Changes in phospholipids and acyl group composition of rat mammary gland during pregnant,lactating, and post-weaning periods

The distribution of phospholipids as well as their fatty acid compositions of rat mammary tissues were examined during pregnant, lactating, and post-weaning periods. There was no apparent change in phospholipids and their acyl groups during the early and late pregnant periods. However, tissue phospholipid composition was different during pregnant, early, and late lactating periods. After parturition, there was a marked increase in the proportion of diacyl-glycerophosphorylcholine in the phospholipids of mammary tissue, but this proportion decreased gradually during lactation. The decrease in diacyl-glycerophosphorylcholine during lactation was marked by a corresponding increase in diacyl-glycerophosphorylethanolamine. Although the shorter chain fatty acids of triglycerides were increased during lactation, only a small proportion of these fatty acids was found in the phosphoglycerides. Marked changes in acyl group composition of individual phospholipids are found during these different physiological stages. In general, there was a rapid decrease in 20∶4 and an increase in 18∶2 in the major phosphoglycerides during parturition. The proportion of 20∶4 in the phosphoglycerides remained low throughout the entire lactating period, while that of 18∶2 continued to increase 2–3 fold. Most of the changes in acyl group of the phosphoglycerides during lactation returned to normal ca. 10 days after weaning. A possible relationship of the variation of phospholipid and acyl group compositions in mammary tissue to changes in hormonal pattern during different physiological stages is discussed.     

Isolation of egg‐yolk phospholipids and enzymatic modification of their acyl chains

Phospholipids (PLs) have many biological and technological functions. Among the main sources of PLs egg yolk is the most attractive because of the high content of phosphatidylcholine (PC). The nutritional value of PLs can be increased by different methods, including enzymatic enrichment with polyunsaturated fatty acids (PUFA). Depending on the enzymes and reaction systems used, a‐linolenic acid (ALA) was introduced into the sn‐1 or sn‐2 position. Useful methods of PL isolation and positional analysis were elaborated.     

Changes in the acyl and alkenyl group composition of cardiac phospholipids in boars fed corn oil or rapeseed oil

Boars fed diets containing rapeseed oil for 8 weeks showed significantly higher levels of neutral lipids and similar levels of phospholipids, compared to those fed corn oil. Erucic and eicosenoic acids were found to be high in ethanolamine phosphoglycerides, and in particular alkenyl acyl-ethanolamine phosphoglyceride. Furthermore, both long chain monoenes were incorporated preferentially in position 2 of the choline and ethanolamine phosphoglycerides. The alkenyl group composition of the cardiac lipids of pigs was influenced by dietary fatty acids. When rapeseed oil was fed, small amounts of 20∶1 and 22∶1 alkenyl constituents were detected. Contribution No. 641 Animal Research Institute     

Elucidation of acyl migration during lipase-catalyzed production of structured phospholipids

Elucidation of acyl migration was carried out in the Lipozyme RM IM (Rhizomucor miehei)-catalyzed transesterification between soybean phosphatidylcholine (PC) and caprylic acid in solvent-free media. A five-factor response surface design was used to evaluate the influence of five major factors and their relationships. The five factors—enzyme dosage, reaction temperature, water addition, reaction time, and substrate ratio—were varied on three levels together with two star points. Enzyme dosage, reaction temperature, and reaction time showed increased effect on the acyl migration into the sn-2 position of PC, whereas increased water addition and substrate ratio had no significant effect in the ranges tested. The best-fitting quadratic response surface model was determined by regression and backward elimination. The coefficient of determination (R ²) was 0.84, which indicates that the fitted quadratic model has acceptable qualities in expressing acyl migration for the enzymatic transesterification. Correlation was observed between acyl donor in the sn-2 position of PC and incorporation of acyl donor into the intermediate lysophosphatidylcholine. Furthermore, acyl migration into the sn-2 position of PC was confirmed by TLC-FID, as PC with caprylic acid was observed on both positions. Under certain conditions, up to 18% incorporation could be observed in the sn-2 position during the lipase-catalyzed transesterification.     

Multiple isotope effects on the acyl group transfer reactions of amides and esters

Acyl group transfer reactions, especially those to amides and esters, are important in biochemistry. Multiple kinetic isotope effects for the atoms at the reactive center of these molecules have provided the most detailed bonding picture of the transition state to date. These kinetic isotope effect studies are reviewed for several reactions of formamide, methyl benzoate, and methyl formate. In these cases all the evidence is consistent with a stepwise mechanism, involving tetrahedral intermediates. In the case of p-nitrophenyl acetate, the change to an excellent leaving group causes the tetrahedral intermediates to become kinetically unstable; the kinetic isotope effects are best fitted to a concerted mechanism.     

