Targeted Metabolomics Revealed a Sex-Dependent Signature for Metabolic Syndrome in the Mexican Population.

Metabolic syndrome (MetS) is a group of several metabolic conditions predisposing to chronic diseases. Individuals diagnosed with MetS are physiologically heterogeneous, with significant sex-specific differences. Therefore, we aimed to investigate the potential sex-specific serum modifications of amino acids and acylcarnitines (ACs) and their relationship with MetS in the Mexican population. This study included 602 participants from the Health Workers Cohort Study. Forty serum metabolites were analyzed using a targeted metabolomics approach. Multivariate regression models were used to test associations of clinical and biochemical parameters with metabolomic profiles. Our findings showed a serum amino acid signature (citrulline and glycine) and medium-chain ACs (AC14:1, AC10, and AC18:10H) associated with MetS. Glycine and AC10 were specific metabolites representative of discrimination according to sex-dependent MetS. In addition, we found that glycine and short-chain ACs (AC2, AC3, and AC8:1) are associated with age-dependent MetS. We also reported a significant correlation between body fat and metabolites associated with sex-age-dependent MetS. In conclusion, the metabolic profile varies by MetS status, and these differences are sex-age-dependent in the Mexican population.

Serum Metabolite Profile Associated with Sex-Dependent Visceral Adiposity Index and Low Bone Mineral Density in a Mexican Population.

Recent evidence shows that obesity correlates negatively with bone mass. However, traditional anthropometric measures such as body mass index could not discriminate visceral adipose tissue from subcutaneous adipose tissue. The visceral adiposity index (VAI) is a reliable sex-specified indicator of visceral adipose distribution and function. Thus, we aimed to identify metabolomic profiles associated with VAI and low bone mineral density (BMD). A total of 602 individuals from the Health Workers Cohort Study were included. Forty serum metabolites were measured using the targeted metabolomics approach, and multivariate regression models were used to test associations of metabolomic profiles with anthropometric, clinical, and biochemical parameters. The analysis showed a serum amino acid signature composed of glycine, leucine, arginine, valine, and acylcarnitines associated with high VAI and low BMD. In addition, we found a sex-dependent VAI in pathways related to primary bile acid biosynthesis, branched-chain amino acids, and the biosynthesis of pantothenate and coenzyme A (CoA). In conclusion, a metabolic profile differs by VAI and BMD status, and these changes are gender-dependent.

Amino Acids and Acylcarnitines Reference Values for Neonatal Screening of Inborn Errors of Metabolism in Colombia by Tandem Mass Spectrometry

Abstract Neonatal screening in Colombia has been carried out since 2000. The problem that most concerns is the absence of expanded screening. To stablish reference values for amino acids and acylcarnitines, in order to provide information to guide the implementation of expanded screening. Samples collected on Whatman 903 filter paper from 10284 newborns were processed by Tandem Mass Spectrometry System (Waters - Perkin Elmer), and the NeoBase™ non-derivatized MS/MS kit (PerkinElmer), which contains controls for 11 amino acids, and 31 acylcarnitine species. For each analyte the upper limit was set above the 99th percentile, while the lower limit was set below the 1st percentile. Comparison of full-term newborn amino acid concentrations with premature ones showed no significant differences in three of them: Glycine p-0.99574, Ornithine p=0.35274, Phenylalanine p=0.13499, neither in levels of 11 of the 31 acylcarnitines. Comparison of analyte concentrations in this study with previous reports showed significant differences for all amino acids and acylcarnitines (<0.05). Experience was gained in the pre-analytic stage of expanded screening and reference values were established, for the implementation of neonatal screening program in the country.

The value of fasting in the diagnosis of medium-chain acyl-CoA dehydrogenase deficiency / O valor do jejum no diagnóstico da deficiência de acil-CoA desidrogenase de cadeia média

ABSTRACT Female patient carrier of medium-chain acyl-CoA dehydrogenase deficiency (MCADD) with recurrent clinical episodes of hypoglycemia and altered level of consciousness, presented changes in blood acylcarnitine profile by tandem mass spectrometry and in the urinary organic acid analysis by gas chromatography/mass spectrometry (GC/MS). This case demonstrates the importance of fasting prior biological sample collection (when possible) when MCADD is suspected, and emphasizes that the time/momentum of biological sample collection is crucial to diagnosis, considering the possibility that MCADD is underdiagnosed in Brazil.

