Composition and fatty acid content of rat ventral prostate phospholipids

The major phospholipids of rat ventral prostate have been separated and examined using thin-layer chromatography, gas chromatography and mass spectrometry. The main phospholipid classes were choline and ethanolamine glycerophospholipids, accounting for 77.9% of total lipid phosphorus. The prostate also contained small amounts of serine glycerophospholipids and sphingomyelin. The relative proportions of fatty acids in the different phospholipid classes were also determined. Arachidonic acid in prostatic phospholipids is contributed primarily by ethanolamine glycerophospholipids. This fraction contained 65-69 mol% plasmalogens, whereas choline and serine glycerophospholipid fractions contained less than 5 mol% plasmalogens. Ethanolamine, choline and serine plasmalogens contained mainly vinyl ethers of palmitic and stearic aldehydes. Ethanolamine plasmalogens also contained the vinyl ether of oleic aldehyde.     

Studies on ether phospholipids. I. A new method of determination using phospholipase A1 from guinea pig pancreas: application to Krebs II ascites cells

A new method for ether phospholipid analysis has been devised, based on the selective destruction of diacyl phospholipids by guinea pig phospholipase A1 and of plasmalogens by acidolysis. The paper describes optimal conditions allowing a specific degradation of diacyl phospholipids by the enzyme(s). This requires the incubation of a total lipid extract in the presence of 2.4 mM sodium deoxycholate, at pH 8.0, at a temperature of 42 degrees C. As shown with various radioactive markers, all the diacyl phospholipids become degraded, whereas sphingomyelin and ether phospholipids remain refractory to phospholipase A1 attack. Phospholipids are then separated by a bidimensional thin-layer chromatography involving the exposure of the plates to HCl fumes between the two runs, in order to hydrolyse plasmalogens. Selectivity of both hydrolytic procedures is further demonstrated upon analysis of acetyl diacylglycerol derived from phospholipids. Various phospholipids can thus be determined by phosphorus measurement using sphingomyelin as an internal standard. By this way, it is shown that Krebs II cells present a very high content of ether phospholipid species (around 25% of total). Among these, about 50% are alkyl forms in ethanolamine phosphoglycerides, whereas this value reaches 70% in choline phosphoglycerides.     

Renal cortical brush-border and basolateral membranes: Cholesterol and phospholipid Composition and relative turnover

Summary A new procedure for the rapid isolation of renal cortical brush-border and basolateral membranes from the same homogenate is described. Brush-border membranes isolated using Mg²⁺-EGTA precipitation were enriched 18-fold for leucine aminopeptidase and had a recovery of 32.5%. Basolateral membrane fractions were isolated using a discontinuous sucrose gradient and showed an enrichment of 10.7-fold and recovery of 12.8% using (Na⁺, K⁺)-ATPase as a marker enzyme. Lipid analysis using two-dimensional TLC separation of phospholipids and gas liquid chromatography for cholesterol showed marked differences in the lipid composition of the brush-border and basolateral membranes. The brush-border membrane had increased sphingomyelin, phosphatidylserine, ethanolamine plasmalogens, and an increased cholesterol-to-phospholipid and sphingomyelin-to-phosphatidylcholine ratio compared to the basolateral membrane. The relative turnover of total membrane and individual phospholipid species using a double isotope ratio method was carried out. Phospholipids were labeled with either phosphorus 32 and 33 or acetate (³H, 1-¹⁴C). The relative turnover of phospholipid species and cholesterol differed strikingly. Phosphatidylcholine showed a high turnover, phosphatidylethanolamine and phosphatidylinositol had intermediate values and sphingomyelin, phosphatidylserine and cholesterol had low relative turnover rates. The order of phospholipid class relative turnover was independent of the labeled precursor used. The brush-border membrane had a significantly reduced relative turnover rate for total membrane phospholipids, sphingomyelin and cholesterol compared to the basolateral membrane. These data show marked differences in the lipid composition and relative turnover rates of the phospholipid species of the brush-border and basolateral membranes. They provide a biochemical basis for the recently reported differences in brush-border and basolateral membrane fluidity and suggest independent cellular regulation of brush-border and basolateral membrane lipids.     

