A monoclonal antibody that recognizes both leb and Y (Ley) antigens

A hemagglutinating monoclonal antibody has been obtained from a mouse/mouse hybridoma after immunisation with the le^b-active oligosaccharide, lacto-N-difucohexaose I, coupled to edestin. The antibody agglutinated human red cells regardless of Lewis phenotype. Blood group O cells were strongly, agglutinated, and progressively weaker agglutination was observed with A₂, B and A₂B cells. Blood group A₁ and A₁B cells were not agglutinated.By examining the binding of the antibody to glycolipids and oligosaccharides it was shown that the Le^b and Y (Le^y)-haptens bind to a similar extent. Full binding activity was dependent on the presence of, both fucosyl residues.Abbreviations LND l lacto-N-difucohexaose l - IV²Fuc,lll⁴FucLcOse₄ LND l-OL, lacto-N-difucohexaitol l     

Expression of Lewis histo-blood group glycolipids in the plasma of individuals of Le(a+b+) and partial secretor phenotypes

Red cell Lewis antigens are carried by glycosphingolipids passively absorbed from plasma. Plasma was collected from a spectrum of individuals with normal and unusual Lewis/secretor phenotypes in order to investigate the glycolipid basis for the unusual phenotypes. Samples were obtained from: a Le(a+b–) ABH nonsecretor who secreted Lewis substances; a Le(a+b–) partial secretor; Le(a+b+) partial secretors; Le(a+b+) secretors; and a full range of normal Lewis/secretor phenotypes as controls. The Le(a+b+) samples represented Polynesian, Asian and Réunion Island ethnic backgrounds. Nonacid glycolipids were prepared, separated by thin-layer chromatography, and then immunostained with potent monoclonal antibodies of known specificity. Despite different serological profiles of the Le(a+b–) and Le(a+b+) Polynesian samples, their plasma glycolipid expressions were very similar, with both Le^a and Le^b co-expressed. The copresence of Le^a and Le^b in Le(a+b+) samples is in marked contrast to Caucasians with normal Lewis phenotypes, who have predominantly either Le^a or Le^b. These results suggest that there is a range of the secretor transferases in different individuals, possibly due to different penetrance or to several weak variants. We also show that Lewis epitopes on longer and/or more complex core chains appear to be predominant in the Polynesian Le(a+b+) samples. The formation of these extended glycolipids is compatible with the concept that in the presence of reduced secretor fucosyltransferase activity, increased elongation of the precursor chain occurs, which supports the postulate that fucosylation of the precursor prevents or at least markedly reduces chain elongation.Abbreviations CBA chromatogram binding assay - TLC thin-layer chromatography     

Isolation and purification of Lea blood-group active and related glycolipids from human plasma of blood-group A Lea individuals

From 8 1 of human plasma of blood-group A Le^a nonsecretors three different Le^a blood-group active ceramide pentasaccharides (a total of 4.65 mg) have been isolated, all revealing glucose, galactose, N-acetylglucosamine and fucose in molar ratios of 1 : 2 : 1 : 1 as determined by gas liquid chromatography. A fourth blood-group active fraction (0.72 mg) represents a mixture of a Le^a active ceramide pentasaccharide and an A active ceramide hexasaccharide (molar ratio 7.7 : 2.3 as calculated from the content of different aminosugars). Additionally, two different globosides, two different hematosides and a new N-acetylglucosamine containing ceramide tetrasaccharide were obtained. All 9 glycolipid fractions demonstrated homogeneity in analytical high performance thin layer chromatography (HPTLC) using 4 different solvent systems. 0.2 μg of each Le^a active glycolipid completely inhibited the agglutination of O Le(a + b ?) erythrocytes by 50 μl of 4 hemagglutinating units of caprine anti Le^a serum. At least 0.04 μg of each Le^a antigen are sufficient for incubation to convert 9 × 10⁷ O Le(a?b?) erythrocytes into Le^a-positive cells. Mainly due to the relatively low content of the blood-group A glycolipid in plasma (0.17 mg/8 1), previously negative erythrocytes readily become agglutinable by anti Le^a sera and not by anti A sera after incubation with appropriate plasma.     

