RESUMO
While selectively quantifying acylcarnitines in thousands of patient samples using UHPLC-MS/MS, we have occasionally observed unidentified branched-chain C8 acylcarnitines. Such observations are not possible using tandem MS methods, which generate pseudo-quantitative acylcarnitine "profiles". Since these "profiles" select for mass alone, they cannot distinguish authentic signal from isobaric and isomeric interferences. For example, some of the samples containing branched-chain C8 acylcarnitines were, in fact, expanded newborn screening false positive "profiles" for medium-chain acyl-CoA dehydrogenase deficiency (MCADD). Using our fast, highly selective, and quantitatively accurate UHPLC-MS/MS acylcarnitine determination method, we corrected the false positive tandem MS results and reported the sample results as normal for octanoylcarnitine (the marker for MCADD). From instances such as these, we decided to further investigate the presence of branched-chain C8 acylcarnitines in patient samples. To accomplish this, we synthesized and chromatographically characterized several branched-chain C8 acylcarnitines (in addition to valproylcarnitine): 2-methylheptanoylcarnitine, 6-methylheptanoylcarnitine, 2,2-dimethylhexanoylcarnitine, 3,3-dimethylhexanoylcarnitine, 3,5-dimethylhexanoylcarnitine, 2-ethylhexanoylcarnitine, and 2,4,4-trimethylpentanoylcarnitine. We then compared their behavior with branched-chain C8 acylcarnitines observed in patient samples and demonstrated our ability to chromographically resolve, and thus distinguish, octanoylcarnitine from branched-chain C8 acylcarnitines, correcting false positive MCADD results from expanded newborn screening.
Assuntos
Acil-CoA Desidrogenase/deficiência , Carnitina/análogos & derivados , Carnitina/metabolismo , Erros Inatos do Metabolismo Lipídico/diagnóstico , Triagem Neonatal/normas , Carnitina/síntese química , Carnitina/isolamento & purificação , Cromatografia Líquida de Alta Pressão/métodos , Reações Falso-Positivas , Humanos , Recém-Nascido , Sensibilidade e Especificidade , Espectrometria de Massas em TandemRESUMO
A validated quantitative method for the determination of free and total carnitine, butyrobetaine, and acylcarnitines is presented. The versatile method has four components: (1) isolation using strong cation-exchange solid-phase extraction, (2) derivatization with pentafluorophenacyl trifluoromethanesulfonate, (3) sequential ion-exchange/reversed-phase (ultra) high-performance liquid chromatography [(U)HPLC] using a strong cation-exchange trap in series with a fused-core HPLC column, and (4) detection with electrospray ionization multiple reaction monitoring (MRM) mass spectrometry (MS). Standardized carnitine along with 65 synthesized, standardized acylcarnitines (including short-chain, medium-chain, long-chain, dicarboxylic, hydroxylated, and unsaturated acyl moieties) were used to construct multiple-point calibration curves, resulting in accurate and precise quantification. Separation of the 65 acylcarnitines was accomplished in a single chromatogram in as little as 14 min. Validation studies were performed showing a high level of accuracy, precision, and reproducibility. The method provides capabilities unavailable by tandem MS procedures, making it an ideal approach for confirmation of newborn screening results and for clinical and basic research projects, including treatment protocol studies, acylcarnitine biomarker studies, and metabolite studies using plasma, urine, tissue, or other sample matrixes.
Assuntos
Betaína/análogos & derivados , Carnitina/análogos & derivados , Carnitina/análise , Músculo Esquelético/química , Animais , Betaína/análise , Betaína/sangue , Betaína/urina , Carnitina/sangue , Carnitina/urina , Cromatografia Líquida de Alta Pressão , Diabetes Mellitus Experimental/diagnóstico , Humanos , Mesilatos/química , Ratos , Extração em Fase Sólida , Espectrometria de Massas por Ionização por Electrospray , Compostos de Trimetilsilil/químicaRESUMO
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.
