M.G.M. de Sain-van der Velden

Acquired multiple Acyl-CoA dehydrogenase deficiency in 10 horses with atypical myopathy

The aim of the current study was to assess lipid metabolism in horses with atypical myopathy. Urine samples from 10 cases were subjected to analysis of organic acids, glycine conjugates, and acylcarnitines revealing increased mean excretion of lactic acid, ethylmalonic acid, 2-methylsuccinic acid, butyrylglycine, (iso)valerylglycine, hexanoylglycine, free carnitine, C2-, C3-, C4-, C5-, C6-, C8-, C8:1-, C10:1-, and C10:2-carnitine as compared with 15 control horses (12 healthy and three with acute myopathy due to other causes). Analysis of plasma revealed similar results for these predominantly short-chain acylcarnitines. Furthermore, measurement of dehydrogenase activities in lateral vastus muscle from one horse with atypical myopathy indeed showed deficiencies of short-chain acyl-CoA dehydrogenase (0.66 as compared with 2.27 and 2.48 in two controls), medium-chain acyl-CoA dehydrogenase (0.36 as compared with 4.31 and 4.82 in two controls) and isovaleryl-CoA dehydrogenase (0.74 as compared with 1.43 and 1.61 nmol min(-1) mg(-1) in two controls). 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 suspected in 10 out of 10 cases as the possible etiology for a highly fatal and prevalent toxic equine muscle disease similar to the combined metabolic derangements seen in human multiple acyl-CoA dehydrogenase deficiency also known as glutaric acidemia type II.

Equine acquired multiple acyl-CoA dehydrogenase deficiency (MADD) in 14 horses associated with ingestion of Maple leaves (Acer pseudoplatanus) covered with European tar spot (Rhytisma acerinum)

This case-series describes fourteen horses suspected of equine acquired multiple acyl-CoA dehydrogenase deficiency (MADD) also known as atypical myopathy of which seven cases were confirmed biochemically with all horses having had access to leaves of the Maple tree (Acer pseudoplatanus) covered with European tar spot (Rhytisma acerinum). Assessment of organic acids, glycine conjugates, and acylcarnitines in urine was regarded as gold standard in the biochemical diagnosis of equine acquired multiple acyl-CoA dehydrogenase deficiency.

Elevated cholesterol precursors other than cholestanol can also be a hallmark for CTX

SummaryCerebrotendinous xanthomatosis (CTX) is an inborn error of bile acid synthesis in which hepatic conversion of cholesterol to cholic and chenodeoxycholic acids is impaired. Patients have abnormal bile alcohols in urine, normal to increased plasma cholesterol concentrations and increased concentrations of plasma cholestanol. Little is known about cholesterol precursors in CTX, however. We studied cholesterol and phytosterol profiles in two siblings with CTX during follow-up. While cholesterol concentrations were low in both patients, plasma cholestanol was 6-fold higher compared to control values. In addition, both siblings had a more than 100-fold increase in 7-dehydrocholesterol (7DHC) and 8-dehydrocholesterol (8DHC). Lathosterol, lanosterol and sitosterol were increased in both patients while concentrations of desmosterol and campesterol were normal. In addition, plasma lathosterol/cholesterol ratios were significantly elevated. After treatment with chenodeoxycholate, both patients showed a marked decrease in cholestanol, 7DHC, 8DHC, lathosterol, lanosterol and sitosterol. In addition, the lathosterol/cholesterol ratio normalized, indicating that overall cholesterol synthesis was sufficiently suppressed. This study shows that elevated cholesterol precursors, other than cholestanol, can be a hallmark for CTX.

Amino acid profile during exercise and training in Standardbreds.

The objective of this study is to assess the influence of acute exercise, training and intensified training on the plasma amino acid profile. In a 32-week longitudinal study using 10 Standardbred horses, training was divided into four phases, including a phase of intensified training for five horses. At the end of each phase, a standardized exercise test, SET, was performed. Plasma amino acid concentrations before and after each SET were measured. Training significantly reduced mean plasma aspartic acid concentration, whereas exercise significantly increased the plasma concentrations of alanine, taurine, methionine, leucine, tyrosine and phenylalanine and reduced the plasma concentrations of glycine, ornithine, glutamine, citrulline and serine. Normally and intensified trained horses differed not significantly. It is concluded that amino acids should not be regarded as limiting training performance in Standardbreds except for aspartic acid which is the most likely candidate for supplementation.

Altered Energetics of Exercise Explain Risk of Rhabdomyolysis in Very Long-Chain Acyl-CoA Dehydrogenase Deficiency.

Rhabdomyolysis is common in very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) and other metabolic myopathies, but its pathogenic basis is poorly understood. Here, we show that prolonged bicycling exercise against a standardized moderate workload in VLCADD patients is associated with threefold bigger changes in phosphocreatine (PCr) and inorganic phosphate (Pi) concentrations in quadriceps muscle and twofold lower changes in plasma acetyl-carnitine levels than in healthy subjects. This result is consistent with the hypothesis that muscle ATP homeostasis during exercise is compromised in VLCADD. However, the measured rates of PCr and Pi recovery post-exercise showed that the mitochondrial capacity for ATP synthesis in VLCADD muscle was normal. Mathematical modeling of oxidative ATP metabolism in muscle composed of three different fiber types indicated that the observed altered energy balance during submaximal exercise in VLCADD patients may be explained by a slow-to-fast shift in quadriceps fiber-type composition corresponding to 30% of the slow-twitch fiber-type pool in healthy quadriceps muscle. This study demonstrates for the first time that quadriceps energy balance during exercise in VLCADD patients is altered but not because of failing mitochondrial function. Our findings provide new clues to understanding the risk of rhabdomyolysis following exercise in human VLCADD.

