Analysis of subcellular phosphatidyl choline in developing rabbit lung

Phosphatidyl choline is a major lung surfactant. Insufficient development of the surfactant in neonates is often associated with the Respiratory Distress Syndrome. The concentration and fatty acid composition of phosphatidyl choline have not been studied in the subcellular organelles of the developing lung. This study has investigated the development of the concentration and fatty acid composition of phosphatidyl choline in subcellular fractions of 28-day and 30-day fetal and maternal New Zealand rabbit lungs. The concentration of total phospholipids in lamellar bodies increased four to five fold from 28-day fetus to 30-day fetus which, in turn, was similar to the maternal level. Total phospholipid content increased only about 50% in mitochondria and microsomes. The percentage of phosphatidyl choline among total phospholipids in lamellar bodies increased successively from 60% at 28 days gestation to 84% at 30 days gestation and leveled at 84% in maternal lamellar bodies. Microsomal PC increased steadily from 52% in the 28-day fetus to 65% in the adult. Analysis of the fatty acid composition of phosphatidyl choline in lamellar bodies confirmed 16∶0 as the major fatty acid, and its content remained constant from 28 days gestation to adult. In contrast, the content of 16∶0 of the microsomal phosphatidyl choline decreased with increasing gestation. Changes of several unsaturated fatty acid components were observed in both lamellar bodies and microsomes in the developing lungs. Maturational development of phosphatidyl choline is reflected in an increase in the concentration of this surfactant, particularly in lamellar bodies, and possibly in remodeling of fatty acid composition in both lamellar bodies and microsomes.     

Phospholipid exchange proteins in rat intestine

The 105,000 g supernatant and pH 5.1 supernatant fractions from rat intestinal homogenates stimulate phosphatidylcholine exchange between [³²P] phosphatidylcholine liposomes and beef heart mitochondria. This active fraction shows the characteristics of a protein. Isoelectric focusing of the intestinal pH 5.1 fraction shows two peaks of phosphatidylcholine exchange activity: one at an acidic pH (4.5–5.3), the other in a basic pH range (8–9). The second peak of activity appears to be a new phospholipid exchange protein. The anatomic distribution of phosphatidylcholine exchange activity in intestine has been investigated. Expressed per mg of protein, phosphatidylcholine exchange activity is higher in mucosa than in the intestinal wall. No significant differences have been found between villi and crypts cells or between jejunal and ileal villi. Furthermore, exchange activity per mg of protein in mucosa is unaffected by fasting or by feeding a high fat or high cholesterol diet. This suggests that phospholipid exchange activity in the absorptive cells is not a rate limiting step in the process of fat absorption. North Atlantic Treaty Organization grant recipient.     

Modification of the fatty acid composition of L1210 leukemia subcellular organelles

We have examined the extent to which it is possible to modify the fatty acid composition of subcellular organelles of L1210 leukemia cells. A polyunsaturated fatty acid, docosahexaenoic acid, or a monounsaturated fatty acid, oleic acid, were added to the culture media. After 48 hr, the cells were ruptured and the subcellular fractions isolated. Fatty acid analysis revealed that nuclei, mitochondria, plasma membranes and microsomes of the cells grown in media supplemented with docosahexaenoic acid contained increased amounts of polyenoic fatty acids, mean number of double bonds and docosahexaenoic acid compared with cells grown in oleic acid. We conclude that it is possible to experimentally modify the lipids of multiple intracellular structures of L1210 cells by the addition of fatty acids to the growth media.     

Phospholipid composition of guinea pig lung lavage

Phospholipids from guinea pig lung lavage were analyzed. The total lavage phospholipid content was 2.65+0.67 mg, per gram of lung, which accounted for 85% of the total lipids in lung wash. Phosphatidylcholine (PC) accounted for over 60% of the total phospholipids. The other phospholipids factions, in order of pedominance, were phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin (SPH), phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and lysophosphatidyl-choline (LPC). Disaturated phosphatidylcholine (DSPC) comprised 80% of the total PC, and it contained ostly palmitic acid. The DSPC content of the lung lavage fluid per square meter of alveolar surface area was 5.76±0.42 mg.     

