Separation of cellular nonpolar neutral lipids by normal-phase chromatography and analysis by electrospray ionization mass spectrometry

Neutral lipids are an important class of hydrophobic compounds found in all cells that play critical roles from energy storage to signal transduction. Several distinct structural families make up this class, and within each family there are numbers of individual molecular species. A solvent extraction protocol has been developed to efficiently isolate neutral lipids without complete extraction of more polar phospholipids. Normal-phase HPLC was used for the separation of cholesteryl esters (CEs), monoalkylether diacylglycerols, triacylglycerols, and diacylglycerols in a single HPLC run from this extract. Furthermore, minor lipids such as ubiquinone-9 could be detected in RAW 264.7 cells. Molecular species that make up each neutral lipid class can be analyzed both qualitatively and quantitatively by on-line LC-MS and LC-MS/MS strategies. The quantitation of >20 CE molecular species revealed that challenging RAW 264.7 cells with a Toll-like receptor 4 agonist caused a >20-fold increase in the content of CEs within cells, particularly those CE molecular species that contained saturated (14:0, 16:0, and 18:1) fatty acyl groups. Longer chain CE molecular species did not change in response to the activation of these cells.     

Cold hardening induces transfer of fatty acids between polar and nonpolar lipid pools in the Arctic collembollan Megaphorura arctica

Cold hardiness in the Arctic Collembola Megaphorura arctica (Tullberg), formerly Onychiurus arcticus, has been the subject of extensive studies over the last decade. This species employs an unusual strategy known as cryoprotective dehydration to survive winter temperatures as low as ?25 °C. To expand knowledge of cryoprotective dehydration in M. arctica, the present study investigates how a reduction in ambient temperature affects the fatty acid composition of the total body lipid content along with polar (mainly membrane phospholipids) and nonpolar (mainly triacylglycerols) lipids. Most ectothermic animals compensate for changes in fluidity by regulating fatty acid composition, a process often described as homeoviscous adaptation. In M. arctica, changes in the fatty acid composition of total body lipid content during cold treatment are only moderate, with no clear pattern emerging. However, the levels of unsaturated fatty acids in the polar lipids increase with cold exposure, largely attributable to 16 : 1(n? 7), 18 : 1(n? 9), 18 : 3(n? 6) and 18 : 3(n? 3), whereas unsaturated fatty acid levels in the nonpolar lipids correspondingly decrease. These results suggest a reallocation of fatty acids between the two lipid pools as a response to a temperature reduction of 6 °C. Because of hypometabolism, a characteristic of cold adaptation, such a mechanism could be less energy demanding than de novo synthesis of fatty acids and may comprise part of an adaptive homeostatic response.     

Simultaneous extraction and preparation for high-performance liquid chromatography of prostaglandins and phospholipids

A method for the maximum recovery of prostaglandins from brain tissue with simultaneous recovery of neutral lipids and phospholipids was developed. Hexane:2-propanol was used to extract lipids from bovine brain. This method, which does not require a washing step to remove nonlipid contaminants, was compared to extraction according to Folch et al. [(1957) J. Biol. Chem. 226, 497-509] for efficiency of lipid extraction. Recoveries of prostaglandins were 12-37% greater with hexane:2-propanol than with the Folch extraction procedure with washing. The ratios of cholesterol to lipid phosphorus and absolute phospholipid recoveries were comparable for the two methods. A new elution sequence was devised for separation of lipid classes on silicic acid columns. The elution sequence was chloroform (neutral lipids and free fatty acids), methyl formate (prostaglandins and cerebrosides), acetone (remaining glycolipids), and methanol (phospholipids). Reverse-phase HPLC of the methyl formate fraction was used to separate the prostaglandins. The method permits simultaneous quantitative recovery of prostaglandins and phospholipids (which contain the 20:4(n-6) precursor for prostaglandin synthesis), and therefore allows changes in phospholipid composition and prostaglandin synthesis to be studied in the same tissue sample.     

Changes in phospholipid extractability and composition accompany mineralization of chicken growth plate cartilage matrix vesicles.

