Fatty acids and aldehydes of brain phospholipids during fetal and infant development in man

Abstract— Brains of human fetuses III, V, VI, VII months) newborn and infants (3, 7 and 13 months old) were investigated and the contents of total lipid, neutral- and phospholipid fractions were estimated. Fatty acids as well as fatty aldehydes of the phosphatides were analysed by gas chromatography. The results showed, that during this period of development the C₁₆-compounds in the fatty acid and aldehyde fractions decrease, while the total C₁₈-derivatives increase. However, the C₁₈-monoenoic fatty acids decrease from the third fetal month until birth and increase during myelination. The same pattern was found for the C₁₈-monoenoic aldehydes. The amounts of C₂₀- and C₂₂-polyenoic fatty acids were relatively constant. Only trace amounts of aldehydes with chain lengths other than C₁₆- and C₁₈-saturated and C₁₈-monoenoic comnniinds were found.     

The catabolism of plasmenylcholine in the guinea pig heart.

The hydrolysis of the alkenyl bonds of plasmenylcholine and plasmenylethanolamine by plasmalogenase, followed by hydrolysis of the resultant lysophospholipid by lysophospholipase, has been postulated as the major pathway for the catabolism of these plasmalogens. However, the postulation was based solely on the presence of plasmalogenase activity towards plasmenylethanolamine and plasmenylcholine in the brain. In this study we have demonstrated the absence of plasmalogenase activity for plasmenylcholine in the guinea pig heart under a wide range of experimental conditions. Plasmenylcholine was hydrolysed by phospolipase A2 activities in cardiac microsomal, mitochondrial and cytosolic fractions. Phospholipase A2 activities in these fractions had an alkaline pH optimum and were enhanced by Ca2+. The enzymes also displayed high specificity for plasmenylcholine with linoleoyl or oleoyl at the C-2 position. Lysoplasmalogenase activity for lysoplasmenycholine was also detected and characterized in the microsomal and mitochondrial fractions. Since the cardiac plasmalogenase is only active towards plasmenylethanolamine but not plasmenylcholine, the catabolism of these two plasmalogens must be different from each other. We postulate that the major pathway for the catabolism of plasmenycholine involves the hydrolysis of the C-2 fatty acid by phospholipase A2, and hydrolysis of the vinyl ether group of the resultant lysoplasmenylcholine by lysoplasmalogenase.     

Biosynthesis of saturated very long chain fatty acids by purified membrane fractions from leek epidermal cells

Elongation of fatty acids by microsomal fractions obtained from leek epidermal cells was measured by the incorporation of [1-¹⁴C]stearate, [1-¹⁴C]stearyl CoA, and [1-¹⁴C]stearyl ACP in the presence of malonyl CoA and NADPH. Stearoyl CoA appears to be the primer of the elongase (s) rather than stearoyl ACP. There are at least two elongases, the first elongating C₁₈ to C₂₀, the second synthesizing C₂₂ to C₃₀ fatty acids from C₂₀. The main site of the elongase (s) is a subcellular fraction enriched in endoplasmic reticulum. The plasma membrane-enriched fraction, which contains large amounts of saturated very long chain fatty acids, synthesizes only minor amounts of them.     

Alcohol dehydrogenase-coupled spectrophotometric assay of plasmalogenase

Plasmalogenase has been assayed by conversion of the fatty aldehydes, released by hydrolysis of the vinyl ether bond of plasmalogens, to long-chain alcohols by horse liver alcohol dehydrogenase. The reaction was followed spectrophotometrically by measuring the oxidation of NADH. The assay is sufficiently sensitive to enable plasmalogenase activity to be determined in isolated oligodendroglia and derived membranes and in brain microsomal membranes using 50-250 micrograms protein.     

Leishmania species: fatty acid composition of promastigotes

The fatty acid composition of the total lipid fractions of five different Leishmania organisms grown on Eagle's medium was determined by gas chromatography. The major fatty acids identified in the total lipid fractions of L. donovani, L. tropica major, L. tropica minor, L. tropica (England strain), and L. enriettii were C_12:0, C_13:0, C_14:0, C_15:0, C_16:0, C_17:0, C_18:0, C_18:1, C_18:2, and C_18:3. The statistical differences among the fatty acid methyl esters of different Leishmania organisms are discussed.Gas chromatographic analysis of the fatty acid methyl esters of the total lipid fractions of the original Eagle's medium and the media after harvesting of various Leishmania species revealed the presence of C_18:3 fatty acid in the total lipid fraction of the medium of L. donovani and the complete absence of 18-carbon unsaturated fatty acids in the total lipid fraction of the medium of L. enriettii. The use of such differences in the differentiation of various Leishmania species is discussed.     

