Enhanced thin-layer chromatographic separation of GM1b-type gangliosides by automated multiple development

Enhancement in separation of gangliosides on silica gel precoated high-performance TLC plates has been obtained by automated multiple development chromatography. A less polar mixture of the standard solvent chloroform-methanol-20 mM aqueous CaCl₂ (120:85:20, v/v) was used. Lowering the water content achieved separation of two complex monosialoganglioside fractions, isolated from murine YAC-1 T lymphoma and MDAY-D2 lymphoreticular cells. Three-fold chromatography in the solvent chloroform-methanol-20 mM aqueous CaCl₂ (120:85:14, v/v) resulted in TLC separation of G_M1b-type gangliosides, substituted with C₂₄ and C₁₆ fatty acids and with Neu5Ac and Neu5Gc as well, which could not be achieved by undirectional standard chromatography. Compared to conventional single chromatography, the technique described allows high-resolution separation of extremely heterogenous ganglioside mixtures and offers a convenient tool for both analytical and preparative TLC.     

A thin-layer chromatographic method for separation of thymidine and deoxyuridine nucleotides

A thin-layer chromatographic method for the separation of thymidine and deoxyuridine nucleotides and nucleosides is described. This procedure involves the following sequence of steps: (i) Ion-exchange thin-layer chromatography to afford separation into fractions of increasing degree of phosphorylation, (ii) conversion of each fraction into an equivalent mixture of thymine and uracil through the combined actions of alkaline phosphatase and thymidine phosphorylase, and (iii) partition thin-layer chromatographic separation of thymine and uracil. A key feature of the method is the specificity afforded by the second step which converts only thymidine and deoxyuridine nucleotides and nucleosides to the corresponding pyrimidine bases. An application of the method to the study of [³H]deoxyuridine metabolism in L1210 cells, as well as the effect of methotrexate on this metabolism is also described.     

A new partition thin-layer chromatographic method for steroid separations

TLC plates with a 25 mu thick polyamide stationary phase were modified for the separation of neutral steroids by impregnation with propylene glycol. A mixture of tritiated 5 alpha-androstan-3 alpha,17 beta-diol, testosterone, 17 beta-hydroxy-5 alpha-androstan-3-one and 4-androstene-3,17-dione was applied to the plate and developed in a toluene mobile phase to a height of 13.6 cm. This resulted in complete resolution of the 4 compounds as detected by a gas flow scanner or imaging analyzer. Cutting and elution of peak areas with methanol resulted in quantitative recovery of all four steroids. The thinness of the layer also permitted a 3-5% counting efficiency on scanning, resulting in good quantitation of recovery without liquid scintillation counting. The high sorptive capacity of the polyamide layer also enabled extracts of normal human serum to be defatted on the TLC plate by development with pure hexane prior to the toluene step. The new method thus offers several advantages over existing methods for steroid separations and should be adaptable to separations of other relatively non-polar compounds.     

Changes of brain gangliosides in chicken and mice during heterothermic development

Developmental profiles of brain gangliosides of chicken and mice were correlated to thermo-biological parameters. In parallel with an increasing body temperature and resistance to cold stress (within the first 11 postnatal days) the brain gangliosides change to a less polar pattern, indicated by a higher proportion of oligosialogangliosides.     

A two-dimensional thin-layer chromatographic procedure for the estimation of plasmalogens

1. The use of two-dimensional thin-layer chromatography is described that allows the rapid and simultaneous determination of phospholipid classes and their constituent plasmalogens. 2. The method is based on the specific hydrolysis of plasmalogens to (2-acyl) lysophospholipid in the presence of a mercuric chloride spray reagent. 3. The proportion of mercuric chloride-labile phospholipid present in each phospholipid class, calculated on the basis of phosphorus recoveries from the charred chromatogram, was compared with the proportion of long-chain aldehyde and of total lipid phosphorus found in small-scale preparations of each class of phospholipid. 4. The method permits the determination of individual plasmalogens on preparations containing as little as 0.2mug.atom of total lipid phosphorus.     

A new reversed phase, paired ion thin-layer chromatographic method for steroid sulfate separations

The sulfoconjugated steroids estrone sulfate (ES) and dehydroepiandrosterone sulfate (DS) were separated in the reversed phase mode on polyamide-coated TLC plates. Baseline resolution was obtained between tritiated ES and DS standards when run with a mobile phase of 20% acetonitrile in 5mM aqueous triethylamine, triethanolamine, tris-hydroxymethylaminomethane, tributylamine or ammonia. ES and DS showed no mobility in the absence of an ion-pair reagent. The radioactive peaks were detected and integrated non-destructively by scanning. Quantitation was confirmed by elution of cut-out peak areas and liquid scintillation counting. Similar results were obtained with washed ethanol extracts of serum labeled with tritiated ES and DS. The extracts were defatted on the plate with hexane: ethyl acetate (1:1) prior to the reversed phase development.     

