Alterations in serum phosphatidylcholine fatty acyl species by eicosapentaenoic and docosahexaenoic ethyl esters in patients with severe hypertriglyceridemia.

A new and sensitive method has been developed to analyze the molecular species of glycerophospholipids. This method was used to examine the effects of hypolipidemic intervention with n-3 fatty acids on the serum phosphatidylcholine species in severely hypertriglyceridemic patients. The drug treated group (n = 19) received 4 g/day of an 85% concentrate of the ethyl esters of eicosapentenoic and docosahexaenoic acids for 6 weeks. Control patients (n = 21) received 4 g/day of ethyl esters of corn oil fatty acids. To evaluate the effects of n-3 fatty acids upon serum phosphatidylcholines (PCs), sera from treated and control patients were analyzed before and after 6 weeks of intervention. PCs isolated from sera were digested with phospholipase C to diglycerides, derivatized with 7-methoxycoumarin-3-carbonyl azide, and analyzed by reverse phase high performance liquid chromatography (HPLC) with fluorescence detection. Pre-intervention serum PC species were, in order of decreasing concentration C16:0,18:2, C16:0,18:1, C18:0,18:2, C16:0,20:1, C16:0,22:0, C18:0,20:4, C16:0,16:0, C18:0,18:1, C18:1,18:2, C16:0,20:5, and C18:1,20:5. In the treated patients, mean increases of 300% in C16:0,20:5 and of 160% in C16:0,22:6 species were observed. There were no significant changes in the molecular species of the serum phosphatidylcholines in the group receiving the corn oil ethyl esters. The cumulative relative percentages for each of the individual fatty acids measured by HPLC were comparable to those determined by gas-liquid chromatography (GLC). In the treated group plasma triglycerides were reduced 26%, while they were increased by 7% in the placebo group. Our data showed that incorporation of eicosapentaenoic and docosahexaenoic acid into the serum PCs occurred within 6 weeks primarily in the C16:0,20:5 and C16:0,22:6 species and were usually accompanied by a reduction in plasma triglyceride.

Free radical-induced alterations in endothelial cell function.

Free radical-induced injury to the arterial wall has been implicated in the pathogenesis and progression of atherosclerosis. To model the in vitro effects of free radicals on endothelial cell function, protein and lipid synthesis were measured after exposing cells to a superoxide generating system of xanthine (X = 100 microM) and xanthine oxidase (XO = 0.2 units). Total protein synthesis, measured by [35S]methionine uptake, decreased by 87.65 +/- 2.04% over 4 hr compared to controls (P < 0.05). Examination of lipid synthesis by high-performance liquid chromatography in cells prelabeled with either [3H]oleic acid or [3H]sodium acetate revealed alterations in all lipid classes. Phospholipid and neutral glyceride synthesis significantly decreased in a time- and dose-dependent fashion compared to controls (two-way ANOVA). In contrast, cholesterol synthesis and lipid peroxidation increased in a time- and dose-dependent fashion. When X = 200 microM and XO = 0.3 units, there was a statistically significant increase in cholesterol synthesis and lipid peroxidation within 24 hr (Tukey's HSD). We conclude that there is evidence of endothelial cell injury as measured by decreases in protein, glyceride, and phospholipid synthesis. The concurrent increases in lipid peroxidation and cholesterol synthesis may explain the relationship between free radical injury and the pathogenesis of atherosclerosis.

Mechanism of cellular phospholipid efflux.

Plasma phospholipid binding to cell-derived cholesterol is important in reverse cholesterol transport, a key step in the regression of atherosclerosis. However, the mechanism by which phospholipids are transferred from cells to plasma remains unclear. [3H]Choline-labeled phospholipid efflux from fibroblasts has been studied using plasma and its components as acceptors. The kinetics were resolved into a fast component (k1 = 0.119 +/- 0.23 min-1) that corresponded to high-affinity binding of high-density lipoproteins (HDL) to the cell surface and a slow component (k2 = 0.0047 +/- 0.0009 min-1) due to protein-mediated desorption (n = 3). Altering the donor charge with heparinase or the acceptor charge by acetylation abolished the fast component, while the slow phase was unchanged. Only HDL displayed biexponential kinetics, comparable to whole plasma. Half-lives for low-density lipoprotein and very-low-density lipoprotein were t1/2 = 278 +/- 22 min and t1/2 = 1003 +/- 147 min, respectively. In the absence of transfer factor, HDL alone significantly reduced phospholipid efflux (t1/2 = 663 min). Phospholipid transfer protein restored biexponential kinetics. We conclude that cell membranes are a potentially important source of plasma phospholipids and that protein-mediated transfer to HDL is the major route for cell-to-plasma transfer. This step represents a locus for anti-atherosclerotic intervention.

Plasma triglycerides determine low density lipoprotein composition, physical properties, and cell-specific binding in cultured cells.

