Adult rat brain synaptic vesicles II. Lipid composition

The lipid composition of synaptic vesicles isolated from adult rat brain was determined. Vesicles contained cholesterol and phospholipid but very little ganglioside, galactolipid, free fatty acid and triglyceride was detected. Ethanolamine and choline phosphoglycerides were the dominant phospholipids. Lysophosphatidyl choline was present in very low amounts. The fatty acid composition of the phosphoglycerides was characterized by high levels of docosahexaenoic acid in the ethanolamine and serine phosphoglycerides, and the absence of long chain fatty acids from the sphingomyelins. All the characteristic features of the lipid composition of the synaptosomal plasma membrane (with the exception of the ganglioside content) were seen in the synaptic vesicle lipids. The results are discussed in terms of the exocytosis mechanism of transmitter release.     

Lipid metabolism in brain tissue explants

Abstract— Tissue explants from frontal lobes of rat brain were used for the study of cerebral fatty acid metabolism. After tissues had been maintained in serum-supplemented medium, a lipid-free medium was substituted and metabolic studies were carried out. Under these conditions explants continued to take up lipid precursors for at least 48 h, as judged by incorporation of dl -[2-¹⁴C]mevalonic acid into cellular lipids. [l-¹⁴C]Stearic acid and [l-¹⁴C]palmitic acid were bound to cells as the free fatty acids, or incorporated into neutral lipids (particularly triglycerides), glycolipids and phospholipids. In the galactolipid fraction, cerebrosides were the principal radioactive lipids. Choline phosphoglycerides, ethanolamine phosphoglycerides, inositol phosphoglycerides and serine phosphoglycerides were the principal radioactive phospholipids. Fatty acids were incorporated into cellular lipids either unchanged or after desaturation, chain elongation, or both. Maximum incorporation of stearate occurred in tissues derived from 3-day-old animals. With increasing age the uptake of fatty acid dropped sharply. When the labelling of lipids as a function of time was followed in 3-day-old animals, triglycerides and choline phosphoglycerides were the first fractions to take up labelled stearate. Labelling of cerebrosides occurred slowly, only becoming evident after 24 h. These studies exemplify the usefulness of tissue explants for prolonged metabolic studies in normal and pathological specimens of brain.     

Composition and fatty acid content of rat ventral prostate phospholipids

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

Effect of temperature acclimatization on the fatty acid composition of goldfish intestinal lipids

1. The fatty acid composition of whole goldfish, whole-intestinal mucosa, intestinal mucosal membranes and individual phospholipids extracted from mucosal membranes were measured, fish adapted to different temperatures being used. 2. Alterations of the adaptation temperature did not noticeably affect the fatty acid composition of the whole-fish lipids, but there were marked changes in the fatty acids of lipids extracted from homogenates of goldfish intestinal mucosa. These changes were more pronounced in a membrane fraction prepared from these homogenates. Raising the adaptation temperature by 20 degrees C halved the percentage of C(20:1), C(20:4) and C(22:6) fatty acids and nearly doubled the percentage of C(18:0) and C(20:3) fatty acids recovered. 3. Choline phosphoglycerides constituted about one-half and ethanolamine phosphoglycerides about one-quarter of the total membrane phospholipids. 4. The fatty acids of choline and ethanolamine phosphoglycerides were more susceptible to temperature-dependent changes than were the phosphoglycerides of inositol or serine. 5. The increase in C(18:0) fatty acid that occurred in membranes of warm-adapted fish was greatest for ethanolamine phosphoglycerides, but increases also occurred in other phospholipid fractions and in membrane neutral lipids.     

Altered Composition of Cerebral Microvessel Membrane Phosphoglycerides from Senescent Mouse

