Preparation of GM1 ganglioside molecular species having homogeneous fatty acid and long chain base moieties

A new procedure is described for preparing the molecular species of GM1 ganglioside that carry a single fatty acid (myristic (C14:0), stearic (C18:0), arachidic (C20:0) or lignoceric (C24:0) acid) and a single long chain base (C18 or C20 sphingosine, C18 or C20 sphinganine, each of them in natural 3D(+)erythro or unnatural 3L(-)threo form). The procedure consisted of the following steps: a) alkaline hydrolysis of GM1 ganglioside in the presence of tetramethylammonium hydroxide, which produces de-N-acylation of the ceramide and de-N-acetylation of the sialic acid residue; b) specific re-N-acylation at the long chain base amino group with a new fatty acid (myristic, stearic, arachidic, or lignoceric) in the presence of 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride; and c) final re-N-acetylation at the level of the sialic acid residue. GM1 ganglioside molecular species, completely homogeneous in the ceramide portion, were prepared by reversed phase high performance liquid chromatography. The GM1 ganglioside molecular species were analyzed for saccharide, fatty acid, and long chain base composition by chemical and spectrometric analyses. Using a combination of the two procedures, 32 different molecular species of GM1 ganglioside, over 99% homogeneous, have been prepared.     

Age-Related Changes in the Ceramide Composition of the Major Gangliosides Present in Rat Brain Subcellular Fractions Enriched in Plasma Membranes of Neuronal and Myelin Origin

Abstract: Age-related changes of the ceramide composition of gangliosides were studied in the synaptosomal and myelin fractions from rat brain, carrying plasma membranes of neuronal and glial origin, respectively. The five major gangliosides (GM1, GD1 a, GD1 b, GT1 b, and GQ1 b) present in these fractions were separated and quantitated by normal-phase HPLC. Each ganglioside was then fractionated by reverse-phase HPLC into the molecular species carrying a single long-chain base (LCB). The largely preponderant LCBs in the synaptosomal and myelin fractions were the C18:1 and C20:1. The content of C20.1 LCB, generally low at 1 month, increased with age in all analyzed gangliosides and in all subcellular fractions and was greater in the "b series" than in the "a series" gangliosides. Remarkably, GM1 was the only ganglioside where the proportion of LCB 20:1 was higher in the synaptosomal fraction than in the myelin fraction. The fatty acid composition of the C18:1 or C20:1 LCB species of the different gangliosides in the synaptosomal and myelin fractions did not undergo appreciable changes with age. Stearic acid was largely predominant in all the gangliosides of the synaptosomal fraction, more in the C18:1 than in the C20:1 LCB species (80–90% vs. 60–70%). The gangliosides of the myelin fraction were characterized by a lower content of 18:0 and a much higher content of 16:0 and 18:1 fatty acids than those of the synaptosomal fraction. Thus, the ceramide composition is different in the gangliosides of neuronal and myelin origin and appears to be subjected to an age-related control.     

GANGLIOSIDE1−COMPOSITION OF BRAIN IN TAY-SACHS DISEASE: INCREASED AMOUNTS OF GD2 AND N-ACETYL-β-D-GALACTOSAMINYL GD1a GANGLIOSIDE

