Phosphocholine Headgroup Research Articles

Structural ordering of lipid bilayers induced by surfactant molecules with small hydrophilic head group

A series of 100 ns-long molecular dynamics calculations for binary bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 33 mol% ethylene-glycol-ethers () were performed under the isothermal–isobaric condition ( K and atm) to investigate systematically changes in physico-chemical properties of the binary bilayer depending on the length of the hydrophilic head group and the hydrophobic tail. Remarkable differences were observed in membrane properties between the bilayers mixed with surfactants with a large hydrophilic head group and a small hydrophilic head group. In the former, the binary bilayers extended laterally with more disorder in lateral arrangement of the lipid molecules and more disordered acyl tail conformation of the DMPC molecules in the binary bilayers than those in pure DMPC bilayer. In the latter, the bilayers shrunk laterally with more order in lateral arrangement of the lipids and more ordered DMPC acyl tail conformation. The molecular mechanism of these changes in membrane properties is discussed on the basis of two-dimensional radial distribution functions of the lipid mass centres, radius of gyration of lipids and void distribution in membranes. The umbrella effect of phosphocholine head groups was investigated.

Aggregation behaviour of a single-chain, phenylene-modified bolalipid and its miscibility with classical phospholipids.

In the present work, we describe the synthesis of a single-chain, phenylene-modified bolalipid with two phosphocholine headgroups, PC-C18pPhC18-PC, using a Sonogashira cross-coupling reaction as a key step. The aggregation behaviour was studied as a function of temperature using transmission electron microscopy (TEM), differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) spectroscopy, and small angle neutron scattering (SANS). We show that our new bolalipid self-assembles into nanofibres, which transform into flexible nanofibres at 27 °C and further to small elongated micelles at 45 °C. Furthermore, the miscibility of the bolalipid with bilayer-forming phosphatidylcholines (DMPC, DPPC, and DSPC) was investigated by means of DSC, TEM, FTIR, and small angle X-ray scattering (SAXS). We could show that the PC-C18pPhC18-PC is partially miscible with saturated phosphatidylcholines; however, closed lipid vesicles with an increased thermal stability were not found. Instead, bilayer fragments and disk-like aggregates are formed.

The cmc-value of a bolalipid with two phosphocholine headgroups and a C24 alkyl chain: Unusual binding properties of fluorescence probes to bolalipid aggregates

Bolalipids with a long alkyl chain and two phosphocholine polar groups self-assemble in water into two different types of aggregate structures, namely helical nanofibers at low temperature and two types of micellar aggregates at higher temperature. We tried to determine the critical aggregation concentration (cac) or critical micellar concentration (cmc) of the bolalipid tetracosane-1,24-bis(phosphocholine) (PC-C24-PC) by using different fluorescent probes. The use of pyrene or pyrene derivatives as fluorophores failed, whereas the probes 1,8-ANS and particularly bis-ANS gave consistent results. The structure of the bolalipid aggregates obviously hinders partitioning or binding of pyrene derivatives into the micellar interior, whereas 1,8-ANS and bis-ANS can bind to the surface of the aggregate structures. The observed large increase in fluorescence intensity of bis-ANS indicates that binding to the hydrophobic surface of the aggregates leads to a reduction of the dye mobility. However, binding of bis-ANS is relatively weak, so that the determination of a cac/cmc-value is difficult. Simulations of the intensity curves for PC-C24-PC lead to estimates of the cac/cmc-value of 0.3–1.0×10−6M, depending on the structure of the aggregates. Single molecule fluorescence correlation spectroscopy was used to determine the mobility of bis-ANS as a function of concentration of PC-C24-PC. The dye diffusion time and the molecular brightness are lower at low bolalipid concentration, when only free dye is present, and increase at higher concentration when bis-ANS is bound to the aggregates. The experimental cac/cmc-values are higher than those estimated, using an incremental method for the change in Gibbs free energy for micellization with n-alkyl-phosphocholines with only one polar group as a comparison. Apparently, for PC-C24-PC in micellar or fibrous aggregates, more CH2 groups are exposed to water than in a conventional micelle of an n-alkyl-phosphocholine.

Model-Free Analysis of Critical Micellar Concentrations for Detecting Demixing in Surfactant Mixtures.

