The role of microscopy in understanding atherosclerotic lysosomal lipid metabolism.

Microscopy has played a critical role in first identifying and then defining the role of lysosomes in formation of atherosclerotic foam cells. We review the evidence implicating lysosomal lipid accumulation as a factor in the pathogenesis of atherosclerosis with reference to the role of microscopy. In addition, we explore mechanisms by which lysosomal lipid engorgement occurs. Low density lipoproteins which have become modified are the major source of lipid for foam cell formation. These altered lipoproteins are taken into the cell via receptor-mediated endocytosis and delivered to lysosomes. Under normal conditions, lipids from these lipoproteins are metabolized and do not accumulate in lysosomes. In the atherosclerotic foam cell, this normal metabolism is inhibited so that cholesterol and cholesteryl esters accumulate in lysosomes. Studies of cultured cells incubated with modified lipoproteins suggests this abnormal metabolism occurs in two steps. Initially, hydrolysis of lipoprotein cholesteryl esters occurs normally, but the resultant free cholesterol cannot exit the lysosome. Further lysosomal cholesterol accumulation inhibits hydrolysis, producing a mixture of cholesterol and cholesteryl esters within swollen lysosomes. Various lipoprotein modifications can produce this lysosomal engorgement in vitro and it remains to be seen which modifications are most important in vivo.     

Uptake and trafficking of mildly oxidized LDL and acetylated LDL in THP-1 cells does not explain the differences in lysosomal metabolism of these two lipoproteins.

Foam cells in the atherosclerotic lesion have substantial cholesterol stores within large, swollen lysosomes. This feature is mimicked by incubating THP-1 macrophages with mildly oxidized low density lipoprotein (LDL). Incubation of THP-1 cells with acetylated LDL produces cytoplasmic cholesteryl ester accumulation rather than lysosomal storage. The differences could be due to differences in uptake and delivery of lipoprotein to lysosomes or to lysosomal and post-lysosomal processing events. We compared uptake and lysosomal trafficking of acetylated and oxidized LDL using colloidal gold-labeled lipoproteins. Labeling did not alter cellular cholesterol accumulation. We found that uptake and delivery to lysosomes are not different for acetylated and oxidized LDL. In fact, both oxidized and acetylated LDL can be delivered to the same lysosomes. Sequential incubation with oxidized LDL followed by acetylated LDL showed that the lipid-engorged lysosomes are long-lived structures, continuously accepting newly ingested lipoprotein. Comparison of acetylated and oxidized LDL in mouse peritoneal macrophages, a cell which does not accumulate substantial lysosomal lipid, also revealed no differences in uptake. This indicates that in THP-1 cells, the differences in metabolism of oxidized and acetylated LDL are due to cell-specific lysosomal or post-lysosomal events not present in B6C3F1 mouse macrophages.     

Effects of Shosaikoto (kampo medicine) on lipid metabolism in macrophages.

We investigated effects of Shosaikoto treatment on cholesterol metabolism in macrophages. Although macrophages, harvested from mice treated with Shosaikoto, took up a small amount of control low density lipoprotein (LDL) (thiobarbituric acid-reactive substance (TBA-RS) value was 0.27 pmol/mg of protein) as control macrophages, they took up more LDL modified with CuSO4 (TBA-RS value was 6.12 pmol/mg of protein) than control macrophages. Degradation of both control LDL and oxidized LDL was enhanced in Shosaikoto treated macrophages. In the presence of control LDL or in the absence of LDL, incorporation of [3H]oleic acid into chlesteryl oleate was significantly reduced in Shosaikoto treated macrophages. This suggests that acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity in macrophages was partly inhibited by Shosaikoto treatment. On the other hand, in the present of oxidized LDL, cholesteryl ester accumulated in Shosaikoto treated macrophages as much as in controls. However, cholesteryl oleate efflux from macrophages in the presence of high density lipoprotein (HDL) was enhanced in Shosaikoto treated macrophages. These result indicate that Shosaikoto facilitates oxidized LDL catabolism in macrophages, resulting in the augmentation of oxidized LDL uptake and the elimination of cholesterol from macrophages by HDL. These Shosaikoto effects may prevent foam cell formation and the progression of atherosclerotic lesions.     

