Specificity of mild periodate oxidation of oligosaccharide-alditols: relevance to the analysis of the core-branching pattern of O-linked glycoprotein oligosaccharides.

The specificity of mild periodate oxidation of 3- and 6-substituted 2-acetamido-2-deoxy-D-galactitols and 4- and 6-substituted D-glucitols has been investigated. The products were reacted with 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine and the derivatives were analysed by application of liquid secondary-ion mass spectrometry directly to the TLC plates. There was > 95% specificity of cleavage of the C-4-C-5 bond (threo-diol) for the GalNAcol derivatives. The major sites of oxidation for the Glcol derivatives also involved threo-diols. For alpha-Neu5Ac-(2-->6)-GalNAcol, approximately 30% of the products of oxidation involved the sialic acid side chain, and approximately 60% were cleaved at the C-4-C-5 bond of the GalNAcol moiety. The mild periodate oxidation reaction forms part of a strategy for determining the patterns of branching of the cores of O-linked glycoprotein oligosaccharides and other oligosaccharide-alditols.

Characterisation by mass spectrometry and 1H-NMR of novel hexasaccharides among the acidic O-linked carbohydrate chains of bovine submaxillary mucin.

The acidic oligosaccharide alditols released from bovine submaxillary-gland mucin by Carlson degradation were investigated by a combination of liquid secondary-ion mass spectrometry, methylation analysis and 1H-NMR. Among the largest structures identified were four branched hexasaccharides, three of them novel, comprising two separate pairs of structures. One pair contained the sequence Fuc(alpha 1-2)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-) (Fuc, L-fucose), at C3 of N-acetylgalactosaminitol and differed only by substitution at C6 by N-acetylneuraminic or N-glycolylneuraminic acid. The other pair also differed in substitution of the sialic acid linked at C6 and contained the GalNAc-(alpha 1-3)[Fuc(alpha 1-2)]Gal(beta 1-4)GlcNAc(beta 1-), sequence at C3 of N-acetylgalactosaminitol. The Lewis(y) and blood-group-A determinants of these sequences have not been found previously in the acidic oligosaccharides of bovine submaxillary-gland mucin, although they have recently been characterised in the neutral chains of bovine submaxillary-gland mucin.

Neutral oligosaccharides of bovine submaxillary mucin. A combined mass spectrometry and 1H-NMR study.

Twenty-two neutral O-linked oligosaccharides ranging from monosaccharides to octasaccharides were identified in bovine submaxillary-gland-mucin glycoprotein by a combination of liquid secondary-ion mass spectrometry, methylation analysis and 1H-NMR. Only five of these have been previously detected in bovine submaxillary-gland mucin although several have been described from other sources of mucin. The structures include short linear sequences 3-linked to N-acetylgalactosaminitol (GalNAcol) and branched structures based on either a GlcNAc(beta 1-6) [Gal(beta 1-3)]GalNAcol or GlcNAc(beta 1-6)[GlcNAc(beta 1-3)]GalNAcol core region. Oligosaccharides not previously characterised from any source were the disaccharide GalNAc alpha 1-6GalNAcol (GalNAc, N-acetylgalactosamine and the hexasaccharide GlcNAc(beta 1-6) [GalNAc(alpha 1-3)( Fuc (alpha 1-2)]Gal(beta 1-4)GlcNAc(beta 1-3)]GalNAcol (Fuc, L-fucose). Oligosaccharides of the blood-group-A type have not been detected previously in bovine submaxillary-gland mucin although their occurrence on bovine gastric-mucosal glycoproteins has been established by classical immunochemical studies.

Oligosaccharide sequence determination using B/E linked field scanning or tandem mass spectrometry of phosphatidylethanolamine derivatives.

Product ion mass spectral data of [M + H]+ ions of oligosaccharides, mainly tetra- and pentasaccharides, as their dipalmitoyl phosphatidylethanolamine derivatives were obtained using both liquid secondary ion mass spectrometry with B/E linked scanning and fast atom bombardment ionization with collision-induced dissociation/tandem mass spectrometry. Both methods give similar positive product ion spectra of equivalent high sensitivity (detection limits of approximately 50 pmol) that principally contain glycosidic cleavage ions retaining the reducing end of the molecule from which monosaccharide sequence can be deduced. A series of ions from fission of the phosphate ester bond together with glycosidic cleavage are present in the tandem mass spectra and B/E linked scan spectra when helium collision gas is used. Monosaccharide linkage position of isomeric molecules is reflected in the intensity of glycosidic fragmentation, without retention of the oxygen atom, with decreasing cleavage in the order 1-3 greater than 1-4 greater than 1-6 linkage. Fucose and N-acetylhexosamines show an increased degree of fragmentation over hexose sugars. The application of product ion spectra of derivatized oligosaccharides is demonstrated for characterizing mixed samples and also the acquisition of spectra directly from the silica surface of high-performance thin-layer chromatography plates.