Characterization of phospholipase A2 from rabbit lung microsomes

A phospholipase A₂ activity associated with the microsomal fraction of rabbit lung homogenates was studied. The enzyme showed specificity for thesn ⁻² ester bond of phosphatidylcholine, had an alkaline pH optimum and required Ca²⁺ for activity. Other divalent cations were unable to support hydrolysis. In the absence of detergents, exogenous phosphatidylethanolamine was deacylated at a rate sevenfold higher than phosphatidylcholine. The activity toward both substrates could be enhanced by sodium deoxycholate or, more effectively, by sodium taurodeoxycholate. Phosphatidylethanolamine required higher detergent/phospholipid molar ratios than phosphatidylcholine. Under these conditions, the preference for the former substrate over the latter was nearly abolished. The zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) and the nonionic detergent Triton X-100 were either ineffective (phosphatidylcholine) or inhibitory (phosphatidylethanolamine). Addition of KCl produced opposite effects on the activity depending on the bile salt used to disperse the substrate. The phospholipase A₂ activity was inhibited byp-bromophenacyl bromide but remained unaffected after treatment with diisopropylfluorophosphate or NaF. N-Ethylmaleimide, but not other thiol reagents, partially inhibited the activity. Presented in part at the 29th CFBS meeting, Guelph, Canada, June 1986, and at the symposium “25 Years Lipids and Biomembranes,” Utrecht, The Netherlands, June 1986.     

Subcellular distribution of disaturated phosphatidylcholine in developing rabbit lung

To determine the subcellular distribution of disaturated phosphatidylcholine (DSPC) in lung tissue during perinatal development, fetal rabbits at 24, 26, 28 and 31 (term) days gestation and newborns were studied. Following alveolar lavage, fractions enriched in nuclei-cellular debris, mitochondria, microsomes, surfactant (lamellar bodies) and cytosol were prepared from the residual tissue homogenate, and their DSPC content was determined. The DSPC content of the unfractionated residual lung tissue homogenate progressively and significantly increased during fetal development, rising from 9.09±0.91 to 17.45±2.88 mg/g dry lung between 24 days gestation, and term. Between 24 and 26 days gestation the overall increase in tissue DSPC was due to a two-fold increase in the mitochondrial, microsomal and cytosolic pools. Lamellar bodies were first isolable at 26 days gestation. The DSPC content of this fraction increased six-fold (from 0.10±0.02 to 0.67±0.15 mg/g dry lung) between 26 and 28 days gestation and a further seven-fold (to 4.63±1.06 mg/g dry lung) by term, accounting for the overall increase in the tissue homogenate value during this time period. By the first postnatal day, microsomal and cytosolic DSPC increased another two-fold, but no significant change occurred in the other subcellular fractions. Alveolar lavage DSPC progressively increased over the time period studied. While there was no change in the lamellar body DSPC/total PC ratio during fetal development, each of the mitochondrial, microsomal and cytosolic ratios decreased between days 26 and 28 of gestation and then increased at term. Our results indicate that in addition to the pulmonary surfactant, for which DSPC is often used as a marker, other subcellular organelles contain significant DSPC pools that undergo dynamic changes in size during perinatal development.     

Phospholipid content of foods

The content of total lipids and total and component phospholipids in ca. 140 foods was compiled in response to frequent requests for data by researchers in nutrition and medicine, and to fill the appar-ent need for a reliable up-to-date tabulation of recent data. Eggs, organ meats, lean meats, fish, shellfish, cereal grains and oilseeds are good sources of phospholipids, especially the choline phosphatides: phosphatidylcholine, sphingomyelin and lysophosphatidylcholine. Leafy vegetables, fruiting parts, roots and tubers are, with few ex-ceptions, relatively poor dietary sources of total lipids and phospho-lipids. Foods or tissues in which the phospholipids perform similar functions also have similar relative phospholipid distributions. The data were tabulated by food group in separate tables with appropri-ate discussion. The use of conversion factors for calculating the total and individual phospholipids, sources of error, and research needs are discussed.     

Ion exchange between two solid-oxide electrolytes

The contact resistances between two samples of zirconia based electrolytes ZrO₂  Y₂O₃ 9 mole% were measured under small direct polarizations and by the impedance diagram technique. The influences of the oxygen pressure and temperature on these contact resistances indicate that no neutral adsorbed oxygen particle is involved in the predominant process of oxygen exchange between the two phases in contact. The direct exchange of ions is likely to prevail. The characteristics of the capacitive effect measured at the contact are also consistent with this assumption.     