Abstract— The ganglioside composition of the brain of a patient with Tay-Sachs disease (TS-brain) was determined by a newly developed ganglioside-mapping procedure and compared with that of an age-matched control brain. GM2 ganglioside was the predominant component in TS-brain and the following gangliosides were also found, GM1, GD1a, GD1b and GT1 (major gangliosides in normal brain), and GM3, GD3, GD2 and GD1a-GAN (minor or undetectable components of normal brain). Individual gangliosides were isolated by column chromatography using a combination of DEAE-Sepharose, Iatrobeads and Silica Gel 60 and their structures were confirmed by comparing them with authentic standards using TLC, analysing their carbohydrate compositions by gas-liquid chromatography and cleaving them sequentially with glycosidases. The amounts of individual components were measured by quantitative densitometric scanning of the thin-layer plates. As a reflection of myelin breakdown, no sialosylgalactosyl ceramide was detectable in TS-brain. Although the total amounts of all gangliosides except GM2 in TS-brain were low, there were normal molar ratios of the main gangliosides in normal brain, that is, GM1, GD1a, GD1b and GT1. In comparison with the amount of GDla ganglioside, the amounts of GM2, GD2 and GD1a-GAN, which contain N-acetylgalactosamine as a terminal carbohydrate residue, were all elevated in TS-brain. The long chain bases of individual gangliosides contained both C-18 and C-20 sphingosine in different ratios and the ratio of C-20 to C-18 increased in the gangliosides in the order: GM2 < GM1 < GD1a < GD1a-GAN < GD1b < GT1 in both normal brain and TS-brain. In contrast, GD2 and GD3 gangliosides consisted mainly of C-18 sphingosine. The C-20 to C-18 ratios of individual gangliosides in the TS-brain were lower than those of age-matched control brain. Hexosaminidase from Turbo cornutus showed the same specific activity and K_m value in catalysing the cleavage of terminal N-acetylgalactosaminyl residues from GM2, GD2 and GD1a-GAN, suggesting that the brain gangliosides that increase in Tay-Sachs disease may be cleaved by the same enzyme.     

Anti-Ganglioside Antibodies Bind with Enhanced Affinity to Gangliosides Containing Very Long Chain Fatty Acids

Gangliosides function in both physiological and pathological molecular recognition. Although much research has focused on the role of ganglioside glycans in recognition, fewer studies have addressed the role of the ceramide moiety. Ceramides of major brain gangliosides are composed predominantly of monounsaturated 18-carbon and 20-carbon long chain bases with a saturated 18-carbon fatty acid amide. In contrast, gangliosides of X-linked adrenoleukodystrophy patients are characterized by abnormal very long chain fatty acids that are proposed to be associated with autoimmune inflammation. In the current study we synthesized and characterized derivatives of the major brain ganglioside GD1a bearing defined very long chain fatty acid amides (C24:0, C24:1, and C26:0). When tested in a solid phase binding assay in the presence of auxiliary membrane lipids, GD1a species with long chain fatty acids were up to 8-fold more potent than normal brain GD1a in binding four different anti-GD1a monoclonal antibodies. These data support the hypothesis that gangliosides bearing very long chain fatty acids are differentially displayed on membranes, which may lead to altered antigenicity.     

Changes in the Ganglioside Long-Chain Base Composition of Rat Cerebellar Granule Cells During Differentiation and Aging in Culture

Abstract: Changes in the ganglioside long-chain base (LCB) composition in rat cerebellar granule cells in culture were studied during differentiation and aging. The total native ganglioside mixtures, extracted from the cells maintained in culture up to 22 days, were fractionated by reversed-phase HPLC, each ganglioside homogeneous in the oligosaccharide chain as well as in the LCB being quantified. Two main LCBs were components of the ganglioside species of cultured cells, the C18:1 LCB and the C20:1 LCB. The content of C20:1 ganglioside molecular species was low and quite constant during differentiation, comprising ∼8% of the total ganglioside species content, the C20:1 LCB appearing to be represented more in the ganglioside of the "b series" (GD1b, GT1b, and GQ1b) than in the "a series" (GM1 and GD1a). During aging in culture, for 8–22 days, the content of the C20:1 species of all gangliosides increased, being more pronounced for GM1 and GD1a.     

