Phospholipid Content Of Cells Research Articles

Analysis of Live Single Cells by Confocal Microscopy and High-Resolution Mass Spectrometry to Study Drug Uptake, Metabolism, and Drug-Induced Phospholipidosis.

The analysis of large numbers of cells from a population results in information that does not reflect differences in cell phenotypes. Individual variations in cellular drug uptake, metabolism, and response to drug treatment may have profound effects on cellular survival and lead to the development of certain disease states, drug persistence, and resistance. Herein, we present a method that combines live cell confocal microscopy imaging with high-resolution mass spectrometry to achieve absolute cell quantification of the drug amiodarone (AMIO) and its major metabolite, N-desethylamiodarone (NDEA), in single liver cells (HepG2 and HepaRG cells). The method uses a prototype system that integrates a confocal microscope with an XYZ stage robot to image and automatically sample selected cells from a sample compartment, which is kept under growth conditions, with nanospray tips. Besides obtaining the distributions of AMIO and NDEA cell concentrations across a population of individual cells, as well as variabilities in drug metabolism, the effect of these on phospholipidosis and cell morphology was studied. The method was suited to identify subpopulations of cells that metabolized less drug and to correlate cell drug concentrations with cell phospholipid content, cell volume, sphericity, and other cell phenotypic features. Using principal component analysis (PCA), the treated cells could be clearly distinguished from vehicle control cells (0 μM AMIO) and HepaRG cells from HepG2 cells. The potential of using multidimensional and multimodal information collected from single cells to build predictive models for cell classification is demonstrated.

Phosphoinositide-Dependent Signaling in Cancer: A Focus on Phospholipase C Isozymes.

Phosphoinositides (PI) form just a minor portion of the total phospholipid content in cells but are significantly involved in cancer development and progression. In several cancer types, phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] play significant roles in regulating survival, proliferation, invasion, and growth of cancer cells. Phosphoinositide-specific phospholipase C (PLC) catalyze the generation of the essential second messengers diacylglycerol (DAG) and inositol 1,4,5 trisphosphate (InsP3) by hydrolyzing PtdIns(4,5)P2. DAG and InsP3 regulate Protein Kinase C (PKC) activation and the release of calcium ions (Ca2+) into the cytosol, respectively. This event leads to the control of several important biological processes implicated in cancer. PLCs have been extensively studied in cancer but their regulatory roles in the oncogenic process are not fully understood. This review aims to provide up-to-date knowledge on the involvement of PLCs in cancer. We focus specifically on PLCβ, PLCγ, PLCδ, and PLCε isoforms due to the numerous evidence of their involvement in various cancer types.

Alteration of lipid fatty acid profile and cationic fluxes in ventricular cardiomyocytes from ω3-depleted rats

The bolus intravenous injection of a medium-chain triglyceride:fish oil emulsion was recently found to increase within 60 min the cell phospholipid content in long-chain polyunsaturated omega3 fatty acids and, hence, proposed as a potential tool to prevent cardiac arrhythmia in subjects with a decreased dietary intake of such fatty acids. In the present study, ventricular cardiomyocytes from second generation rats depleted in omega3 fatty acids were found to display the same changes in the phospholipid fatty acid pattern as that previously documented in the cardiac muscle and endothelium of such rats, altered 86Rb and 45Ca fluxes with emphasis on a decrease in both K+ inflow and K+ content and an increase in both Ca2+ inflow and content. The alteration of K+ inflow could not be attributed to a decrease in ouabain-sensitive Na+,K+-ATPase activity as measured in cell homogenates. The cationic alterations were corrected, in part at least, by the prior intravenous injection of the medium-chain triglyceride:fish oil emulsion 60 min before sacrifice of the omega3-depleted rats.

Preclinical investigations of a medium-chain triglyceride:fish oil emulsion III. Experiments in cultured endothelial cells

The bolus intravenous injection of a novel medium-chain triglyceride:fish oil emulsion was recently reported to provoke a rapid and sustained increase in the cell phospholipid content of long-chain polyunsaturated omega3 fatty acids in both rats and human subjects. This report deals mainly with a comparison between this and other emulsions, as well as albumin-bound omega3 fatty acids, in terms of the time course, reversibility and concentration dependency for the incorporation of the omega3 fatty acids in the phospholipids of cultured endothelial cells. The results document that the new emulsion is quite efficient for a rapid and sustained enrichment of phospholipids in long-chain polyunsaturated omega3 fatty acids. The potential beneficial effects of such an enrichment in terms of aortic endothelial and cardiac function are emphasized.

