Formation Of Lecithin Research Articles

Particle Formation of Lecithin Process with Particles from Gas-Saturated Solutions using Supercritical Carbon Dioxide

The particles from gas-saturated solutions process was performed to micronize lecithin isolated by ethanol from deoiled mackerel by Supercritical Carbon Dioxide (SC-CO2). The particle formation of lecithin with biodegradable polymer, polyethylene glycol (PEG) was carried out by PGSS process using SC-CO2. Conditions were optimized by applying different temperatures (40 and 50°C) and pressures (15-30MPa) for PGSS process for 1 hr. The particles were characterized by scanning electron microscope (SEM) and Particle size analyzer (PSA) to determine their shape. The average diameter of lecithin particle was high at higher temperature and pressure. The average diameter of the particles was about 0.37-2.42μm. The inclusion of lecithin in PEG was qualitative measured by HPLC.

Thermodynamics on Penetration of Amino Acid Surfactant into Lipid Spread Films

This review was introduced on the recent studies of thermodynamic arguments for equilibrium penetration. Two penetration systems, especially, which were composed of L-α, β-dimyristoyllecithinN-dodecyl-β-alanine (NDA) surfactant and α, α'-dilaurin NDA were described in detail. The characteristics in investigation of these systems was that adsorbed amount of surfactant was directly measured by a radiotracer technique using tritium-labeled compounds at solution surface with and without the spread monolayers. The molecular complex formation of lecithin and NDA was precisely explained how to be analyzed from the enthalpy changes of spread films and adsorbed surfactant in penetration at water surface.

1-acyl-lysolecithin acyltransferase and synthesis of biliary lecithins in rat liver

The role of lysolecithin acyltransferase activities in biliary lecithin formation was investigated, using livers perfused in the presence of labeled palmitoyl-lysolecithin and albumin, overloaded or not with linoleic acid. At the end of liver perfusion, the lecithins extracted from microsomes, mitochondria and plasma membranes displayed the same specific activity. Double-labeled lysolecithin was used to prove that labeled lecithins were synthesized by lysolecithin acylation. In the absence or presence of a linoleic acid overload, the level of lysolecithin incorporation into linoleyl and arachidonyl containing lecithin was identical. Hence fatty acids did not influence phosphatidylcholine synthesis by the acylation pathway. In vitro the rate of linoleyl lecithin synthesis was the same in plasma membranes, mitochondria and microsomes provided the linoleyl-CoA concentration was lower than 3 μ M Taurocholate was essential to the excretion of lecithin synthesized from lysolecithin and stimulated its synthesis. The specific activities of the two lecithin molecular species excreted in bile (linoleyl and arachidonyl) were not significantly different. These results enabled us to evaluate the contribution of the lysolecithin pathway to the synthesis of lecithin in liver and bile : this contribution in bile was less than 2 % under the perfusion condition used.

Biosynthesis of phosphatidylcholine from serum phospholipids in Chinese hamster ovary cells deprived of choline.

We report that Chinese hamster ovary cells assemble membrane phospholipids from choline-linked lipid present in fetal calf serum. This was examined by testing the ability of various serum preparations to satisfy the choline requirement of the cells. Chinese hamster ovary cells divided in growth medium containing lipoprotein-deficient serum and approximately 8 microM lysolecithin. Identical results were obtained in growth medium supplemented with solvent-extracted (delipidated) serum reconstituted with purified egg lysolecithin and the uptake of lipid was inhibited by the addition of bovine serum albumin. Analysis of the phospholipid composition of cells incubated with 32Pi and egg lysolecithin in place of choline revealed that approximately 30% of the phosphorus moieties of the cellular phospholipids were derived from the added lipid, while in the presence of choline less than 10% arose in this fashion. Choline starvation enhanced the formation of lecithin from [32P]lysolecithin without affecting phospholipid turnover and labeled lecithin was converted to other phospholipids, especially sphingomyelin and phosphatidylserine. Unlike endogenous serum lysolecithin, lipoproteins obtained from human and fetal calf sera failed to satisfy the choline requirement of Chinese hamster ovary cells, even though 95% of the lipoprotein phospholipid was phosphatidylcholine and sphingomyelin. Together, these results demonstrate that animal cells can derive all of the choline required for membrane phospholipid synthesis from serum lysolecithin and that its conversion to lecithin within the cell is regulated by the availability of choline. In contrast, serum lipoproteins do not normally serve as a major source of choline moieties.

