Twenty-two neutral 0-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 'H-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(p1-6)[Gal(p1-3)IGalNAcol or GlcNAc(B1 -6)[GlcNAc(pl-3)IGalNAcol core region. Oligosaccharides not previously characterised from any source were the disaccharide GalNAcal -6GalNAcol (GalNAc, N-acetylgalactosamine and the hexasaccharide GlcNAc(p1-6){GalNAc (al-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.Bovine submaxillary-gland mucin (BSM) was among the first mucin glycoproteins to be purified and studied [l -51. Early structural investigations of its 0-linked carbohydrate chains (E 70% of the mass) indicated a predominance of the acidic disaccharide NeuAc(a2 -6)GalNAc [6 -81. Later, the presence of galactose, L-fucose (Fuc) and/or N-acetylglucosamine [9] was detected in addition to N-acetylneuraminic acid and N-acetylgalactosaminitol, consistent with the occurrence of longer chains and more complex oligosaccharides in BSM. This has been confirmed by studies on acidic oligosaccharides [lo-131.Neutral oligosaccharides comprise approximately one fifth of the total oligosaccharides and five structures have been characterised previously [lo]. In the present study a more comprehensive analysis of neutral oligosaccharides released from BSM by Carlson degradation [14] has been made by a combination of liquid secondary-ion mass spectrometry (LSIMS), GC-MS and 'H-NMR, to establish the range of structures present and allow comparison with other mucins. The results show greater diversity of chains than previously reported, among which are a series of oligosaccharides with blood-group-A-rela ted sequences. MATERIALS AND METHODS Preparation and isolation of BSM neutral oligosaccharide alditolsBSM, prepared by a procedure essentially as described by Tettamanti and Pigman [3], was purchased from Sigma Chemical Co., Dorset, England (Type 1-S).Oligosaccharides were released from BSM by the digestion procedure of Carlson [14]. In brief, BSM (450 mg) was incubated with 0.05 M NaOH/1.0 M NaBH, (20 ml) for 16 h at 45°C. After acidification to pH 5 by addition of acetic acid/ H 2 0 (1 : 1, by vol.), the reaction mixture was passed through a column of ion-exchange resin (Bio-Rad) with a 15 ml lower bed containing AG 50W-X2 (H' form) and a 20 ml upper bed containing AG 50W-X8 (H' form), and washed with four column volumes of HzO. The combined effluent and washes were lyophilized and the boric acid removed by co-ev...

Mucins from the pooled gastric juice of Lewis-positive secretors were investigated to establish their glycosylation patterns with particular reference to the type and abundance of the glycan-core structures. Following reductive P-eliminination, the neutral glycan alditols from these mucins were fractionated by ion exchange and size-exclusion chromatographies and subjected to structural analyses. It was possible to gain insights into the core sequences of the neutral 0-linked glycan alditols by matching (a) composition data from liquid secondary-ion mass spectrometry of the native alditol fractions, (b) specific structural information on the core sequences by thin-layer-chromatography mass spectrometry of alditol-derived neoglycolipids and (c) data from electron-impact mass spectrometry of permethylated glycan alditols or their partially methylated alditol acetates. The predominant core structures detected among the neutral glycans representing about 77% (by mass) of the total carbohydrates released from gastric mucins were core 1, Galpl-3GalNAc (Ac, acetyl) and core 2, Galjl1-3(GlcNAc~l-6)GalNAc in the approximate ratio 1 : 2. Core 3, GlcNAcj?l-3GalNAc, and core 4, GlcNAc~l-3(GlcNAc~1-6)GalNAc, were much less abundant (< lo%), while core 5, GalNAcal-3GalNAc, core 6, GlcNAcPl-6GalNAc, and a recently described sequence GalNAcal-6Gal-NAc (core 7) were not detected. This investigation also addressed the question of the presence of the sequence GalPl-6GalNAc which has been reported previously to occur as a core-structure element in gastric mucins. This was greatly assisted by the availability of the authentic chemically synthetised disaccharide alditol which, when converted into a neoglycolipid after mild periodate oxidation, gives diagnostic ions in mass spectrometry and can be detected with high sensitivity. No evidence was found for the presence of this unusual sequence among the oligosaccharides in gastric mucins.Biosynthesis of the core regions of 0-linked glycans is thought to proceed in an organ-characteristic manner, reflecting varying activities of the competing glycosyltransferases and their mode of compartmentalization, but also, as postulated recently, being influenced by the much peptide sequence in the near vicinity of putative glycosylation sites [l].