PHOTOOXIDATIVE DAMAGE TO LYSOSOMES OF CULTURED MACROPHAGES BY ACRIDINE ORANGE

Cultured cells accumulate acridine orange (AO), which is a weak basic dye and a photosensitizer, in lysosomes and other acidic compartments. During exposure to blue light, AO-loaded macrophages show decreasing red granular fluorescence and increasing green diffuse fluorescence. This is hypothesized to represent peroxidative damage to lysosomal membranes resulting in an impaired proton gradient with deprotonation of the AO to its uncharged form and subsequent leakage of the dye. Further damage to the lysosomal membranes will result in release of lytic enzymes from the lysosomal compartment into the cytosol, leading to degeneration and finally cell death. The survival of AO-loaded and light-exposed macrophages is controllable by varying the exposure times to blue light. Inhibition of lysosomal proteases by E-64 results in increased cell survival after AO and blue light-mediated damage, indicating a role of proteolytic enzymes in this type of damage. Morphological analysis shows 'rounding up' with formation of retraction fibrils and pronounced plasma membrane blebbing. The formation of autophagic vacuoles is an early and pronounced event. After protease inhibition, however, all these phenomena are inhibitable to a considerable degree. We have thus directed photooxidative damage selectively to lysosomal membranes and their contents. This technique will allow further detailed studies of the role of lysosomes in degeneration-regeneration processes.     

Alteration of the endocytotic pathway by photosensitization with fluoroquinolones

The endocytotic pathway is profoundly altered by the UVA-induced photosensitization of HS 68 fibroblasts by the fluoroquinolone (FQ) antibiotics lomefloxacin, BAYy 3118, norfloxacin and ciprofloxacin, which preferentially localize in lysosomes. The endocytosis of low-density lipoproteins (LDL) loaded with two carbocyanine dyes compatible for effective Forster-type resonance energy transfer (FRET), namely 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO) as the donor and 1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) as the acceptor, has been used as a model system. Binding of LDL to their cell surface receptors is impaired by irradiation with 10 J cm(-2) of UVA and/or treatment with 250 microM BAYy 3118 during 2 h. Perturbation of the plasma membrane by the FQ is revealed by the change in the rate of exchange of DiO from the LDL to the cell membrane as compared to untreated cells. The lysosomal degradation of LDL, demonstrated by the disappearance of FRET between DiO and DiI, is partly inhibited by the FQ. The actin filament network, involved in the fusion of mature endosomes with lysosomes, is readily destroyed upon photosensitization with the four FQ. However, actin depolymerization can be avoided by incubation of the cells with trans-epoxysuccinyl-1-leucylamido-(4-guanidino)butane, an inhibitor of lysosomal cathepsins prior to FQ photosensitization. All these data suggest that several components of the endocytotic pathway are impaired by photosensitization with these FQ.     

Oxidation-dependent effects of oxidized LDL: proliferation or cell death

Oxidized low-density lipoprotein (oxLDL) induces a wide range of cellular responses to produce atherosclerotic lesion, but key factors determining the response are not understood. In this study, purified LDL was oxidized with copper sulfate, and its physical properties and the related biological responses were investigated. The average hydrodynamic diameter of the lightly oxidized LDL was approximately 25 nm and its Rf value relative to nLDL on agarose gel was between 1.0 and 1.25. The diameter of the extensively oxidized LDL was over 30 nm, the Rf value was over 2.0. A 24 h-exposure of resting RAW264.7 macrophage cells to 100 microg/ml of the lightly oxidized LDL induced proliferation and macrophage activation whereas the extensively oxidized LDL induced cell death at the same concentration. In contrast, 200 microg/ml of oxLDL caused cell death regardless of oxidation degree. Short incubation (4-6 h) of the highly oxidized LDL (100 microg/ml) also resulted in cell proliferation. OxLDL-induced cell death showed mixed characteristics of apoptosis and/or necrosis depending on the strength and duration of the insult. These results suggest that cellular responses induced by oxLDL be dependent on the oxidation degree, the duration of exposure, and the concentration of oxLDL.     

