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Sander M Houten - One of the best experts on this subject based on the ideXlab platform.
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slc22a5 haploinsufficiency does not aggravate the phenotype of the long chain acyl coa dehydrogenase ko mouse
Journal of Inherited Metabolic Disease, 2020Co-Authors: Pablo Ranearobles, Frédéric M. Vaz, Naomi Van Vlies, Sander M HoutenAbstract:Secondary carnitine deficiency is commonly observed in inherited metabolic diseases characterised by the accumulation of Acylcarnitines such as mitochondrial fatty acid oxidation (FAO) disorders. It is currently unclear if carnitine deficiency and/or Acylcarnitine accumulation play a role in the pathophysiology of FAO disorders. The long-chain acyl-CoA dehydrogenase (LCAD) KO mouse is a model for long-chain FAO disorders and is characterised by decreased levels of tissue and plasma free carnitine. Tissue levels of carnitine are controlled by SLC22A5, the plasmalemmal carnitine transporter. Here, we have further decreased carnitine availability in the LCAD KO mouse through a genetic intervention by introducing one defective Slc22a5 allele (jvs). Slc22a5 haploinsufficiency decreased free carnitine levels in liver, kidney, and heart of LCAD KO animals. The resulting decrease in the tissue long-chain Acylcarnitines levels had a similar magnitude as the decrease in free carnitine. Levels of cardiac deoxycarnitine, a carnitine biosynthesis intermediate, were elevated due to Slc22a5 haploinsufficiency in LCAD KO mice. A similar increase in heart and muscle deoxycarnitine was observed in an independent experiment using Slc22a5jvs/jvs mice. Cardiac hypertrophy, fasting-induced hypoglycemia and increased liver weight, the major phenotypes of the LCAD KO mouse, were not affected by Slc22a5 haploinsufficiency. This may suggest that secondary carnitine deficiency does not play a major role in the pathophysiology of these phenotypes. Similarly, our data do not support a major role for toxicity of long-chain Acylcarnitines in the phenotype of the LCAD KO mouse.
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the impact of altered carnitine availability on Acylcarnitine metabolism energy expenditure and glucose tolerance in diet induced obese mice
Biochimica et Biophysica Acta, 2016Co-Authors: Marieke G Schooneman, Ronald J.a. Wanders, Sander M Houten, Frédéric M. Vaz, Maarten R Soeters, Riekelt H Houtkooper, Carla E M HollakAbstract:Abstract Aim Acylcarnitines are fatty acid oxidation (FAO) intermediates, which have been implicated in diet-induced insulin resistance. Elevated Acylcarnitine levels are found in obese, insulin resistant humans and rodents, and coincide with lower free carnitine. We hypothesized that increasing free carnitine levels by administration of the carnitine precursor γ-butyrobetaine (γBB) could facilitate FAO, thereby improving insulin sensitivity. Methods C57BL/6N mice were fed with a high fat or chow diet with or without γBB supplementation (n = 10 per group). After 8 weeks of diet, indirect calorimetry, glucose tolerance and insulin sensitivity tests were performed. AC profiles and carnitine biosynthesis intermediates were analyzed in plasma and tissues by tandem mass spectrometry (MS) and liquid chromatography tandem MS. Results γBB supplementation did not facilitate FAO, was unable to curb bodyweight and did not prevent impaired glucose homeostasis in the HFD fed mice in spite of marked alterations in the Acylcarnitine profiles in plasma and liver. Remarkably, γBB did not affect the Acylcarnitine profile in other tissues, most notably muscle. Administration of a bolus acetylcarnitine also caused significant changes in plasma and liver, but not in muscle Acylcarnitine profiles, again without effect on glucose tolerance. Conclusion Altogether, increasing carnitine availability affects Acylcarnitine profiles in plasma and liver but does not modulate glucose tolerance or insulin sensitivity. This may be due to the lack of an effect on muscle Acylcarnitine profiles, as muscle tissue is an important contributor to whole body insulin sensitivity. These results warrant caution on making associations between plasma Acylcarnitine levels and insulin resistance.
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transorgan fluxes in a porcine model reveal a central role for liver in Acylcarnitine metabolism
American Journal of Physiology-endocrinology and Metabolism, 2015Co-Authors: Marieke G Schooneman, Sander M Houten, Naomi Van Vlies, Gabriella Ten A M Have, Nicolaas E P Deutz, Maarten R SoetersAbstract:Acylcarnitines are derived from mitochondrial acyl-CoA metabolism and have been associated with diet-induced insulin resistance. However, plasma Acylcarnitine profiles have been shown to poorly reflect whole body Acylcarnitine metabolism. We aimed to clarify the individual role of different organ compartments in whole body Acylcarnitine metabolism in a fasted and postprandial state in a porcine transorgan arteriovenous model. Twelve cross-bred pigs underwent surgery where intravascular catheters were positioned before and after the liver, gut, hindquarter muscle compartment, and kidney. Before and after a mixed meal, we measured Acylcarnitine profiles at several time points and calculated net transorgan Acylcarnitine fluxes. Fasting plasma Acylcarnitine concentrations correlated with net hepatic transorgan fluxes of free and C2- and C16-carnitine. Transorgan Acylcarnitine fluxes were small, except for a pronounced net hepatic C2-carnitine production. The peak of the postprandial Acylcarnitine fluxes was between 60 and 90 min. Acylcarnitine production or release was seen in the gut and liver and consisted mostly of C2-carnitine. Acylcarnitines were extracted by the kidney. No significant net muscle Acylcarnitine flux was observed. We conclude that liver has a key role in Acylcarnitine metabolism, with high net fluxes of C2-carnitine both in the fasted and fed state, whereas the contribution of skeletal muscle is minor. These results further clarify the role of different organ compartments in the metabolism of different Acylcarnitine species.