Evidence for Nonrandom Distribution of GD1 a Ganglioside in Rabbit Brain Microsomal Membranes

GD1a is the major ganglioside of rabbit brain microsomal membranes and occurs mainly with two molecular species, containing the C18:1 (62.3%) and C20:1 (37.7%) long-chain bases. The membranes were exposed to Vibrio cholerae (VC) sialidase under conditions where the enzyme hydrolyzed only GD1a (approximately 9%), producing GM1 ganglioside, whereas the other gangliosides remained virtually unaffected. The long-chain-base analysis showed that newly-formed GM1 contained approximately 68% of the C20:1 molecular species. This indicates that VC sialidase did not randomly affect the two molecular species of GD1a but hydrolyzed preferentially the C20:1 one. In similar experiments, GD1a was inserted into the external layer of phosphatidylcholine vesicles and incubated with VC sialidase under conditions producing approximately 10% hydrolysis. Long-chain-base analysis showed that the proportion of C20:1 species in GM1 was 25.1% using vesicles composed of dipalmitoylphosphatidylcholine and 42.3% with egg phosphatidylcholine, whereas it was 39.2% in the starting GD1a. Therefore, in artificial membranes, VC sialidase acted preferentially on the C18:1 or C20:1 molecular species, depending on the length and unsaturation of the phospholipid fatty acids. Because VC sialidase is known to affect molecular dispersions more easily than packed aggregations of the gangliosidic substrate, the data suggest that in rabbit brain microsomal membranes the GD1a ganglioside molecular species carrying C20:1 long-chain base are more molecularly dispersed than those containing C18:1 long-chain base.     

Different Ceramide Compositions of Gangliosides Between Human Motor and Sensory Nerves

Ganglioside analysis of human motor and sensory nerves revealed that ceramide compositions of sensory nerve GD1a, GD1b, and GM1 differed apparently from those in the motor nerve. These gangliosides from sensory nerve contained a large amount of long-chain fatty acids and d18:1 as a major long chain base. On the contrary, the motor nerve gangliosides contained C16-18 fatty acids and a large amount of d20:1 besides d18:1. Furthermore, these gangliosides were enriched more in the axon fraction than in the myelin fraction. LM1, which was a major ganglioside in myelin from human peripheral nerve, was composed of similar ceramide compositions in the two nerves. The present findings suggest that the characteristic ceramide species of nerve gangliosides may reflect in part properties of their own neurons.     

Influence of glycolipid oligosaccharide and long-chain base composition on the thermotropic properties of dipalmitoylphosphatidylcholine large unilamellar vesicles containing gangliosides

The thermotropic behavior of dipalmitoylphosphatidylcholine large unilamellar vesicles containing gangliosides has been studied by high-sensitivity heating and cooling differential scanning calorimetry. These studies have been directed to identify and evaluate the influence of both the ganglioside lipidic portion and oligosaccharide moiety on the physical properties of phospholipid bilayers containing gangliosides. The influence of the ganglioside lipidic portion has been evaluated by studying the behavior of vesicles containing different GD1a molecular species carrying homogeneous lipid moieties (C20 or C18 sphingosine or sphinganine and stearic acid). The influence of the ganglioside saccharide portion was evaluated by investigating the thermotropic behavior of vesicles containing different gangliosides (GM1, Fuc-GM1, GD1a, GT1b) carrying the same homogeneous long-chain base moiety (C20 sphingosine and stearic acid). These studies, in conjunction with previous studies using homogeneous lipidic portion ganglioside GM1 and phosphatidylcholines of various chain lengths [Masserini, M., & Freire, E. (1986) Biochemistry 25, 1043-1049], indicate that, for a given oligosaccharide composition, gangliosides exhibit lateral phase separation in an extent dependent upon the length and unsaturation difference between the ganglioside long-chain base and phosphatidylcholine acyl chains. For a given ganglioside lipidic composition the extent of phase separation is dependent upon the number of sugar units present in the glycolipid. The addition of Ca2+ induces or enhances phase separation in a manner dependent on the long-chain base and oligosaccharide composition. Cooling differential scanning calorimetry experiments showed that the ganglioside property to form aggregates within the membrane is independent of the initial physical state of the bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of major gangliosides from frog liver