Phenylpropanoid glucosides from Tadehagi triquetrum inhibit oxLDL-evoked foam cell formation through modulating cholesterol homeostasis in RAW264.7 macrophages

The phenylpropanoid glucosides from Tadehagi triquetrum were found to be beneficial to glucose and lipid metabolism in vitro. Herein, we investigated the effects of these compounds on oxidised low-density lipoprotein (oxLDL)-induced foam cell formation in RAW264.7 macrophages, aiming to evaluate their potential utility in prevention of atherosclerosis. Our results showed that three out of seven phenylpropanoid glucosides significantly inhibited oxLDL-evoked foam cell formation. These three compounds remarkably inhibited cholesterol influx and enhanced cholesterol efflux. Treatment with compounds 3, 4 and 7 significantly down-regulated the expression of scavenge receptors 1 (SR-1) and cluster of differentiation 36 (CD36) and increased the expression ATP-binding cassette transporters A1 and G1 (ABCA1 and ABCG1). Analyses of structure–activity relationships revealed that cinnamyl group was the most pivotal group for their activities. This work provided phenomenon that these phenylpropanoid glucosides are effective regulator of cholesterol influx/efflux and may be useful in leading for development of anti-atherosclerotic agents.     

Inhibition of acyl-coenzyme A:cholesterol acyltransferase stimulates cholesterol efflux from macrophages and stimulates farnesoid X receptor in hepatocytes

We investigated the mechanism of spontaneous cholesterol efflux induced by acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibition, and how an alteration of cholesterol metabolism in macrophages impacts on that in HepG2 cells. Oleic acid anilide (OAA), a known ACAT inhibitor reduced lipid storage substantially by promotion of cholesterol catabolism and repression of cholesteryl ester accumulation without further increase of cytotoxicity in acetylated low-density lipoprotein-loaded THP-1 macrophages. Analysis of expressed mRNA and protein revealed that cholesterol 7alpha-hydroxylase (CYP7A1), oxysterol 7alpha- hydroxylase (CYP7B1), and cholesterol 27-hydroxylase (CYP27) were highly induced by ACAT inhibition. The presence of a functional cytochrome P450 pathway was confirmed by quantification of the biliary cholesterol mass in cell monolayers and extracelluar medium. Notably, massively secreted biliary cholesterol from macrophages suppressed the expression of CYP7 proteins in a farnesoid X receptor (FXR)-dependent manner in HepG2 cells. The findings reported here provide new insight into mechanisms of spontaneous cholesterol efflux, and suggest that ACAT inhibition may stimulate cholesterol-catabolic (cytochrome P450) pathway in lesion-macrophages, in contrast, suppress it in hepatocyte via FXR induced by biliary cholesterol (BC).     

Quantification of major lipid classes in human triacylglycerol-rich lipoproteins by high-performance liquid chromatography with evaporative light-scattering detection

Triacylglycerol-rich lipoproteins (TRL), comprising chylomicrons (CM) and very-low-density lipoproteins (VLDL), have been associated with cardiovascular disease. The lipid class content in the remnant particles of these lipoproteins is a determinant for the accumulation of lipids in macrophages and their transformation into foam cells. We have optimized a method for the simultaneous determination of cholesteryl esters (CE), triacylglycerols (TG), free cholesterol (FC), monoacylglycerols (MG), and phospholipids (PL) by HPLC coupled to a light-scattering detector (ELSD). A diol column and a ternary gradient of hexane, 2-propanol, and methanol were applied to CM and VLDL of human origin (n = 10), with excellent precision in terms of repeatability of peak areas and retention times. All peaks were baseline resolved although the resolution of CE and TG was compromised for the sake of simplicity of the solvent gradient. The ELSD response was fitted to second-order equations, with correlation coefficients (r2) higher than 0.999 for a wide range of concentrations (0.25-10 microg of lipid injected). TG were the major lipid class detected in human TRL, accounting for 62% in CM obtained 2 h after the oil intake. In addition we recorded a depletion of TG and CE in CM obtained 2 h after the oil intake of about 60%. We conclude that the method reported here is suitable for a rapid and precise determination of lipid classes in human TRL and, therefore, may be a useful tool for investigations on the atherogenicity of these lipoproteins.     

Sites of photodamage induced by photodynamic therapy with a chlorin e6 triacetoxymethyl ester (CAME)

The acetoxymethyl ester of chlorin e6 (CAME) was initially designed to be a hydrophobic photosensitizing agent that would be recognized by an endocytic pathway and initially accumulated in lysosomes. This was expected to lead to hydrolysis of the ester groups, followed by redistribution of the free chlorin to other subcellular sites. In this study, we examined the patterns of localization of CAME and of subsequent photodamage in murine leukemia L1210 cells. The drug was initially localized at intracellular sites, yielding a pattern similar to that obtained with a fluorescent probe for acidic intracellular vesicles and endosomes. A brief (30 min) incubation with 10 microM CAME followed by irradiation led to mitochondrial photodamage and apoptotic cell death. At a higher drug level, or with a longer incubation time, we observed additional photodamage to the plasma membrane and to lysosomes. The higher photodynamic therapy dose led to inhibition of apoptosis, with cell death likely occurring via a necrotic process. Distribution of CAME among the components of human plasma was to albumin > high-density lipoprotein > low-density lipoprotein. These results have implications concerning the likely mechanism of CAME accumulation and subcellular distribution.     

