Abstract
Background: Acylcarnitines play an important role in fuel metabolism in skeletal muscle.
Objective: To assess acylcarnitine ester utilization by the hindlimb of horses at rest and following low intensity exercise and carnitine supplementation.
Animals and methods: Acylcarnitine ester 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 a standardized treadmill protocol consisting of 5 minutes of walk, 20 minutes of trot and thereafter another 5 minutes of walk. At the end of the first exercise day, three horses were given carnitine supplementation (100 mg/kg bodyweight), whereas the other horses received saline. The next day the exercise was repeated and blood samples collected similarly. Free carnitine and acylcarnitines were analyzed as their butyl ester derivatives in heparinized plasma by electrospray tandem mass spectrometry. Statistical analysis was performed using a general linear mixed model.
Results: C3-carnitine, C6-carnitine and C14:1-carnitine showed the largest average extraction by the hindlimb at rest and C3-carnitine, C5:1-carnitine and C16-carnitine immediately after low-intensity exercise. Carnitine supplementation significantly increased free carnitine, C5-carnitine and C8-carnitine extraction.
Conclusion: Carnitine supplementation altered the extraction of acylcarnitines by the hindlimb in horses exercising at low intensity.
Clinical importance: Findings might aid in optimizing performance and myopathy prevention of the equine athlete.
1. Introduction
Fatty acid oxidation in the mitochondrial matrix is a major source of energy, especially when physiological energy demand is increased and exceeds the amount which can be provided through glycolysis (Liang & Nishino Citation2010). Carnitine is an amino acid derivative found in high energy demanding tissues (skeletal muscles, myocardium, the liver and the suprarenal glands). It is indispensable for beta-oxidation of long-chain fatty acids in the mitochondria by acting as a carrier of fatty acyl groups from the cytoplasm to the mitochondrion, but also regulates CoA concentration and removal of the produced acyl groups. Acyl-CoAs act as restraining factor for several enzymes participating in intermediary metabolism. Transformation of acyl-CoA into acylcarnitine is an important system also for removing the toxic acyl groups (Hoppel Citation1982; Evangeliou & Vlassopoulos Citation2003). Long-chain acyl-CoA derivatives do not penetrate the mitochondrial inner membrane. Carnitine palmitoyltransferase I (CPT-I), located on the external surface of the inner membrane, catalyzes the conversion of cytoplasmic long-chain acyl-CoA and carnitine into acylcarnitine. The acylcarnitine is reconverted to intramitochondrial acyl-CoA by the action of carnitine palmitoyltransferase II located in the inner membrane. Now, the acyl-CoA is available for beta-oxidation in the matrix. An inner membrane carnitine–acylcarnitine translocase exchanges carnitine and acylcarnitine across the inner membrane. In liver, malonyl-CoA, an intermediate in fatty acid synthesis, is proposed to regulate the activity of CPT-I (Hoppel Citation1982).
L-carnitine is found in virtually all cells of animals, and also in some micro-organisms and plants. In animals, it is synthesized almost exclusively in the liver. Two essential amino acids, lysine and methionine, serve as primary substrates for its biosynthesis and L-carnitine is released into the circulation by the liver primarily as acetylcarnitine. Animal-derived feeds are rich in L-carnitine, whereas plant-derived feeds contain usually very little or no L-carnitine. As a consequence, typical horse feeds can be expected to be low in L-carnitine (Zeyner and Harmeyer Citation1999). Carnitine supplementation has been hypothesized to improve exercise performance in healthy humans through various mechanisms, including enhanced muscle fatty acid oxidation, altered glucose homeostasis, enhanced acylcarnitine production, modification of training responses, and altered muscle fatigue resistance (Brass Citation2000).
As has been shown previously, exercise in Standardbred horses significantly decreased plasma C3-carnitine and C4-carnitine concentrations, whereas plasma C2-carnitine concentration significantly increased. Remarkably, training did not affect plasma free fatty acids and acylcarnitine ester concentrations in Standardbred geldings (Westermann et al. Citation2008b).
In the current experiment, we aimed to study acylcarnitine ester metabolism in more detail by utilizing the arteriovenous difference technique as published previously (Pethick et al. Citation1993) in order to assess acylcarnitine ester uptake by the hindlimb at rest and following low intensity exercise and carnitine supplementation.
