Globoid cell leukodystrophy (Krabbe's disease): isolation of myelin with normal glycolipid composition

Myelin was isolated from the brain of a patient with Krabbe's globoid cell leukodystrophy at 0.4% of the normal yield. Despite the exceedingly low yield, the fraction appeared morphologically clean, and consisted mostly of well-preserved myelin lamellae and few contaminating structures. Total lipid and cholesterol were slightly lower than in normal myelin. Total phospholipid was normal, but the ratio of ethanolamine phospholipid to lecithin was reversed. Total galactolipid was normal, and consisted only of cerebroside and sulfatide in normal proportions. The only sugar in cerebroside and sulfatide was galactose. The fatty acid composition of cerebroside and sulfatide was essentially normal with no deficiency of long-chain fatty acids and only with a reversed ratio of C(24:0) to C(24:1) in cerebroside. These data appear to exclude the previous postulate that abnormally rapid breakdown of myelin occurs in this disorder as the result of the formation of chemically abnormal myelin, deficient in sulfatide.     

THE ISOLATION AND LIPID COMPOSITION OF MYELIN-FREE AXONS FROM RAT CNS1

—Myelin-free axons were isolated from rat CNS using a modification of the method of De Vries et al. (1972). On a dry weight basis, the axons contained 15·2% lipid composed of 19·4% cholesterol, 56·9% phospholipid and 23·7% galactolipid with a weight ratio of cerebroside to sulfatide of 3·6-1. The phospholipid was composed of 11·0% ethanolamine phosphatides (44·4% in the plasmalogen form), 21·0% choline phosphatides (9·3% in the plasmalogen form), 4·5% sphingomyelin, 4·5% phosphatidyl serine, 4·3% phosphatidyl inositol, 3·0% diphosphatidyl glycerol and 8·5% unidentified phospholipid. The rat axons contained 0·18 μg ganglioside NeuNAc/mg dry wt. In addition to the 4 major brain gangliosides, the rat axons contained gangliosides G_D2 and G_D3. The axonal galactolipid could not be accounted for by myelin contamination as revealed by electron microscopy, absence of the characteristic ratio of myelin specific proteins in the axonal protein profile as shown by polyacrylamide gel electrophoresis, and the axonal level of the myelin marker enzyme 2′,3′-cyclic nucleotide-3′-phosphohydrolase. The relationship between lipids of axons isolated from rat and bovine CNS, and rat whole brain and CNS myelin is discussed.     

Lipid composition of rat brain myelin in triethyl tin-induced edema

Chronic triethyl tin intoxication was induced in young adult rats by oral feeding of triethyl tin sulfate. Progressively severe brain edema developed during the 3-month experimental period. The yield of myelin from the brains of the experimental animals decreased to almost half normal per brain, but the isolated myelin appeared morphologically normal. The analysis of whole brain showed corresponding decreases in proteolipid protein and total lipid, particularly galactolipids. The proportions of the major constituents of isolated myelin (chloroform-methanol-insoluble residue, proteolipid protein, and total lipid) were unchanged despite the low yield. However, the proportion of cholesterol increased from 16 to 21% dry weight, and that of total galactolipid decreased from 21 to 15%, as the yield of myelin decreased. This decrease of total galactolipid was mainly due to the decrease in cerebroside. Total phospholipid remained constant initially but showed a slight decrease toward the end of the experiment, due mostly to decreased ethanolamine phospholipid. There was no preferential loss or preservation of phosphatidalethanolamine. The fatty acid composition of sulfatide showed statistically significant shifts to less long-chain fatty acids and less monoenoic acids, but cerebroside and sphingomyelin did not show significant changes in the fatty acid composition. There was no increase in esterified cholesterol. These findings generally support our hypothesis of nonspecific chemical abnormalities of the myelin sheath undergoing secondary degeneration. In an acute experiment, a single intraperitoneal injection of triethyl tin sulfate produced acute and transient brain edema. There were slight decreases in the yield of myelin, but no detectable changes in the chemical composition.     