Matrix vesicles are lipid bilayer-enclosed structures that initiate extracellular mineral formation. Little attention has been given to how newly formed mineral interacts with the lipid constituents and then emerges from the lumen. To explore whether specific lipids bind to the incipient mineral and if breakdown of the membrane is involved, we analyzed changes in lipid composition and extractability during vesicle-induced calcification. Isolated matrix vesicles were incubated in synthetic cartilage lymph to induce mineral formation. At various times, samples of the lipids were taken for analysis, extracted both before and after demineralization to remove deposited mineral. Phosphatidylserine and phosphatidylinositol both rapidly disappeared from extracts made before decalcification, indicating rapid degradation. However, extracts made after demineralization revealed that phosphatidylserine had become complexed with newly forming mineral. Concomitantly, its levels actually increased, apparently by base-exchange with phosphatidylethanolamine. Though partially complexed with the mineral, phosphatidylinositol was nevertheless rapidly broken down. Sphingomyelin and phosphatidylethanolamine also underwent rapid breakdown, but phosphatidylcholine was degraded more slowly, all accompanied by a buildup of free fatty acids. The data indicate that phosphatidylserine forms complexes that accompany mineral formation, while degradation of other membrane phospholipids apparently enables egress of crystalline mineral from the vesicle lumen.     

Lipid composition of mitochondria from bovine heart, liver, and kidney

Highly purified preparations of mitochondria from bovine heart, liver, and kidney were isolated and characterized by electron microscopy, oxidative phosphorylation ability, cytochrome c reductase activity, and cytochrome content. Components of lipid extracts of the preparations were determined by thin-layer chromatography, diethylaminoethyl-cellulose column chromatography, and spectrophotometric procedures. The major phospholipids were identified by their chromatographic behavior, IR spectrometry, and paper chromatography of their hydrolysis products. The lipid content of the mitochondria paralleled that of the components of the electron transfer chain, heart mitochondria being richest and liver mitochondria poorest in lipid. Heart mitochondria contain equal concentrations of coenzyme Q and cholesterol (1%); the highest cholesterol content (4.7%) was found in mitochondria from kidney. The phospholipids of mitochondria from the three organs were qualitatively and quantitatively very similar. The major polar lipid components (cardiolipin, choline glycerophosphatides, and ethanolamine glycerophosphatides) were present in a molar ratio of 1:4:4. It is suggested that mitochondria from different sources contain characteristic lipids, mainly phospholipids, of which cardiolipin is particularly diagnostic of the source of the mitochondria.     

A monophasic extraction strategy for the simultaneous lipidome analysis of polar and nonpolar retina lipids

Lipid extraction using a monophasic chloroform/methanol/water mixture, coupled with functional group selective derivatization and direct infusion nano-ESI-high-resolution/accurate MS, is shown to facilitate the simultaneous analysis of both highly polar and nonpolar lipids from a single retina lipid extract, including low abundance highly polar ganglioside lipids, nonpolar sphingolipids, and abundant glycerophospholipids. Quantitative comparison showed that the monophasic lipid extraction method yielded similar lipid distributions to those obtained from established “gold standard” biphasic lipid extraction methods known to enrich for either highly polar gangliosides or nonpolar lipids, respectively, with only modest relative ion suppression effects. This improved lipid extraction and analysis strategy therefore enables detailed lipidome analyses of lipid species across a broad range of polarities and abundances, from minimal amounts of biological samples and without need for multiple lipid class-specific extractions or chromatographic separation prior to analysis.     

Complete separation of lipid classes on a single thin-layer plate

A double-development procedure employing first a polar and then a nonpolar solvent system is described for the complete separation by thin-layer chromatography of the main lipid classes encountered in natural lipids. For better quantification, long plates (34 cm) are employed. Diglycerides were separated from cholesterol, 1,2- from 1,3-diglycerides, and monoglycerides from phospholipids.     

Lipid Composition of Citrus Leaves from Plants Resistant and Susceptible to Citrus Bacterial Canker

The contents and composition of lipids in citrus leaves in relation to their general resistance to infection by strains of Xanthomonas campestris pv. citri (Xcc) were determined. The composition and contents of total polar lipids and phospholipids and the degree of fatty acid unsaturation were significantly different between resistant and susceptible species. Leaves from resistant plants had less phospholipids, but more free sterols than those from susceptible plants. The predominant fatty acids in the phospholipids were palmitic (16:0), linoleic (18:2) and α-linolenic acid (18:3). The degree of fatty acid unsaturation was higher in susceptible plants than in resistant plants. Major phospholipids in citrus leaves were phosphatidylchloline (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and phosphatidylinositol (PI). β-Sitosterol, campesterol and lanosterol were major sterols in the leaves of citrus plants with resistant species having a higher ratio of free sterols to total phospholipids than susceptible species. Differences in lipid metabolism may contribute to differences in Xcc-resistance of citrus leaves.     