Four major gangliosides isolated from frog liver were characterized by compositional analysis involving GLC and GC-MS, methylation analysis, chromium trioxide oxidation, and enzymatic hydrolysis. The results revealed that the most major ganglioside in the tissue was GM4 containing N-acetylneuraminic acid and the others were GM4 containing N-glycolylneuraminic acid, GD1a, and a fucosyl ganglioside which was tentatively assigned to be alpha-galactosyl alpha-fucosyl GM1. This is the first report describing the presence of GM4 containing N-glycolylneuraminic acid. The fatty acids in both GM4 were mainly alpha-hydroxylated, and those in the fucosyl ganglioside were exclusively nonhydroxy fatty acids. The GD1a contained both nonhydroxy and alpha-hydroxy fatty acids in a ratio of about 3:2. The predominant species were 22:0, 23:0, 24:0, and 24:1 in both species of the fatty acids. The long-chain bases of these four gangliosides consisted of C18-sphingosine and C18-phytosphingosine together with significant amounts of C16 to C19 dihydroxy and trihydroxy bases with iso and anteiso structures.     

Changes in the Ceramide Composition of Rat Forebrain Gangliosides with Age

Five major gangliosides (GM1, GD1a, GD1b, GT1b, and GQ1b) were extracted and isolated by normal-phase HPLC from the forebrain of Sprague-Dawley rats of ages ranging from 3 days to 24 months. Each ganglioside was fractionated by reverse-phase HPLC into the molecular species carrying a single long-chain base moiety. At all ages, the C18:1 and C20:1 long-chain base species predominated, whereas the C18:0 and C20:0 ones represented 1-3% of the total. The C18:1 long-chain base species, predominant at 3 days (91-96%), diminished with age and reached, at 2 years, 73%, 65%, 61%, 59%, and 45% of the total for GD1a, GM1, GT1b, GD1b, and GQ1b, respectively. The content of the C20:1 long-chain base species, low at birth (4-9%), increased with age in all gangliosides and reached, at 2 years, 27-55% of the total. The developmental behavior of the ganglioside species containing the C18:1 long-chain base was characterized by the following: (a) a biphasic profile with a maximum around 15 days for GD1a, the most abundant ganglioside at all ages; (b) an increase until 6 months for GM1; (c) a sharp decrease until 30 days, followed by leveling for GT1b; and (d) a low, constant level for GD1b and GQ1b. All the ganglioside species containing the C20:1 long-chain base showed a constant increase during development, the increase being more marked in the first 30 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tissue-specific expression of GM3(NeuGc) and GD3(NeuGc) in epithelial cells of the small intestine of strains of inbred rats. Absence of NeuGc in intestine and presence in kidney gangliosides of brown Norway and spontaneously hypertensive rats

The ganglioside composition of the epithelial cells of the small intestine was investigated in 15 strains of inbred rats. Most of these strains had GM3 as the only detectable ganglioside. In addition to GM3, small amounts of GD3 were found in four strains, AVN, BN, DA, and LE. The fatty acid content of the ceramide portion was composed of a large, although variable, percentage of 2-hydroxy fatty acids. The sphingoid base was always C18-4D-hydroxysphinganine. The highly prominent sialic acid was N-glycolylneuraminic acid (NeuGc) in most strains. However in two strains, Brown Norway (BN) and spontaneously hypertensive rats (SHR), NeuAc was the only sialic acid of the gangliosides of the intestinal epithelium. The analysis of the ganglioside composition of the epithelium of the small intestine of the first generation hybrids of SHR with DA and BN, respectively, demonstrated that the expressions of GM3 (NeuGc) and GD3 were genetically transmitted as dominant traits and that BN and SHR were likely to carry the same deficient gene that led to the expression of GM3(NeuAc) instead of GM3(NeuGc) in the small intestine. For comparison, the sialic acid composition of kidney gangliosides was analyzed in some strains. 21-23% of the kidney gangliosides was GM3(NeuGc) in all tested strains, including BN and SHR. Therefore, the ganglioside composition of the intestinal epithelium could vary in the rat species, and the defect of N-glycolylneuraminic acid was not only strain-specific but also occurred in a tissue-specific way among strains of inbred rats.     

