High-performance thin-layer chromatography and densitometric determination of brain ganglioside compositions of several species.
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Biomedical subjects
Publications and source records attributed to S Ando.
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We reported the case of a 40-year-old man who was hospitalized to our department on November 16, 1975 with a year history of neuralgia in the saddle region and vesicorectal dysfunction. Examination of CSF on lumbar puncture at L4-5 revealed xanthochromia with a protein content of 2,560 mg/dl. Myelography revealed a tortuous filling defect at the level of L2 coupled with complete blockade at L3 level. Selective spinal angiography of the left 12th intercostal artery demonstrated the enlarged Adamkiewicz's artery. This blood vessel was found to enter a diffuse uniform density, where no distinct blood vessels were visible. Within one second after the injection of contrast agent an efferent vein appeared from the right side of the density, ascended to the level of Th12 and then, upon turning, descended to the pelvic canal. These findings suggested a hemangioblastoma of the spinal cord. Laminectomy was performed at levels of L2 through L4 and a hemangioblastoma of the cauda equina was totally excised at L3 level. Although the postoperative course was complicated by a transient dysuria and hypesthesia at S1-S5 levels, complete cure was achieved in a month. In the present paper clinical features and neuroradiological examination of a hemangioblastoma of the spinal cord were described.
The monosialoganglioside hematoside (GM3) is an important precursor in the synthesis of the more complex gangliosides. To obtain large quantities of GM3 for use in ganglioside biosynthetic experiments, we have devised a column chromatographic procedure for the isolation and purification of GM3 from human liver. A total ganglioside mixture was obtained from a 550 g sample of normal human liver. Quantitative analysis by gas-liquid chromatography indicated about 66 microgram of lipid-bound N-acetylneuraminic acid per gram of fresh tissue. Hematoside appeared as double bands on thin-layer plates and represented 86.4% of the total sialic acid content. Additional ganglioside species, notably GD3, were also seen. Hematoside was separated from the other gangliosides by GM3 obtained was 120 mg, which represented a 90% recovery. Although the long-chain base and sugar compositions of the upper and lower GM3 fractions were similar, striking fatty acid differences were detected. The upper fraction contained predominantly unsubstituted fatty acids, while the lower fraction showed a preponderance of alpha-hydroxy fatty acids. The unsubstituted fatty acid and hydroxy fatty acid composition of the total GM3 fraction was calculated to be 56.9% and 43.1%, respectively.
Gangliosides were isolated from whole spinal cords and cord myelin of human, cat, and rabbit by a revised methodology. The method included the sequential application of DEAE-Sephadex column chromatography, base treatment, Sephadex G-50 column chromatography, and finally Iatrobeads column chromatography. The human whole spinal cord was found to contain about one-tenth of the ganglioside concentration as in cerebral gray matter and about one-third of that in cerebral white matter. Low levels of gangliosides were also found in cat and rabbit whole cords. Only N-acetyl neuraminic acid could be detected in the ganglioside fractions of all three species. The whole cords also possessed unique ganglioside patterns when compared with the patterns of cerebral tissues. The most prominent and consistent features were the reduced concentration of Gd1a and increased amounts of Gm3 and Gd3. Human, but not cat and rabbit, spinal cord also contained Gm4 as one of the major gangliosides. Myelin prepared from the spinal cords of all three species also contained gangliosides. The amounts were only about half of those in the respective cerebral white matter myelin. The cord myelin ganglioside pattern was generally similar to the cerebral white matter myelin within the same species. Gm1 was the most abundant ganglioside in the cord myelin. Gm4 was found to be highly enriched only in myelin prepared from human sources.
A novel trisialoganglioside has been isolated from normal adult human brain in a yield of 0.6% of the total gangkioside. By graded neuraminidase treatment, mild acid hydrolysis and periodate oxidation analysis, the ganglioside was identified as GT1a having the following structure: NeuAc(alpha, 2-8)NeuAc(alpha, 2-3)Gal(beta, 1-3)GalNAc(beta, 1-4) [NeuAc(alpha, 2-3)]Gal(beta, 1-4)Glc(1-1)ceramide.
Chemical ionization (CI) mass spectra with isobutane and ammonia for the oligosaccharides obtained from sphingoglycolipids were compared with their electron impact (EI) mass spectra. The oligosaccahride moieties were liberated from the parent glycolipids and were further reduced with sodium borohydride. They were analyzed as their permethyl peracetyl and pertrimethylsilyl derivatives. In the CI spectra, peaks corresponding to QM+ and/or [M-59]+ were observed in all of the peracetylated oligosaccharides examined. In CI with ammonia as the reagent, H+ was transferred to nitrogen-containing saccharides to produce [MH]+ and NH4 was transferred to nitrogen-free saccharides to yield [M+NH4]+ as QM+. Non-reducing ends yielded very intense peaks in CI spectra. On the other hand, the reduced end, glucitol, produced rather prominent peaks in EI spectra. Fragment ions due to cleavage of glycosidic bonds were major ones under the CI conditions, and they could be used for elucidating the sugar sequence in the oligosaccharides. An additional characteristic feature in the CI spectra was that ions due to scission of hexosaminyl glycosidic linkages were observed with very high intensities.
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The effect of methysergide (MES), a serotoninergic antagonist on prolactin (PRL) blood levels was studied in five hyperprolactinemic amenorrheic women. The drug was administered for five days at a daily dosage of 11.2 mg. MES decreased significantly the PRL blood levels in all subjects (p less than 0.01). Since the MES has been shown to have antiserotoninergic effects and since serotonin has been thought to be involved in the control of PRL release, the effects of MES in lowering PRL might be due to a decrease of serotonin tone.
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A new high-resolution preparative column chromatographic system was developed for efficient and rapid isolation of ganglioside molecular species. The system involved a combination of ion-exchange and adsorption chromatographies using DEAE-Sephadex A-25 and the newly developed, totally porous silica spheres, Iatrobeads. Using this system the brain gangliosides, GM1, GD1a, GD1b and GT1 were obtained in high purity and in milligram amounts, in a relatively short time, by simple procedures. The presence of a number of unidentified molecular species of gangliosides, which are present only in small amounts, was also demonstrated.
Good preparative separation of neutral glycolipids of human erythrocytes was achieved by column chromatography using the totally porous silica spheres, Iatrobeads. The solvent flow through the Iatrobeads column was rapid under atmospheric pressure, and so elution bands migrated with minimal diffusion in the column. Ceramide dihexoside and ceramide trihexoside were both separated into two fractions of different molecular species on the Iatrobeads column. Two ceramide tetrasaccharides, globoside I and paragloboside, were also clearly separated on the Iatrobeads column. Chemical analysis showed that separation of these molecular species was due to differences in their fatty acid and long chain base compositions. Sphingadienine and phytosphingosine were found as minor components of long chain bases in human erythrocyte glycolipids.
A novel glycolipid containing N-acetylglucosamine (lacto-N-triose II ceramide, Amino CTH-I) was isolated as a minor component from the ceramide trihexoside fraction of human erythrocyte lipids. This glycolipid was purified by column chromatography and preparative high-speed liquid chromatography on Iatrobeads. Its structure was found to be N-acetylglucosaminyl(beta 1-3)galactosyl(beta 1-4)glucosyl-(beta 1-1)ceramide, so it is suggested that it may be a precursor of paragloboside and blood group-active glycolipids.
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