Involvement of the Saccharomyces cerevisiae hydrolase Ldh1p in lipid homeostasis

Here, we report the functional characterization of the newly identified lipid droplet hydrolase Ldh1p. Recombinant Ldh1p exhibits esterase and triacylglycerol lipase activities. Mutation of the serine in the hydrolase/lipase motif GXSXG completely abolished esterase activity. Ldh1p is required for the maintenance of a steady-state level of the nonpolar and polar lipids of lipid droplets. A characteristic feature of the Saccharomyces cerevisiae Δldh1 strain is the appearance of giant lipid droplets and an excessive accumulation of nonpolar lipids and phospholipids upon growth on medium containing oleic acid as a sole carbon source. Ldh1p is thought to play a role in maintaining the lipid homeostasis in yeast by regulating both phospholipid and nonpolar lipid levels.     

Paired helical filaments contain small amounts of cholesterol, phosphatidylcholine and sphingolipids

By using qualitative and quantitative high-performance thin layer chromatography (hpTLC) we found lipids associated with purified Alzheimer's (AD) paired helical filaments (PHF) in an amount of 1.4+/-0.2% of the total anhydrous mass. Compared to normal brain tissue these lipids have an unusual lipid class composition. The most prominent lipid classes were phosphatidylcholine (PC), cholesterol (CH), galactocerebrosides (GC) and sphingomyelin (SM). In addition, the use of micro high-performance liquid chromatography (HPLC) in combination with matrix-assisted laser desorption and ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) allowed the determination of the molecular species of the polar membrane lipid classes present in PHF. The lipid pattern of intracellular PHF shows many characteristics of the conserved lipid pattern previously described for extracellular amyloid fibrils, suggesting similarities in their pathway of formation.     

Lipid analysis of Greek walnut oil (Juglans regia L.)

The walnut oil (Juglans regia L.) total lipids (TL) were extracted by the Bligh-Dyer method and the lipid classes have been isolated by chromatographic techniques and they were analyzed by high performance thin layer chromatography (HPTLC)/FID and GC-MS. The oil was found to be rich in neutral lipids (96.9% of total lipids) and low in polar lipids (3.1% of total lipids). The neutral lipid fraction consisted mainly of triacylglycerides whereas the polar lipids mainly consisted of sphingolipids. GC-MS data showed that the main fatty acid was linoleic acid. Unsaturated fatty acids were found as high as 85%, while the percentage of the saturated fatty acids was found 15%. Two types of liposomes were prepared from the isolated walnut oil phospholipids and characterized as new formulations. These formulations may have future applications for encapsulation and delivery of drugs and cosmetic active ingredients.     

Separation and identification of lipids and fatty acids of the marine algaFucus vesiculosus by TLC and GC—MS

Ten fractions of polar and nonpolar lipids (phospholipids, glyeolipids and neutral lipids) were isolated from the brown marine algaFucus vesiculosus by TLC. In each fraction the quality and quantity of fatty acids were determined by GC—MS.F. vesiculosus is another type of marine alga suitable as a source of polyene fatty acids.     

Composition of adrenal lipids from some domestic and wild ruminants.

1. Lipids from the whole adrenal glands of the ox and the African buffalo (Syncerus caffer) were extracted and fractionated into neutral and phospholipids. Both species revealed the presence of considerable quantities of cholesterol but only very small quantities of cholesteryl esters. 2. Fatty acids from various fractions of bovine and buffalo adrenal glands were investigated by gas-chromatography. They showed a remarkably low content of higher unsaturated fatty acids and a very high content of stearic acid. 3. Mitochondrial and microsomal fractions were isolated from the adrenal glands of the impala antelope (Aepyceros melampus), their lipids extracted, analyzed and compared with the composition of the mitochondrial lipids from bovine adrenal glands. 4. Subcellular fractions of the bovine and impala adrenal glands contain only very small quantities of esterified cholesterol. Most of the lipid fractions were characterized by the absence of adrenic acid (C22:4 omega 6) and a low content of arachidonic acid.     

