Jorma Kärkkäinen

Analysis of disaccharides as permethylated disaccharide alditols by gas—liquid chromatography—mass spectrometry

Abstract Gas-liquid chromatography (g.l.c.) and combined gas—liquid chromatography—mass spectrometry (g.l.c.—m.s.) have been used for the analysis of disaccharides containing simple hexoses and amino sugars. The disaccharides were reduced to the corresponding alditols with borodeuteride, methylated, and separated by g.l.c. The molecular weight of the monosaccharide units and the position of the glycosidic linkage could be determined on the basis of the mass spectra. Differentiation of (1→4)- and (1→3)-linked alditols was not possible without deuterium labelling, whereas (1→6)-linked alditols could be distinguished by the presence of a fragment containing carbon atoms 1–4 of the alditol moiety. The glycosidic linkage was cleaved by methanolysis after isolation of the disaccharide alditol methyl ethers by preparative g.l.c., and the products were identified by g.l.c.—m.s. Methylation (methyl iodide in the presence of methylsulphinyl carbanion) of disaccharide alditols containing N -acetylated amino sugars resulted in the formation of a derivative in which the nitrogen atom carried both a methyl and an acetyl substituent.

Neutral and acidic glycopeptides in adult and developing rat brain

Abstract 1. 1. Glycopeptides were released from the lipid-free residue of rat brain by proteolytic digestion. Glycosaminoglycans and nucleic acids were removed from the digest by precipitation with cetyl pyridinium chloride. The glycopeptides were purified in a quantitative yield by gel filtration. 2. 2. A simple method was developed for fractionation of the glycopeptides by ion-exchange chromatography. Two distinct groups of glycopeptides were separated. 3. 3. The glycopeptide groups were characterized by gel filtration, ion-exchange chromatography and gas-liquid chromatographic analysis. The two groups of glycopeptides differed clearly in molecular size and sugar composition. The neutral glycopeptides, which account for 45% of the glycopeptide carbohydrate in rat brain, mostly contain mannose and N- acetylglucosamine in a ratio of 5:2, and have an average composition of 9–12 sugar residues per molecule. About 55% of the glycopeptide carbohydrate is found in the acidic glycopeptides, which contain fucose, mannose, galactose, N- acetylglucosamine , N- acetylgalactosamine and N- acetylneuraminic acid. The acidic glycopeptides, on average, are composed of 15–20 sugar residues per molecule. 4. 4. Qualitative and quantitative changes in the sugars composing the neutral and acidic glycopeptides and glycosaminoglycans were followed during the postnatal development of the rat brain between the ages of 0 and 60 days. There was a rise of about 50% in the glycopeptide content during the first four weeks to the adult level. The increase in the acidic glycopeptides was less great than in the neutral glycopeptides. N- acetylneuraminic acid and N- acetylgalactosamine differed from all other glycoprotein sugars in that their levels were highest between the ages of five and ten days. The amount of glycosaminoglycans decreased during development. This decrease was for the most part due to hyaluronic acid.

Determination of the structure of disaccharides as O-trimethylsilyl derivatives of disaccharide alditols by gas-liquid chromatography-mass spectrometry

A method is described for the structural analysis of disaccharides by gas-liquid chromatography (g.l.c.)-mass spectrometry. A disaccharide mixture is reduced to the corresponding alditols, and analyzed as the O-trimethylsilyl derivatives by g.l.c. Simultaneous recording of the mass spectra allows the determination of the position of the intermolecular bond, the molecular weight, and certain other structural details. The use of borodeuteride instead of borohydride in the reduction stage is essential for the interpretation of the mass spectra. The monosaccharide components are identified by g.l.c. after isolation of the O-trimethylsilyl derivatives of the disaccharide alditols by small-scale, preparative g.l.c. The method is applied to the analysis of disaccharides containing both simple and amino sugars.

Methylation analysis of neuraminic acids by gas chromatography-mass spectrometry.

The permethylated methyl glycoside methyl esters of N-acetyl- and N-glycolyl-neuraminic acids and of N-acetylneuraminic acid 8-acetate have been analyzed by g.1.c.-mass spectrometry. Fragmentation of the neuraminic acid derivatives in electron-impact mass spectrometry has been studied by deuterium labelling. The results were applied in the methylation analysis of neuraminic acids from gangliosides of brain and kidney.

Methylation techniques in the structural analysis of glycoproteins and glycolipids.

Publisher Summary This chapter describes the methylation techniques used in the structural analysis of glycoproteins and glycolipids. The methylation reaction can be directly applied to the analysis of glycolipids, whereas glycoproteins are not generally methylated directly due to their low solubility in dimethyl sulfoxide. The carbohydrate moiety of the glycoprotein is first isolated as a glycopeptide after extensive proteolytic digestion, or as a reduced oligosaccharide after treatment with alkaline sodium borohydride. Completeness of the methylation reaction is a prerequisite for successful methylation analysis. The critical step in the permethylation procedure is the generation of the sugar alkoxides catalyzed by methylsulfinyl carbanion. It is to be expected that the extensive formation of the alkoxide species has occurred if an excess of the carbanion can still be detected in the mixture after the carbohydrates have reacted. Commercial tert -butoxide salt can be dissolved in dimethyl sulfoxide without any further steps. When this method is used, the intensities of the nonsugar signals in gas liquid chromatography (GLC) tend to be lowering than those seen when the reagent is prepared with the aid of sodium hydride. Methanolysis is also preferred in the analysis of N -acetyl- and N -glycolylneuraminic acid residues, which are commonly found in animal glycolipids and glycoproteins. The quantitation of the methylated monosaccharides is also elaborated in the chapter.

