Estrategia para el diagnóstico de las dislipidemias. Recomendación 2018 / Strategy for the diagnosis of dyslipidaemias

Las dislipidemias son alteraciones del metabolismo lipídico que cursan con concentraciones de lípidos alteradas, tanto por exceso como por defecto. Estas alteraciones están fuertemente asociadas con el proceso aterosclerótico, y se ha demostrado que el control de dichas alteraciones consigue disminuir la incidencia de episodios de origen isquémico. Diagnosticar las dislipidemias desde un punto de vista etiológico es muy importante, ya que el riesgo cardiovascular al que predispone cada una de ellas es diferente, dependiendo del tipo de lipoproteína que esté alterada y de su concentración. Por ello es de gran utilidad disponer de algoritmos diagnósticos sencillos que incluyan magnitudes del metabolismo lipídico disponibles en la mayoría de los laboratorios clínicos, con el fin de realizar el diagnóstico inicial del tipo de dislipidemia, en caso de poseer las herramientas diagnósticas adecuadas identificarla y, en caso contrario, disponer de la información apropiada para recomendar la ampliación del estudio en otro centro que disponga de los recursos necesarios para establecer el diagnóstico

Dyslipidaemias are alterations in lipid metabolism that involve an excess, as well as a deficit, in lipid concentrations. These alterations are strongly associated with atherosclerosis, and it has been shown that its control reduces the incidence of episodes of ischaemic origin. Diagnosing dyslipidaemias from an aetiological point of view is very important, since the cardiovascular risk to which each one predisposes is different, and depends on the type of lipoprotein that is altered and its concentration. For this reason, it is very useful to have simple diagnostic algorithms that include the measurements of lipid metabolism that are available in most clinical laboratories in order to make the initial diagnosis of the type of dyslipidaemia. In the case of having the right diagnostic tools, identify it; and if not, to have the appropriate information to recommend the extension of the study in another centre with resources to establish the diagnosis

Recomendaciones para la estandarización de la medida de lípidos y lipoproteínas. Recomendación (2018) / Recommendations for the standardisation of lipids and lipoproteins measurements. Recommendation (2018)

Este documento describe recomendaciones para la estandarización de la medida de las magnitudes lipídicas, puesto que resultan críticas para la toma de decisiones clínicas. Deben emplearse métodos recomendados validados frente a un método de referencia o definitivo, y materiales de control que cumplan con la Directiva Europea sobre Diagnóstico in vitro; deben cumplir también los objetivos recomendados por el National Cholesterol Education Program (NCEP) y la Sociedad Española de Medicina del Laboratorio (SEQCML). La determinación de colesterol de HDL por métodos homogéneos en equipos automatizados se considera aceptable para la práctica rutinaria, y la fórmula de Friedewald utilizable para estimar la concentración de colesterol de LDL siempre que las concentraciones de triglicéridos sean iguales o inferiores a 200mg/dL (2,3mmol/L); en otro caso, se recomienda utilizar la concentración de colesterol-no-HDL. La cuantificación rutinaria de apolipoproteínas A1 y B, y lipoproteína (a), puede efectuarse por métodos de inmunonefelometría e inmunoturbidimetría, con calibradores trazables a materiales de referencia

Some recommendations are presented for standardising the measurement of lipids and lipoproteins, as they are critical for clinical decisions making. Recommended methods validated against a reference or definitive method should be employed, as well as the use of control materials that comply with European Directives on in vitro diagnostics. Additionally, the chosen methods must comply with the objectives set forth by the National Cholesterol Education Program (NCEP) and by the Spanish Society of Laboratory Medicine (SEQCML). Determination of HDL cholesterol using automatic homogenous methods is considered acceptable for normal clinical practice, and the Friedewald Formula is considered to be usable to estimate LDL cholesterol concentration when triglyceride concentrations are below 200mg/dL (2.3mmol/L). If this should not be the case, the use of non-HDL cholesterol is recommended. Routine quantification of apolipoproteins A1 and B, and lipoprotein (a) can be measured using immunonephelometric or immunoturbidimetric methods, with calibrators that are traceable to reference materials

Removing Lipemia in Serum/Plasma Samples: A Multicenter Study.

