Hemolytic anemia and methemoglobinemia in a preterm baby as a complication of antenatal intraamnial injection of toluidine blue.

A late preterm infant was born 4.5 h after intraamniotic injection of 90 mg of Toluidine blue to confirm premature rupture of membranes. Due to the fetal exposition to the dye, the entire body of the patient was blue stained and the baby suffered from methemoglobinemia, Heinz' body positive hemolytic anemia and hyperbilirubinaemia requiring exchange transfusion. These complications underline that antenatal exposition of toluidine blue may result in considerable postnatal infant morbidity. Therefore intraamniotic application of toluidine blue should be discouraged.

[A simple clavicle score. An effective and reliable classification for outcome assessments of midclavicular fractures]. / Ein einfacher Klavikulascore. Bewertungssystem zur Beurteilung der Behandlungsergebnisse nach Frakturen des mittleren Klavikuladrittels bei Erwachsenen.

BACKGROUND: To compile an evaluation system (score) for post-treatment outcomes of midclavicular fractures, 172 patients were studied on average 15 months post-injury. As a control group 45 healthy volunteers were examined. The most relevant elements were filtered out for use in a new classification system, the Clavicle Score (CS). METHODS: The CS is based on a system of three partnered objective/subjective items as well as radiographic assessment of fracture healing. For the partnered items, subjective responses with the most significant correlation to the specific objective parameters were selected. Total score cutoff values (very good, good, moderate, poor) were established to keep interpretation simple. To validate the system, linear regression analysis was performed comparing the CS to two established assessment systems (Constant Score and the DASH Score). ERGEBNISSE: The correlation coefficients R=0.756 (Constant) and R=0.687 indicated that the conclusions were comparable and therefore valid. The reliability coefficient Cronbach's alpha was calculated at 0.8241, indicating high reliability. CONCLUSION: The CS is a simple, valid and reliable instrument to assess outcomes post-midclavicular fracture.

[ Intramedullary nailing (ESIN) in clavicular pseudoarthroses. Results of a prospective clinical trial]. / Die intramedulläre Nagelung (ESIN) von Klavikulapseudarthrosen. Ergebnisse einer prospektiven Anwendungsbeobachtung.

This prospective clinical trial was performed to assess healing, clinical outcome and complications after intramedullary nailing of midshaft clavicular nonunions. Over 2.5 years, 14 patients were included. Exclusion criteria were pathological fractures, bony defects, previous operative therapy, atrophic and infection pseudarthrosis and the patient's age: <18 years or >70 years. Results were evaluated after 3, 6, 12 and 18 months. Beginning 3 months after the operation, pain (VAS), subjective satisfaction, Constant score and DASH score were significantly better than preoperatively during the follow-up period (p<0.001). There were no infections, no implant displacements or refractures.Intramedullary fixation of midshaft clavicular nonunions with an elastic titanium nail is a safe, minimally invasive surgical technique, producing excellent functional and cosmetic results without additional bone grafting.

Isolation and structural characterization of five oligosaccharide alpha1-phosphates and one sulfated glycopeptide from hemofiltrate.

Five oligosaccharide alpha1-phosphates and one sulfated glycopeptide have been isolated from the hemofiltrate of one patient with end-stage renal disease. Isolation of these compounds has been achieved using reverse osmosis, ion-exchange and size-exclusion chromatography and high performance liquid chromatography. The structures were predominantly elucidated by one- and two-dimensional 1H and 31P NMR spectroscopy. The chemical structures were determined to be: 1 NeuAc alpha2-3Gal alpha1-OPO3H2; 2 NeuAc alpha2-6Galbeta1-4GlcNAc alpha1-OPO3H2; 3 NeuAc alpha2-3Galbeta1-3GalNAc alpha1-OPO3H2; 4 NeuAc alpha2-3Galbeta1-3[NeuAc alpha2-6]GalNAc alpha1-OPO3H2 (proposed structure); 5 Fuc alpha1-2Galbeta1-4[Fuc alpha1-3]GlcNAc alpha1-OPO3H2; 6 HOSO3-4Fuc alpha1-6GlcNAcbeta1-NAsn. While 2 and 3 have been previously characterized as compounds of urine and hemofiltrate, the oligosaccharide alpha1-phosphates 1, 4, and 5 could be isolated--to our knowledge--for the first time from biological material. Compound 6 is the first glycopeptide reported to contain a 4-sulfated fucose residue.

The asparagine-linked oligosaccharides at individual glycosylation sites in human thyrotrophin.

