A New Fluorescence Method for the Continuous Determination of Surface Lipid Oxidation in Lipoproteins and Plasma

We report on a new method for the determination of lipid oxidation in lipoproteins and plasma. The biological lipid system is preloaded with a fluorescent analog of phosphatidylcholine containing diphenylhexatriene (DPH) propionic acid covalently linked to the sn-2 position. When externally added, the respective phospholipid label (DPHPC) localizes to the surface monolayer of a lipoprotein. Under oxidative conditions (e.g. in the presence of Cu²⁺ ions) the fluorophore undergoes decomposition, resulting in a continuous decrease of fluorescence intensity which reflects the oxidation of a chemically defined phospholipid molecule with well defined localization. When incorporated into LDL particles, the kinetics of the decrease in DPHPC fluorescence intensity upon exposure to Cu²⁺ is very similar to that of conjugated diene accumulation. Furthermore, our assay can be applied to follow the oxidation of lipids in diluted serum and may also be developed into a suitable test system for clinical studies of susceptibility of plasma lipids to oxidation.     

Effects of copper and cadmium ions on the physicochemical properties of lipids of the marine bacterium <Emphasis Type="Italic">Pseudomonas putida</Emphasis> IB28 at different growth temperatures

The phospholipid and fatty acid composition and thermotropic behavior of total lipids were studied in the metal-accumulating marine strain Pseudomonas putida IB28 grown in the presence of Cu²⁺ and Cd²⁺ at 4 and 24°C. Despite the changes in acidic lipid content, unsaturated/saturated fatty acid ratio, and cyclopropane fatty acid level, the temperature range of calorimetric phase transitions of bacterial total lipids was slightly altered under these factors. The suppressive action of heavy metals on bacterial growth is attributable to the phase separation of lipids and, as a consequence, to a sharp increase in the ion permeability of the lipid bilayer. The increase in acidic phospholipid level under the influence of Cu²⁺ and Cd²⁺, especially at 24°C, is likely to be indicative of their complexation with heavy metal ions.     

The Effect of Copper and Lead Ions on Growth and Lipid Composition of the Fern Matteuccia sthruthiopteris

In the present study, the effect of copper (Cu²⁺) and lead (Pb²⁺) ions on the growth and lipid composition of various parts of the fern, Matteuccia sthruthiopteris, was examined. Plants were incubated in the presence or absence of 1, 10, 100 μM of Cu(NO₃)₂ or Pb(NO₃)₂. Cu²⁺ and Pb²⁺ ions at concentrations of 1 and 10 μM caused an increased growth of the roots and leaves. A higher concentration of Pb²⁺ did not show any effect on growth, whereas that of Cu²⁺ slowed down the growth of the whole plants. The roots accumulated more than 700 μg of Cu²⁺ and 400 μg of Pb²⁺ per 1 g dry weight when the plants were incubated with the higher concentrations of metals, whereas in the leaves the concentration of Cu²⁺ was much lower and did not exceed 12 μg/g dry weight. No accumulation of Pb²⁺ ions by leaves was detected. The lipid composition of photosynthetic leave tissues was shown to be affected by the presence of metal ions in the root medium at either concentration studied. Various changes in lipid classes were noted as responsive reactions of M. sthruthiopteris to the heavy metal ions in nutrient medium. Cu²⁺ ions decreased the content of total lipids, total phospholipids, and individual phosphatidylcholines and phosphatidylethanolamines, whereas Pb²⁺ ions caused a decrease in the content of total lipids and glycolipids. Changes in the lipid composition were more pronounced in the mature leaves than in the scrolls of the studied fern.     