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plasma Acylcarnitines inadequately reflect tissue Acylcarnitine metabolism
Biochimica et Biophysica Acta, 2014Co-Authors: Marieke G Schooneman, Sander M Houten, Niki Achterkamp, Carmen Argmann, Maarten R SoetersAbstract:Acylcarnitines have been linked to obesity-induced insulin resistance. However the majority of these studies have focused on Acylcarnitines in plasma. It is currently unclear to what extent plasma levels of Acylcarnitines reflect tissue Acylcarnitine metabolism. We investigated the correlation of plasma Acylcarnitine levels with selected tissue Acylcarnitines as measured with tandem mass spectrometry, in both fed and fasted BALB/cJ (BALB) and C57BL/6N (Bl6) mice. Fasting affected Acylcarnitine levels in all tissues. These changes varied substantially between the different tissue compartments. No significant correlations were found between plasma Acylcarnitine species and their tissue counterparts in both mouse strains, with the exception of plasma C4OH-carnitine in BALB mice. We suggest that this lack of correlation is due to differences in Acylcarnitine turnover rates between plasma and tissue compartments and the fact that the plasma Acylcarnitine profile is a composition of Acylcarnitines derived from different compartments. Therefore, plasma Acylcarnitine levels do not reflect tissue levels and should be interpreted with caution. A focus on tissue Acylcarnitine levels is warranted in metabolic studies.
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substrate specificity of human carnitine acetyltransferase implications for fatty acid and branched chain amino acid metabolism
Biochimica et Biophysica Acta, 2013Co-Authors: S Violante, Lodewijk Ijlst, Jos P N Ruiter, Janet Koster, Henk Van Lenthe, M Duran, Isabel Tavares De Almeida, Sander M Houten, F V VenturaAbstract:Carnitine acyltransferases catalyze the reversible conversion of acyl-CoAs into Acylcarnitine esters. This family includes the mitochondrial enzymes carnitine palmitoyltransferase 2 (CPT2) and carnitine acetyltransferase (CrAT). CPT2 is part of the carnitine shuttle that is necessary to import fatty acids into mitochondria and catalyzes the conversion of Acylcarnitines into acyl-CoAs. In addition, when mitochondrial fatty acid β-oxidation is impaired, CPT2 is able to catalyze the reverse reaction and converts accumulating long- and medium-chain acyl-CoAs into Acylcarnitines for export from the matrix to the cytosol. However, CPT2 is inactive with short-chain acyl-CoAs and intermediates of the branched-chain amino acid oxidation pathway (BCAAO). In order to explore the origin of short-chain and branched-chain Acylcarnitines that may accumulate in various organic acidemias, we performed substrate specificity studies using purified recombinant human CrAT. Various saturated, unsaturated and branched-chain acyl-CoA esters were tested and the synthesized Acylcarnitines were quantified by ESI-MS/MS. We show that CrAT converts short- and medium-chain acyl-CoAs (C2 to C10-CoA), whereas no activity was observed with long-chain species. Trans-2-enoyl-CoA intermediates were found to be poor substrates for this enzyme. Furthermore, CrAT turned out to be active towards some but not all the BCAAO intermediates tested and no activity was found with dicarboxylic acyl-CoA esters. This suggests the existence of another enzyme able to handle the acyl-CoAs that are not substrates for CrAT and CPT2, but for which the corresponding Acylcarnitines are well recognized as diagnostic markers in inborn errors of metabolism.
F V Ventura - One of the best experts on this subject based on the ideXlab platform.
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substrate specificity of human carnitine acetyltransferase implications for fatty acid and branched chain amino acid metabolism
Biochimica et Biophysica Acta, 2013Co-Authors: S Violante, Lodewijk Ijlst, Jos P N Ruiter, Janet Koster, Henk Van Lenthe, M Duran, Isabel Tavares De Almeida, Sander M Houten, F V VenturaAbstract:Carnitine acyltransferases catalyze the reversible conversion of acyl-CoAs into Acylcarnitine esters. This family includes the mitochondrial enzymes carnitine palmitoyltransferase 2 (CPT2) and carnitine acetyltransferase (CrAT). CPT2 is part of the carnitine shuttle that is necessary to import fatty acids into mitochondria and catalyzes the conversion of Acylcarnitines into acyl-CoAs. In addition, when mitochondrial fatty acid β-oxidation is impaired, CPT2 is able to catalyze the reverse reaction and converts accumulating long- and medium-chain acyl-CoAs into Acylcarnitines for export from the matrix to the cytosol. However, CPT2 is inactive with short-chain acyl-CoAs and intermediates of the branched-chain amino acid oxidation pathway (BCAAO). In order to explore the origin of short-chain and branched-chain Acylcarnitines that may accumulate in various organic acidemias, we performed substrate specificity studies using purified recombinant human CrAT. Various saturated, unsaturated and branched-chain acyl-CoA esters were tested and the synthesized Acylcarnitines were quantified by ESI-MS/MS. We show that CrAT converts short- and medium-chain acyl-CoAs (C2 to C10-CoA), whereas no activity was observed with long-chain species. Trans-2-enoyl-CoA intermediates were found to be poor substrates for this enzyme. Furthermore, CrAT turned out to be active towards some but not all the BCAAO intermediates tested and no activity was found with dicarboxylic acyl-CoA esters. This suggests the existence of another enzyme able to handle the acyl-CoAs that are not substrates for CrAT and CPT2, but for which the corresponding Acylcarnitines are well recognized as diagnostic markers in inborn errors of metabolism.