Substrate-specificities of acid and alkaline ceramidases in fibroblasts from patients with Farber disease and controls

The specific activity of acid ceramidase (N-acylsphingosine deacylase, EC 3.5.1.23) was measured at pH4.5 in normal fibroblasts and in fibroblasts from patients with Farber disease and obligate heterozygotes. Greater activity was found when the synthetically made ceramide substrates contained shorter-chain fatty acids or higher content of double bonds. Acid ceramidase activities towards N-lauroyl- (C_12:0), N-myristoyl- (C_14:0) and N-palmitoyl- (C_16:0) sphingosine (C_18:1) were respectively about 38, 26 and 6 times higher than the activity towards the N-stearoyl (C_18:0) substrate. The activity towards N-linolenoylsphingosine (C_18:3/C_18:1), N-linoleoylsphingosine (C_18:2/C_18:1) and N-oleoylsphingosine (C_18:1/C_18:1) were respectively about 5, 4 and 3 times higher than the activity towards N-stearoylsphingosine (C_18:0/C_18:1). The activity towards N-stearoyldihydrosphingosine (C_18:0/C_18:0) was about 40% of that towards N-stearoylsphingosine. Fibroblast alkaline ceramidase possessed significant activity only towards ceramides of unsaturated fatty acids, with a pH optimum of about 9.0. Deficiency of acid ceramidase activity in fibroblasts from patients with Farber disease and intermediate activities in obligate heterozygotes were demonstrated with all ceramides examined except for N-hexanoylsphingosine (C_6:0/C_18:1), whereas alkaline ceramidase activity was unaffected. Comparative kinetic studies of acid ceramidase activity with N-lauroylsphingosine and N-oleoylsphingosine demonstrated about 5 (2–12)-fold and 7 (4–17)-fold higher K_m values in fibroblasts from patients with Farber disease as compared with normal controls. N-Lauroylsphingosine, towards which acid ceramidase activity in control fibroblasts was about 10 times higher than that towards N-oleoylsphingosine, may serve as a better substrate for enzymic diagnosis of Farber disease as well as for further characterization of the catalytically defective acid ceramidase.     

The alkane oxidation system of Pseudomonas oleovorans: induction of the alk genes in Escherichia coli W3110(pGEc47) affects membrane biogenesis and results in overexpression of alkane hydroxylase in a distinct cytoplasmic membrane subtraction

The alkane hydroxylase system of Pseudomonas oleovorans, which catalyses the initial oxidation of aliphatic substrates, is encoded by three genes. One of the gene products, the alkane hydroxyiase AlkB, is an integral cytoplasmic membrane protein. Induction leads to the synthesis of 1.5–2% AlkB relative to the total cell protein, both in P. oleovorans and in recombinant Escherichia coli DH1. We present a study on the Induction and localization of the alkane hydroxylase in E. coli W3110, which appears to be an interesting host strain because it permits expression levels of AlkB of up to 10–15% of the total cell protein. This expression level had negative effects on cell growth. The phospholipid content of such cells was about threefold higher than that of wild-type W3110. Freeze-fracture electron microscopy showed that induction of the alk genes led to the appearance of membrane vesicles in the cytoplasm; these occurred much more frequently in cells expressing alkB than in the negative control, which contained all of the alk genes except for alkB. Isolation and separation of the membranes of cells expressing alkB by density gradient centrifugation showed the customary cytoplasmic and outer membranes, as well as a low-density membrane fraction. This additional fraction was highly enriched in AlkB, as shown both by SDS-PAGE and enzyme activity measurements. A typical cytoplasmic membrane protein, NADH oxidase, was absent from the low-density membrane fraction, alkB expression in W3110 changed the composition of the phospholipid headgroup in the membrane, as well as the fatty acid composition of the membrane. The major changes occurred in the unsaturated fatty acids: C_16:1 and C_18:1 increased at the expense of C_17:0cyc and C_19:0cyc*     

