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PubMed1983

Methylation of phospholipids in microsomes of the rat aorta.

The methylation of phospholipids by S-adenosyl-L-methionine was characterized in microsomes prepared from strips of rat aorta. In the presence of 0.5 microM S-adenosyl-L-methionine, endogenous phosphatidylethanolamine was methylated to form three products: phosphatidyl-N-monomethylethanolamine, phosphatidyl-N,N-dimethylethanolamine and phosphatidylcholine. In the presence of 150 microM S-adenosyl-L-methionine the methylation activity increased more than 50-fold and the principal radioactive product was phosphatidylcholine. Optimal activity was at pH 9 and no magnesium requirement was detected. Exogenous phosphatidylethanolamine, phosphatidyl-N-monomethylethanolamine and phosphatidyl-N,N-dimethylethanolamine served as substrates for the enzyme. The methylation of exogenous phosphatidyl-N,N-dimethylethanolamine proceeded at a slower rate. Incubation of trypsin with the aorta microsomes reduced the enzymatic activity and reduced the relative yield of phosphatidyl-N-monomethylethanolamine. Phospholipase C degraded the methylated phospholipids, but phosphatidyl-N,N-dimethylethanolamine appeared to be less accessible to the phospholipase. The phospholipid methylation activity was inhibited by the addition of S-adenosyl-L-homocysteine or by L-homocysteinethiolactone. When intact strips of rat aorta were incubated with L-[methyl-3H]methionine, [3H]methyl groups were incorporated into phospholipids. This incorporation was inhibited when L-homocysteinethiolactone was added to the incubation. Polarized fluorescence of diphenylhexatriene in aorta microsomes was measured to determine the apparent membrane fluidity. When intact strips of aorta were incubated with methionine or with L-homocysteinethiolactone, methionine enhanced and L-homocysteinethiolactone decreased apparent fluidity of the microsomal membranes. Phospholipid methylation activity was examined in aorta microsomes prepared from genetically spontaneous hypertensive SHR strain rats. Phospholipid methylation activity was substantially greater in the SHR aorta microsomes than in microsomes prepared from Wistar-Kyoto WKY control strain aorta. Membrane fluidity was greater in the SHR aorta microsomes than in the WKY aorta microsomes. The hypothesis that phospholipid methylation activity influences fluidity of membranes and the possible involvement of methylated phospholipids in aorta membrane functions are discussed.

Animals↗
PubMed1987

Compartmentation of phosphorylated precursors of phospholipid biosynthesis in cultured neuroblastoma cells.

The continuous turnover of membrane phospholipids requires a steady supply of biosynthetic precursors. We evaluated the effects of decreasing extracellular Na+ concentration on phospholipid metabolism in cultured neuroblastoma (N1E 115) cells. Incubating cultures with 145 to 0 mM NaCl caused a concentration-dependent inhibition of [32P]phosphate uptake into the water-soluble intracellular pool and incorporation into phospholipid. Phospholipid classes were differentially affected; [32P]phosphate incorporated into phosphati-dylethanolamine (PE) and phosphatidylcholine (PC) was consistently less than into phosphatidylinositol (PI) and phosphatidylserine (PS). This could not be attributed to decreased phospholipid synthesis since under identical conditions, there was no effect on arachidonic acid or ethanolamine incorporation, and choline utilization for PC synthesis was increased. The effect of Na+ was highly specific since reducing phosphate uptake to a similar extent by incubating cultures in a phosphate-deficient medium containing Na+ did not alter the relative distribution of [32P]phosphate in phospholipid. Of several cations tested only Li+ could partially (50%) replace Na+. Incubation in the presence of ouabain or amiloride had no effect on [32P]phosphate incorporation into phospholipid. The differential effects of low Na+ on [32P]phosphate incorporation into PI relative to PC and PE suggests preferential compartmentation of [32P]phosphate into ATP in pools used for phosphatidic acid synthesis and relatively less in ATP pools used for synthesis of phosphocholine and phosphoethanolamine, precursors of PC and PE, respectively. This suggestion of heterogeneous and distinct pools of ATP for phospholipid biosynthesis, and of potential modulation by Na+ ion, has important implications for understanding intracellular regulation of metabolism.

