Formation of acetylcarnitine in muscle of horse during high intensity exercise

To study the changes in carnitine in muscle with spring exercise, two Thoroughbred horses performed two treadmill exercise tests. Biopsies of the middle gluteal were taken before, after exercise and after 12 min recovery. Resting mean muscle total carnitine content was 29.5 mmol.kg-1 dry muscle (d.m.). Approximately 88% was free carnitine, 7% acetylcarnitine and acylcarnitine was estimated at 5%. Exercise did not affect total carnitine, but resulted in a marked fall in free carnitine and almost equivalent rise in acetylcarnitine. The results are consistent with a role for carnitine in the regulation of the acetyl-CoA/CoA ratio during sprint exercise in the Thoroughbred horse by buffering excess production of acetyl units.     

Changes in muscle free carnitine and acetylcarnitine with increasing work intensity in the Thoroughbred horse

Treadmill exercise in Thoroughbred horses of 2 min duration and increasing intensity resulted in increased formation and accumulation of acetylcarnitine in the working middle gluteal muscle. At high work intensities a plateau in acetylcarnitine formation was reached corresponding to approximately 70% of the total carnitine pool (approx. 30 mmol.kg-1 dry muscle). Formation of acetylcarnitine was mirrored by an equal fall in the free carnitine content, which stabilised, at the highest work intensities, at around 8 mmol.kg-1 dry muscle. Acetylcarnitine and carnitine reached their point of maximum change at a work intensity just below that resulting in the rapid production and accumulation of lactate and glycerol 3-phosphate. It is possible that the formation of acetylcarnitine is important in the regulation of the intramitochondrial acetyl CoA/CoA ratio; equally these changes may represent a blocking mechanism aimed at preventing the transfer of unwanted free fatty acids (as acylcarnitines) into the mitochondria at work intensities where they could contribute little to energy production.     

Malonyl-CoA and carnitine in regulation of fat oxidation in human skeletal muscle during exercise

Intracellular mechanisms regulating fat oxidation were investigated in human skeletal muscle during exercise. Eight young, healthy, moderately trained men performed bicycle exercise (60 min, 65% peak O2 consumption) on two occasions, where they ingested either 1) a high-carbohydrate diet (H-CHO) or 2) a low-carbohydrate diet (L-CHO) before exercise to alter muscle glycogen content as well as to induce, respectively, low and high rates of fat oxidation. Leg fat oxidation was 122% higher during exercise in L-CHO than in H-CHO (P < 0.001). In keeping with this, the activity of alpha2-AMP-activated protein kinase (alpha2-AMPK) was increased twice as much in L-CHO as in H-CHO (P < 0.01) at 60 min of exercise. However, acetyl-CoA carboxylase (ACC)beta Ser221 phosphorylation was increased to the same extent (6-fold) under the two conditions. The concentration of malonyl-CoA was reduced 13% by exercise in both conditions (P < 0.05). Pyruvate dehydrogenase activity was higher during exercise in H-CHO than in L-CHO (P < 0.01). In H-CHO only, the concentrations of acetyl-CoA and acetylcarnitine were increased (P < 0.001), and the concentration of free carnitine was decreased (P < 0.01), by exercise. The data suggest that a decrease in the concentration of malonyl-CoA, secondary to alpha2-AMPK activation and ACC inhibition (by phosphorylation), contributes to the increase in fat oxidation observed at the onset of exercise regardless of muscle glycogen levels. They also suggest that, with high muscle glycogen, the availability of free carnitine may limit fat oxidation during exercise, due to its increased use for acetylcarnitine formation.     

PDC activity and acetyl group accumulation in skeletal muscle during prolonged exercise.

