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.