L.W.E. Peters et al.
ORIGINAL ARTICLE
Amino acid utilization by the hindlimb of warmblood horses at rest and following low
intensity exercise
L.W.E. Petersa, E. Smieta, M.G.M. de Sain-van der Veldenb, and J.H. van der Kolkc,*
a
Department of Equine Sciences, Medicine Section, Faculty of Veterinary Medicine, Utrecht
University, Utrecht, the Netherlands; bDepartment of Metabolic Diseases, University Medical
Centre Utrecht, Utrecht, the Netherlands; cSection of Equine Metabolic and Genetic Diseases,
Euregio Laboratory Services, Maastricht, the Netherlands
Background: In particular branched-chain amino acids might limit muscle protein loss in
pathological conditions. Little is known on basic amino acid utilization of muscle in horses.
Objective: To assess amino acid utilization by the hindlimb of horses at rest and following
low intensity exercise.
Animals & Methods: Amino acid uptake by the hindlimb was investigated using the
arteriovenous difference technique. Blood from six warmblood mares (mean age 12±3 (SD)
years and weighing 538±39 kg) was collected simultaneously from the (transverse) facial
artery and from the caudal vena cava. Food was withheld for 12 hours prior to exercise.
Exercise comprised of a standardized treadmill protocol consisting of 5 minutes of walk, 20
minutes of trot, and thereafter another 5 minutes of walk. Amino acids were determined
quantitatively by means of anion exchange chromatography. Statistical analysis was
performed using a general linear mixed model.
Results: Amino acids with the largest average extraction at rest were citrulline (11.1±9%),
cystine (8.3±36%), serine (7.9±11%), and leucine (5.9±9%). Of the 25 amino acids studied,
none showed a significant difference following exercise. Glycine (485±65 µmol/L), glutamine
(281±40 µmol/L), valine (183±26 µmol/L), and serine (165±22 µmol/L) showed highest
plasma concentrations. The average extraction for α-aminobutyric acid at rest was 18.2±26%.
Arterial plasma citrulline concentration was higher than venously.
Conclusion: Citrulline, cystine, serine, and leucine might be regarded as most important
amino acids at rest in warmblood mares.
Clinical importance: Further investigation is necessary into the specific role of leucine
supplementation to preserve or restore body protein in horses.
Keywords: equine, horse, amino acid, ketogenesis, arteriovenous difference, exercise
Title Page Footnote:
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*Corresponding author. Email: jh.vdkolk@euregio-lab.nl
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1. Introduction
Amino acids are important nutrients during rest and exercise in horses. Various studies
assessed the normal plasma concentration of (free) amino acids as well as the muscle content
of amino acids in horses. These studies showed that plasma concentrations of amino acids in
horses are influenced by many factors like physical activity, diet, age, and various hormones
(Johnson and Hart 1974; Poso et al. 1987; Miller-Graber et al. 1990; Hanzawa et al. 1992;
Pethick et al. 1993; Poso et al. 1993; Cabrera et al. 1996; Casini et al. 2000; McGorum and
Kirk 2001; Trottier et al. 2002; Essen-Gustavsson and Jensen-Waern 2002; Berhane et al.
2004; Hackl et al. 2009; Graham-Thiers and Bowen 2010; Westermann et al. 2011). Insulin
stimulates amino acid uptake and protein synthesis leading to decreasing plasma
concentrations in man (Fukagawa et al. 1985; Fukagawa et al. 1986), whereas cortisol induces
protein breakdown and de novo synthesis of protein in muscle (Gelfand et al. 1984), but the
magnitude of effect of these factors is limited as physiologically amino acid concentrations in
blood are tightly regulated within fixed limits. Plasma concentrations of amino acids reflect
absorption via the digestive tract, production and secretion by various tissues as well as
elimination due to metabolism and excretion. The way in which the concentration of one
specific amino acid reacts on e.g. protein intake or exercise is quite different given the amino
acid involved. For example, glutamine and alanine are mainly synthesized from other amino
acids in muscles, whereas others are more dependent on protein intake and subsequent
digestion and absorption from the small intestine (Cynober 2002). Nine of the 20 classic
amino acids are regarded essential among which the branched-chain amino acids (leucine,
isoleucine and valine). Essential (or indispensable) amino acids cannot be synthesized de novo
by the organism itself. The major pathway through which essential amino acids induce
anabolic responses involves the mammalian target of rapamycin (mTOR) Complex 1, a
signaling pathway that is especially sensitive to regulation by the branched-chain amino acid
leucine. Recent evidence suggests that muscle of older individuals require increasing
concentrations of leucine to maintain robust anabolic responses through the mTOR pathway
(Katsanos et al. 2006; Dillon 2012). The role of leucine supplementation to preserve or restore
body protein has not been fully delineated, but animal studies suggest potential benefit
(McNurlan 2012).