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carnitine palmitoyltransferase 2 and carnitine Acylcarnitine translocase are involved in the mitochondrial synthesis and export of Acylcarnitines
The FASEB Journal, 2013Co-Authors: Sara Violante, Lodewijk Ijlst, Ronald J.a. Wanders, Isabel Tavares De Almeida, F V Ventura, Heleen Te Brinke, Sander M HoutenAbstract:Acylcarnitines are commonly used in the diagnosis of mitochondrial fatty acid β-oxidation disorders (mFAODs). It is generally assumed that this plasma Acylcarnitine profile reflects the mitochondrial accumulation of acyl-CoAs. The identity of the enzymes and the mitochondrial and plasmalemmal transporters involved in the synthesis and export of these metabolites have remained undefined. We used lentiviral shRNA to knock down the expression of medium-chain acyl-CoA dehydrogenase (MCAD) in control and carnitine palmitoyltransferase 2 (CPT2)-, carnitine/Acylcarnitine translocase (CACT)-, and plasmalemmal carnitine transporter (OCTN2)-deficient human fibroblasts. These cell lines, including mock-transduced controls, were loaded with decanoic acid and carnitine, followed by the measurement of the Acylcarnitine profile in the extracellular medium. In control fibroblasts, MCAD knockdown markedly increased the production of octanoylcarnitine (3-fold, P<0.01). OCTN2-deficient cell lines also showed extracellular accumulation of octanoylcarnitine (2.8-fold, P<0.01), suggesting that the cellular export of Acylcarnitines does not depend on OCTN2. In contrast, in CPT2- and CACT-deficient cells, the accumulation of octanoylcarnitine in the medium did not significantly increase in the MCAD knockdown. Similar results were obtained using pharmacological inhibition of CPT2 in fibroblasts from MCAD-deficient individuals. This shows that CPT2 and CACT are crucial for mitochondrial Acylcarnitine formation and export to the extracellular fluids in mFAOD.
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carnitine palmitoyltransferase 2 and carnitine Acylcarnitine translocase are involved in the mitochondrial synthesis and export of Acylcarnitines
The FASEB Journal, 2013Co-Authors: S Violante, Lodewijk Ijlst, Ronald J.a. Wanders, Isabel Tavares De Almeida, F V Ventura, Heleen Te Brinke, Sander M HoutenAbstract:Acylcarnitines are commonly used in the diagnosis of mitochondrial fatty acid β-oxidation disorders (mFAODs). It is generally assumed that this plasma Acylcarnitine profile reflects the mitochondrial accumulation of acyl-CoAs. The identity of the enzymes and the mitochondrial and plasmalemmal transporters involved in the synthesis and export of these metabolites have remained undefined. We used lentiviral shRNA to knock down the expression of medium-chain acyl-CoA dehydrogenase (MCAD) in control and carnitine palmitoyltransferase 2 (CPT2)-, carnitine/Acylcarnitine translocase (CACT)-, and plasmalemmal carnitine transporter (OCTN2)-deficient human fibroblasts. These cell lines, including mock-transduced controls, were loaded with decanoic acid and carnitine, followed by the measurement of the Acylcarnitine profile in the extracellular medium. In control fibroblasts, MCAD knockdown markedly increased the production of octanoylcarnitine (3-fold, P<0.01). OCTN2-deficient cell lines also showed extracellular a...
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quantitative Acylcarnitine profiling in fibroblasts using u 13c palmitic acid an improved tool for the diagnosis of fatty acid oxidation defects
Clinica Chimica Acta, 1999Co-Authors: F V Ventura, Lodewijk Ijlst, Jos P N Ruiter, M Duran, Isabel Tavares De Almeida, C G Costa, Edward A Struys, Paul Allers, Cornelis JakobsAbstract:A method was developed for the investigation of mitochondrial fatty acid β-oxidation in cultured fibroblasts. Monolayer cultures were incubated without foetal calf serum with commercially available [U-13C] palmitic acid and l-carnitine for 96 h. The Acylcarnitines produced by the cells were extracted from the cell suspension and analysed either by quantitative stable isotope dilution gas chromatography chemical ionization mass spectrometry, or by fast atom bombardment mass spectrometry. Characteristic Acylcarnitine profiles were obtained for all the different enzyme deficiencies investigated, with the exception of carnitine palmitoyltransferase II deficiency and carnitine/Acylcarnitine carrier deficiency which showed similar patterns. Comparison between this method and the 3H-myristate and 3H-palmitate tritium release assays revealed that the method described here is superior, allowing unequivocal identification of patients.