Phospholipids from Bacillus stearothermophilus

The lipids of Bacillus stearothermophilus strain 2184 were extracted with chloroform-methanol and separated into neutral lipid and three phospholipid fractions by chromatography on silicic acid columns. The phospholipids were identified by specific staining reactions on silicic acid-impregnated paper, by chromatography of alkaline and acid hydrolysis products, and by determination of acyl ester:glycerol:nitrogen:phosphorus molar ratios. The total extractable lipid was 8% of the dry weight of whole cells and consisted of 30 to 40% neutral lipid and 60 to 70% phospholipid. The phospholipid consisted of diphosphatidyl glycerol (23 to 42%), phosphatidyl glycerol (22 to 39%), and phosphatidyl ethanolamine (21 to 32%). The concentrations of diphosphatidyl glycerol and phosphatidyl glycerol were lower in 2-hr cells than in 4- and 8-hr cells. Whole cells were fractionated by sonic treatment and differential centrifugation. The total lipid content, expressed in per cent of dry weight of each fraction was: whole protoplasts, 10%; membrane fraction, 18%; 30,000 x g particulate fraction, 22%; and 105,000 x g particulate fraction, 26%. The relative phospholipid concentrations in each fraction were about the same. As had been previously reported, none of the phospholipid was stable to alkaline hydrolysis.     

Lipid composition of purified transverse tubule membranes isolated from amphibian skeletal muscle

The level and proportion of lipids and their fatty acid composition were analyzed in highly purified transverse tubule membranes of amphibian skeletal muscle. Tubule membranes show (a) a higher content of lipids, (b) a higher phospholipid/cholesterol ratio and (c) a different phospholipid composition from other subcellular fractions, such as the light and heavy membranes from sarcoplasmic reticulum, which are similar in lipid profile. Transverse tubule membranes are characterized by a high percentage of phosphatidylserine and sphingomyelin and a low proportion of phosphatidylcholine compared with the other membranes. All three show a high proportion of ethanolamine plasmalogens (50% of the total ethanolamine glycerophospholipid). Transverse tubule membrane lipids contain a high proportion of 20- and 22-carbon polyunsaturated fatty acids, predominantly 20:4, 20:5, 22:5 and 22:6. Arachidonate predominates in phosphatidylinositol, eicosapentaenoate and docosahexaenoate in ethanolamine and serine glycerophospholipids.     

Phospholipid and nucleic acid gradients in the developing amphibian embryo

Rana pipiens embryos at the end of the blastula stage were dissociated and the cell suspension was separated into presumptive ectoderm, mesoderm, light endoderm, and heavy endoderm cells by a discontinuous density gradient centrifugation technique. The isolated germ layers were analyzed for total lipid, lipid phosphorus, plasmalogen, RNA, and DNA. Per gram dry weight, DNA showed a threefold decrease from ectoderm to heavy endoderm. On the same basis, the RNA content of the mesoderm was 34 per cent higher than that of ectoderm, and 320 and 570 per cent higher than that of light and heavy endoderm, respectively. In addition to the RNA and DNA gradients, there were at least two superimposed lipid gradients: a neutral lipid gradient decreasing from ectoderm to endoderm, and a total phospholipid gradient increasing from ectoderm to endoderm. In contrast to total phospholipid, a specific phospholipid class, ethanolamine plasmalogen, decreased from ectoderm to endoderm. The total lipid content per gram dry weight was the same in all the germ layers. Total phospholipids were analyzed quantitatively by thin layer chromatography. Phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and inositol phospholipid constituted 34, 13, 12, and 34 per cent, respectively, of the total lipid phosphorus. The phospholipid composition was different in each germ layer. The possible role of specific lipids in embryonic induction and differentiation is discussed.     

