Biosynthesis of phospholipids in subcellular particles from cultured cells of human tissue

A time study of the incorporation of³²P_i into the phospholipids of HeLa, KB, human heart, and liver tissue-culture cell lines has been carried out. The incorporation of³²P_i at various time-intervals into the phospholipids of nuclei, mitochondria, and microsomes of HeLa and KB cells was investigated. The labeling of the isotope into the phospholipids was divided into three groups. The first had two components: phosphatidyl inositol and polyglycerol phosphatides, which showed the greatest incorporation of the isotope as demonstrated in the specific activity values and the percentage of total radioactivity after 15 to 30 minutes of incubation. A second group was composed of the major phospholipids of all tissue-culture cell lines studied, phosphatidyl choline, and phosphatidyl ethanolamine. At first, there was a delayed labeling of these phospholipids; however, after one hour of incubation, a rapid increase was shown in the incorporation of³²P_i. A third group of lipids containing sphingomyelin and phosphatidyl serine demonstrated low specific activity values. The phospholipids of the subcellular fractions, nuclei, mitochondria, and microsomes, had a high degree of incorporation of the isotope into the individual phospholipids and probably represented an active process in the membranes of these cellular units or a renewal of the biological membrane structures. Part of a thesis submitted to the Graduate School of the University of North Dakota in partial fulfillment for the degree of Doctor of Philosophy.     

Phospholipid composition of cultured human endothelial cells

Detailed analyses of the phospholipid compositions of cultured human endothelial cells are reported here. No significant differences were found between the phospholipid compositions of cells from human artery, saphenous and umbilical vein. However, due to the small sample sizes, relatively large standard deviations for some of the phospholipid classes were observed. A representative composition of endothelial cells is: phosphatidylcholine 36.6%, choline plasmalogen 3.7%, phosphatidylethanolamine 10.2%, ethanolamine plasmalogen 7.6%, sphingomyelin 10.8%, phosphatidylserine 7.1%, lysophosphatidylcholine 7.5%, phosphatidylinositol 3.1%, lysophosphatidylethanolamine 3.6%, phosphatidylinositol 4,5-bisphosphate 1.8%, phosphatidic acid 1.9%, phosphatidylinositol 4-phosphate 1.5%, and cardiolipin 1.9%. The cells possess high choline plasmalogen and lysophosphatidylethanolamine contents. The other phospholipids are within the normal biological ranges expected. Phospholipids were separated by high-performance liquid chromatography and quantified by lipid phosphorus assay.     

Lipids of subcellular particles

Methods for isolation and characterization of subcellular particles as well as procedures for analysis of lipid class composition are discussed. The literature on distribution of lipids in subcellular particles is then reviewed. Pertinent new data from our laboratories are presented as well. The isolated particles are related to the organelles to which they correspond in the cell and are discussed with regard to heterogeneity and morphological integrity. Confusion can arise with regard to subcellular particles unless it is appreciated that: 1) preparation of particles of high purity generally requires more than the classical differential centrifugation scheme (both differential and gradient centrifugation may be required); 2) it is hazardous to apply exactly the same procedure for all tissues; 3) all subcellular fractions must be thoroughly characterized. The more recently devised DEAE cellulose column and thin-layer chromatographic procedures for analysis of lipid class composition are more reliable than the older hydrolytic or silicie acid column or paper chromatographic techniques. The chief lipid components of mitochondria from all organs and species are lecithin, phosphatidyl ethanolamine, and cardiolipin (diphosphatidyl glycerol). Despite the fact that reports in the literature are in agreement that phosphatidyl inositol is a major component of mitochondria, it is concluded on the basis of new data obtained from highly purified mitochondria and improved analytical methods that phosphatidyl inositol is not a major component of mitochondria. The presence of a relatively large amount of phosphatidyl inositol in mitochondrial preparations is probably related in part to contamination with other particles. Some analytical procedures are demonstrated to give erroneous values for this lipid class. It is also concluded that phosphatidyl serine, phosphatidic acid, sphingomyelin, cerebrosides, and lysophosphatides, reported to occur in mitochondria, are not characteristic mitochondrial components and furthermore that the large amount of uncharacterized mitochondrial phospholipid reported is actually an analytical artifact. Microsomes appear to be similar to mitochondria except that cardiolipin is either low in or absent from microsomes. Available data indicate nuclei to be rather similar to mitochondria and microsomes, at least in some organs. Studies of the fatty acids of subcellular particles indicate that different particles from one organ have very similar fatty acid compositions. It is clear that there are marked variations in fatty acid composition of particles from different organs and from different species. Differences in dietary fat may be associated with marked changes in fatty acid composition, although brain mitochondrial lipids are largely unchanged. Each lipid class from mitochondria of most organs appears to have a fairly characteristic fatty acid composition. Cardiolipin from some organs contains primarily linoleic acid, phosphatidyl ethanolamine contains large amounts of linoleic and higher polyunsaturates, and lecithin is similar to phosphatidyl ethanolamine except that it does not contain as much arachidonic acid and/or other highly unsaturated fatty acids. New data, the first to be reported, are presented for heart mitochondrial cardiolipin, phosphatidyl ethanolamine, and lecithin. It is concluded that there are two basically different types of membranous structures. Myelin is the chief representative of the metabolically stable type of membrane structure while mitochondria represent the more labile type. The two types of membranes have very different in vivo properties and very different lipid compositions. Myelin is characterized by a high content of cholesterol and sphingolipids with more long chain saturated or monoenoic fatty acids while mitochondria are characterized by a low cholesterol content, little or no sphingolipid, and highly unsaturated fatty acids. It is clear that formulations of the myelin type membrane structure such as that of Vandenheuvel cannot apply to mitochondria. It is postulated that membrane structures intermediate between the extremes represented by myelin and mitochondria exist.     