Abstract— Phosphoglyceride and fatty acid composition was determined in the cellular membranes of isolated cerebral microvessels and brain parenchymal cells (neurons and glia) taken from 10-, 20-, and 27–30-month-old C57BL6/NNIA mice. Lipids were extracted from each fraction and the fatty acid profiles of ethanolamine, cho-line, serine, and inositol phosphoglycerides analyzed by gas chromatography. The results suggest that membrane phosphoglycerides from cerebral microvessels are significantly more affected by the aging process than are those of the brain parenchyma. Relative percentage for fatty acids in cerebral microvessels indicate an overall decline in membrane unsaturation with a concomitant elevation in the level of saturation. The decline in unsaturation is reflected primarily in the loss of precursor fatty acids for arachidonic (18:2n-6 and 20:3n-6) and docosahexaenoic (20:5n-3 and 22:5n-3) acids. Levels of arachidonic (20:4n-6) and docosahexaenoic (22:6n-3) acids in each phos-phoglyceride remained unchanged with age; however, mol% for ethanolamine plasmalogen, a major source of these fatty acids, was significantly reduced in 27–30-month-old mice. Conversely, mol% for choline phospho-glyceride increased with age. The age-related changes in fatty acid profile for microvessel membrane phosphoglycerides are reflected by increased saturation/unsaturation ratios and decreased unsaturation indices. These parameters were not affected by aging in parenchymal membranes.     

The alk-1-enyl group content of mammalian myelin phosphoglycerides by quantitative two-dimensional thin-layer chromatography

Myelin phospholipids have been examined by a separation-reaction-separation procedure for two-dimensional thin-layer chromatography on silica gel. After separation in one dimension, alk-1-enyl groups are cleaved by exposure of the plates to HCl fumes. Development in the second dimension quantitatively separates acid-labile and acid-stable phosphoglycerides as well as the aldehydes released from the acid-labile phosphoglycerides. Myelin phospholipids from the central nervous systems of the rhesus monkey, squirrel monkey, ox, and mouse contain 32-36% acid-labile ethanolamine phosphoglycerides (ethanolamine plasmalogens) and 8-14% acid-stable ethanolamine phosphoglycerides. Acid-labile choline and serine phosphoglycerides account for less than 1% of the myelin phospholipids.     

Changes in fatty acid composition of human brain myelin lipids during maturation

Abstract— The variation with age of the fatty acid composition of the major lipids in human brain myelin was compared with that of cerebral white matter from the same region. The myelin was isolated from the semiovale centre of the cerebrum of 27 subjects neonatal to old aged. The phospholipid, cholesterol and galactolipid concentrations were determined in all the samples, as were the proportions of the major phospholipid classes. The proportions of cholesterol and especially of the galactolipids increased in myelin during the first 6 months, and in cerebral white matter up to 2 years. During this period the individual phospholipids also varied substantially. Serine phosphoglycerides and especially sphingomyelins increased, and choline phosphoglycerides decreased. The fatty acid patterns of ethanolamine phosphoglycerides (EPG) and sphingomyelins underwent the largest changes. The proportions of saturated fatty acids in EPG diminished rapidly, and there was an increase of monoenoic acids. Fatty acids of the linoleic acid series showed a peak between 4 and 12 months, after which time their proportion slowly diminished to old age. The major fatty acid of this series was docosatetraenoic acid, 22:4 (n-6), which constituted more than 25% of total fatty acids at the maximum level. The fatty acid changes were larger in cerebral white matter, but from 2 years of age the EPG fatty acid pattern in myelin was similar to that in white matter. The fatty acid changes in serine and choline phosphoglycerides of myelin with maturation were much less striking than in EPG but of a similar type. In myelin sphingomyelin the proportion of saturated long-chain fatty acids, C₁₆-C₂₂, diminished, while that of monoenoic acids increased and continued to do so up to old age. From 2 years of age the fatty acid patterns in myelin and cerebral white matter were quite similar. Also the fatty acid patterns of cerebrosides and sulphatides in cerebral white matter and myelin were the same except for the first 2 months of life. The same fatty acid changes occurred in cerebrosides and sulphatides as in the sphingomyelins, i.e. increased proportions of unsaturated (monoenoic) acids. The proportions of 24:1 and 24h:1 and of the odd-numbered fatty acids 25:1 and 23h:1 continued to increase to old age. The variations of the individual lipid fatty acid patterns were small except in the youngest age classes, in which the variations were presumably ascribable to the difficulty in determining the gestational age.     

Antagonistic effects of isovalerate and glycine on plasma choline levels in rabbits

Infusion into rabbits of glycine increased the concentration of plasma choline while infusion of neutralized isovaleric acid which conjugates glycine caused decreased levels. It is suggested that these effects are due to differences in the availability of glycine, convertible to serine which subsequently displaces choline from phospholipids in the base exchange reaction.     

Lipid Composition and Temperature Adaptation of the Nervous System of the Leech Hirudo medicinalis L.