RESUMEN Paciente portadora de deficiencia de acil-CoA deshidrogenasa de cadena media (MCADD) con episodios clínicos recurrentes de hipoglucemia y alteración de consciencia presentó mudanzas en el perfil de acilcarnitinas en la sangre con técnicas de espectrometría de masas en tándem y en el análisis de ácidos orgánicos urinarios mediante cromatografía de gases acoplada a espectrometría de masas. Este caso demuestra la importancia de la toma de muestras biológicas en ayunas (se posible) cuando se sospecha de MCADD y destaca que el tiempo/momento de extracción de la muestra biológica es valioso para el diagnóstico, considerando la posibilidad de que la MCADD es subdiagnosticada en Brasil.

RESUMO Paciente portadora de deficiência de acil-CoA desidrogenase de cadeia média (MCADD), com episódios clínicos recorrentes de hipoglicemia e alteração de consciência, apresentou alterações no perfil de acilcarnitinas em sangue por espectrometria de massas em tandem e na análise de ácidos orgânicos urinários por cromatografia gasosa acoplada à espectrometria de massa. Este caso demonstra a importância da coleta de amostra biológica em jejum (se possível) quando há suspeita de MCADD e ressalta que o tempo/momento de coleta da amostra biológica é importante para o diagnóstico, considerando a possibilidade de a MCADD ser subdiagnosticada no Brasil.

Optimization of kidney dysfunction prediction in diabetic kidney disease using targeted metabolomics.

AIMS: Metabolomics have been used to evaluate the role of small molecules in human disease. However, the cost and complexity of the methodology and interpretation of findings have limited the transference of knowledge to clinical practice. Here, we apply a targeted metabolomics approach using samples blotted in filter paper to develop clinical-metabolomics models to detect kidney dysfunction in diabetic kidney disease (DKD). METHODS: We included healthy controls and subjects with type 2 diabetes (T2D) with and without DKD and investigated the association between metabolite concentrations in blood and urine with eGFR and albuminuria. We also evaluated performance of clinical, biochemical and metabolomic models to improve kidney dysfunction prediction in DKD. RESULTS: Using clinical-metabolomics models, we identified associations of decreased eGFR with body mass index (BMI), uric acid and C10:2 levels; albuminuria was associated to years of T2D duration, A1C, uric acid, creatinine, protein intake and serum C0, C10:2 and urinary C12:1 levels. DKD was associated with age, A1C, uric acid, BMI, serum C0, C10:2, C8:1 and urinary C12:1. Inclusion of metabolomics increased the predictive and informative capacity of models composed of clinical variables by decreasing Akaike's information criterion, and was replicated both in training and validation datasets. CONCLUSIONS: Targeted metabolomics using blotted samples in filter paper is a simple, low-cost approach to identify outcomes associated with DKD; the inclusion of metabolomics improves predictive capacity of clinical models to identify kidney dysfunction and DKD-related outcomes.

Orange juice affects acylcarnitine metabolism in healthy volunteers as revealed by a mass-spectrometry based metabolomics approach.

Citrus juices, especially orange juice, constitute rich sources of bioactive compounds with a wide range of health-promoting activities. Data from epidemiological and in vitro studies suggest that orange juice (OJ) may have a positive impact on lipid metabolism. However, the effect of orange juice intake on blood lipid profile is still poorly understood. We have used two different blood samples, Dried Blood Spots (DBS) and plasma, to assess the effect of two-week orange juice consumption in healthy volunteers by a mass-spectrometry based metabolomics approach. DBS were analysed by liquid chromatography mass spectrometry (LC-MS) and plasma samples were analysed by the gas chromatography mass spectrometry (GC-MS). One hundred sixty-nine lipids including acylcarnitines (AC), lysophosphatidylcholines (LysoPC), (diacyl- and acyl-alkyl-) phosphatidylcholines (PC aa and PC ae) and sphingomyelins (SM) were identified and quantified in DBS. Eighteen fatty acids were identified and quantified in plasma. Multivariate analysis allowed to identify an increase in C3:1, C5-DC(C6-OH), C5-M-DC, C5:1-DC, C8, C12-DC, lysoPC18:3, myristic acid, pentadecanoic acid, palmitoleic and palmitic acid and a decrease in nervonic acid, C0, C2, C10, C10:1, C16:1, C16-OH, C16:1-OH, C18-OH, PC aa C40:4, PC ae C38:4, PC ae C42:3, PC ae C42:4 and cholesterol levels after orange juice intake. A two-week period of orange juice intake could affect fatty acids ß-oxidation through mitochondrial and peroxisomal pathways, leading to an increase of short-chain acylcarnitines and a decrease of medium and long-chain acylcarnitines. This is the first report analyzing the effect of orange juice intake in healthy volunteers using a dried blood spot-based metabolomics approach.