Chemical composition and mass emission factors of candle smoke particles

The aim of this study is to investigate the physical and chemical properties of particle emissions from candle burning in indoor air. Two representative types of tapered candles were studied during steady burn, sooting burn and smouldering (upon extinction) under controlled conditions in a walk-in stainless steel chamber. Steady burn emits relatively high number emissions of ultrafine particles dominated by either phosphates or alkali nitrates. The likely source of these particles is flame retardant additives to the wick. Sooting burn in addition emits larger particles mainly consisting of agglomerated elemental carbon. This burning mode is associated with the highest mass emission factors. Particles emitted during smouldering upon extinction are dominated by organic matter. A mass closure was illustrated for the total mass concentration, the summed mass concentration from chemical analysis and the size-integrated mass concentration assessed from number distribution measurements using empirically determined effective densities for the three particle types.     

Fatty acid composition of subcellular particles from sheep platelets and topological distribution of phosphatidylethanolamine fatty acids in the plasma membrane

The fatty acid composition of individual phospholipids in subcellular fractions of sheep platelets and the asymmetrical distribution of phosphatidylethanolamine (PE) fatty acyl chains across the plasma membrane were examined. The main fatty acids of total lipid extracts were oleic (18∶1; 32–41%), linoleic (18∶2, 10–17%), stearic (18∶0; 13–15%), palmitic (16∶0; 11–15%) and arachidonic (20∶4; 8–12%) acids, with a saturated/unsaturated ratio of about 0.4. Each phospholipid class had a distinct fatty acid pattern. Sphingomyelin (SM) showed the highest degree of saturation (50%), with large proportions of behenic (22∶0), 18∶0 and 16∶0 acids. The main fatty acid in PE, phosphatidylserine (PS) and phosphatidylcholine (PC) was 18∶1n−9. Our findings suggest that fatty acids are asymmetrically distributed between thecholineversus the non-choline phospholipids, and also between plasma membranes and intracellular membranes. The transbilayer distribution of PE fatty acids in plasma membranes from non-stimulated sheep platelets was investigated using trinitrobenzenesulfonic acid (TNBS). A significant degree of asymmetry was found, which is a new observation in a non-polar cell. The PE molecules from the inner monolayer contained higher amounts of 18∶2 and significantly less 18∶1 and 20∶5 than those found in the outer monolayer, although no major differences were detected in the transbilayer distribution of total unsaturatedversus saturated PE acyl chains.     

Chemical composition and bond structure of aerosol particles of amorphous hydrogenated silicon forming from thermal decomposition of silane

Aerosol particles of amorphous hydrogenated silicon resulting from thermal decomposition of silane were investigated by hydrogen evolution, IR-, EPR-, NMR spectroscopy, and transmission electron microscopy.

The experimental data show that aerosol particles contain to a various extent {SiH₂}_n polymer structures and two types of monohydride groups SiH- “clustered” and “dilute” monohydride groups. The hydrogen atoms of the “clustered” monohydride groups are located close to each other. The “clustered” monohydride groups are inaccessible to the ambient because they are embedded in the amorphous network. The “dilute” monohydride groups are relatively isolated from each other. The majority of “dilute” monohydride groups are open to the ambient. They are located on the surface of preferentially interconnected microchannels and microvoids.