纯磷脂单分子膜压缩过程中磷脂分子的构象和取向(英文)

On the basis of energy conservation law and surface pressure isotherm, the conformation energy changes of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) in pure phospholipid monolayer at the air/water interface during compression are derived. The optimized conformations of phospholipids at absolute freedom state are simulated by Gaussian 98 software. Based on following assumptions: (1)the conformation energy change is mainly caused by the rotation of one special bond; (2)the atoms of glycerol near the water surface are active; (3)the rotation is motivated by hydrogen-bond action; (4)the rotation of bond is inertial, one simplified track of conformational change is suggested and the conformations of DPPC and DPPG at different states are determined by the plots of conformation energy change vs. dihedral angle. The thickness of the simulated phospholipid monolayer is consistent with published experimental result. According to molecular areas at different states, the molecular orientations in the compressing process are also developed.     

Phospholipid reactivation of plasmalogen metabolism

This report is concerned mainly with the properties of an enzyme from rat liver microsomes which hydrolyzes the alkenyl ether bond of 1-(1′-alk-1′-enyl)-glycero-3-phosphoryl-choline (alkenyl-GPC hydrolase). Destruction of the normal environment of the microsomes by treatment with phospholipases A or C caused inactivation of the alkenyl-GPC hydrolase, which was then partially reactivated by the addition of exogenous phospholipids. Both sphingomyelin and diacyl-GPC were efficient in restoring activity; diacyl-GPE was less effective; and monoacyl-GPC and monoacyl-GPE were ineffective. The presence of two long hydrocarbon chains in the lipid activator is apparently required for reactivation, suggesting that interaction of hydrophobic areas of the enzyme with the phospholipid is necessary for maximal activity. High concentrations of sucrose mimicked the effect of phospholipids, and because the sucrose and diacyl-GPC did not show an additive effect, they may reactivate the enzyme in a similar manner. Disrupting the enzyme's environment by freezing and thawing the preparation also resulted in a loss of enzymatic activity, which was restored by added exogenous phospholipids. The alkenyl-GPC hydrolase was inhibited by imidazole and some of its derivatives. Histidine and N-acetyl histidine did not inhibit the enzyme, presumably due to the presence of a negative charge on the carboxyl group rather than the steric bulk of that group, since histidine methyl ester did inhibit the enzyme. Kinetic evidence showed imidazole to be a competitive inhibitor. The enzymatic activity of imidazole-treated microsomes also increased following addition of exogenous phospholipids. Imidazole inhibition differed from the phospholipase A-inactivation in that it was partially reversed by KCl, but not by sucrose. Imidazole did not inhibit other microsomal enzymes tested, indicating that it is not a general inhibitor of membrane-associated enzymes.     

Synthesis of platelet activating factor by cholinephospho-transferase in developing fetal rabbit lung

Developing fetal lung is a possible source of the platelet activating factor (PAF, 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) present in amniotic fluid of women in labor. We have assayed the microsomal activities of a specific enzyme for the de novo synthesis of PAF in developing fetal and neonatal rabbit lung, 1-alkyl-2-acetyl-glycerol-dependent dithiothreitol-insensitive cholinephosphotransferase. The specific activity of this enzyme increased from 0.92 to 3.60 nmol×min⁻¹×mg⁻¹ protein between day 21 and day 31 of gestation. In contrast, during this same period the activity of the PAF-biosynthetic cholinephos-photransferase in developing rabbit kidney did not change significantly. The specific activity of the diacyl-glycerol-dependent, dithiothreitol-sensitive cholinephos-photransferase that catalyzes the final step in phosphatidylcholine biosynthesis was not altered during late gestation in either fetal lung or kidney. Previously, increased amounts of pulmonary PAF had been found during the latter stages of gestation (Hoffman, Truong and Johnston (1986)Biochim. Biophys. Acta 879, 88–96) and may be attributed to increased activity of the PAF biosynthetic enzymes found in this investigation. This elevated level of PAF in fetal lung may serve to facilitate breakdown of glycogen that provides, in part, the carbon and energy source for surfactant biosynthesis. In addition, PAF may be secreted in association with surfactant into amniotic fluid in which it may interact with amnion tissue and subsequently participate in the events associated with the initiation of parturition.     

Electrodialysis processes using ion exchange resins between membranes

Ion exchange resins were introduced between permselective membranes in a laboratory scale electrodialysis unit. It was found that by this method polarization is substantially decreased, and high electrical efficiency can be obtained even when working on highi/c values (1000–5000) and at low linear velocity of solution (1–2 cm/sec) in the cell. Different ion exchange resins were introduced in the diluate and brine cells and the boundary conditions of such a stack were determined.     