High-sensitivity structural analyses of oligosaccharide probes (neoglycolipids) by liquid-secondary-ion mass spectrometry.

The sensitivity of detection and extent of information on structure obtainable by liquid-secondary-ion mass spectrometry (l.s.i.-m.s.) of neoglycolipids on the conventional target probe and directly from the surface of silica plates following t.l.c. has been assessed. Neoglycolipids were derived from malto-oligosaccharides, chitin oligosaccharides, and a range of deoxyhexose-, hexose-, 2-acetamido-2-deoxyhexose-, and sialic acid-containing mammalian oligosaccharides by reductive amination using phosphatidylethanolamine dipalmitoate (PPEADP). Sub-pmol amounts of the maltopentaose-PPEADP derivative applied directly to the target probe provided information on molecular weight, whereas approximately 1 pmol was required when analysed on the silica gel t.l.c. plate. With a biantennary octasaccharide derivative, the sensitivity of detection was 20-50 times lower and the other oligosaccharides had intermediate sensitivities. Information on composition and sequence was obtained readily from fragment ions, using 5 pmol of the maltopentaose derivative and 50 pmol of the octasaccharide derivative on the target probe, and 50 and 200 pmol, respectively, on the silica gel chromatogram. The optimised conditions formed the basis for characterising the structures of the components of mixtures of oligosaccharides generated from glycoproteins.

Characterisation by mass spectrometry and 500-MHz proton nuclear magnetic resonance spectroscopy of penta- and hexasaccharide chains of human foetal gastrointestinal mucins (meconium glycoproteins).

Structural studies using liquid secondary ion mass spectrometry, gas liquid chromatography/mass spectrometry and 500-MHz 1H NMR are described of the major penta- and hexasaccharides of a fraction of human foetal gastrointestinal mucins. Glycoproteins from a blood group H active meconium pool were studied after depletion of Ii antigenic activities by immunoaffinity chromatography and treatment with mild acid hydrolysis to reduce the chain heterogeneity. Oligosaccharides were released by mild alkali/borohydride degradation and purified by Bio-Gel P4 chromatography and HPLC. Eleven penta- and hexasaccharides have been fully characterised as a result of this study and one previous report [Hounsell et al. (1988) Biochem. J. 256, 397-401] and information obtained on additional oligosaccharides present in small amounts. These oligosaccharides show the following features: (table; see text) Sequences in these oligosaccharides not commonly found in mucins so far studied are chain-terminating GlcNAc alpha 1-4Gal, repeating-type-I (Gal beta 1-3GlcNAc) backbones, the backbone branch GlcNAc beta 1-6(GlcNAc beta 1-3)Gal and the backbone sequence GlcNAc beta 1-6Gal beta 1- in the absence of a substituent at C3 of galactose.

Identification of a novel oligosaccharide backbone structure with a galactose residue monosubstituted at C-6 in human foetal gastrointestinal mucins.

An oligosaccharide purified from a major penta- to hexa-saccharide fraction of human meconium glycoproteins has been shown by m.s. and n.m.r. analysis to have a novel backbone structure containing an internal galactose residue monosubstituted at C-6 by N-acetylglucosamine: (Formula: see text). This oligosaccharide may represent a biosynthetic product of a previously unrecognized N-acetylglucosaminyltransferase catalysing formation of a linear GlcNAc beta 1-6Gal sequence.

On the homogeneity of stools with respect to bile acid composition and normal day-to-day variations: a detailed qualitative and quantitative study using capillary column gas chromatography-mass spectrometry.