Seven subjects cycled to exhaustion [58 +/- 7 (SE) min] at approximately 75% of their maximal oxygen uptake (VO2max). Needle biopsy samples were taken from the quadriceps femoris muscle at rest, after 3, 10, and 40 min of exercise, at exhaustion, and after 10 min of recovery. After 3 min of exercise, a nearly complete transformation of the pyruvate dehydrogenase complex (PDC) into active form had occurred and was maintained throughout the exercise period. The total in vitro activated PDC was unchanged during exercise. The muscle concentration of acetyl-CoA increased from a resting value of 8.4 +/- 1.0 to 31.6 +/- 3.3 mumol/kg dry wt at exhaustion and that of acetylcarnitine from 2.9 +/- 0.7 to 15.6 +/- 1.6 mmol/kg dry wt. This was accompanied by corresponding decreases in reduced CoA (CoASH) from 45.3 +/- 3.1 to 25.9 +/- 3.1 mumol/kg dry wt and in free carnitine from 18.8 +/- 0.7 to 5.7 +/- 0.5 mmol/kg dry wt. Acetyl group accumulation, in the form of acetyl-CoA and acetylcarnitine, was maintained throughout exercise to exhaustion while the glycogen content decreased by 90%. This suggests that availability of acetyl groups was not limiting to exercise performance despite the nearly total depletion of the glycogen store. The increased acetyl-CoA-to-CoASH ratio during exercise caused inhibition of neither the PDC transformation nor the calculated catalytic activity of active PDC.     

Influence of carnitine supplementation on muscle substrate and carnitine metabolism during exercise

We examined 1) the effect of L-carnitine supplementation on free fatty acid (FFA) utilization during exercise and 2) exercise-induced alterations in plasma levels and skeletal muscle exchange of carnitine. Seven moderately trained human male subjects serving as their own controls participated in two bicycle exercise sessions (120 min, 50% of VO2max). The second exercise was preceded by 5 days of oral carnitine supplementation (CS; 5 g daily). Despite a doubling of plasma carnitine levels, with CS, there were no effects on exercise-induced changes in arterial levels and turnover of FFA, the relation between leg FFA inflow and FFA uptake, or the leg exchange of other substrates. Heart rate during exercise after CS decreased 7-8%, but O2 uptake was unchanged. Exercise before CS induced a fall from 33.4 +/- 1.6 to 30.8 +/- 1.0 (SE) mumol/l in free plasma carnitine despite a release (2.5 +/- 0.9 mumol/min) from the leg. Simultaneously, acylated plasma carnitine rose from 5.0 +/- 1.0 to 14.2 +/- 1.4 mumol/l, with no evidence of leg release. Consequently, total plasma carnitine increased. We concluded that in healthy subjects CS does not influence muscle substrate utilization either at rest or during prolonged exercise and that free carnitine released from muscle during exercise is presumably acylated in the liver and released to plasma.     

Sensitivity of CPT I to malonyl-CoA in trained and untrained human skeletal muscle

The present study examined the sensitivity of carnitine palmitoyltransferase I (CPT I) activity to its inhibitor malonyl-CoA (M-CoA), and simulated metabolic conditions of rest and exercise, in aerobically trained and untrained humans. Maximal CPT I activity was measured in mitochondria isolated from resting human skeletal muscle. Mean CPT I activity was 492.8 +/- 72.8 and 260.8 +/- 33.6 micromol. min(-1). kg wet muscle(-1) in trained and untrained subjects, respectively (pH 7.0, 37 degrees C). The sensitivity to M-CoA was greater in trained muscle; the IC(50) for M-CoA was 0.17 +/- 0.04 and 0.49 +/- 0.17 microM in trained and untrained muscle, respectively. The presence of acetyl-CoA, free coenzyme A (CoASH), and acetylcarnitine, in concentrations simulating rest and exercise conditions did not release the M-CoA-induced inhibition of CPT I activity. However, CPT I activity was reduced at pH 6.8 vs. pH 7.0 in both trained and untrained muscle in the presence of physiological concentrations of M-CoA. The results of this study indicate that aerobic training is associated with an increase in the sensitivity of CPT I to M-CoA. Accumulations of acetyl-CoA, CoASH, and acetylcarnitine do not counteract the M-CoA-induced inhibition of CPT I activity. However, small decreases in pH produce large reductions in the activity of CPT I and may contribute to the decrease in fat metabolism that occurs during moderate and intense aerobic exercise intensities.     