Aqueous mixtures of two or more surfactants are often employed for research or industrial purposes because such mixtures offer advantages over single-surfactant systems. This is particularly true for mixtures of fluorocarbon (FC) and hydrocarbon (HC) surfactants, which display a broad range of mutual miscibilities in mixed micelles. Unfortunately, the prediction and even the experimental elucidation of the micellar mixing behavior of surfactant mixtures remain challenging, as evidenced by conflicting results and conclusions derived from diverse, and often complex, mixing models. One of the most intriguing questions is whether certain combinations of FC and HC surfactants form only one type of mixed micelle or rather demix into two micelle populations, namely, FC-rich and HC-rich ones. Here, we demonstrate a novel approach to the model-free analysis of critical micellar concentrations (CMCs) of surfactant mixtures that is based on a fit of the experimental data with cubic splines using a stringent thermodynamic criterion for mixing. As a proof of principle, we analyze CMC values determined by isothermal titration calorimetry and confirm the conclusions with the aid of combined 1H- and 19F-NMR spectroscopy. Specifically, we show that aqueous mixtures of an FC maltoside and an HC maltoside conform with the assumption of only one type of micelle regardless of the mixing ratio, whereas combining the same FC surfactant with an HC surfactant carrying a zwitterionic phosphocholine headgroup gives rise to two coexisting micelle populations at high mole fractions of the FC maltoside.

Mechanosensitivity of polydiacetylene with a phosphocholine headgroup

We have demonstrated colorimetric and fluorescence detection of a peptide, melittin, based on polydiacetylene (PDA) made of 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC(8,9)PC). The PDA used in this work has a phosphocholine headgroup, which mimics peptide-cell membrane interactions better than the conventional PDAs with carboxyl headgroups. The dose curve (colorimetric response vs. melittin concentration) showed a half maximum response at the melittin concentration of 0.1 mg ml-1, which is similar to that reported in traditional PDA assays. It suggests that the replacement of the headgroup was achieved without sacrificing the sensitivity. From the dose curve, the Hill coefficient was extracted as αHill = 2.1. The value is in agreement with the results from previous melittin studies with phospholipids, which reflects the benefit of having a biologically relevant headgroup. In addition, we found an unexpectedly slow spectral change when DC(8,9)PC-PDA was incubated with melittin. The origin of the time-dependent signal was studied by combining UV/VIS spectroscopy, fluorescence spectroscopy and dynamic light scattering.

Normal and Frictional Interactions between Liposome-Bearing Biomacromolecular Bilayers.

Highly efficient lubricating boundary layers at biosurfaces such as cartilage have been proposed to comprise phospholipids complexed with biomacromolecules exposed at the surfaces. To gain insight into this, a systematic study on the normal and frictional forces between surfaces bearing a sequentially deposited model alginate-on-chitosan bilayer, bearing different adsorbed phosphatidylcholine (PC) liposomes, was carried out using a surface force balance. Structures of the resulting surface complexes were determined using atomic force microscopy (AFM) and cryo-scanning electron microscopy (cryo-SEM). The liposome/lipid-polymer complexes could maintain their integrity up to high pressures in terms of both normal and shear interactions between the surfaces, which were repeatable, reproducible, and revealed very low friction (coefficient of friction μ down to 10(-3)-10(-4), depending on the PC used) up to pressures of hundreds of atm. We attribute this remarkable lubrication capability ultimately to hydration lubrication acting at the hydrated phosphocholine headgroups of the PC lipids, either exposed at the liposome surfaces or through complexation with the polyelectrolyte bilayer. Values of μ, while low, were roughly an order of magnitude higher than for the same PC vesicles adsorbed on bare mica, a difference attributed to their lower density on the bilayer; the bilayer, however, stabilized the PC-vesicles far better than bare mica against rupture and shear at high compressions and sliding.

Structural Characterization of Lecithin-Stabilized Tetracosane Lipid Nanoparticles. Part I: Emulsions.

The structure of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)-stabilized colloidal tetracosane emulsions was investigated by photon correlation spectroscopy and small-angle X-ray and neutron scattering, using emulsions with different neutron scattering contrasts. Special emphasis was placed on the structure of the DMPC stabilizer layer covering the emulsion droplets. A monolayer, structurally similar to a half DMPC bilayer, with a thickness of 16 Å is found. Thereby, the phosphocholine headgroups arrange flat at the oil-water interface. A deep penetration of the tetracosane oil into the stabilizer layer can be ruled out.

Comparative atomic-scale hydration of the ceramide and phosphocholine headgroup in solution and bilayer environments.