Fecal bile acid excretion was determined using recently developed techniques in order to investigate: the extent of the homogeneity in composition and concentration of individual bile acids in a single stool sample, the detailed qualitative and quantitative day-to-day variations in total and individual bile acids in the typical healthy adult, information on the relative proportions of conjugated bile acids in healthy stools, and inter-individual variations in fecal bile acid excretion. Bile acids were extracted from feces and separated into groups based upon their mode of conjugation using lipophilic gel chromatography, prior to analysis by capillary column gas chromatography-mass spectrometry. The majority of bile acids were excreted in the unconjugated form, while in all samples, conjugated bile acids accounted for less than 6% of the total fecal bile acids excreted, of which sulphated bile acids represented less than 3% of the total. Quantitative total and individual bile acid excretion, determined from single daily collections exhibited wide variations in values from day-to-day, and in accordance with early findings, indicates the need to use a minimum of 3- to 5-day collections for a more reliable index of bile acid excretion in feces. Examination of frozen and sectioned single stools revealed wide variations in water content and in quantitative bile acid concentration and composition within the stool. These data indicate random stool samples, which are commonly used in clinical studies, and data expressed as concentrations to be unsatisfactory for the accurate determination of fecal bile acid excretion.

A comparative study of zinc, copper, cadmium, and lead levels in fertile and infertile men.

Eighty infertile men and 38 men of known fertility were studied for investigation of both the importance of zinc, copper, cadmium, and lead to fertility and the possible interrelationships between these trace elements. The infertile men had higher mean concentrations of plasma copper than those of proven fertility. The difference was statistically significant (P less than 0.01) but was of small magnitude (approximately 1.5 mumol mean difference). The concentrations of plasma zinc, erythrocyte zinc, whole blood lead and cadmium, and seminal plasma zinc and copper did not differ significantly between infertile and fertile men. There was a significant positive relationship between sperm density and seminal plasma zinc concentration in the fertile, but not in the infertile, men. The infertile men with antisperm antibodies or counts greater than 20 million/ml had significantly higher mean levels of seminal plasma zinc than infertile men with oligospermia. The higher semen zinc in these two groups may reflect an abnormal fragility of the spermatozoa, resulting in the release of zinc, but the absence of significant overall differences between fertile and infertile men suggests that measurement of the concentration of zinc in plasma or zinc and copper in seminal plasma has little value in the routine investigation of infertility.

Plasma cortisone in man: its determination, physiological variation, and significance.

In a method for the simultaneous determination of cortisone, cortisol, and corticosterone, the extracted steroids are separated using paper chromatography and assayed by competitive protein binding. Evidence of the reliability of the method, particularly with respect to the determination of cortisone, is presented. Only trivial changes in cortisone level were detected in response to exercise, surgery, and ACTH, even though marked changes in cortisol level were simultaneously recorded. It is suggested that the inertia in plasma cortisone level may be related to its lesser degree of binding to plasma proteins, relative to cortisol, which would permit more rapid hepatic uptake and possible accumulation of a large extravascular pool of cortisone.

Adrenocortical response to one-leg and two-leg exercise on a bicycle ergometer.

Twelve healthy male subjects exercised for 30 min on a bicycle ergometer, using only one leg, at a work load that was nearly exhausting. Nine subjects (group A) showed a marked rise in plasma cortisol concentration (240 +/- 50 nmol x l-1), but in three subjects no such rise occurred (group B). All group B subjects had elevated cortisol levels prior to the period of exercise, which may have inhibited the adrenocortical response. Seven subjects of group A also performed the same work load using both legs. Under this regime, increases in plasma cortisol and aldosterone levels, blood lactate level and heart rate, but not plasma potassium, were significantly smaller than for one-leg exercise. These findings are consistent with the hypothesis that activation of the hypothalamic-pituitary-adrenal (HPA) system during exercise could be mediated through the stimulation of chemoreceptors in the exercising muscles. This effect could be reinforced by HPA stimulation in response to the greater acceleration of heart rate in one-leg exercise. The marked rises in plasma potassium levels might be responsible for the elevation of plasma aldosterone concentration particularly in those experiments when this occurs earlier than, or in the absence of, rises in cortisol concentration.

The reduction of soiling behaviour in an 11-year-old boy with the parent as therapist.

An 11-year-old boy who had soiled daily for six years since entering school had threatened to commit suicide. Following an initial assessment, a parent was trained to monitor an experimental procedure that effectively reduced soiling to a level where the only occurrence noted in two months resulted from a bout of diarrhoea.

Reliability of plasma cortisol determination by a simple competitive protein binding method.

The method investigated used (3H) cortisol as the ligand, diluted human plasma as the binding protein, and florisil to separate free and bound cortisol. The precision and specificity were found to be adequate for routine use. The effects of such factors as delay in separation of plasma, storage of plasma, provenance of the binding protein, venepuncture, and venous occlusion have been studied. The extent and significance of short term, inter-daily, and longer term variation in plasma cortisol level are discussed.