Apolipoprotein-lipid association in oxidatively modified HDL and LDL

We investigated the effect of Cu²⁺ catalyzed peroxidation on the status of tryptophan (Trp) in protein moieties in HDL and LDL together with its effect on apolipoprotein-lipid association. Incubation of HDL with Cu²⁺ resulted in a rapid decrease of Trp fluorescence intensity with time with a concomitant increase in Trp maximum emission wavelength (λ_max). LDL incubated with Cu²⁺ also showed a rapid decrease in Trp fluorescence intensity with time, but with no associated increase in λ_max. The status of apo HDL and apo LDL was investigated after 4 h oxidation (4h-oxHDL and 4h-oxLDL respectively). With 4h-oxHDL, the shift in λ_max was not associated with protein dissociation but rather with protein crosslinking and formation of larger HDL species. Progressive increase in λ_max was observed in 4h-oxHDL with increase in guanidine hydrochloride (GuHCl) concentration; this was not due to protein dissociation. Although oxidation of LDL did not produce an increase in λ_max, a significant increase in wavelength was observed when 4h-oxLDL was exposed to increasing concentration of GuHCl. SDS-polyacrylamide gel electrophoresis and nondenaturing gradient gel electrophoresis of the 4h-oxLDL indicated formation of smaller molecular weight protein fragments that were still associated with LDL. Ultracentrifugation of oxidized LDL in the presence and absence of GuHCl showed no dissociated protein. In summary, these data indicate the following: (a) lipid peroxidation has a direct effect on Trp residues in both HDL and LDL, (b) oxidation of HDL is associated with conformational change in apo HDL, crosslinking and formation of larger particles, (c) oxidized HDL have a more stable apolipoprotein-lipid association than native HDL, (d) oxidation of LDL is associated with changes in apo B, that by fluorescence are apparent only in presence of GuHCl and results in fragmentation of apo B without dissociation of protein or change in particle size, and (e) stability of apolipoprotein-lipid association is comparable in oxidized and native LDL.     

α-Tocopherol mediated peroxidation in the copper (II) and met myoglobininduced oxidation of human low density lipoprotein: The influence of lipid hydroperoxides

The principal antioxidant in human LDL, α-tocopherol, is converted to the α-tocopheroxyl radical after reaction with peroxyl radicals or Cu²⁺, and, if it does not terminate with peroxyl radicals, could initiate lipid peroxidation; a phenomenon called ‘tocopherol mediated peroxidation’. Only in the presence of Cu²⁺ and low levels of lipid hydroperoxides was an α-tocopherol dependent decrease in the resistance of LDL to oxidation detected. This suggests that tocopherol mediated peroxidation will probably not contribute significantly as a pro-oxidant process in those individuals most at risk of developing atherosclerosis through an oxidative mechanism.     

Spectroscopic Analysis of the Cu<Superscript>2+</Superscript>-Induced Fluorescence Quenching of Fluorescent Proteins AmCyan and mOrange2

Fluorescent proteins show fluorescence quenching by specific metal ions, which can be applied towards metal biosensing applications. In order to develop metal-biosensor, we performed spectroscopic analysis of the fluorescence quenching of fluorescent protein AmCyan and mOrange2 by various metal ions. The fluorescence intensity of AmCyan was reduced to 48.54% by Co²⁺ and 67.77% by Zn²⁺; Cu²⁺ reduced the fluorescence emission of AmCyan to 19.30% of its maximum. The fluorescence intensity of mOrange2 was quenched by only Cu²⁺, to 11.48% of its maximum. When analyzed by Langmuir equation, dissociation constants for AmCyan and mOrange2 were 56.10 and 21.46 µM, respectively. The Cu²⁺ quenching of AmCyan and mOrange2 were reversible upon treatment with the metal chelator EDTA, indicating that the metal ions were located on the protein surface. Their model structures suggest that AmCyan and mOrange2 have novel metal-binding sites.     