Animals↗
PubMed1988

Phospholipid asymmetry in cardiac sarcolemma. Analysis of intact cells and 'gas-dissected' membranes.

The investigation focuses on the phospholipid composition of the sarcolemma of cultured neonatal rat heart cells and on the distribution of the phospholipid classes between the two monolayers of the sarcolemma. The plasma membranes are isolated by 'gas-dissection' technique and 38% of total cellular phospholipid is present in the sarcolemma with the composition: phosphatidylethanolamine (PE) 24.9%, phosphatidylcholine (PC) 52.0%, phosphatidylserine/phosphatidylinositol (PS/PI) 7.2%, sphingomyelin 13.5%. The cholesterol/phospholipid ratio of the sarcolemma is 0.5. The distribution of the phospholipids between inner and outer monolayer is defined with the use of two phospholipases A2, sphingomyelinase C or trinitrobenzene sulfonic acid as lipid membrane probes in whole cells. The probes have access to the entire sarcolemmal surface and do not produce detectable cell lysis. The phospholipid classes are asymmetrically distributed: (1) the negatively charged phospholipids, PS/PI are located exclusively in the inner or cytoplasmic leaflet; (2) 75% of PE is in the inner leaflet; (3) 93% of sphingomyelin is in the outer leaflet; (4) 43% of PC is in the outer leaflet. The predominance of PS/PI and PE at the cytoplasmic sarcolemmal surface is discussed with respect to phospholipid-ionic binding relations between phospholipids and exchange and transport of ions, and the response of the cardiac cell on ischemia-reperfusion.

Animals↗
PubMed1994

The relationship between phospholipid content and Ca2+-ATPase activity in the sarcoplasmic reticulum.

The relationship between the phospholipid composition of sarcoplasmic reticulum and the activity of the Ca2+, Mg2+-stimulated ATPase was analyzed by digestion of membrane phospholipids with phospholipase C and A2 enzymes of diverse specificity and by detergent extraction. Phospholipase C of Clostridium perfringens and Clostridium welchii, that hydrolyze preferentially phosphatidylcholine (PC), inhibited the Ca2+-ATPase activity parallel with the depletion of phosphatidylcholine from the membrane. Phospholipase C of Bacillus cereus hydrolyzed in addition to PC, phosphatidylethanolamine (PE) and phosphatidylserine (PS), causing complete inhibition of Ca2+-stimulated ATPase activity. Digestion of sarcoplasmic reticulum with the phospholipase A2 of snake or bee venom produced similar effects. The phosphatidylinositol (PI)-specific phospholipases of B. cereus and Bacillus thuringiensis caused less than 10% inhibition of the Ca2+-ATPase, accompanied by the hydrolysis of more than 70% of the phosphatidylinositol content of the membrane, without significant change in PC, PE and PS content. The inhibition of ATPase activity by the C type phospholipases was nearly completely reversed by octaethyleneglycol dodecyl ether (C12E8). These experiments suggest that the full phospholipid content of native sarcoplasmic reticulum (congruent to 100 mol phospholipid per mol Ca2+-ATPase), is required for ATPase activity and there is no indication that PE, PS, and PI play a specific role in ATP hydrolysis. Extraction of sarcoplasmic reticulum phospholipids by detergents such as deoxycholate, cholate and C12E8 also caused proportional inhibition of ATPase activity with the decrease in phospholipid content; the parallel extraction of PC, PE and PI left the phospholipid composition largely unchanged during delipidation. These observations do not support the requirement for a 'lipid annulus' of congruent to 30 phospholipid molecules/Ca2+-ATPase as proposed by Hesketh et al. ((1976) Biochemistry 15, 4145-4151) or the specific interaction of phosphatidylethanolamine with the ATPase molecule proposed by Bick et al. ((1991) Arch. Biochem. Biophys. 286, 346-352).

Animals↗
PubMed1996

The water adsorption characteristics of charged phospholipids.