Effect of sports activity on carnitine metabolism: Measurement of free carnitine, γ-butyrobetaine and acylcarnitines by tandem mass spectrometry

The effects of sports activity on carnitine metabolism were studied using mass spectrometry. Serum levels of free carnitine, acylcarnitines (acetylcarnitine, propionylcarnitine, C4-, C5- and C8-acylcarnitine) and γ-butyrobetaine, a carnitine precursor, were determined by tandem mass spectrometry in liquid secondary ion mass ionization mode. The coefficients of variation at three different concentrations were 2.8∼7.9% for γ-butyrobetaine, and 1.2∼6.7% for free carnitine. The recoveries added to serum were 109.1% for γ-butyrobetaine, 89.3% for free carnitine. Sports activity caused increased serum levels of γ-butyrobetaine, acetylcarnitine, C4- and C8-acylcarnitines and decreased serum levels of free carnitine. This method requires a small amount of sample volume (20 μl of serum) and short total instrumental time for the analysis (1 h for preparation, 2 min per sample for mass spectrometric analysis). Therefore, this method can be applied to study carnitine metabolism under various conditions that affect fatty acid oxidation.     

Muscle carnitine after strenuous endurance exercise.

The effect of very long endurance exercise on muscle carnitine was studied. Eighteen cross-country skiers took part in a race in the Alps (average inspired partial pressure of O2 100-110 Torr) that lasted on average 13 h 26 min. Carnitine intake, evaluated for 2 wk before the event, was 50 +/- 4 (SE) mg/day. Muscle (vastus lateralis) total carnitine concentration, measured twice with a 2-yr interval on eight rested subjects, did not change with time (17 vs. 16 mumol/g dry wt, NS) but showed consistent interindividual differences (range 12-22, P = 0.001) with no correlation with intake. After exercise, total muscle carnitine was unaltered (from 17.9 +/- 1.0 at rest to 18.3 +/- 0.8 mumol/g dry wt postexercise in the 15 subjects who completed the race, NS), but muscle free carnitine decreased 20% (from 14.9 +/- 0.8 mumol/g, P = 0.01) and short-chain acylcarnitine increased 108% (from 3.5 +/- 0.4 mumol/g, P = 0.01). These results suggest that carnitine deficiency will probably not result from strenuous aerobic exercise in trained subjects who consume a moderate amount of carnitine in their food.     

Metabolism and excretion of carnitine and acylcarnitines in the perfused rat kidney

Rat kidneys were perfused for 30 min with a Krebs-Henseleit bicarbonate buffer with 5 mM glucose. Albumin proved superior to pluronic polyols as oncotic agent with regard to carnitine reabsorption in the perfused kidney. The reabsorption of 30 μM (−)-[methyl-³H]carnitine was approx. 96% during the first 10 min. At 750 μM the reabsorption decreased to 40%. The tubular reabsorptive maximum (T_max) was approx. 170 nmol/min per kidney. The fractional reabsorption and clearance of (+)-carnitine, γ-butyrobetaine, and carnitine esters did not deviate significantly from that of (−)-carnitine. (+)-Carnitine was not metabolized by the perfused kidney. In perfusions with (−)-carnitine or (−)-carnitine plus 10 mM α-ketoisocaproate or α-ketoisovalerate increased amounts of acetylcarnitine, isovalerylcarnitine and isobutyrylcarnitine were found. Propionate (5 mM) inhibited acetylcarnitine formation. Isovalerylcarnitine, isobutyrylcarnitine and propionylcarnitine were actively degraded to free (−)-carnitine. In urine, we found a disproportionally high excretion of carnitine or carnitine esters formed in the kidney, compared to the same derivatives when ultrafiltrated. Leakage of metabolites formed in the kidney into preurine may explain this phenomenon.     

