, wasting, soreness and peripheral fatigue
(Macintosh and Rassier, 2002; Niblett, 2007; Phillips, 2004).
The provision of an amino acid supplement that did not
require digestion was provided in this study for ingestion
immediately after exercise. In this way amino acids could
be delivered directly to the circulation minimising potential
demands on muscle protein stores during recovery. The
provision of amino acids immediately after exercise
resulted in a significant increase in resting total levels of
amino acids in plasma from 2,293±68 µmol/l (Cohort 1)
and 2,044±135 µmol/l (Cohort 2) to 2,674±41 µmol/l and
2,663±124 µmol/l, respectively. The consistent increase in
resting plasma levels of amino acids may potentially reflect
reduced daily demands on muscle protein stores during
the supplementation period with an ultimate outcome of
the ability to maintain a higher availability of amino acids
in plasma to support exercise and recovery. The horses
that were not in work relied entirely on foraging grass
pastures with no additional dietary protein but these horses
had an average total plasma concentration of 3,450±61
µmol/l (Table 3). There was no evaluation of the dietary
protein intake of these horses in rest but it was assumed
that the protein intake would be less without the carefully
managed protein loading supplied for each of the animals
in work. The results were consistent with previous research
indicating that exercise leads to higher rates of protein
turnover and nitrogen/energy demands (Martin-Rosset,
2008) which also appears to result in lower plasma levels of
amino acids. It was proposed that extended periods of high
intensity training involving increased daily utilisation and
replenishment of muscle stores could lead to diminished
amino acid reserves where the body’s demands are not
met via dietary intake and/or de novo synthesis, resulting
in lower resting plasma levels of amino acids.
In the current study, increased plasma amino acid levels
were accompanied by corresponding increased levels in
sweat following supplementation. The higher levels of
amino acids in sweat were largely attributable to differences
in serine, glycine, alanine, leucine, lysine and glutamic
acid which were major components of the amino acid
supplements. These results supported the hypothesis that
amino acids measured in the sweat collected from the
horses reflected metabolic homeostasis and the nutritional
status of the horses. This result does not necessarily
indicate that sweat composition directly reflects dietary
contributions, but rather reflects the nutritional status of
the animals. Specific differences in sweat composition from
the various locations tested (front, back and rear) indicated
an interesting trend where sweat was progressively more
concentrated in samples taken from the front compared
with samples taken from the mid-section and rear of the
animal. These results suggested that the composition
of sweat may have been regulated in regard to localised
metabolism and local muscle activities.
Serine was persistently measured as the most abundant
amino acid lost in the sweat from the horses consistent
with previous findings for sweat loss in humans
(Kutyshenko et al., 2011; Liappis and Hungerland, 1972).
In addition to its requirements for protein synthesis,
serine and its derivatives form functional groups in key
membrane phospholipids such as phosphatidylserine,
phosphatidylcholine and phosphatidylethanolamine.
Serine is the immediate precursor for the synthesis of
glycine which is the most abundant amino acid in the
horse plasma. The formation of glycine from serine also
generates methylene-tetrahydrofolate from tetrahydrofolate
which is essential for the synthesis of nucleic acids (De
Koning et al., 2003). Thus substantial sweat facilitated
losses of serine could lead to a broad range of metabolic
deficits with impact on performance and recovery if the
body’s supply is unable to meet demands (De Koning et
al., 2003; Lacey and Wilmore, 2009). The levels of serine,
alanine, glycine, glutamic acid and aspartic acid were also
high in sweat representing a group of amino acids that
can be synthesised de novo. However, under conditions
of prolonged exercise or exposure to heat, these amino
R.H. Dunstan et al.
210 Comparative Exercise Physiology 11 (4)
acids may become conditionally essential if synthesis
does not equal demand (Booth and Watson, 1985; Lacey
and Wilmore, 2009). Prior to supplementation, aspartic
acid was not detected in the plasma. However, following
the supplementation of Cohort 2, which were provided
with higher daily doses of amino acids, aspartic acid was
detected in the plasma. This finding was consistent with
aspartic acid becoming conditionally essential during high
intensity training.
The major plasma components, including glycine,
glutamine, valine, serine, alanine, leucine, threonine and
lysine measured in the current set of resting animals not
in work, were found at similar levels to those recorded in
a previous study (McGorum and Kirk, 2001). The mean
glycine concentrations in the plasma of animals in training
at baseline (588 and 608 µmol/l) and post-supplementation
(736 and 795 µmol/l) were substantially higher compared
with the levels for animals not in work (423 µmol/l) and
from previous research (487 µmol/l) (McGorum and Kirk,
2001). The implication of this observation is not clear,
but the relatively higher levels of glycine in the plasma of
working animals may reflect a physiological adjustment to
support the elevated levels of physical activity.