Interaction between the “dilute” SiH groups and atmospheric oxygen results in formation of OSiH groups in which hydrogen and oxygen are bonded to a common silicon atom. Evidently, the interaction occurs throw the oxygen reaction with weak bonds associated with “dilute” monohydride groups. There is no interaction between oxygen and both “clustered” SiH groups and {SiH₂}_n chain because the former are inaccessible to atmospheric oxygen and the latter has presumably no weak bonds in the chains.     

Lipid composition of subcellular membranes from larvae and prepupae ofDrosophila melanogaster

Subcellular membranes were analyzed for their lipid composition and protein content at two developmental points representing the third instar wandering larvae and prepupal stages ofDrosophila. At both stages, phosphatidylethanolamine (PE) and phosphatidylcholine (PC) were the major constituents with phosphatidylinositol (PI), phosphatidylserine (PS), diphosphatidylglycerol (DPG) and phosphatidic acid (PA) being relatively minor components. In total homogenates and in the nuclear-enriched fraction there was no significant difference in the phospholipid composition of the wandering larvae and prepupae. In mitochondria only a significant increase in the minor component PS was observed in the prepupae. In lysosomal membranes on the other hand, the relative abundance of the major components PE and PC increased in the prepupae although the molar ratios of the two lipids remained almost constant. The fatty acid composition of the phospholipids remained virtually unchanged in all of the fractions examined, including the lysosomes, and there was no evidence of lipid peroxidation. With regard to cellular degeneration and the involvement of lysosomes, we conclude that mechanisms other than gross modification of the lipid and/or lipid/protein ratio of their membranes are involved in the liberation of the acid phosphatase contents.     

Lipid composition and biosynthesis of human omental tissue

Two groups of five males each were selected for total lipid analyses of their omental tissue. One of these groups had been subjected to a severe caloric restriction and had undergone total weight reduction of about 20%. The other group served as control. Both of these groups of patients required elective surgical procedures during which it was possible to obtain small samples of omental tissue, adipose pad, and/or mesenteric tissue. Total lipid analyses were performed on all of the materials. A distinctive positional distribution of the acyl groups was maintained in the triglycerides of omental tissue for all the patients regardless of dietary state. Patients under caloric restriction showed a reduction in their total triglyceride content, a reduction in their content of unsaturated fatty acyl groups, and a relative increase in phospholipid content. The de novo lipid biosynthetic capacity of omental tissues, as determined by 1-[¹⁴C]-acetate incorporation, showed an inverse proportionality to the lipid content of the samples. Omental tissue is biosynthetically a very versatile material capable of yielding rapidly many types of fatty acids. This ability, among others, could account for the usefulness of omental tissue as a supporting base in many types of restorative surgery.     

Chemical composition of dusts from cottonseed oil mills

The proximate chemical composition of total dust samples from five Texas cottonseed oil mills was determined. Dust compositions were highly variable and were affected more by the processing step than by differences among mills. Composition of cleaning room dusts reflected differences in soil minerals. Typical inorganic values were: silicon (12%), aluminum (0.5%), and iron (0.7%). Delintering dust was highest in cellulosic materials (40%), whereas the 23% protein composition of the hulling dust showed the influence of kernel fragments. Baling, dust contained large amounts of cellulose (38%) and noncellulosic organic constituents (36%).     

Lipid composition of cultured endothelial cells in relation to their growth

Human endothelial cells in culture were examined in different growth conditions. The human endothelial cell line, EA.hy 926 cell line, was used and cells were studied either in exponential growth phase, at confluence, or growth-arrested by serum deprivation. Phospholipids were separated and analyzed by high-performance thin-layer chromatography, and their fatty acids were quantified by gas-liquid chromatography. No significant differences in the phospholipid distributions were found between exponentially growing and confluent endothelial cells in which phosphatidylcholine (PC) represented the major phospholipid. In comparison, serum-deprived cells exhibited higher proportions of sphingomyelin and lower content of PC. We also found that among the total lipids, cholesterol level for dividing endothelial cells was lower than for cells growth-arrested either by serum deprivation or by contact inhibition at confluence. The global fatty acid distribution was not affected by the growth conditions. Thus, oleate (18∶1n−9 and 18∶1n-7), palmitate (C_16∶0), and stearate (C_18∶0) were the main components of endothelial cell membranes. However, the fatty acid distributions obtained from each phospholipid species differed with the growth status. Altogether, the data indicated that subtle modulations of endothelial cell metabolism appear upon cell growth. The resulting membrane-dependent cellular functions such as cholesterol transport and receptor activities can be expected to be relevant for lipid trafficking within the vessel wall in vitro and in vivo.     