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 'H-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(al-2)Gal(~1-4)[Fuc(al-3)]GlcNAc(~l-) (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- Recent reports [l, 21 on the neutral 0-linked oligosaccharides in bovine submaxillary-gland mucin (BSM) have revealed a greater diversity of structure than previously recognised [3 -61. Seventeen sugar chains ranging from disaccharides to heptasaccharides have been identified [l, 21, including oligosaccharides having blood-group-A, blood-group-H and LewisY determinants.In addition to these, there are also acidic oligosaccharides which represent about 85% of the carbohydrate in BSM and a total of 12 monosialylated oligosaccharides have been isolated so far [6 -101. The present study describes the characterisation of an additional number of mainly larger, acidic structures released from BSM by Carlson degradation. These have been characterised by a combination of liquid secondary-ion mass spectrometry (LSIMS), methylation analysis and 'H-NMR after chromatography on Bio-Gel P-4 and HPLC separation and include four hexasaccharides containing N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic acid (NeuGc) which extend the range of known structures on BSM. MATERIALS AND METHODS Isolation of BSM acidic oligosaccharide alditolsBSM, prepared by a procedure essentially the same as described by Tettamanti and Pigman [l I], was purchased from Sigma Chemical Co., Dorset, England (Type I-S).Oligosaccharides were released as alditols as previously described [2]. After removal of salt and borate, the oligosaccharide mixture was dissolved in 2 mM pyridinel2 mM acetic acid, pH 5.0, ( 2 ml) and applied to a Bio-Rad AG 1-X2 (acetate form, 10-ml bed volume) anion-exchange column equilibrated with the same buffer. The column was washed with 50 ml equilibration buffer to elute the neutral oligosaccharide alditol fraction (23 mg, dry mass from 450 mg BSM) investigated previously [2]. The column was then eluted with 50 ml of 0.3 M and 50 m10.5 M formic acid. After lyophilization of the combined formic acid eluent, 138 mg acidic oligosaccharide alditols were obtained.The acidic oligosaccharides were fractionated on a BioGel P-4 (200 -400 mesh, 1.6 cm x 90 cm) column at room temperature with elution by 50 mM pyridinel25 mM acetic acid buffer (pH 5.3) to give A1 -7 (Fig. 1) in the following amounts: Al, 16.7 mg; A2,73 mg; A3, 14.3 mg; A4, 19.1 mg; AS, 3.6 mg; A6, 1.5 mg; A7, 7.3 mg. HPLC purificationFractions AS and A6 were chromatographed on a APSHypersil-2 column (NH2...

Summary Analgesia mediated by a direct spinal action of narcotic drugs administered via the epidural route may have considerable advantages over conventional (conduction block) epidural analgesia in labour. The efficacy, mode of action and placental transfer of epidurally administered narcotics in labour has not yet been established. We have compared the systemic absorption, analgesia and other effects on the mothers and transfer of drug to the fetus in primigravidae who received epidural or intramuscular pethidine 100 mg in labour. The superior analgesia following epidural pethidine did not appear to be attributable to a selective spinal action but rather to higher plasma concentrations of pethidine together with a possible weak regional conduction block due to local anaesthetic action of 1 % pethidine solution. Epidural pethidine is not an advantageous method for providing analgesia in labour.

Plasma homocysteine levels are lowered by insulin and can be elevated in insulin-resistant states. However, it is uncertain whether homocysteine and insulin resistance or components of the metabolic (insulin resistance) syndrome are related in healthy individuals. Total homocysteine concentrations were measured by gas chromatography-mass spectrometry in samples from 100 male participants in the second follow-up cohort of the Heart Disease and Diabetes Risk Indicators in a Screened Cohort Study. Members of this cohort have each undergone an iv glucose tolerance test with measurement of insulin sensitivity by minimal model analysis. Age ranged from 31--62 yr (mean, 46.8), body mass index from 20.6--36.5 kg/m(2) (mean, 26.3), insulin sensitivity from 0.0--9.6 min/mU.L (mean, 2.32), and homocysteine concentrations from 7.5--30.6 micromol/L (mean, 12.2). In univariate correlation, homocysteine concentrations were unrelated to insulin sensitivity or to components of the metabolic syndrome, including fasting serum triglycerides, high density lipoprotein cholesterol, high density lipoprotein subfraction 2 cholesterol, blood pressure, uric acid, systolic blood pressure, or body mass index. These measures were, nevertheless, highly intercorrelated. These findings strengthen the possibility that in healthy humans, homocysteine metabolism is not substantially affected by insulin action.