Liposomal formulations from phospholipids of Greek almond oil. Properties and biological activity

The seeds of the almond tree [(Prunus dulcis (Mill.) D. A. Webb. (syn. Prunus amygdalus)] were collected in two different periods of maturity and were studied for their lipid content. The total lipids (TL) were extracted by the Bligh-Dyer method and the lipid classes have been isolated by chromatographic techniques and were analyzed by HPTLC coupled with a flame ionization detector (HPTLC/FID) and GC-MS. The oils were found to be rich in neutral lipids (89.9% and 96.3% of total lipids) and low in polar lipids (10.1% and 3.7% of total lipids) for the immature and mature seed oils, respectively. The neutral lipid fraction consisted mainly of triacylglycerides whereas the polar lipids mainly consisted of phospholipids. GC-MS data showed that the main fatty acid for both oils was 9-octadecenoic acid (oleic acid). The unsaturated fatty acids were found as high as 89.4% and 89.7%, while the percentage of the saturated fatty acids was found 10.6% and 10.3% for the immature and mature seed oils, respectively. Liposomes were prepared from the isolated phospholipids using the thin lipid film methodology, and their physical properties were characterized. Cytotoxicity was found absent when assayed against normal and cancerous cell lines. These new formulations may have future applications for encapsulation and delivery of drugs and cosmetically active ingredients.     

Modeling of matrix vesicle biomineralization using large unilamellar vesicles

Stable, large unilamellar vesicles (LUV) have been constructed that model matrix vesicles (MV) in inducing de novo mineral formation when incubated in synthetic cartilage lymph (SCL). Using a dialysis method for incorporation of predetermined pure lipid, electrolyte and protein constituents, the detergent n-octyl beta-D-glucopyranoside enabled formation of stable, impermeable LUV with a diameter ( approximately 300 nm), lipid composition (phosphatidylcholine-phosphatidylserine-cholesterol, 7:2:2, molar ratio) and enclosed inorganic phosphate level (25-100 mM) similar to that of native MV. Mineral formation by these LUVs was measured by 45Ca(2+) uptake and FTIR analysis following incubation in SCL. Addition of the ionophore A23187 to SCL enabled 45Ca(2+) uptake comparable to that of native MV. FTIR analysis revealed that crystalline mineral formed in the LUV during incubation in SCL, but not in the absence of ionophore. This mineral had an IR absorption spectrum like that of the acid-phosphate-rich, octacalcium phosphate-like mineral formed by native MV. Perturbing the LUV membrane with either detergents or phospholipase A(2) following prior incubation in SCL enabled egress of mineral crystallites from the vesicle lumen, stimulating further mineral formation. Annexin V, a major protein in native MV with known Ca(2+) channel activity, incorporated into the LUV lumen or added to the external medium, induced only limited 45Ca(2+) uptake. This indicates that additional factors are required for annexin V to form Ca(2+) channels. Nevertheless for the first time, stable LUVs have been constructed with MV-like lipid, electrolyte, and protein composition and size that induce formation of mineral like that formed by native MV.     

Whole-cell and membrane lipids of the methylotrophic bacterium Methylosinus trichosporium.

The lipid composition of the methylotrophic bacterium Methylosinus trichosporium was examined. Whole-cell lipid distribution was 39.1% neutral lipids, 34.5% polar lipids, and 26.4% poly-beta-hydroxybutyric acid. Membrane lipids were 83% phospholipids, with phosphatidylethanolamine and phosphatidylglycerol accounting for over 94% of the total. All the phospholipids had similar fatty acid compositions, with 18:1 accounting for about 87% of the total and most of the rest consisting of 16:1. Similarities between the lipid composition of this bacterium and other bacteria are discussed.     

Oxidized phospholipids as biomarkers of tissue and cell damage with a focus on cardiolipin