Degradation of oligo(lactone) branches linked to poly(methacrylate) networks

Partially resorbable composite materials were developed for bone applications and were prepared by crosslinking copolymerization of oligo(lactone) macromonomers with selected comonomers in the presence of hydroxy apatite. The composites were incubated in aqueous solution of different pH and electrolyte content over a period up to 300 days. The percentage of the released hydroxycarboxylic acid from the oligo(lactone) branches was determined by the titration or by high performance liquid chromatography (HPLC). A catalytic influence of cholesterol esterase (CE) on the hydrolysis of the materials was observed. Determination of CE activity in the presence of the different composite materials showed that the half-life time of CE was the lowest in the presence of the material undergoing the fastest degradation. After degradation the composite materials have preserved their coherence and have shown higher glass transition temperatures in the dried state than the materials before degradation.     

Composition,structure and substrate properties of reconstituted discoidal HDL with apolipoprotein A-I and cholesteryl ester

To investigate the influence of lipid unsaturation and neutral lipid on the maturation of high density lipoproteins, the discoidal complexes of apoA-I, phosphatidylcholine and cholesteryl ester (CE) were prepared. Saturated dipalmitoylphosphatidylcholine (DPPC) and unsaturated palmitoyllinoleoylphosphatidylcholine (PLPC), palmitoyloleoylphosphatidylcholine (POPC), and fluorescent probe cholesteryl 1-pyrenedecanoate (CPD) that forms in a diffusion- and concentration-dependent manner short-lived dimer of unexcited and excited molecules (excimer) were used. The apoA-I/DPPC/CPD complexes were heterogeneous by size, composition and probe location. CPD molecules incorporated more efficiently into larger complexes and accumulated in a central part of the discs. The apoA-I/POPC(PLPC)/CPD were also heterogeneous, however, probe molecules distributed preferentially into smaller complexes and accumulated at disc periphery. The kinetics of CPD transfer by recombinant cholesteryl ester transfer protein (CETP) to human plasma LDL is well described by two-exponential decay, the fast component with a shorter transfer time being more populated in PLPC compared to DPPC complexes. The presence of CE molecules in discoidal HDL results in particle heterogeneity. ApoA-I influences the CETP activity modulating the properties of apolipoprotein–phospholipid interface. This may include CE molecules accumulation in the boundary lipid in unsaturated phosphatidylcholine and cluster formation in the bulk bilayer in saturated phosphatidylcholine.     

Reconstituted low density lipoprotein: a vehicle for the delivery of hydrophobic fluorescent probes to cells.

Previous studies have shown that the cholesteryl ester core of plasma low density lipoprotein (LDL) can be extracted with heptane and replaced with a variety of hydrophobic molecules. In the present report we use this reconstitution technique to incorporate two fluorescent probes, 3-pyrenemethyl-23, 24-dinor-5-cholen-22-oate-3 beta-yl oleate (PMCA oleate) and dioleyl fluorescein, into heptane-extracted LDL. Both fluorescent lipoprotein preparations were shown to be useful probes for visualizing the receptor-mediated endocytosis of LDL in cultured human fibroblasts. When normal fibroblasts were incubated at 37 degrees C with either of the fluorescent LDL preparations, fluorescent granules accumulated in the perinuclear region of the cell. In contrast, fibroblasts from patients with the homozygous form of familial hypercholesterolemia (FH) that lack functional LDL receptors did not accumulate visible fluorescent granules when incubated with the fluorescent reconstituted LDL. A fluorescence-activated cell sorter was used to quantify the fluorescence intensity of individual cells that had been incubated with LDL reconstituted with dioleyl fluorescein. With this technique a population of normal fibroblasts could be distinguished from a population of FH fibroblasts. The current studies demonstrate the feasibility of using fluorescent reconstituted LDL in conjunction with the cell sorter to isolate mutant cells lacking functional LDL receptors.     