BACKGROUND: Lipemia, a significant source of analytical errors in clinical laboratory settings, should be removed prior to measuring biochemical parameters. We investigated whether lipemia in serum/plasma samples can be removed using a method that is easier and more practicable than ultracentrifugation, the current reference method. METHODS: Seven hospital laboratories in Spain participated in this study. We first compared the effectiveness of ultracentrifugation (108,200×g) and high-speed centrifugation (10,000×g for 15 minutes) in removing lipemia. Second, we compared high-speed centrifugation with two liquid-liquid extraction methods-LipoClear (StatSpin, Norwood, USA), and 1,1,2-trichlorotrifluoroethane (Merck, Darmstadt, Germany). We assessed 14 biochemical parameters: serum/plasma concentrations of sodium ion, potassium ion, chloride ion, glucose, total protein, albumin, creatinine, urea, alkaline phosphatase, gamma-glutamyl transferase, alanine aminotransferase, aspartate-aminotransferase, calcium, and bilirubin. We analyzed whether the differences between lipemia removal methods exceeded the limit for clinically significant interference (LCSI). RESULTS: When ultracentrifugation and high-speed centrifugation were compared, no parameter had a difference that exceeded the LCSI. When high-speed centrifugation was compared with the two liquid-liquid extraction methods, we found differences exceeding the LCSI in protein, calcium, and aspartate aminotransferase in the comparison with 1,1,2-trichlorotrifluoroethane, and in protein, albumin, and calcium in the comparison with LipoClear. Differences in other parameters did not exceed the LCSI. CONCLUSIONS: High-speed centrifugation (10,000×g for 15 minutes) can be used instead of ultracentrifugation to remove lipemia in serum/plasma samples. LipoClear and 1,1,2-trichlorotrifluoroethane are unsuitable as they interfere with the measurement of certain parameters.

Síntesis y purificación de apolipoproteína apo A-I Zaragoza (L144R) recombinante / Synthesis and purification of recombinant apolipoprotein apo A-I Zaragoza (L144R)

Introducción La apolipoproteína A-I Zaragoza (apo A-I Z, mutación L144R) es una variante de la apolipoproteína A-I cuyos portadores tienen muy baja concentración de colesterol-HDL (c-HDL) pero, aunque presenten factores de riesgo aterogénicos adicionales, paradójicamente no muestran signos de aterosclerosis a nivel subclínico. Estudios metabólicos in vivo realizados en los portadores heterocigotos de esta variante de apo A-I revelaron que tiene una tasa de catabolismo que duplica la de la proteína nativa sugiriendo la posibilidad de que tuviera un efecto favorable sobre el proceso global de transporte reverso de colesterol.Objetivo Establecer un sistema de expresión y purificación de apo A-I nativa y apo A-I Z recombinantes de alto rendimiento y elevada pureza con el fin de comparar sus propiedades en distintos aspectos relativos al transporte reverso de colesterol, así como otras características antiaterogénicas de la apo A-I. Metodología Partiendo del cDNA de apo A-I nativa se amplificó mediante PCR de la secuencia de la apo A-I madura y se diseñaron los oligonucleótidos cebadores que permitieron el clonaje en un plásmido de expresión inducible pET-45 que incorpora una cola de Histidinas en la posición N-terminal del péptido expresado. Mediante mutagénesis dirigida se introdujo la mutación L144R en la secuencia madura de apo A-I para expresarla con el mismo sistema. Bacterias E. coli de la cepa BL21(DE3) fueron transformadas con los plásmidos preparados, la expresión de las correspondientes proteínas fue inducida y su purificación realizada en condiciones no desnaturalizantes mediante cromatografía de afinidad en columnas de níquel. Resultados (..) (AU)

Introduction Apolipoprotein A-I Zaragoza (apo A-I Z, L144R mutation) is an apolipoprotein A-I variant whose carriers have low HDL-cholesterol (HDL-c) concentrations but, paradoxically, no atherosclerotic symptoms despite the presence of additional atherogenic risks. In vivo metabolic studies performed on heterozygous carriers of this apo A-I variant revealed a two-fold increased fractional catabolic rate compared to the wild type protein, suggesting an enhanced effect on the overall reverse cholesterol transport process. Objectives To establish an expression and purification system of recombinant wild type apo A-I and apo A-I Z with high yield and purity in order to compare their properties related to reverse cholesterol transport as well as other anti-atherogenic characteristics. MethodsA cDNA clone of wild type apo A-I was used as a PCR template to amplify the mature peptide sequence. Specially designed primers allowed the cloning of the sequence into an inducible expression pET-45 plasmid adding a Histidine tag in the N-terminal expressed peptide. Site directed mutagenesis was used to produce the L144R mutation in the apo A-I sequence to be expressed in the same system. E. coli BL21(DE3) were transformed with the prepared plasmids, peptide expression was induced and purification was performed in non-denaturing conditions by nickel affinity chromatography. Results Expression and purification of both proteins was achieved and verified by SDS-PAGE and immunochemical procedures. Actual yields were over 30mg of purified protein per litre of culture and a 94% purity grade for the wild type protein and 93% for the mutant protein were obtained. Conclusions A system for the expression and purification of wild type apo A-I and apo A-I Z with high yield and purity grade has been set up. This will be the basis for future structural and functional characterization of the L144R apo A-I mutant allowing the study of its anti-atherogenic properties (AU)