Some relationships between pulmonary prostaglandins and hepatocyte lecithin formation

A hepatocyte lipogehic system which uses 14C-acetate as a lipogenic precursor was used. Phosphatidylethanolamine is biosynthesized the most in this newly established in vitro lipogenic system. Cholesterol, triacylglycerols, free fatty acids, lecithin and other phospholipids are also biosynthesized in the system. When a lung mince is added to hepatocytes biosynthesizing lipids from 14C-acetate, an increase in lecithin (PC) formation greater than that of lung mince alone and hepatocyte alone is detected. This study not only supports the concept that the lung has the capacity to stimulate the liver for lecithin synthesis but also indicates that lung minces have the ability to produce a prostaglandin (s) which is/are inhibitory to lipogenesis in hepatocytes. Lung minces from rats given indomethacin in vivo also stimulated lecithin formation in hepatocytes.

Passage of molecules through the wall of the gastrointestinal tract. Increased passive permeability in rat ileum after exposure to lysolecithin.

The interaction between lysolecithin and mucosal cells in the distal ileum has been studied. Using a rat experimental model, we determined the intestinal permeability to fluorescent dextran 3000 after exposure to different amounts of lysolecithin. At pH 7.3, lysolecithin, 10 mg/ml, significantly enhanced the transmural passage, and at pH 3.5, even 1 mg/ml markedly increased the permeability. However, when lysolecithin was incubated with homogenized mucosal cells, a rapid disappearance of the compound occurred; this was accompanied by formation of free fatty acids and minor formation of lecithin. It appears, therefore, that high concentrations of lysolecithin can impair the intestinal barrier function in the distal part of ileum but that the mucosal cells are well equipped with activities for the rapid removal of such high concentrations. This could be physiologically significant, since the mucosal cells could otherwise be faced with large amounts of lysolecithin that might facilitate the absorption of potentially antigenic and toxic compounds.

Thermolabile CDP-choline synthetase in an animal cell mutant defective in lecithin formation.

Thermolabile CDP-choline synthetase in an animal cell mutant defective in lecithin formation.

Fetal lung development and the influence of glucocorticoids on pulmonary surfactant

The fetal lung undergoes extensive anatomic and histologic differentiation during gestation in preparation for its role as an organ for gas exchange in the neonate. To successfully adapt to the air breathing state, it must also mature physiologically and biochemically and develop the capacity to produce essential pulmonary surfactants. The surfactants are composed primarily of phospholipids, especially lecithins, and serve the vital function of maintaining alveolar stability on expiration. This role is demonstrated experimentally with the classical pressure-volume apparatus. It has been observed that when a fetal lung is inflated with air, a threshold pressure of approximately 15 cm water is required to increase lung volume, and that maximum inflation is achieved between approximately 30 and 35 cm water. During the deflation phase, an immature lung will readily collapse, whereas a mature lung will show resistance to collapse when the pressure is lowered. With advancing gestation, progressively improved deflation stability is achieved, signalling the presence of adequate pulmonary surfactant in the fetal airway. Biochemical approaches to the study of lung development depend on assessment of phospholipid synthesis by determination of enzyme activities, pathway rates, and the concentration of phospholipid products. The attention of investigators has centered specifically on the biosynthesis of lung lecithin. There are two pathways for de novo formation of lecithin: the choline incorporation pathway (I) and phosphatidylethanolamine methylation (II). Recent studies with isotopic techniques have demonstrated that pathway I is the predominant mechanism. There are three enzymes in this pathway, each of which shows significantly increased activity during late gestation in fetal rats. Shortly after the rise in enzyme activity, an increase in overall choline pathway rate has been identified, and subsequently, the concentration of lung lecithin is likewise increased. Hormonal regulation of fetal lung development has been studied with a number of techniques in several species of lower animals. Using the anatomic approach, investigators have demonstrated that 48–72 h following administration of glucocorticoids, increased potential air space and greater “alveolarization” is apparent. Physiologic evidence of enhanced maturation following corticosteroid administration to fetal animals includes greater distensibility, greater deflation stability, and an earlier appearance of surface active material in lung extracts. The biochemical effects of glucocorticoids with respect to lung development are an increased synthesis of lecithin, an enhanced rate of choline incorporation, and increased activities of various enzymes. The net biochemical effect is to enhance the capacity of the fetal lung to produce the surface active phospholipids of the alveolar lining layer. Because of exogenous corticosteroid, this capacity is developed at an earlier time in gestation than would normally be found. The hormone therefore acts as a stimulus capable of changing the timing of lung development such that the maturation process is accelerated.

