Periodate-thiobarbituric Acid Research Articles

Clinical evaluation of total and lipid bound sialic acid levels in oral precancer and oral cancer

Aims:Special attention has been given to define the biochemical changes in cell-surface glycoproteins and glycolipids that take place during malignant transformation. This study was thus designed to explore the clinical utility of total and lipid bound sialic acid (LSA) in patients with oral precancer and oral cancer.Materials and Methods:Blood samples were obtained from 95 subjects divided into three groups, namely healthy individuals, oral cancer, and precancer. Serum total and LSA levels were determined using periodate-thiobarbituric acid method and Katopodis et al. method, respectively. Finally, spectrophotometricreadings were obtained.Results:Mean values of serum sialic acid (total and lipid bound) in oral cancer were significantly higher than control and the precancer group (P<0.001). The multivariate analysis data revealed progressive rise in total and LSA, which was statistically significant with clinical stage of the cancer.Conclusion:Serum sialic acid levels can differentiate between patients with oral precancer and oral cancer. It could be used as an adjunct to diagnosis, monitor response to therapy, and assess the staging of cancer.

Structure and Mechanism of 3-Deoxy-d-manno-octulosonate 8-Phosphate Synthase

3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the condensation of phosphoenolpyruvate (PEP) with arabinose 5-phosphate (A5P) to form KDO8P and inorganic phosphate. KDO8P is the phosphorylated precursor of 3-deoxy-D-manno-octulosonate, an essential sugar of the lipopolysaccharide of Gram-negative bacteria. The crystal structure of the Escherichia coli KDO8P synthase has been determined by multiple wavelength anomalous diffraction and the model has been refined to 2.4 A (R-factor, 19.9%; R-free, 23.9%). KDO8P synthase is a homotetramer in which each monomer has the fold of a (beta/alpha)(8) barrel. On the basis of the features of the active site, PEP and A5P are predicted to bind with their phosphate moieties 13 A apart such that KDO8P synthesis would proceed via a linear intermediate. A reaction similar to KDO8P synthesis, the condensation of phosphoenolpyruvate, and erythrose 4-phosphate to form 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P), is catalyzed by DAH7P synthase. In the active site of DAH7P synthase the two substrates PEP and erythrose 4-phosphate appear to bind in a configuration similar to that proposed for PEP and A5P in the active site of KDO8P synthase. This observation suggests that KDO8P synthase and DAH7P synthase evolved from a common ancestor and that they adopt the same catalytic strategy.

α-KDOase Activity in Oyster and Synthesis of α- and β-4-Methylumbelliferyl Ketosides of 3-Deoxy-d-manno-octulosonic Acid (KDO)

Although alpha- and beta-linked 3-deoxy-D-manno-octulosonic acid (KDO) is found in lipopolysaccharides (LPSs) of Gram-negative bacteria, capsular polysaccharides of microorganisms, and plants, very little is known about its degradation. Using both thin-layer chromatography and the periodate-thiobarbituric acid reaction, we found that the hepatopancreas of oyster (Crassostrea virginica) contained an enzyme (alpha-KDOase) capable of releasing alpha-linked KDO from LPSs. To facilitate the studies of alpha-KDOase, we have carried out the synthesis of 4-methylumbelliferyl-alpha-KDO (alpha-KDO-MU) by conjugating the glycosyl chloride of the per-O-acetylated methylester of KDO with methylumbelliferone by the SN2 type reaction and the catalyzed phase-transfer. In both cases, the beta-anomer was obtained as the major product with a yield of about 80%, whereas the yield of alpha-anomer was only about 7%. Attempts to increase the yield of alpha-anomer were not successful. alpha-KDO-MU was used as substrate to follow the purification of alpha-KDOase from oyster hepatopancreas. The pH optimum for oyster alpha-KDOase was determined to be 4.5 using Re-LPS as substrate and 3.0 using alpha-KDO-MU as substrate. The enzyme was found to be stable in the pH range of 3-8. This enzyme released KDO from different LPSs, including Re-LPS from Escherichia coli and Salmonella minnesota, Rd-LPS from S. minnesota, and de-O-acyl-Re-LPS (Kiang, J., Szu, S. C., Wang, L.X., Tang, M., and Lee, Y. C. (1997) Anal. Biochem. 245, 97-101).