Preparation of precipitating antigens by coupling oligosaccharides to polylysine

Oligosaccharides isolated from human milk when coupled to polylysine by a mixed anhydride procedure are effective precipitating antigens. The lacto-N-fucopentaose II conjugate specifically precipitates antibody directed against the human Le^a blood group antigen while the lacto-N-difucohexaose I conjugate specifically precipitates antibody directed against the human Le^b blood group antigen. The derivatives were used to define the specificity of a human anti-I cold agglutinin.     

Immunochemistry of the Lewis-blood-group system: Partial characterization of Lea-, Leb-, and H-type 1 (LedH)-blood-group active glycosphingolipids from human plasma

Four different H-type 1 (Le^dH) blood-group-active glycosphingolipids (Le^dH-I–IV) have been isolated from the plasma of blood-group O Le(a?b?) secretors. The agglutination of O Le(a?b?) erythrocytes from secretors by 50 μl of 4 hemagglutinating units of caprine anti-Le^dH (anti-H-type 1) serum was inhibited by 0.02 μg of each of all four glycolipids. No Le^a or Le^b activities or reaction against Ulex europaeus lectin could be found. Le^dH-I, -II, -III, and -IV at 0.05, 0.01, 0.01, and 0.02 μg each are sufficient for incubation in order to convert 9 × 10⁷ O Le(a?b?) erythrocytes from nonsecretors into H-type 1 (Le^dH)-positive cells. Structural analysis of the H-type 1 glycolipids was performed in comparison to that of Le^a- and Le^b-blood-group-active glycolipids from human plasma isolated previously: Gas chromatography of peracetylated alditols revealed sugar composition. Combined gas chromatography-mass spectrometry established the glycosidic linkages. Together with the results obtained by direct inlet mass spectrometry of permethylated glycosphingolipids and by 360-MHz ¹H nuclear magnetic resonance spectroscopy (Egge, H., and Hanfland, P., 1981, Arch. Biochem. Biophys., 210, 396–404; Dabrowski, J., Hanfland, P., Egge, H., and Dabrowski, U., 1981, Arch. Biochem. Biophys., 210, 405–411) the complete structures of the oligosaccharide chains of the Le^a-, Le^b-, and H-type 1-active glycolipids were established: Galβ1 → 3GlcNAc(4 ← 1αFuc)β1 → 3Galβ1 → 4Glcβ1 → 1 Cer for the Le^a antigens; Fucα1 → 2Galβ1 → 3GlcNAc(4 ← 1αFuc)β1 → 3Galβ1 → 4Glcβ1 → 1 Cer for the Le^b antigens; and Fucα1 → 2Galβ1 → 3GlcNAcβ1 → 3Galβ1 → 4Glcβ1 → 1 Cer for the H-type 1 (Le^dH) glycolipids. The diverse antigens of the same blood-group specificity obviously differ from one another in their lipid residue. In addition, plasmatic neolactotetraosylceramide could be identified, differing from that of human erythrocytes by a slower migration behavior in thin-layer chromatography.     

Immunochemical and immunohistological expression of Lewis histo-blood group antigens in small intestine including individuals of the Le(a+b+) and Le(a−b−) nonsecretor phenotypes

Histological samples and total non-acid glycosphingolipids were prepared from small intestine of human cadavers with the Le(a+b+) and Le(a–b–) nonsecretor phenotypes and contrasted with the more common Lewis phenotypes. Glycolipid fractions were analysed by thin-layer chromatography and tested for Lewis activity with monoclonal antibodies reactive to Lewis epitopes. Paraffin-embedded small intestine sections were also fluorescently immunostained with anti-Lewis antibodies. Unlike the common Lewis positive phenotypes, we were immunochemically able to demonstrate the copresence of large amounts of Le^a and Le^b glycolipids in the Le(a+b+) sample. In addition we demonstrated increased formation of extended Lewis structures in this phenotype. By immunohistochemistry Le^a, Le^b and type 1 precursor chain epitopes could be demonstrated in the brush border. These results show that the expression of the Le(a+b+) phenotype at the erythrocyte phenotyping level parallels the small intestinal expression of this phenotype, and the patterns of Lewis antigen expressions are unique to this phenotype. By immunohistochemistry and immunochemistry we also demonstrated the presence of trace amounts of Lewis active glycoconjugates in the small intestine of the Le(a–b–) nonsecretor and Le(a+b–) samples. In the Le(a–b–) nonsecretor Le^a and Le^b activity was absent and type 1 precursor was present in brush border, while Le^b activity was immunohistologically demonstrated in the Golgi apparatus of the deep glands. Trace amounts of both Le^a and Le^b glycolipids were identified in this sample. In parallel trace Le^b activity could also be detected in the glycolipids of the Le(a+b–) sample and could be immunohistologically demonstrated to be fully expressed in occasional cells in the deep glands of the small intestine, a pattern quite dissimilar to that of the Le(a–b–) nonsecretor. The results in this paper show that the expression of Lewis glycoconjugates in the small intestine parallel the expression of Lewis erythrocyte phenotypes. However, inappropriate Lewis activity is also seen in individuals of other phenotypes and the mechanisms by which these Lewis antigens are made appears to be different for different phenotypes.Abbreviations FITC fluorescein isothiocyanate - HPLC high-performance liquid chromatography - NeuAc N-acetyl-d-neuraminic acid - RBC red blood cell - TLC thin-layer chromatography - TRITC tetramethyl rhodamine isothiocyanate     