Inelastic light scattering at the second critical micellar formation of lecithin

Quasielastic light scattering provides a direct probe of the diffusional mobility of large particles in solution. Analysis of the light scattering spectra of natural lecithin as a function of scattering angle and concentration in hydrophilic and lipophilic solvents supports the conclusion that the second critical micelle transition is associated with the elongation of spherical molecular aggregates into cylindrical or ellipsoidal particles. The effective radius of the scattering entity below the second critical point is significantly less in nonpolar solvents. The latter result is explained in terms of amphiphilic lipid-solvent interactions.

Biosynthesis of Lecithin by the CDP-Choline Pathway in Liver Microsomes of Rainbow Trout, Salmo gairdneri

The enzymatic synthesis of lecithin from CDP-choline (14C) was characterized in microsomal preparations from the livers of rainbow trout (Salmo gairdneri). Optimal activity was obtained with the addition of 1,2-digylceride and Mg++ to the incubation medium. There was a 290% stimulation in cholinephosphotransferase activity when the incubation temperature was raised from 15 to 37 C. The results indicate that the CDP-choline pathway is operative in the formation of lecithin in the liver of rainbow trout. The possible function of this reaction in the acclimation offish to environmental temperature is discussed.

Biosynthesis of lipids in golgi complex and other subcellular fractions from rat liver

1. 1. Golgi complex, rough and smooth microsomes, plasma membranes, mitochondria and nuclei from rat liver were isolated and their purity assessed using specific marker enzymes. 2. 2. The various subcellular fractions were assayed for the following processes: biosynthesis of sphingomyelin, CDPdiglycerides, phosphatidylinositol, phosphatidylserine, the conversion of phosphatidylserine into phosphatidylethanolamine, the formation of lecithin via N-methylation, and the activation of palmitic and octanoic acids. 3. 3. None of these processes were found to be present in Golgi complex. 4. 4. The endoplasmic reticulum appears to be the principal site in the cell for the synthesis of sphingomyelin, CDPdiglycerides, phosphatidylinositol, phosphatidylserine and the formation of lecithin. Interestingly, the biosynthesis of phosphatidylserine appears to be four times more active in rough than in smooth microsomes, which might suggest a ribosomal localization of this process. 5. 5. Except for CDPdiglyceride synthesis, mitochondria do not contain any of the synthesizing activities described in 4. Mitochondria are, however, the only site in the cell where phosphatidylserine is decarboxylated. This activity appears to be localized in the inner membrane. 6. 6. The activation of palmitate is localized predominantly in endoplasmic reticulum and mitochondria, though some activity was detected in plasma membranes as well. All other cell organelles, including Golgi and probably nuclei, did not contain significant palmitoyl-CoA synthetase activity. The subcellular distribution of octanoyl-CoA synthetase resembled that of palmitoyl-CoA synthetase except that the former enzyme is more active in mitochondria than in microsomes.

Endoplasmic reticulum as the site of lecithin formation in castor bean endosperm.

The properties of a discrete membranous fraction isolated on sucrose gradients from castor bean endosperm have been examined. This fraction was previously shown to be the exclusive site of phosphorylcholine-glyceride transferase. The distribution of NADPH-cytochrome c reductase and antimycin insensitive NADH-cytochrome c reductase across the gradient followed closely that of the phosphorylcholine-glyceride transferase. This fraction also had NADH diaphorase activity and contained cytochromes b(5) and P 450. On sucrose gradients containing 1 mM EDTA this fraction had a mean isopycnic density of 1.12 g/cm(3) and sedimented separately from the ribosomes; electron micrographs showed that it was comprised of smooth membranes. When magnesium was included in the gradients to prevent the dissociation of membrane-bound ribosomes, the isopycnic density of the membrane fraction with its associated enzymes was increased to 1.16 g/cm(3) and under these conditions the electron micrographs showed that the membranes had the typical appearance of rough endoplasmic reticulum. Together these data show that the endoplasmic reticulum is the exclusive site of lecithin formation in the castor bean endosperm and establish a central role for this cytoplasmic component in the biogenesis of cell membranes.

The conversion of lysolecithin to lecithin in supernatant fractions of various rat tissues: on the distribution of the so-called Marinetti's enzyme.