Enrichment of very-long-chain mono-unsaturated fatty acids by lipase-catalysed hydrolysis and transesterification

Partial hydrolysis of triacylglycerols of high-erucic-acid seed oils from white mustard (Sinapis alba), oriental mustard (Brassica juncea) and honesty (Lunaria annua), catalysed by lipases from Candida cylindracea and Geotrichum candidum, leads to enrichment of erucic acid and other very-long-chain mono-unsaturated fatty acids (VLCMFA) in the acylglycerols (mono-, di- and triacylglycerol) while the C₁₈ fatty acids (oleic, linoleic and linolenic) are enriched in the fatty acid fraction. Partial hydrolysis of the high-erucic-acid triacylglycerols, catalysed by lipases from porcine pancreas, Chromobacterium viscosum, Rhizopus arrhizus and Rhizomucor miehei yields fatty acids with substantially higher levels of VLCMFA, as compared to the starting material, while the C₁₈ fatty acids are enriched in the acylglycerol fraction. Lipases from Penicillium sp. and Candida antarctica are ineffective for the fractionation of either group of fatty acids. Transesterification of the high-erucic-acid triacylglycerols with ethyl, propyl or butyl acetate or with n-butanol, catalysed by the lipase from R. miehei, leads to enrichment of VLCMFA in the alkyl (ethyl, propyl or butyl) esters, whereas the C₁₈ fatty acids are enriched in the acetylacylglycerols and acylglycerols.     

Determination of structure and composition of suberin from the roots of carrot, parsnip, rutabaga, turnip, red beet, and sweet potato by combined gas-liquid chromatography and mass spectrometry

Suberin from the roots of carrots (Daucus carota), parsnip (Pastinaca sativa), rutabaga (Brassica napobrassica), turnip (Brassica rapa), red beet (Beta vulgaris), and sweet potato (Ipomoea batatas) was isolated by a combination of chemical and enzymatic techniques. Finely powdered suberin was depolymerized with 14% BF₃ in methanol, and soluble monomers (20-50% of suberin) were fractionated into phenolic (<10%) and aliphatic (13-35%) fractions. The aliphatic fractions consisted mainly of ω-hydroxyacids (29-43%), dicarboxylic acids (16-27%), fatty acids (4-18%), and fatty alcohols (3-6%). Each fraction was subjected to combined gas-liquid chromatography and mass spectrometry. Among the fatty acids very long chain acids (>C₂₀) were the dominant components in all six plants. In the alcohol fraction C₁₈, C₂₀, C₂₂, and C₂₄ saturated primary alcohols were the major components. C₁₆ and C₁₈ dicarboxylic acids were the major dicarboxylic acids of the suberin of all six plants and in all cases octadec-9-ene-1, 18-dioic acid was the major component except in rutabaga where hexadecane-1, 16-dioic acid was the major dicarboxylic acid. The composition of the ω-hydroxyacid fraction was quite similar to that of the dicarboxylic acids; 18-hydroxy-octadec-9-enoic acid was the major component in all plants except rutabaga, where equal quantities of 16-hydroxyhexadecanoic acid and 18-hydroxyoctadec-9-enoic acid (42% each) were found. Compounds which would be derived from 18-hydroxyoctadec-9-enoic acid and octadec-9-ene-1, 18-dioic acid by epoxidation, and epoxidation followed by hydration of the epoxide, were also detected in most of the suberin samples. The monomer composition of the six plants showed general similarities but quite clear taxonomic differences.     

The ceramide structure of GM1 ganglioside differently affects its recovery in low-density membrane fractions prepared from HL-60 cells with or without triton-X100

Gangliosides are characteristically enriched in various membrane domains that can be isolated as low density membrane fraction insoluble in detergents (detergent-resistant membranes, DRMs) or obtained after homogenization and sonication in 0.5 M sodium carbonate (low-density membranes, LDMs). We assessed the effect of the ceramide structure of four [³H]-labeled GM1 ganglioside molecular species (GM1s) taken up by HL-60 cells on their occurrence in LDMs, and compared it with our previous observations for DRMs. All GM1s contained C18 sphingosine, which was acetylated in GM1(18:1/2) or acylated with C₁₄, C₁₈ or C_18:1 fatty acids (Fas)     

Early alterations induced by carbon tetrachloride in the lipids of the membranes of the endoplasmic reticulum of the liver cell II. Distribution of the alterations in the various lipid fractions