On the phospholipids ofCulex pipiens fatigans

The lipids of different developmental stages ofCulex pipiens fatigans, vector of bancroftian filariasis, have been investigated. The phospholipid composition of the developmental stages and of the subcellular fractions of fourth instar larvae of the insects were analyzed. The composition of fatty acids and their positional distribution have also been examined in the major phospholipids of the larvae. The insect eggs contained higher amounts of lipids than larvae suggesting that they were utilized during embryogenesis. Phosphatidyl ethanolamine (PE) and phosphatidyl choline (PC) comprised over 75% of the insect phospholipids. Of these, PE was present in the greatest amounts during all stages of growth and in the subcellular fractions of larvae. An ethanolamine containing sphingolipid was found as a component of the phospholipids of the insects. About 50% of the lipids of the larvae were localized in the cell debris and nuclei fraction which also contained most of the lysolipids of the insects. As in other Diptera 16∶0, 16∶1 and 18∶1 were the major fatty acids present in the insect lipids of which the fatty acid found in greatest amounts was 16∶1. Similar to the phospholipids of animal species, saturated fatty acids were predominantly linked to the 1 position of the major phospholipids of the insects while the unsaturated fatty acids were in higher amounts at the 2 position.     

Nucleoside-based phospholipids and their liposomes formed in water

Phospholipids and liposomes have been the subjects of considerable attention because of their importance in biological systems. We have efficiently synthesized novel nucleoside-based phospholipids in six-step sequences starting from their corresponding nucleosides. These nucleoside-based phospholipids self-assemble into liposome-like structures in aqueous solutions. We have analyzed the structures of these liposomes by dynamic light scattering, transmission electron microscopy, and confocal microscopy.     

Effect of radiotherapy and chemotherapy on composition of tumor membrane phospholipids

Phospholipid extracts were made of a murine mammary adenocarcinoma implanted in the dorsum of the foot of C3H/He mice before and 96 h after 17 Gy irradiation or 150 mg/kg cyclophosphamide. Extracts of untreated tumors, which had grown for a further 96 h, were also studied. Although previous studies have shown significant changes in the precursors and catabolites of phosphatidylcholine and phosphatidylethanolamine following therapy, ³¹P nuclear magnetic resonance analysis of extracts showed no changes in these membrane constituents and other observed phospholipid species. A significant decrease in 1-alkyl-2-acyl-sn-glycero-3-phosphocholine, however, was observed 96 h following cyclophosphamide treatment.     

The role of neutral glycolipids and phospholipids in myxovirus-induced membrane fusion

Myxoviruses (influenza virus and paramyxovirus) enter host cells by two successive steps consisting of attachment and fusion between viral and cellular membranes. The initial attachment is known to occur through specific binding of the viruses with the neuraminic acid-containing receptors of cellular membranes. Evidence is presented here that, in the following step of membrane fusion, neutral glycolipids terminating in galactose and certain phospholipids (primarily lecithin and sphingomyelin) interact with the viral envelopes and that this interaction may be fundamental to the fusion process.     

Phospholipid acyl group composition in normal and tumoral nerve cells in culture

We have studied the fatty acid composition of total phosphoglycerides from various types of nerve cells in culture. Primary cell cultures were compared with tumoral cell strains. Glial cells exhibited no characteristic pattern when compared to neurons. Tumoral cell phosphogly cerides contained much higher levels of octadecenoic acid and lower levels of C-20 to C-22 polyunsaturated fatty acids than normal cell phosphoglycerides. This observation seems to be a general feature in tumoral cell membranes. It could be of interest in respect to the membrane fluidity of cancer cells.     

Aggregation and fusion of vesicles composed of N-palmitoyl derivatives of membrane phospholipids