Major gangliosides in normal and pathological human thyroids

The major gangliosides of human thyroids were extracted, purified and then analyzed by gas-liquid chromatography. In normal thyroid, GM3 and GD3 represented about 80% of lipid-bound sialic acid. GM3 contained more than 50% of long-chain fatty acids, whereas GD3 contained mostly short ones. 4D hydroxy sphinganine represented 20% of long-chain base content in both cases. In pathological thyroids (Graves' disease, cancer, toxic adenoma), GM3 represented about 60% of lipid-bound sialic acid; its fatty acid content was mostly short chain fatty acids, as in normal GD3. 4D hydroxy sphinganine proportion was decreased.     

Composition of gangliosides from ovine testis and spermatozoa

Gangliosides were extracted and purified from ovine testis and ejaculated spermatozoa which contained, respectively, 57 and 9 nmol lipid-bound sialic acid per gram wet weight. Fourteen gangliosides were resolved by thin-layer chromatography of testicular gangliosides, of which eleven were purified in sufficient quantity to enable a complete compositional analysis of the carbohydrate residues to be performed. None of the gangliosides contained fucose, but several contained N-glycolylneuraminic acid as a component of the sialic acid species. Relative migration on thin-layer chromatograms relative to known standards, compositional analysis, and selective degradation by specific enzymes were used as the basis for identification. Testis contained members of the ganglio series (GM1, GD1a, GD1b, GT1b, GQ1b), hematoside series (GM3, GD3), and sialosylparagloboside in the molar ratio of 54:40:6, respectively. Testicular GM3, GM1, GD3, GD1a, GD1b and GT1b ran as double bands on thin-layer chromatography which could be accounted for by observed differences in the fatty acid moiety. In addition, the slower migrating band of each pair contained some or all of its sialic acid residues as N-glycolylneuraminic acid, whereas the faster migrating band contained exclusively N-acetylneuraminic acid, except for GM3 where N-acetylneuraminic acid was the sole species in both bands. Thin-layer chromatography of sperm gangliosides revealed seven bands comigrating with equivalent testicular gangliosides. These coincided with the slower migrating bands of testicular GM3, GM1, GD3, GD1a, both bands of GD1b, and possibly both bands of GT1b. Sperm contained only trace amounts of sialosylparagloboside but, in addition, two unidentified bands which were absent from testis were also observed. The molar ratio of the ganglio series to the hematoside series in sperm was 42:58 with GM3 accounting for 42% of total gangliosides.     

Induction of GM1a/GD1b synthase triggers complex ganglioside expression and alters neuroblastoma cell behavior; a new tumor cell model of ganglioside function

Neuroblastoma is the most common extracranial solid tumor in children and tumor ganglioside composition has been linked to its biological and clinical behavior. We recently found that high expression of complex gangliosides that are products of the enzyme GM1a/GD1b synthase predicts a more favorable outcome in human neuroblastoma, and others have shown that complex gangliosides such as GD1a inhibit metastasis of murine tumors. To determine how a switch from structurally simple to structurally complex ganglioside expression affects neuroblastoma cell behavior, we engineered IMR32 human neuroblastoma cells, which contain almost exclusively (89%) the simple gangliosides (SG) GM2, GD2, GM3, and GD3, to overexpress the complex gangliosides (CG) GM1, GD1a, GD1b and GT1b, by stable retroviral-mediated transduction of the cDNA encoding GM1a/GD1b synthase. This strikingly altered cellular ganglioside composition without affecting total ganglioside content: There was a 23-fold increase in the ratio of complex to simple gangliosides in GM1a/GD1b synthase-transduced cells (IMR32-CG) vs. wild type (IMR32) or vector-transfected (IMR32-V) cells with essentially no expression of the clinical neuroblastoma marker, GD2, confirming effectiveness of this molecular switch from simple to complex ganglioside synthesis. Probing for consequences of the switch, we found that among functional properties of IMR32-CG cells, cell migration was inhibited and Rho/Rac1 activities were altered, while proliferation kinetics and cell differentiation were unaffected. These findings further implicate cellular ganglioside composition in determining cell migration characteristics of tumor cells. This IMR32 model system should be useful in delineating the impact of ganglioside composition on tumor cell function.     

