Methods on LDL particle isolation, characterization, and component fractionation for the development of novel specific oxidized LDL status markers for atherosclerotic disease risk assessment

Abstract

The present study uses simple, innovative methods to isolate, characterize and fractionate LDL in its main components for the study of specific oxidations on them that characterize oxidized low-density lipoprotein (oxLDL) status, as it causatively relates to atherosclerosis-associated cardiovascular disease (CVD) risk assessment. These methods are: (a) A simple, relatively time-short, low cost protocol for LDL isolation, to avoid shortcomings of the currently employed ultracentrifugation and affinity chromatography methodologies. (b) LDL purity verification by apoB100 SDS-PAGE analysis and by LDL particle size determination; the latter and its serum concentration are determined in the present study by a simple method more clinically feasible as marker of CVD risk assessment than nuclear magnetic resonance. (c) A protocol for LDL fractionation, for the first time, into its main protein/lipid components (apoB100, phospholipids, triglycerides, free cholesterol, and cholesteryl esters), as well as into LDL carotenoid/tocopherol content. (d) Protocols for the measurement, for the first time, of indicative specific LDL component oxidative modifications (cholesteryl ester-OOH, triglyceride-OOH, free cholesterol-OOH, phospholipid-OOH, apoB100-MDA, and apoB100-DiTyr) out of the many (known/unknown/under development) that collectively define oxLDL status, which contrasts with the current non-specific oxLDL status evaluation methods. The indicative oxLDL status markers, selected in the present study on the basis of expressing early oxidative stress-induced oxidative effects on LDL, are studied for the first time on patients with end stage kidney disease on maintenance hemodialysis, selected as an indicative model for atherosclerosis associated diseases. Isolating LDL and fractionating its protein and main lipid components, as well as its antioxidant arsenal comprised of carotenoids and tocopherols, paves the way for future studies to investigate all possible oxidative modifications responsible for turning LDL to oxLDL in association to their possible escaping from LDL’s internal antioxidant defense. This can lead to studies to identify those oxidative modifications of oxLDL (after their artificial generation on LDL), which are recognized by macrophages and convert them to foam cells, known to be responsible for the formation of atherosclerotic plaques that lead to the various CVDs.

Novel oxidized LDL-based clinical markers in peritoneal dialysis patients for atherosclerosis risk assessment

“…In the present study, 39 PD patients (22–92 years old) and 40 healthy, sex and age matched, control adults (23–67 years old) were recruited from the Department of Nephrology and Kidney Transplantation of the University Hospital of Patras, Greece. Additionally, data from 61 HD patients participating in a recent study of our group [ 28 ] were also used. Demographic and clinical data for all PD, HD and control group subjects are presented in Table 1 .…”

“…Some of these studies measure oxLDL levels directly in the blood of PD patients [ 3 , 10 , 13 , [16] , [17] , [18] , [19] , [20] , [21] ], while the others measure the generated in the blood autoantibodies against oxLDL (anti-oxLDL Ab) [ 4 , [20] , [21] , [22] , [23] , [24] , [25] , [26] , [27] ]. However, all these studies are not in unison in their oxLDL marker related conclusions, with the main problem being the fact that they report contradictory results due to the use of non-specific and non-standardized methodologies for evaluating oxLDL status [ 28 ]. Furthermore, anti-oxLDL Ab levels are questionable as atherosclerotic marker, since some studies consider them as atheroprotective while others as atherogenic molecules [ 11 ].…”

“…In this study, we evaluated oxLDL in PD patients (versus a control group) by the quantification of specific oxidative modifications on LDL lipid and protein components and by the quantification of LDL carotenoid content, using novel, simple and clinically applicable methods previously developed [ 28 ]. These methods include the isolation of LDL particles (with >90% recovery and at 90% purity [ 28 ]) by a heparin/MgCl 2 -based precipitation method in order to avoid the time-consuming and cumbersome ultracentrifugation alternative.…”

“…In this study, we evaluated oxLDL in PD patients (versus a control group) by the quantification of specific oxidative modifications on LDL lipid and protein components and by the quantification of LDL carotenoid content, using novel, simple and clinically applicable methods previously developed [ 28 ]. These methods include the isolation of LDL particles (with >90% recovery and at 90% purity [ 28 ]) by a heparin/MgCl 2 -based precipitation method in order to avoid the time-consuming and cumbersome ultracentrifugation alternative. Subsequently, LDL particles are for the first-time fractionated into their main components [apolipoprotein B100 (apoB100), cholesteryl esters, triglycerides, free cholesterol, phospholipids, carotenoids and tocopherols] using an organic solvent extraction methodology.…”

“…In the present study, the oxidative markers lipid hydroperoxides (-OOH), on the four LDL lipid fractions, as well as malondialdehyde (MDA) and dityrosines (DiTyr) on apoB100 were quantified using simple photometric and fluorometric methods, respectively, while LDL carotenoids were also quantified by a photometric method instead of the cumbersome HPLC alternative. Furthermore, the levels of the tested oxLDL markers in PD patients were also compared with those of CKD on HD patients, which have been previously studied by our group [ 28 ]. Our goal was to investigate possible correlation between oxLDL levels and PD and to evaluate possible use of oxLDL as a more reliable clinical marker for CVD prevalence in patients on PD.…”