The asparagine-linked carbohydrate structures at each of the three glycosylation sites of human thyrotrophin were investigated by 400 MHz 1H-NMR spectroscopy. Highly purified, biologically active human thyrotrophin (hTSH) was dissociated into its subunits hTSH alpha (glycosylated at Asn 52 and Asn 78) and hTSH beta (glycosylated at Asn 23). The alpha-subunit was further treated with trypsin which gave two glycopeptides that were subsequently separated by reverse-phase HPLC and identified by amino acid sequence analysis. The oligosaccharides were liberated from hTSH alpha glycopeptides and from intact hTSH beta by hydrazinolysis, and were fractionated as alditols by anion-exchange and ion-suppression amine-adsorption HPLC preparatory to structural analysis. The N-glycans present on hTSH were mainly diantennary complex-type structures with a common Man alpha 1-3 branch that terminated with 4-O-sulphated GalNAc. The Man alpha 1-6 branch displayed structural heterogeneity in the terminal sequence, with chiefly alpha 2-3-sialylated Gal and/or 4-O-sulphated GalNAc. The relative amounts of the two major complete diantennary oligosaccharides and their core fucosylation differed according to glycosylation site; the sulphated/sialylated diantennary oligosaccharide was most abundant at the two sites on the alpha-subunit, whereas the disulphated, core-fucosylated oligosaccharide was more plentiful on the beta-subunit. Some interesting structural features, not previously reported for the N-glycans of hTSH, included 3-O-sulphated galactose (SO4-3Gal) and peripheral fucose (Fuc alpha 1-3GlcNAc) in the Man alpha 1-6 branch of some diantennary structures; the former suggests the presence of a hitherto uncharacterized galactose-3-O-sulphotransferase in thyrotroph cells of the human anterior pituitary gland.

Site-specific N-glycosylation of human chorionic gonadotrophin--structural analysis of glycopeptides by one- and two-dimensional 1H NMR spectroscopy.

Glycopeptides representing individual N-glycosylation sites of the heterodimeric glycoprotein hormone human chorionic gonadotrophin (hCG) were obtained from subunits hCG alpha (N-glycosylated at Asn-52 and Asn-78) and hCG beta (N-glycosylated at Asn-13 and Asn-30) by digestion with trypsin and chymotrypsin, respectively. Following purification by reverse-phase HPLC and identification by amino acid sequencing, the glycopeptides were analysed by one- and two-dimensional 1H NMR spectroscopy. The results are summarized as follows: (i) oligosaccharides attached to Asn-52 of hCG alpha comprised monosialylated 'monoantenary' NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3[Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc (N1-4'), disialylated diantennary NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3[NeuAc alpha 2-3-Gal beta 1-4GlcNAc beta 1-2Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc (N2), and the monosialylated hybrid-type structures NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3[Man alpha 1-3Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc (N1-A) and NeuAc alpha 2-3Gal-beta 1-4GlcNAc beta 1-2Man alpha 1-3[Man alpha 1-3(Man alpha 1-6)Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc (N1-AB) in a ratio approaching 5:2:2:1; (ii) Asn-78 of hCG alpha carried N2 and N1-4' almost exclusively (ratio approximately 3:2); (iii) both N-glycosylation sites of hCG beta contained predominantly component N2, partially (approximately 25%) and completely alpha 1-6-fucosylated at the N-acetylglucosamine linked to Asn-13 and Asn-30, respectively. The distinct site-specific distribution of the oligosaccharide structures among individual N-glycosylation sites of hCG appears to reflect primarily the influence of the surrounding protein structure on the substrate accessibility of the Golgi processing enzymes alpha-mannosidase II, GlcNAc transferase II and alpha 1,6-fucosyltransferase.

NMR investigations of the N-linked oligosaccharides at individual glycosylation sites of human lutropin.

Human lutropin or luteinizing hormone (hLH) is a heterodimeric glycoprotein, composed of two subunits. hLH alpha (N-glycosylated at Asn52 and Asn78) and hLH beta (N-glycosylated at Asn30). The sugar chains were liberated by hydrazinolysis from intact hLH beta and from glycopeptides obtained after tryptic digestion of hLH alpha, subsequently reduced and fractionated as alditols by anion-exchange and ion-suppression amine-adsorption HPLC and identified mainly by one-dimensional (1D) and two-dimensional (2D) 1H-NMR spectroscopy. The results indicate predominantly diantennary. N-acetyllactosamine-type structures at all three glycosylation sites. The oligosaccharides attached to Asn52 (hLH alpha) and Asn30 (hLH beta) show a remarkably similar pattern, with mainly chain-terminating 4-sulphated 2-deoxy-2-N-acetylamino-D-galactose (GalNAc) and a sulphated/sialylated structure as the major single component. However, virtually all N-glycans on the beta subunit bear a fucose residue alpha 1-6-linked to the proximal GlcNAc, whereas those at Asn52 (and Asn78) of the alpha subunit are predominantly non-fucosylated. The oligosaccharides at Asn78 (hLH alpha) are sialylated rather than sulphated and contain the unique sequence NeuAc alpha 2-6 GalNAc beta 1-4GlcNAc beta 1-2 Man alpha 1-3 as part of the majority of mono- and disialylated compounds. The major single constituent at Asn78 has the following structure: [formula, see text]

Fast-atom-bombardment mass spectrometry of sulphated oligosaccharides from ovine lutropin.