The relationship between the plasma triglycerides and the LDL triglycerides of 30 normal and 48 hypertriglyceridemic subjects has been quantified; the data fit a simple adsorption isotherm, LDL triglyceride/(LDL triglyceride+LDL cholesterol ester) = 0.65 plasma triglyceride/(464 + plasma triglyceride). In vitro transfer of triglyceride from concentrated VLDL to VLDL-depleted plasma produced triglyceride-rich LDL that had similar properties. LDL uptake by HepG2 cells increased with LDL triglyceride content whereas the reverse was found with skin fibroblasts. At 37 degrees C, the cores of both normal and hypertriglyceridemic LDL were isotropic liquids. Circular dichroic spectra revealed no difference in the secondary structure of normal and triglyceride-rich LDL. The affinity of monoclonal antibody MB47, which binds to the receptor ligand of apo B-100 was independent of LDL triglyceride content. MB3, which binds near residue 1022 of apo B-100, showed a triglyceride-dependent decrease in affinity for LDL from hypertriglyceridemic subjects and from in vitro incubations. LDL with an elevated triglyceride content formed in vitro had reduced proteolytic cleavage of apo B-100 by Staphylococcus aureus V8 protease. From these data, we infer that (a) LDL triglyceride is a predictable function of plasma triglyceride, (b) triglyceride induces subtle changes in apo B-100 structure at a site that is remote from the putative receptor binding ligand, and (c) the triglyceride-dependent receptor-binding determinants of apo B-100 are recognized differently by fibroblasts and HepG2 cells.

Guanidine hydrochloride stabilization of a partially unfolded intermediate during the reversible denaturation of protein disulfide isomerase.

The reversible denaturation of protein disulfide isomerase proceeds through intermediates that are stabilized by interaction with guanidine hydrochloride. At pH 7.5, the equilibrium denaturation by urea is completely reversible and the transition can be reasonably well-described by a two-state model involving only native and denatured forms. In comparison, the equilibrium denaturation by guanidine hydrochloride occurs in two distinct steps. In the presence of a low constant amount of guanidine hydrochloride (0.5-1.4 M), urea denaturation also becomes biphasic, suggesting the accumulation of an intermediate species that is stabilized by specific interaction with guanidine hydrochloride but not by high concentrations of other salts or other denaturants.

Apolipoproteins C-I, C-II, and C-III: kinetics of association with model membranes and intermembrane transfer.

The apoproteins (apo) C-I, C-II, and C-III are low molecular weight amphiphilic proteins that are associated with the lipid surface of the plasma chylomicron, very low density lipoprotein (VLDL), and high-density lipoprotein (HDL) subfractions. Purified apoC-I spontaneously reassociates with VLDL, HDL, and single-bilayer vesicles (SBV) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. ApoC-I also transfers reversibly from VLDL to HDL and from VLDL and HDL to SBV. The kinetics of association of the individual apoC proteins with SBV are second order overall and first order with respect to lipid and protein concentrations. At 37 degrees C, the rates of association were 2.5 x 10(10), 4.0 x 10(10) and 3.8 x 10(10) M-1 s-1 for apoC-I, apoC-II, and apoC-III, respectively. Arrhenius plots of association rate vs temperature were linear and yielded activation energies of 11.0 (apoC-I), 9.0 (apoC-II), and 10.6 kcal/mol (apoC-III). The kinetics of vesicle to vesicle apoprotein transfer are biexponential for intermembrane transfer, indicating two concurrent transfer processes. Rate constants at 37 degrees C for the fast component of dissociation were 11.7, 9.5, and 9.9 s-1, while rate constants for the slow component were 1.3, 0.6, and 0.9 s-1 for apoC-I, apoC-II, and apoC-III, respectively. The dissociation constants, Kd, of apoC-I, apoC-II, and apoC-III bound to the surface monolayer of phospholipid-coated latex beads were 0.5, 1.4, and 0.5 microM, respectively. These studies show that the apoC proteins are in dynamic equilibrium among phospholipid surfaces on a time scale that is rapid compared to lipolysis, lipid transfer, and lipoprotein turnover.

Ether phosphatidylcholines: comparison of miscibility with ester phosphatidylcholines and sphingomyelin, vesicle fusion, and association with apolipoprotein A-I.

Nonhydrolyzable matrices of ether-linked phosphatidylcholines (PCs) and sphingomyelin have been used to study the mechanism of action of lipolytic enzymes. Since ether PCs, sphingomyelin, and ester PCs vary in the number of hydrogen bond donors and acceptors in the carbonyl region of the bilayer, we have examined several physical properties of ether PCs and sphingomyelin in model systems to validate their suitability as nonhydrolyzable lipid matrices. The intermolecular interactions of ether PCs with ester PCs, sphingomyelin, and cholesterol were investigated by differential scanning calorimetry. Phase diagrams constructed from the temperature dependence of the gel to liquid-crystalline phase transition of 1,2-O-dihexadecyl-sn-glycero-3-phosphocholine (DPPC-ether) and 1,2-O-ditetradecyl-sn-glycero-3-phosphocholine (DMPC-ether) with both 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) demonstrated complete lipid miscibility in the gel and liquid-crystalline phases. Additionally, phase diagrams of egg yolk sphingomyelin (EYSM) with DMPC or DMPC-ether and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1,2-O-dioctadecyl-sn-glycero-3-phosphocholine (DSPC-ether) demonstrated no major differences in miscibility of EYSM in ester and ether PCs. The effect of 10 mol % cholesterol on the thermal transitions of mixtures of ester and ether PCs also indicates little preference of cholesterol for either lipid. The fusion of small single bilayer vesicles of DMPC, DMPC-ether, DPPC, and DPPC-ether to larger aggregates as determined by gel filtration indicated that the ester PC vesicles were somewhat more stable.(ABSTRACT TRUNCATED AT 250 WORDS)