Cholesterol efflux capacity measurement from j774a.1 macrophage cell line to human hdl

Cholesterol Efflux Capacity Measurement from J774a.1 Macrophage Cell Line to Human HDL SUMMARY Atheroslerotic plaque formation is induced by accumulation of cholesterol and its esters in arterial wall. This process leads to various cardiovascular diseases which are globally one of the leading causes of death. At the moment, there is no detailed clinical analysis, which would allow to determine individuals‘ atheroslerotic risk. One of the most promising individual predictors for atherosclerotic risk is cholesterol efflux capacity (CEC). CEC can be described as an index of resident arterial wall immune system cells‘ – macrophages‘ ability to efflux and HDLs‘, circulating in bloodstream, ability to influx cholesterol. During this process, the amount of cholesterol in arterial wall is diminished and the organism is protected from atherosclerotic plaque formation. At this moment, CEC measurement is still in experimental stages, because there is no cheap, efficient and universal method to assess CEC. The main aim of the study was to create and evaluate a method for CEC measurement by comparing its‘ results to commercially available CEC measurement kits‘. A measurement system was created which allows to simulate cholesterol efflux process occuring in an organism. In this system J774a.1 cell line macrophage cells were enriched with fluorescent cholesterol and incubated with specially prepared cholesterol acceptors from human blood – HDLs. After incubation CEC was assessed by measuring the fluorescence intensity. For this trial a sample of patients was selected so that it would reflect a natural heterogenic population with healthy and dyslipidemic individuals. After putting to test the measurement system it was concluded that it is suitable for CEC measurement. Even though the system worked, cAMP concentration, which was used for positive control, was not effective in inducing cholesterol efflux. For future trials cAMP concentration should be raised to check if cAMP is suitable for use as cholesterol efflux inducer or cAMP should be replaced with other agent. After measuring and calculating CEC values by commercially available kit and our measurement method, the results were compared by Bland-Altman method to evaluate if these methods could be used interchangeably. Prior to the trials, clinical assumption was made, that these methods could be used interchangeably if their mean of result differences would not be more than 5%. Using Bland-Altman method it was concluded that averagely measurements differ by -11,6761 %. It means that our measurement method yelds values that are averagely 11,67611 % greater than those of commercial kits‘. This difference leads to conclusion that even though our measurement method is proven to be able to assess CEC, it cannot be used as an alternative for comercially available kit for CEC assessement