Composition and incorporation of [3H]arachidonic acid into molecular species of phospholipid classes by cultured human endothelial cells

Based on quantitative high-performance liquid chromatographic analyses of molecular species in selected phospholipid subclasses from culture human umbilical vein endothelial cells, the relative degree of unsaturation was ethanolamine plasmalogens greater than phosphatidylethanolamine greater than phosphatidylcholine. A total of 36 different molecular species were identified in the phosphatidylcholine fraction. Interestingly, the phosphatidylcholine contained a significant amount (11.7%) of the dipalmitoyl species, a lipid normally associated with lung surfactant. The arachidonoyl-containing molecular species of phosphatidylserine/inositol were labeled to the highest extent and the ethanolamine plasmalogens contained the lowest specific radioactivity after incubating [3H]arachidonic acid with human endothelial cells for 4 h. Within each phospholipid subclass the arachidonoyl species where both acyl groups of the phospholipid are unsaturated (20:4-20:4, 18:2-20:4 + 16:1-20:4, and 18:1-20:4) had higher specific radioactivities, after labeling with [3H]arachidonic acid, than those that contained saturated aliphatic chains (16:0-20:4 and 18:0-20:4). This indicates that the unsaturated species have higher turnover rates.     

Lipid requirement of the membrane sodium-plus-potassium ion-dependent adenosine triphosphatase system.

The dependence of the (Na-++K-+)-dependent ATPase (adenosine triphosphatase) (EC 3.6.1.3) on lipid has been examined in a number of different ways, with the use of various preparations from kidney tissue. The main findings were as follows. (1) The ATPase activities of the preparations examined were closely correlated with their total phospholipid content. (2) Extraction of the ATPase with deoxycholate or Lubrol W, combined with suitable salt-fractionation and washing procedures, removed phospholipid, cholesterol and enzymic activity in parallel; but activity was completely lost before all lipid had been removed. (3) The loss of activity could not be attributed to inhibition by residual detergent. (4) No selective removal of any particular phospholipid class by detergent could be detected. (5) Consistent reactivation of the Lubrol-extracted enzymes was obtained by adding dispersions of exogenous phospholipid, but only some, bearing a net negative charge, such as phosphatidylserine and phosphatidylglycerol, were effective. (6) The degree of reactivation was correlated with the amount of residual activity remaining after lipid depletion. (7) Partial purification of the ATPase, giving a 50-fold increase in specific activity, was not accompanied by selective enhancement of any particular class of phospholipid. We conclude that although the ATPase is dependent on phospholipid, only the reactivation results provide evidence for specificity.     

Lipids of human leukocytes: relation to celltype

Significant differences in lipid composition have been found between normal human lymphocytes and polymorphonuclear leukocytes (isolated from blood by means of glass-bead columns), abnormal leukocytes from patients with acute and chronic leukemia, and leukocytes from peritoneal exudates. Lipid extracts of isolated leukocytes were analyzed for total lipid, phosphorus, cholesterol, and plasmalogens. Individual phospholipids and neutral lipids were separated by thin-layer chromatography. The major phospholipids were phosphatidyl choline, ethanolamine glycerophosphatides, sphingomyelin, phosphatidyl serine, and phosphatidyl inositol. Plasmalogen was found mainly as phosphatidal ethanolamine. The neutral lipid fractions contained free cholesterol and various amounts of triglyceride, but little esterified cholesterol. Normal lymphocytes contained about half as much total lipid per cell as normal polymorphonuclear leukocytes, with a similar cholesterol:-lipid-P ratio but relatively more lecithin and less ethanolamine glycerophosphatide. Normal mature leukocytes, compared with immature cells of the same morphological series, had a higher total lipid content per cell, more cholesterol, and a higher ratio of cholesterol to lipid-P. Little difference was found in total lipid-P per cell, but mature cells contained relatively less lecithin and more sphingomyelin. These findings may reflect differences in the relative content of various intracellular organelles as well as possible differences in the quantity and composition of the plasma membrane.     

