Rajendra Varma

Simultaneous determination of neutral sugars and hexosamines in glycoproteins and acid mucopolysaccharides (glycosaminoglycans) by gas-liquid chromatography

A reliable and reproducible method for the simultaneous determination of neutral sugars and hexosamines in glycoproteins and acid mucopolysaccharides has been described. It involves the following steps: the release of neutral sugars and hexosamines from biopolymers by resin-catalysed hydrolysis, the nitrous acid deamination of resin-bound hexosamines in this hydrolysate to aldoses, and the determination of these newly formed anhydroaldoses together with the pre-existing neutral sugars as aldononitrile acetates by gas chromatography. Applications of this method to analyses of glycoprotein from urine of schizophrenic patients, bovine thyroglobulin, human brain glycoprotein, chondroitin sulfates from bovine vitreous humor, and veal brain, and human umbilical cord hyaluronic acid are given.

Conformational analysis of peracetylated hexononitriles

Abstract The conformations of six peracetylated hexononitriles in solution have been investigated by Fourier-transform, proton n.m.r. spectroscopy at 90 MHz, with iterative analysis and simulation of many of the spectra. The conformation of tetra- O -acetyl- L -arabinononitrile has been re-examined by the same methods. A shift reagent [Eu(fod) 3 - d 30 ] and spectra at 220 MHz were used to improve spectral dispersion, where necessary. For practically all of the derivatives studied, the vicinal, proton-proton coupling-constants are consistent with a zigzag conformation in which the cyano group lies in the plane of the other carbon atoms of the chain, unless this conformation contains a parallel 1,3-interaction of substituents. Other conformers that are also consistent with the coupling constants observed are discussed, including rotamers about chain-terminal, carbon-carbon bonds.

Mechanism of the cyanohydrin (Kiliani-Fischer) synthesis

Abstract Polarimetry, paper chromatography, gas-liquid chromatography, and infrared and nuclear magnetic resonance spectroscopy have been used in the elucidation of the mechanism of the cyanohydrin synthesis as applied to D -arabinose. D -Arabinose was allowed to react with various proportions of potassium cyanide in aqueous sodium carbonate, and various compounds formed in the reaction mixture were identified. Polarimetry indicated that the cyanohydrin synthesis involves at least two steps: presumably, a condensation and a hydrolysis. Paper and gas-liquid chromatography showed the presence of D -gluconate, D -mannonate, D -gluconolactone, D -mannonolactone, D -gluconamide, D -mannonamide, D -gluconoimidolactone, D -mannonoimidolactone, D -glucononitrile, D -mannononitrile, and unchanged D -arabinose. The main pathway suggested by the time course of the intermediates is: D -arabinose→ D -hexononitrile (cyanohydrin) → D -hexonoimidolactone → D -hexonolactone → D -hexonic acid. The acyclic hexonamide, usually postulated as the initial hydrolysis product of the cyanohydrin, is probably only a side product that is in equilibrium with the cyclic hexonoimidolactone.

A simple procedure for combined gas chromatographic analysis of neutral sugars, hexosamines and alditols. Determination of degree of polymerization of oligo- and polysaccharides and chain weights of glycosaminoglycans.

A reliable and reproducible method that allows the combined, simultaneous gas chromatographic (GC) determination of neutral sugars, hexosamines, alditols, identification and quantitation of the reducing aldose end-group in oligo and polysaccharides and glycosaminoglycans has been described. It involves the following steps: release of the reducing end-group from its protein linkage in glycosaminoglycans and reduction of this reducing end-group into alditol, release of the components of the reduced polymer by resin-catalysed hydrolysis, nitrous acid deamination of the resin-bound hexosamines in this hydrolysate into anhydroaldoses and a combined derivatization and GC determination of the neutral sugars as aldononitrile acetates, anhydroaldoses as peracetylated oximes and alditols as alditol acetates. Application of the method to determination of degree of polymerization of oligo-and polysaccharides and chain weights of proteoglycans has been described. This method has several advantages over the previous methods.

Serum glycoproteins in schizophrenia

A comparative investigation of the individual protein-bound carbohydrate components and the distribution of carbohydrates in the electrophoretic glycoprotein fractions was carried out in sera from 20 normal subjects and 30 schizophrenic patients matched for age and sex. The mean concentration of each of the protein-bound carbohydrate components was significantly elevated in schizophrenics. The electrophoretic patterns for serum glycoprotein showed increases in alpha-2 and beta globulins in schizophrenics. The serum glycoproteins contained glucose and L-arabinose, in addition to mannose, galactose, fucose, sialic acid, and a trace of xylose. The identity of glucose and arabinose was confirmed by g.l.c.-electron-impact mass spectrometry and by specific enzymic reactions. The contents of glucose and arabinose were higher in serum glycoproteins from schizophrenic patients. This elevation of serum glycoprotein paralleled serum glycosaminoglycan elevation previously reported by us, but was opposite to decrease of urinary glycoprotein in schizophrenics.

