Molar Ratio Of Phosphatidylcholine Research Articles

The effect of non-receptor-mediated uptake of cholesterol on intracellular cholesterol metabolism in human skin fibroblasts.

Unilamellar lipid vesicles of various cholesterol:phosphatidylcholine molar ratios were used to alter, via passive exchange at the plasma membrane, the cellular free cholesterol content of cultured human skin fibroblasts which had been preincubated in lipoprotein-deficient serum. The effects of these net surface transfers of cholesterol on cellular cholesterol biosynthesis, cholesterol esterification and low density lipoprotein (LDL) binding were determined and were compared with the effects of cholesterol delivered to the cell interior via the receptor-mediated endocytosis of LDL. Both LDL and cholesterol-rich lipid vesicles increased cell cholesterol within 6 h. Cells exposed to LDL also showed, within 6 h, decreased cholesterol synthesis, decreased LDL binding and increased cholesterol esterification. Cells incubated with the cholesterol-rich vesicles showed similar changes but these were delayed and did not occur until 24 h. Fibroblasts incubated with cholesterol-free phosphatidylcholine vesicles had decreased cell cholesterol, increased cholesterol synthesis, increased LDL binding, and decreased esterification, but only after 24 h of incubation. These results suggest that passive net transfers of cholesterol occurring at the cell surface can with time modulate intracellular cholesterol metabolism. These findings are consistent with the idea that the movement of cholesterol from the cell surface to the cell interior is a limited and relatively slow process.

Serum albumin enhances the uptake of [3H]cholesterol from phosphatidylcholine vesicles by cultured human fibroblasts.

Cultured human lung fibroblasts, incubated with cholesterol/phosphatidylcholine vesicles (cholesterol: phosphatidylcholine molar ratio 1:1) incorporated vesicle [3H]-cholesterol linearly for at least 48 h by an exchange process without gaining sterol mass. The incorporation of [3H]cholesterol by the cells was markedly enhanced in the presence of purified bovine serum albumin. A fraction of the incorporated vesicle [3H]cholesterol was esterified by the cells.

Binding of cyclosporine by human lymphocytes and phospholipid vesicles.

The purpose of this investigation was to identify and characterize a possible plasma membrane receptor for cyclosporine (CsA) on human lymphoid cells. Binding of 3H-CsA by normal human lymphocytes and purified lymphoid subpopulations was examined using ligand concentrations ranging from 5 X 10(-7) to 10(-10) M. Specificity was determined using a 500-fold excess of unlabeled drug. No differences were observed in the uptake of 3H-CsA by B cells or T cells using Cartesian or semilogarithmic analyses. Scatchard analysis of the specific binding of CsA by normal peripheral blood lymphocytes yielded two dissociation constants: a high affinity site with a KD of 2 to 6 X 10(-9) M and a low affinity site with a KD of about 10(-7) M CsA. Scatchard analysis of specific CsA uptake by purified splenic T cells and B cells showed both populations to exhibit a low affinity site (KD = 3 to 6 X 10(-7) M and 5 to 8 X 10(-7) M, respectively), whereas only B cells bore a high affinity site (KD = 2 X 10(-9) M). Analysis of specific CsA uptake by cultured human kidney cells and phospholipid vesicles (1:1 molar ratio of phosphatidylcholine:cholesterol) showed both these targets to display a single, low affinity binding site (KD = 1 X 10(-7) M and 2 X 10(-8) M, respectively). The relevance of the low affinity site to CsA-mediated in vitro immunosuppression was suggested by the need for 10(-7) M CsA to achieve 50% suppression of lymphoproliferation during mixed lymphocyte culture. Taken together, these data argue against the existence of a specific CsA receptor on the surface of human T cells. Although a role for the high affinity B cell receptor in some types of CsA-induced immunomodulation cannot be excluded, the present study suggests that immunosuppression is achieved by the partitioning of the hydrophobic CsA molecule into the membrane lipid bilayer, thereby perturbing homeostatic control of membrane function.

Characterization of discoidal complexes of phosphatidylcholine, apolipoprotein A-I and cholesterol by gradient gel electrophoresis

Complexes of egg yolk phosphatidylcholine and apolipoprotein A-I were prepared by a detergent (sodium cholate)-dialysis method and characterized by gradient gel electrophoresis, gel filtration, electron microscopy and chemical analysis. Multicomponent electrophoretic patterns were obtained indicating formation of at least eight classes of discoidal complexes. The relative contribution of the different classes to the electrophoretic pattern was a function of the molar ratio of phosphatidylcholine: apolipoprotein A-I in the interaction mixture. Molar ratios of phosphatidylcholine:apolipoprotein A-I in isolated complexes were strongly and positively correlated with disc diameter obtained by electron microscopy. Incorporation of unesterified cholesterol into phosphatidylcholine/apolipoprotein A-I interaction mixtures also resulted in formation of unique complexes but with considerably different particle size distributions relative to those observed in the absence of cholesterol. One common consequence of cholesterol incorporation into interaction mixtures of 87.5:1 and 150:1 molar ratio of phosphatidylcholine: apolipoprotein A-I was the disappearance of a major complex class with diameter of 10.8 nm and the appearance of a major component with diameter of approximately 8.8 nm. Electrophoretic patterns of cholesterol-containing complexes showed a strong similarity to patterns recently published for high density lipoproteins from plasma of lecithin: cholesterol acyltransferase-deficient subjects, suggesting that the complexes formed in vitro by the detergent-dialysis method may serve as appropriate models for investigation of the origins of the HDL particle size distribution.