A fluorescence polarization study of calcium and phase behaviour in synaptosomal lipids

Steady-state fluorescence polarization of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene reported temperature-dependent lipid order in l-α-dimyristoylphosphatidylcholine, egg phosphatidylcholine and synaptosomal membranes. No change in lipid order was detected after depolarization of synaptosomes by veratridine (150 μM) even in the presence of 2 mM CaCl₂. However, Ca²⁺ reduced the mobility of a second probe, dansylated dipalmitoylphosphatidylethanolamine, in dispersions of synaptosomal lipids. This effect, which was seen at a Ca²⁺/total phospholipid ratio as low as 0.1, may represent an interaction between the cation and negatively-charged phospholipids. It is suggested that Ca²⁺ promotes a phase separation in synaptosomal lipids which may be relevant to the process of neurotransmitter release.     

The Role of Lipids in the Function of the Acetylcholine Receptor

Abstract

The composition of membranes containing acetylcholine receptor was altered in order to examine the possible role of lipids in receptor function. Polyethylene glycol was used to fuse AcChR-rich membranes with an excess of lipid vesicles of defined composition. By this procedure, the cholesterol composition was reduced to as little as 20% of that found in native membranes. Using a TI⁺ flux assay it was possible to measure receptor function in such altered membrane environments. The apparent K_d for carbamylcholine was found to decrease as the cholesterol content was reduced. This result was confirmed by measuring the agonist-induced fluorescence change of the covalently attached probe, 4-[N-(iodoacetoxy)-ethyl-N-methyl] amino-7-nitrobenz-2-oxa-1,3-diazole. When the phospholipid composition was manipulated by membrane fusion, ion flux was found to be optimal when the lipid composition resembled that of native receptor membranes. These results indicate that membrane lipids potentially play a role in the regulation of acetylcholine receptor function.     

The effect of histidine modification on copper-dependent lipid peroxidation in human low-density lipoprotein

Summary

Lipid peroxidation and subsequent oxidative modification of low-density lipoprotein (LDL) have been implicated as causal events in atherosclerosis. Cu²⁺ may play an important role in LDL oxidation by binding to histidine residues of apolipoprotein B-100 (apo B) and initiating and propagating lipid peroxidation. To investigate the role of histidine residues, we used diethylpyrocarbonate (DEPC), a lipid-soluble histidine-specific modifying reagent. When LDL (0.1 mg protein/ml, or 0.2 µM) was incubated with DEPC (1 mM), at least 76 ± 7% of the histidine residues in apo B were modified. Treatment of LDL with DEPC led to an increase in the rate of Cu²⁺-induced initiation of lipid peroxidation (R_i), but a significant decrease in the rate of propagation. These changes resulted in an overall increased resistance of LDL to oxidation, with a significantly increased lag phase preceding the propagation phase of lipid peroxidation. In contrast to DEPC, ascorbate completely prevented the initiation of LDL oxidation (R_i = 0). Our data indicate that there are two types of copper/histidine binding sites on apo B: those facing the lipid core of the LDL particle, which mediate the propagation of lipid peroxidation and are modified by DEPC; and those found on the surface of the LDL particle exposed to the aqueous environment, which are responsible for mediating the initiation of lipid peroxidation and are modifiable by ascorbate in the presence of Cu²⁺.     

A highly selective fluorescence and absorption sensor for rapid recognition and detection of Cu2+ ions in aqueous solution and film

A fluorescence and absorption chemosensor (SAAT) based on 5-(hydroxymethyl)-salicylaldehyde (SA) and o-aminothiophenol (AT) was designed and synthesized. SAAT in DMSO–HEPES (20.0 mM, v/v, 1:99, pH = 7.0) solution shows a highly selective and sensitive absorption and an ‘on–off’ fluorescence response to Cu²⁺ ions in aqueous solutions over all other competitive metal ions including Na⁺, Ag⁺, Ba²⁺, Ca²⁺, Cd²⁺, Mg²⁺, Zn²⁺, Cr³⁺, Al³⁺, Hg²⁺, K⁺, Mn²⁺, Ni²⁺, Sr²⁺, Tb³⁺ and Co²⁺. SAAT exhibits ratiometric absorption sensing ability for Cu²⁺ ions. Importantly, SAAT also can sense Cu²⁺ ions using fluorescence quenching, the fluorescence intensity of SAAT showed a good linear relationship with Cu²⁺ concentration, and the detection limit of Cu²⁺ was 0.34 μM. The results of Job's plot, Benesi–Hildebrand plot, mass spectra, and density functional theory calculations confirmed that the selective absorption and fluorescence response were attributed to the formation of a 1:1 complex between SAAT and Cu²⁺. SAAT in test film could identify Cu²⁺ in water samples using the intuitive fluorescence colour change under a UV lamp. SAAT has great application value as a selective and sensitive chemosensor to discriminate and detect Cu²⁺ ions.     