Diffusion-controlled reaction kinetics of the binding of carbon monoxide to the heme undecapeptide of cytochrome c (microperoxidase 11) in high viscosity solvents

Glycosphingolipids from human plasma with Le^a, Le^b, and H-type 1 (Le^dH) Lewis-blood-group activity have been analyzed after permethylation by electron impact mass spectrometry using an indirectly heated direct insertion probe. The spectra obtained are compared with that of permethylated neo-lactotetraosyl ceramide (Gl-3) from human plasma. The fragmentation patterns presented show clearly, that Le^a and H-type 1 glycosphingolipids are ceramide pentasaccharides while Le^b is a ceramide hexasaccharide. All Lewis-blood-group-active compounds investigated produced ions specific for type 1 carbohydrate chains. It is therefore concluded, that all compounds are derivatives of lacto-N-tetraose. The obtained spectra support the following sequences: Hexose-1→3-hexosamine[4←1-deoxyhexose]-hexose-hexose ceramide for the Le^a derivatives; deoxyhexose-hexose-1→3-hexosamine4←1-deoxyhexose]-hexose-hexose ceramide for the Le^b derivatives; and deoxyhexose-hexose-1→3-hexosamine-hexose-hexose ceramide for all H-type 1 (Le^dH) derivatives. In the case of the H-type 1 glycosphingolipids four subfractions were analyzed separately. While all four fractions contained the same carbohydrate sequence, significant differences were observed in the ceramide residues. Specific fragmentation patterns indicate the presence of sphingosine, icosasphingosine, and 4-hydroxysphinganine besides normal, unsaturated, and hydroxylated fatty acids in all Lewis-blood-group-active glycolipids.     

Red cell H-deficient,salivary ABH secretor phenotype of Reunion island. Genetic control of the expression of H antigen in the skin

Based on the genetic model proposing thatH andSe are two structural genes, we predicted that the red cell H-deficient, salivary ABH secretor phenotype should be found on Reunion island, where a large series of H-deficient non-secretor families have been previously described. Two such Reunion individuals are now reported. POU [A_h, Le(a–b+), secretor of A, H, Le^a and Le^b in saliva] and SOU [O_h, Le(a–b+), secretor of H, Le^a and Le^b in saliva]. Both are devoid of H -2-fucosyltransferase activity in serum. In addition, the preparation of total non-acid glycosphingolipids from plasma and red cells of POU revealed the type 1ALe^b heptaglycosylceramide and small amounts of the monofucosylated type 1 A hexaglycosylceramide. Both glycolipids possess an H structure probably synthesised by the product of theSe gene. No other blood group A glycolipids, with types 2, 3 or 4 chains, normally present in the presence of the product of theH gene, were found on red cells or plasma of POU.TheH,Se andLe genetic control of the expression of ABH and related antigens in different tissue structures of the skin is described in 54 H-normal individuals of known ABO, secretor and Lewis phenotypes; in one red cell H-deficient salivary secretor (SOU); and in one H-deficient non-secretor (FRA). Sweat glands express ABH under the control of theSe gene. Sweat ducts express ABH under the control of bothH andSe genes and Lewis antigens under the control ofLe and bothH andSe genes. Epidermis, vascular endothelium and red cells express ABH under the control of theH gene. The products ofH andSe genes are usually expressed in different cells. However, the results illustrate that in some structures, like the epithelial cells of sweat ducts, both the products ofH andSe genes can contribute to the synthesis of the same Le^b structure.     