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broad specificity of carnitine palmitoyltransferase ii towards long chain acyl coa beta oxidation intermediates and its practical approach to the synthesis of various long chain Acylcarnitines
Journal of Inherited Metabolic Disease, 1997Co-Authors: F V Ventura, Lodewijk Ijlst, M Duran, C G Costa, L Dorland, C Jakobs, I T De Almeida, R J A WandersAbstract:Long-chain fatty acid β-oxidation defects involving 3-hydroxyacyl-coenzyme A dehydrogenase (LCHAD) or mitochondrial trifunctional protein (MTP) (Pollitt 1995) are characterized by abnormal urinary organic acids and specific plasma Acylcarnitine profile during crises (Dorland et al 1995). The same atypical Acylcarnitine esters have also been found in in vitro studies comprising the incubation of patient's cells (Nada et al 1995; Schaefer et al 1995) with long-chain fatty acids. An explanation for the finding of these abnormalities might be that the different acyl-CoA esters that accumulate within the mitochondrial matrix are exported to the cytosol in the Acylcarnitine form. The mechanism associated with this process is unclear but may primarily involve carnitine palmitoyltransferase (CPT) II catalysing the conversion of the intramitochondrial long-chain acyl-CoA esters into the corresponding Acylcarnitines followed by export from the mitochondria via the Acylcarnitine/carnitine carrier. While studying the specificity of CPT towards palmitoyl-CoA and its β-oxidation intermediates, we found that CPT II accepts as substrates not only acyl-CoA esters but also 2,3-unsaturated, 3-hydroxy and 3-keto acyl-CoA esters. In the present paper we have made use of the reactivity of CPT II towards 3-hydroxy-palmitoyl-CoA to synthesize 3-hydroxypalmitoylcarnitine enzymatically. The synthesis of this compound and other 3-hydroxyAcylcarnitines is important for the qualitative and quantitative analysis of the Acylcarnitine profile in LCHAD and MTP deficiencies. These substances may also be useful for experiments directed towards solving the problems of the peculiar clinical findings in LCHAD deficiency.
Ronald J.a. Wanders - One of the best experts on this subject based on the ideXlab platform.
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the impact of altered carnitine availability on Acylcarnitine metabolism energy expenditure and glucose tolerance in diet induced obese mice
Biochimica et Biophysica Acta, 2016Co-Authors: Marieke G Schooneman, Ronald J.a. Wanders, Sander M Houten, Frédéric M. Vaz, Maarten R Soeters, Riekelt H Houtkooper, Carla E M HollakAbstract:Abstract Aim Acylcarnitines are fatty acid oxidation (FAO) intermediates, which have been implicated in diet-induced insulin resistance. Elevated Acylcarnitine levels are found in obese, insulin resistant humans and rodents, and coincide with lower free carnitine. We hypothesized that increasing free carnitine levels by administration of the carnitine precursor γ-butyrobetaine (γBB) could facilitate FAO, thereby improving insulin sensitivity. Methods C57BL/6N mice were fed with a high fat or chow diet with or without γBB supplementation (n = 10 per group). After 8 weeks of diet, indirect calorimetry, glucose tolerance and insulin sensitivity tests were performed. AC profiles and carnitine biosynthesis intermediates were analyzed in plasma and tissues by tandem mass spectrometry (MS) and liquid chromatography tandem MS. Results γBB supplementation did not facilitate FAO, was unable to curb bodyweight and did not prevent impaired glucose homeostasis in the HFD fed mice in spite of marked alterations in the Acylcarnitine profiles in plasma and liver. Remarkably, γBB did not affect the Acylcarnitine profile in other tissues, most notably muscle. Administration of a bolus acetylcarnitine also caused significant changes in plasma and liver, but not in muscle Acylcarnitine profiles, again without effect on glucose tolerance. Conclusion Altogether, increasing carnitine availability affects Acylcarnitine profiles in plasma and liver but does not modulate glucose tolerance or insulin sensitivity. This may be due to the lack of an effect on muscle Acylcarnitine profiles, as muscle tissue is an important contributor to whole body insulin sensitivity. These results warrant caution on making associations between plasma Acylcarnitine levels and insulin resistance.
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carnitine palmitoyltransferase 2 and carnitine Acylcarnitine translocase are involved in the mitochondrial synthesis and export of Acylcarnitines
The FASEB Journal, 2013Co-Authors: S Violante, Lodewijk Ijlst, Ronald J.a. Wanders, Isabel Tavares De Almeida, F V Ventura, Heleen Te Brinke, Sander M HoutenAbstract:Acylcarnitines are commonly used in the diagnosis of mitochondrial fatty acid β-oxidation disorders (mFAODs). It is generally assumed that this plasma Acylcarnitine profile reflects the mitochondrial accumulation of acyl-CoAs. The identity of the enzymes and the mitochondrial and plasmalemmal transporters involved in the synthesis and export of these metabolites have remained undefined. We used lentiviral shRNA to knock down the expression of medium-chain acyl-CoA dehydrogenase (MCAD) in control and carnitine palmitoyltransferase 2 (CPT2)-, carnitine/Acylcarnitine translocase (CACT)-, and plasmalemmal carnitine transporter (OCTN2)-deficient human fibroblasts. These cell lines, including mock-transduced controls, were loaded with decanoic acid and carnitine, followed by the measurement of the Acylcarnitine profile in the extracellular medium. In control fibroblasts, MCAD knockdown markedly increased the production of octanoylcarnitine (3-fold, P<0.01). OCTN2-deficient cell lines also showed extracellular a...