To the authors’ knowledge, plasma amino acid concentrations in horses were assessed almost
exclusively in peripheral venous blood (Gelfand et al. 1984; Cabrera et al. 1996; McGorum
and Kirk 2001; Westermann et al. 2011), but it is important to realize that peripheral plasma
amino acid concentrations reflect complex uptake and release processes by tissues upstream.
Studies in man revealed that amino acid profiles in venous blood differ clearly from that in
arterial blood. For example, alanine and glutamine levels show marked arteriovenous
differences (Abumrad and Miller 1983; Elia et al. 1985).
The introduction of the so-called arteriovenous differences technique has generated new
insights in human physiology. For instance, regarding the effect of growth hormone
administration on venous plasma amino acid concentrations at the femoral level (Mjaaland et
al. 1993). As a consequence, the arteriovenous differences technique provided useful
additional information.
The arteriovenous differences technique has also been applied to Thoroughbred horses in
order to study nutrient utilization by the hindlimb at rest comprising few selected nutrients
other than amino acids. The technique for measuring nutrient uptake across the hindlimb
using the arteriovenous difference turned out to be relatively simple and was deemed valuable
in investigating fuel use by muscle during exercise (Pethick et al. 1993). More recently, a
similar technique was used to study movement of ions across erythrocyte cell membranes in
endurance horses (Meyer et al. 2010).
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The aim of the current study was to assess amino acid utilization by the hindlimb of
warmblood horses at rest and following low intensity exercise comprising the almost total
profile of 25 different amino acids.
2. Materials and methods
2.1. Animals
Six warmblood mares (with a mean age of 12±3 (SD) years and weighing 538±39 kg) were
used. The horses were adapted to frequent handling, trained on a moderate exercise intensity
level and were accustomed to treadmill exercise. Prior to the experiment, the horses were kept
in a group on pasture. Before commencement of the study, the horses were individually
housed in boxes and food was withheld 12 hours prior to initial blood sampling. The animals
had free access to water. Arterial blood was obtained from a 20G catheter (Mila, Erlanger,
KY, USA) inserted into the (transverse) facial artery depending on accessibility. Venous
blood was simultaneously collected from a catheter placed into the caudal vena cava via the
medial saphenous vein using a human cardiac catheter (‘Swan-Ganz 111F7’ catheter,
Edwards Lifesciences, Unterschleissheim, Germany). This procedure was first validated by
inserting the Swan-Ganz 111F7 catheter via the medial saphenous vein in a warmblood horse
cadaver of similar body mass. Necropsy revealed the tip of the catheter indeed being
positioned in the caudal vena cava just cranial to the femoral branch. The day before the start
of the experiment both catheters were positioned and removed immediately after ending the
experiment. After removing the catheters, the horses were monitored an extra night in their
box and then returned to pasture.
The Institutional Animal Care and Use Committee of Utrecht University had approved the
experiment.
2.2. Exercise
Exercise comprised of 5 minutes walking, 20 minutes trotting and another 5 minutes walking
on a treadmill (Karga, Graber AG, Fahrwagen, Switzerland) with food withheld for 12 hours
prior to the exercise. To assess workload and check for any abnormal rhythm or aberrant beats
heart rate was monitored during exercise using a telemetric device (Televet 100 version 4.0,
Offenbach am Main, Germany).
2.3.Sample collection and analyses
Just prior to and immediately following exercise blood was collected from each catheter into a
heparinized syringe and without delay analyzed for the concentration of haemoglobin and
oxygen saturation of haemoglobin. Another blood sample was collected from each catheter
into tubes containing lithium heparin as anticoagulant (Becton, Dickinson and Company,
Franklin Lakes, NJ, USA). The tubes were centrifuged (Hettich Zentrifugen, Tuttlingen,
Germany) for 10 minutes at 6,000 x g and plasma was harvested and stored at -20 °C for
future analysis. The haemoglobin content and oxygen saturation of haemoglobin were
assessed by means of an automated analyzer (ABL-605 Radiometer, Radiometer Copenhagen,
Copenhagen, Denmark). Amino acids and α-aminobutyric acid were analyzed quantitatively
by means of automated ion exchange chromatography with post column ninhydrin
derivatization (JEOL AminoTac, JLC-500/V, Tokyo, Japan).
2.4. Calculations and statistics
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The content of oxygen was calculated from the haemoglobin content and oxygen saturation of
haemoglobin as follows: O2 = [Hb] x [O2]SAT. The fractional extraction (E) of a nutrient by the
hindlimb was calculated (according to Pethick et al. 1993) by the formula E = [A] – [V] / [A]
where [A] and [V] are the concentration (µmol/liter) of the metabolite in the (transverse)
facial artery and the caudal vena cava, respectively either at rest or following exercise. Data
were analyzed by means of a general linear model with random horse effects for pre and post
exercise differences. Differences between arterial and venous concentrations were analyzed
using a paired t-test. Results are presented as mean ± SD values. Differences were considered
significant at values of P < 0.05.
3. Results
Mean speed during trot was 4.1±0.23 m/s associated with a mean heart rate of 108±6.4
beats/min.