Previous studies have used neutron diffraction to elucidate the hydration of the ceramide and the phosphatidylcholine headgroup in solution. These solution studies provide bond-length resolution information on the system, but are limited to liquid samples. The work presented here investigates how the hydration of ceramide and phosphatidylcholine headgroups in a solution compares with that found in a lipid bilayer. This work shows that the hydration patterns seen in the solution samples provide valuable insight into the preferential location of hydrating water molecules in the bilayer. There are certain subtle differences in the distribution, which result from a combination of the lipid conformation and the lipid-lipid interactions within the bilayer environment. The lipid-lipid interactions in the bilayer will be dependent on the composition of the bilayer, whereas the restricted exploration of conformational space is likely to be applicable in all membrane environments. The generalized description of hydration gathered from the neutron diffraction studies thus provides good initial estimation for the hydration pattern, but this can be further refined for specific systems.

Infrared and fluorescence spectroscopic studies of a phospholipid bilayer supported by a soft cationic hydrogel scaffold

Polarized attenuated total reflection (ATR-IR) spectroscopy and fluorescence microscopy techniques were used to characterize a 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) membrane supported on porous, cationic hydrogel beads. Fluorescence microscopy images showed that the DPhPC coated the external surface of the hydrogel scaffold. In addition, a fluorescence assay of the emission intensity of the Tb3+/dipicolinic acid complex demonstrated that the DPhPC coating acted as a barrier to Tb3+ efflux from the scaffolded vesicle and successfully sealed the porous hydrogel bead. Fluorescence quenching and ATR-IR spectroscopic measurements revealed that the lipid coating has a bilayer structure. The phytanoyl chains were found to exhibit significant trans-gauche isomerization, characteristic of the fluid liquid phase. However, no lipid lateral mobility was observed by fluorescence recovery after photobleaching (FRAP) measurements. The phosphocholine headgroup was found to be well hydrated and oriented such that the cationic choline group tucked in behind the anionic phosphate group, consistent with an electrostatic attraction between the cationic scaffold and zwitterionic lipid. The absence of lipid lateral mobility may be due to the strength of this attraction.

Analysis of Sphingolipid Synthesis and Transport by Metabolic Labeling of Cultured Cells with [³H]Serine.

Analysis of lipid biosynthesis by radioactive precursor incorporation provides information on metabolic rates and the identity of rate-limiting enzymes and transporters. The biosynthesis of sphingolipids in cultured cells is initiated in the endoplasmic reticulum (ER) by the formation of a sphingoid base from serine and palmitoyl-CoA. N-acylation of the sphingoid base produces ceramide, which is transported to the Golgi apparatus where phosphocholine or carbohydrate headgroups are added to form sphingomyelin (SM) and complex glycosphingolipids (GSLs), respectively. Herein is described a protocol to measure ceramide and SM biosynthesis in cultured cells based on [(3)H]serine incorporation at the first step in the pathway. The method can be used to assay the effect of pharmacological and genetic manipulations on ceramide synthesis and transport to the Golgi apparatus.

Driving Adsorbed Gold Nanoparticle Assembly by Merging Lipid Gel/Fluid Interfaces.

Surface forces between inorganic nanoparticles and lipid bilayer is of great relevance to biophysics, medicine, and nanobiotechnology. Adsorbed nanoparticles may influence the fluidity of the underlying lipids, which may in turn influence nanoparticle assembly. Herein three types of lipids (DOPC, Tc = -20 °C; DMPC, Tc = 23 °C; and DPPC, Tc = 41 °C) are used, all with the same phosphocholine (PC) headgroup. Gold nanoparticle (AuNP) color change is monitored as a function of lipid phase transition temperature (Tc), surface ligands on AuNPs, and temperature. Liposomes with higher fluidity induce much faster aggregation of AuNPs. Aside from the kinetic aspect of faster diffusion on fluid bilayers, this faster color change is attributed to the local lipid gelation and merging of gelled regions to eliminate the interface between different lipid phases.