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carnitine palmitoyltransferase 2 and carnitine Acylcarnitine translocase are involved in the mitochondrial synthesis and export of Acylcarnitines
The FASEB Journal, 2013Co-Authors: Sara Violante, Lodewijk Ijlst, Ronald J.a. Wanders, Isabel Tavares De Almeida, F V Ventura, Heleen Te Brinke, Sander M HoutenAbstract:Acylcarnitines are commonly used in the diagnosis of mitochondrial fatty acid β-oxidation disorders (mFAODs). It is generally assumed that this plasma Acylcarnitine profile reflects the mitochondrial accumulation of acyl-CoAs. The identity of the enzymes and the mitochondrial and plasmalemmal transporters involved in the synthesis and export of these metabolites have remained undefined. We used lentiviral shRNA to knock down the expression of medium-chain acyl-CoA dehydrogenase (MCAD) in control and carnitine palmitoyltransferase 2 (CPT2)-, carnitine/Acylcarnitine translocase (CACT)-, and plasmalemmal carnitine transporter (OCTN2)-deficient human fibroblasts. These cell lines, including mock-transduced controls, were loaded with decanoic acid and carnitine, followed by the measurement of the Acylcarnitine profile in the extracellular medium. In control fibroblasts, MCAD knockdown markedly increased the production of octanoylcarnitine (3-fold, P<0.01). OCTN2-deficient cell lines also showed extracellular accumulation of octanoylcarnitine (2.8-fold, P<0.01), suggesting that the cellular export of Acylcarnitines does not depend on OCTN2. In contrast, in CPT2- and CACT-deficient cells, the accumulation of octanoylcarnitine in the medium did not significantly increase in the MCAD knockdown. Similar results were obtained using pharmacological inhibition of CPT2 in fibroblasts from MCAD-deficient individuals. This shows that CPT2 and CACT are crucial for mitochondrial Acylcarnitine formation and export to the extracellular fluids in mFAOD.
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Carnitine-Acylcarnitine translocase deficiency, clinical, biochemical and genetic aspects.
Molecular Aspects of Medicine, 2004Co-Authors: M. E. Rubio-gozalbo, Hans R. Waterham, Jaap A. Bakker, Ronald J.a. WandersAbstract:The carnitine-Acylcarnitine translocase (CACT) is one of the components of the carnitine cycle. The carnitine cycle is necessary to shuttle long-chain fatty acids from the cytosol into the intramitochondrial space where mitochondrial beta-oxidation of fatty acids takes place. The oxidation of fatty acids yields acetyl-coenzyme A (CoA) units, which may either be degraded to CO(2) and H(2)O in the citric acid cycle to produce ATP or converted into ketone bodies which occurs in liver and kidneys. Metabolic consequences of a defective CACT are hypoketotic hypoglycaemia under fasting conditions, hyperammonemia, elevated creatine kinase and transaminases, dicarboxylic aciduria, very low free carnitine and an abnormal Acylcarnitine profile with marked elevation of the long-chain Acylcarnitines. Clinical signs and symptoms in CACT deficient patients, are a combination of energy depletion and endogenous toxicity. The predominantly affected organs are brain, heart and skeletal muscle, and liver, leading to neurological abnormalities, cardiomyopathy and arrythmias, skeletal muscle damage and liver dysfunction. Most patients become symptomatic in the neonatal period with a rapidly progressive deterioration and a high mortality rate. However, presentations at a later age with a milder phenotype have also been reported. The therapeutic approach is the same as in other long-chain fatty acid disorders and includes intravenous glucose (+/- insulin) administration to maximally inhibit lipolysis and subsequent fatty acid oxidation during the acute deterioration, along with other measures such as ammonia detoxification, depending on the clinical features. Long-term strategy consists of avoidance of fasting with frequent meals and a special diet with restriction of long-chain fatty acids. Due to the extremely low free carnitine concentrations, carnitine supplementation is often needed. Acylcarnitine profiling in plasma is the assay of choice for the diagnosis at a metabolite level. However, since the Acylcarnitine profile observed in CACT-deficient patients is identical to that in CPT2-deficient patients, definitive identification of CACT-deficiency in a certain patient requires determination of the activity of CACT. Subsequently, mutational analysis of the CACT gene can be performed. So far, 9 different mutations have been identified in the CACT gene.