Changes in lipid composition in the tissues of fresh-water plant <Emphasis Type="Italic">Hydrilla verticillata</Emphasis> induced by accumulation and elimination of heavy metals

Qualitative and quantitative composition of lipids was investigated in fresh-water vascular plant Hydrilla verticillata (L. fil.) Royle in the course of the accumulation and elimination of heavy metals (HM). The plants were incubated in 100μM solutions of metal nitrates for 10 days. The accumulation of Cu²⁺, Zn²⁺, and Pb²⁺ and their elimination from the plants depended on the duration of exposure and chemical nature of the metal. Accumulation of lead and copper salts was the greatest on the 3rd day, and zinc, on the 10th day. It was associated with changes in the composition of total lipids, polar lipids, and fatty acid (FA). Copper ions suppressed lipid metabolism stronger than other metals. Zn²⁺ and Pb²⁺ induced the accumulation of biomass and elevated the content of some phospholipids and glycolipids. The detected changes (decrease or increase) were observed both during the incubation with HM and within an afterstress period when the plants recovered in the medium free of metals. Judging by their effect on the content of lipids and FA, HM form a series: Cu²⁺ > Zn²⁺ > Pb²⁺. The responses of plant lipid metabolism to the metals of various chemical nature are discussed.     

Increased oxidazability of plasma low density lipoprotein from patients with coronary artery disease

Oxidative modification of lipoproteins may play a crucial role in the pathogenesis of atherosclerosis. This study was designed to examine whether increased lipid peroxides and/or oxidative susceptibility of plasma lipoproteins occur in patients with coronary artery disease. The levels of lipid peroxides, estimated as thiobarbituric acid-reactive substances (TBARS), were significantly greater in the plasma and very low density lipoprotein (VLDL) of symptomatic patients with coronary artery disease than in those of healthy persons, but the TBARS levels of low density lipoprotein (LDL) and high density lipoprotein (HDL) showed insignificant difference between patients and normals. To evaluate the oxidative susceptibility of lipoproteins, we employed in vitro Cu²⁺ oxidation of lipoproteins monitored by changes in fluorescenece, TBARS level, trinitrobenzene sulfonic acid (TNBS) reactivity, apolipoprotein immunoreactivity and agarose gel electrophoretic mobility. While VLDL and LDL of normal controls were oxidazed at 5–10 μM Cu²⁺, pooled VLDL and LDL of patients with coronary artery disease were oxidized at 1–2.5 μM Cu²⁺, i.e., at relatively lowver oxidative stress. At 5 μM Cu²⁺, VLDL and LDL of patients with coronary artery disease still showed at faster oxidation rate, judged by the rate of fluorescence increase, higher TBARS level, less TNBS reactivity, greater change in apo B immunoreactivity and higher electrophoretic mobility than those of normal controls. However, the difference on the oxidizability of HDL was insignificant for patients vs. normals. In conclusion, we have shown that plasm VLDL and LDL of patients with coronary artery disease are more susceptible to in vitro oxidative modification than those of health persons. The data suggest that enhanced oxidizability of plasma lipoproteins may be important factor influencing the development of coronary artery disease.     