N-Acylphosphatidylethanolamines and N-acylphosphatidylserines have been isolated from mammalian cells and have been associated with some tissue degenerative changes, although the relationship between their synthesis and the uncontrolled sequence of events that ends in irreversible tissue damage is not completely established. Our results show that monovalent and divalent cations induce aggregation and fusion of liposomes constituted by N-palmitoylphosphatidylethanolamine (NPPE) and N-palmitoylphosphatidylserine (NPPS). The effectiveness of cations to induce the aggregation of NPPE and NPPS liposomes is Ca²⁺>Mg²⁺>>Na⁺. NPPS liposomes aggregate at lower concentrations of divalent cations than NPPE liposomes, but with sodium NPPE liposomes aggregate to a higher extent than NPPS liposomes. The reaction order for the aggregation processes depends on the lipid and the cation nature and range from 1.04 to 1.64. Dynamic light scattering shows an irreversible increase of the size of the aggregates in the presence of all cations tested. The irreversibility of the aggregation process and the intermixing of bilayer lipids, as studied by resonance energy transfer assay, suggest that fusion, rather than aggregation, occurs. The existence of a real fusion was demonstrated by the coalescence of the aqueous contents of both NPPS and NPPE liposomes in the presence of either monovalent or divalent cations. The different binding sensitivity of Ca²⁺ to NPPS and NPPE liposomes, determined by ş potential measurements, agrees with the results obtained in the aggregation and fusion assays. Our results suggest that the synthesis in vivo of N-acylated phospholipids can introduce important changes in membrane-mediated processes.     

Synthesis of supercritical carbon dioxide-philic phospholipids and determination of their solubility

The supercritical technology is a promising green process in the fields of medicine, cosmetics, food, and feed industries. However, the poor solubility of phospholipid in supercritical carbon dioxide (ScCO₂) limits its application in some processes. In order to enhance the solubility, a modified phospholipid with ScCO₂-philic group (eg, polyvinyl acetate), is designed and successfully synthesized. The solubility of the ScCO₂-philic phospholipids in ScCO₂ with ethanol as cosolvent (0.5, 1.0, 2.0 mol%) is determined at the temperatures of 313.15, 318.15, 323.15, 328.15 K, and the pressure range of 9 to 15 MPa. The determination results indicate that the solubility of the modified phospholipid in ScCO₂ is much higher than that of the soybean phospholipids. Furthermore, five semiempirical equations, namely, Chrastil-G, MST-S, Sodeifian-Sajadian, Pérez, and Reddy, are used to correlate and predict the experimental results. The average absolute relative deviations of the correlation equations are 6.91%, 7.08%, 6.06%, 2.62%, and 6.15%, respectively, indicating that the solubility data can be well predicted.     

Plasma membrane lipids of bovine adrenal chromaffin cells

The lipid composition of plasma membranes isolated from bovine adrenal chromaffin cells has been determined. Choline and ethanolamine phosphatides were predominant; the level of lyso compounds was very low. The amount of cholesterol and the cholesterol/phospholipid molar ratio was low compared to those of the other subcellular fractions of chromaffin cells. A complex pattern of neutral glycolipids was observed in contrast to that of gangliosides.     

Incorporation ofcis-octadecenoic acids into the rat liver mitochondrial membrane phospholipids and adipose tissue triglycerides

The incorporation of the dietarycis 18∶1 (n−12) andcis 18∶1 (n−10) into liver mitochondrial membrane phospholipids and adipose tissue trigly cerides was studied in 4 groups of rats fed diets containing 10 weight percent (wt%) of fat with the following contents of octadecenoic acids: 50%cis 18∶1(n−12) +9%cis 18∶1 (n−9); 25%cis 18∶1 (n−12)+32%cis 18∶1 (n−9); 50%cis 18∶1 (n−10)+10%cis 18∶1 (n−9); or 54%cis 18∶1 (n−9). Dietary linoleic acid was 3 wt% in all 4 groups. In the mitochondrial membranes, the isomeric octadecenoic acids were primarily incorporated into the 1-position of phosphatidylcholines and phosphatidylethanolamines at the expense of saturated fatty acids. The maximal incorporations observed in the 1-position of phosphatidylethanolamines were 4.8% 18∶1 (n−12) and 8.9% 18∶1 (n−10). No effects on the contents of polyunsaturated fatty acids in the phospholipids were seen. In the adipose tissue, the isomeric octadecenoic acids were incorporated at a level of 13%cis 18∶1 (n−12) or 23%cis 18∶1 (n−10), paralleled by a reduction in the content of oleic acid. Presented in part at the 9th Scandinavian Symposium on Lipids, Visby, Sweden, June 1977.     