Characterisation of 2-amino-2-deoxy-d-glucose, 2-amino-2-deoxy-d-galactose, and related compounds, as their trimethylsilyl derivatives by gas-liquid chromatography-mass spectrometry

Abstract Combined gas-liquid chromatography-mass spectrometry (GC-MS) has been used for the analysis of 2-amino-2-deoxy- d -glucose, 2-amino-2-deoxy- d -galactose, their methyl glycosides, and the corresponding alditols, as well as of the N -acetylated forms. The compounds were analyzed as trimethylsilyl (TMS) derivatives synthesized by using chlorotrimethylsilane and hexamethyldisilazane in pyridine, or using these reagents together with bis(trimethylsilyl)acetamide. In the latter procedure, the free amino groups, in addition to the hydroxyl groups, are also trimethylsilylated. Identification of the derivatives was based on their retention times in gas-liquid chromatography and their mass-spectral characteristics. The value of GC-MS of TMS derivatives in the analysis of polysaccharides is discussed.

Quantitative determination of neuraminic acid by gas chromatography — mass spectrometry

Neuraminic acid is widely distributed in nature, occurring generally as a component of the oligosaccharide units of glycoproteins, gangliosides and of the milk and urinary oligosaccharides. Increased attention has recently been paid to its presence in the membranes of normal and malignant cell surfaces. Several methods have been developed for the determination of neuraminic acid; most of them are calorimetric [l-3] . Gas chromatographic [4,5], radioisotopic [6] and enzymatic [7] methods have also been described. These methods, however, require relatively pure samples and estimation of amounts under 1 pg from samples of biological origin is often difficult. In the present work neuraminic acid is determined with gas chromatography-mass spectrometry using the multiple-ion-detection technique. The compound is liberated by methanolysis, re-iV-acetylated and trimethylsilylated before gas chromatographic analysis. A derivative labelled with deuterium in the methyl groups is used as an internal standard. For quantitation by mass fragmentography two ions (m/e 298 and m/e 420) are recorded, which makes the method highly specific. The method allows an accurate determination of neuraminic acid from 1 ng (3.3 pmdl) up to micrograms. As far as the authors are aware, no similar method for the determination of a monosaccharide has previously been described. The method was applied to the analysis of neuraminic acid in a crude protein fraction from rat brain.

Urinary excretion of free bufotenin by psychiatric patients

According to the methylation hypothesis of mental disorders, the human body is capable of producing toxic methylated substances that play a part in the patbogenesis and symptomatology of psychiatric disease. Earlier attempts to show that the methylated indoleamines bufotenin (N,Ndimethylserotonin) and DMT (N.N-dimethyltryptamine) are present in increased quantities in schizophrenia (Wyatt et al. 1971) were largely hampered by difficulties in analytical methodology. These indoleamines can now be determined reliably by gas chromatography-mass spectrometry (GC-MS) (Walker et al. 1973; Riiisanen and K&&k&en 1979). Using GC-MS, we have shown that bufotenin is also excreted into urine in healthy humans as a free form and as a conjugate (R%istien and K&rkkainen 1979; Rg;isanen 1984). Comparable levels have also been found by liquid chromatography (Riceberg and Van Vunakis 1978; Sitaram et al. 1983).

Plasma renin substrate and oestrogens in normal pregnancy

Relationship between concentrations of serum oestrogens, plasma renin substrate and plasma renin activity were studied in six women throughout pregnancy. There was a significant positive correlation between serum oestradiol-17 beta and plasma renin substrate concentrations (r=0.60). Serum oestriol concentrations also correlated significantly with plasma renin substrate concentrations (r = 0.68). Correlation coefficients calculated separately for each subject throughout pregnancy were higher than those for the whole group. Also, there was much individual variation in dose-response of serum oestrogens to plasma renin substrate concentrations. There was no significant correlation between serum oestrogens and plasma renin activity. Our results support the view that oestrogens cause the increase in plasma renin substrate concentration during pregnancy, and emphasize the individual variation in response of renin substrate concentration to serum level of oestrogens.

Plasma endogenous opioids and dexamethasone suppression test in depression

Plasma levels of beta-endorphin plus beta-lipotropin were determined in 35 hospital patients with depression and in 23 controls before and after administration of 1 mg of dexamethasone (dxm). Dxm suppressed opioid secretion in both groups. The opioid levels of the patients were significantly higher than those of the controls both before and after dxm. All the controls were cortisol suppressors. Among the patients the post-dxm opioid levels of cortisol nonsuppressors (n = 14) were higher than those of cortisol suppressors (n = 21). A significant correlation between the opioid and cortisol levels was found in the patients. There was a significant association between the use of neuroleptics and high opioid levels, but the difference between the patients and the controls was not explained by the effect of any single class of drugs. The results support the concept of hypersecretion of corticotropin-releasing factor in depression.