New derivatization and separation procedures for reducing oligosaccharides

4-Trifluoroacetamidoaniline was reacted with reducing oligosaccharides in the presence of sodium cyanoborohydride to give aminoalditol derivatives, useful for linkage to proteins or solid matrices. A mixture of reducing oligosaccharides, difficult to separate by HPLC, was treated in the same way. The resulting derivatives were easily separated by HPLC.Abbreviations TFAN 4-trifluoroacetamidoaniline - LcOse₄ lacto-N-tetraose - IV²Fuc-LcOse₄ lacto-N-fucopentaose l - III⁴Fuc-LcOse₄ lacto-N-fucopentaose II - III³Fuc-nLcOse₄ lacto-N-fucopentaose III - IV²Fuc, III⁴Fuc-LcOse₄ lacto-N-difucohexaose I - II⁶Galß1-4GlcNAc-LcOse₄ lacto-N-hexaose - II³NeuAc-Lac 3-sialyllactose - GlcNAcß1-4GlcNAcß1-4GlcNAc chitotriose - GalNac1-3|Fuc1-2|Galß1-4Glc A-tetrasaccharide     

Immunochemistry of the Lewis-blood-group system: Proton nuclear magnetic resonance study of plasmatic Lewis-blood-group-active glycosphingolipids and related substances

The Le^a-, Le^b-, and H-type 1 (Le^dH)-blood-group-active glycosphingolipids, as well as H-I-type 2 glycolipid, lactotetraosyl ceramide, and neo-lactotetraosyl ceramide were examined by ¹H nuclear magnetic resonance at 360 MHz in dimethyl-d₆ sulfoxide as solvent. The resonances of almost all protons of the sugar rings were assigned with the aid of spin decoupling and nuclear Overhauser difference spectroscopy. The latter technique was also applied to establish the sequences and sites of glycosidic linkage. This information, combined with the chemical shift-structure correlations established in our previous work, led to an independent identification of those six glycolipids. Type 1 (Galβ1 → 3GlcNAc) and type 2 (Galβ1 → 4GlcNAc) saccharide chains can be distinguished by this approach. Some deviations from additivity in chemical shifts, calculated for oligosaccharides from the data on their constituent sugar residues, furnished information on the conformational changes in crowded glycolipid molecules.     

Differentiation of isomeric Lewis blood groups by positive ion electrospray tandem mass spectrometry

Lewis histo-blood group antigens are one of the major classes of biologically active oligosaccharides. In this work, underivatized Lewis blood groups were studied by electrospray tandem mass spectrometry (ESI-MSⁿ) in the positive mode with three different mass analyzers: Q-TOF (quadrupole time-of-flight), QqQ (triple quadrupole), and LIT (linear ion trap). It was observed that, under collision-induced fragmentations, type 1 Lewis antigens (Le^a and Le^b) could be distinguished from type 2 (Le^x and Le^y) on the basis of specific fragmentations of the GlcNAc unit. Whereas O-4-linked sugars of the GlcNAc are lost as residues, the O-3-linked sugars undergo fragmentation both as sugar units and as sugar residues (unit −18 Da). Type 2 Lewis antigens also showed a characteristic cross-ring cleavage ^0,2A₂ of the GlcNAc. As a result, the product ions at m/z 388 and 305, characteristic of Le^x, and m/z 372, characteristic of Le^a, are proposed to distinguish the trisaccharide isomers Le^x/Le^a. Also, the product ions at m/z 534 and 305, characteristic of Le^y, and m/z 372, characteristic of Le^b, are proposed to distinguish the tetrasaccharide isomers Le^b/Le^y. These diagnostic fragment ions were further applied in the identification of Lewis type 2 antigens (Le^x and Le^y) in the lipopolysaccharide of the human gastric pathogen, Helicobacter pylori.     