On arabinose as a component of brain hyaluronate. Confirmation by chromatographic, enzymatic and chemical ionization-mass spectrometric analyses.

The controversy about the presence of the pentose arabinose in brain hyaluronate was reinvestigated using modern analytical tehcnics. The purified bovine brain hyaluronate contained the neutral sugars: arabinose, 0.18%; glucose, 0.05%; and fucose, 0.22%. The confirmation of the presence of arabinose was obtained by paper and thin layer chromatography of the neutral sugars in deionized hyaluronate hydrolysate. Gas-liquid chromatography of the aldononitrile peracetate of the pentose isolated by preparative paper chromatography gave a single distinct peak, corresponding to standard arabinose on three columns packed with three different phases. Chemical ionization data and mass spectrum of the aldononitrile peracetate derivative agreed with those of the authentic arabinonitrile tetracetate. Analysis of the isolated pentose with the help of the enzymes L-arabinose isomerase and L-ribulose kinase, which are specific for their substrates, further established its identity as L-arabinose.

On the carbohydrate—protein linkage group in vitreous humor hyaluronate

Abstract Fractional molar ratios of serine, threonine and aspartic acid to neutral sugars in the purified bovine vitreous humor hyaluronate, and a 4–5-fold increase in the percentage of these amino acids and the absence of sugar alditols in hyaluronate reduced with NaBH4PdCl2 after alkali treatment indicated the absence of a carbohydrate—protein linkage. Gel filtration behavior, a decrease in intrinsic viscosity of reduced hyaluronate to about one-half and a significant decrease in its specific rotation suggested that the two antiparallel chains of the hyaluronate double helix may come apart upo reduction. The vitreous humor hyaluronate contained 109.2 ppm of “bound” silicon. It is suggested that the bound silicon may bridge the two antiparallel chains through the neutral sugars and/or through the hydroxyl group of the uronic acid moiety.

Human umbilical cord hyaluronate. Neutral sugar content and carbohydrate-protein linkage studies.

Paper chromatography of neutral sugars and gas chromatography of their aldononitrile acetates indicated the presence of fucose, arabinose and a small amount of glucose in purified human umbilical cord hyaluronate. The molar ratios of serine, threonine and aspartic acid to neutral sugars were not unity, suggesting the non-involvement of the neutral sugars and the amino acids in a carbohydrate-protein linkage. The same was indicated by an increase in the percentage of the aforementioned amino acids and by the absence of sugar alditols in umbilical cord hyaluronate reduced eith NaBH4 -PdCl2, after alkali treatment. This reduction caused a decrease in the intrinsic viscosity and molecular wieght to about one-half and an appreciable decrease in the specific rota tion of hyaluronate, suggesting a separation of the two antiparallel chains o the double helical hyaluronate. The umbilical cord hyluronate containe contained bound silicon and it is possible that this bound silicon may cross-link the two chains at interspersed intervals through the uronic acid moiety and/or through neutral sugars.

Urinary excretion of acid mucopolysaccharides in schizophrenia

Abstract 1. (1) The 24-hr urinary excretion of acid mucopolysaccharides measured as glucuronic acid was found to be significantly lower in male schizophrenics as compared to normal male subjects of a corresponding age group. 2. (2) No hyaluronic acid was detected in normal urine. About one-third of total AMPS was present as hyaluronic acid in schizophrenic urine. 3. (3) Hyaluronic acid in schizophrenic urine was identified by electrophoresis, ion-exchange chromatographic behavior, infrared spectroscopy and by the presence of glucosamine in its hydrolysate.

Urinary glycoproteins in schizophrenia.

Abstract A comparative study of the urinary glycoprotein from 30 normal and 60 schizophrenic male subjects of the same age group revealed three remarkable features: (1) a significant decreased excretion of glycoprotein in 24-hr urine of schizophrenic patients, (2) the presence of a significant amount of protein-bound rhamnose in schizophrenic urine in the form of a small molecular weight glycoprotein, and (3) a significantly higher glucosamine/galactosamine ratio in patients urine glycoprotein along with other differences in molar ratios of other classes of carbohydrate components. Since the amounts and types of glycoproteins and glycosaminoglycans of urine, in general, reflect the metabolism of these macromolecules in connective tissue, the synchronicity in decreased excretion and of higher glucosamine/galactosamine ratios in both glycoproteins observed in this study and also in glycosaminoglycans, reported in our previous study (27) in urine of schizophrenic patients, probably results from an altered metabolism involving a stress on metabolism of glucosamine-containing macromolecules in the connective tissue of the schizophrenic patients.