Aspects of carnitine ester metabolism in sheep liver

1. Carnitine acetyltransferase (EC 2.3.1.7) activity in sheep liver mitochondria was 76nmol/min per mg of protein, in contrast with 1.7 for rat liver mitochondria. The activity in bovine liver mitochondria was comparable with that of sheep liver mitochondria. Carnitine palmitoyltransferase activity was the same in both sheep and rat liver mitochondria. 2. The [free carnitine]/[acetylcarnitine] ratio in sheep liver ranged from 6:1 for animals fed ad libitum on lucerne to approx. 1:1 for animals grazed on open pastures. This change in ratio appeared to reflect the ratio of propionic acid to acetic acid produced in the rumen of the sheep under the two dietary conditions. 3. In sheep starved for 7 days the [free carnitine]/[acetylcarnitine] ratio in the liver was 0.46:1. The increase in acetylcarnitine on starvation was not at the expense of free carnitine, as the amounts of free carnitine and total acid-soluble carnitine rose approximately fivefold on starvation. An even more dramatic increase in total acid-soluble carnitine of the liver was seen in an alloxan-diabetic sheep. 4. The [free CoA]/[acetyl-CoA] ratio in the liver ranged from 1:1 in the sheep fed on lucerne to 0.34:1 for animals starved for 7 days. 5. The importance of carnitine acetyltransferase in sheep liver and its role in relieving ;acetyl pressure' on the CoA system is discussed.     

Association between muscle acetyl-CoA and acetylcarnitine levels in the exercising horse

Treadmill exercise of 2-min duration and increasing intensity resulted in increased formation of acetyl-CoA and acetylcarnitine in working muscle of Thoroughbred horses. At high work intensities a plateau was reached for both acetyl-CoA (approximately 50 mumols/kg dry muscle) and acetylcarnitine (approximately 20 mmol/kg dry muscle). Postexercise concentrations were significantly (P less than 0.001) correlated; [acetylcarnitine] = 349.[acetyl-CoA] + 2.4. The results indicate that approximately 350 mumols acetylcarnitine were accumulated for every 1 mumol acetyl-CoA. Under the conditions of exercise used it is probable that most of the acetyl-CoA formed is generated through the intramitochondrial decarboxylation of pyruvate. The acetyl groups of acetyl-CoA are apparently redistributed throughout the whole cell through formation of acetylcarnitine, which readily transverses the mitochondrial membrane. Despite the redistribution, however, the close correlation between acetylcarnitine and acetyl-CoA would indicate that equilibrium was maintained and that neither acetylcarnitine transferase nor carnitine/acetylcarnitine translocase were rate limiting. There is some question as to whether the changes observed relate directly to exercise itself or to the state in muscle 10 s or more after exercise.     

An animal model of systemic carnitine deficiency produced by haemodialysis of sheep

1. Sheep, which had previously been surgically prepared with cannulae in various vessels to monitor substrate and metabolite exchanges across all the major organs, were connected to a haemodialysis machine and their blood was dialysed at an average rate of 6.23 ml/min/kg body weight. 2. Dialysis for 4 hr reduced the blood free carnitine concentrations to approx. 50% of the initial values and the concentrations returned to the initial values after 18 hr recovery. 3. Carnitine balance studies showed that approx. twice the amount of carnitine lost from the blood during dialysis passed into the dialysate indicating that carnitine was also lost from the extracellular fluid. 4. The average blood concentration of short-chain acylcarnitines did not vary significantly during dialysis or during the recovery phase. However, an output of short-chain acylcarnitines by the liver at 3 and 18 hr recovery and an uptake by the hind-body at 18 hr recovery was observed. 5. These results suggest that haemodialysis of sheep provides a useful model of systemic carnitine deficiency and suggest that treatment with acetylcarnitine or propionylcarnitine could be an efficient means of supplying carnitine in carnitine replacement therapy.     