MALDI-TOF "fingerprint" phospholipid mass spectra allow the differentiation between ruminantia and feloideae spermatozoa

The detailed comparative analysis of sperm lipids could essentially contribute to a better understanding of membrane function in the context of fertilization and, moreover, of sperm preservation. The application of sensitive analytical methods is particularly necessary for endangered species as the available amount of spermatozoa (and, accordingly, extractable lipids) is strongly limited. It will be shown that matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a fast, simple and sensitive method for the determination of the phospholipid composition of spermatozoa from several ruminantia (cattle, roe deer, Klipspringer) and feloideae species (domestic cat, Siberian tiger, fosa). Characteristic “fingerprints” are obtained from the positive ion spectra that allow the differentiation between both animal groups. In contrast to the lipid extracts of ruminantia spermatozoa which predominantly contain ether lipids including essential amounts of plasmalogens, the more complex phospholipid composition of feloideae spermatozoa is clearly dominated by diacyl phospholipids and contains only marginal amounts of plasmalogens. It will also be shown that the lipid compositions of ejaculated, electroejaculated and cauda epididymal spermatozoa of the same species are very similar and give comparable data. Therefore, the analysis of ejaculated spermatozoa is not an absolute must.     

Influence of plasmalogen deficiency on membrane fluidity of human skin fibroblasts: a fluorescence anisotropy study

The influence of plasmalogen deficiency on membrane lipid mobility was determined by measuring fluorescence anisotropy of trimethylammoniumdiphenylhexatriene (TMA-DPH) and diphenylhexatrienylpropanoylhydrazylstachyose (glyco-DPH) inserted in the plasma membranes of human skin fibroblasts deficient in plasmalogens. The cells used were from patients affected with cerebrohepatorenal (Zellweger) syndrome (CHRS) or rhizomelic chondrodysplasia punctata. Their plasmalogen content (0-5% of total phospholipid) is significantly reduced compared with that of control cells from healthy donors (13-15% of total phospholipid) or of CHRS fibroblasts supplemented with the plasmalogen precursor, hexadecylglycerol. Plasmalogen-deficient cells consistently showed lower fluorescence anisotropies of membrane-bound DPH fluorophores corresponding to higher membrane lipid mobilities as compared to controls. However, very similar lipid mobilities were found for sonicated aqueous dispersions of phospholipids extracted either from CHRS or control cells. Therefore, the differences observed with living cells are not due to differences in the overall physical properties of the membrane lipid constituents. Other phenomena such as lipid asymmetry and/or plasmalogen-protein interactions may be responsible for the effects observed in the biomembranes.     

Lipid Properties of Carex aquatilis from Hot Spring and Permafrost-Dominated Sites in Alaska: Implications for Nutrient Requirements

Selected lipid properties were compared between populations of Carex aquatilis occupying adjacent hot spring and permafrost-dominated sites in interior Alaska. Analyses were carried out on a variety of plant parts and results correlated with prevailing ambient temperature. Phospholipid concentrations were higher in plants from permafrost than from hot spring areas, even though there was no consistent interpopulation difference in total lipid content. The population differences in phospholipid content were greatest in those plant parts that experienced the greatest interpopulation temperature difference. Despite lower soil phosphorus availability, permafrost plants had higher total phosphorus concentrations, and a larger proportion of their phosphorus was contained in phospholipid than in hot spring plants. There was no consistent relationship between the proportion of various classes of phospholipids present and prevailing ambient temperature. Fatty acid chain length and degree of unsaturation of the phospholipid fraction were inversely correlated with temperature both between populations and among plant parts. No such relationship was found in the fraction containing neutral lipids. We suggest that the high tissue phosphorus content that characterizes northern plants may be necessary in part to maintain large quantities of phospholipid-containing membrane. Increased amounts of membrane in turn may be a prerequisite for the high rates of membrane-associated processes characteristic of plants in the cold subarctic environment.     

Naturally derived commercial surfactants differ in composition of surfactant lipids and in surface viscosity