Factors influencing resumption of meiotic maturation and cumulus expansion of porcine oocyte-cumulus cell complexes in vitro

The present study was undertaken to examine effects of various combinations of epidermal growth factor (EGF), transforming growth factor-b?₁ (TGF-b?₁), follicle-stimulating hormone (FSH), luteinizing hormone (LH), androstenedione (A₄), and estradiol-17b? (E₂) on meiotic maturation and cumulus expansion in the pig using an in vitro model system. Oocyte-cumulus cell complexes (OCC) were cultured in the media containing the abovementioned agents for 24 hr and were observed for germinal vesicle breakdown (GVBD), indicative of initiation of meiotic maturation, and for expansion of their cumulus cells. Treatment with EGF significantly increased (P < 0.05) incidence of GVBD, with maximal stimulation occurring at 1 ng/ml (55% vs. 12% in the control). Concentrations of EGF as low as 100 pg/ml significantly stimulated GVBD over control (37% vs. 12%). Addition of EGF (1 ng/ml) and FSH (1.5 μg/ml) together and LH (2 μg/ml) and FSH (1.5 μg/ml) together resulted in significantly higher (P < 0.01) GVBD levels than were observed in response to EGF, FSH, or LH alone. Addition of E₂ (1 μg/ml) had no effect by itself but significantly decreased the incidence of GVBD in the presence of FSH and of LH + FSH. Addition of A₄ (1 μg/ml) significantly reduced the percentage of oocytes undergoing GVBD when added alone or with FSH. Although both EGF and LH stimulated cumulus expansion, FSH was more effective in stimulating cumulus expansion than EGF or LH. TGF-b?₁ had no effect on GVBD or cumulus expansion. These studies indicate that these hormones may have differing roles in oocyte maturation and that their interactions may be part of an intricate system regulating the maturation of oocytes during follicular development in vivo. © 1993 Wiley-Liss, Inc.     

Expression and localization of Lewisx glycolipids and GD1a ganglioside in human glioma cells

We analysed the glycolipid composition of glioma cells (N-370 FG cells), which are derived from a culture of transformed human fetal glial cells. The neutral and acidic glycolipid fractions were isolated by column chromatography on DEAE-Sephadex and analysed by high-performance thin-layer chromatography (HPTLC). The neutral glycolipid fraction contained 1.6 µg of lipid-bound glucose/galactose per mg protein and consisted of GlcCer (11.4% of total neutral glycolipids), GalCer (21.5%), LacCer (21.4%), Gb4 (21.1%), and three unknown neutral glycolipids (23%). These unknown glycolipids were characterized as Lewis^x (fucosylneolactonorpentaosyl ceramide; Le^x), difucosylneolactonorhexaosyl ceramide (dimeric Le^x), and neolactonorhexaosyl ceramide (nLc6) by an HPTLC-overlay method for glycolipids using specific mouse anti-glycolipid antibodies against glycolipid and/or liquid-secondary ion (LSI) mass spectrometry. The ganglioside fraction contained 0.6 µg of lipid-bound sialic acid per mg protein with GD1a as the predominant ganglioside species (83% of the total gangliosides) and GM3, GM2, and GM1 as minor components. Trace amounts of sialyl-Le^x and the complex type of sialyl-Le^x derivatives were also present. Immunocytochemical studies revealed that GD1a and GalCer were primarily localized on the surface of cell bodies. Interestingly, Le^x glycolipids and sialyl-Le^x were localized not only on the cell bodies but also on short cell processes. Especially, sialyl-Le^x glycolipid was located on the tip of fine cellular processes. The unique localization of the Le^x glycolipids suggests that they may be involved in cellular differentiation and initiation of cellular growth in this cell line.     

Determination of N-(trans-4-isopropylcyclohexylcarbonyl)-d-phenylalanine in human plasma by solid-phase extraction and column-switching high-performance liquid chromatography with ultraviolet detection

A column-switching high-performance liquid chromatography method with ultraviolet detection at 210 nm has been developed for the determination of N-(trans-4-isopropylcyclohexylcarbonyl)-d-phenylalanine (AY4166, I) in human plasma. Plasma samples were prepared by solid-phase extraction with Sep-Pak Light tC₁₈, followed by HPLC. The calibration graph for I was linear in the range 0.1–20 μg/ml. The limit of quantitation of I, in plasma, was 0.05 μg/ml. The recovery of spiked I (0.5 μg/ml) to drug-free plasma was over 92% and the relative standard deviation of spiked I (0.5 μg/ml) compared to drug-free plasma was 4.3% (n = 8).     