Muscle malonyl-CoA decreases during exercise

Malonyl-CoA, the inhibitor of carnitine acyltransferase I, is an important regulator of fatty acid oxidation and ketogenesis in the liver. Muscle carnitine acyltransferase I has previously been reported to be more sensitive to malonyl-CoA inhibition than is liver carnitine acyltransferase I. Fluctuations in malonyl-CoA concentration may therefore be important in regulating the rate of fatty acid oxidation in muscle during exercise. Male rats were anesthetized (pentobarbital via venous catheters) at rest or after 30 min of treadmill exercise (21 m/min, 15% grade). The gastrocnemius/plantaris muscles were frozen at liquid N2 temperature. Muscle malonyl-CoA decreased from 1.66 +/- 0.17 to 0.60 +/- 0.05 nmol/g during the exercise. This change was accompanied by a 31% increase in cAMP in the muscle. The decline in malonyl-CoA occurred before muscle glycogen depletion and before onset of hypoglycemia. Plasma catecholamines, corticosterone, and free fatty acids were all significantly increased during the exercise. This exercise-induced decrease in malonyl-CoA may be important for allowing the increase in muscle fatty acid oxidation during exercise.     

Carnitine, acetylcarnitine and the activity of carnitine acyltransferases in seminal plasma and spermatozoa of men, rams and rats.

The concentration of total carnitine (i.e. carnitine plus acetylcarnitine) was measured in seminal plasma and spermatozoa of men and rams. In ram semen, there was a close correlation between the concentration of spermatozoa and that of total carnitine in the seminal plasma, indicating that the epididymal secretion was the sole source of seminal carnitine. The percentage of total carnitine present as acetylcarnitine was 40% in seminal plasma and 70-80% in spermatozoa. The acetylation state of carnitine in seminal plasma was apparently not influenced by the metabolic activity of spermatozoa in ejaculated ram semen as no change was found in the plasma concentration of carnitine or acetylcarnitine up to 45 min after ejaculation. In spermatozoa, the activity of carnitine acetyltransferase (EC 2.3.1.7) was approximately equivalent to that of carnitine palmitoyltransferase (EC 2.3.1.21); and the activity of these enzymes was similar in ram and human spermatozoa but greater in rat spermatozoa. It is concluded that there is no correlation between the content of either total carnitine or the carnitine acyltransferases and the respiratory capacity of spermatozoa.     

Metabolic changes induced by maximal exercise in human subjects following L-carnitine administration

In double-blind cross-over experiments, ten moderately trained male subjects were submitted to two bouts of maximal cycle ergometer exercise separated by a 3 day interval. Each subject was randomly given either L-carnitine (2 g) or placebo orally 1 h before the beginning of each exercise session. At rest L-carnitine supplementation resulted in an increase of plasma-free carnitine without a change in acid-soluble carnitine esters. Treatment with L-carnitine induced a significant post-exercise decrease of plasma lactate and pyruvate and a concurrent increase of acetylcarnitine. The determination of the individual carnitine esters in urine collected for 24 h after the placebo exercise trial revealed a decrease of acetyl carnitine and a parallel increase of a C4 carnitine ester, probably isobutyrylcarnitine. Conversely, acetylcarnitine was strongly increased and C4 compounds were almost suppressed in the L-carnitine loading trial. These results suggest that L-carnitine administration prior to high-intensity exercise stimulates pyruvate dehydrogenase activity, thus diverting pyruvate from lactate to acetylcarnitine formation.     

Skeletal muscle metabolism during high-intensity sprint exercise is unaffected by dichloroacetate or acetate infusion.