The distribution of carbon tetrachloride-induced alterations of membrane lipids in various fractions of liver microsomal lipids was studied. The chromatographic spot (referred to as the “D” spot in the previous paper [1]) which has been shown to contain the compounds responsible for the diene conjugation absorption [1], was found in the fatty acid methyl esters prepared from the fraction containing phosphatidylethanolamine (PE) and also in those obtained from the fraction containing phosphatidylserine (PS) and phosphatidylinositol (PI). The absorption of conjugated dienes was very marked in PE and less intense in PS and PI. The fatty acid methyl esters prepared from the fraction containing phosphatidylcholine (PC) showed no presence of the “D” spot and minimal absorption of conjugated dienes.A decrease in arachidonic acid content was found in the fraction containing PE, while no change in content of this fatty acid was found in the fraction containing PC. Results similar to those observed for PC were also found for neutral lipids (NL).Analysis of the fatty acid methyl esters of the various lipid fractions by gas-liquid chromatography (GLC) with an electron capture detector (ECD) gave a qualitative index of the free radical attack by CCl₄ metabolites. Quantitative estimation was attained by study of the irreversible binding of ¹⁴C from ¹⁴CCl₄ to the various lipid fractions. It was found that the fraction containing PS had the highest specific activity, while the fraction containing PC had the lowest specific activity of all the phospholipids. Thin layer chromatography (TLC) of the fraction containing PS revealed that only 11% of the radioactivity was associated with the pure PS moiety, while the remainder was associated with uncharacterized lipids (probably oxidation products).The possible relevance of the alterations induced by carbon tetrachloride in the various phospholipid fractions of liver microsomes to functional changes is discussed.     

Interrelationships among the ethanolamine phosphatides in myelinating rat brain

Abstract— —The ethanolamine phosphatide fraction was isolated from rat brain at 17, 19, and 22 days of age. Analysis by gas-liquid chromatography of the liberated fatty aldehydes and alkyl glyceryl ethers demonstrated a chain length composition quite distinct from that of the fatty acids in the comparable 1(3)-position of the diacyl phosphatides. [1-¹⁴C]-Acetate was administered intraperitoneally to 17-day-old rats. With the exception of the polyunsaturated fatty acids, isotope was readily incorporated into the individual side chains of the 1- and 2-positions of the glycerol moiety. Time studies revealed no readily discernible precursor-product relationships among the linkages in question. Therefore, although the long chain precursors for the alkenyl and alkyl ethers may be related by biosynthetic interconversion, the isotope data are suggestive of independent pathways of biosynthesis for the alkenyl ether, alkyl ether, and ester linkages.     

Plasmalogenase activity in normal and demyelinating tissue of the central nervous system

1. The plasmalogenase activity of brain was found to be associated with the white matter but was absent from myelin fractions. 2. Increased enzyme activity was found in demyelinating spinal cords from vitamin B₁₂-deficient monkeys and in white matter from a patient with multiple sclerosis.     

S1P and plasmalogen derived fatty aldehydes in cellular signaling and functions

Long-chain fatty aldehydes are present in low concentrations in mammalian cells and serve as intermediates in the interconversion between fatty acids and fatty alcohols. The long-chain fatty aldehydes are generated by enzymatic hydrolysis of 1-alkyl-, and 1-alkenyl-glycerophospholipids by alkylglycerol monooxygenase, plasmalogenase or lysoplasmalogenase while hydrolysis of sphingosine-1-phosphate (S1P) by S1P lyase generates trans ∆2-hexadecenal (∆2-HDE). Additionally, 2-chloro-, and 2-bromo- fatty aldehydes are produced from plasmalogens or lysoplasmalogens by hypochlorous, and hypobromous acid generated by activated neutrophils and eosinophils, respectively while 2-iodofatty aldehydes are produced by excess iodine in thyroid glands. The 2-halofatty aldehydes and ∆2-HDE activated JNK signaling, BAX, cytoskeletal reorganization and apoptosis in mammalian cells. Further, 2-chloro- and 2-bromo-fatty aldehydes formed GSH and protein adducts while ∆2-HDE formed adducts with GSH, deoxyguanosine in DNA and proteins such as HDAC1 in vitro. ∆2-HDE also modulated HDAC activity and stimulated H3 and H4 histone acetylation in vitro with lung epithelial cell nuclear preparations. The α-halo fatty aldehydes elicited endothelial dysfunction, cellular toxicity and tissue damage. Taken together, these investigations suggest a new role for long-chain fatty aldehydes as signaling lipids, ability to form adducts with GSH, proteins such as HDACs and regulate cellular functions.     