The hydration isotherms of the negatively charged phospholipids, egg phosphatidic acid, bovine heart cardiolipin and two phosphatidylserines as well as one positively charged phospholipid, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, have been obtained gravimetrically. The presence of an electrical charge on these phospholipids does not, in itself, determine whether the water binding to the phospholipids is 'strong' or 'weak'. Interestingly, hysteresis effects were present for certain charged phospholipids suggesting some rearrangement of the lipid molecular organization upon hydration and dehydration, perhaps due to the presence of the ionizable moiety. The hydration isotherms of the charged phospholipids have been analyzed by BET theory, although, of the charged lipids studied, all may not be amenable to the application of BET theory. The hydration isotherms and the resulting parameters obtained from the BET analysis are compared to those found previously for zwitterionic phospholipids, especially egg phosphatidylcholine. The water adsorption characteristics of phospholipids are found to depend mainly on the total head-group structure including the presence of hydrophobic groups as well as electrical charge on the head group.

Adsorption↗
PubMed1976

[Phospholipid composition in thermosensitive mutants of Escherichia coli (author's transl)].

Quantitative phospholipid composition was investigated on various thermo-sensitive mutants of Escherichia coli K 12. Phosphatidylethanolamine was the major component. Three mutants (T1, T46 and T5) grown at 40 degrees C, had more anionic phospholipids (phosphatidylglycerol and diphosphatidylglycerol) and less bipolar phospholipids (phosphatidylethanolamine and lysophosphatidylethanolamine) than the wild type. Two mutants strains T46 and T83 which have the same Dna A mutation and identical membrane proteins alterations, have different phospholipid composition. Other mutants, such as T46, exhibit a blocking o? THE INITIATION OF DNA synthesis or morphological alterations under restrictive growth conditions (40 degrees C); however, the phospholipid composition of these mutants is normal and differs from the phospholipid composition of T46.T1 and T5 mutants have the same alterations in the phospholipid composition as T46 although they do not possess Dna A and Div A mutations of the T46 strain. Mutations leading to the alteration of the phospholipid composition are unrelated to other mutations leading to changes in the morphology and cellular division.

DNA, Bacterial↗
PubMed1976

Phospholipid acyl group stability in cultured fibroblasts. Differences between human cell lines of fetal and adult origin.

Human fibroblasts of both fetal and adult origin incorporated [1-14C] acetate primarily into phospholipid acyl groups (70-80% of total radioactivity). When these labeled cells were replated in non-radioactive medium, there was continuous loss of 14C from steroids, triacylglycerols and non-lipid material. In contrast, after some initial loss, cell lines of fetal origin completely retained 14C in cellular phospholipids during continued cell division. Unlike cells of fetal origin, fibroblasts of adult origin continued to lose radioactivity from their phospholipid acyl groups during growth in unlabeled medium. Loss of radioactivity from [1-3H] acetate incorporated into phospholipids of adult cells cannot be attributed to cell death since it was not accompanied by any loss of previously incorporated [ME-14C] thymidine. If cellular phospholipids were labeled with [U-14C] glycerol, both fetal and adult fibroblasts continued to lose radioisotope from the cells during growth in nonradioactive medium. Thus, there is turnover of the phospholipid molecules themselves in fetal human fibroblasts grown in vitro, but their acyl groups are retained within cellular phospholipids. In this respect, fibroblasts of fetal origin resemble established cell lines such as the L fibroblast. Fibroblasts of adult origin do not exhibit this complete conservation of their phospholipid acyl groups.

Acetates↗
PubMed1977

Fatty acid synthesis and metabolism of phospholipid acyl groups in strain L mouse fibroblasts.

Strain L mouse fibroblasts grown in medium supplemented with 2.5% delipidized horse serum were found capable of desaturating oleic and linoleic acid to dienoic and trienoic acid(s), respectively. Although 40-60% of de novo fatty acid synthesis from [2-3H]acetate was inhibited by the administration of exogenous oleic or linoleic acid, sterole synthesis was only slightly affected. Within 24-48 h after incorporation, phospholipid fatty acyl groups could undergo active exchange between phospholipids. After this dynamic transition period was over, not only were the phospholipid acyls retained but some vicinal fatty acyl pairs of phospholipid also appeared to be stable and remained together throughout the depletion period. At any time in the experiment, however, introduction of exogenous fatty acid perturbed this phospholipid acyl retention, delayed the time at which the phospholipid acyl groups no longer moved between phospholipids and also decreased the ultimate number of phospholipid acyl groups retained by strain L mouse fibroblasts.