Detection and characterization of cholesteryl ester hydroperoxides in oxidized LDL and oxidized HDL by use of an Orbitrap mass spectrometer

Oxidation of cholesteryl esters in lipoproteins by reactive oxygen species yields cholesteryl ester hydroperoxides (CEOOH). In this study, we developed a novel method for identification and characterization of CEOOH molecules in human lipoproteins by use of reversed-phase liquid chromatography with an hybrid linear ion trap-Orbitrap mass spectrometer (LC-LTQ Orbitrap). Electrospray ionization tandem mass spectrometric analysis was performed in both positive-ion and negative-ion modes. Identification of CEOOH molecules was completed by use of high-mass-accuracy (MA) mass spectrometric data obtained by using the spectrometer in Fourier-transform (FT) mode. Native low-density lipoproteins (nLDL) and native high-density lipoproteins (nHDL) from a healthy donor were oxidized by CuSO(4), furnishing oxidized LDL (oxLDL) and oxidized HDL (oxHDL). No CEOOH molecules were detected in the nLDL and the nHDL, whereas six CEOOH molecules were detected in the oxLDL and the oxHDL. In positive-ion mode, CEOOH was detected as [M + NH(4)](+) and [M + Na](+) ions. In negative-ion mode, CEOOH was detected as [M + CH(3)COO](-) ions. CEOOH were more easily ionized in positive-ion mode than in negative-ion mode. The LC-LTQ Orbitrap method was applied to human plasma and six species of CEOOH were detected. The limit of detection was 0.1 pmol (S/N = 5:1) for synthesized CEOOH.     

Enantioselective capillary electrophoresis for the assessment of CYP3A4-mediated ketamine demethylation and inhibition in vitro

Enantioselective CE with sulfated cyclodextrins as chiral selectors was used to determine the CYP3A4-catalyzed N-demethylation kinetics of ketamine to norketamine and its inhibition in the presence of ketoconazole in vitro. Ketamine, a chiral phencyclidine derivative, was incubated with recombinant human CYP3A4 from a baculovirus expression system as racemic mixture and as single enantiomer. Alkaline liquid/liquid extracts of the samples were analyzed with a pH 2.5 buffer comprising 50 mM Tris and phosphoric acid together with either multiple isomer sulfated β-cyclodextrin (10 mg/mL) or highly sulfated γ-cyclodextrin (2%, w/v). Data obtained in the absence of ketoconazole revealed that the N-demethylation occurred stereoselectively with Michaelis-Menten (incubation of racemic ketamine) and Hill (separate incubation of single enantiomers) kinetics. Data generated in the presence of ketoconazole as the inhibitor could best be fitted to a one-site competitive model and inhibition constants were calculated using the equation of Cheng and Prusoff. No stereoselective difference was observed, but inhibition constants for the incubation of racemic ketamine were found to be larger compared with those obtained with the incubation of single ketamine enantiomers.     

Selecting selective suppressors of selective uptake

Scavenger receptor BI (SR-BI) is a high-density lipoprotein (HDL) receptor that mediates the selective uptake of HDL cholesteryl ester (CE) and the bidirectional flux of free cholesterol (FC). The identification of selective uptake inhibitors holds promise for mechanistic studies of SR-BI and for discovery of pharmaceuticals useful in therapy of atherosclerosis.     

Secreted phospholipase A<Subscript>2</Subscript>, lipoprotein hydrolysis,and atherosclerosis: integration with lipidomics

Phospholipase A₂ (PLA₂) is a group of enzymes that hydrolyze the sn-2 position of glycerophospholipids to yield fatty acids and lysophospholipids. Of many PLA₂s or related enzymes identified to date, secreted PLA₂s (sPLA₂s) comprise the largest family that contains 10 catalytically active isozymes. Besides arachidonic acid released from cellular membranes for eicosanoid synthesis, several if not all sPLA₂s have recently been implicated in hydrolysis of phospholipids in lipoprotein particles. The sPLA₂-processed low-density lipoprotein (LDL) particles contain a large amount of lysophospholipids and exhibit the property of “small-dense” or “modified” LDL, which facilitates foam cell formation from macrophages. Transgenic overexpression of these sPLA₂s leads to development of atherosclerosis in mice. More importantly, genetic deletion or pharmacological inhibition of particular sPLA₂s significantly attenuates atherosclerosis and aneurysm. In this article, we will give an overview of current understanding of the role of sPLA₂s in atherosclerosis, with recent lipidomics data showing the action of a subset of sPLA₂s on lipoprotein phospholipids.     