The feedback from the trainer from both study cohorts
provided subjective evidence that supplementation
provided benefits in regard to the general condition of
the horses including the development of muscle mass,
glossy coats, bright eyes and a capacity to work hard for
longer. The higher levels of plasma amino acids would
potentially be able to better support provision of energy
during work and protein synthesis supporting processes of
oxygen delivery, muscle growth and recovery from exercise.
The effect sizes produced indicated that haematocrit and
haemoglobin increases occurred after supplementation
with amino acids which was consistent with an improved
capacity to support high intensity training and recovery.
It should be noted that the elevations in haematocrit and
haemoglobin may also be attributed as a direct outcome of
the training period over the course of the study for which
there were no control samples for comparison. However,
improved training efficiency, quicker recovery times and
haematopoiesis have previously been reported in human
athletes provided BCAA formulations during a training
period (Ohtani et al., 2006). Evidence has been provided
that histidine was essential for human adults (Kopple and
Swendseid, 1975) on the basis that subjects with histidine-
deficient diets, developed a negative nitrogen balance and
had reduced haematocrit levels (Clemens et al., 1984).
After replenishment of histidine, their nitrogen balance
became positive and haematocrit levels increased to normal
(Cooperman and Lopez, 2002; Kopple and Swendseid,
1975). Histidine is a significant amino acid component of
haemoglobin and both human and animal studies have
demonstrated that histidine deficiency results in reductions
in haemoglobin and haematocrit values indicative of
reduced erythropoiesis (Clemens et al., 1984). During
prolonged periods of training and racing the availability
of amino acids for haematopoiesis may potentially become
limiting. In the present study, histidine was found to be
consistently lost in sweat at concentrations more than
four times greater than that found in plasma. Provision of
additional histidine, together with the other amino acids
in the supplement may enhance erythropoiesis.
To date, most research involving sweat has focussed on
electrolyte and fluid loss during exercise and training,
particularly in hotter climes. However, the losses of
amino acids through sweat can produce an immediate
and significant deficit to the circulating amino acid pool
placing demands on muscle proteins to supply amino acids
for the metabolic processes required to support exercise.
The results from this study indicated that amino acid
supplementation immediately following exercise raised
amino acid concentrations to a higher operating level in
plasma and that this new level was consistent for both
cohorts of horses. Maintaining a higher resting base level
of amino acid content in circulating plasma was deemed
to provide more resources which would be immediately
available to support exercise, muscle growth and repair
and to minimise depletion of muscle proteins.
5. Conclusions
It was found that the sweat facilitated losses of amino acids
were sufficiently high in regards to the plasma loadings
and that this process has the potential to contribute to the
depletion of amino acid resources in Standardbred horses
during exercise. The concentrations of five amino acids,
including serine and histidine were highly concentrated in
the sweat relative to the plasma. The remaining amino acids
measured were either isotonic or conserved in the plasma.
These findings suggest the presence of mechanisms for
controlling the loading of sweat with amino acids at higher
concentrations than plasma while other mechanisms are in
place to restrict losses of key amino acids from the plasma.
The process of providing amino acid supplementation
immediately after exercise resulted in increased resting
levels of amino acids in plasma to support exercise
and recovery.
Acknowledgements
The 2013 project work was funded jointly by Top Nutrition
Pty Ltd. (Newcastle, NSW Australia) and Enterprise
Connect (an Australian Government Initiative) via a
Researchers in Business Grant. Agricure Pty Ltd. is thanked
for providing the mid-chain trigyceride oil for the 2013
study. Hy Gain Pty Ltd. (Officer, Victoria, Australia)
provided the Hygain Omina R3 amino acids for the 2014
project work. Mr Ray Harkness from Berry Park Equine
Sweat amino acid losses in Standardbred horses
Comparative Exercise Physiology 11 (4) 211
(Berry Park, NSW, Australia) is thanked for his cooperation,
assistance and access to horses and training facilities under
his control which was vital for this project to proceed.
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... High levels of exercise activity would increase the requirement for protein intake and for protein turnover in order to offset the increased losses of free amino acids arising from demands for energy metabolism, tissue repair and recovery processes [8,9] as well as excretory losses via sweating [10,11]. Similar increased demands for amino acids would be seen during illness and recovery from trauma. ...
... It has been shown that the levels of several key amino acids including serine, glycine, histidine, alanine and ornithine are present in sweat at much higher concentrations than occur in the plasma [10,11,14,15]. It was proposed that these levels of amino acids in sweat, as has been found to be the case with sweat electrolytes [16], could be achieved by a process of leaching of the amino acids from the natural moisturising factor in the stratum corneum of the skin to combine with the quantities excreted in sweat [10,11,15]. ...
