INTRODUCTION: Adiponectin is a circulating hormone that is produced exclusively by adipocytes and has antiinflammatory and anti-atherogenic properties. The hypothesis that there are differences in adiponectin levels between stable and unstable coronary-artery disease patients remains controversial. Furthermore, the potential relationships between the plasma adiponectin level and the inflammatory and non-inflammatory markers (oxidized low density lipoprotein and nitric oxide) in patients with stable and unstable coronary-artery disease relative to normal subjects have not been assessed. OBJECTIVES: To assess whether plasma adiponectin levels differ among patients with stable and unstable coronary-artery disease and among control subjects, and to correlate plasma adiponectin level with inflammatory and clinical risk factors (such as oxidized-LDL and nitric oxide) in these patients. METHODS: This study included 50 control subjects, 50 stable angina patients and 50 unstable angina patients with angiographically documented coronary-artery disease. Plasma adiponectin and oxidized-LDL levels were determined using an enzyme immunoassay. Plasma nitric oxide, high sensitivity C-reactive protein and lipid profile levels were also measured. RESULTS: Plasma adiponectin levels were lower in the unstable angina patients (4.9 ± 1.30 µg/mL) than in the stable angina patients (6.34 ± 1.0 µg/mL) or in the controls (9.25 ± 1.8 µg/mL); these levels were also significantly lower in stable angina patients versus controls (p<0.001). Plasma adiponectin levels were negatively correlated with oxidized-LDL, high sensitivity C-reactive protein, lipid profile and other clinical risk factors but positively correlated with nitric oxide. CONCLUSION: Plasma adiponectin levels were found to be lower in both stable and unstable angina patients relative to control subjects, and the correlation between plasma adiponectin and cardiovascular markers is weakened in these patients.

Objetivo: Evaluar el porcentaje de carnitina y acilcarnitinas que permanece en los glóbulos rojos, luego de lavarlos con solución salina a diferentes concentraciones. Materiales y métodos: Muestras de sangre humana fueron centrifugadas y los glóbulos rojos fueron lavados con solución salina a diferentes concentraciones. El pellet obtenido, fue resuspendido en PBS para preparación de tarjetas y análisis por espectrometría de masa en tandem. Resultados: La carnitina, así como las acilcarnitinas de cadena corta, cadena media y cadena larga, permanecen en los glóbulos rojos a porcentajes promedio de 19,3; 34; 34; and 32%, respectivamente. Se encontró diferencia significativa al comparar los niveles de carnitina y acilcarnitinas de sangre lavada con una solución salina isotónica vs. varias hipotónicas (p<0.05). Conclusión: debido a que la carnitina y las acilcarnitinas permanecen en los glóbulos rojos, se recomienda el uso de sangre entera para medir los niveles de estos metabolitos.

Objective: To evaluate the percentage of carnitine and acylcarnitines remaining in red blood cells after washing them with different concentrations of saline solution. Materials and methods: Human blood samples were centrifuged and the blood cells were washed with different saline solutions. The final pellet was resuspended in PBS for card preparation and tandem mass spectrometry analysis. Results: It was found that carnitine, as well as short-chain, medium-chain, and long-chain acylcarnitines remain in red blood cells at average percentages of 19.3; 34; 34; and 32%, respectively. Significant differences were found for carnitine and acylcarnitine levels in blood washed with an isotonic solution compared to their levels using several hypotonic solutions (p<0.05). Conclusion: Because carnitine and acylcarnitines remained associated with the blood cells, we recommend using whole blood to measure these metabolites.

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.

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.

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.

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.

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.