Fatty Acids↗
PubMed1978

Effect of exogenous fatty acids on the retention of phospholipid acyl groups by mouse L fibroblasts.

Exogenous oleic or linoleic acid, given at a high but nontoxic level (1 mg fatty acid/day for 20 . 10(6) cells in 50 ml medium), caused substantial redistribution of the otherwise permanently retained phospholipid acyls in mouse L fibroblasts. 18--40% of the preformed phospholipid acyls were shifted to triglycerides but most returned to phospholipids when the supply of exogenous fatty acid was removed. The phospholipid acyls could be reshuttled back to triglycerides again whenever an adequate amount of exogenous fatty acid was provided. Daily changes of medium containing oleic acid bound to bovine serum albumin caused a still greater total loss of phospholipid acyls into the medium. The removal of the prelabeled phospholipid acyls also occurred with phospholipid acyls which had been synthesized from [1-(14C)]acetate 3 days earlier. The results demonstrate the fact that the apparent permanently retained phospholipid acyl groups found in L-cells could in fact be displaced through experimental manipulations.

L Cells↗
PubMed1981

Phospholipid synthesis in fetal lung organotypic cultures and isolated type II pneumocytes.

Type II pneumocytes from fetal rabbit lungs were grown in an organotypic system and used to study surfactant phospholipid synthesis. This organotypic system was further employed as a means for isolating purified type II cells which were grown in monolayer cultures. Phospholipid synthetic properties for these purified type II cells at different stages of culture were studied using the radioactivity labeled substances: palmitate, choline and acetate. Additionally, fibroblasts were recovered from the organotypic system and grown in isolated cultures or in combination with type II cells. A comparison of the phospholipid composition and biosynthesis for these cell preparations indicated that in all instances, with the exception of fetal lung fibroblasts, the cultures were capable of producing saturated phosphatidylcholine as a major phospholipid product. The relative molar incorporation into phospholipids for the substrates studied differed depending on the type cell preparation being examined. The formation of surfactant phospholipids appeared most similar for the organotypic system and mixed fibroblast/type II cell preparations. Furthermore, a mixed culture of fibroblasts and type II cells produced larger proportions of surfactant phospholipids than type II cells alone. These observations indicate that the organotypic system is a useful model for examining fetal lung surfactant phospholipid synthesis and may also be employed as a simple means or isolating fetal type II pneumocytes.

Acetates↗
PubMed1982

Accumulation and depletion of trans octadecenoic acid in rat peripheral nerve phospholipid.

The accumulation and depletion of trans 18:1 acids in the sciatic nerve phospholipids was studied in rats fed a diet containing 18% partially hydrogenated soybean oil of which 45% was trans 18:1. The nerve phospholipid of rats fed partially hydrogenated soybean oil beginning at weaning age reached a maximum incorporation of trans 18:1 or 0.8% in 4 weeks; whereas the nerve phospholipid of 18-day-old pre-weanling pups of mothers fed the partially hydrogenated soybean oil diet since their weaning contained 3.3% trans 18:1. The trans content decreased rapidly after the weaning of these second-generation rats to a steady-state level 50% higher than the maximum level reached in the first generation. Upon removal of partially hydrogenated soybean oil from the diet, the trans 18:1 of nerve phospholipid decreased much more slowly than from liver or heart phospholipid. Essential fatty acid deficiency had no influence on incorporation or removal of trans 18:1 in nerve phospholipid. The accumulation and depletion of trans 18:1 in nerve phospholipid of second-generation partially hydrogenated soybean oil-fed rats paralleled the changes of polyunsaturated fatty acids and was quite different from the changes observed in 18:0 and cis 18:1. Polyunsaturated fatty acids increased in nerve phospholipid from second but not first-generation rats fed partially hydrogenated soybean oil and declined rapidly when partially hydrogenated soybean oil was removed from the diet.