... It has been shown that the levels of several key amino acids including serine, glycine, histidine, alanine and ornithine are present in sweat at much higher concentrations than occur in the plasma [10,11,14,15]. It was proposed that these levels of amino acids in sweat, as has been found to be the case with sweat electrolytes [16], could be achieved by a process of leaching of the amino acids from the natural moisturising factor in the stratum corneum of the skin to combine with the quantities excreted in sweat [10,11,15]. The majority of amino acids which constitute the natural moisturising factor are thought to be derived from the protein filaggrin which is primarily composed of glutamine/glutamic acid, arginine/ornithine, serine, proline, glycine, histidine, and aspartic acid/alanine [17]. ...
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Background The excretion of amino acids in urine represents an important avenue for the loss of key nutrients. Some amino acids such as glycine and histidine are lost in higher abundance than others. These two amino acids perform important physiological functions and are required for the synthesis of key proteins such as haemoglobin and collagen. Methods Stage 1 of this study involved healthy subjects (n?=?151) who provided first of the morning urine samples and completed symptom questionnaires. Urine was analysed for amino acid composition by gas chromatography. Stage 2 involved a subset of the initial cohort (n?=?37) who completed a 30?day trial of an amino acid supplement and subsequent symptom profile evaluation. Results Analyses of urinary amino acid profiles revealed that three groups could be objectively defined from the 151 participants using k-means clustering. The amino acid profiles were significantly different between each of the clusters (Wilks? Lambda?=?0.13, p?<?0.0001). Cluster 1 had the highest loss of amino acids with histidine being the most abundant component. Cluster 2 had glycine present as the most abundant urinary amino acid and cluster 3 had equivalent abundances of glycine and histidine. Strong associations were observed between urinary proline concentrations and fatigue/pain scores (r?=?.56 to .83) for females in cluster 1, with several other differential sets of associations observed for the other clusters. Conclusions Different phenotypic subsets exist in the population based on amino acid excretion characteristics found in urine. Provision of the supplement resulted in significant improvements in reported fatigue and sleep for 81% of the trial cohort with all females reporting improvements in fatigue. Trial registration The study was registered on the 18th April 2011 with the Australian New Zealand Clinical Trials Registry (ACTRN12611000403932).
... The cytoplasmic lysate was filtered by transferring to QIAgen spin columns and centrifuging at 15,000×g for 5 min. The filtered lysate (100 µL) was added to 200 µL Milli-Q H 2 O with 100 μL norvaline as the internal standard and processed using EZ:Faast™ (Phenomenex ® Inc.) derivatisation kits for amino acid analysis by gas chromatography with flame ionization detection (GC/FID) as previously described (Dunstan et al. 2015). The EZ:Faast™ kit has been designed for rapid and efficient analyses of amino acids in plasma (Badawy 2019;Badawy et al. 2008). ...
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Erythrocytes have a well-defined role in the gaseous exchange of oxygen and carbon dioxide in the mammalian body. The erythrocytes can contain more than half of the free amino acids present in whole blood. Based on measures showing that venous erythrocyte levels of amino acids are much less than arterial erythrocyte levels, it has previously been proposed that erythrocytes also play a role in the delivery of amino acids to tissues in the body. This role has been dismissed because it has been assumed that to act as an amino acid transport vehicle, the erythrocytes should release their entire amino acid content in the capillary beds at the target tissues with kinetic studies showing that this would take too long to achieve. This investigation set out to investigate whether the equine erythrocytes could rapidly take up and release smaller packages of amino acids when exposed to high or low external concentrations of amino acids, because it seemed very unlikely that cells would be able to release all of their amino acids without serious impacts on osmotic balance. Freshly prepared erythrocytes were placed in alternating solutions of high and low amino acid concentrations in PBS to assess the capacities of these cells to rapidly take up and release amino acids depending on the nature of the external environment. It was found that amino acids were rapidly taken up and released in small quantities in each cycle representing 15% of their total load in equine erythrocytes and 16% in human erythrocytes. The capacity for rapid uptake/release of amino acids by equine and human erythrocytes provided evidence to support the theory that mammalian erythrocytes have a significant role in transport of amino acids from the liver to tissues, muscles and organs.
... Athletes can lose 1-2 L per hour of fluid through sweating with estimates of losses of amino acids at 5-23 mmol per hour of exercise in warm conditions (Dunstan et al. 2016). Such losses would place a considerable demand on protein turnover in the body to maintain homeostatic levels of amino acids in the plasma to support exercise while nutrients cannot be provided via ingestion (Dunstan et al. 2015(Dunstan et al. , 2016. ...
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