Differences in liver ganglioside patterns in various inbred strains of mice

The ganglioside patterns in the liver of different inbred and hybrid strains of mice were investigated. The inbred strains were Balb/cAnNCr1BR, C57BL/6NCr1BR, DBA/2NCr1BR. C3H/HeNCr1BR; the hybrid strain was the Swiss albino. The following major gangliosides were found to be present in mouse liver: GM3-NeuAc; GM3-NeuGl, GM2 [a mixture of one species carrying N-acetylneuraminic acid (NeuAc) and one carrying N-glycollylneuraminic acid (NeuGl)], GM1 and GD1a-(NeuAc,NeuGl). The qualitative and quantitative patterns of liver gangliosides were markedly different in the various inbred strains of mice; in Balb/cAnNCr1BR strain, ganglioside GM2 was preponderant (99.2% of total ganglioside content); in C57BL/6NCr1BR, the major ganglioside was GM2 (90.4%), followed by GM3-NeuAc (5.6%) and GM3-NeuGl (4.0%); in DBA/2NCr1BR, GM2 accounted for 77.1%, GD1a-(NeuAc,NeuGl) 18.9% and GM1 3.1% of gangliosides; in C3H/HeNCr1BR, GM2 constituted 50.6%, GM1 22.8% and GD1a-(NeuAc,NeuGl) 22.1%. In the hybrid Swiss albino mice, liver ganglioside composition markedly varied from one animal to another, GM3-NeuGl, GM2 and GD1a-(NeuAc,NeuGl) being the predominant gangliosides in the various cases.     

Synthesis of lysogangliosides

The synthesis of gangliosides GM3, GM2, GM1, and GD1a solely lacking the fatty acid moiety, and thus called lysogangliosides in analogy to lysophospholipids, is described. Since a selective elimination of the fatty acid residue has not been achieved as yet, the gangliosides were first subjected to alkaline hydrolysis. By this procedure the fatty acyl as well as the acetyl groups of the sialic acid residue(s) were completely removed. The acetamido group of the N-acetylgalactosamine moiety of the gangliosides GM2, GM1, and GD1a was very little (congruent to 10%) hydrolyzed. In a two-phase system composed of water and ether, the selective protection of the sphingoid amino group was accomplished with a hydrophobic protective group (9-fluorenylmethoxycarbonyl). Lysogangliosides were obtained after re-N-acetylation of the sialooligosaccharide amino group(s) followed by removal of the protecting group. The overall yield was about 30%. The structures of the lysogangliosides were confirmed by chemical analysis as well as negative ion FAB mass spectrometry and 1H NMR spectroscopy. By simple re-N-acylation of lysogangliosides with any labeled fatty acid, labeled gangliosides are now obtainable that are identical with their parent gangliosides except for their labeled fatty acid residue. This has been demonstrated by the synthesis of GM1 with a [1-13C]palmitic acid moiety in its ceramide portion. If desired, double-labeled gangliosides may be obtained by use of labeled acetic anhydride in the synthesis of the lysogangliosides.     