Pulmonary surfactant biophysical properties are best described by surface tension and surface viscosity. Besides lecithin, surfactant contains a variety of minor lipids, such as plasmalogens, polyunsaturated fatty acid-containing phospholipids (PUFA-PL), and cholesterol. Plasmalogens and cholesterol improve surface properties of lipid mixtures significantly. High PUFA-PL and plasmalogen content in tracheal aspirate of preterm infants reduces the risk of developing chronic lung disease. Different preparations are available for exogenous surfactant substitution; however, little is known about lipid composition and surface viscosity. Thus lipid composition and surface properties (measured by oscillating drop surfactometer) of three commercial surfactant preparations (Alveofact, Curosurf, Survanta) were compared. Lipid composition exhibited strong differences: Survanta had the highest proportion of disaturated PL and total neutral lipids and the lowest proportion of PUFA-PL. Highest plasmalogen and PUFA-PL concentrations were found in Curosurf (3.8 +/- 0.1 vs. 26 +/- 1 mol%) compared with Alveofact (0.9 +/- 0.3 vs. 11 +/- 1) and Survanta (1.5 +/- 0.2 vs. 6 +/- 1). In Survanta samples, viscosity increased >8 x 10(-6) kg/s at surface tension of 30 mN/m. Curosurf showed only slightly increased surface viscosity below surface tensions of 25 mN/m, and viscosity did not reach 5 x 10(-6) kg/s. By adding defined PL to Survanta, we obtained a Curosurf-like lipid mixture (without plasmalogens) that exhibited biophysical properties like Curosurf. Different lipid compositions could explain some of the differences in surface viscosity. Therefore, PL pattern and minor surfactant lipids are important for biophysical activity and should be considered when designing synthetic surfactant preparations.     

Protein-Associated Lipid of Bacillus stearothermophilus

The composition and patterns of metabolism of phospholipids isolated as part of a lipid-depleted membrane fragment (LDM fragment) and associated with the membrane adenosine triphosphatase complex have been compared with those of the bulk membrane phospholipid. The bulk lipid was extracted from washed membranes with sodium cholate. The LDM fragments, which contained a portion of the electron transport system and the membrane adenosine triphosphatase complex, were purified by chromatography with Sepharose 6B. The LDM fragment preparations contained 0.10 +/- 0.02 mumol of lipid phosphorus per mg of protein, compared with 0.54 +/- 0.05 mumol of lipid phosphorus per mg of protein for washed membranes. The phospholipid associated with the LDM fragments consisted of 78 +/- 4% cardiolipin, 7 +/- 1% phosphatidylglycerol, and 15 +/- 3% phosphatidylethanolamine. Changes in the total membrane lipid composition (produced by culture conditions) did not alter the phospholipid composition of the LDM fragments. The adenosine triphosphate complex was separated from the other components of the LDM fragments by suspension of the fragments in 1% Triton X-100 and precipitation with antibody specific for the F(1) component of the adenosine triphosphatase complex. The phospholipid isolated with the adenosine triphosphatase complex consisted of 86% cardiolipin, 8% phosphatidylglycerol, and 6% phosphatidylethanolamine. In pulse-chase experiments with (32)P and [2-(3)H]glycerol, the labeling patterns of the phosphatididylglycerol and phosphatidylethanolamine associated with the LDM fragments were different from those of the bulk membrane phosphatidylglycerol and phosphatidylethanolamine. It was concluded that at least a portion of the phospholipid isolated with the LDM fragments was part of a native lipid-protein complex.     

Hemagglutinating Property of Haemophilus aegyptius

Extracts possessing the capacity to hemagglutinate normal human erythrocytes were recovered from Haemophilus aegyptius by treatment with either diethylene glycol or acetone. Antisera prepared against these extracts or the unextracted bacterial cell inhibited hemagglutination by homologous and heterologous antigens. Microgel diffusions indicated the presence of identical components in each extract as expressed by lines of identity between antisera to each fraction. The hemagglutinin was identified as a lipopolysaccharide, 42% lipid and 57% carbohydrate. The determination of 6% phosphorus in the lipid fraction identified it as containing phospholipid.     

High plasmalogen and arachidonic acid content of canine myocardial sarcolemma: a fast atom bombardment mass spectroscopic and gas chromatography-mass spectroscopic characterization

Canine myocardial sarcolemma was purified, and its phospholipid constituents were determined by gas chromatography-mass spectrometry, fast atom bombardment mass spectrometry, and conventional techniques. Canine myocardial sarcolemma contained 2.7 mumol of lipid Pi/mg of protein which was comprised predominantly of choline glycerophospholipids (47%), ethanolamine glycerophospholipids (28%), and sphingomyelin (11%). Sarcolemmal phospholipids contained 40% plasmalogen which was quantitatively accounted for by choline (57% of choline glycerophospholipid) and ethanolamine (64% of ethanolamine glycerophospholipid) plasmalogens. Choline plasmalogens contained predominantly the vinyl ether of palmitic aldehyde though ethanolamine plasmalogens were composed predominantly of the vinyl ethers of stearic and oleic aldehydes. The majority of sarcolemmal ethanolamine glycerophospholipids (75%) contained arachidonic acid esterified to the sn-2 carbon. Sphingomyelin was composed predominantly of long-chain saturated fatty acids (stearic and arachidic) as well as substantial amounts (8%) of odd chain length saturated fatty acids. The possible functional role of these unusual phospholipid constituents is discussed.     