Assuntos
Acetil-CoA C-Aciltransferase/deficiência , Acil-CoA Desidrogenase/deficiência , Erros Inatos do Metabolismo dos Aminoácidos/diagnóstico , Carbono-Carbono Ligases/deficiência , Carnitina/análogos & derivados , Erros Inatos do Metabolismo Lipídico/diagnóstico , Distúrbios Congênitos do Ciclo da Ureia/diagnóstico , Acetil-CoA C-Aciltransferase/sangue , Acetil-CoA C-Aciltransferase/líquido cefalorraquidiano , Acetil-CoA C-Aciltransferase/urina , Acil-CoA Desidrogenase/sangue , Acil-CoA Desidrogenase/líquido cefalorraquidiano , Acil-CoA Desidrogenase/urina , Erros Inatos do Metabolismo dos Aminoácidos/sangue , Erros Inatos do Metabolismo dos Aminoácidos/líquido cefalorraquidiano , Erros Inatos do Metabolismo dos Aminoácidos/urina , Betaína/análogos & derivados , Betaína/sangue , Betaína/líquido cefalorraquidiano , Betaína/urina , Carbono-Carbono Ligases/sangue , Carbono-Carbono Ligases/líquido cefalorraquidiano , Carbono-Carbono Ligases/urina , Carnitina/sangue , Carnitina/líquido cefalorraquidiano , Carnitina/urina , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida de Alta Pressão/normas , Feminino , Humanos , Recém-Nascido , Isomerismo , Erros Inatos do Metabolismo Lipídico/sangue , Erros Inatos do Metabolismo Lipídico/líquido cefalorraquidiano , Erros Inatos do Metabolismo Lipídico/urina , Masculino , Triagem Neonatal , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Massas em Tandem/normas , Distúrbios Congênitos do Ciclo da Ureia/sangue , Distúrbios Congênitos do Ciclo da Ureia/líquido cefalorraquidiano , Distúrbios Congênitos do Ciclo da Ureia/urinaRESUMO
Mitochondrial fission is a critical cellular event to maintain organelle function. This multistep process is initiated by the enhanced recruitment and oligomerization of dynamin-related protein 1 (Drp1) at the surface of mitochondria. As such, Drp1 is essential for inducing mitochondrial division in mammalian cells, and homologous proteins are found in all eukaryotes. As a member of the dynamin superfamily of proteins (DSPs), controlled Drp1 self-assembly into large helical polymers stimulates its GTPase activity to promote membrane constriction. Still, little is known about the mechanisms that regulate correct spatial and temporal assembly of the fission machinery. Here we present a cryo-EM structure of a full-length Drp1 dimer in an auto-inhibited state. This dimer reveals two key conformational rearrangements that must be unlocked through intramolecular rearrangements to achieve the assembly-competent state observed in previous structures. This structural insight provides understanding into the mechanism for regulated self-assembly of the mitochondrial fission machinery.
Assuntos
GTP Fosfo-Hidrolases , Dinâmica Mitocondrial , Animais , GTP Fosfo-Hidrolases/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Dinaminas/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Mamíferos/metabolismoRESUMO
Mitochondrial enoyl-CoA isomerase (ECI1) is an auxiliary enzyme involved in unsaturated fatty acid oxidation. In contrast to most of the other enzymes involved in fatty acid oxidation, a deficiency of ECI1 has yet to be identified in humans. We used wild-type (WT) and Eci1-deficient knockout (KO) mice to explore a potential presentation of human ECI1 deficiency. Upon food withdrawal, Eci1-deficient mice displayed normal blood ß-hydroxybutyrate levels (WT 1.09 mM vs. KO 1.10 mM), a trend to lower blood glucose levels (WT 4.58 mM vs. KO 3.87 mM, P=0.09) and elevated blood levels of unsaturated acylcarnitines, in particular C12:1 acylcarnitine (WT 0.03 µM vs. KO 0.09 µM, P<0.01). Feeding an olive oil-rich diet induced an even greater increase in C12:1 acylcarnitine levels (WT 0.01 µM vs. KO 0.04 µM, P<0.01). Overall, the phenotypic presentation of Eci1-deficient mice is mild, possibly caused by the presence of a second enoyl-CoA isomerase (Eci2) in mitochondria. Knockdown of Eci2 in Eci1-deficient fibroblasts caused a more pronounced accumulation of C12:1 acylcarnitine on incubation with unsaturated fatty acids (12-fold, P<0.05). We conclude that Eci2 compensates for Eci1 deficiency explaining the mild phenotype of Eci1-deficient mice. Hypoglycemia and accumulation of C12:1 acylcarnitine might be diagnostic markers to identify ECI1 deficiency in humans.
Assuntos
Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Ácidos Graxos Insaturados/metabolismo , Mitocôndrias/enzimologia , Animais , Glicemia/metabolismo , Isomerases de Ligação Dupla Carbono-Carbono/genética , Carnitina/análogos & derivados , Carnitina/sangue , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Dodecenoil-CoA Isomerase , Immunoblotting , Espectrometria de Massas , Camundongos , Camundongos Knockout , Oxirredução , Reação em Cadeia da Polimerase em Tempo RealRESUMO
O-GlcNAcylation is a prevalent form of glycosylation that regulates proteins within the cytosol, nucleus, and mitochondria. The O-GlcNAc modification can affect protein cellular localization, function, and signaling interactions. The specific impact of O-GlcNAcylation on mitochondrial morphology and function has been elusive. In this manuscript, the role of O-GlcNAcylation on mitochondrial fission, oxidative phosphorylation (Oxphos), and the activity of electron transport chain (ETC) complexes were evaluated. In a cellular environment with hyper O-GlcNAcylation due to the deletion of O-GlcNAcase (OGA), mitochondria showed a dramatic reduction in size and a corresponding increase in number and total mitochondrial mass. Because of the increased mitochondrial content, OGA knockout cells exhibited comparable coupled mitochondrial Oxphos and ATP levels when compared to WT cells. However, we observed reduced protein levels for complex I and II when comparing normalized mitochondrial content and reduced linked activity for complexes I and III when examining individual ETC complex activities. In assessing mitochondrial fission, we observed increased amounts of O-GlcNAcylated dynamin-related protein 1 (Drp1) in cells genetically null for OGA and in glioblastoma cells. Individual regions of Drp1 were evaluated for O-GlcNAc modifications, and we found that this post-translational modification (PTM) was not limited to the previously characterized residues in the variable domain (VD). Additional modification sites are predicted in the GTPase domain, which may influence enzyme activity. Collectively, these results highlight the impact of O-GlcNAcylation on mitochondrial dynamics and ETC function and mimic the changes that may occur during glucose toxicity from hyperglycemia.