Reference values of amino acids, acylcarnitines and succinylacetone by tandem mass spectrometry for use in newborn screening in southwest Colombia.

INTRODUCTION: Inborn errors of metabolism (IEM) represent an important public health problem due to current diagnosis and treatment limitations, poor life quality of affected patients, and consequent untimely child death. In contrast to classical methods, tandem mass spectrometry (MS/MS) has allowed simultaneous evaluation of multiple metabolites associated with IEM offering higher sensitivity, low false positive rates and high throughput. AIMS: Determine concentration levels for amino acids and acylcarnitines in blood of newborns from Colombia, to establish reference values for further use in diagnosis of IEM. METHODS: Implementation of a method to determine amino acids, acylcarnitines and succinylacetone in newborn dried blood spots using MS/MS, and its application in a cross-sectional study conducted in 891 healthy neonates from Cali and Quibdo cities is described. RESULTS: fifty-seven analytes that allow the diagnosis of more than 40 different pathologies were tested. The method showed to be linear, precise and accurate. Healthy neonates 1-18 days of age were included, 523 from Cali and 368 from Quibdo; 52% male and 48% female. Age-related differences on the concentration levels of amino acids and acylcarnitines were observed whereas no significant differences by gender were found. CONCLUSION: The study has contributed to reveal the usual concentration levels of amino acids, acylcarnitines and succinylacetone that could be used as reference for the establishment of a newborn metabolic screening program in Colombia.

INTRODUCCIÓN: Los Errores Innatos del metabolismo (EIM) representan un importante problema de salud pública debido a limitaciones en el tratamiento y diagnóstico oportuno, la pobre calidad de vida de los pacientes afectados, así como la muerte infantil prematura. Comparada con los métodos clásicos, la espectrometría de masas en tándem (MS/MS) ha permitido la evaluación simultánea de múltiples metabolitos asociados con EIM, con una alta sensibilidad, baja proporción de falsos positivos y alto rendimiento. OBJETIVOS: Determinar los niveles de concentración de aminoácidos y acilcarnitinas en sangre de recién nacidos de Colombia, para establecer los valores normales para usarlos como referencia en el diagnóstico de EIM. MÉTODOS: Aquí, se describe la implementación de un método para determinar aminoácidos, acilcarnitinas y succinilacetona en gotas de sangre seca de recién nacidos usando MS/MS, y su aplicación en un estudio de corte transversal realizado en 891 neonatos sanos de las ciudades de Cali y Quibdó. RESULTADOS: Se evaluaron 57 analitos que permiten el diagnóstico de más de 40 patologías diferentes. El método mostró ser lineal, preciso y exacto. Se incluyeron neonatos sanos de 1-18 días de edad, 523 de Cali y 368 de Quibdó, 52% hombres y 48% mujeres. Se observaron diferencias en los niveles de concentración de aminoácidos y acilcarnitinas relacionadas con la edad, mientras que no se encontraron diferencias significativas por sexo. CONCLUSIÓN: El estudio ha contribuido a revelar los niveles usuales de concentración de aminoácidos, acilcarnitinas y succinilacetona que pueden ser usados como referencia para el establecimiento del programa de tamizaje neonatal metabólico en Colombia.