Radical-induced fragmentation of phospholipid cations using metastable atom-activated dissociation mass spectrometry (MAD-MS)

The fragmentation pattern of several protonated 1+ phosphatidylcholines (PCs) was studied using low energy collision induced dissociation (CID) and helium metastable atom-activated dissociation (He-MAD). He-MAD of the protonated compounds produced a dominant phosphocholine head group at m/z 184 as well as typical sn-1 and sn-2 glycerol fragments such as [M+H-Rx−1CHCO]+ and [M+H-Rx−1CO2H]+. Within the aliphatic chain, He-MAD showed fragments consistent with high-energy collision induced dissociation (HE-CID) and products/pathways consistent with Penning ionization of the 1+ precursor ions to their respective radical dications. These Penning ionization products included both singly and doubly charged radical fragments, and the fragment ions are related to the number and position of double bonds in the acyl chains. Fragments created through HE-CID-like fragmentation followed classic charge remote fragmentation pathways including ladder-like fragmentation along the acyl chain, except for additional or missing peaks due to predictable rearrangement reactions. He-MAD therefore shows utility in being able to effectively fragment singly charged lipids into a variety of useful product ions using both radical and high-energy processes in the confines of a 3D ion trap.

Probing the position of resveratrol in lipid bilayers: A neutron reflectivity study

The effect of the natural antioxidant resveratrol on the structure of solid supported di-palmitoyl-phosphatidyl-choline (DPPC) bilayers in their fluid state was investigated by neutron reflectometry. Results reveal an accumulation of resveratrol (up to 25%, mol/mol) inside the headgroups and they exclude its presence in the hydrophobic core. The presence of resveratrol induces an increase of the average thickness and of the interfacial roughness of the headgroup layer. This may be due to a change of the tilt angle of the phosphocholine headgroups residing next to the resveratrol to a more upright orientation and leading to a reduction of the projected area per headgroup. This effect is propagated into the hydrophobic core, where the chain packing is modified despite the absence of resveratrol. When interacting with a DPPC/cholesterol membrane, resveratrol has a similar effect on the neighboring PC headgroups as in the cholesterol free membrane. The almost precise 1:1 insertion ratio (resveratrol:cholesterol) suggests that resveratrol is most probably inserted on top of the hydroxyl group of the cholesterol in between the PC headgroups. The ordering effect of cholesterol on the hydrophobic core is absent when both cholesterol and resveratrol are present. Most probably, the interaction of resveratrol with lipid membranes is non-specific.

Lymphocytes in atherosclerosis.

Atherosclerosis is the major cause of cardiovascular disease, and cardiovascular disease remains the primary cause of death in the United States. The primary complication for patients with type 2 diabetes mellitus (T2D) is cardiovascular disease.1–8 Because the incidences of T2D and obesity are drastically increasing in the United States and now worldwide, understanding how atherosclerosis is accelerated in these diseases becomes even more important to discern. Although many scientific advances in research on atherosclerosis have been made by multiple groups during the past 30 years, particularly about the function of endothelial and smooth muscle biology and macrophage foam cell formation in atherosclerotic plaque development, the roles of immune cells in regulating atherosclerosis onset and progression are still being elucidated. It was widely thought that immune cells played little part in atherogenesis until 1986, when Hansson et al9–12 reported the presence of lymphocytes within atherosclerotic lesions; yet it was many years later until it was widely accepted that immune cells play an important role in atherogenesis. Since 1986, the viewpoint has changed significantly, but details on lymphocyte subsets and mechanisms are still emerging. Moreover, controversy still exists on the roles that some lymphocyte subsets play in atherosclerosis. These discrepancies in findings are at least partly because of differences in mouse models used, diet composition, and length of time on diet for atherosclerosis measurements. However, despite a few ongoing controversies, there are many studies on immune cells in atherogenesis in mice that show clear findings.10,12–23 For example, CD4+ T lymphocytes have been shown to accelerate atherosclerosis.18,24–29 Activated CD4 T cells can be distinguished based on their phenotype, and the predominant CD4+ effector T cells studied to date in the context of atherosclerosis are Th1, …

Micelle-catalyzed domain swapping in the GlpG rhomboid protease cytoplasmic domain.

Three-dimensional domain swapping is a mode of self-interaction that can give rise to altered functional states and has been identified as the trigger event in some protein deposition diseases, yet rates of interconversion between oligomeric states are usually slow, with the requirement for transient disruption of an extensive network of interactions giving rise to a large kinetic barrier. Here we demonstrate that the cytoplasmic domain of the Escherichia coli GlpG rhomboid protease undergoes slow dimerization via domain swapping and that micromolar concentrations of micelles can be used to enhance monomer-dimer exchange rates by more than 1000-fold. Detergents bearing a phosphocholine headgroup are shown to be true catalysts, with hexadecylphosphocholine reducing the 26 kcal/mol free energy barrier by >11 kcal/mol while preserving the 5 kcal/mol difference between monomer and dimer states. Catalysis involves the formation of a micelle-bound intermediate with a partially unfolded structure that is primed for domain swapping. Taken together, these results are the first to demonstrate true catalysis for domain swapping, by using micelles that work in a chaperonin-like fashion to unfold a kinetically trapped state and allow access to the domain-swapped form.