The effect of dimethylsulfoxide on the lipid composition of inducible and non inducible Friend leukemia cells

Changes in phospholipids and in fatty acid composition were observed when Friend leukemia cells (FLC), but not the resistant one (RFLC) were exposed to DMSO. As a result of these changes, the molar ratio of phosphatidyl choline (PC) to phosphatidyl ethanolamine (PE) and the relative amount of arachidonic acid (20:4) isolated from total lipids, PC and PE increased in differentiated cells. We therefore conclude that erythroid differentiation induced by DMSO is related to changes in the lipid components of FLC membranes.

Liposomally trapped AraCTP to overcome AraC resistance in a murine lymphoma in vitro.

Two cell lines, one sensitive and one resistant to the cytotoxic effects of cytosine arabinoside (AraC) were studied in vitro as a drug-resistance model. The sensitivity of these cell lines, to the effects of free and liposomally trapped AraC and AraCTP as well as empty liposomes alone and mixed with free drug, was studied. This was done by following the inhibition of [3H]-dT incorporation into cellular DNA during exposure to the various drugs and liposomes. Some of the liposomal-lipid compositions inhibited [3H]-dT incorporation at very low concentrations, which made them unsuitable for further study. Liposomes composed of a 7:2:1 molar ratio of phosphatidylcholine:cholesterol:phosphatidic acid were selected as a suitable non-inhibitory carrier. Sensitivity of the two cell lines to free AraC differed by 3 logs, when compared in the [3H]-dT-incorporation assay. The resistant cell line was studied further, and was found to be up to 2 logs more sensitive to AraCTP when given in liposomes than to either the free drug alone or mixed with empty liposomes. It appears from these studies that liposomes are able to help overcome drug resistance in this cell line in vitro.

Influence of environmental temperature on mitochondrial membranes

Mitochondrial phospholipids from goldfish lateral line muscle were analysed with respect to polar and apolar groups. Groups of 20 goldfish, acclimated to 5, 20 and 30°C, were used. Temperature-induced shifts of both polar and apolar groups of the mitochondrial phospholipids were observed. The fatty acid composition of mitochondrial phospholipids is characterized by a large amount of polyenoic acids, dominated by docosahexaenoic acid and by octadecadienoic acid. At the higher acclimation temperatures, a significant decrease in docosahexaenoic acid is found. However, the resultant effect of environmental temperature on the degree of unsaturation is small, in contrast to the marked effect on mean chain length. Pronounced changes in the molar ratio of phosphatidylcholine and phosphatidylethanolamine are seen; a decrease in mitochondrial phosphatidylcholine is observed at low acclimation temperature, which is compensated for by a nearly equal increase in phosphatidylethanolamine. The main phospholipids are, apparently, phosphatidylcholine, phosphatidylethanolamine and cardiolipin, comprising 90% of the total pool of 12 species. It is found that the anionic nature of the phospholipids is increased at low acclimation temperatures. We discuss this effect and its probable importance in the stabilization of the surface potential of the mitochondrial membranes.

Tissue distribution and tumour localization of 99m-technetium-labelled liposomes in cancer patients.

The possible use of liposomes (phospholipid vesicles) to direct cytotoxic drugs to tumours has led us to investigate the tissue localization of i.v. injected 99m-Tc-labelled liposomes in cancer patients. Twenty mg or 300 mg doses of liposomal lipid (7:2:1 molar ratio of phosphatidylcholine : cholesterol : phosphatidic acid) were used in a study of 13 patients with advanced cancer and one with polycythaemia rubra vera (PRV). In all cases except the patient with PRV the major site of uptake of the label was the liver and spleen. In the patient with PRV the liver uptake was greatly reduced and the major site of uptake was found in regions corresponding to marrow. With the exception of one patient with a primary hepatoma, there was no significant tumour uptake of the label.

The activity of phospholipase D on aggregates of phosphatidylcholine, dodecylsulfate and Ca2+.