This study investigated whether increased provision of oxidative substrate would reduce the reliance on nonoxidative ATP production and/or increase power output during maximal sprint exercise. The provision of oxidative substrate was increased at the onset of exercise by the infusion of acetate (AC; increased resting acetylcarnitine) or dichloroacetate [DCA; increased acetylcarnitine and greater activation of pyruvate dehydrogeanse (PDH-a)]. Subjects performed 10 s of maximal cycling on an isokinetic ergometer on three occasions after either DCA, AC, or saline (Con) infusion. Resting PDH-a with DCA was increased significantly over AC and Con trials (3.58 +/- 0.4 vs. 0.52 +/- 0.1 and 0.74 +/- 0.1 mmol. kg wet muscle(-1). min(-1)). DCA and AC significantly increased resting acetyl-CoA (35.2 +/- 4.4 and 22.7 +/- 2.9 vs. 10.2 +/- 1.3 micromol/kg dry muscle) and acetylcarnitine (12.9 +/- 1.4 and 11.0 +/- 1.0 vs. 3.3 +/- 0.6 mmol/kg dry muscle) over Con. Resting contents of phosphocreatine, lactate, ATP, and glycolytic intermediates were not different among trials. Average power output and total work done were not different among the three 10-s sprint trials. Postexercise, PDH-a in AC and Con trials had increased significantly but was still significantly lower than in DCA trial. Acetyl-CoA did not increase in any trial, whereas acetylcarnitine increased significantly only in DCA. Exercise caused identical decreases in ATP and phosphocreatine and identical increases in lactate, pyruvate, and glycolytic intermediates in all trials. These data suggest that there is an inability to utilize extra oxidative substrate (from either stored acetylcarnitine or increased PDH-a) during exercise at this intensity, possibly because of O(2) and/or metabolic limitations.     

Adrenergic response to intense muscular activity in sedentary subjects as a function of emotivity and training]

Seven male sedentary human subjects were studied during intense muscular work (80% of maximal oxygen uptake) performed either for 15 min or until exhaustion (mean duration: 47 +/- 2 min). Plasma catecholamines were estimated before and after the experiment by means of an original fluorimetric assay. Epinephrine or norepinephrine were individually isolated from plasma and assayed in single extracts by a highly sensitive fluorimetric method. Epinephrine and norepinephrine levels as low as 15 ng per liter were detectable by this procedure in human plasma. The adrenergic pattern was found to be greatly different from one subject to another and related to emotivity: the effect of this factor was revealed by the predominance of epinephrine in plasma at rest or under exercise (ratio NA/A less than 1). In nonemotive subjects (ratio NA/A greater than 1 at rest) plasma epinephrine and norepinephrine increased progressively during exercise. Increments after exercise were higher for norepinephrine changes; however, the fact that epinephrine concentrations correlated significantly with norepinephrine suggests a simulataneous and coordinated stimulation of adrenal glands and orthosympathetic nervous system. In emotive subjects (ratio NA/A less than 1 at rest) the apprehension of muscular work promoted a difference in catecholamine responses: norepinephrine release was not affected by subject's anxiety, while epinephrine secretion, already elevated before the test, reached a high degree of magnitude in the first minutes of muscular work, remaining nearly constant until exhaustion. Physical training of nonemotive subjects, during 2 months with two intense exercises by a week, reduced strongly norepinephrine release after exhaustive muscular work. In the same conditions, the adrenal-medullary response was not significantly modified when compared with untrained subjects. Our results suggest that the adrenergic behaviour during exercise is a function of effort intensity to be supplied; catecholamines seem to be important factors in regulating body homeostasy during muscular work in man. In addition, emotive subjects exhibit amplified adrenal-medullary response, which may be related to psychological stimuli.     

Carnitine metabolism during prolonged exercise and recovery in humans

Lennon et al. (J. Appl. Physiol. 55: 489-495, 1983) have recently reported a large loss of muscle total carnitine (TC) after 40 min of moderate exercise. These authors have also suggested that elevations in plasma esterified carnitine (EC) were due to the release of these carnitine esters from muscle during exercise. After 10 male subjects underwent 90 min of cycle egometry we found no alteration in muscle TC from preexercise values. Plasma EC progressively increased above resting values during exercise and remained elevated above rest at 0.75 and 1.5 h into recovery. Elevations of plasma EC were largely due to a decrement in free carnitine (FC) in both conditions. Immediately postexercise the urinary fractional reabsorbsion of EC and FC were similar to that at rest. These results suggest that a net loss of TC from exercising muscle does not occur. As in other conditions marked by falling insulin concentrations, elevations in plasma EC could result from an exchange of carnitine with the hepatic carnitine pool.     