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 used to frequent handling including treadmill exercise and were trained at a low exercise intensity level. Prior to the experiment, the horses were kept in a group on pasture. Before the start 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. 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 immediately after ending the experiment they were removed. At the end of the exercise day, three of the six horses were given carnitine supplementation [100 mg/kg bodyweight (BW) (L-carnitine fumarate, Sigma-tau S.p.A., Roma, Italy) with 58% of the dose intravenously and 42% orally], whereas the other three (control) horses received saline. The next day the exercise was repeated and blood samples were collected similarly. 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 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 to 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 hemoglobin and oxygen saturation of hemoglobin. 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 6000× g and plasma was harvested and stored at −20 °C for future analysis. The glucose concentration, lactate concentration, hemoglobin content and oxygen saturation of hemoglobin were assessed by means of an automated analyzer (ABL-605 Radiometer, Radiometer Copenhagen, Copenhagen, Denmark).
Analyses of free carnitine and acylcarnitines in plasma were carried out as their butyl ester derivatives by electrospray tandem mass spectrometry (Micromass Quattro Ultima system, Waters, Vernon Hills, Illinois, USA) validated for use in horses (Westermann et al. Citation2007).
2.4. Calculations and statistical analysis
The content of oxygen was calculated from the hemoglobin content and oxygen saturation of hemoglobin as follows: O2 = [Hb] × [O2]SAT. The fractional extraction (E) of a metabolite by the hindlimb was calculated (according to Pethick et al. Citation1993) by the formula E = [A] − [V]/[A], where [A] and [V] are the concentrations (µmol/L) of the metabolite in the transverse facial artery and the caudal vena cava, respectively, either at rest or following exercise and supplementation. The fractional contribution of a metabolite to the oxygen consumption by the hindlimb (C) was calculated (according to Pethick et al. Citation1993) by the formula C = ([A] − [V])m × Q/([A] − [V])O2, where ([A] − [V])m and ([A] − [V])O2 represent the arteriovenous difference (µmol/L) for the metabolite and oxygen, respectively. Q is the number of moles of metabolite required to account for one mole of oxygen uptake assuming complete oxidation. 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 and absence of an increase in average plasma lactate concentration (). As expected, both at rest and following low-intensity exercise, lactate was released into the circulation by the hindlimb musculature instead of extracted.
Table 1. Mean percent hind limb extraction of acylcarnitines (±SD) during rest and immediately following low intensity exercise in six warmblood mares as reflected by positive values. Negative values indicate net release into the blood. The right column shows the mean (±SD) fractional contribution of a metabolite to the oxygen consumption by the hindlimb at rest. Values at rest compared to post-exercise were not statistically significant.
C3-carnitine, C6-carnitine and C14:1-carnitine showed the largest average extraction by the hindlimb at rest and C3-carnitine, C5:1-carnitine and C16-carnitine immediately after low-intensity exercise (). The fractional contribution of various acylcarnitines to the oxygen consumption by the hindlimb at rest was not substantial in contrast to glucose with 53% (). Of note, following low-intensity exercise free carnitine was released into the circulation rather than extracted by the hindlimb at rest with highest concentration in arterial blood (). On the other hand, carnitine supplementation significantly increased free carnitine, C5-carnitine and C8-carnitine extraction by the hindlimb ().
Table 2. Mean arterial and venous plasma concentrations (µmol/L) of acylcarnitines (±SD) during rest and immediately following low intensity exercise in six warmblood mares. Arterial samples were collected from the transverse facial artery and venous samples from the caudal vena cava. Different superscripts indicate statistical significance.
Table 3. Mean percent hind limb extraction of acylcarnitines (±SD) during rest and immediately following low intensity exercise in warmblood mares with (n = 3) or without (n = 3) single supplementation of carnitine (100 mg/kg BW). Hind limb extraction of acylcarnitines is reflected by positive values. Negative values indicate net release into the blood. Different superscripts indicate statistical significance at P < 0.05.