Reference values of amino acids, acylcarnitines and succinylacetone by tandem mass spectrometry for use in newborn screening in southwest Colombia / Valores de referencia de aminoácidos, acilcarnitinas y succinilacetona por espectrometría de masas en tándem para su uso en el tamizaje neonatal en el suroccidente de Colombia

Abstract Introduction: Inborn errors of metabolism (IEM) represent an important public health problem due to current diagnosis and treatment limitations, poor life quality of affected patients, and consequent untimely child death. In contrast to classical methods, tandem mass spectrometry (MS/MS) has allowed simultaneous evaluation of multiple metabolites associated with IEM offering higher sensitivity, low false positive rates and high throughput. Aims: Determine concentration levels for amino acids and acylcarnitines in blood of newborns from Colombia, to establish reference values for further use in diagnosis of IEM. Methods: Implementation of a method to determine amino acids, acylcarnitines and succinylacetone in newborn dried blood spots using MS/MS, and its application in a cross-sectional study conducted in 891 healthy neonates from Cali and Quibdo cities is described. Results: fifty-seven analytes that allow the diagnosis of more than 40 different pathologies were tested. The method showed to be linear, precise and accurate. Healthy neonates 1-18 days of age were included, 523 from Cali and 368 from Quibdo; 52% male and 48% female. Age-related differences on the concentration levels of amino acids and acylcarnitines were observed whereas no significant differences by gender were found. Conclusion: The study has contributed to reveal the usual concentration levels of amino acids, acylcarnitines and succinylacetone that could be used as reference for the establishment of a newborn metabolic screening program in Colombia.

Resumen Introducción: Los Errores Innatos del metabolismo (EIM) representan un importante problema de salud pública debido a limitaciones en el tratamiento y diagnóstico oportuno, la pobre calidad de vida de los pacientes afectados, así como la muerte infantil prematura. Comparada con los métodos clásicos, la espectrometría de masas en tándem (MS/MS) ha permitido la evaluación simultánea de múltiples metabolitos asociados con EIM, con una alta sensibilidad, baja proporción de falsos positivos y alto rendimiento. Objetivos: Determinar los niveles de concentración de aminoácidos y acilcarnitinas en sangre de recién nacidos de Colombia, para establecer los valores normales para usarlos como referencia en el diagnóstico de EIM. Métodos: Aquí, se describe la implementación de un método para determinar aminoácidos, acilcarnitinas y succinilacetona en gotas de sangre seca de recién nacidos usando MS/MS, y su aplicación en un estudio de corte transversal realizado en 891 neonatos sanos de las ciudades de Cali y Quibdó. Resultados: Se evaluaron 57 analitos que permiten el diagnóstico de más de 40 patologías diferentes. El método mostró ser lineal, preciso y exacto. Se incluyeron neonatos sanos de 1-18 días de edad, 523 de Cali y 368 de Quibdó, 52% hombres y 48% mujeres. Se observaron diferencias en los niveles de concentración de aminoácidos y acilcarnitinas relacionadas con la edad, mientras que no se encontraron diferencias significativas por sexo. Conclusión: El estudio ha contribuido a revelar los niveles usuales de concentración de aminoácidos, acilcarnitinas y succinilacetona que pueden ser usados como referencia para el establecimiento del programa de tamizaje neonatal metabólico en Colombia.

Skeletal Muscle Acute and Chronic Metabolic Response to Essential Amino Acid Supplementation in Hypertriglyceridemic Older Adults.