The Formation of Phosphatidylcholine Oxidation Products by Stimulated Phagocytes

Phagocytic cells produce a variety of oxidants as part of the immune defence, which react readily both with proteins and lipids, and could contribute to the oxidation of low density lipoprotein in atherosclerosis. We have investigated the oxidation of phospholipid vesicles by neutrophils and mononuclear cells, to provide a model of lipid oxidation in the absence of competing protein. Phorbol 12-myristate 13-acetate-stimulated neutrophils were incubated with phospholipid vesicles containing dipalmitoyl phosphatidylcholine, palmitoyl-arachidonoyl phosphatidylcholine (PAPC) and stearoyl-oleoyl phosphatidylcholine, before extraction of the lipids for analysis by HPLC coupled to electrospray mass spectrometry. The formation of monohydroperoxides (814?m/z) and bishydroperoxides (846?m/z) of PAPC was observed. However, the major oxidized product occurred at 828?m/z, and was identified as 1-palmitoyl-2-(5,6-epoxyisoprostane E²)-sn-glycero-3-phosphocholine. These products were also formed in incubations where the neutrophils were replaced by mononuclear cells, and the amounts produced per million cells were similar. These results show that following oxidative attack by phagocytes stimulated by PMA, intact phospholipid oxidation products can be detected. The identification of an epoxyisoprostane phospholipid as the major product of phagocyte-induced phospholipid oxidation is novel, and in view of its inflammatory properties has implications for phagocyte involvement in atherogenesis.     

Prooxidant and antioxidant properties of Trolox C,analogue of vitamin E,in oxidation of low-density lipoprotein

Trolox C (Trolox), a water-soluble analogue of vitamin E lacking the phytyl chain, was investigated with respect to its effect on the oxidation of low-density lipoprotein (LDL). Trolox was added at different time points of LDL oxidation induced by Cu²⁺ and aqueous peroxyl radicals. In the case of Cu²⁺ -induced LDL oxidation, the effect of Trolox changed from antioxidant to prooxidant when added at later time points during oxidation; this transition occurred whenever α-tocopherol was just consumed in oxidizing LDL. Thus, in the case of Cu²⁺-dependent LDL oxidation, the presence of lipophilic antioxidants in the LDL particle is likely to be a prerequisite for the antioxidant activity of Trolox.

When oxidation was induced by peroxyl radicals, as a model of metal-independent oxidation, the effect of Trolox was always antioxidant, suggesting the importance of Cu²⁺/Cu⁺ redox-cycling in the prooxidant mechanism of Trolox. Our data suggest that, in the absence of significant amounts of lipophilic antioxidants, LDL becomes highly susceptible to oxidation induced by transition metals in the presence of aqueous reductants.     

A novel optical probe based on d‐penicillamine‐functionalized graphene quantum dots: Preparation and application as signal amplification element to minoring of ions in human biofluid

In this study, d ‐penicillamine‐functionalized graphene quantum dots (DPA‐GQD) has been synthesized, which significantly increases the fluorescence intensity of GQD. We used this simple fluorescent probe for metal ions detection in human plasma samples. Designed DPA‐GQD respond to Hg²⁺, Cu²⁺, Au²⁺, Ag⁺, Co²⁺, Zn²⁺, and Pb²⁺ with high sensitivity. The fluorescence intensity of this probe decreased significantly in the presence of metal ions such as, Hg²⁺, Cu²⁺, Au²⁺, Ag⁺, Co²⁺, Zn²⁺, and Pb²⁺. In this work, a promising probe for ions monitoring was introduced. Moreover, DPA‐GQD probe has been tested in plasma samples. The functionalized DPA‐GQDs exhibits great promise as an alternative to previous fluorescent probes for bio‐labeling, sensing, and other biomedical applications in aqueous solution.     