The positive- and negative-ion f.a.b.-mass spectra and the fragmentation of sulphated oligosaccharides derived from ovine lutropin are described. Negative-ion f.a.b.-m.s. of methylated derivatives offers a sensitive and rapid method for screening glycans for sulphation, for defining the location of sulphated residues, and for sequencing sulphated branches. Positive-ion f.a.b.-m.s. gives complementary data on non-sulphated branches in both complex and hybrid-type sulphated structures.

Site-specific N-glycosylation of ovine lutropin. Structural analysis by one- and two-dimensional 1H-NMR spectroscopy.

The Asn-linked carbohydrate structures of the heterodimeric glycoprotein hormone lutropin from ovine pituitary glands have been investigated at each of its three glycosylation sites using one- and two-dimensional 400-MHz 1H-NMR spectroscopy. Highly purified, biologically active ovine lutropin (oLH) was dissociated and separated into its alpha and beta subunits (oLH alpha, glycosylated at Asn56 and Asn82; oLH beta glycosylated at Asn13). Oligosaccharides from intact oLH beta and from glycopeptides obtained after tryptic digestion of oLH alpha were released by hydrazinolysis and subsequently fractionated according to charge and size by anion-exchange and ion-suppression amine-adsorption HPLC, respectively. 1H-NMR analysis revealed, that monosulphated, mostly hybrid-type, oligosaccharides predominate at both glycosylation sites of oLH alpha, whereas a disulphated, diantennary N-acetyllactosamine-type structure accounts for more than 60% of total oligosaccharides in the beta subunit. Furthermore, the saccharides attached to the beta subunit are almost completely fucosylated (Fuc alpha 1-6) at the reducing terminal GlcNAc, whereas the sugar chains in oLH alpha are either approximately 50% fucosylated (Asn82) or contain fucose only to a minor extent (Asn56). The results clearly indicate a distinct subunit- and site-specific synthesis of oligosaccharides in ovine lutropin and suggest that biosynthesis is effectively influenced by the surrounding polypeptide chain(s) at a given site.

Isolation of unusually composed sialyl-compounds from hemofiltrate.

Sialyl compounds are essential components of various biological fluids but relatively little is known about their occurrence in the extracellular fluid of patients with end-stage renal disease. As we have developed a macropreparative method for concentrating and desalting a wide range of fractions from diluted biological fluids we have been able to isolate and identify 5 sialooligosaccharides, 3 sialosugarphosphates, 2 monosialoglycopeptides and 1 disialoglycopeptide. The structures have been elucidated predominantly by one and two-dimensional NMR spectroscopy, enzymatic degradation and FAB mass spectrometry. The accumulation of these compounds in uremic sera may be of particular interest as they may interact in the molecular biology of diseases typically associated with the uremic state, e.g., immune deficiency, neurological disorders, receptor binding abnormalities, complement system disturbances and cell membrane alterations.

[Cleavage and synthesis of sialic acids with aldolase. NMR studies of stereochemistry, kinetics, and mechanisms]. / Spaltung und Synthese von Sialinsäuren mit Aldolase. 1H-NMR-Untersuchungen zur Stereochemie, zur Kinetik und zum Mechanismus.

1H-NMR spectroscopy was used to study cleavage and synthesis of N-acetyl- and N-glycoloyl-D-neuraminic acid by Clostridium perfringens aldolase. Whereas the alpha-anomers of Neu5Ac and Neu5Gc serve as substrate in the cleavage reaction, alpha-ManNAc and alpha-ManNGc are its primary products. The same alpha-anomers are needed by the aldolase for the synthesis of Neu5Ac and Neu5Gc. During the enzyme reaction in D2O both H-atoms at C-3 of Neu5Ac are exchanged by deuterium, H-3e reacting faster than H-3a. Rate constants and concentrations at equilibrium of reactants are temperature- and pH-dependent: The amount of Neu5Ac in equilibrium increases with decreasing temperature and increasing pH-value. Based on these results a mechanism of aldolase action is discussed.

[Sialic acid containing compounds in the hemofiltrate of patients with chronic renal insufficiency. II. Isolation and structure determination of sialoglycopeptides]. / Sialinsäurehaltige Verbindungen im Hämofiltrat von Patienten mit chronischer Niereninsuffizienz, II. Isolierung und Strukturaufklärung von Sialoglycopeptiden.