Hyperphagia modifies FA profiles of plasma phospholipids,plasma FFA,and adipose tissue TAG

Hyperphagia was achieved by continuous intracerebroventricular infusion of a melanocortin receptor antagonist (HS024; Neosystem, Strasbourg, France) in rats. The effects of hyperphagia on FA composition and concentration of plasma phospholipids (PL), plasma FFA, and adipose tissue TAG were studied in rats for 8 d [short-term hyperphagia (STH); n=8], or 28 d [longterm hyperphagia (LTH); n=9]. The control rats were treated with artificial cerebrospinal fluid for 8 d (n=8) or 28 d (n=10). The rats were fed the same regular diet. In STH rats the plasma PL and fasting plasma FFA contained higher concentrations of saturated FA (SFA) and monounsaturated FA (MUFA), and plasma FFA contained lower n−6 PUFA than in the control rats. In LTH rats the plasma PL contained higher concentrations of SFA, MUFA, and n−3 PUFA and higher proportions of 16∶1n−7 and 18∶1n−9 at the expense of 18∶2n−6 than in the control rats. In LTH rats the abundant dietary intake of 18∶2n−6 did not enrich 18∶2n−6 of the plasma PL or adipose tissue TAG. In LTH rats the fasting plasma FFA contained more than twofold higher concentrations of SFA and MUFA, and higher proportions of 16∶1n−7 and 18∶1n−9 at the expense of 18∶2n−6 than in the control rats. This animal obesity model shows that LTH affects the FA composition and concentration of plasma PL, plasma FFA, and adipose tissue TAG, a result consistent with changes associated with increased risk of various diseases in humans. These results also demonstrate that LTH alters the FA composition of plasma PL and adipose tissue TAG in a way that does not reflect the FA composition of dietary fat.     

Distribution of cholesteryl esters and other lipid in subcellular fractions of the adrenal gland of the pig

Total lipids from whole pig adrenal glands as well as from their mitochondria, microsomes, liposomes, and cell sap were extracted and fractionated first into neutral lipids and phospholipids. The highest percentage of neutral lipids was found in the cell sap, and the lowest in the microsomal fraction. Neutral lipids were subfractionated into cholesteryl esters, free cholesterol, triglycerides, and free fatty acids. Cholesteryl esters were distributed throughout the liposomes. Free fatty acids represented a substantial part of cell sap lipids, but were present also in the mitochondria, microsomes, and liposomes. Fatty acids of all fractions were analyzed by gas liquid chromatography. Free fatty acids and cholesteryl ester fatty acids from all cellular fractions were similar in composition and were characterized by considerable quantities of linoleic and arachidonic acid. Triglycerides were characterized by an increased percentage of palmitic and a low content of arachidonic acid. Phosphatidyl choline, phosphatidyl ethanolamine, diphosphatidyl glycerol, and sphingomyelin plus phosphatidyl inositol were isolated from the lipids by preparative thin layer chromatography, and their fatty acids analyzed by gas liquid chromatography. Phosphatidyl choline and phosphatidyl ethanolamine from mitochondria, microsomes, and cell sap were very similar in respect of their fatty acid composition. Sphingomyelin plus phosphatidyl inositol was characterized by a high content of C22:2omega6. Diphosphatidyl glycerol was present in mitochondria and in the cell sap.     

Fatty acids of bovine milk glycolipids and phospholipids and their specific distribution in the diacylglycerophospholipids

Milk lipids were fractionated by silicic acid column chromatography and preparative thinlayer chromatography (TLC). Ceramide monohexoside (CMH), ceramide dihexoside (CDH), phosphatidyl ethanolamine (PE), phosphatidyl choline (PC), phosphatidyl serine (PS), and sphingomyelin (Sph) were isolated, and the purity of each was checked by infrared spectroscopy and TLC. The diacylphospholipids were hydrolyzed with phospholipase A and the products separated by TLC. Fatty acid methyl esters were prepared from the various fractions and analyzed by gas chromatography. The glycolipids, CMH and CDH, and Sph contained large amounts of long-chain saturated fatty acids, especially C_22:0, C_23:0, and C_24:0, PE, PS, and PC contained C₁₀-C₂₂ normal and branched-chain saturated fatty acids, and C₁₅-C₂₀ unsaturated fatty acids (mainly monoenes). The distributions of saturated acids between the α′- and β-positions were respectively: PE, 46 and 11%; PS, 65 and 19%; and PC, 72 and 53%. PC was exceptional in that there was 10.8% myristic acid in the β-position and only 5.6% in the α′-position. PE and PS were similar in composition except that in PE oleic acid was evenly distributed, and in PS was largely in the β-position. In general, PC was much more saturated than PE or PS, and there was no overall pattern governing the specific distribution of the fatty acids in the three diacylphospholipids. Comparison with PC from other bovine tissues and from egg lecithin showed that fatty acids are located much less specifically in milk phospholipids than in PC from other sources. Presented at the AOCS Meeting, Houston, Texas, April, 1965.     