The headgroup (a)symmetry strongly determines the aggregation behavior of single-chain phenylene-modified bolalipids and their miscibility with classical phospholipids.

In the present work, we describe the synthesis of two single-chain phenylene-modified bolalipids, namely PC-C17pPhC17-PC and PC-C17pPhC17-OH, with either symmetrical (phosphocholine) or asymmetrical (phosphocholine and hydroxyl) headgroups using a Sonogashira cross-coupling reaction as key step. The temperature-dependent aggregation behavior of both bolalipids in aqueous suspension was studied using transmission electron microscopy (TEM), differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) spectroscopy, small angle neutron scattering (SANS), and X-ray scattering. We show that different headgroup symmetries lead to a change in the aggregation behavior: Whereas PC-C17pPhC17-PC forms nanofibers with a diameter of 5.7 nm that transform into small ellipsoidal micelles at 23 °C, the PC-C17pPhC17-OH self-assembles into lamellae with bolalipid molecules in an antiparallel orientation up to high temperatures. Furthermore, the mixing behavior of both bolalipids with bilayer-forming phospholipids (DPPC and DSPC) was studied by means of DSC and TEM. The aim was to stabilize bilayer membranes formed of phospholipids in order to improve these mixed lipid vesicles for drug delivery purposes. We show that the symmetrical PC-C17pPhC17-PC is miscible with DPPC and DSPC; however, closed lipid vesicles are not observed, and elongated micelles and bilayer fragments are found instead. In contrast, the asymmetrical PC-C17pPhC17-OH shows no miscibility with phospholipids at all.

Nanoscale Packing Differences in Sphingomyelin and Phosphatidylcholine Revealed by BODIPY Fluorescence in Monolayers: Physiological Implications

Phosphatidycholines (PC) with two saturated acyl chains (e.g., dipalmitoyl) mimic natural sphingomyelin (SM) by promoting raft formation in model membranes. However, sphingoid-based lipids, such as SM, rather than saturated-chain PCs have been implicated as key components of lipid rafts in biomembranes. These observations raise questions about the physical packing properties of the phase states that can be formed by these two major plasma membrane lipids with identical phosphocholine headgroups. To investigate, we developed a monolayer platform capable of monitoring changes in surface fluorescence by acquiring multiple spectra during measurement of a lipid force–area isotherm. We relied on the concentration-dependent emission changes of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)-labeled PC to detect nanoscale alterations in lipid packing and phase state induced by monolayer lateral compression. The BODIPY-PC probe contained an indacene ring with four symmetrically located methyl (Me) substituents to enhance localization to the lipid hydrocarbon region. Surface fluorescence spectra indicated changes in miscibility even when force–area isotherms showed no deviation from ideal mixing behavior in the surface pressure versus cross-sectional molecular area response. We detected slightly better mixing of Me4-BODIPY-8-PC with the fluid-like, liquid expanded phase of 1-palmitoyl-2-oleoyl-PC compared to N-oleoyl-SM. Remarkably, in the gel-like, liquid condensed phase, Me4-BODIPY-8-PC mixed better with N-palmitoyl-SM than dipalmitoyl-PC, suggesting naturally abundant SMs with saturated acyl chains form gel-like lipid phase(s) with enhanced ability to accommodate deeply embedded components compared to dipalmitoyl-PC gel phase. The findings reveal a fundamental difference in the lateral packing properties of SM and PC that occurs even when their acyl chains match.

Effect of millimetre waves on phosphatidylcholine membrane models: a non-thermal mechanism of interaction.

The nonthermal biological effects of millimeter waves have been mainly attributed to the interaction with biological membranes. Several data on biomimetic membrane systems seem to support this conclusion. In this paper a mechanistic hypothesis is evaluated to explain such an interaction taking into account experimental NMR data on deuterium-labeled phospholipid vesicles. These data showed that millimeter waves induce a time and a hydration-dependent reduction of the water ordering around the phosphocholine headgroups. This effect is here interpreted as a change in membrane water partitioning, due to the coupling of the radiation with the fast rotational dynamics of bound water molecules, that results in a measurable relocation of water molecules from the inner to the outer binding regions of the membrane interface. When millimeter wave exposure is performed in the vicinity of the transition point, this effect can lead to an upward shift of the membrane phase transition temperature from the fluid to the gel phase. At a macroscopic level, this unique sensitivity may be explained by the universal dynamic behaviour of the membranes in the vicinity of the transition point, where a pretransitional increase of membrane area fluctuations, i.e., of the mean area per phospholipid headgroup, is observed. Exposure to millimeter waves increases the above fluctuations and enhances the second order character of the transition.