Background: Supplementation with essential amino acids (EAAs) + arginine is a promising nutritional approach to decrease plasma triglyceride (TG) concentrations, which are an independent risk factor for ischemic heart disease. Objective: The objective of this study was to examine the effects of 8 wk of EAA supplementation on skeletal muscle basal metabolite concentrations and changes in metabolic response to acute EAA intake, with an emphasis on mitochondrial metabolism, in adults with elevated TGs to better understand the mechanisms of lowering plasma TGs. Methods: Older adults with elevated plasma TG concentrations were given 22 g EAAs to ingest acutely before and after an 8-wk EAA supplementation period. Skeletal muscle biopsy samples were collected before and after acute EAA intake, both pre- and postsupplementation (4 biopsy samples), and targeted metabolomic analyses of organic acids and acylcarnitines were conducted on the specimens. Results: Acute EAA intake resulted in increased skeletal muscle acylcarnitine concentrations associated with oxidative catabolism of the supplement components, with the largest increases found in acylcarnitines of branched-chain amino acid oxidative catabolism, including isovaleryl-carnitine (2200%) and 2-methylbutyryl-carnitine (2400%). The chronic EAA supplementation resulted in a 19% decrease in plasma TGs along with accumulation of long-chain acylcarnitines myristoyl- (90%) and stearoyl- (120%) carnitine in skeletal muscle and increases in succinyl-carnitine (250%) and the late-stage tricarboxylic acid cycle intermediates fumarate (44%) and malate (110%). Conclusions: Supplementation with EAAs shows promise as an approach for moderate reduction in plasma TGs. Changes in skeletal muscle metabolites suggest incomplete fatty acid oxidation and increased anaplerosis, which suggests a potential bottleneck in fatty acid metabolism.

cis-4-Decenoic and decanoic acids impair mitochondrial energy, redox and Ca(2+) homeostasis and induce mitochondrial permeability transition pore opening in rat brain and liver: Possible implications for the pathogenesis of MCAD deficiency.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is biochemically characterized by tissue accumulation of octanoic (OA), decanoic (DA) and cis-4-decenoic (cDA) acids, as well as by their carnitine by-products. Untreated patients present episodic encephalopathic crises and biochemical liver alterations, whose pathophysiology is poorly known. We investigated the effects of OA, DA, cDA, octanoylcarnitine (OC) and decanoylcarnitine (DC) on critical mitochondrial functions in rat brain and liver. DA and cDA increased resting respiration and diminished ADP- and CCCP-stimulated respiration and complexes II-III and IV activities in both tissues. The data indicate that these compounds behave as uncouplers and metabolic inhibitors of oxidative phosphorylation. Noteworthy, metabolic inhibition was more evident in brain as compared to liver. DA and cDA also markedly decreased mitochondrial membrane potential, NAD(P)H content and Ca(2+) retention capacity in Ca(2+)-loaded brain and liver mitochondria. The reduction of Ca(2+) retention capacity was more pronounced in liver and totally prevented by cyclosporine A and ADP, as well as by ruthenium red, demonstrating the involvement of mitochondrial permeability transition (mPT) and Ca(2+). Furthermore, cDA induced lipid peroxidation in brain and liver mitochondria and increased hydrogen peroxide formation in brain, suggesting the participation of oxidative damage in cDA-induced alterations. Interestingly, OA, OC and DC did not alter the evaluated parameters, implying lower toxicity for these compounds. Our results suggest that DA and cDA, in contrast to OA and medium-chain acylcarnitines, disturb important mitochondrial functions in brain and liver by multiple mechanisms that are possibly involved in the neuropathology and liver alterations observed in MCAD deficiency.

Aplicación de la espectrometría de masas en tandem en el tamiz neonatal de los errores innatos del metabolismo / Use of tandem mass spectrometry for the newborn screening of inborn errors of metabolism / AplicaþÒo da espectrometria de massas em tandem no screening tamiz neonatal dos erros inatos do metabolismo

La aplicación de la espectrometría de masas en tandem en el diagnóstico de los errores innatos del metabolismo ofrece la posibilidad de ampliar el número de enfermedades que son tamizadas durante el periodo neonatal. Esta tecnología permite detectar, con gran sensibilidad, especificidad y rapidez, más de 30 enfermedades metabólicas en un mismo ensayo a partir de un único disco de sangre seca sobre papel de filtro. Para esto se realiza el análisis combinado del perfil de aminoácidos y acilcarnitinas sin requerir, generalmente, de sistemas de cromatografía adicionales. El procesamiento analítico que actualmente se utiliza en los diferentes laboratorios de tamiz es relativamente homogéneo.(AU)

The use of tandem mass spectrometry in the diagnosis of inborn errors of metabolism has the potential to expand newborn screening programmes to include many different diseases. This technique can quickly detect, with great specificity and sensibility, more than 30 diseases using the same punch of dried blood in filter paper through the combined analysis of acylcarnitines and amino acid mass spectra profile. The detection of a particular disease this way could be made without the need of a chromatographic system. The analytical analyses in current use by different screening laboratories are very similar.(AU)