Studies on the localization and properties of rat duodenal HCO3−-ATPase with special relation to alkaline phosphatase

Brush-border membrane fractions were isolated from rat duodenum. Purity and integrity of the fraction was confirmed by electron microscopy, enzymic analysis and demonstration of Na⁺-dependent glucose uptake. The membranes were enriched 15-fold in alkaline phosphatase and α-glucosidase and 6-fold in HCO₃⁻-ATPase activities. Assays of latent activity indicated that these enzymes were predominantly localised to the external aspect of the microvillus membrane. The enzymes were solubilised and subjected to analysis by gel filtration, ion exchange and phenylboronate chromatography. No separation of alkaline phosphatase and HCO₃⁻-ATPase was obtained and it is suggested that they reflect the same enzyme activity. The apparent activation by HCO₃⁻ was investigated, and was found to be due to shifts in the pH dependency of the activity due to changes in ionic strength.     

In vitro antiherpes effects of a C‐glycosylflavonoid‐enriched fraction of Cecropia glaziovii Sneth*

Aims: To investigate the in vitro antiherpes effects of the crude aqueous extract obtained from Cecropia glaziovii leaves and their related fractions, the n‐butanol fraction (n‐BuOH) and the C‐glycosylflavonoid‐enriched fraction (MeOH_AMB), and to determine the viral multiplication step(s) upon which this C‐glycosylflavonoid‐enriched fraction acts. Methods and Results: The antiviral activity was evaluated against human herpes virus types 1 and 2 (HHV‐1, HHV‐2) by plaque reduction assay. The mode of action of the most active fraction was investigated by a set of assays, and the results demonstrated that MeOH_AMB fraction exerts anti‐herpes action by the reduction of viral infectivity (only against HHV‐2); by the inhibition of virus entry into cells; by the inhibition of cell‐to‐cell virus spread as well as by the impaired levels of envelope proteins of HHV‐1. The high‐performance liquid chromatography (HPLC)–photo‐diode array (PDA) analysis showed that the C‐glycosylflavonoids are the major constituents of this fraction. Conclusions: These data showed that the MeOH_AMB fraction has an antiviral activity against HHV types 1 and 2. The C‐glycosylflavonoids are the major constituents of this fraction, which suggests that they could be one of the compounds responsible for the detected anti‐herpes activity. Significance and Impact of the Study: The MeOH_AMB fraction can be regarded as a phytopharmaceutical candidate for the treatment of herpetic infections.     

Diacylglycerol kinase and lipase activities in rat brain subcellular fractions

Phosphatidic acid synthesis via diacylglycerol kinase and free fatty acid release via diacylglycerol lipase were investigated in rat brain subcellular fractions using membrane-bound [I-¹⁴C]arachidonoyl-diacylglycerol as substrate. Labeled diacylglycerol was generated by incubating brain membranes containing [I-¹⁴C]arachidonoyl-phosphatidylinositols in the presence of deoxycholate and Ca²⁺. Incubation of the prelabeled synaptosomes enriched in [1-¹⁴C]arachidonoyl-diacylglycerols or incubation of brain subcellular fractions with heat-treated prelabeled membranes resulted in the release of free fatty acids from the diacylglycerols. When incubations were carried out in the presence of ATP, MgCl₂ and NaF, both free fatty acid release and conversion of diacylglycerols to phosphatidic acids were observed. The conversion of diacylglycerols to phosphatidate or their hydrolysis to free fatty acids were linear with time for at least 15 min. In three brain subcellular fractions examined, diacylglycerol kinase activity indicated a pH maximum of 7.4. The free fatty acid release was enhanced slightly by Ca²⁺ (1 mM), but Ca²⁺ (0.5–4 mM) in the presence of Mg²⁺ (10 mM) was inhibitory to the diacylglycerol kinase reaction. Phosphatidate formation was also inhibited by an excessive amount of deoxycholate added to the incubation mixture. Among the brain subcellular fractions, diacylglycerol kinase was more active in synaptic vesicles and cytosol than in the microsomal fraction, whereas diacylglycerol lipase activity was higher in the cytosol fraction than in the membrane fractions. Upon washing the membranes by centrifugation, a substantial portion of the diacylglycerol kinase activity was removed after the first washing, whereas the diacylglycerol lipase activity remained essentially unchanged. The metabolic role of arachidonoyl-diacylglycerols in brain membranes in relation to the biosynthesis of phosphatidate and the release of arachidomic acid is discussed.     