The fatty acid composition of human gliomas differs from that found in nonmalignant brain tissue

To compare the fatty acid composition of tumor tissue from glioma patients with that of normal brain tissue, tissue samples were obtained from 13 glioma patients and from 3 nonmalignant patients. Following lipid extraction, total fatty acid composition was measured using gas-liquid chromatography. Samples were further separated into phospholipids and neutral lipids. Representative samples were then separated into phospholipid classes by thin-layer chromatography and the fatty acid composition assayed. Levels of the polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA), were significantly reduced (P=0.029) in the glioma samples compared with normal brain samples; mean values were 4.8±2.9% and 9.2±1.0%, respectively. This reduction in glioma DHA content was also observed in terms of phospholipids (4.6±2.1% vs. 9.6±0.8%,P=0.002). The phosphatidylserine and phosphatidylethanolamine phospholipid classes were reduced in the glioma samples. Differences were also noted in the n-6 PUFA content between glioma and normal brain samples. The glioma content of the n-6 PUFA linoleic acid was significantly greater (P<0.05) than that observed in the control samples in terms of total lipids. Thus, the fatty acid composition of human gliomas differs from that found in nonmalignant brain tissue.     

Abnormal lipid composition of fat tissue in human mesenteric panniculitis

Mesenteric fat tissue obtained at autopsy from 6 patients with mesenteric panniculitis (MP) were found to contain significant amounts of cholesteryl esters (CE). In addition, samples from 3 of these cases were found to contain 0.5–1.3% free cholesterol, 0.9–1.9% free fatty acids (FFA), 0.6–2.5% 1-alkyl glyceryl ether diesters and small amounts of squalene. Two of these tissues also contained alk-1-enyl glyceryl ether diesters. The fatty acid compositions of the CE, FFA, triacylglycerides and glyceryl ether diesters (GEDE) were determined and oleic acid (18∶1) was found to be the major fatty acid. The alkyl group composition of the GEDE consisted essentially of 16∶0 and 18∶0 and 18∶1 carbon atoms in both types of ethers.     

Biofuels from waste fish oil pyrolysis: Chemical composition

In a previous study, waste fish oil was converted into bio-oil by a fast pyrolysis process at 525 °C in a continuous pilot plant reactor with 72-73% yield. The bio-oil was distilled to obtain light bio-oil and heavy bio-oil and these biofuels were characterized in terms of their physico-chemical properties. In this study, the chemical composition of light bio-oil and heavy bio-oil was determined using GC-FID, GC-MS, ¹H and ¹³C NMR techniques. The GC-MS analysis of waste fish oil showed the main composition of fatty acids to be the following: C_16:0 (15.87%), C_18:2 (20.96%), C_18:1 (17.29%), C_20:5 (5.11%), C_20:1 (7.59%), C_22:6 (4.53%), C_22:1 (10.42%) and others. The GC-FID analysis of the light bio-oil showed 482 compounds that were PIONA classified as paraffins (4.48%), iso-paraffins (8.31%), olefins (26.56%), naphthenes (6.07%) and aromatics (16.86%). The heavy bio-oil had a similar chromatographic profile as diesel oil, with a high content of carboxylic acids and olefins. These results are in good agreement with those for the gasoline and diesel oil fractions of petroleum.     

Site-specific differences in the fatty acid composition of human adipose tissue

The fatty acid composition of triacylglycerols from fifteen distinct adipose depots taken from each of seven adult male human subjects was compared. Oleic, palmitic, linoleic, stearic, myristic, palmitoleic and vaccenic acids accounted for more than 90% of the triacylglycerol fatty acids in all sites from all subjects; a number of other fatty acids were also identified and quantified. There were large differences in theaverage fatty acid composition between individual subjects. There were no site-specific differences in the proportions of myristic (3.8–4.7% of triacylglycerol fatty acids), palmitic (23–29%), linoleic (6.7–9.8%) or vaccenic (4.1–4.7%) acids or in the proportions of any of the less abundant fatty acids. There were some significant site-specific differences in the proportions of palmitoleic, oleic and stearic acids. The calf depot contained more palmitoleic acid (6.41±1.09%) than the trapezius (3.12±0.55%), perirenal (3.59±0.50%) and mesenteric (3.70±0.43%) depots, more oleic acid (42.13±1.27%) than the trapezius (36.03±2.18%), perirenal (36.50±1.56%) and breast (37.13±1.55%) depots and less stearic acid (5.18±0.89%) than the trapezius (8.57±0.97%), perirenal (8.49±0.75%), mesenteric (7.87±0.42%), breast (8.02±0.75%) and clavicular (8.34±0.78%) depots. The buttock depot contained less stearic acid (6.06±0.65%) than the perirenal, mesenteric and clavicular depots, while the anterior thigh depot contained less stearic acid (6.07±0.70%) than the perirenal depot. These findings indicate that, while most human adipose depots differ little in fatty acid composition, some sites, in particular the calf, perirenal, trapezius and mesenteric depots, have site-specific properties.     