The lipid composition of the nervous system of the leech Hirudo medicinalis was investigated following acclimatization of animals at 25 degrees C and 5 degrees C. Choline, ethanolamine, and serine plus inositol phosphoglycerides are the major phospholipid classes of the leech ganglionic chain; minor amounts of lysophosphatidylcholine, phosphatidic acid, and sphingomyelin are also present. Neither the phospholipid pattern nor the cholesterol to total phospholipid molar ratio was dependent on the acclimatization temperature, whereas the fatty acid patterns of choline and serine plus inositol phosphoglycerides were significantly affected. Both for choline and serine plus inositol phosphoglycerides, a significant increase of the unsaturation index and a decrease of saturated to unsaturated fatty acid ratio was observed in animals acclimatized at 5 degrees C in comparison with those acclimatized at 25 degrees C. These observations, which point to increased lipid fluidity of the nervous system of cold-adapted leeches, are strengthened by results obtained by the fluorescence polarization method using 1,6-diphenyl-1,3,5-hexatriene as a probe: a decrease of the fluorescence polarization value was observed throughout the temperature range selected (5-40 degrees C).     

Ether lipid metabolism. Incorporation of O-hexadecyl ethanediol into rat brain lipids.

1-O-[1'-14C]Hexadecyl ethanediol was administered intracerebrally to myelinating rat brain, and incorporation of radioactivity into brain lipids was followed over a 48-h period: (1) O-Hexadecyl ethanediol was metabolized primarily through oxidative ether bond cleavage, and much of the label was recovered in phospholipid acyl groups. (2) Substantial amounts of radioactivity were also found in choline and ethanolamine phospholipids having an O-hexadecyloxyethyl glycerol backbone. This means that alkyl ethanediol was used in glycerol ether biosynthesis as are long-chain primary alcohols. (3) Acidic hydrolysis of the ethanolamine glycerophosphatide fraction yielded also labeled hexadecanol which may indicate desaturation of 1-O-hexadecyloxyethyl 2-acyl glycerophosphoryl ethanolamine to the plasmalogen analogue. (4) Small amounts of the substrate were oxidized to O-hexadecyl glycolic acid and incorporated into the phospholipids. The substrate did not serve as precursor of O-hexadecyl ethanediol phosphorylcholine or phosphorylethanolamine in the brain.     

Polyunsaturated fatty acid metabolism in neuroblastoma cells in culture.

Neuroblastoma cell cultures took up linoleic and linolenic acids at approximately equal rates, and incorporated them into a variety of lipid fractions, principally cellular phospholipids. Linoleic acid was preferentially incorporated into choline phosphoglycerides, while most of the radioactivity derived from linolenic acid entered ethanolamine phosphoglycerides. There was no evidence for direct transfer of fatty acids between these two phosphoglyceride fractions. When, after the addition of cytosine arabinoside, cell division was arrested, the entry of labelled fatty acids into ethanolamine and serine phosphoglycerides was reduced, suggesting that these lipids are involved in the formation of new cell membranes. In the ethanolamine phosphoglyceride fraction, phosphatidal ethanolamine (plasmalogen) was the principal acceptor for the higher polyunsaturated fatty acids of the φ 3 series. The ratio of labelled fatty acids entering ethanolamine plasmalogens to that entering ethanolamine phosphoglycerides increased following the addition of cytosine arabinoside, suggesting plasmalogens to be involved in formation of cell processes. The first step in the metabolism of both linoleic and linolenic acid was the addition of a two-carbon unit. Conversion of linoleic acid to higher polyunsaturated fatty acids was slower than the conversion of linolenic acid to its higher analogues. This contrasted with the behaviour of dissociated cultures of normal brain cells which were able to form higher analogues of linoleic and linolenic acids at nearly equal rates.     

COMPOSITION OF MOUSE BRAIN MYELIN DURING DEVELOPMENT

Myelin was isolated from the brains of mice at ages of 14, 24, 41, 44, 47, and 182 days and the contents of lipid phosphorus, cholesterol, lipid galactose, alkenyl groups, ethanolamine phosphoglycerides, choline phosphoglycerides, sphingomyelin, and serine and inositol phosphoglycerides were determined. Significant differences in the composition relative to total lipid phosphorus were found in the myelin. At 14 days of age, the myelin had lower relative amounts of cholesterol, galactolipids, alkenyl groups, and ethanolamine phosphoglycerides and a higher relative amount of choline phosphoglycerides.     