Effect of Cu2+-ascorbic acid on lipid peroxidation,Mg2+-ATPase activity and spectrin of RBC membrane and reversal by erythropoietin

The effect of erythropoietin (Ep), a glycoprotein hormone, has been studied on lipid peroxidation induced by Cu²⁺ and ascorbate in vitro, Mg²⁺ ATPase activity and spectrin of RBC membrane. Our present investigation reveals that Cu²⁺ and ascorbic acid increases lipid peroxidation of RBC membrane significantly. It has further been observed that under the same experimental condition spectrin, a major cytoskeleton membrane protein, and Mg²⁺-ATPase activity of RBC membrane decrease significantly. However, exogenous administration of Ep completely restores lipid peroxidation and Mg²⁺-ATPase activity and partially recovers spectrin of RBC membrane.     

Application of 2,4,5-tris(2-pyridyl)imidazole as ‘turn-off’ fluorescence sensor for Cu(II) and Hg(II) ions and in vitro cell imaging

The 2,4,5-tris(2-pyridyl)imidazole ( L ) molecule has been evaluated as a probe for dual sensing of Hg²⁺ and Cu²⁺ ions in EtOH/HEPES buffer medium (5 mM, pH = 7.34, 1:1, v/v). Probe L shows a good sensitive and selective turn-off response in the presence of both Hg²⁺ and Cu²⁺ ions, which is comprehensible under long UV light. The probe can detect Cu²⁺ ion in the pH range 3–11 and Hg²⁺ ion in pH 6–8. The limit of detection for Cu²⁺ (0.77 μM) is well under the allowable limit prescribed by the United States Environmental Protection Agency. Two metal (Cu²⁺/Hg²⁺) ions are needed per L for complete fluorescence quenching. The probe shows marked reversibility on treatment with Na₂EDTA, making the protocol more economical for practical purposes. Paper strip coated with the L solution of EtOH can detect the presence of Cu²⁺ and Hg²⁺ ions in the sample using visible quenching of the fluorescence intensity. Density functional theory–time-dependent density functional theory (DFT–TDDFT) calculations support experimental observations, and d-orbitals of Cu²⁺/Hg²⁺ provide a nonradiative decay pathway. Cell imaging study using HDF and MDA-MB-231 cells also supported the viability of L in detecting Cu²⁺ and Hg²⁺ ions in living cells.     

Absence of an effect of vitamin E on protein and lipid radical formation during lipoperoxidation of LDL by lipoxygenase

Low-density lipoprotein (LDL) oxidation is the primary event in atherosclerosis, and LDL lipoperoxidation leads to modifications in apolipoprotein B-100 (apo B-100) and lipids. Intermediate species of lipoperoxidation are known to be able to generate amino acid-centered radicals. Thus, we hypothesized that lipoperoxidation intermediates induce protein-derived free radical formation during LDL oxidation. Using DMPO and immuno-spin trapping, we detected the formation of protein free radicals on LDL incubated with Cu²⁺ or the soybean lipoxidase (LPOx)/phospholipase A₂ (PLA₂). With low concentrations of DMPO (1 mM), Cu²⁺ dose-dependently induced oxidation of LDL and easily detected apo B-100 radicals. Protein radical formation in LDL incubated with Cu²⁺ showed maximum yields after 30 min. In contrast, the yields of apo B-100 radicals formed by LPOx/PLA₂ followed a typical enzyme-catalyzed kinetics that was unaffected by DMPO concentrations of up to 50 mM. Furthermore, when we analyzed the effect of antioxidants on protein radical formation during LDL oxidation, we found that ascorbate, urate, and Trolox dose-dependently reduced apo B-100 free radical formation in LDL exposed to Cu²⁺. In contrast, Trolox was the only antioxidant that even partially protected LDL from LPOx/PLA₂. We also examined the kinetics of lipid radical formation and protein radical formation induced by Cu²⁺ or LPOx/PLA₂ for LDL supplemented with α-tocopherol. In contrast to the potent antioxidant effect of α-tocopherol on the delay of LDL oxidation induced by Cu²⁺, when we used the oxidizing system LPOx/PLA₂, no significant protection was detected. The lack of protection of α-tocopherol on the apo B-100 and lipid free radical formation by LPOx may explain the failure of vitamin E as a cardiovascular protective agent for humans.     