A aplicaþÒo da espectrometria de massas em tandem no diagnóstico dos erros inatos do metabolismo oferece a possibilidade de ampliar os programas de screening neonatal para incluir maior número de doenþas. Esta tecnologia permite detectar, com grande sensibilidade, especificidade e rapidez, mais de 30 doenþas metabólicas em um mesmo ensaio a partir de um único disco de sangue seco sobre papel de filtro. Para isso é realizada a análise combinada do perfil de aminoácidos e acilcarnitinas sem precisar, geralmente, de sistemas de cromatografia adicionais. O processamento analítico que atualmente Þ utilizado nos diferentes laboratórios de screening é relativamente homogÛneo.(AU)

Aplicación de la espectrometría de masas en tandem en el tamiz neonatal de los errores innatos del metabolismo / Use of tandem mass spectrometry for the newborn screening of inborn errors of metabolism / Aplicação da espectrometria de massas em tandem no screening tamiz neonatal dos erros inatos do metabolismo

La aplicación de la espectrometría de masas en tandem en el diagnóstico de los errores innatos del metabolismo ofrece la posibilidad de ampliar el número de enfermedades que son tamizadas durante el periodo neonatal. Esta tecnología permite detectar, con gran sensibilidad, especificidad y rapidez, más de 30 enfermedades metabólicas en un mismo ensayo a partir de un único disco de sangre seca sobre papel de filtro. Para esto se realiza el análisis combinado del perfil de aminoácidos y acilcarnitinas sin requerir, generalmente, de sistemas de cromatografía adicionales. El procesamiento analítico que actualmente se utiliza en los diferentes laboratorios de tamiz es relativamente homogéneo.

The use of tandem mass spectrometry in the diagnosis of inborn errors of metabolism has the potential to expand newborn screening programmes to include many different diseases. This technique can quickly detect, with great specificity and sensibility, more than 30 diseases using the same punch of dried blood in filter paper through the combined analysis of acylcarnitines and amino acid mass spectra profile. The detection of a particular disease this way could be made without the need of a chromatographic system. The analytical analyses in current use by different screening laboratories are very similar.

A aplicação da espectrometria de massas em tandem no diagnóstico dos erros inatos do metabolismo oferece a possibilidade de ampliar os programas de screening neonatal para incluir maior número de doenças. Esta tecnologia permite detectar, com grande sensibilidade, especificidade e rapidez, mais de 30 doenças metabólicas em um mesmo ensaio a partir de um único disco de sangue seco sobre papel de filtro. Para isso é realizada a análise combinada do perfil de aminoácidos e acilcarnitinas sem precisar, geralmente, de sistemas de cromatografia adicionais. O processamento analítico que atualmente è utilizado nos diferentes laboratórios de screening é relativamente homogêneo.

Effect of anticaagulant type during the blood smple collection process on the determination of the acylcarnitine profile in blood and plasma / Efcto del tipo ddeanticoagulante utilizado en la toma de muestrass, sobre la determinación del perfil de acilcarnitinas en sangre y plamma

Background: the collection process for blood sampling is a very important factor that can influence the final results of some laboratory tests. The acylcarnitine determinations in blood are usually used for diagnosis and follow up of patients suffering certain inherited metabolic illnesses. Objective: this study analyzed the possible influence of the type of anticoagulant used in blood sampling on the acylcarnitine profile. Methodology: blood and plasma samples were collected using anticoagulants such as: lithium heparin, potassium EDTA, sodium fluoride-potassium oxalate, and sodium citrate. Samples were also collected without the use of anticoagulants. Acylcarnitine levels were assessed for each sample via tandem mass spectrometry. Results: no significant differences were found among the anticoagulant used. Conclusion: the type of anticoagulant has no effect on the acylcarnitine determination on blood or plasma. For this type of analysis any of the above mentioned anticoagulants can be used. Abbreviations: ESI-MS/MS, electrospray injection tandem mass spectrometry; IEM, innate errors of metabolism.

