A Simple Fluorescent Cholesterol Labeling Method to Cryoprotect and Detect Plasma Lipoprotein-X

SIMPLE SUMMARY: Lipoprotein-X is an abnormal toxic particle in blood that is highly enriched in cholesterol. Lipoprotein-X forms in patients lacking an enzyme in blood called lecithin-cholesterol-acyl-transferase. With time, lipoprotein-X causes kidney disease in these patients, resulting in death at 40–50 years of age. Lipoprotein-X also forms, at very high levels, in the blood of patients with several different types of liver disease. Such high levels of lipoprotein-X cause additional painful and debilitating problems in these patients that can also be fatal. Currently, difficult and time-consuming tests only available in research laboratories can identify lipoprotein-X in blood. Unfortunately, lipoprotein-X in patient blood samples is unstable outside the body, and so with time becomes undetectable, even more so if it is frozen for evaluation at a later time. We have developed a simple method to label blood-derived lipoprotein-X so that it can be easily detected, and this method also stabilizes lipoprotein-X particles when frozen, enabling its detection after thawing. This methodology can easily be developed into a simple clinical test to identify both types of diseases where lipoprotein-X particles form in the blood and can be used to monitor how well treatments are able to reduce toxic lipoprotein-X in people with these diseases. ABSTRACT: Lipoprotein-X (LpX) are abnormal nephrotoxic lipoprotein particles enriched in free cholesterol and phospholipids. LpX with distinctive lipid compositions are formed in patients afflicted with either familial LCAT deficiency (FLD) or biliary cholestasis. LpX is difficult to detect by standard lipid stains due to the absence of a neutral lipid core and because it is unstable upon storage, particularly when frozen. We have recently reported that free cholesterol-specific filipin staining after agarose gel electrophoresis sensitively detects LpX in fresh human plasma. Herein, we describe an even more simplified qualitative method to detect LpX in both fresh and frozen–thawed human FLD or cholestatic plasma. Fluorescent cholesterol complexed to fatty-acid-free BSA was used to label LpX and was added together with trehalose in order to cryopreserve plasma LpX. The fluorescent cholesterol bound to LpX was observed with high sensitivity after separation from other lipoproteins by agarose gel electrophoresis. This methodology can be readily developed into a simple assay for the clinical diagnosis of FLD and biliary liver disease and to monitor the efficacy of treatments intended to reduce plasma LpX in these disease states.