Aging↗
PubMed1986

Turnover of phospholipid fatty acyl chains in cultured neuroblastoma cells: involvement of deacylation-reacylation and de novo synthesis in plasma membranes.

Cultured neuroblastoma cells (NIE-115) rapidly incorporated the essential fatty acid, linoleic acid (18:2 (n = 6), into membrane phospholipids. Fatty acid label appeared rapidly (2-10 min) in plasma membrane phospholipids without evidence of an initial lag. Specific activity (nmol fatty acid/mumol phospholipid) was 1.5-2-fold higher in microsomes than in plasma membrane. In these membrane fractions phosphatidylcholine had at least 2-fold higher specific activity than other phospholipids. With 32P as radioactive precursor, the specific activity of phosphatidylinositol was 2-fold higher compared to other phospholipids in both plasma membrane and microsomes. Thus a differential turnover of fatty acyl and head group moieties of both phospholipids was suggested. This was confirmed in dual-label (3H fatty acid and 32P), pulse-chase studies that showed a relatively rapid loss of fatty acyl chains compared to the head group of phosphatidylcholine; the opposite occurred with phosphatidylinositol. A high loss of fatty acyl chain relative to phosphorus indicated involvement of deacylation-reacylation in fatty acyl chain turnover. The patterns of label loss in pulse-chase experiments at 37 and 10 degrees C indicated some independent synthesis and modification of plasma membrane phospholipids at the plasma membrane. Lysophosphatidylcholine acyltransferase and choline phosphotransferase activities were demonstrated in isolated plasma membrane in vitro. Thus, studies with intact cells and with isolated membrane fractions suggested that neuroblastoma plasma membranes possess enzyme activities capable of altering phospholipid fatty acyl chain composition by deacylation-reacylation and de novo synthesis at the plasma membrane itself.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1986

Modification of the fatty acid composition of individual phospholipids and neutral lipids after infection of the simian erythrocyte by Plasmodium knowlesi.

Using capillary gas-liquid chromatography, we have analyzed the alteration in the total fatty acid, phospholipid and neutral lipid compositions of the monkey erythrocyte, after infection by the malarial parasite Plasmodium knowlesi. Data based on fatty acid quantitation show that the phospholipid composition is altered, with particularly large increases in phosphatidylcholine (PC) and phosphatidylethanolamine (PE), the most abundant phospholipids in normal and P. knowlesi-schizont-infected cells. Unesterified fatty acids were found to be less abundant in infected cells. The total fatty acid content of the cell is increased 6-fold during infection, and total fatty acid composition is also changed: the infected cells are richer in palmitate (+23%), oleate (+29%) and linoleate (+89%), but contained less stearate (-27%) and arachidonate (-40%). The determination of the fatty acid composition of individual phospholipids, neutral lipids and unesterified fatty acids showed that choline-containing phospholipids (PC and sphingomyelin) were not as altered in their fatty acid pattern as anionic phospholipids (PE, phosphatidylserine (PS) and phosphatidylinositol (PI) and lysophosphatidylcholine (lysoPC). Specific alterations in the fatty acid compositions of individual phospholipids were detected, whereas the rise in linoleic acid was the only change during infection that was recovered in each phospholipid (except PC), neutral lipid and unesterified fatty acids. The fatty acid composition of the neutral lipids and unesterified fatty acids was particularly modified: the only rise in arachidonic acid level was observed in these lipid classes after infection. The total plasmalogen level of the erythrocyte is decreased in infected cells (-60%), but their level is increased in PI.

Animals↗
PubMed1990

Effects of intravenous infusions of commercial fat emulsions (Intralipid 10 or 20%) on rat plasma lipoproteins: phospholipids in excess are the main precursors of lipoprotein-X-like particles.