Phospholipase D from peanut seeds catalyzes the hydrolysis of phosphatidylcholine (PC) to phosphatidic acid and choline. The catalytically active enzyme has a molecular weight of 200,000. Smaller subunits were obtained upon dilution, low pH or in the presence of certain detergents. Activators and Ca2+ ions are required for the hydrolysis; the amphipath sodium dodecylsulfate (SDS) is the most effective in activating the reaction. The dependence of the reaction on SDS is complex. Some physical properties of the PC — SDS mixed aggregates, as a function of their concentration ratio, were studied. Proton PMR, turbidity and ultracentrifugal data in the absence or presence of Ca2+, indicate that “dispersible” PC — SDS mixed micelles are formed at any SDS to PC molar ratio below 4. Upon the addition of Ca2+ however, optimal dispersibility was demonstrated at SDS to PC molar ratio ranging from 0.25–0.65, probably because high concentrations of the insoluble calcium dodecylsulfate causes a precipitation of the mixed micelles. Kinetic experiments showed that PC in low speed precipitated aggregates is not available for hydrolysis by phospholipase D. On the other hand PC aggregates obtained by high speed centrifugation were degraded by the enzyme. These observations serve as a basis for a partial explanation of the dependence of the enzymatic activity on the presenca of SDS.

Nuclear magnetic resonance studies of amphiphile hydration: Effects of cholesterol on phosphatidyl choline hydration

Water which remains unfrozen at −25 °C in the presence of phosphatidyl choline (PC) gives rise to a proton magnetic resonance signal which can be used to measure the hydration of single-walled vesicles and multilamellar liposomes of PC. The proton magnetic resonance signal of the unfrozen water in these systems is strongly dependent upon the nature of the molecular domain in which the water is situated. For example, at cholesterol to PC molar ratios below 35 mol%, the vesicle hydration signal consists of a relatively narrow symmetric peak (line width, ~150 Hz). At higher molar ratios, however, rather broad asymmetric signals appear (line widths, ~300–1000 Hz) which indicate that when significant quantities of cholesterol are packed in the bilayer there must be regions in which there is a preferred direction for motion of the unfrozen water. It is possible to solubilize significant quantities of cholesterol by sonicating it in concentrated solutions of sodium dodecyl sulfate. Addition of cholesterol to PC vesicles via these sodium dodecyl sulfate-cholesterol complexes caused hydration changes in the PC, which, at high cholesterol to PC molar ratios, paralleled the effects of cholesterol on PC hydration in homogeneous vesicles in which the cholesterol and PC were simply cosonicated.

Phospholipids and detergents as effectors in the liver microsomal hydroxylase system

When rat liver microsomes were delipidated with phospholipases and organic solvent extractions, 80–85% of the phospholipids and essentially all neutral lipids were removed. After delipidation, the measurable amount of cytochrome P-450, determined by the CO-difference spectra, represented only 20–25% of the amount originally present. However, when the delipidated microsomes were treated with different phospholipids (phosphatidylcholine being most effective) or non-ionic detergents, the measurable amount of cytochrome P-450 increased by up to three times. This renaturation of cytochrome P-450 was inhibited if cholesterol was added together with phospholipid. Also purified delipidated rabbit liver microsomal cytochrome P-450 (10–15 nmol per mg of protein in the original preparation) was renatured after addition of phosphatidylcholine. In these experiments NaIO 4-supported 6β-hydroxylation of androstenedione was stimulated 2.5-fold; the activity of the hydroxylase was well correlated to the amount of renatured cytochrome P-450 present. Maximal stimulation of the hydroxylase by phosphatidylcholine was obtained at a molar ratio of phosphatidylcholine: cytochrome P-450 of about 10 which in contrast to the corresponding ratio of 1000 needed to obtain saturation of stimulation of the 6β-hydroxylase by phosphatidylcholine in NADPH-supported hydroxylation reactions. It is suggested that the phospholipid participates in the liver microsomal hydroxylase system by: (1) specific binding to cytochrome P-450 so that the enzyme assumes the active conformation, and (2) formation of a fluid environment to facilitate electron transport from NADPH to cytochrome P-450.

CHROMATOGRAPHICALLY HOMOGENEOUS LECITHIN FROM EGG PHOSPHOLIPIDS.

AbstractChromatographically homogeneous egg lecithin, as determined by TLC on Silica Gel G, has been isolated from crude egg phosphatides by column chromatography on alumina through modification of existing, lengthy methods. The modified method involved application of crude egg phosphatides to a column of alumina in the proportion of 1 g phosphatide/25 g alumina, and elution of the lecithin fraction with the 2‐component solvent system chloroform:methanol, 9:1 by vol. This method of purification separated lecithin from other choline and non‐choline components of crude phosphatides, avoided overloading of the alumina column, and made unnecessary the need for a second chromatographic fractionation of partially purified lecithin on silicic acid, which is needed in existing methods of purification of lecithin.The use of fresh yolks permitted easier removal of pigment from the final product than was possible with commercially dried yolks.Phosphatides extracted from dried yolks were much more highly colored than were the phosphatides extracted from fresh yolks and the color presisted through chromatography on alumina.The fatty acid/phosphorus molar ratio of the purified lecithin was 2.00, which is the theoretical FA/P molar ratio of phosphatidylcholine; other materials with this ratio were not present.