Composition of the phospholipids and their fatty acids in the ROC-1 oligodendroglial cell line

ROC-1 cells are a hybrid of C−6 rat glioma and rat oligodendroglia cells. Biochemically these cells resemble the oligodendroglia parent, but their lipid composition is unknown. The phospholipid composition in mole % was: cardiolipin, 1.0; phosphatidylglycerol, 1.2; ethanolamine glycerophospholipids, 27.6; phosphatidylinositol, 5.8; lysophosphatidylethanolamine, 0.8; phosphatidylserine, 5.6; choline glycerophospholipids, 43.7; sphingomyelin, 13.7; phosphatidylinositol-4-monophosphate, 0.8; and lysophosphatidylcholine, 0.6. The choline and ethanolamine plasmalogens made up 7.2 and 18.4% of the total phospholipids, respectively. The phospholipid composition reflects that of both parental cells. The cells had moderate to high levels of 20∶3n−9 indicating n−6 series fatty acid deficiency. The phosphatidylinositol had very high 20∶3n−9 levels with a 20∶3n−9/20∶4n−6 ratio of 2.1 compared to 0.44 and 0.58 for ethanolamine glycerophospholipids (EtnGpl) and choline glycerophospholipids (ChoGpl) respectively. The saturated/polyenoic fatty acid ratios were 0.40 for EtnGpl, 3.38 for ChoGpl and 1.48 for phosphatidylinositol.     

Destruction of polyunsaturated alkyl/acyl and alkenyl/acyl glycerophosphocholine of plasma lipoproteins during incubation with group V and X secretory phospholipase A2s

Plasma lipoproteins are carriers of various glycerophospholipids including diacyl, alkenyl/acyl, and alkyl/acyl glycerophosphocholines (GPCs), which become distributed among cells and tissues during metabolism. For metabolic function, these phospholipids require hydrolysis by phospholipases, but the responsible enzymes have not been identified. We had previously shown that after complete digestion of lipoprotein diacyl- and oxo-diacyl-GPCs, degradation of residual alkyl/acyl and alkenyl/acyl GPCs continues, despite the fact that ether lipids are resistant to hydrolysis by Ca²⁺-activated secretory PLA₂s and require the presence of the Ca²⁺-independent PLA₂. In the course of further investigation, we came across a report by Khaselev and Murphy in which the autoxidative degradation of plasmalogens in the presence of 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) proceeded beyond the formation of dihydroperoxides, hydroxides and epoxides, and led to an attack on the enyl bond of the plasmalogen, resulting in formation of 1-OH/2-20:4-GPC and 1-formyl/2-20:4-GPC. Our preliminary investigation indicated that lipoprotein 16:0p/20:4ω6-GPC yielded the same autoxidation products as those reported for synthetic 16:0p/20:4ω6-GPC in the presence of AAPH. Such autoxidative degradation of lipoprotein plasmalogens had not been previously reported with or without AAPH. Subsequent study led to the conclusion that this reaction was not limited to arachidonates, but extended to other polyunsaturated eicosanoids, docosanoids, and tetracosanoids, as well as oligounsaturated octadecanoids. These observations led to a hypothesis that the autoxidative cleavage of the lipoprotein plasmalogens proceeded under the influence of apo-protein-derived free radicals as intermediates of oxidative processes.     

Effect of epinephrine on the oxidative desaturation of fatty acids in the rat adrenal gland

Delta-6 and Δ5 desaturation activity of rat adrenal gland microsomes was studied to determine the effect of microsomal protein and the substrate saturation curves. This tissue has a very active Δ6 desaturase for linoleic and α-linoleic acids and a Δ5 desaturase for eicosa-8,11,14-trienoic acid. The administration of epinephrine (1 mg/kg body weight) 12 hr before killing, produced approximately a 50% decrease in desaturation of [1-¹⁴C]linoleic acid to γ-linolenic acid, [1-¹⁴C]α-linolenic acid to octadeca-6,9,12,15-tetraenoic acid and [1-¹⁴C]eicosa-8,11,14-trienoic acid to arachidonic acid. A 30% decrease in Δ5 desaturation activity was also shown after 7 hr of epinephrine treatment. The changes on the oxidative desaturation of the same fatty acids in liver microsomes were similar. No changes were observed in the total fatty acid composition of adrenal microsomes 12 hr after epinephrine treatment. Mechanisms of action of the hormone on the biosynthesis of polyunsaturated fatty acids in the adrenal gland are discussed.