X-ray structure, thermodynamics, elastic properties and MD simulations of cardiolipin/dimyristoylphosphatidylcholine mixed membranes

Cardiolipins (CLs) are important biologically for their unique role in biomembranes that couple phosphorylation and electron transport like bacterial plasma membranes, chromatophores, chloroplasts and mitochondria. CLs are often tightly coupled to proteins involved in oxidative phosphorylation. The first step in understanding the interaction of CL with proteins is to obtain the pure CL structure, and the structure of mixtures of CL with other lipids. In this work we use a variety of techniques to characterize the fluid phase structure, material properties and thermodynamics of mixtures of dimyristoylphosphatidylcholine (DMPC) with tetramyristoylcardiolipin (TMCL), both with 14-carbon chains, at several mole percentages. X-ray diffuse scattering was used to determine structure, including bilayer thickness and area/lipid, the bending modulus, KC, and SXray, a measure of chain orientational order. Our results reveal that TMCL thickens DMPC bilayers at all mole percentages, with a total increase of ∼6Å in pure TMCL, and increases AL from 64Å2 (DMPC at 35°C) to 109Å2 (TMCL at 50°C). KC increases by ∼50%, indicating that TMCL stiffens DMPC membranes. TMCL also orders DMPC chains by a factor of ∼2 for pure TMCL. Coarse grain molecular dynamics simulations confirm the experimental thickening of 2Å for 20mol% TMCL and locate the TMCL headgroups near the glycerol-carbonyl region of DMPC; i.e., they are sequestered below the DMPC phosphocholine headgroup. Our results suggest that TMCL plays a role similar to cholesterol in that it thickens and stiffens DMPC membranes, orders chains, and is positioned under the umbrella of the PC headgroup. CL may be necessary for hydrophobic matching to inner mitochondrial membrane proteins. Differential scanning calorimetry, SXray and CGMD simulations all suggest that TMCL does not form domains within the DMPC bilayers. We also determined the gel phase structure of TMCL, which surprisingly displays diffuse X-ray scattering, like a fluid phase lipid. AL=40.8Å2 for the ½TMCL gel phase, smaller than the DMPC gel phase with AL=47.2Å2, but similar to AL of DLPE=41Å2, consistent with untilted chains in gel phase TMCL.

Determinants of binding of oxidized phospholipids on apolipoprotein (a) and lipoprotein (a)

Oxidized phospholipids (OxPLs) are present on apolipoprotein (a) [apo(a)] and lipoprotein (a) [Lp(a)] but the determinants influencing their binding are not known. The presence of OxPLs on apo(a)/Lp(a) was evaluated in plasma from healthy humans, apes, monkeys, apo(a)/Lp(a) transgenic mice, lysine binding site (LBS) mutant apo(a)/Lp(a) mice with Asp(55/57)→Ala(55/57) substitution of kringle (K)IV10)], and a variety of recombinant apo(a) [r-apo(a)] constructs. Using antibody E06, which binds the phosphocholine (PC) headgroup of OxPLs, Western and ELISA formats revealed that OxPLs were only present in apo(a) with an intact KIV10 LBS. Lipid extracts of purified human Lp(a) contained both E06- and nonE06-detectable OxPLs by tandem liquid chromatography-mass spectrometry (LC-MS/MS). Trypsin digestion of 17K r-apo(a) showed PC-containing OxPLs covalently bound to apo(a) fragments by LC-MS/MS that could be saponified by ammonium hydroxide. Interestingly, PC-containing OxPLs were also present in 17K r-apo(a) with Asp(57)→Ala(57) substitution in KIV10 that lacked E06 immunoreactivity. In conclusion, E06- and nonE06-detectable OxPLs are present in the lipid phase of Lp(a) and covalently bound to apo(a). E06 immunoreactivity, reflecting pro-inflammatory OxPLs accessible to the immune system, is strongly influenced by KIV10 LBS and is unique to human apo(a), which may explain Lp(a)'s pro-atherogenic potential.