The effect of long-term oral L-carnitine administration on insulin sensitivity, glucose disposal, plasma concentrations of leptin and acylcarnitines, and urinary acylcarnitine excretion in warmblood horses

Background: Insulin resistance in horses is an emerging field of interest as it is thought to be a contributing factor in the pathogenesis of many equine conditions. Objectives: The objectives of the present study were to determine the effects of long-term oral administration of L-carnitine on insulin sensitivity, glucose disposal, plasma leptin concentrations and acylcarnitine spectrum both in plasma and urine. Animals and methods: Six 3-year-old healthy warmblood geldings were used. In a double blind 2 × 2 Latin square design at a dosage of 100 mg/kg body weight (BW)/day for 28 days the effects of oral supplementation of L-carnitine (as fumarate) were assessed. Glucose disposal and insulin sensitivity were measured by means of the euglycemic-hyperinsulinemic clamp technique. Radioimmunoassays were used to determine plasma leptin and insulin concentrations. Electrospray tandem mass spectrometry was used to assess acylcarnitines both in plasma and urine. Statistical analysis was performed using a linear mixed-effects model and P values <0.05 were considered significant. Results: Long-term L-carnitine administration did not affect insulin sensitivity. Plasma leptin and free carnitine concentrations in plasma and urine increased significantly (P = 0.047 and 0.000, respectively) following L-carnitine administration as well as short-chain acylcarnitines in plasma and urinary excretion of short- and medium-chain acylcarnitines. Conclusion and clinical relevance: Given the effects of oral administration of L-carnitine further clinical study is necessary in order to assess the potential beneficial effects in equine patients suffering from metabolic myopathies such as acquired multiple acyl-CoA dehydrogenase deficiency. Impact for human medicine: The current study supports the treatment rationale of short-chain acyl-CoA dehydrogenase deficiency in humans with L-carnitine at an oral dosage of 100 mg/kg BW/day.

Some notes on fatal acquired multiple acyl-CoA dehydrogenase deficiency (MADD) in a two-year-old warmblood stallion and European tar spot (Rhytisma acerinum)

Some notes on fatal acquired multiple acyl-CoA dehydrogenase deficiency (MADD) in a two-year-old warmblood stallion and European tar spot (Rhytisma acerinum) J. H. van der Kolk a , R. Boelens b , S. B.A. Halkes c , I. D. Wijnberg d , M. G.M. de Sainvan der Velden e & J. H. Ippel b a Section Equine Metabolic and Genetic Diseases , Euregio Laboratory Services , Maastricht , the Netherlands b NMR Spectroscopy Research Group, Bijvoet Centre for Biomolecular Research , Utrecht University , Utrecht , the Netherlands c PhytoGeniX BV/Medicinal Chemistry and Chemical Biology, Department of Pharmaceutical Sciences , Faculty of Sciences, Utrecht University , Utrecht , the Netherlands d Department of Equine Sciences , Medicine Section, Faculty of Veterinary Medicine, Utrecht University , Utrecht , the Netherlands e Department of Metabolic Diseases , University Medical Center , Utrecht , the Netherlands Accepted author version posted online: 20 Dec 2012.Published online: 16 Jan 2013.

Amino acid utilization by the hindlimb of warmblood horses at rest and following low intensity exercise

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 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.

Acylcarnitine ester utilization by the hindlimb of warmblood horses at rest and following low intensity exercise and carnitine supplementation.

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.

Comment on Zwickler et al.: Usefulness of biochemical parameters in decision-making on the start of emergency treatment in patients with propionic acidemia

Dear Editor, Treatment of patients with (acutely) decompensated propionic acidemia (PA) is a medical emergency. We read with great interest the paper by Zwickler et al. (2014), who reported that ammonia, acid–base balance, and anion gap are important biochemical parameters by which to identify an (impending) metabolic decompensation and can be used to assess its severity in PA patients. Moreover, Zwickler et al. suggested that presently available metabolic parameters, such as organic acids and acylcarnitines, were not discriminative and can be omitted. Based on our study results, we fully agree with this conclusion. In addition, we quantified methylcitrate (MCA) in plasma of patients with PA (n=54) using ultraperformance liquid chromatography–tandem mass spectrometry. The analysis of MCA in plasma was based on the method described by Fu et al. (2013), with some modifications. MCA in PA patients is formed as a consequence of propionyl–coenzyme A (CoA) accumulation and subsequent condensation with oxaloacetate. MCA was consistently increased in all patients, irrespective of their metabolic condition (range 6.7– 107.5 μmol/L, median 30.6 μmol/L, normal <0.4 μmol/ L). Our results further show that when metabolic control in patients with PA becomes unstable, ammonia levels increase concurrently with plasma MCA (Fig. 1, y=0.450x+ 1.184, r=0.75, P<0.0001, n=28). A direct link between ammonia and MCA has also been reported in the study by Jafari et al. (2013), who showed parallel changes in ammonium after exposure to MCA (dose–response studies in an in vitro model). The authors show that exposure to MCA causes morphological changes in neuronal and glial cells and suggest that ammonium accumulation is secondary to cell suffering and/ or death. We suggest further investigation of MCA in plasma as a marker for PA decompensation. This stable compound may be favorable, as plasma ammonia concentrations may artificially increase by contamination and deterioration of blood components during specimen handling and storage. In conclusion, MCA measurements in plasma, along with the other suggested parameters, may provide information on decompensation in PA.