Protection of aortic endothelial function in both normal and diabetic rats by intravenous bolus injection of a medium-chain triglyceride: fish oil emulsion

The bolus intravenous injection of a novel medium-chain triglyceride:fish oil emulsion (MCT:FO) was recently proposed as a tool to provoke a rapid and sustained increase of cell phospholipid content in long-chain polyunsaturated omega3 fatty acids, for instance in selected subjects prior to anesthesia and surgery. In this study, therefore, the possible protective effect of MCT:FO upon aortic endothelial function was investigated in both normal and diabetic rats. The animals were injected intravenously 20 h before sacrifice with 1.0 ml of either saline, MCT:FO or a control medium-chain triglyceride:long-chain triglyceride emulsion. The vasomotor response of isolated aortic rings was then explored by assessing the relaxation provoked by increasing concentrations of acetylcholine in rings contracted with phenylephrine. Such measurements were performed before and after exposure of the aortic rings to suitable concentrations of oxidized LDL. In both normal and diabetic rats, the prior injection of the MCT:FO emulsion protected the aortic rings against the deleterious effect of oxidized LDL. In the diabetic rats, a beneficial effect of the MCT:FO emulsion was even observed prior to exposure of the aortic rings to oxidized LDL. These findings support the view that this novel procedure is indeed appropriate to protect endothelial function against oxidative stress.

The Fatty Acid Profile of Buccal Cheek Cell Phospholipids Is a Noninvasive Marker of Long-Chain Polyunsaturated Fatty Acid Status in Piglets

The fatty acid pattern of cheek cell phospholipids has been proposed as a noninvasive marker of long-chain polyunsaturated fatty acid (PUFA) status. However, the cheek cell phospholipid fatty acid pattern has been compared only with that of plasma and erythrocytes. The objective of this study was to assess the extent to which the fatty acid profile of cheek cell phospholipids reflects that of tissue phospholipids. Piglets (n = 31; 6 d old) were fed five formula diets differing in total fat and fatty acid composition. After 14 d of consuming the assigned diets, cheek cell plasma, erythrocyte, liver, muscle, adipose tissue, retina and brain samples were collected for determination of the phospholipid fatty acid patterns. There were significant correlations between the cheek cell phospholipid content of most PUFA and the content of these fatty acids in tissue phospholipids (r = 0.509-0.951, P < 0.01). The cheek cell phospholipid content of most of the PUFA, except 20:4(n-6), reflected that of other tissue phospholipids as well as, or nearly as well as the contents of plasma and/or erythrocyte phospholipids. The correlations between the 22:6(n-3) contents of cheek cell, plasma, or erythrocyte phospholipids and those of brain and retina phospholipids were relatively poor (r = 0.596-0.737, P < 0.001). We conclude that the fatty acid pattern of cheek cell phospholipid can be used as a noninvasive marker of PUFA status, but it is not a better index than the pattern of plasma or erythrocyte phospholipids, particularly for assessing the fatty acid pattern of organs with slow fatty acid incorporation and/or turnover rates.

Phospholipid Composition and Phospholipase Activity of Euglena

The phospholipid content of Euglena cells and changes in lipid components during the preservation of harvested cells were investigated. Analysis of phospholipid content of cells cultured under heterotrophic conditions in the dark indicated phosphatidylinositol as well as phosphatidylcholine to be the major components of pospholipid, phosphatidylserine to be absent.When cells were aseptically preserved at 25°C for 7 d, the phospholipid fraction decreased and free fatty acid fraction increased, possibly due to phospholipase reactions. Phospholipase activity was thus determined using phosphatidylcholine, which markedly decreased during the course of preservation, as the substrate. It was thus possible to detect phospholipase A, C, and D activity.

Erythrocyte cation transport systems and membrane lipids in insulin-dependent diabetes.