4. Discussion
This study was designed to allow assessment of acylcarnitine ester utilization by the hindlimb musculature of the warmblood horse reflecting an important aspect of fat metabolism. Due to the catheters in the transverse facial artery and the caudal vena cava, we were able to compare the concentrations of acylcarnitine esters in both vessels and subsequently calculate the extractions of various acylcarnitine esters by the hindlimb musculature. C3-carnitine showed the largest average extraction both at rest and immediately after exercise similar to the decreased plasma concentration reported earlier in Standardbred horses following exercise (Westermann et al. Citation2008b). As a consequence, C3-carnitine might be regarded as an important acylcarnitine ester in equine hindlimb musculature. The increase in the plasma concentration of C2-carnitine in Standardbred geldings following much strenuous exercise (80% of the HRmax equivalent to a treadmill speed of 7.5–8.5 m/s with a 1%–4% incline for 20 minutes) seems in accord to the (non-significantly) decreased extraction of this acylcarnitine ester in this study. Furthermore, an elevation of the C2-carnitine content in the middle gluteal muscle in exercising Thoroughbreds has been reported (Foster & Harris Citation1987; Foster & Harris Citation1992). Although the study by Westermann et al. (Citation2008b) also showed a decrease in the plasma concentration of C4-carnitine in Standardbred geldings following exercise, this was not reflected by an extraction of this acylcarnitine ester in this study in warmblood mares at low intensity exercise.
The fractional contribution of various acylcarnitines to the oxygen consumption by the hindlimb at rest was not substantial. This is in line with the fact that – contrarily to what is reported for the human athlete – in the horse during high speed exercise, the aerobic pathway (i.e. oxidation of carbohydrate and free fatty acids) is the dominant way of adenosinetrifosfaat (ATP) production. (Eaton et al. Citation1995; Art & van Erck Citation2003). In accord, values of extractions of various acylcarnitine esters by the hindlimb musculature at rest compared to post-exercise were not statistically significant perhaps due to the low intensity of exercise in this study.
In healthy humans, supplementation of L-carnitine (without insulin) via oral or intravenous routes either at rest or during prolonged exercise has failed to increase muscle carnitine concentration (Soop et al. Citation1988; Brass Citation2000). In contrast to the findings in man, the bioavailability of carnitine in horses is much greater. Carnitine is absorbed in the small intestine probably by a sodium-dependent active transport mechanism and at higher carnitine concentrations this transport is paralleled by passive diffusion (Zeyner & Harmeyer Citation1999). Following absorption, equine muscle carnitine content can be increased (Rivero et al. Citation2002). Carnitine is transported into skeletal muscle against a considerable concentration gradient via a saturable Na+-dependent, high affinity, active transport process via the organic cation transporter OCTN2 (Inano et al. Citation2003). In line, a single high dose of carnitine (100 mg/kg BW) increased the extraction of (free) carnitine as well as C5-carnitine and C8-carnitine by the hindlimb musculature in warmblood mares at low intensity exercise. The dosage of the L-carnitine in this study was chosen on the basis of the highest dosage found in literature for oral supplements for humans being 100 mg/kg BW (Brass & Hiatt Citation1998; Brass Citation2000; Brass Citation2004) and on the highest dosage for horses reported as 60 g daily (Foster et al. Citation1988). In comparison, long-term oral supplementation of L-carnitine (as fumarate at a dosage of 100 mg/kg BW/day for 28 days) to warmblood geldings enhanced free carnitine concentration in plasma as well as short-chain acylcarnitines (acyl groups less than six carbon atoms) plus C18-OH-carnitine and C18:1-DC-carnitine (Kranenburg et al. Citation2014).
A deficiency of several mitochondrial dehydrogenases that utilize flavin adenine dinucleotide as cofactor including the acyl-CoA dehydrogenases of fatty acid beta-oxidation, and enzymes that degrade the CoA-esters of glutaric acid, isovaleric acid, 2-methylbutyric acid, isobutyric acid and sarcosine was described in horses similar to the combined metabolic derangements seen in human multiple acyl-CoA dehydrogenase deficiency (MADD) also known as glutaric acidemia type II. This acquired equine MADD is associated with a substantial elevation of C2-, C3-, C4-, C5-, C6-, C8-, C8:1-, C10:1- and C10:2-carnitine concentrations in plasma in the majority of cases (Westermann et al. Citation2008a). As single carnitine supplementation increases the utilization of C5- and C8-carnitne in healthy horses, it is a plausible idea to use it in horses affected with MADD and other potential fatty acid oxidation disorders (Wijnberg et al. Citation2008).
It is concluded that a single high dose carnitine supplementation to warmblood horses increases utilization by the hindlimb musculature of free carnitine, C5-carnitine and C8-carnitine extraction following low intensity exercise. These findings might aid in optimizing performance and myopathy prevention of the equine athlete.
Acknowledgements
The authors acknowledge support from Utrecht University, Utrecht, the Netherlands, for the use of some facilities.
Disclosure statement
No potential conflict of interest was reported by the authors.
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