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首页> 外文期刊>Cellular Physiology and Biochemistry >The Measurement of High-Density Lipoprotein Mediated Cholesterol Efflux from Macrophage Cells by Liquid Chromatography Tandem Mass Spectrometry
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The Measurement of High-Density Lipoprotein Mediated Cholesterol Efflux from Macrophage Cells by Liquid Chromatography Tandem Mass Spectrometry

机译：液相色谱-串联质谱法测定巨噬细胞中高密度脂蛋白介导的胆固醇流出

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biBackground /i/bStudies have shown a negative association between macrophage cholesterol efflux and atherosclerotic cardiovascular diseases (CVD). However, the current methods for measuring cholesterol efflux require a radioactive tracer and involve a variety of cell treatments, making the measurement of macrophage cholesterol efflux impractical for use in clinical laboratories. In this study, we developed a non-radioactive and precise LC/MS/MS method for the measurement of high-density lipoprotein (HDL) mediated cholesterol efflux from J774 macrophages. biMethods /i/bJ774 cells were seeded on 12-well plates at a density of 1.5×10sup5/sup cells/ml in H-DMEM medium, and when the cells were approximately 80% confluent, they were incubated with H-DMEM medium containing 2% FBS, 0.5 μg/ml ACAT inhibitor Sandoz 58-035, and 20 μg/ml [3,4-sup13/supC]cholesterol for 6 h. After washing and equilibrating the cells, HDL samples were added at a final concentration of 7% and incubated for 8 h. The cells were lysed, and [3,4-sup13/supC]cholesterol and cholesterol were measured by LC/MS/MS. Cholesterol efflux was expressed as the percent decrease of cell [3,4-sup13/supC]cholesterol mass during the incubation. biResults /i/bWhen incubated with [3,4-sup13/supC]cholesterol enriched J774 cells, HDL mediated higher cell cholesterol efflux than influx compared to serum and isolated LDL; therefore, HDL was used as the extracellular acceptor. The results from healthy volunteers showed that the rate of cholesterol efflux was negatively correlated with weight, BMI, blood pressure, and FERsubHDL/sub and positively correlated with HDL-C, HDL2-C, and apoAI levels. biConclusions /i/bA LC/MS/MS method for the measurement of HDL mediated cholesterol efflux from macrophage cells has been established. This method is non-radioactive, precise and reliable and is potentially useful for the assessment of HDL function and cardiovascular disease risks.
机译：背景 研究表明巨噬细胞胆固醇外流与动脉粥样硬化性心血管疾病（CVD）之间存在负相关关系。但是，当前的测量胆固醇流出的方法需要放射性示踪剂，并且涉及多种细胞治疗，使得巨噬细胞胆固醇流出的测量不适用于临床实验室。在这项研究中，我们开发了一种非放射性且精确的LC / MS / MS方法，用于测量J774巨噬细胞介导的高密度脂蛋白（HDL）介导的胆固醇流出。 方法 J774细胞在H-DMEM培养基中以1.5×10 5 细胞/ ml的密度接种在12孔板上，细胞约80％汇合，将它们与含有2％FBS，0.5μg/ ml ACAT抑制剂Sandoz 58-035和20μg/ ml的H-DMEM培养基一起孵育[3,4- 13 C]胆固醇6小时。洗涤并平衡细胞后，以7％的终浓度添加HDL样品，并孵育8小时。裂解细胞，并通过LC / MS / MS测量[3,4- 13 C]胆固醇和胆固醇。胆固醇外流表示为在培养过程中细胞[3,4- 13 C C]胆固醇质量减少的百分比。 结果 与富含[3,4- 13 C]胆固醇的J774细胞一起孵育时，与血清和血清相比，HDL介导的细胞胆固醇外流率高于流入量。隔离的LDL;因此，HDL被用作细胞外受体。健康志愿者的结果表明，胆固醇外流率与体重，BMI，血压和FER HDL 呈负相关，与HDL-C，HDL2-C和apoAI水平呈正相关。 结论 已建立了一种LC / MS / MS方法，用于测量巨噬细胞中HDL介导的胆固醇流出。该方法是非放射性的，精确且可靠的，并且可能对HDL功能和心血管疾病风险的评估有用。

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