Regulation of phospholipid metabolism in differentiating cells from rat brain cerebral hemispheres in culture. Serine incorporation into serine phosphoglycerides: base exchange and decarboxylation patterns.

The patterns of serine metabolism into phospholipids of cultured brain cells was examined. Labeled serine was incorporated predominantly into serine- ad ethanolamine-containing phospholipids and sphingolipids. The highest rates of labeling were observed in the (1)acyl-(2)acyl- and (1)alkyl-(2)acyl-serine phosphoglyceride fractions. Serine incorporation into both compounds appears to proceed via a base exchange mechanism. A decrease in the rate of serine phosphoglycerides labeling and a depletion of the ATP levels were observed when oligomycin or the calcium ionophore A23187 was added to the incubation medium. The inhibition of serine incorporation by A23187 could be partially reversed following addition of 10 mM CaCl2. Based on these findings it is suggested that in addition to demonstrating the energy-independent calcium-stimulated pathway, there may also be an energy related pathway. Formation of ethanolamine phosphoglycerides, as a result of serine phosphoglycerides decarboxylation, has been analyzed by using a simplified compartmental model. Of the 0.67 nmol/mg of protein turned over per h in the diacylserine phosphoglyceride compartment, 0.14 nmol/mg of protein are converted into the ethanolamine phosphoglycerides. In a similar manner, of the 0.09 nmol/mg of protein turned over per h in the (1)alkyl-(2)acyl-serine phosphoglyceride compartment, 0.014 nmol/mg of protein is converted into the (1)alkyl-(2)acyl-ethanolamine phosphoglyceride. These figures provide a first indication that a considerable portion of the ethanolamine phosphoglycerides in cultured brain cells is formed via a direct decarboxylation of the serine phosphoglycerides. In estimating the rates of (1)alkenyl-(2)acyl-ethanolamine phosphoglyceride formation from (1)alkyl-(2)acyl-ethanolamine phosphoglyceride the precursor-product specific activity crossover point could not be established. Mathematical analysis, however, enabled us to estimate the flux from the former into the latter as 0.04 nmol/mg of protein per h. A scheme for the possible metabolic interconversions of the ether bond containing serine and ethanolamine phosphoglycerides is proposed.     

THE FATTY ACID COMPOSITION OF PHOSPHOLIPIDS AND GLYCOLIPIDS IN JIMPY MOUSE BRAIN

Abstract— Phospholipids and sphingolipids from brains of normal and Jimpy mice were isolated in a pure form by thin-layer chromatographic procedures. The fatty acid composition of the major phospholipids, i.e. ethanolamine glycerophospholipids, serine glycerophospholipids, choline glycerophospholipids and inositol glycerophospholipids, as well as sphingomyelin, cerebrosides and sulphatides was determined by gas-liquid chromatography. A specific fatty acid pattern for each of the four glycerophospholipids was found. The fatty acid composition of inositol glycerophospholipid, which has not previously been studied in mouse brain, was characterized by a high concentration of arachidonic acid. After 16 days of age, fatty acid analysis showed definite differences between the phospholipids from normal and mutant brains. A small increase of polyunsaturated fatty acids in glycerophospholipids of ethanolamine, serine and choline from the Jimpy central nervous system was found, which has been explained by the myelin deficiency. Sphingomyelin, cerebrosides and sulphatide analyses showed a wide distribution of saturated and mono-unsaturated fatty acids in both normal and mutant mice. A reduction in the amount of long-chain fatty acids was demonstrated in mutant brain sphingolipids; in sulphatides and cerebrosides, the amount of non-hydroxy fatty acids was reduced to a greater extent than in sphingomyelin. The distribution of fatty acids in sphingolipids from the myelin and microsomal fractions was also investigated in both types of mice. Cerebrosides were characterized by a high content of long-chain fatty acids in myelin as well as in microsomes. Sulphatides and sphingomyelin, on the other hand, showed a higher content of medium-chain fatty acids in microsomes than in myelin. In the mutant brain, the amount of long-chain fatty acids was reduced in both subcellular fractions. The deviation from normal in the pattern of fatty acid distribution in Jimpy brain is discussed in relation to the current concepts of glycolipid biosynthesis.     