Preparation of asymmetric,cerebroside sulfate-containing phospholipid vesicles

A procedure is described which inserts asymmetrically cerebroside sulfate (‘sulfatide’) into the outer leaflet of bilayered phospholipid vesicles. Cerebroside sulfate is adsorbed onto a cellulose, filter-paper support and, when incubated with phosphatidylcholine vesicles is transferred to and inserted into the outer leaflet of these vesicles. This transfer occurs at, or above the transition temperature of the phospholipid and follows a similar pattern with small or larger (‘fused’) unilamellar vesicles. The transfer is linear with time for 1–2 h and is maximal after about 6 h, when the sulfatide content reaches about 6 mol% of the total quantity of phospholipid, corresponding to about 10 mol% of the phospholipids present in the outer layer. Initial rates of sulfatide transfer were somewhat increased when the vesicles contained a positively charged lipid (e.g. stearylamine) and decreased when this lipid was negatively charged (e.g. dicetyl phosphate) or hydrophobic (e.g. cholesterol). Divalent ions markedly inhibited sulfatide transfer and monovalent ions did so to a lesser degree. Once incorporated into the outer leaflet of the vesicle, the sulfatide could not be removed by washing with buffer, 1 M NaCl or 1 M urea.     

Neuronal perikarya and astroglia of rat brain: chemical composition during myelination

Cells isolated by a new technique from 10-, 20-, and 30-day-old rat brains have been analyzed for total lipid, cholesterol, galactolipid, individual phospholipids, gangliosides, DNA, and RNA. The lipid composition does not vary appreciably in either neurons or astrocytes during this period of rapid myelination. Moreover, the lipid compositions of the two cell types are surprisingly similar, both having very low galactolipid concentrations, high phospholipid content, and cholesterol concentrations lower than whole brain. Astrocytes have a higher ganglioside content than neuronal perikarya, a finding ascribed to the higher ratio of surface membrane to mass in the astrocytes, and considered as evidence that gangliosides are normal glial constituents. Compared with an average astrocyte, the individual neuron soma has less mass, a lower total lipid content, and a much higher RNA content.     

Phospholipids from Bacillus stearothermophilus

The lipids of Bacillus stearothermophilus strain 2184 were extracted with chloroform-methanol and separated into neutral lipid and three phospholipid fractions by chromatography on silicic acid columns. The phospholipids were identified by specific staining reactions on silicic acid-impregnated paper, by chromatography of alkaline and acid hydrolysis products, and by determination of acyl ester:glycerol:nitrogen:phosphorus molar ratios. The total extractable lipid was 8% of the dry weight of whole cells and consisted of 30 to 40% neutral lipid and 60 to 70% phospholipid. The phospholipid consisted of diphosphatidyl glycerol (23 to 42%), phosphatidyl glycerol (22 to 39%), and phosphatidyl ethanolamine (21 to 32%). The concentrations of diphosphatidyl glycerol and phosphatidyl glycerol were lower in 2-hr cells than in 4- and 8-hr cells. Whole cells were fractionated by sonic treatment and differential centrifugation. The total lipid content, expressed in per cent of dry weight of each fraction was: whole protoplasts, 10%; membrane fraction, 18%; 30,000 x g particulate fraction, 22%; and 105,000 x g particulate fraction, 26%. The relative phospholipid concentrations in each fraction were about the same. As had been previously reported, none of the phospholipid was stable to alkaline hydrolysis.     

Differences in neuronal lipid composition between superior cervical ganglia and nodose ganglia of the rat

The lipid content and composition of rat superior cervical ganglia containing sympathetic motor neurons and nodose ganglia containing parasympathetic sensory neurons were studied for the first time to elucidate the mechanism of the different effects of exogenous gangliosides on these neurons in the culture medium. The ganglioside content of the superior cervical ganglia was almost 3-times that of the nodose ganglia. Although both ganglia contained GM3, GD3, GD1b and GT1b as major gangliosides, the nodose ganglia additionally contained a significant amount of sialosyllactoneotetraosylceramide LM1 (10% of total sialic acids). Contrasting with nodose ganglia, vagus fiber and dorsal root ganglia of rats, superior cervical ganglia had a higher content of sulfatide than galactosylceramide. The phospholipid content was lower in superior cervical ganglia than in nodose ganglia. Superior cervical ganglia contained less ethanolamine plasmalogen and more phosphatidylcholine than nodose ganglia. Sphingomyelin in superior cervical ganglia contained mainly medium-chain fatty acids, while that in nodose ganglia contained mainly longer-chain fatty acids. Differences in the fatty acid composition of glycerophospholipids were also observed. The results indicate that the properties of neuronal cell membranes from superior cervical ganglia and nodose ganglia are quite different, and that the differences may reflect the physiological roles of these ganglia.     