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Characteristic Acylcarnitine Profiles in Inherited Defects of Peroxisome Biogenesis: A Novel Tool for Screening Diagnosis Using Tandem Mass Spectrometry
Pediatric Research, 2003Co-Authors: Cristiano Rizzo, Ronald J.a. Wanders, Sara Boenzi, Marinus Duran, Ubaldo Caruso, Carlo Dionisi-viciAbstract:Patients with inherited defects of peroxisomal metabolism, a class of diseases with marked clinical and genetic heterogeneity, show a characteristic phenotype in most cases with severe neurologic impairment, craniofacial abnormalities, and hepatic and kidney dysfunction. For the differential diagnosis of clinically suspected cases, a complex biochemical and genetic approach is required. Analysis of plasma very-long-chain fatty acids is a reliable screening method to detect most but not all peroxisomal disorders. To study the potential presence of abnormal Acylcarnitine species in plasma and blood, we screened by tandem mass spectrometry a series of patients affected by a peroxisome biogenesis disorder (PBD) and compared the results with those obtained in patients with isolated peroxisomal defects ( e.g. D-bifunctional protein deficiency, X-linked adrenoleukodystrophy) and mitochondrial long-chain fatty acid oxidation defects. The most relevant finding observed in plasma of patients with PBD was a significant increase of long-chain dicarboxylic C16- and C18-carnitine, i.e. hexadecanedioyl- and octadecanedioyl-carnitine, with high dicarboxylycarnitine/monocarboxylylcarnitine ratio. Elevation of very long-chain Acylcarnitines C24- and C26-, i.e. lignoceroyl- and cerotoyl-carnitine, was detected in some PBDs and in D-bifunctional protein deficiency. Similar abnormalities were also found in neonatal screening blood spots. Detection of these compounds alone, in the absence of other shorter-chain Acylcarnitines, is highly specific and characteristic of PBD, as confirmed by the differing profiles observed in patients with adrenoleukodystrophy and mitochondrial long-chain fatty acid oxidation defects. Our study adds a novel method to the diagnosis of PBD, which may also be of benefit for future neonatal mass screening programs based on Acylcarnitine profiling.
Ute Spiekerkoetter - One of the best experts on this subject based on the ideXlab platform.
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Pre-exercise medium-chain triglyceride application prevents Acylcarnitine accumulation in skeletal muscle from very-long-chain acyl-CoA-dehydrogenase-deficient mice
Journal of Inherited Metabolic Disease, 2010Co-Authors: Sonja Primassin, Sara Tucci, Diran Herebian, Annette Seibt, Lars Hoffmann, Frank Veld, Ute SpiekerkoetterAbstract:Dietary modification with medium-chain triglyceride (MCT) supplementation is one crucial way of treating children with long-chain fatty acid oxidation disorders. Recently, supplementation prior to exercise has been reported to prevent muscular pain and rhabdomyolysis. Systematic studies to determine when MCT supplementation is most beneficial have not yet been undertaken. We studied the effects of an MCT-based diet compared with MCT administration only prior to exercise in very-long-chain acyl-CoA dehydrogenase (VLCAD) knockout (KO) mice. VLCAD KO mice were fed an MCT-based diet in same amounts as normal mouse diet containing long-chain triglycerides (LCT) and were exercised on a treadmill. Mice fed a normal LCT diet received MCT only prior to exercise. Acylcarnitine concentration, free carnitine concentration, and acyl-coenzyme A (CoA) oxidation capacity in skeletal muscle as well as hepatic lipid accumulation were determined. Long-chain Acylcarnitines significantly increased in VLCAD-deficient skeletal muscle with an MCT diet compared with an LCT diet with MCT bolus prior to exercise, whereas an MCT bolus treatment significantly decreased long-chain Acylcarnitines after exercise compared with an LCT diet. C8-carnitine was significantly increased in skeletal muscle after MCT bolus treatment and exercise compared with LCT and long-term MCT treatment. Increased hepatic lipid accumulation was observed in long-term MCT-treated KO mice. MCT seems most beneficial when given in a single dose directly prior to exercise to prevent Acylcarnitine accumulation. In contrast, continuous MCT treatment produces a higher skeletal muscle content of long-chain Acylcarnitines after exercise and increases hepatic lipid storage in VLCAD KO mice.
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Carnitine Supplementation Induces Acylcarnitine Production in Tissues of Very Long-Chain Acyl-CoA Dehydrogenase-Deficient Mice, Without Replenishing Low Free Carnitine
Pediatric research, 2008Co-Authors: Sonja Primassin, Ertan Mayatepek, Frank Ter Veld, Ute SpiekerkoetterAbstract:Deficiency of very long-chain acyl-CoA dehydrogenase (VLCAD) results in accumulation of C14-C18 Acylcarnitines and low free carnitine. Carnitine supplementation is still controversial. VLCAD knockout (VLCAD(+/-)) mice exhibit a similar clinical and biochemical phenotype to those observed in humans. VLCAD(+/-) mice were fed with carnitine dissolved in drinking water. Carnitine, Acylcarnitines, and gamma-butyrobetaine were measured in blood and tissues. Measurements were performed under resting conditions, after exercise and after 24 h of regeneration. HepG2 cells were incubated with palmitoyl-CoA and palmitoyl-carnitine, respectively, to examine toxicity. With carnitine supplementation, Acylcarnitine production was significantly induced. Nevertheless, carnitine was low in skeletal muscle after exercise. Without carnitine supplementation, liver carnitine significantly increased after exercise, and after 24 h of regeneration, carnitine concentrations in skeletal muscle completely replenished to initial values. Incubation of hepatic cells with palmitoyl-CoA and palmitoyl-carnitine revealed a significantly reduced cell viability after incubation with palmitoyl-carnitine. The present study demonstrates that carnitine supplementation results in significant accumulation of potentially toxic Acylcarnitines in tissues. The expected prevention of low tissue carnitine was not confirmed. The principle mechanism regulating carnitine homeostasis seems to be endogenous carnitine biosynthesis, also under conditions with increased demand of carnitine such as in VLCAD-deficiency.