Skeletal muscle lipids. II. Changes in phospholipid composition in man from fetal to middle age

Phospholipid compositions were determined in samples of gastrocnemius muscles of individuals from fetal stage to the age of 55 yr. The lipids were separated by thin-layer chromatography. In order to enable characterization of the individual phospholipids, a lipid extract was prepared from pooled samples of gastrocnemius muscles from adult males and separated by ion-exchange chromatography on TEAE-cellulose. The individual phosphoglycerides were purified by thin-layer chromatography and then characterized according to their content of fatty acids, aldehydes, phosphorus, and the identity of the bases. The relative amounts of the major phospholipids, choline and ethanolamine phosphoglycerides, changed little with age, and in adult males they constituted 47% and 24%, respectively. Cardiolipin increased from 3% in the fetal stage to 9% by the end of the first year of life. Sphingomyelin and serine phosphoglyceride decreased with increasing age, while inositol phosphoglyceride increased. In adult males, cardiolipin constituted 10% of total lipid phosphorus, inositol phosphoglyceride constituted 9%, serine phosphoglyceride 3%, and sphingomyelin 7%.     

Diacyl,Alkylacyl, and Alkenylacyl Phospholipids of Meloidogyne javanica Females

The phospholipid composition and acyl, alkyl, and alkenyl group compositions of diacyl, alkylacyl, and alkenylacyl phosphoglycerides of M. javanica were investigated. Phospholipid was comprised of 61.7% choline phosphoglyceride, 22.0% ethanolamine phosphoglyceride, and smaller quantities of six other lipids. Phospholipid fatty acid was more unsaturated than neutral lipid fatty acid and contained 61.3% octadecenoic (18:1) acid. Fatty acid at the 1-position of diacyl phospholipids was shorter and more saturated than that at the 2-position. Compared to choline phosphoglyceride, ethanolantine phosphoglyceride contained less 18:1 and 20:5 and more 18:0 and 20:0 acid. Alkenylacyl and alkylacyl compounds comprised 34.6% and 9.3%, respectively, of the ethanolamine phosphoglyceride but only 0.5% and 0.6% of the choline phosphoglyceride. Alkenylacyl and alkylacyl ethanolamine phosphoglycerides contained a smaller percentage of 20-carbon polyunsaturated acid at their 2-positions than did their diacyl analogue. At least 95% of the alkenyl and alkyl groups were 18:0 compounds. Tomato roots did not contain alkenylacyl or alkylacyl phosphoglycerides; their occurrence in M. javanica is a significant biochemical difference between the nematode and its host.     

CHANGES IN LIPID CONCENTRATIONS AND FATTY ACID COMPOSITIONS IN RAT CEREBRUM DURING MATURATION

Abstract— Rat cerebrum was analysed at 20 different ages from birth to 45 days of age, for its concentration of protein, cholesterol, cerebrosides, phospholipids and gangliosides, and for the concentration of fatty acids of the linoleic and linolenic acid series. The fatty acid patterns of choline phosphoglycerides and ethanolamine phosphoglycerides were determined at the same ages. Phases of rapid accretion were found for protein, phospholipids, gangliosides and cholesterol. The accretion of the fatty acids of the linoleic acid series ceased at 20 days of age, while that of the fatty acids of the linolenic acid series continued. The fatty acid composition of the phosphoglycerides changed during the maturation of rat cerebrum and these changes consisted of chain elongation, increased unsaturation and variation in the pattern of the polyenoic acids. These changes varied irregularly with age and each developmental stage had characteristic fatty acid patterns of choline and ethanolamine phosphoglycerides.     

Can mitochondria and synaptosomes of guinea-pig brain synthesize phospholipids?