Studies on lipid and fatty acid composition of human hepatoma tissue

Studies are reported on the composition of the lipids of human liver and hepatoma tissues from male adults. Liver tissues were obtained from individuals who died from causes other than liver disease or cancer. The hepatoma tissues were obtained from individuals shortly after they succumbed to cancer. The total lipid of each tissue was fractionated quantitatively by silicic acid column chromatography into neutral lipid, glycolipid, and phospholipid fractions. These fractions were analyzed by thin layer chromatography and converted to methyl esters for analysis of their constituent fatty acids by gas liquid chromatography. In comparison to liver tissue, the total amount of lipid in the hepatoma tissues was generally higher and more variable; the lipid of one hepatoma was ca. 92% of the dry wt of the tissue. The greater lipid content of the hepatoma tissues was due to the high percentage of neutral lipid. Except for one specimen, there was ca. the same amount of glycolipid in the hepatoma as in the liver tissues, but the composition of the glycolipid fraction of the hepatoma lipid differed considerably, particularly in the ganglioside fraction. The phospholipid fraction of hepatoma lipid was much lower than that of liver but exhibited only quantitative differences in composition. No glyceryl ether diesters and only traces of plasmalogens of phosphatidyl choline or phosphatidyl ethanolamine were detected in the liver and hepatoma lipids. The levels of monoenoic acids were higher and those of linoleic and polyunsaturated fatty acids lower in the hepatoma lipids. Positional isomers of trienoic acids not normally present in liver tissue were detected in hepatoma lipids. The abnormalities observed in lipid composition indicated interferences in the regulatory processes of lipid metabolism in human hepatoma similar to those observed in animals.     

Chemical characterization of nanometer-sized elemental carbon particles emitted from diesel vehicles

In order to obtain the exact chemical structure and further discuss the global warming effect of elemental carbon (EC) particles, the morphology and the chemical structure of EC particles emitted from diesel vehicles were first investigated in detail using scan electron microscopy equipped with an energy dispersive X-ray spectrometer (SEM-EDX), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), Fourier Transformation Infrared (FT-IR) spectrum and ¹³C Nuclear Magnetic Resonance (NMR) spectrum in this study. The SEM-EDX results showed that EC particles emitted from diesel bus are 50 nm spherical particles with smooth surface and an O/C ratio (mass ratio) of 0.07±0.01. The 50 nm EC particles were further deduced to be an aggregate of new fullerene C₃₆(OH)₂ according to the results of MALDI-TOF-MS, FT-IR and ¹³C NMR. Fullerenes smaller than ⁶⁰C were considered to be unstable since they cannot fulfill the so-called isolated pentagon rule. So far, most of our knowledge regarding small fullerenes comes from gas-phase experiments and theoretical investigations, the production and separation of small fullerene solids in the macroscopic quantity has been reported. The present report is the first discovery of C₃₆(OH)₂-based solid.     

Chemical composition of Athabasca bitumen

A detailed analysis has been carried out on the deasphaltened Athabasca bitumen using two different series of Chromatographic separations followed by i.r., u.v., n.m.r. and computerized GC/MS studies of the separated fractions: saturates (22–25%), monoaromatics (10.3–10.8%), diaromatics (4.6–5.3%), polyaromatics and non-specific polar compounds (28.6%), acids (16.5%), bases (7.8%), and neutral nitrogen compounds (1.6%). The acyclic paraffin content of the maltene is low: straight-chain paraffins and the isoprenoids, phytane and pristane, are present in very low concentrations. Polycyclic saturates represent about 90% of the saturate fraction. The presence of C-27 and C-29–C-35 hopanes of the 17(h):21β(H) series and C-21–C-30 steranes was established. The mono- and diaromatic fractions were analysed by computerized mass spectrometry. The monoaromatic fraction contains alkylbenzenes, naphthenebenzenes and dinaphthenebenzenes in a ratio of 1.0:1.7:1.3. The ratio for naphthalenes, acenaphthenes + dibenzofurans and fluorenes in the diaromatic fraction is 1:0.9:0.5.     

Chemical composition of acacia seeds

Several plants of Acacieae (family Leguminosae) have been recommended under aforestation programs. The seeds of some such plants have been examined for their fatty acid composition with special reference to epoxy acids. Epoxy 18:1 in Acacia auriculiformis, A. catechu, A. coriacea and A. mellifera was 4.9, 0.1, 2.1, and 0.6%,respectively.