STALOSYLGALACTOSYL CERAMTDE AS A SPECIFIC MARKER FOR HUMAN MYELIN AND OLIGODENDROGLIAL PERIKARYA: GANGLIOSIDES OF HUMAN MYELIN, OLIGODENDROGLIA AND NEURONS

Gangliosides were isolated from human brain myelin, oligodendroglia, and neurons. Quantitative analysis revealed the following ganglioside contents: myelin, 2.0; neurons, 1.3; and oligodendroglia, 0.35 μg ganglioside sialic acid per mg protein. Myclin had a relatively simple ganglioside pattern with G_M4 and G_M1 as the predominant ganglioside species. The ganglioside pattern of oligodendroglia was quite complex and it resembled that of whole white matter rather than that of myelin. A high concentration of G_M4 was found in oligodendroglial fractions in addition to G_M1, G_D1a, G_D1b, and G_T1b. The usually- minor brain gangliosides G_M3, G_M2, and G_M3 were also enriched in oligodendroglia. The neuronal ganglioside pattern was generally similar to the pattern of whole gray matter. Both neurons and whole gray matter contained very low amounts of G_M4. These results indicate that G_M4 is specifically localized in myelin and oligodendroglia of the CNS. Evidence is also presented that myelin, but not oligodendroglia, is the major reservoir of human white matter G_M1 and G_M4.     

Analysis of membrane fractions from Mycoplasma gallisepticum

Membrane fractions have been isolated from Mycoplasma gallisepticum following a procedure derived from that described by Maniloff, J. and Quinlan, D.C. (J. Bacteriol. (1974) 120, 495–501). A light fraction F₁ was obtained which contained structures resembling the bleb-infrableb apparatus characteristic of M. gallisepticum. It was enriched in DNA and had an electrophoretic profile different from that of unfractionated membranes. Cholesterol-to-phospholipid ratios higher than two and elevated values of the ratio of saturated to unsaturated fatty acids were other characteristics of this fraction. The two other fractions isolated (F_II and F_IV) also differed from intact membranes by their cholesterol and phospholipid content as well as by their saturation ratios. The membrane fluidity of F_II and F_IV, estimated by fluorescence polarization, was similar to that of unfractionated membranes while a slight but significant difference was recorded for the light fraction. Possible relationships between the lateral heterogeneity of the M. gallisepticum membrane and the obtainment of fractions are discussed.     

THE BIOSYNTHESIS AND CONCENTRATION OF GALACTOSYL DIGLYCERIDE IN GLIAL AND NEURONAL ENRICHED FRACTIONS OF ACTIVELY MYELINATING RAT BRAIN

—In continuation of our studies on the association of the galactosyl diglycerides of brain with myelination, we have measured the biosynthesis and concentration of these glyceride glycolipids, in oligodendroglial, astroglial, neuronal, and myelin enriched fractions from brains of rats of postnatal age 16, 19 and 29 days. The relative purity of cell fractions and myelin derived from 50 to 60 brains of each age-group was checked by phase contrast microscopy and 2′,3′-cyclic nucleotide-3′-phosphohydrolase activity. The relative purity was comparable to that reported by other investigators for cell fractions from bovine brain. Of the three cell types, the oligodendroglia had the highest and the neurons had the lowest capacity to enzymatically synthesize and to accumulate monogalactosyl diglyceride. The amount of monogalactosyl diglyceride found in myelin compared to that found in oligodendroglial fraction greatly increased during development between 16 and 29 days of age. The biosynthesis of galactosyl ceramide but not glucosyl ceramide was highest in oligodendroglial enriched cell fraction. However, ceramide glucosyl-transferase activity, which was greatly affected by the method used for cellular separation, was highest in a microsomal fraction derived from grey matter. Our results support the contention that the oligodendroglial cells are the site of synthesis of myelin constituents of the central nervous system, and that there is a temporal relationship between this site of synthesis and the site of deposition (myelin).