Four sialyl glycopeptides have been isolated from the hemofiltrate of a patient with end stage renal disease using reverse osmosis, gel filtration and ion-exchange chromatography. Structural studies including one- and two-dimensional 1H-NMR spectroscopy, FAB mass spectrometry and enzymatic degradation indicated the following structures: (Formula: see text). While the disialyl glycopeptide has been previously characterized from normal and pregnancy urine, the three other sialyl glycopeptides could be isolated for the first time from biological material. The origin of these compounds and their possible clinical relevance is subject to further investigations.

[Isolation and NMR spectroscopic characterization of sialic acid compounds and hemofiltrate of chronic uremia patients]. / Isolierung und NMR-spektroskopische Charakterisierung sialinsäurehaltiger Verbindungen aus dem Hämofiltrat chronisch urämischer Patienten.

Eight sialyloligosaccharides have been isolated from the hemofiltrate of a patient with end stage renal disease using reverse osmosis, gel filtration, ion-exchange and high-performance liquid chromatography. The structures were predominantly elucidated by one- and two-dimensional 1H- and 13C-NMR spectroscopy: 1 NeuAc alpha 2-3Gal beta 1-4Glc; 2 NeuAc alpha 2-6Gal beta 1-4Glc; 3 NeuAc alpha 2-3Gal beta 1-4GlcNAc; 4 NeuAc-alpha 2-6Gal beta 1-4GlcNAc; 5 NeuAc alpha 2-3Gal beta 1-4-GlcNAc alpha 1-P; 6 NeuAc alpha 2-6Gal beta 1-4GlcNAc alpha 1-P; 7 NeuAc alpha 2-3Gal beta 1-3GalNAc alpha 1-P; 8 NeuAc alpha 2-8NeuAc. While compounds 1-7 are also components of normal human urine, di-N-acetyl-D-neuraminic acid (8) could be isolated for the first time from biological material. The origin and possible clinical relevance of these compounds have to be proved in further investigations.

[H-NMR spectroscopy. Specificity of microbial sialidases against complex substrates]. / 1H-NMR-Spektroskopie. Spezifität mikrobieller Sialidasen gegenüber komplexen Substraten.

The specificities of one viral and five bacterial sialidases were investigated by 1H-NMR-spectroscopy with substrates or substrate mixtures containing two sialic acid residues of different linkage types. This technique allows - in contrast to the methods used before - the simultaneous determination of the rates of hydrolysis of both NeuAc linkages in a single experiment. The substrate specificities of the enzymes are discussed on the basis of the relation of the rate constants k/k'. The data obtained are more exact and more informative than those of separate experiments as reported previously. Among the enzymes investigated, i.e. sialidases of fowl plague virus (FPV = VKH), Clostridium perfringens (CP), Vibrio cholerae (VC), Bifidobacterium bifidum var. pennsylvanicum (BBif), Bifidobacterium lactentis (BLac), and Arthrobacter ureafaciens (AU), the activity of the viral sialidase VKH shows the highest, the activities of the Bifidobacterium sialidases the lowest dependence on the nature and on the linkage type of the different substrates. All sialidases preferentially cleave the NeuAc alpha 2-3-Gal linkage with the exception of the enzyme of Arthrobacter ureafaciens (AU) which shows a higher affinity to alpha 2-6 linkages. However, this does not apply to the side-arm-linked NeuAc alpha 2-6 structure in NeuAc alpha 2-3 Gal beta 1-3 (NeuAc alpha 2-6)-GlcNAc beta 1-3Gal beta 1-4Glc (Substrate B). This substrate in generally cleaved very slowly and is hardly affected by the viral enzyme. After the alpha 2-3 linkage, the alpha 2-8 bond in NeuAc alpha 2-8 NeuAc alpha 2-3 Gal beta 1-4Glc(Substrate A) is most susceptible for the sialidases VKH, CP and VC. An elongation of the carbohydrate chain (Substrate D) is accompanied by a reduction of the rate of cleavage for all enzymes. The experiments with alpha 1-acid glycoprotein, fetuin, and with the glycopeptides obtained by proteolytic degradation of the latter, revealed the same specificity towards the alpha 2-3 and the alpha 2-6 linkages as the oligosaccharides. Influenced by the chemical nature and the size of the substrate, NeuAc is released from the native alpha 1-acid glycoprotein more quickly than from the corresponding glycopeptide. All sialidases investigated so far are strictly exo-enzymes as could be demonstrated by the cleavage of NeuAc alpha 2-8 NeuAc alpha 2-3 Gal beta 1-4Glc (Substrate A).