Like most commercial parenteral emulsions, Intralipid contains the same amount of phospholipids (12 mg/ml) to stabilize 100 or 200 mg of soybean oil (10 or 20% formula, respectively). By centrifugation, 10 or 20% Intralipid was separated into a supernatant, fat particles containing the bulk of triacylglycerols stabilized by a fraction of phospholipids and an infranatant--called mesophase--consisting mainly of phospholipids used in excess as emulsifier. We observed that the initial triacylglycerol/phospholipid ratio of the emulsion (100/12 and 200/12, respectively) determines the size of the triacylglycerol-rich particles (260 and 350 nm) as well as the phospholipid content of the mesophase (6.02 and 4.67 mg/ml). To understand the mechanism of the lipoprotein-X (LPX) accumulation generally reported after intravenous fat infusions, plasma lipid levels and lipoprotein profiles were first compared in the rats after infusion (at a constant rate of 0.5 or 1 ml/h for 43 h) of Intralipid 10 or 20%. For the same intravenous triacylglycerol load (100 mg/h), rats infused with Intralipid 10% at 1 ml/h displayed higher triacylglycerol levels than rats infused with the 20% emulsion at 0.5 ml/h, suggesting that the size of exogenous fat particles modulated the catabolic rate of their triacylglycerols. The plasma levels of LPX varied according to the infusion rate of phospholipids not associated with triacylglycerol-rich particles of the emulsion. Moreover, an apo E and apo B enrichment of plasma and an elevation of the apo B48/apo B100 ratio was always observed after Intralipid infusions. In order to confirm that phospholipids of the mesophase are the main LPX precursors, lipoprotein profiles were then compared in the rats after intravenous infusion, at a constant rate of 1 ml/h, of either the mesophase or a suspension of triacylglycerol-rich particles isolated from Intralipid 20%. As expected, significant LPX amounts were only detected in rats infused with the pure mesophase of the emulsion. It was concluded that products of the lipolysis of exogenous fat particles play only a minor role in the formation of LPX. In fact these abnormal lipoproteins are generated by phospholipids of the mesophase which, like infused liposomes, actively mobilize endogenous free cholesterol. Consequently, in order to be considered as true chylomicron models for safe fat delivery in parenteral nutrition and in order to prevent some detrimental effects on cholesterol metabolism, commercial emulsions should be cleared of phospholipid excess.

Animals↗
PubMed1994

Steady-state fluorescence polarization study of structurally defined phospholipids from liver mitochondria of rats fed elaidic acid.

In vivo-modified phospholipids from rat liver mitochondria were used to study the effect of trans-fatty acid incorporation into phospholipids on the steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) embedded in phospholipid liposomes. Pure fractions of mitochondria phospholipids were prepared and analyzed for their fatty acid compositions and fatty acid positional distribution. In rats fed a diet enriched with trielaidin, elaidic acid (trans-9 18:1 acid) was extensively incorporated in position 1 of phosphatidylcholine (PC; 31% of fatty acids esterified to this position), phosphatidylethanolamine (PE; 42.5%) and phosphatidylinositol (PI; 43%). Less than 10% of the incorporated elaidic acid was esterified to position 2 of these phospholipids. More than 90% of elaidic acid esterified to position 1 displaced saturated acids. Consequently, about one-third of PC molecules and two-fifths of PE and PI molecules contained one molecule of elaidic acid instead of one saturated fatty acid molecule in their 1-position. On the other hand, cardiolipin, which is naturally practically devoid of saturated acids, was particularly resistant to elaidic acid incorporation. The fluorescence polarization of DPH incorporated in liposomes made of PC-PI and of PC-PI-PE from liver mitochondria of rats fed or not fed elaidic acid was measured. No significant differences between phospholipids containing or not containing elaidic acid could be detected. Values of DPH fluorescence polarization for all samples were comprized between 0.133 and 0.135 at 25 degrees C. We thus conclude that when elaidic acid replaces saturated fatty acids in phospholipids, even in a high proportion (one-third), the physical state of acyl chains in the hydrophobic core of membranes is not grossly modified. Thus, elaidic acid seems to behave like a saturated fatty acid, not only biochemically for the acylation of phospholipids, but also physically.

Animals↗
PubMed1988

Acylation and deacylation of phospholipids in isolated bovine rod outer segments.