MYELIN SUBFRACTIONS IN DEVELOPING RAT BRAIN: CHARACTERIZATION AND SULPHATIDE METABOLISM

Centrifugation of isolated myelin on discontinuous sucrose gradients resulted in a separation into three bands and a pellet. The three bands were morphologically identical to myelin, whereas the pellet consisted primarily of vesicular membranes. These four fractions differed from one another in their lipid-to-protein ratios and in molar ratios of cholesterol:phospholipid:galactolipid. All of the fractions contained proteins typical of myelin, although the proportions of the proteins varied, with the pellet being the lowest in basic protein and proteolipid protein. High activity of 2′,3′-cyclic nucleotidase and low activity of cerebroside sulphotransferase further distinguished these fractions from the microsomal fraction. Distribution of radioactive sulphatide in the subfractions at 15 min after intracranial injection of radioactive sulphate indicated that newly-labelled sulphatide first appeared in the lipid-poor fractions, followed by the lipid-rich fractions; results of pulse-chase experiments also suggested this relationship. Several days or weeks after the injection of radioactive sulphate, most of the radioactive sulphatide was in the lipid-rich 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.     

The lipid composition of axons from bovine brain

Abstract— Bovine axons derived from myelinated CNS axons were found to have 13.5% lipid. This lipid was composed of 20.1% cholesterol, 20.1% galactolipid, 14.6% ethanolarhine phosphatides (56.4% in the plasmalogen form), 18.3% choline phosphatides (10.0% in the plasmalogen form), 9.3% sphingomyelin, 5.6% phosphatidyl serine and 3.4% phosphatidyl inositol. The bovine axons had 0.33 μg ganglioside NeuNAc/mg dry weight. The axon gangliosides were found to contain the four major types of bovine gangliosides, as well as gangliosides G_M2 and G_D3. The latter two amounted to 20.9 and 15.8 mole per cent respectively, of total gangliosides. On a molar basis, about one half of the gangliosides were monosialogangliosides, with a decreased contribution by gangliosides G_T1 and GD_1b relative to ganglioside distributions which have been reported for most other CNS components. The relationship of the bovine axonal lipid composition to bovine white matter and its constituents, as well as to other CNS and PNS axonal preparations is discussed.     

Lipid composition of Mycoplasma neurolyticum

The total lipid content of Mycoplasma neurolyticum comprises about 14% of the dry weight of the organisms and is about equally distributed between the phospholipid and the neutral-glycolipid fractions. The neutral lipids were identified as triglycerides, diglycerides, and cholesterol. The glycolipid fraction contained 1-O-beta-glucopyranosyl-d-2,3-diglyceride and 1-[O-beta-d-glycopyranosyl-(1-->6)-O-beta-d-glucopyranosyl]-d-2,3-diglyceride. The latter lipid is structurally identical to the diglucosyl diglyceride which occurs in Staphylococcus aureus. The phospholipids of the organism consist of a fully acylated glycerophosphoryl-glycerophosphoryl glycerol, phosphatidic acid, diphosphatidyl glycerol, phosphatidyl glycerol, and amino acyl esters of phosphatidyl glycerol. Phosphatidic acid and phosphatidyl glycerol account for greater than 90% of the phospholipids of organisms in the exponential phase of growth. The predominant fatty acids found in all of the acyl lipids were palmitic, stearic, and oleic acids.     

Isolation and some chemical properties of oligodendroglia from calf brain

Abstract— The method of Norton and Poduslo (1970) for isolating brain cells has been adapted for the isolation of oligodendroglia from the white matter of calf brain. The cells were obtained in greater than 90 per cent purity, and in a yield of 11 × 10⁶ cells/g of white matter. This number of cells represented a recovery of 11 per cent of the total cells in the tissue and therefore a considerably higher recovery of the original number of oligodendroglia. The average cell contained 5, 2 pg of DNA, 2–0 pg of RNA and 6, 7 pg of lipid. The lipid comprised cholesterol, galactolipid (both cerebroside and sulphatide) and phospholipid in the molar ratio of 1:0, 45:2, 3. Gangliosides were present in a concentration similar to that found in isolated rat neurons, The myelin-specific enzyme, 2′, 3′-cyclic nucleotide 3′-phosphohydrolase, was present at a level nearly equal to that in myelin, and eight-fold higher than the levels in rat neurons or astrocytes. The isolated oligodendroglia differed considerably from isolated astrocytes in size, morphology and chemical composition.     