The relationship between erythrocyte cation transport systems and membrane and plasma lipids was examined in normal men and patients with insulin-dependent diabetes mellitus (IDDM). Different measurements of erythrocyte transport systems were obtained in patients with IDDM and in age- and weight-matched healthy men: Na+:Li+ countertransport activity or Li(+)-stimulated Na+ efflux, Na+:K+ cotransport activity or bumetanide-sensitive Na+ efflux, Na+:K+ pump activity or ouabain-sensitive Na+ efflux, and the ouabain- and bumetanide-resistant Na+ and K+ fluxes or the ground membrane permeability for Na+ and K+ as well as the intraerythrocyte Na+, K+, and Mg2+ concentrations. Plasma cholesterol, triglycerides, phospholipids, low- and high- density lipoprotein cholesterol, and erythrocyte membrane cholesterol and phospholipid content were obtained from the fasting subjects. The patients with IDDM had an elevated (P < .05 or less) erythrocyte Na+:Li+ countertransport activity, ground membrane leak for K+, intraerythrocyte K+ concentration, and erythrocyte membrane cholesterol content, but a lower red blood cell phospholipid content. In single regression analysis, the erythrocyte Na+:Li+ countertransport, Na+:K+ cotransport, and Na+:K+ pump activity and ground membrane leak for Na+ and K+ were inversely related to the red blood cell membrane lipid content. Our data in patients with IDDM show that a decreased erythrocyte membrane lipid content was accompanied by a higher erythrocyte Na+:Li+ countertransport, Na+:K+ cotransport, and Na+:K+ pump activity.

Time-course of altered islet phospholipids and of calcium binding and ionophoretic properties of islet lipids following glucose stimulation

The time-course of alteration in islet cell phospholipid content following d-glucose exposure in islet cells and in islet cell membranes was related to the ability of lipids extracted from both cultured pancreatic islet cells and from plasma membranes isolated from the islet cells to translocate calcium in two model membrane systems. The first model system (bulk-phase system) detected lipid species with the ability to bind calcium, irrespective of their ability to enhance calcium transport across cell membranes. The second system (multilamellar membrane system) detected lipid species with the ability to both bind calcium and to enhance calcium transport across cell membranes (true ionophores). Pre-exposure to high d-glucose concentration led to a rapid (within 1 min) fall in membrane phosphoinositides. This was partially blocked by mannoheptulose. A concurrent fall in calcium bindig activity of lipids from the plasma membrane was observed. In the whole islet cell fraction, d-glucose induced a marked increase in Ca 2+ ionophoretic activity. Unlike the fall in membrane polyphosphoinositides and membrane Ca 2+ binding activity, these changes were dependent on the presence of added extracellular calcium. l-Glucose was without effect on membrane phosphoinositide content. It is concluded that altered membrane and intracellular phospholipids may contribute to the increased availability of intracellular Ca 2+ following d-glucose stimulation by virtue of theie Ca 2+ binding and ionophoretic properties.

Lipid composition of a thermotolerant yeast, Hansenula polymorpha.

A thermotolerant yeast, Hansenula polymorpha CK-1, which had an optimum growth temperature of 40°C, was isolated from bagasse. The fatty acid composition of the yeast grown at 40°C was characterized by the presence of oleic (18:1, 25 to 27%), linoleic (18:2, 42 to 43%) and linolenic (18:3, 3 to 6%) acids. An increase in the growth temperature from 20 to 50°C resulted in a decrease in the phospholipid content of cells from 2.84 to 1.24% on a dry weight basis, and reduced the proportion of 18:3 acid from 17.5 to 1.3% in phospholipids. The sterol content ranged between 0.34 and 0.53% of the dry weight of the cells. An increase in the molar ratio of sterol/phospholipid from 0.26 to 0.54 was observed with increasing temperature from 20 to 40°C, although no further increase in this molar ratio was found at 50°C. Since the decrease in fatty acid unsaturation in the phospholipids was not significant, the increase in the molar ratio of sterol/phospholipid may play an important role in the adaptation of H. polymorpha CK-1 to high temperatures.

Phospholipid composition of human monocytes and alterations occurring due to culture and stimulation by C3b