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changes in blood carnitine and Acylcarnitine profiles of very long chain acyl coa dehydrogenase deficient mice subjected to stress
European Journal of Clinical Investigation, 2004Co-Authors: Ertan Mayatepek, Ute Spiekerkoetter, M Duran, C Tokunaga, Udo Wendel, Vernat Exil, Frits A Wijburg, R J A WandersAbstract:Background In humans with deficiency of the very long-chain acyl-CoA dehydrogenase (VLCAD), C14–C18 Acylcarnitines accumulate. In this paper we have used the VLCAD knockout mouse as a model to study changes in blood carnitine and Acylcarnitine profiles under stress. Design VLCAD knockout mice exhibit stress-induced hypoglycaemia and skeletal myopathy; symptoms resembling human VLCADD. To study the extent of biochemical derangement in response to different stressors, we determined blood carnitine and Acylcarnitine profiles after exercise on a treadmill, fasting, or exposure to cold. Results Even in a nonstressed, well-fed state, knockout mice presented twofold higher C14–C18 Acylcarnitines and a lower free carnitine of 72% as compared to wild-type littermates. After 1 h of intense exercise, the C14–C18 Acylcarnitines in blood significantly increased, but free carnitine remained unchanged. After 8 h of fasting at 4 °C, the long-chain Acylcarnitines were elevated 5-fold in knockout mice in comparison with concentrations in unstressed wild-type mice (P < 0·05), and four out of 12 knockout mice died. Free carnitine decreased to 44% as compared with unstressed wild-type mice. An increase in C14–C18 Acylcarnitines and a decrease of free carnitine were also observed in fasted heterozygous and wild-type mice. Conclusions Long-chain Acylcarnitines in blood increase in knockout mice in response to different stressors and concentrations correlate with the clinical condition. A decrease in blood free carnitine in response to severe stress is observed in knockout mice but also in wild-type littermates. Monitoring blood Acylcarnitine profiles in response to different stressors may allow systematic analysis of therapeutic interventions in VLCAD knockout mice.
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Selective and accurate C5 Acylcarnitine quantitation by UHPLC-MS/MS: Distinguishing true isovaleric acidemia from pivalate derived interference.
Journal of chromatography. B Analytical technologies in the biomedical and life sciences, 2017Co-Authors: Paul E. Minkler, Maria S.k. Stoll, Stephen T. Ingalls, Charles L. HoppelAbstract:Tandem MS Acylcarnitine "profiles" are extremely valuable. Although used appropriately in newborn screening programs to identify patients with possible diseases, their inadequate quantitative accuracy and lack of selectivity is problematic for confirmatory testing. In this report, we show the application of our validated, selective, accurate, precise, and robust UHPLC-MS/MS method for quantitation of Acylcarnitines, specifically to C5 Acylcarnitines: pivaloyl-, 2-methylbutyryl-, isovaleryl-, and valerylcarnitine. Standardized calibrants were used to generate 13-point, 200-fold concentration range calibration curves. Samples were isolated by solid-phase extraction and derivatized with pentafluorophenacyl trifluoromethanesulfonate. Acylcarnitine pentafluorophenacyl esters were eluted in 14min chromatograms. Data demonstrating quantitative stability and method robustness over a five year time period are shown and these results validate the method's accuracy and robustness. Urine from patients with isovaleric acidemia (with the disease marker isovalerylcarnitine) and with pivaloylcarnitine present are shown. These results demonstrate the method's ability to distinguish true isovaleric acidemia from pivalate derived interference. Our method for Acylcarnitine quantitation is shown to be accurate, precise, and robust for selective quantitation of isovalerylcarnitine, and thus is recommended for confirmatory testing of suspected isovaleric acidemia patients.