Sulfatides in Prenatal Metachromatic Leukodystrophy

In one 21-week-old fetus with prenatally diagnosed metachromatic leukodystrophy, galactolipid contents were determined in the forebrain cortex, cerebellum, brainstem, spinal cord, and kidney and compared to an appropriate control. Spinal cord and kidney showed the highest sulfatide accumulation as a consequence of deficient cerebroside sulfatase activity. No increase, but a measurable amount of sulfatide, was detected in the forebrain. The prenatal neural sulfatides contained a high proportion of the hydroxy fatty acid component. The galactosyl ceramides were not reduced in the early stage of the demyelinating disease.     

Studies on the brush border membrane on mouse duodenum: lipids

Lipids were extracted from purified mouse duodenal brush border membranes. Lipid: protein ratios in different membrane preparations varied from 0.58 to 0.68. Both the chloroform and non-chloroform phases were quantitatively analysed for lipids. Chloroform extracts were composed of cholesterol, triglycerides and phospholipids. The major neutral lipid was cholesterol. Rechromatrography of the phospholipid spot showed sphingomyelin (7.9%), phosphatides of ethanolamine (61.7%), inositol (14.2%) and serine (16.2%). The average molar ratio of cholesterol to phospholipid was 1.39. Lipids in the non-chloroform phase were all glycosylated, being cerebrosides (69.3%), cerebroside sulphates (28.8%) and galgliosides (1.9%). Overal membrane lipid composition was neutral lipid 24%, phospholipid 33%, and glycolipid 43%.     

Studies on plasma membranes

Summary Plasma membranes were isolated from rat and mouse livers, one rat hepatoma (and its subline) and two mouse hepatomas, and their lipid class compositions were determined. Lipids accounted for 30 to 35% of the dry weight of the membranes of livers and mouse hepatomas, and for 45% in the case of rat hepatoma-subline. Of the total lipids of rat-liver plasma membranes, 60% consisted of phospholipids, the corresponding values for mouse-liver and rat-hepatoma plasma membranes amounting to 55% and for both mouse-hepatoma plasma membranes to about 50%. The free cholesterol and cholesteryl ester contents of all hepatoma plasma membranes were significantly increased as compared with normal. Evidence is presented that the increase of free cholesterol was not a preparative artefact. The major phospholipid classes in all plasma membranes were phosphatidyl choline, sphingomyelin, phosphatidyl ethanolamine and phosphatidyl serine. The relative proportions in each plasma membrane species could differ appreciably, the mouse- and rat-liver membranes showing the closest resemblance. Possible reasons for (a) the higher level of phosphatidyl serine as compared with published values, and (b) the wide divergencies which may be found among the phospholipid profiles of rat-liver plasma membranes reported by other authors, are presented. Cardiolipin was absent from liver plasma membranes, but some could be found in the hepatoma membranes due to mitochondrial contamination. No consistent phospholipid profile characterized hepatoma as distinct from liver plasma membranes, nor did the hepatoma data-including plasmalogens-resemble the few available data on other hepatomas.     

Membrane Lipids of Adult Human Brain: Lipid Composition of Frontal and Temporal Lobe in Subjects of Age 20 to 100 Years

Abstract: The membrane lipid composition of human frontal and temporal cortices and white matter has been studied in 118 subjects, age 20–100 years. The brain specimens were selected from subjects who lived a normal social life and died suddenly and unexpectedly with no history of neurologic or psychiatric disease. Macroscopic and microscopic examinations ruled out any signs of organic brain disorder. The sudden death eliminated all risk of changes over a long agonal stage. The data for total solids and major lipids are summarized in graphic form. Total solids, phospholipids, and cholesterol diminished linearly from 20 years of age in frontal and temporal cortices, whereas total solids phospholipids, cholesterol, cerebroside, and sulfatide showed a curvilinear diminution in frontal and temporal white matter. Gangliosides differed from the other lipids, showing an almost constant concentration between 20 and 70 years of age with a slight peak around 50 years of age. The ganglioside pattern showed continuous change with aging, with decreasing proportions of GM1 and GD1a and increasing proportions of GD1b, GM3, and GD3. Equations are given that can be used to calculate the lipid composition of normal human frontal and temporal cortices and white matter at any age between 20 and 100 years of age. These data can be used where data by direct analysis are not available for comparison with values for various pathological states.     