Glycolipids of the bovine pineal organ and retina

Neutral and acidic glycolipids from the bovine pineal organ and neutral glycolipids from the bovine retina were characterized. The chemical structures of the isolated glycolipids were determined by means of carbohydrate analysis, methylation analysis, enzyme treatment, fatty acid analysis, long chain base analysis, mass spectrometry, NMR spectroscopy, and IR spectroscopy. GM3, GD3, and GT1 were the major bovine pineal organ gangliosides, GD3 accounting for 75% of the total gangliosides. Galactosylceramide, glucosylceramide, and lactosylceramide were found in both the bovine pineal organ and retina. Sulfatide was also present in both tissues. It had already been reported that the major bovine retina ganglioside was GD3 (Handa, S. & Burton, R.M. (1969) Lipids 4, 205-208). The glycolipid patterns of the two tissues were very similar to each other and quite different from those of other tissues.     

Specific gangliosides increase rapidly in rat liver following partial hepatectomy

Rat liver gangliosides (sialic acid containing glycosphingolipids) were analyzed by HPTLC and HPLC following either partial hepatectomy or sham operation. Analysis of whole liver gangliosides by HPTLC demonstrated that within 6 h after partial (68%) hepatectomy, there was a significant increase in GM1 compared to both sham and control animals. By 48 h, GM1 was further increased and the polysialylgangliosides GD1a, GD1b and GT1b had also risen significantly, whereas changes in GM3 were negligible. Gangliosides associated with the plasma membrane were increased up to 3.5-fold in regenerating liver compared to sham-hepatectomized controls as assessed by HPLC. Although elevations in membrane gangliosides were associated with hepatocyte proliferation, they did not closely follow the growth curve. The time course of changes in ganglioside biosynthesis suggests differential upregulation of GM3 synthase and GD3 synthase in regenerating livers.     

Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis of glycosphingolipids including gangliosides.

Long chain base compositions of gangliosides containing mainly stearic acid could be determined without any chemical modification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry with delayed ion extraction (DE MALDI-TOF MS). The analytical results for the long chain base compositions of various samples of GM1 from the brain tissues of patients with different diseases at different ages confirmed that the proportion of d20:1 (icosasphingosine) and d20 (icosa-sphinganine) of the total sphingosine bases increased quickly until adolescent or adult age and then remained constant slightly exceeding 50%; this value was evidently higher than the proportion of d20:1 and d20 of GM1 in various adult mammalian brains. A long chain base composition of GM1 from the brain tissue of a patient with infantile type of GM1-gangliosidosis at 4y2m was abnormal and so was in two sibling patients with Spielmeyer-Vogt type of juvenile amaurotic idiocy at 19y and 21y in spite of that in the latter there was no accumulation of GM1 in the brain tissue. On the other hand, a patient with adult type of GM1 gangliosidosis at 66y showed a local accumulation of GM1 in the putamen and caudate nucleus, but its long chain base composition was found to be normal. It was of interest that the white matter of Eker rat with hereditary renal carcinoma contained a large amount of plasmalocerebroside as compared with the amount of cerebroside and sphingomyelin. The individual molecular species of plasmalocerebroside were identified by DE MALDI-TOF MS.     

Gangliosides of human thyroid gland

The ganglioside composition of adult human thyroid gland was examined in autopsy material obtained from patients who died of circulatory diseases but who showed no signs of thyroid disorders. The concentrations of phospholipids, cholesterol and gangliosides (lipid-bound sialic acid) in the whole glands were 5.2, 4.3 and 0.12 mmol/kg fresh tissue weight and, in dissected follicular material, 7.0, 3.4 and 0.24 mmol/kg tissue, respectively. The molar ratio of phospholipids/cholesterol/gangliosides in the follicular material was 1.00:0.49:0.034. Twelve molecular species of gangliosides were isolated and identified. Gangliosides GM3 and GD3 were most abundant, but GD1a, GD1b, GT1b and 3'-LM1 were also present in quantities greater than 5% of the total gangliosides. N-Acetylneuraminic acid and an alkali labile sialic acid, probably N-acetyl-9-O-acetylneuraminic acid, were found to occur in human thyroid.