The activity of lecithin formation from lysolecithin (lysolecithin: lysolecithin acyl-transferase) in 105000 xg supernatant fraction of various rat tissues was deter-mined with lysolecithin labeled with 3H-glycerol as substrate. The highest values of the activity (n moles/mg protein/min.) found in supernatant fractions in lung, intestine, liver and spleen were 9, 8, 5.7, 4.9 and 2.3, respectively. The activity was mainly localized in the supernatant fraction in spleen and in lung but it was rather lower in the supernatant fraction than in the particulate fraction in intestine and in liver. These tissues also showed a high activity of lysolecithinase in the following descending order; intestine, lung, liver and spleen. Low activiti-es of both enzymes were found in four tissues; heart, skeletal muscle, kidney and brain.

The formation of lecithin from lysolecithin in rat lung supernatant

The enzyme activity of lecithin formation from lysolecithin was studied in rat lung supernatant. This activity was independent of the addition of ATP and CoA, or palmitoyl-CoA. The 14 C/ 3 H ratio of the lecithin product was twice as much as that of lysolecithin substrate, using double-labeling with [ 14C]palmitate and [ 3H]glycerol. Most of the lecithin synthesized in lung supernatant was a disaturated species. From these data, it is most probable that lecithin formation from lysolecithin in rat lung supernatant depends on acyl transfer between two molecules of lysolecithin.

Intracellular distribution of enzymes of the cytidine diphosphate choline pathway in castor bean endosperm.

The occurrence and subcellular distribution of enzymes of the cytidine diphosphate choline pathway of lecithin synthesis have been examined. Choline kinase (EC 2.7.1.32) was completely soluble, while phosphorylcholine-cytidyl transferase (EC 2.7.7.15) and phosphorylcholine-glyceride transferase (EC 2.7.8.2) were associated with particulate fractions. Although components sedimenting at 10,000 to 100,000 x g contained both enzymes, phosphorylcholine-cytidyl transferase and particularly phosphorylcholine-glyceride transferase were present in the 10,000 x g pellet, which contained the major organelles, mitochondria, and glyoxysomes. When the crude homogenate was centrifuged on a sucrose density gradient, four major bands of particulate protein were recovered. A band at density 1.24 g/cm(3) contained the glyoxysomes and was devoid of phosphorylcholine-cytidyl transferase and phosphorylcholine-glyceride transferase activity. Enzyme activity was barely detectable in the mitochondria, at density 1.18 g/cm(2). Phosphorylcholine-glyceride transferase was found almost exclusively in a sharp band at density 1.12 g/cm(3), and phosphorylcholinecytidyl transferase was found in the uppermost band at density 1.08 g/cm(3). Thus, for the synthesis of lecithin in their membranes, the glyoxysomes and mitochondria depend on enzymes elsewhere in the cell; the final two steps in lecithin formation occur, apparently exclusively, in separate particulate cell components.

Metabolic fate of lysolecithin injected into rats.

The incorporation of labeled lysolecithin into various rat organs was studied. Lysolecithin injected was rapidly taken up by various organs and it was found that the conversion into lecithin proceeded more rapidly in the liver and intestine than in other organs. The mode of formation of lecithin from lysolecithin in various organs was also studied in vivo. Using lysolecithin doubly labeled with the glycerol and fatty acid portions, it was shown that lysolecithin injected was preferentially converted to tetraenoic lecithin in all organs examined by Lands' pathway, while the saturated+monoenoic species of the lung and brain was formed with 14C/3H of approximately 2.0 by Marrinetti's pathway. These results suggest that the lysolecithin-lecithin cycle between liver and plasma may serve to retain polyunsaturated fatty acids, and the saturated lecithin in the lung known as a surfactant may only in some parts be derived from exogenous lysolecithin.

The interaction in vitro between polymorphonuclear leukocytes and mycoplasma.

The interaction, between mycoplasma (PPLO) and human or rabbit leukocytes was examined in vitro. Upon incubation of M. hominis or M. arthritidis for 2 hr with rabbit peritoneal exudate granulocytes or leukocytes from human peripheral blood, no killing of mycoplasma was observed either in the presence or absence of type-specific antiserum. However, (14)CO(2) production from glucose-1-(14)C was stimulated up to 10-fold in the presence of live or heat-killed PPLO. The extent of stimulation depended upon the number of organisms and the presence of type-specific antiserum. The stimulation of (14)CO(2) production seems not because of tight adherence of PPLO to the leukocytes, since PPLO were quantitatively recovered in the medium after sedimenting the granulocytes. The enhanced conversion of medium lysolecithin to cellular lecithin that accompanies phagocytosis of polystyrene particles was significantly reduced when PPLO were also present. Mycoplasma alone elicited no stimulation of lecithin formation. Killing of E. coli, a microorganism readily engulfed and killed by leukocytes in vitro, was diminished when the leukocytes were preincubated with mycoplasma. These findings indicate that M. hominis and M. arthritidis are not ingested by granulocytes to any detectable extent, but that these organisms affect the leukocytes' metabolism and also impair phagocytosis of E. coli.

