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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: _______________________________________________________________ *Corresponding author. Email: [email protected] 1 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). 2 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 3 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 4 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. References Abumrad NN, Miller B. 1983. 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