Assuntos
Acilação/genética , Acilação/fisiologia , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/fisiologia , N-Acetilglucosaminiltransferases/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Dinaminas/genética , Dinaminas/metabolismo , Glucose/genética , Glucose/metabolismo , Glicosilação , Células HCT116 , Humanos , Camundongos , Camundongos Knockout , Mitocôndrias Cardíacas/genética , Dinâmica Mitocondrial/genética , Dinâmica Mitocondrial/fisiologia , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , N-Acetilglucosaminiltransferases/genética , Fosforilação Oxidativa , Processamento de Proteína Pós-Traducional/genética , Transdução de Sinais/genéticaRESUMO
Several mouse models for mitochondrial fatty acid beta-oxidation (FAO) defects have been developed. So far, these models have contributed little to our current understanding of the pathophysiology. The objective of this study was to explore differences between murine and human FAO. Using a combination of analytical, biochemical and molecular methods, we compared fibroblasts of long chain acyl-CoA dehydrogenase knockout (LCAD(-/-)), very long chain acyl-CoA dehydrogenase knockout (VLCAD(-/-)) and wild type mice with fibroblasts of VLCAD-deficient patients and human controls. We show that in mice, LCAD and VLCAD have overlapping and distinct roles in FAO. The absence of VLCAD is apparently fully compensated, whereas LCAD deficiency is not. LCAD plays an essential role in the oxidation of unsaturated fatty acids such as oleic acid, but seems redundant in the oxidation of saturated fatty acids. In strong contrast, LCAD is neither detectable at the mRNA level nor at the protein level in men, making VLCAD indispensable in FAO. Our findings open new avenues to employ the existing mouse models to study the pathophysiology of human FAO defects.
Assuntos
Ácidos Graxos/metabolismo , Mitocôndrias/metabolismo , Acil-CoA Desidrogenase de Cadeia Longa/deficiência , Animais , Carnitina/análogos & derivados , Carnitina/química , Carnitina/metabolismo , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Fibroblastos/enzimologia , Humanos , Camundongos , Oxirredução , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Analysis of carnitine and acylcarnitines by tandem mass spectrometry (MS/MS) has limitations. First, preparation of butyl esters partially hydrolyzes acylcarnitines. Second, isobaric nonacylcarnitine compounds yield false-positive results in acylcarnitine tests. Third, acylcarnitine constitutional isomers cannot be distinguished. METHODS: Carnitine and acylcarnitines were isolated by ion-exchange solid-phase extraction, derivatized with pentafluorophenacyl trifluoromethanesulfonate, separated by HPLC, and detected with an ion trap mass spectrometer. Carnitine was quantified with d(3)-carnitine as the internal standard. Acylcarnitines were quantified with 42 synthesized calibrators. The internal standards used were d(6)-acetyl-, d(3)-propionyl-, undecanoyl-, undecanedioyl-, and heptadecanoylcarnitine. RESULTS: Example recoveries [mean (SD)] were 69.4% (3.9%) for total carnitine, 83.1% (5.9%) for free carnitine, 102.2% (9.8%) for acetylcarnitine, and 107.2% (8.9%) for palmitoylcarnitine. Example imprecision results [mean (SD)] within runs (n = 6) and between runs (n = 18) were, respectively: total carnitine, 58.0 (0.9) and 57.4 (1.7) micromol/L; free carnitine, 44.6 (1.5) and 44.3 (1.2) micromol/L; acetylcarnitine, 7.74 (0.51) and 7.85 (0.69) micromol/L; and palmitoylcarnitine, 0.12 (0.01) and 0.11 (0.02) micromol/L. Standard-addition slopes and linear regression coefficients were 1.00 and 0.9998, respectively, for total carnitine added to plasma, 0.99 and 0.9997 for free carnitine added to plasma, 1.04 and 0.9972 for octanoylcarnitine added to skeletal muscle, and 1.05 and 0.9913 for palmitoylcarnitine added to skeletal muscle. Reference intervals for plasma, urine, and skeletal muscle are provided. CONCLUSIONS: This method for analysis of carnitine and acylcarnitines overcomes the observed limitations of MS/MS methods.