Molecular composition of the submicrosomal membrane lipid of rat brain

Rough-surfaced and light and heavy smooth-surfaced microsomes were isolated from rat brain by means of discontinuous sucrose gradient centrifugation. Electron microscopically, the rough-surfaced microsomes were characterized by vesicles with ribosomes and the light and heavy smooth-surfaced microsomes by fairly homogeneous membrane features without ribosomes. The rough-surfaced microsomal membranes were distinguished by the absence of glycolipids, such as ganglioside, cerebroside, and sulfatide. Cerebroside was exclusively recovered in the light smooth-surfaced microsomal membranes. Ganglioside and Na,K-ATPase were contained in larger amounts in the heavy smooth-surfaced microsomal membranes than in the light smooth-surfaced microsomal membranes in terms of protein content. Among the three submicrosomal membranes, cholesterol and phospholipid were found in the largest amounts in the light smooth-surfaced microsomal membranes, where the molar ratio of cerebroside-cholesterol-phospholipid was about 1:10:10. The membranes of rough- and smooth-surfaced microsomes were very similar in regards to the composition of phospholipid classes, although the fatty acid composition of the former contained a greater proportion of unsaturated fatty acids than that of the latter. When the membrane proteins were analyzed by sodium dodecyl sulfate gel electrophoresis, some differences were observed between the light and heavy smooth-surfaced microsomal membranes.     

Characterization of membrane components of the flask-shaped mycoplasma Mycoplasma mobile

The cell membrane of Mycoplasma mobile was isolated by either ultrasonic or French press treatment of intact cells. The membrane fraction contained all of the cellular lipids, but only one-third of cellular proteins and had a density of 1.14 g ml-1. The soluble fraction contained the NADH dehydrogenase activity of the cells, as well as a protein with an apparent molecular mass of 55 kDa that was phosphorylated in the presence of ATP. Lipid analyses of M. mobile membranes revealed that membrane lipid could be labelled by radioactive glycerol, oleate and to a much higher extent by palmitate but not by acetic acid. The membrane lipid fraction was composed of 54% neutral and 46% polar lipid. The major constituents of the neutral lipid fraction were free fatty acid, free cholesterol and cholesterol esters (45, 25 and 20%, respectively, of total neutral lipid fraction). The free cholesterol count was 13% (w/w) of total membrane lipids with a cholesterol:phospholipid molar ratio of about 0.9. Among the polar lipids, both phospho- and glycolipids were detected. The phospholipid fraction consisted of a major de novo-synthesized phosphatidylglycerol (approximately 63% of total phospholipids), plus exogenous phosphatidylcholine and sphingomyelin incorporated in an unchanged form from the growth medium. The glycolipid fraction was dominated by a single glycolipid (approximately 90% of total glycolipids) that was preferentially labelled by palmitic acid and showed a very high saturated:unsaturated fatty acids ratio.     

Characteristic Constituents of Glycolipids from Frog Brain and Sciatic Nerve

Cerebroside, sulfatide, monoglycosyl glyceride, and ester cerebroside were isolated from frog brain and sciatic nerve, and their distribution and chemical constituents were determined. The long-chain base compositions of cerebroside, sulfatide, and ester cerebroside were unique in the presence of branched-base components (5-15% of the total bases) and in the abundance of saturated dihydroxy base components (15-45% of the total). The amount of branched long-chain bases was greater in sciatic nerve than in brain. The hexose composition of the glycolipids consisted entirely of galactose except for brain cerebroside, in which a small amount of glucose was detected. Monogalactosyl glyceride consisted of the diacyl and alkylacyl forms, in a molar ratio of 81:19 for brain and 62:38 for sciatic nerve. The fatty acid composition of glycosphingolipids was characterized by the predominance of hydroxy and nonhydroxy 24:1 acids, and the concentration of 24:0 was extremely low. The proportion of unsaturated fatty acids accounted for 80% of the total. Major fatty acids of monogalactosyl glyceride were palmitic, oleic, stearic, and palmitoleic acids; the highest concentration was that of palmitic acid. Ester cerebroside was separated into three subfractions mainly on the basis of the proportion of hydroxy and nonhydroxy components in the amide-linked fatty acids.