Antecedentes: la toma de muestras de sangre es un factor importante que puede influir los resultados finales de algunas pruebas de laboratorio. Las determinaciones de acilcarnitinas en sangre son utilizadas corrientemente para el diagnóstico y seguimiento de pacientes que sufren enfermedades metabólicas hereditarias. Objetivo: el presente estudio analizó la posible influencia del tipo de anticoagulante utilizado en la toma de la muestra de sangre, sobre el perfil de acilcarnitinas obtenido. Metodología: muestras de sangre y plasma fueron obtenidas utilizando heparina, potasio-EDTA, fluoruro de sodio-oxalato de potasio, y citrato de sodio y sin anticoagulante. Resultados: no fue encontrada diferencia estadísticamente significativa entre los anticoagulantes utilizados. Conclusión: el tipo de anticoagulante no tiene efecto sobre la determinación de acilcarnitinas en sangre o plasma. Para este tipo de análisis, puede ser utilizado cualquier anticoagulante de los antes mencionados. Abreviaturas: IEE-MS/MS, inyección de electrospray espectrometría de masas en tándem; EIM, errores innatos del metabolismo.

Effect of addin the internal standard to bloodsamples, prior to the preparatiion of blood spots for acylcarnitine aaalysis / Efeco de la adicción del estandar inerno a muestras de sangre antess de la preparación de manchas de sangre para el analisis de acylcarnitinas

Background: some general factors can influence when determining acylcarnitines through tandem mass spectrometry. Objective: to study the effect of adding the internal standard to blood samples before the preparation of filter paper cards compared with the addition of internal standard after having the filter paper cards prepared for determining acylcarnitines in blood for tandem mass spectrometry. Methodology: two groups of blood samples were prepared: group one without adding internal standard before the preparation of filter paper cards, and group two adding internal standard prior to the preparation of filter paper cards. Subsequently the acylcarnitines profile was measured using tandem mass spectrometry. Results: the recovery of acylcarnitines during extraction for the samples which were not added the internal standard before the preparation of the filter paper cards was 48% while for the samples that were added the internal standard the recovery percentage was 96% which shows a significant difference between the two groups. Conclusion: the ideal extraction process showing the highest acylcarnitines recovery level happens by adding the internal standard to the sample prior to the preparation of filter paper cards. However, for diagnostic purposes, filter paper cards containing samples without internal standard are normally received for the acylcarnitines analysis and, therefore, the method used to add the internal standard after preparing the samples on filter paper without internal standard is appropriate for determining acylcarnitines in blood. Abbreviations: ESI-MS/MS, Electrospray and tandem mass spectrometry.

Antecedentes: algunos factores generales pueden influir en la determinación de acilcarnitinas por espectrometría de masas en tándem. Objetivo: estudiar el efecto de agregar el estándar interno a las muestras de sangre antes de la preparación de las tarjetas en papel de filtro, comparado con la adición del estándar interno luego de preparar las tarjetas en papel de filtro sobre la determinación de acilcarnitinas en sangre por espectrometría de masas en tándem. Metodología: dos grupos de muestras de sangre fueron preparados: al grupo uno no le fue adicionado el estándar interno antes de la preparación de las tarjetas en papel de filtro, mientras que al grupo dos sí, y posteriormente se midió el perfil de acilcarnitinas por espectrometría de masas en tándem. Resultados: la recuperación de acilcarnitinas durante la extracción para las muestras a las cuales no se les agregó el estándar interno antes de la preparación de las tarjetas en papel de filtro fue del 48%, mientras que con las muestras a las cuales se les adicionó el estándar interno se obtuvo un porcentaje de recuperación del 96%, observándose una diferencia significativa entre los dos grupos. Conclusión: el proceso de extracción ideal que presenta el máximo nivel de recuperación de acilcarnitinas se da agregando el estándar interno a las muestras de sangre antes de preparar las tarjetas en papel de filtro. Sin embargo, con fines diagnósticos, las tarjetas de papel de filtro que contienen muestras de sangre sin estándar interno son recibidas para el análisis de acilcarnitinas y, por lo tanto, el método mediante el cual se agrega el estándar interno luego de preparar las muestras en papel de filtro sin estándar interno es adecuado para la determinación de acilcarnitinas en sangre. Abbreviations: ESI-MS/MS, Electrospray tandem mass spectrometry.