Isolated bovine rod outer segments (ROS) were incubated under different conditions with radiolabeled fatty acid-Coenzyme A (CoA) compounds, fatty acids and phospholipids in order to further investigate the rates, mechanisms and function of phospholipid metabolism within that organelle. ROS contain acyl CoA synthetase, acyl transferase, acyl CoA hydrolase, and phospholipase A activities. Although different radiolabeled fatty acid CoAs were esterified to the major ROS phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine) at the same rate, different free fatty acids were esterified at different rates. There was no correlation between these estimates of in vitro rates of incorporation of fatty acids and the fatty acid composition of ROS phospholipids. Both the deacylation of radiolabeled phospholipids (phospholipase A activity) and the acylation of endogenous phospholipids (acyl transferase activity) were maximally stimulated when ATP, CoA, Mg2+ and Ca2+ were present, and both processes were stimulated by pro-oxidizing conditions and exposure to light. Under phospholipase A-stimulatory conditions, there was preferential hydrolysis of polyenoic fatty acids from endogenous ROS phospholipids. Both the acylation and deacylation reactions were primarily at the sn-2 position of ROS phospholipids.

Acylation↗
PubMed1992

Simultaneous determination of amounts of major phospholipid classes and their fatty acid composition in erythrocyte membranes using high-performance liquid chromatography and gas chromatography.

A method for the simultaneous determination of amounts of major phospholipid classes and their fatty acid composition in erythrocyte membranes is described. The method consists in extraction of phospholipids from erythrocyte membranes, separation of phospholipid classes by high-performance liquid chromatography, methylation of phospholipids and determination of phospholipid-bound fatty acids by capillary gas chromatography. The amounts of phospholipid classes are calculated from the total weight of phospholipid-bound fatty acids and their average molecular weights. The method was applied to erythrocytes from rats. The results show that the method is reproducible and is useful for the determination of amounts of phospholipid classes and their fatty acid composition in small blood samples.

Animals↗
PubMed1992

Effect of amiodarone therapy on the time course of myocardial phospholipid hydrolysis during in vitro total ischaemia in cat hearts.

The effects of chronic amiodarone therapy on myocardial phospholipid hydrolysis induced by total in vitro ischaemia were investigated in cat hearts. Chronic treatment of cats with amiodarone (30 mg/kg/day, orally) for 6 weeks resulted in a sufficient uptake of the drug reaching tissue levels of 83 +/- 13 & 122 +/- 22 microM (n = 12) for amiodarone and its principle metabolite, desethylamiodarone, respectively. This was accompanied by a significant increase (37%, P less than 0.001) in total phospholipid content of heart in treated as compared to untreated animals. Upon in vitro total ischaemia, these endogenous drug levels were sufficient to attenuate significantly hydrolysis of membrane phospholipid. The degree of attenuation was dependent upon the duration of ischaemic insult. In this regard, protection against phospholipid losses by amiodarone treatment was significantly more in the later irreversible phase of ischaemic injury whether studied in an in vitro total ischaemia model or in an isolated perfused heart preparation. Similar trend was observed in the relative accumulation of lysophospholipid and non-esterified fatty acid levels during ischaemia, i.e. both were significantly attenuated by amiodarone treatment. However, in contrast to the fatty acid data, the net changes in lysophospholipids per gram tissue wet weight were similar in treated and untreated animals, suggesting that the protective effects of amiodarone may have involved other enzymes including phospholipase C and D. Also, during the entire time course studied, all the phospholipid classes appeared to be affected to more or less a similar degree, indicating that the effects of the drug may have manifested in other subcellular compartments besides lysosomes. However, at all time periods studied, the net release of eicosatetraenoic and docosahexaenoic acid (fatty acids occupying primarily sn-2 position of phospholipids) was different, release of the former fatty acid being inhibited more than the latter, suggesting specific interaction of amiodarone with the molecular species of phospholipid. The data suggest that amiodarone attenuates ischaemia-induced membrane lipid abnormalities in part through modulation of phospholipid metabolism, and that this effect may be one of the key determinants which contribute to its antiarrhythmic properties during acute ischaemia.

Amiodarone↗