SUBCELLULAR LOCALIZATION OF AND PHOTOSENSITIZATION BY PROTOPORPHYRIN IX IN HUMAN KERATINOCYTES AND FIBROBLASTS CULTIVATED WITH 5-AMINOLEVULINIC ACID

Abstract— The subcellular localization of protoporphyrin (PP) has been studied by microspectrofluo-rometric techniques in NCTC 2544 keratinocytes incubated with 5-aminolevulinic acid (ALA) for times up to 42 h. Whereas the plasma membrane shows strong staining, fluorescent spots are observed within the cytoplasm especially in the perinuclear region. Although the topographic pattern of the PP distribution does not change much with the incubation time with ALA, the fluorescence spectra suggest that the PP microenvironments are quite different at short and long incubation times. Addition of 18 uJW desferoxamine almost doubles the ALA-induced PP concentration. Colocalization experiments with rhodamine 123, a mitochondrial probe, and lucifer yellow (LY) or neutral red (NR), two lysosome probes, demonstrate that at least some of these spots are of lysosomal origin. Study of the time evolution of the NR fluorescence under irradiation with visible light in the presence and absence of ALA demonstrates that lysosomes are damaged in cells that have synthesized PP. No PP fluorescence can be detected in mitochondria after incubation with ALA. However, photosensitization of mitochondria occurs under irradiation with visible light. Very little formation of lipofuscins by photosensitization with exogenous PP or ALA-induced PP is observed with the NCTC 2544 keratinocytes, as compared to normal human fibroblasts.     

Inhibitor screening of pharmacological chaperones for lysosomal β-glucocerebrosidase by capillary electrophoresis

Pharmacological chaperones (PCs) represent a promising therapeutic strategy for treatment of lysosomal storage disorders based on enhanced stabilization and trafficking of mutant protein upon orthosteric and/or allosteric binding. Herein, we introduce a simple yet reliable enzyme assay using capillary electrophoresis (CE) for inhibitor screening of PCs that target the lysosomal enzyme, β-glucocerebrosidase (GCase). The rate of GCase-catalyzed hydrolysis of the synthetic substrate, 4-methylumbelliferyl-β-d-glucopyranoside was performed using different classes of PCs by CE with UV detection under standardized conditions. The pH and surfactant dependence of inhibitor binding on recombinant GCase activity was also examined. Enzyme inhibition studies were investigated for five putative PCs including isofagomine (IFG), ambroxol, bromhexine, diltiazem, and fluphenazine. IFG was confirmed as a potent competitive inhibitor of recombinant GCase with half-maximal inhibitory concentration (IC ₅₀ ) of 47.5 ± 0.1 and 4.6 ± 1.4 nM at pH 5.2 and pH 7.2, respectively. In contrast, the four other non-carbohydrate amines were demonstrated to function as mixed-type inhibitors with high micromolar activity at neutral pH relative to acidic pH conditions reflective of the lysosome. CE offers a convenient platform for characterization of PCs as a way to accelerate the clinical translation of previously approved drugs for oral treatment of rare genetic disorders, such as Gaucher disease.     

Charge density profiling of circulating human low-density lipoprotein particles by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) has been utilized to profile the low-density (LDL) particles in human blood serum in this study. A 5 mM sodium phosphate buffer, pH 7.40, was chosen as the most suitable CE buffer and an extensive ultrafiltration (UF) procedure was applied to purify the LDL sample. Two LDL particle species, LDL with lower mobility and LDL- with higher mobility were observed. The electropherograms were highly reproducible with good precision of effective mobilities, corrected peak areas (CPAs) and CPA ratio of LDL-/LDL. LDL particles shown on the electropherogram were also characterized by several procedures. The applications of Sigma HDL cholesterol reagent and CE on-line 2-propanol precipitation indicated that the two particle species shown in the electropherogram belong to LDL. The LDL particles were found to associate with the buoyant LDL fraction and the LDL- particles associate with the dense LDL fraction. This study utilizes CZE for the profiling of LDL isoforms and provides a new analytical method for the resolution of LDL subspecies. It demonstrates a high-mobility LDL particle which circulates in healthy subjects and diminishes in atherosclerotic patients. Diminution of the high-mobility LDL subspecies may be linked to minimal formation of arterial plaque in atherosclerotic patients.