1. The use of ;marker' enzymes for investigating the contamination by endoplasmic reticulum of mitochondrial and synaptosomal (nerve-ending) fractions isolated from guinea-pig brain was examined. NADPH-cytochrome c reductase appeared to be satisfactory. With the synaptosomal preparation there was a non-occluded enzymic activity believed to arise from contaminating microsomes and an occluded form released by detergent, which probably was derived from some type of intraterminal smooth endoplasmic reticulum. 2. Isolated brain mitochondria, both intact and osmotically shocked, could not synthesize more labelled phosphatidylcholine from CDP-[Me-(14)C]choline or phosphoryl[Me-(14)C]choline than could be accounted for by microsomal contamination. They could synthesize only phosphatidic acid and diphosphatidylglycerol from a [(32)P]P(i) precursor and not nitrogen-containing phosphoglycerides or phosphatidylinositol. 3. The synaptosomal outer membrane and the intraterminal mitochondria could not synthesize phosphatidylcholine from CDP-[Me-(14)C]choline but the synaptic vesicles and probably the intraterminal ;endoplasmic reticulum' appeared to be capable of catalysing the incorporation of label from this substrate into their phospholipids. 4. Microsomal fractions and synaptosomes from guinea-pig brain could incorporate [Me-(14)C]choline into their phospholipids by a non-energy-requiring exchange process, which was catalysed by Ca(2+). Fractionation of the synaptosomes after such an exchange had taken place revealed that the label was predominantly in the intraterminal mitochondria and not associated with membranes containing NADPH-cytochrome c reductase. 5. On the intraperitoneal injection of [(32)P]P(i) into guinea pigs, incorporation of radioactivity into phosphatidylinositol and phosphatidic acid was much faster than into the nitrogen-containing phosphoglycerides. Mitochondria and microsomal fractions showed a roughly equivalent incorporation into individual phospholipids, and that into synaptosomes was appreciably less, whereas the phospholipids of myelin showed little (32)P incorporation up to 10h.     

Changes in the fatty acid profiles of red blood cell membrane phospholipids in human neonates during the first month of life

We have studied the changes in the fatty acid profiles of red blood cell membrane phospholipids in 47 infants who were exclusively fed human milk from birth to 1 month of life. Twenty blood samples were obtained from cord, 15 at 7 days and 12 at 30 days after birth. Membrane phospholipids were obtained from erythrocyte ghosts by thin-layer chromatography and fatty acid composition was determined by gas liquid chromatography. Phosphatidylcholine showed the most important changes during early life; stearic, w6 eicosatrienoic and arachidonic acids decreased whereas oleic and linoleic acids increased. In phosphatidylethanolamine, palmitic and stearic acid declined and oleic, linoleic and docosahexenoic acids increased with advancing age. Small changes were noted for individual fatty acids in phosphatidylserine. In sphingomyelin stearic acid increased from birth to 1 month and linoleic, arachidonic and nervonic acids decreased. Total polyunsaturated fatty acids of the w6 series greater than 18 carbon atoms increased with advancing age in phosphatidylethanolamine and decreased in choline and serine phosphoglycerides and in sphingomyelin. Long chain fatty acids derived from linoleic acid decreased in phosphatidylcholine but increased in ethanolamine and serine phosphoglycerides. The different behavior in the changes observed in fatty acid patterns for each erythrocyte membrane phospholipid may be a consequence of its different location in the cell membrane bilayer and specific exchange with plasma lipid fractions.     

The molecular organization of nerve membranes

Summary The lipid content and composition from an axolemma-rich preparation isolated from squid retinal axons was analyzed.The lipids, which accounted for 45.5% of the dry weight of this membrane, were composed of 22% cholesterol, 66.7% phospholipids and 5.2% free fatty acids. The negatively charged species phosphatidyl ethanolamine (37%), phosphatidyl serine (10%) and lysophosphatidyl ethanolamine (4%) made up 51% of the phospholipids. The amphoteric phosphatidyl choline and sphingomyelin accounted for 39% and 4%, respectively.The relative distribution of fatty acids in each of the isolated phospholipids was studied. The most remarkable feature of these phospholipids was the large proportion of long-chain polyunsaturated fatty acids. The 226 acyl chain accounted for 37% in phosphatidyl ethanolamine, 21.7% in phosphatidyl choline, 17.5% on phosphatidyl serine and 20.3% in sphingomyelin (all expressed as area %).The molar fraction of unsaturated fatty acids reached 65% in phosphatidyl ethanolamine and 42.0 and 44.8% in phosphatidyl choline and phosphatidyl serine, respectively. The double bond index in these species varied between 1.0 and 2.6.The lipid composition of the axolemma-rich preparation isolated from squid retinal axons appears to be similar to other excitable plasma membranes in two important features: (a) a low cholesterol/phospholipid molar ratio of 0.61; and (b) the polyunsaturated nature of the fatty acid of their phospholipids.This particular chemical composition may contribute a great deal to the molecular unstability of excitable membranes.The preceding papers of this series were published inArchives of Biochemistry and Biophysics.