Assuntos
Carnitina/análise , Carnitina/química , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Acetilação , Calibragem , Carnitina/metabolismo , Humanos , Estrutura MolecularRESUMO
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.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/urina , Carnitina/análogos & derivados , Cromatografia Líquida de Alta Pressão/métodos , Isovaleril-CoA Desidrogenase/deficiência , Espectrometria de Massas em Tandem/métodos , Carnitina/urina , Humanos , Isovaleril-CoA Desidrogenase/urina , Limite de Detecção , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Although correctly used in expanded newborn screening programs to identify patients with possible diseases, flow-injection tandem mass spectrometry (MS/MS) acylcarnitine "profiles" are inadequate for standard clinical uses owing to their limited quantitative accuracy and lack of selectivity. We report the application of our selective, accurate, and precise method for quantification of acylcarnitines, applied to urine glutarylcarnitine from a patient with glutaric acidemia type I (GAI). METHODS: A previously validated acylcarnitine ultra-HPLC-MS/MS method was used, with a focus on analysis of glutarylcarnitine. Calibrants and samples were isolated by solid-phase extraction and derivatized with pentafluorophenacyl trifluoromethanesulfonate. Acylcarnitine pentafluorophenacyl esters were eluted in 14-min chromatograms. Standardized calibrants and a 13-point, 200-fold concentration range calibration curve were used for accurate quantification of glutarylcarnitine. Quality control samples validated method accuracy and long-term analytic stability. RESULTS: Quantification of glutarylcarnitine in urine from a patient with GAI is reported. Long-term analytical stability of the method over a 5-year period is shown. CONCLUSIONS: Our method for acylcarnitine quantification is shown to be selective, accurate, and precise; thus, we recommend it for confirmatory testing and monitoring of plasma and urine samples from patients with GAI.
RESUMO
Aging selectively decreases the rate of oxidative phosphorylation in the interfibrillar population of cardiac mitochondria (IFM) located between the myofibers. In contrast, subsarcolemmal mitochondria (SSM), located below the plasma membrane, remain unaffected. IFM from elderly (24-month-old) Fischer 344 rats have a decreased specific activity of complexes III and IV. Complexes III and IV require an inner mitochondrial membrane lipid environment enriched in the oxidatively sensitive phospholipid cardiolipin for maximal activity. We asked if aging decreases the content or alters the composition of cardiolipin as a potential mechanism of the aging defect in IFM. The content and composition of mitochondrial phospholipids were measured in SSM and IFM from adult and aging rat hearts. Aging did not alter the content of mitochondrial phospholipids, including cardiolipin, in either population of mitochondria. The composition of cardiolipin based on characterization of both acyl group and the individual molecular species of cardiolipin was also unaltered by aging. Lipid-mediated oxidative modification of complex III subunits was not detected, making cardiolipin-derived oxidative damage to complex III unlikely. Thus, alterations in cardiolipin are not the mechanism for the aging defect in IFM in Fischer 344 rats.
Assuntos
Envelhecimento/metabolismo , Cardiolipinas/metabolismo , Mitocôndrias Cardíacas/metabolismo , Miofibrilas/metabolismo , Animais , Cardiolipinas/química , Cromatografia Líquida de Alta Pressão , Metabolismo dos Lipídeos , Masculino , Oxirredução , Fosfolipídeos/metabolismo , Ratos , Ratos Endogâmicos F344 , Sarcolema/metabolismo , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Ischemia and reperfusion result in mitochondrial dysfunction, with decreases in oxidative capacity, loss of cytochrome c, and generation of reactive oxygen species. During ischemia of the isolated perfused rabbit heart, subsarcolemmal mitochondria, located beneath the plasma membrane, sustain a loss of the phospholipid cardiolipin, with decreases in oxidative metabolism through cytochrome oxidase and the loss of cytochrome c. We asked whether additional injury to the distal electron chain involving cardiolipin with loss of cytochrome c and cytochrome oxidase occurs during reperfusion. Reperfusion did not lead to additional damage in the distal electron transport chain. Oxidation through cytochrome oxidase and the content of cytochrome c did not further decrease during reperfusion. Thus injury to cardiolipin, cytochrome c, and cytochrome oxidase occurs during ischemia rather than during reperfusion. The ischemic injury leads to persistent defects in oxidative function during the early reperfusion period. The decrease in cardiolipin content accompanied by persistent decrements in the content of cytochrome c and oxidation through cytochrome oxidase is a potential mechanism of additional myocyte injury during reperfusion.