Physico-chemical Properties of Marine Phospholipid Emulsions

Many studies have shown that marine phospholipids (PL) have better bioavailability, better resistance towards oxidation and contain higher polyunsaturated fatty acids such as eicosapentaenoic (EPA) and docosahexaenoic acids (DHA) than triglycerides (TAG) present in fish oil. The objective of this study was to investigate the emulsifying properties of various commercial marine PL and the feasibility of using them to prepare stable emulsions prepared with or without addition of fish oil. In addition, this study also investigated the relationship between chemical composition of marine PL and the stability of their emulsions. Physical stability was investigated through particle size distribution (PSD), zeta potential, microscopy inspection and emulsion separation (ES); while the oxidative and hydrolytic stability of emulsions were investigated through peroxide value (PV) and free fatty acids value (FFA) after 32?days storage at room temperature and at 2?°C. In conclusion, marine PL showed good emulsifying properties and it was possible to prepare marine PL emulsions with and without addition of fish oil. Emulsion with both good oxidative stability and physical stability could be prepared by using marine PL of high purity, less TAG, more PL, cholesterol and higher antioxidant content.     

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.     

Alveolar lavage and lavaged lung tissue phosphatidylcholine composition during fetal rabbit development

Pulmonary surfactant, the major surface-active component of which in the adult is dipalmitoylglycerophosphocholine, can be obtained by lavaging lungs with physiological saline. We have previously shown that there is an increase in the amount of phosphatidylcholine in fetal rabbit lung lavage during the latter part of gestation. We have now measured the amount of disaturated phosphatidylcholine as well as the fatty acid composition of phosphatidylcholine in lung lavage from fetal rabbits during the period 27 days’ gestation to full term (31 days). There was no developmental change in the amount of disaturated phosphatidylcholine during the period examined. About 50% of the total phosphatidylcholine was disaturated which is approximately the same as in adult rabbit lung lavage. The fatty acid composition, however, did change. There was an increase in the amount of 16∶0 from about 20% of the total fatty acids in phosphatidylcholine at 27–28 days to about 60% at full term, after birth, and in the adults. There was a corresponding decrease in the amounts of 14∶0, 18∶0, and longer chain fatty acids, most of which were saturated. In the lavaged lung tissue, there was a 2.6-fold increase in the percentage of phosphatidylcholine which was disaturated during the period 27–31 days’ gestation. It had not decrease to the adult value 24 hr after birth.     

Solids exchange between adjacent fluid beds without gas mixing

A new reactor system, in which two adjacent fluidized beds exchange solids but not gases, has been developed, and patented. The partition between the two fluidized beds is designed to permit the movement of solids in both directions, but to provide an effective seal against gas mixing. There are no moving parts. High circulation rates can be achieved, and can easily be controlled. Gas mixing at less than the 2% level can be achieved without a neutral blanketing gas to seal the interface. It should be possible to achieve zero leakage with a blanketing gas. The reactor system was developed as an oil shale retort in which the combustor exchanges solids with the retort — but in which the combustion gases and product gases are kept separate. However, the system has many potential applications including gasification and pyrolysis of coal, hydrogen production, heat exchange in difficult conditions and SO₂ removal by absorption or adsorption.     

Interactions between large organic cations and cation exchange membranes

Interactions between cation exchange membranes and large organic cations have been studied; namely (1) adsorption and electrodialytic desorption of cationic surface active agents, and (2) change in transport properties of the membranes by adsorption.N-dodecyl pyridyl compounds, such as N-dodecyl pyridinium bromide (C₁₂-Py), N-dodecyl dipyridyl bromide (C₁₂-Dipy) and N-dodecyl tripyridyl bromide (C₁₂-Tripy) were used for the adsorption and electrodialytic desorption; and for the study on the change in transport properties of membranes, N-dodecyl pyridinium chloride, C₁₂-Py, C₁₂-Dipy, C₁₂-Tripy and hexadecyl trimethylammonium chloride were used. It was confirmed that the higher the molecular weight of the cation the longer the time required to reach adsorption equilibrium, and that the amount of adsorption exceeded the ion-exchange capacity of the membrane. After equilibrium was attained, electrodialytic desorption from the membranes was difficult.The adsorption or ion-exchange of large organic cations was effective to the permselectivity of the cation exchange membrane to the sodium ion in electrodialysis of a solution of sodium chloride and calcium chloride. The relationship between the permselectivity and the properties of the cations was examined.