Determination of N-(trans-4-isopropylcyclohexylcarbonyl)-d-phenylalanine in human plasma by solid-phase extraction and column-switching high-performance liquid chromatography with ultraviolet detection

A column-switching high-performance liquid chromatography method with ultraviolet detection at 210 nm has been developed for the determination of N-(trans-4-isopropylcyclohexylcarbonyl)-d-phenylalanine (AY4166, I) in human plasma. Plasma samples were prepared by solid-phase extraction with Sep-Pak Light tC₁₈, followed by HPLC. The calibration graph for I was linear in the range 0.1–20 μg/ml. The limit of quantitation of I, in plasma, was 0.05 μg/ml. The recovery of spiked I (0.5 μg/ml) to drug-free plasma was over 92% and the relative standard deviation of spiked I (0.5 μg/ml) compared to drug-free plasma was 4.3% (n = 8).     

Characterization of the binding specificity ofAnguilla anguilla agglutinin (AAA) in comparison toUlex europaeus agglutinin I (UEA-I)

Using immunochemical and immunohistochemical methods, the binding site ofAnguilla anguilla agglutinin (AAA) was characterized and compared with the related fucose-specific lectin fromUlex europaeus (UEA-I). In solid-phase enzyme-linked immunoassays, the two lectins recognized Fuc1-2Gal-HSA. AAA additionally cross-reacted with neoglycolipids bearing lacto-N-fucopentaose (LNFP) I [H type 1] and II [Le^a] and lactodifucotetraose (LDFT) as glycan moieties. UEA-I, on the other hand, bound to a LDFT-derived neoglycolipid but not to the other neoglycolipids tested. Binding of AAA to gastric mucin was competitively neutralized by Le^a-specific monoclonal antibodies. UEA-I binding, on the other hand, was reduced after co-incubation with H type 2- and Le^y-specific monoclonal antibodies. According to our results, AAA reacts with fucosylated type 1 chain antigens, whereas UEA-I binds only to the 1-2-fucosylated LDFT-derived neoglycolipid. In immunohistochemical studies, the reactivity of AAA and UEA-I in normal pyloric mucosa from individuals with known Lewis and secretor status was analysed. AAA showed a broad reaction in the superficial pyloric mucosa from secretors and non-secretors, but AAA reactivity was more pronounced in Le^(a+b-) individuals. On the other hand, UEA-I stained the superficial pyloric mucosa only from secretor individuals. A staining of deep mucous glands by the lectins was found in all specimens. Both reacted with most human carcinomas of different origin. Slight differences in their binding pattern were observed and may be explained by the different fine-specificities of the lectins.     

Interaction of the Laburnum anagyroides lectin with fucoantigens

We studied interaction of the lectin from the bark of Golden Rain shrub (Laburnum anagyroides, LABA) with a number of basic fucose-containing carbohydrate antigens by changes in its tryptophan fluorescence. The strongest LABA binding was observed for the trisaccharide H of type 6 [α-L-Fucp-(1-2)-β-D-Galp-(1-4)-D-Glc, K _a = 4.2 × 10³ M^?1]. The following antigens were bound with a weaker affinity: H-disaccharide α-L-Fucp-(1-2)-D-Gal, a glucoanalogue of tetrasaccharide Le^y α-L-Fucp-(1-2)-β-D-Galp-(1-4)-[α-L-Fucp-(1-3)]-D-Glc, and 6-fucosyl-N-acetylglucosamine, a fragment of core of the N-glycans family (K _a 1.1?1.7 × 10³ M^?1). The lowest binding was observed for L-fucose (K _a = 2.7 × 10² M^?1) and trisaccharide Le^a, (β-Galp-(1-3)-[α-L-Fucp-(1-4)]-GlcNAc (K _a = 6.4 × 10² M^?1). The Le^d, Le^a, and Le^x pentasaccharides and Le^b hexasaccharide were not bound to LABA.     