Metabolism of phosphorylcholine and lecithin in normal and choline-deficient rats

1. 1. The metabolism of phosphorylcholine and lecithin in liver and plasma of normal rats and rats in the early stages of choline-deficiency has been studied. 2. 2. A striking reduction (over 70%) of phosphorylcholine levels in choline-deficient livers and a greatly decreased labelling from 32P i and [1,2- 14C 2]choline was found. 3. 3. Levels of lecithin were slightly depressed in choline-deficient livers but there was increased incorporation of 32P i, [1,2- 14C 2]-choline and [ Me- 14C]methionine into liver lecithin. Increased radioactivity from the three precursors was found also in plasma phospholipids. 4. 4. The labelling of phosphorylcholine and lecithin from 32P i at different time intervals suggested that, in choline-deficient livers, although phosphorylcholine synthesis was depressed, it was not rate-limiting for lecithin formation and that there was, in fact, an increased conversion of 32P-labelled phosphorylcholine into lecithin. 5. 5. The labelling of liver phosphorylcholine and lecithin from [1,2- 14C 2]choline in the control animals was also consistent with a precursor-product relationship. However in the choline-deficient animals the specific activity of lecithin was almost equal to that of phosphorylcholine in the early intervals after injection of the tracer and it is suggested that [1,2- 14C 2]choline may, in part, be incorporated into liver lecithin by a pathway independent of phosphorylcholine. 6. 6. The possible relationship between these findings and the accumulation of liver fat in the choline-deficient animals is discussed.

The biosynthesis of glycerides and glycerophosphatides by rabbit reticulocytes

1. 1. When rabbit reticulocytes were incubated with [1- 14C]glycerol, radioactivity was incorporated into glycerides and glycerophosphatides but no radioactivity was present in their fatty acid moieties or in cholesterol. 2. 2. Lecithin and ethanolamine phosphatide together contained about 70% and the neutral lipids about 20% of the total radioactivity. The triglyceride, which comprised less than 2% of the lipids, had the highest specific activity of all the lipids. 3. 3. Experiments with other radioactive precursors of the phosphatides gave results which indicate that the minor phosphatides are metabolically much less active than lecithin and that the formation of lecithin from serine phosphatide or ethanol-amine phosphatide is quantitatively unimportant in these cells. 4. 4. Incubation of rabbit reticulocytes with [2- 3H]inositol resulted in the formation of radioactive phosphoinositides. Most of this radioactivity was present in a monophosphoinositide.

Phospholipid metabolism by phagocytic cells: I. A comparison of conversion of [ 32P]lysolecithin to lecithin and glycerylphosphorylcholine by homogenates of rabbit polymorphonuclear leukocytes and alveolar macrophages

A comparison has been made of the metabolism of lysolecithin by homogenates of two types of phagocytic cells which differ in their intermediary metabolic pathways, polymorphonuclear leukocytes and alveolar macrophages obtained from rabbits. Using lysolecithin labeled with 32P and [1- 14C]labeled fatty acid, evidence was obtained for the direct conversion of lysolecithin to lecithin by two synthetic mechanisms: At physiologic pH and at low lysolecithin concentration acylation of lysolecithin accounts for lecithin formation. The extent and properties of this enzymatic activity appear closely similar in homogenates of leukocytes and macrophages. In addition, confirming an earlier report, at acid pH and at higher lysolecithin concentration, the reaction 2 lysolecithin → lecithin and glycerylphosphorylcholine operates in the leukocyte homogenates. This activity, in contrast to the acylating activity, is not sedimented by centrifugation at 100000 × g for 1 h, does not require addition of ATP and CoA and produces much more lecithin. Compared with leukocytes, macrophage homogenates have a smaller capacity for conversion of lysolecithin to lecithin at higher lysolecithin concentrations. At all concentrations studied, lecithin-forming activity was dependent on added ATP and CoA and was sedimented by centrifugation at 100000 × g for 1 h. No definite conclusion was reached concerning the separate occurrence in macrophage homogenates of the transfer reaction (2 lysolecithin → lecithin and glycerylphosphorylcholine). As has previously been shown for leukocytes, macrophages also contain phospholipase A (EC 3.1.1.4) and lysolecithinase (EC 3.1.1.5) activities.