Hidrólisis de acilcarnitinas durante el análisis en sangre y plasma por espectrometría de masas en tandem / Hydrolysis of acylcarnitines during measurement in blood and plasma by tandem mass spectrometry

La determinación de acilcarnitinas en sangre es una herramienta importante para el diagnóstico de algunas enfermedades hereditarias, así como deficiencias metabólicas secundarias. Bajo las condiciones acídicas y la alta temperatura utilizadas durante el proceso de derivatización, es posible que pueda ocurrir algún grado de hidrólisis de acilcarnitinas, lo cual puede potencialmente interferir con las determinaciones de la carnitina libre. El objetivo del presente estudio fue investigar la hidrólisis de las acilcarnitinas (de cadena corta, media y larga) durante el proceso de derivatización y analizar su efecto sobre la determinación de carnitina libre. El porcentaje de hidrólisis fue de 27% para acilcarnitinas de cadena corta, 17% para acilcarnitinas de cadena media y 5% para acilcarnitinas de cadena larga. Estos resultados pueden ocasionar un incremento en los niveles de carnitina libre de las muestras analizadas.

The measurement of acylcarnitines in blood is an important tool for diagnosis of some inherited metabolic diseases and secondary metabolic deficiencies. Under the acidic conditions and the high temperature used for the derivatisation process, it is feasible that some degree of hydrolysis of acylcarnitines to free carnitine may occur and therefore potentially interfere with free carnitine measurements. The objective of the present study was to investigate the hydrolysis of acylcarnitines (short-chain-, medium-chain, and long chain acylcarnitines) during derivatisation process and to analyse its effect on free carnitine measurement. The average percentage of hydrolysis was 27% for short-chain acylcarnitines, 17% for medium-chain acylcarnitines, and 5% for long-chain acylcarnitines. These results can increase the free carnitine levels in the analysed samples.

Tráfico de acyl- CoA de membrana y niveles remanentes en glóbulos rojos, plasma y suero, analizados por espectrometría de masas en tandemso de la espectrometría de masas en tándem / Membrane ACYL-CoA traffic and remaning levels in red blood cells, plasma and serum analysed

Existe un cuestionamiento permanente acerca del fluido ideal para el análisis de carnitina y acilcarnitina por medio de la espectrometría de masas en tándem. El presente estudio evalúa el porcentaje de carnitina y acilcarnitinas en glóbulos rojos y la relación con el contenido de carnitina y acilcarnitina en la sangre, plasma y suero. Se centrifugaron muestras de sangre humana, se extrajeron plasma y suero, y se lavaron los glóbulos rojos con diferentes soluciones isotónicas. Se resuspendió el pellet en PBS para la preparación de tarjetas y análisis por espectrometría de masas en tandem. Se encontró que la carnitina y las acilcarnitinas de cadenas corta, media y larga, permanecen en los glóbulos rojos en porcentajes promedio de 43,4; 48;49; y 70% respectivamente. Se encontró una diferencia significativa entre los niveles de carnitina y acilcarnitina en la sangre comparado con sus niveles en plasma o suero (p<0,05). Dada la asociación de la carnitina y las acilcarnitinas con los glóbulos rojos, parece ser que ni la plasma ni el suero son el material ideal para el análisis de carnitina y acilcarnitinas.

There has been a permanent question about the ideal fluid for carnitine and acylcarnitine analysis by tandem mass spectrometry. The present study evaluates the percentage of carnitine and acylcarnitines in red blood cells and the relationship with the carnitine and acylcarnitines content in whole blood, plasma, and serum. Human blood samples were centrifuged, plasma or serum extracted, and blood cells were washed with different isotonic solutions. The final pellet was resuspended in PBS for card preparation and tandem mass spectrometry analysis. It was found that carnitine, short-chain, medium-chain and longchain acylcarnitines remain in red blood cells at average percentages of 43.4; 48; 49; and 70% respectively. A significant difference was found between carnitine and acylcarnitine levels in whole blood compare to its levels in plasma or serum (p<0.05). As carnitine and acylcarnitines remained associated with the blood cells, it seems therefore that plasma (or serum) is not the ideal material for the analysis of carnitine and acylcarnitines.