Differential pulse polarographic determination of plasma menadione

A differential pulse polarographic assay for plasma vitamin K₃ (menadione) has been developed. Details of the assay are (i) lipid-soluble material is extracted from plasma into ether by the method of Bjornsson et al. [(1978) Thromb. Haemostas.2, 466–473]; (ii) ether is evaporated under nitrogen and the residue is dissolved in the supporting electrolyte, methanol: 0.2 m borate buffer (9:1), pH 6.8; (iii) current height is measured at ?0.32 V vs SCE on the differential pulse polarogram. The lower sensitivity limit of this technique after addition of standard vitamin K₃ to plasma is 0.3 μm; the calibration curve is linear from 0.6 through 10 μm. Two patients treated with a single dose of menadiol sodium diphosphate, 20 mg/M² i.m., achieved measurable plasma vitamin K₃ levels at 0.5 to 1.0 h ranging between 0.5 (0.08 μg/ml) and 2 μm (0.3 μg/ml).     

Structural and immunochemical identification of Leb glycolipids in the plasma of a group O Le(a-b-) secretor

Total non-acid glycosphingolipids were isolated from the plasma of a healthy red blood cell group O Le(a-b-) salivary ABH secretor individual. Glycolipids were fractionated by HPLC and combined into eight fractions based on chromatographic and immunoreactive properties. These glycolipid fractions were analysed by thin-layer chromatography and tested for Lewis activity with antibodies reactive to the type 1 precursor (Le^c), H type 1 (Le^d), Le^a and Le^b epitopes. Fractions were structurally characterized by mass spectrometry (EI-MS and LSIMS) and proton NMR spectroscopy. Expected blood group glycolipids, such as H type 1, (Fuc1-2Gal1-3GlcNAc1-3Gal1-4Glc1-1Cer) were immunochemically and structurally identified. Inconsistent with the red cell phenotype and for the first time, small quantities of Le^b blood group glycolipids (Fuc1-2Gal1-3(Fuc1-4)GlcNAc1-3Gal1-4Glc1-1Cer) were immunochemically and structurally identified in the plasma of a Lewis-negative individual. These findings confirm recent immunological evidence suggesting the production of small amounts of Lewis antigens by Lewis negative individuals. Abbreviations: HPLC, high performance liquid chromatography; TLC, (high performance) thin layer chromatography; EI-MS, electron impact ionisation mass spectrometry; LSIMS, liquid secondary ion mass spectrometry; NMR, nuclear magnetic resonance spectroscopy. The sugar types are abbreviated to Hex for hexose, HexNAc forN-acetylhexosamine and dHex for deoxyhexose (fucose). The ceramide types are abbreviated to d for dihydroxy and t for trihydroxy base, n for non-hydroxy and h for hydroxy fatty acids; LCB, long chain base.     

Blood group glycosphingolipid expression in kidney of an individual with the rare blood group A1 Le(a−b+) p phenotype: absence of blood group structures based on the globoseries

Total neutral glycolipid fractions were isolated from kidney and ureter tissue obtained at autopsy of an individual of the rare blood group A₁ Le(a–b+) p. The amount of glycolipids isolated were 3.7 and 2.5 mg g^–1 dry tissue weight for the kidney and ureter tissue, which is in the range of reference blood group P kidneys. Part of the kidney glycolipid fraction was subfractionated by HPLC. Glycolipid compounds were structurally characterized by thin-layer chromatography (chemical detection and immunostaining with monoclonal antibodies), proton NMR spectroscopy and mass spectrometry. Globotriaosyl- and globotetraosyl-ceramides, which are the major compounds in kidneys of P individuals, were absent in the p kidney, and a comparatively increased amount of monoglycosyland lactosylceramides was found. A shift to longer fatty acyl chains in the ceramide part of lactosylceramides was noted. Elongated globoseries compounds with five to seven sugar residues, including the blood group A type 4 chain structure, were lacking. A slight increase in neolactotetraosyl- and blood group X pentaglycosyl-ceramides was noticed. The study confirms an enzymatic block in the conversion of lactosylceramide to elongated globoseries compounds in the kidney tissue similar to that of erythrocytes of p individuals.Abbreviations: for blood group glycolipid antigens the short hand designation stands for: blood group — number of sugar residues — type of carbohydrate chain. Thus A-7-4 means a blood group A heptaglycoconjugate on a type 4 chain. The sugar types are abbreviated for mass spectrometry to Hex for hexose, HexNAc forN-acetylhexosamine and dHex for deoxyhexose. HPLC, high-performance liquid chromatography; HPTLC, high performance thin layer chromatography; EI, electron impact ionisation; LSI, liquid secondary ion; MS, mass spectrometry; NMR, nuclear magnetic resonance.