Lipid composition of the spinal cord in the fruit bat Rousettus aegyptiacus

Spinal cords were removed from ten Egyptian fruit bats Rousettus aegyptiacus and the lipids analysed. The major phosphatides were choline glycerophosphatide (32.3%), ethanolamine glycerophosphatide (29.3%), serine glycerophosphatide (15.2%) and phosphatidylinositol (8.4%). Sphingomyelin accounted for 13.8% of the phospholipid. Glycosphingolipids amounted to 43.2 mol/100 mol lipid phosphorus, plasmalogens 32.5 mol/100 mol P and cholesterol 159.5 mol/100 mol P. The fatty acid composition of whole cord, sphingomyelin and non-hydroxy cerebroside were determined. The results were compared with data from other species.     

Phospholipids of Thiobacillus thiooxidans

Cells and spent growth media from sulfur- and thiosulfate-grown cultures of Thiobacillus thiooxidans were analyzed. The phosphatides were examined by thinlayer chromatography, and the products of their hydrolysis by hydrochloric acid and methanolic potassium hydroxide were separated by paper chromatography. The phospholipids in both cells and spent growth media were identified as phosphatidyl ethanolamine, phosphatidyl-N-monomethylethanolamine, phosphatidyl glycerol, and diphosphatidyl glycerol. These comprised about 97% of the total lipid phosphorus. Lyso-phosphatidyl-N-monomethylethanolamine and lysophosphatidyl glycerol accounted for the remaining 3%. The percentage of the total lipid phosphorus accounted for by each phospholipid depended on the age of the culture.     

An apolipoprotein preferentially enriched in cholesteryl ester-rich very low density lipoproteins

Phosphatidyl glycerol is present in lamellar bodies and in the material obtained by alveolar wash representing 12.3 and 11.5%, respectively, of the total phospholipid phosphorus. Lung microsomes catalyze the formation of phosphatidyl glycerol from the known precursors, L-glycerol 3-phosphate and CDP-diglyceride. The rate of [¹⁴C]L-glycerol 3-phosphate incorporation into phosphatidyl glycerol was 30% higher in microsomes as compared to mitochondria. The addition of mercuric chloride inhibited the synthesis of phosphatidyl glycerol, and stimulated the incorporation into another as yet incompletely identified lipid. After pulse labeling of microsomal phosphatidyl glycerol $\text{in vitro}$, further incubation of microsomes with lamellar bodies or alveolar wash resulted in nearly quantitative appearance of label in surfactant.     

Characterization of lipid composition in stimulated human lymphocytes by 1H-NMR

Recent in vivo NMR studies have raised interest in the structural changes of cellular lipids during proliferative activity. We investigated the changes in plasma membrane lipid and total cell lipid during mitogenically-stimulated proliferation of human peripheral blood lymphocytes by extraction of lipids and assay by 500 MHz 1H-NMR. Resonances were assigned using one- and two-dimensional spectroscopic techniques, and signals unique to certain species of lipid were identified. Choline and ethanolamine-containing lipids, glycerophospholipid backbones, sphingolipids, cholesterol, plasmalogens and triacylglycerols were readily detected. Resolution of a number of lipid species was not possible, despite the use of high-resolution techniques. NMR values for proliferation-induced changes in the most easily determined parameters, namely the total cholesterol to total phospholipid molar ratio, and phosphatidylcholine, phosphatidylethanolamine and sphingolipid composition, were found to agree with traditional methods. Differences in phospholipid and fatty acid profiles were found between plasma membranes and total cell lipid for resting values and for response to mitogen.