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Acylcarnitines as markers of exercise associated fuel partitioning xenometabolism and potential signals to muscle afferent neurons
Experimental Physiology, 2017Co-Authors: Jie Zhang, Charles L. Hoppel, Alan R Light, Caitlin Campbell, Carol J Chandler, Dustin J Burnett, Elaine Souza, Gretchen A Casazza, Ronald W Hughen, Nancy L KeimAbstract:New Findings What is the central question of this study? Does improved metabolic health and insulin sensitivity following a weight-loss and fitness intervention in sedentary, obese women alter exercise-associated fuel metabolism and incomplete mitochondrial fatty acid oxidation (FAO), as tracked by blood Acylcarnitine patterns? What is the main finding and its importance? Despite improved fitness and blood sugar control, indices of incomplete mitochondrial FAO increased in a similar manner in response to a fixed load acute exercise bout; this indicates that intramitochondrial muscle FAO is inherently inefficient and is tethered directly to ATP turnover. With insulin resistance or type 2 diabetes mellitus, mismatches between mitochondrial fatty acid fuel delivery and oxidative phosphorylation/tricarboxylic acid cycle activity may contribute to inordinate accumulation of short- or medium-chain Acylcarnitine fatty acid derivatives [markers of incomplete long-chain fatty acid oxidation (FAO)]. We reasoned that incomplete FAO in muscle would be ameliorated concurrent with improved insulin sensitivity and fitness following a ∼14 week training and weight-loss intervention in obese, sedentary, insulin-resistant women. Contrary to this hypothesis, overnight-fasted and exercise-induced plasma C4–C14 Acylcarnitines did not differ between pre- and postintervention phases. These metabolites all increased robustly with exercise (∼45% of pre-intervention peak oxygen consumption) and decreased during a 20 min cool-down. This supports the idea that, regardless of insulin sensitivity and fitness, intramitochondrial muscle β-oxidation and attendant incomplete FAO are closely tethered to absolute ATP turnover rate. Acute exercise also led to branched-chain amino acid Acylcarnitine derivative patterns suggestive of rapid and transient diminution of branched-chain amino acid flux through the mitochondrial branched-chain ketoacid dehydrogenase complex. We confirmed our prior novel observation that a weight-loss/fitness intervention alters plasma xenometabolites [i.e. cis-3,4-methylene-heptanoylcarnitine and γ-butyrobetaine (a co-metabolite possibly derived in part from gut bacteria)], suggesting that host metabolic health regulated gut microbe metabolism. Finally, we considered whether Acylcarnitine metabolites signal to muscle-innervating afferents; palmitoylcarnitine at concentrations as low as 1–10 μm activated a subset (∼2.5–5%) of these neurons ex vivo. This supports the hypothesis that in addition to tracking exercise-associated shifts in fuel metabolism, muscle Acylcarnitines act as signals of exertion to short-loop somatosensory–motor circuits or to the brain.
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Quantitative Acylcarnitine determination by UHPLC-MS/MS - Going beyond tandem MS Acylcarnitine "profiles"
Molecular Genetics and Metabolism, 2015Co-Authors: Paul E. Minkler, Maria S.k. Stoll, Stephen T. Ingalls, Jon Kerner, Charles L. HoppelAbstract:Tandem MS "profiling" of Acylcarnitines and amino acids was conceived as a first-tier screening method, and its application to expanded newborn screening has been enormously successful. However, unlike amino acid screening (which uses amino acid analysis as its second-tier validation of screening results), Acylcarnitine "profiling" also assumed the role of second-tier validation, due to the lack of a generally accepted second-tier Acylcarnitine determination method. In this report, we present results from the application of our validated UHPLC-MS/MS second-tier method for the quantification of total carnitine, free carnitine, butyrobetaine, and Acylcarnitines to patient samples with known diagnoses: malonic acidemia, short-chain acyl-CoA dehydrogenase deficiency (SCADD) or isobutyryl-CoA dehydrogenase deficiency (IBD), 3-methyl-crotonyl carboxylase deficiency (3-MCC) or ß-ketothiolase deficiency (BKT), and methylmalonic acidemia (MMA). We demonstrate the assay's ability to separate constitutional isomers and diastereomeric Acylcarnitines and generate values with a high level of accuracy and precision. These capabilities are unavailable when using tandem MS "profiles". We also show examples of research interest, where separation of Acylcarnitine species and accurate and precise Acylcarnitine quantification is necessary.
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plasma Acylcarnitine profiles suggest incomplete long chain fatty acid β oxidation and altered tricarboxylic acid cycle activity in type 2 diabetic african american women
Journal of Nutrition, 2009Co-Authors: Sean H. Adams, Paul E. Minkler, Charles L. Hoppel, Ling Zhao, Scott W Wong, Daniel H Hwang, John W Newman, Timothy W GarveyAbstract:Inefficient muscle long-chain fatty acid (LCFA) combustion is associated with insulin resistance, but molecular links between mitochondrial fat catabolism and insulin action remain controversial. We hypothesized that plasma Acylcarnitine profiling would identify distinct metabolite patterns reflective of muscle fat catabolism when comparing individuals bearing a missense G304A uncoupling protein 3 (UCP3 g/a) polymorphism to controls, because UCP3 is predominantly expressed in skeletal muscle and g/a individuals have reduced whole-body fat oxidation. MS analyses of 42 carnitine moieties in plasma samples from fasting type 2 diabetics (n = 44) and nondiabetics (n = 12) with or without the UCP3 g/a polymorphism (n = 28/genotype: 22 diabetic, 6 nondiabetic/genotype) were conducted. Contrary to our hypothesis, genotype had a negligible impact on plasma metabolite patterns. However, a comparison of nondiabetics vs. type 2 diabetics revealed a striking increase in the concentrations of fatty Acylcarnitines reflective of incomplete LCFA β-oxidation in the latter (i.e. summed C10- to C14-carnitine concentrations were ∼300% of controls; P = 0.004). Across all volunteers (n = 56), acetylcarnitine rose and propionylcarnitine decreased with increasing hemoglobin A1c (r = 0.544, P < 0.0001; and r = −0.308, P < 0.05, respectively) and with increasing total plasma Acylcarnitine concentration. In proof-of-concept studies, we made the novel observation that C12-C14 Acylcarnitines significantly stimulated nuclear factor κ-B activity (up to 200% of controls) in RAW264.7 cells. These results are consistent with the working hypothesis that inefficient tissue LCFA β-oxidation, due in part to a relatively low tricarboxylic acid cycle capacity, increases tissue accumulation of acetyl-CoA and generates chain-shortened Acylcarnitine molecules that activate proinflammatory pathways implicated in insulin resistance.