Acute pantothenic acid and cysteine supplementation does not affect muscle coenzyme A content, fuel selection, or exercise performance in healthy humans

Reduced skeletal muscle free coenzyme A (CoASH) availability may decrease the contribution of fat oxidation to ATP production during high-intensity, submaximal exercise or, alternatively, limit pyruvate dehydrogenase complex (PDC) flux and thereby carbohydrate oxidation. Here we attempted to increase the muscle CoASH pool in humans, via pantothenic acid and cysteine feeding, in order to elucidate the role of CoASH availability on muscle fuel metabolism during exercise. On three occasions, eight healthy male volunteers (age 22.9 ± 1.4 yr, body mass index 24.2 ± 1.5 kg/m(2)) cycled at 75% maximal oxygen uptake (Vo(2max)) to exhaustion, followed by a 15-min work output performance test. Muscle biopsies were obtained at rest, and after 60 min and 91.3 ± 3.1 min of exercise (time to exhaustion on baseline visit) on each occasion. Two weeks following the first visit (baseline), 1 wk of oral supplementation with either 3 g/day of a placebo control (glucose polymer; CON) or 1.5 g/day each of d-pantothenic acid and l-cysteine (CP) was carried out prior to the second and third visits in a randomized, counterbalanced, double-blind manner, leaving a 3-wk gap in total between each visit. Resting muscle CoASH content was not altered by supplementation in any visit. Following 60 min of exercise, muscle CoASH content was reduced by 13% from rest in all three visits (P < 0.05), and similar changes in the respiratory exchange ratio, glycogenolysis (～235 mmol/kg dry muscle), PCr degradation (～57 mmol/kg dry muscle), and lactate (～25 mmol/kg dry muscle) and acetylcarnitine (～12 mmol(.)kg/dry muscle) accumulation was observed during exercise when comparing visits. Furthermore, no difference in work output was observed when comparing CON and CP. Acute feeding with pantothenic acid and cysteine does not alter muscle CoASH content and consequently does not impact on muscle fuel metabolism or performance during exercise in humans.     

Tandem mass spectrometric assay for the determination of carnitine palmitoyltransferase II activity in muscle tissue

Carnitine palmitoyltransferase II (CPT-II) mediates the import of long-chain fatty acids into the mitochondrial matrix for subsequent beta-oxidation. Defects of CPT-II manifest as a severe neonatal hepatocardiomuscular form or as a mild muscular phenotype in early infancy or adolescence. CPT-II deficiency is diagnosed by the determination of enzyme activity in tissues involving the time-dependent conversion of radiolabeled CPT-II substrates (isotope-exchange assays) or the formation of chromogenic reaction products. We have established a mass spectrometric assay (MS/MS) for the determination of CPT-II activity based on the stoichiometric formation of acetylcarnitine in a coupled reaction system. In this single-tube reaction system palmitoylcarnitine is converted by CPT-II to free carnitine, which is subsequently esterified to acetylcarnitine by carnitine acetyltransferase. The formation of acetylcarnitine directly correlates with the CPT-II activity. Comparison of the MS/MS method (y) with our routine spectrophotometric assay (x) revealed a linear regression of y = 0.58x + 0.12 (r = 0.8369). Both assays allow one to unambiguously detect patients with the muscular form of CPT-II deficiency. However, the higher specificity and sensitivity as well as the avoidance of the drawbacks inherent in the use of radiolabeled substrates make this mass spectrometric method most suitable for the determination of CPT-II activity.