The phospholipid composition of human peripheral blood monocytes has not been previously reported, due to difficulty in isolating these cells in a purified state. In this study, monocytes were purified by counterflow centrifugation without selective adherence, and were characterized with the use of fluorescent monoclonal antibodies to T and B lymphocytes and monocytes by flow cytometry. These platelet-free cell preparations contained less than 5% T cells and less than 3% B cells. Isolated monocytes, which were rapidly frozen after isolation, contained phospholipids (in order of decreasing concentrations) as follows: phosphatidylcholine > phosphatidylethanolamine > sphingomyelin > phosphatidylserine > phosphatidylinositol > cardiolipin. A small amount of lyso-PC, but no lyso-PE, phosphatidic acid or lyso-PI, was found. The effect of culturing these cells in the presence or absence of a known stimulant of monocyte prostaglandin E and thromboxane release, the C3b fragment of the third component of human complement (C3), was studied with regard to phospholipid composition. Monocytes cultured without stimulant for 24 h contained 3–4% more sphingomyelin than did uncultured cells, and lyso-PC concentrations were consistently elevated. The addition of the stimulant C3b to cultured cells resulted in enhancement of release of immunoreactive prostaglandin E into culture supernatants, without affecting the release of lysosomal enzymes. Analysis of the phospholipid content of cells cultured in the presence of C3b revealed that there was a significant decrease in total PI compared to cells cultured in the absence of C3b, in addition to an increased concentration of sphingomyelin and lyso-PC when compared to freshly isolated cells. These changes occurred in the absence of elevated concentrations of phosphatidic acid.

Abnormalities in erythrocyte membrane phospholipids in patients with liver cirrhosis

The cholesterol:phospholipid molar ratio, the fatty acid composition, and the distribution of individual phospholipids within erythrocyte membranes were studied in eight patients with alcoholic liver cirrhosis and seven healthy subjects. The total red cell phospholipid content was significantly ( P < 0.02) lower in cirrhotics than in normal subjects, resulting in increased membrane cholesterol:phospholipid molar ratio in the former. The percent of arachidonic acid was significantly ( P < 0.02) lower in the patient than in the control group. Cirrhotic patients displayed a significantly ( P < 0.001) higher percentage of phosphatidylserine and phosphatidylinositol and a lower percentage of phosphatidylethanolamine than the control subjects. No significant differences were observed in the phosphatidylcholine:sphingomyeline molar ratio between the two groups. The results suggest that the altered lipid composition of erythrocyte plasma membranes could be, at least in part, related to changes in the phospholipid distribution within the membranes.

Expansion of phospholipid pool size of rat intestinal villus cells during fat absorption.

The effect of fat absorption upon the phospholipid pool size of the intestinal mucosal cells was determined in rats receiving fatty emulsions as a bolus by stomach tube or as multiple meals in the form of fat-laden laboratory chow. The phospholipid content of the mucosal scrapings and of the isolated villus cells was determined 3 to 34 h after the meals and was compared with the phospholipid content of cells from similar animals receiving water alone or 10% sucrose in water. It was shown that continuously fed animals averaged 5-10% and single meal fed animals up to 40% higher phospholipid content in their mucosal cells than the corresponding controls, when compared per milligram cell protein. The expansion of the phospholipid pool involved all phospholipid classes and correlated well with the phospholipid composition of prechylomicrons and of microsomal membranes, which undergo a significant proliferation during fat absorption. The apparent lower expansion of the phospholipid pool in the continuously fed animals correlated with the lower triacylglycerol content of the lumen and of the cells at these times.

Effect of oxygen limitation on cellular L-lysine pool and lipid spectrum of Corynebacterium glutamicum

During submerged cultivation of L-lysine producing Corynebacterium glutamicum 9366, oxygen limitation caused a three-fold increase in the intracellular pool of L-lysine while its excretion into the medium cased. At the same time, the phospholipid content of cells increased markedly whereas the proportion of oleic acid in the fatty acids of phospholipids decreased.

Structure and synthesis of a lipid-containing bacteriophage: V. Phospholipids of the host BAL-31 and of the bacteriophage PM2

The major phospholipids of Pseudomonas BAL-31 grown in synthetic medium are phosphatidylethanolamine (75%) and phosphatidylglycerol (23%). The phospholipid composition remains relatively constant during different growth stages and in different growth media. Bacteriophage PM2 contains the same phospholipids as the host BAL-31, but with an altered percent composition (28% phosphatidylethanolamine and 65–68% phosphatidylglycerol). Phosphatidic acid is a trace component in uninfected cells, and appears to be present in virus (2–4%). The cellular phospholipid composition shifts during infection with bacteriophage PM2. Lysophosphatidylethanolamine and phosphatidic acid increase from trace amounts to as much as 18–22% of the infected cell phospholipid content, and at the same time phosphatidylglycerol increases from 23% to 36%. Phosphatidylethanolamine decreases from 75% to about 43% of the total phospholipid in infected cells. The dissimilarity between bacteriophage and host cell phospholipid compositions is discussed in terms of the probable biogenesis of the viral membrane.