Oxidized phospholipid species are important, biologically relevant, lipid signaling molecules that usually exist in low abundance in biological tissues. Along with their inherent stability issues, these oxidized lipids present themselves as a challenge in their detection and identification. Often times, oxidized lipid species can co-chromatograph with non-oxidized species making the detection of the former extremely difficult, even with the use of mass spectrometry. In this study, a normal-phase and reverse-phase two dimensional high performance liquid chromatography (HPLC)-mass spectrometric system was applied to separate oxidized phospholipids from their non-oxidized counterparts, allowing unambiguous detection in a total lipid extract. We have utilized bovine heart cardiolipin as well as commercially available tetralinoleoyl cardiolipin oxidized with cytochrome c (cyt c) and hydrogen peroxide as well as with lipoxygenase to test the separation power of the system. Our findings indicate that oxidized species of not only cardiolipin, but other phospholipid species, can be effectively separated from their non-oxidized counterparts in this two dimensional system. We utilized three types of biological tissues and oxidative insults, namely rotenone treatment of lymphocytes to induce mitochondrial damage and cell death, pulmonary inhalation exposure to single walled carbon nanotubes, as well as total body irradiation, in order to identify cardiolipin oxidation products, critical to the cell damage/cell death pathways in these tissues following cellular stress/injury. Our results indicate that selective cardiolipin (CL) oxidation is a result of a non-random free radical process. In addition, we assessed the ability of the system to identify CL oxidation products in the brain, a tissue known for its extreme complexity and diversity of CL species. The ability of the two dimensional HPLC-mass spectrometric system to detect and characterize oxidized lipid products will allow new studies to be formulated to probe the answers to biologically important questions with regard to oxidative lipidomics and cellular insult. This article is part of a Special Issue entitled: Oxidized phospholipids - their properties and interactions with proteins.     

Lipidomics reveals that adiposomes store ether lipids and mediate phospholipid traffic

Lipid droplets are accumulations of neutral lipids surrounded by a monolayer of phospholipids and associated proteins. Recent proteomic analysis of isolated droplets suggests that they are part of a dynamic organelle system that is involved in membrane traffic as well as packaging and distributing lipids in the cell. To gain a better insight into the function of droplets, we used a combination of mass spectrometry and NMR spectroscopy to characterize the lipid composition of this compartment. In addition to cholesteryl esters and triacylglycerols with mixed fatty acid composition, we found that approximately 10-20% of the neutral lipids were the ether lipid monoalk(en)yl diacylglycerol. Although lipid droplets contain only 1-2% phospholipids by weight, >160 molecular species were identified and quantified. Phosphatidylcholine (PC) was the most abundant class, followed by phosphatidylethanolamine (PE), phosphatidylinositol, and ether-linked phosphatidylcholine (ePC). Relative to total membrane, droplet phospholipids were enriched in lysoPE, lysoPC, and PC but deficient in sphingomyelin, phosphatidylserine, and phosphatidic acid. These results suggest that droplets play a central role in ether lipid metabolism and intracellular lipid traffic.     

Metabolic characteristics and lipid composition of yeastlike cells and mycelium of Mucor circinelloides var. lusitanicus INMI grown at a high glucose content in the medium

It is shown that the fungus Mucor circinelloides var. lusitanicus INMI grown under aerobic conditions in a medium with a high glucose concentration (20%) is capable of both yeastlike and mycelial growth. In the mycelium, the activity of NAD-dependent isocitrate dehydrogenase was more than twice as high as in yeastlike cells, whereas the isocitrate lyase activity was lower. A number of significant differences were found in the lipid composition of the cells of two different morphological variants. Yeastlike cells contained more polar lipids and free fatty acids and less principal reserve lipids (triacylglycerides) than mycelial cells; the content of gamma-linolenic acid and the degree of lipid unsaturation were significantly lower in these cells than in the mycelium. In yeastlike cells, glycolipids composed the bulk of polar lipids; the proportion of phospholipids (primarily phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, and cardiolipin) was lower. The relationship between cellular metabolism and the lipid composition of fungal cells of different morphotypes grown at high concentrations of glucose, one of the main inducers of dimorphic growth, is discussed.     

Lipid phase transitions measured in intact cells with Fourier transform infrared spectroscopy

Lipid phase transitions in membranes are thought to be a major damaging event during cooling of cells prior to cryopreservation or during warming after freeze-thaw has been completed. Although there is abundant evidence that such transitions occur in isolated phospholipids, the evidence that they are found in membranes in intact cells is less clear, due largely to technical difficulties in detecting such transitions in the complex mixtures of lipids and proteins found in natural membranes. We show here that Fourier transform infrared spectroscopy provides a rapid, convenient method for detecting these transitions in intact cells. We have used intact pollen grains of cattail (Typha latifolia) as a primary experimental subject. Spectra taken of the intact pollen grains show most of the features commonly seen in natural membrane vesicles or pure phospholipids. Shifts in the vibrational frequency and width of the CH2 bands with temperature can be used to detect lipid phase transitions. Biochemical analysis, coupled with the spectroscopy, was used to assign transitions to nonpolar and polar lipids. Finally, although assignment of the melting lipid unambiguously in other cells has not yet been made, we show that the transitions can nevertheless be detected in other intact cells, including those of four plant species and sperm of three animals.