The content and composition of gangliosides in brain subcellular fractions of the frog Rana temporaria

Ganglioside distribution in various frog brain subcellular fractions (myelin, microsomes, mitochondria, synaptosomes, plasma membranes of nerve endings and synaptic vesicles) was investigated. The synaptosomes and plasma membranes of nerve endings were found to be the main places of ganglioside localization, ganglioside concentration being 2.42 and 1.79 times higher than that in homogenates. Gangliosides were shown to be present in synaptic vesicles. The characteristic features of gangliosides from frog brain and its subcellular fractions are the predominance of polysialogangliosides with 3-5 sialic acid residues (up to 57.4%), low content of monosialogangliosides (not more than 7%) and the presence of disialogangliosides with short carbohydrate chain. The increase of ganglioside content per one nerve cell during phylogenetic development of vertebrates is discussed.     

Gas--liquid chromatographic assay of lipid-bound sialic acids: measurement of gangliosides in brain of several species

A method is described for analysis of gangliosides by GLC assay of the sialic acid component. Mild acid treatment in methanol converted the latter to methyl ketoside methyl ester, which was then chromatographed as the TMS derivative. The major methanolysis product was shown to be the beta-anomer, and its chromatographic peak was used for quantification. NANA and NGNA could be analyzed simultaneously, while an O-acetylated derivative of NGNA was detected qualitatively. Standard curves were obtained for the three following representative samples: (a) a mixture of beef brain gangliosides, (b) Tay-Sachs ganglioside, and (c) hematoside-NANA. These had different slopes which reflected the variation in yield of beta-NANA obtained from methanolysis. The smallest sample analyzed in the present study contained 0.3 micro g of NANA. The advantage of GLC in solving the problem of false chromogens is illustrated in a comparative study with two colorimetric procedures. Two columns are described whose combined use is highly effective in establishing identity and in eliminating false peaks when they arise. The GLC method has been applied to analysis of the total brain ganglioside content of several species, and a general trend was observed toward decreasing levels in the lower vertebrates. In addition, NGNA was detected and quantified in several of these samples.     

A thin-layer chromatographic method for the quantitative separation and estimation of S-adenosylmethionine and S-adenosylethionine in rat liver

A procedure is described for the simultaneous isolation, separation, and quantitation of S-adenosylmethionine (AdoMet) and S-adenosylethionine (AdoEt) in rat liver. These compounds are isolated by precipitation with ammonium reineckate, are separated by thin-layer chromatography, and are quantitated by an isotope dilution determination. The procedure was tested with commercial standards added to liver homogenate ranging up to 160 μg AdoMet/g liver and 173 μg AdoEt/g liver; at all of the concentrations tested, the recoveries were linear and accurate. AdoMet recoveries were linear in the presence of 11.6 or 307 μg AdoEt/g liver, and AdoEt recoveries were linear in the presence of 37.8 or 191 μg AdoMet/g liver. AdoMet and AdoEt levels were measured in the livers of rats fed diet containing 0 or 0.3% dl-ethionine for 2 weeks. In the ethionine-treated animals, AdoMet concentrations were lower than in the controls; and, conversely, AdoEt increased from 0 to 259 μg/g liver.     

A novel heptasialosyl c-series ganglioside in embryonic chicken brain: its structure and stage-specific expression

A ganglioside of unknown structure (ganglioside X) was purified from chicken brain at embryonic day 12 (E12) and characterized for its structure. Ganglioside X was reactive with a monoclonal antibody A2B5 and migrated below GH1c on thin-layer chromatography (TLC). Extensive treatment of ganglioside X with Clostridium perfringens sialidase produced a single ganglioside product. This ganglioside was identified as GM1 based upon its chromatographic mobility and reactivity to cholera toxin B subunit and anti-GM1 antibody. Partial hydrolysis of ganglioside X by sialidase generated several degradation products including GH1c, GP1c, and GQ1c. Electrospray ionization (ESI)-mass spectrometry (MS) of the permethylated derivative of ganglioside X produced a triple-charged parent ion peak at m/z 1355, which corresponded with the gangliotetraose oligosaccharide structure having seven sialic acids and ceramide with the molecular mass of 566 (as non-methylated form). Collision-induced dissociation (CID)-MS(2) showed fragment ions including those at m/z 1066 and 1931; these two ions matched the structures of (NeuAc)(3)-Gal-Glc-Cer and (NeuAc)(4)-Gal-GalNAc, respectively. These structures were confirmed by CID-MS(3) of the corresponding peaks. Based upon these findings, the structure of ganglioside X was identified as NeuAc-NeuAc-NeuAc-NeuAc-Galbeta1-3GalNAcbeta1-4(NeuAc-NeuAc-NeuAcalpha2-3)Galbeta1-4Glcbeta1-1'Cer. This ganglioside was designated as GS1c. A developmental study demonstrated that GS1c was expressed in chicken brain during a period from E6 to E13 and thereafter decreased rapidly in its concentration. The present study suggests that GS1c may play a specific role in early development of chicken brain.     

Cell-to-cell contacts in primary cultures of dissociated chicken embryonic brain

Summary In the elasmobranch fish, Scyllium stellare, a complex group of cells protrudes into the cavity of the mesencephalic ventricle of the optic tectum. It consists of six to seven large spherical perikarya which resemble neurons of the mesencephalic nucleus of the Vth cranial nerve. The bundled processes of these cells form a stalk connecting the protrusion with the brain tissue. The protrusion is located in the region where the mesencephalic ventricle joins the cerebral aqueduct. This complex was not found in all specimens examined in the present study. The functional role of this peculiar group of cells, which contain dense core granules and are bathed in the cerebrospinal fluid, is open to discussion.