Amino acids with the largest average percent extraction at rest were citrulline (11.1±9%),
cystine (8.3±36%), serine (7.9±11%), and leucine (5.9±9%), as well as α-aminobutyric acid
(18.2±26%)(Table 1). Of the 25 amino acids studied, none showed a significantly altered
percent extraction following low intensity exercise. The amino acids glycine (485±65
µmol/L), glutamine (281±40 µmol/L), valine (183±26 µmol/L), and serine (165±22 µmol/L)
showed highest absolute plasma concentrations. The high mean extraction of α-aminobutyric
acid was associated with a very low plasma concentration (4±1 µmol/L). Only citrulline
showed significantly higher concentrations in arterial blood (49±1 µmol/L) compared to
venous blood (44±5 µmol/L) at rest.
4. Discussion
Various studies on plasma concentrations of amino acids as well as amino acid content of
skeletal muscle and the influence of diet and exercise have been performed in horses (Casini
et al. 2000; Essen-Gustavsson et al. 2002; Graham-Thiers and Bowen 2010). Some of these
studies focused on alterations of particular amino acids during different types of exercise
(Johnson and Hart 1974; Poso et al. 1987; Casini et al. 2000; Essen-Gustavsson and JensenWaern 2002; Trottier et al. 2002), whereas more recent studies investigated a large panel of
amino acids (Hackl et al. 2009; Westermann et al. 2011). Besides, only few studies addressed
the changes in skeletal muscle content of amino acids in horses before and after (fatiguing)
exercise (Miller-Graber et al. 1990). In the current study, the utilization of a large panel of
amino acids and α-aminobutyric acid by the tissues in the hindlimb was addressed by means
of the arteriovenous differences technique. The catheters simultaneously inserted into the
(transverse) facial artery and the caudal vena cava made it possible to precisely measure the
amino acid use by hind limb (muscles) prior to and immediately following low intensity
exercise. Results revealed that amino acids with the largest average percent extraction in the
current study at rest were citrulline, cystine, serine, and leucine. Of these 4 amino acids, serine
had the largest concentration at rest in plasma. Glycine, glutamine, valine, and serine were
most abundant in plasma in warmblood mares similar to the findings in Standardbreds
(Westermann et al. 2011). Of the 25 amino acids studied, none showed a significantly altered
extraction following low intensity exercise. It has been stated that amino acids should not be
regarded as limiting training performance in Standardbreds except for aspartic acid seen as the
most likely candidate for supplementation (Westermann et al. 2011). Plasma aspartic acid
concentration was similarly low in the current study. However, its extraction was almost nil.
In Thoroughbred horses, the extraction of the ketone bodies acetate and D-3-hydroxybutyrate
were large (41±6 and 28±4%, respectively). A 52% oat grain diet significantly increased D-3hydroxybutyrate extraction to 51±5%. In addition, D-3-hydroxybutyrate and acetate taken
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together contributed 39% to hindlimb oxidation (Pethick et al. 1993). In accord, the very low
plasma α-aminobutyric acid concentration seen in warmblood mares in the current study was
associated with a rather high extraction of 18.2±26%. This reflects substantial ketogenesis in
the equine species despite the high muscle glycogen content characteristic of horses.
Previous research showed that plasma concentrations of these amino acids were not affected
by exercise as well (Poso et al. 1987; Hackl et al. 2009), neither were their concentrations in
the middle gluteal muscle (Miller-Graber et al. 1990). In contrast, significant decreases in
plasma concentrations of citrulline and serine were reported 60 minutes after a standardized
exercise test in Standardbreds (Westermann et al. 2011) in agreement with the large mean
extractions found in the current study. However, it should be realized that the timing of blood
sampling in relation to the bout of exercise possibly has an impact on the concentrations of
various amino acids (Westermann et al. 2011). As a consequence, this might greatly influence
the ability to compare various studies. Furthermore, it has been assumed that a genetic
variation in the amino acid concentrations in erythrocytes of Thoroughbreds affects post
exercise amino acid plasma concentrations (Hanzawa et al. 1992), but this assumption has
been questioned in a recent report showing that the red blood cell amino acid pool only
slightly contributed to plasma amino acid concentrations following short intensive exercise in
Standardbreds. However, plasma amino acid concentrations showed a poor repeatability, but
the general pattern of changes was comparable on both sampling days (Hackl et al. 2009).
Both at rest and immediately after exercise cystine, a dimeric amino acid formed by the
oxidation of two cysteine residues, showed one of the largest extractions. However, it should
be realized that especially plasma cystine concentration is largely dependent on sample
conditions. It has been stated that the role of cystine in equine veterinary science is not well
appreciated especially in relationship to the pathophysiology of laminitis (Berhane et al.
2004). Based on the results of the current study one might conclude that cystine is a potential
important amino acid in equine hindlimb musculature besides citrulline, serine, and leucine.
Further investigation is necessary into the specific role of leucine supplementation to preserve
or restore body protein in horses.
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