The role of Ca2+ ions in modulating changes induced in bean plants by an excess of Cu2+ ions. Chlorophyll fluorescence measurements

Ca²⁺-dependent influence of excess Cu²⁺ on the photosynthetic akpparatus monitored through chlorophyll fluorescence measurements was investigated in runner bean plants (Phaseolus coccineus L. cv. Pie kny Ja?) at three different growth stages. It was observed that the toxic effect of excess Cu²⁺ on plants depends both on their growth stages and the Ca²⁺ content in the medium. Increased Ca²⁺ content limits Cu²⁺ action on plants at their initial growth stage (I) through: stabilization of the PSII complex (increase of the ratio of variable to minimal fluorescence [F_v/F₀]), improved electron flow and reoxidative processes of the quinone primary electron acceptor of PSII (Q_A) (increase of quantum yield of PSII electron transport [φ_e] and photochemical quenching of fluorescence [q_P] values) and elimination of nonphotochemical energy dissipation (decrease of nonphotochemical fluorescence quenching from the Stern-Volmer equation [NPQ] and fraction of the absorbed light energy not used for photochemistry [LNU] values). At this growth stage excess Cu²⁺ decreases the rates of Q_A reduction as a result of decreased PSII activity at its donor side only at lower Ca²⁺ level. At the intermediate growth stage (II) the plants were less sensitive to Cu²⁺ treatment and also to changed Ca²⁺ content. A weakening of some photochemical processes by excess Cu²⁺ could be observed only at a higher Ca²⁺ dose. At the final growth stage of plants (III) Ca²⁺ ions exerted a decisively different effect on the mechanism of excess Cu²⁺ action on bean plants, visualized by decreased PSII stabilization and utilization of absorbed light energy at increased Ca²⁺ content in the medium.     

Mechanisms involved in the in vitro modification of low density lipoprotein by human umbilical vein endothelial cells and copper ions

In this study we evaluated the time course and mechanism of low density lipoprotein (LDL) oxidation induced by human umbilical vein endothelial cells (HUVECs), cell-free medium (CFM) and Cu²⁺. After incubating LDL (200 μg/ml) with HUVECs, CFM and Cu²⁺ (concentration adjusted to obtain the same degree of LDL modification as with HUVECs), the extent of LDL lipid peroxidation and apoprotein B modification was monitored at different times from 0 to 24 h. This involved evaluating the time course of LDL conjugated diene, peroxide, malonyldialdehyde (MDA), fluorescence, relative electrophoretic mobility (REM), vitamin E and monounsaturated and polyunsaturated fatty acids. After incubation with HUVECs, the LDL REM was significantly higher than that obtained in CFM (p < 0.01). When balanced for the same degree of LDL modification as obtained with HUVECs, Cu²⁺ gave a REM similar to that obtained with HUVECs. At the different times of incubation there was no statistical difference between conjugated diene and peroxide values after incubation with HUVECs and with CFM. The values obtained with Cu²⁺ were significantly higher than those obtained with HUVECs and CFM (p < 0.01). MDA and LDL fluorescence were significantly higher after exposure to HUVECs than to CFM (p<0.01), values being similar to those obtained with Cu²⁺. There was no statistical difference between the values of LDL oleic, linoleic, arachidonic and eicosapentaenoic acids after incubation with HUVECs and CFM. Eicosatetraynoic acid (ETYA), a lipoxygenase inhibitor, determined dose-dependent reduction of MDA formation induced by the incubation of LDL with HUVECs; it did not affect LDL conjugated diene. ETYA did not have any effect on the MDA derived from LDL after incubation with Cu²⁺ or CFM. The results of this study demonstrate that, unlike Cu²⁺, the contribution of HUVECs to LDL modification does not involve only lipid peroxidation of the lipoprotein; it also includes intracellular radical and non-radical processes.