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.

Keywords: HDL-C; LDL lipid fractions; LDL-C; apoB100; atherosclerosis; cardiovascular diseases; clinical markers; oxidized LDL.

FIGURE 1

Oxidized LDL as depicted by possible oxidative modifications of its main components, possibly recognized by macrophages (left and right panel, respectively) for oxLDL uptake during the course of atherosclerosis development. Starting with the oxidative modifications of the LDL lipid components, clinically important are those of cholesterol (free or esterified) and particularly the following: (i) Those carrying keto-groups (-C=O; i.e., in 7-keto-chol, and secosterol-B and -A with 1 and 2 keto-groups, respectively), to be quantified by a method under development by our lab. (ii) Those having a hydroperoxy group (HOO-; i.e., 5α-HOO-chol, 5β-HOO-chol, 6α-HOO-chol, and 6β-HOO-chol) (85); there is also the 7-HOO-chol, which is converted to the 7-keto-chol (86). Oxidatively modified cholesterol can also exist in its ester form, and is expected to translocate to the surface of the LDL particle as being polar (shown with large red arrows). Moreover, 5-oxovaleroyl-chol (5-OVC) and 9-oxononanoyl-chol (9-ONC) represent important oxidative modifications on the polyunsaturated fatty acid (PUFA) part of the cholesterol esters (87), which are also expected to translocate to the LDL’s surface due to their polar aldehyde end group [see (5-OVC/9-ONC)-CH=O], as will also translocate their O=C-/HOO-chol modifications [see O=C-/HOO-(5-OVC/9-ONC)-CH=O]. Additionally, these oxidative modifications of 5-OVC and 9-ONC can cross-link with apoB100 due to their very reactive aldehyde end (87). All these aldehydic product can be collectively quantified by a method under development by our lab. PUFA can be also oxidized in lipid hydroperoxides and keto/aldehydes (LOOH and L=O, respectively), and when present in the LDL phospholipids/triglycerides are expected to translocate to the LDL lipid monolayer surface due to their polarity (depicted by the small red arrows); LOOH can further oxidize to malondialdehyde (MDA), which could accumulate on the LDL surface as being polar (see depicted as red circles), or react with certain amino acids (88) in apoB100 (depicted as red oval). L=O can be quantified by a method under development by our lab. Extending to apoB100 oxidative modifications, of particular interest are the following: the OS-induced formation of dityrosines (89), and disulfides (between two Cys; -S-S-) (90), hydroperoxides (apoB100-OOH) (50), and carbonyls (apoB100-C=O) (91, 92), the last three by methods developed by our lab. As illustrated in (right) panel, macrophages recognize and bind oxLDL with several scavenger receptors (SR, e.g., SR-A1, CD36, and LOX-1). Future studies, could address quantitatively oxLDL uptake by macrophages by flow cytometry in order to be used as an ex vivo clinical marker for CVD risk assessment and for studies to identify the oxidative modifications on oxLDL recognized by macrophages. In such studies, oxLDL from patients with diseases prone to atherosclerosis development, and LDL from healthy subjects to be used either as control or artificially turned to oxLDL with known oxidative modifications, can be labeled with, e.g., fluorescein isothiocyanate [FITC; preliminarily achieved by our lab as described elsewhere (93, 94)]. Under normal (healthy) conditions of low OS, lysosomal acid lipase (LAL) in late endosomes/lysosomes, degrades LDL cholesteryl esters (CE) to free cholesterol (chol) and fatty acids, while acyl coenzyme A:cholesterol acyltransferase-1 (ACAT1), contributes to formation of CE from the chol in the endoplasmic reticulum (ER) where they accumulate. Neutral CE hydrolase (NCEH) converts CE to chol that is transported outside the cells via the ATP-binding cassette (ABC) transporters ABCA1 and ABCG1, and also SR-BI, with the latter two passing chol to HDL via apolipoprotein A-1 (ApoA-1), thereby ensuring cholesterol homeostasis control. In high OS-promoted atherosclerosis, this control is deregulated, leading to increased SR expression and subsequent oxLDL elevated uptake. In contrast, expression of the chol transporters ABCA1 and ABCG1 is suppressed, which diminishes cholesterol efflux and promotes its deposition in macrophages. At the same time, ACAT1 and NCEH are upregulated and downregulated, respectively, which leads to accumulation of CE. Concurrent operation of these mechanisms results in excessive deposition of lipids and transformation of macrophages to foam cells, as also outlined elsewhere (95). LDL and macrophage draws are major modifications from Berg et al. (96).

FIGURE 2

Methods outline protocols for the development of new clinical markers for CVD risk assessment based on oxLDL. Numbers in panels designate protocols’ sub-sections and steps in section “Methods”. CHCl₃/CH₃OH, chloroform/methanol; C₈H₁₈/C₄H₈O₂, iso-octane/ethyl acetate; C₃H₆O, acetone.

FIGURE 3

SDS-PAGE electrophoresis of human LDL apoB100. The gel shows apoB100 isolated from LDL (sample band in right gel column) against pure apoB100 (control band in left gel column).

FIGURE 4

Frequency histogram of LDL-P diameter in CKD-5d patients participating in the present study; 32 subjects with LDL-P 20–25 nm, 22 with 15–20 nm, and 7 with 25–30 nm, giving an average 21 nm.

FIGURE 5

Thin layer chromatography of LDL lipid sub-fractions. (A) LDL cholesteryl esters (lane 1: 1× moles, lane 2: 2× moles); (B) LDL triglycerides (lane 3: 1× moles, lane 4: 2× moles); and (C) LDL free cholesterol (lane 5: pure cholesterol standard solution, lane 6: 1× moles LDL free cholesterol, lane 7: 2× moles LDL free cholesterol).

FIGURE 6

Low-density lipoprotein carotenoid absorbance spectrum. It is composed of two peaks, 448 and 470 nm, which are typical of carotenoids.

FIGURE 7

Correlation of the clinical biochemical markers LDL-C and HDL-C with LDL triglyceride-OOH and free cholesterol-OOH levels in CKD-5d patients. (A) LDL-C with free cholesterol-OOH; (B) LDL-C with triglyceride-OOH; (C) HDL-C with free cholesterol-OOH; and (D) HDL-C with triglyceride-OOH.

FIGURE 8

Receiver operator characteristic curve for LDL-P diameter (nm) and serum concentration (nmoles/L). The test hypothesis is if (A) smaller or (B) larger LDL-P particles, and (C) smaller or (D) larger concentration of LDL-P particles correlate with high risk for CVDs development.