Analysis of the enzymatic cleavage (beta elimination) of the capsular K5 polysaccharide of Escherichia coli by the K5-specific coliphage: reexamination.

The capsular K5 polysaccharide of Escherichia coli is the receptor of the capsule-specific coliphage K5, which harbors an enzyme that degrades the capsular K5 polysaccharide to a number of oligosaccharides. Analysis of the degradation products using gel permeation chromatography, the periodate-thiobarbituric acid and bicinchoninic acid reactions, and nuclear magnetic resonance spectroscopy showed that the major reaction products are hexa-, octa-, and decasaccharides with 4,5-unsaturated glucuronic acid (delta4,5GlcA) at their nonreducing end. Thus, the bacteriophage enzyme is a K5 polysaccharide lyase and not, as we had reported previously, an endo-N-acetylglucosaminidase.

Sialic acid and oxidizability of low density lipoprotein subfractions of hyperlipidemic patients

Objectives: Low density lipoprotein (LDL) does not constitute an homogenous fraction and it is known that the heavy LDL subfraction is potentially more atherogenic than the light one. Because concentration of LDL. subfractions tend to be different in hyperlipidemias, it was verified whether these subfractions can also differ in sialic acid and neutral sugar content, as well as their resistance to oxidation. Design and Methods: Two subfractions of low density lipoprotein (light LDL, density 1.019–1.034 g/mL and heavy LDL, density 1.034–1.063 g/mL) were isolated from the plasma of 17 patients with hypercholesterolemia, 11 with combined hyperlipidemia, 7 with hypertriglyceridemia, and 19 normolipidemic subjects. The content of sialic acids and neutral sugars of apo B was determined, respectively, by the periodate-thiobarbituric acid method and by reaction with phenol. The oxidation of LDL subfractions was determined by exposure to 5 μM copper (II) followed by the measurement of lipid hydroperoxides production by high performance liquid chromatography with electrochemical detection. Results: The study groups did not differ in the neutral sugar content of LDL subfractions. However, compared to normolipidemic subjects, the sialic acid concentration of both LDL subfractions was lower in patients with hypercholesterolemia and hypertriglyceridemia and higher in those with combined hyperlipidemia ( p < 0.05). In the hypercholesterolemia and combined hyperlipidemia groups, the lipid hydroperoxide content (μM) of heavy LDL was higher than in normolipidemic subjects ( p < 0.05). Conclusions: The heavy LDL subfraction was more susceptible to oxidation in the patients with combined hyperlipidemia compared to controls and the other hyperlipidemic groups. The effect of sialic acids on heavy LDL oxidizability seems to vary according to the type of hyperlipidemia.

A chemotaxonomic study of Vibrio fluvialis based on the sugar composition of the polysaccharide portion of the lipopolysaccharides.

A chemotaxonomic study was carried out with a new serotyping scheme comprising 35 O-antigen groups of Vibrio fluvialis on the basis of the sugar composition of the polysaccharide portion of their lipopolysaccharide (LPS). A previously developed rapid method of preparing samples for compositional sugar analysis was employed. The 35 O-antigen groups were divided into 21 chemotypes. It is noted that a rarely occurring component sugar of gram-negative bacterial LPS, D-glycero-D-manno-heptose, and two kinds of uronic acids, i.e., galacturonic acid of a weakly bound type and glucuronic acid of a strongly bound type, were found in common in all the 21 chemotypes. A characteristic sugar component of gram-negative bacterial LPS, 2-keto-3-deoxyoctonate (KDO), was not detectable in any of the 21 chemotypes. Instead, three kinds of "KDO-like substances" were found, one in each of three chemotypes (III, XI and XVII). They were strongly positive in Weissbach's periodate-thiobarbituric acid test for KDO, but definitely not identical to it in high-voltage paper electrophoresis (HVPE); the substance present in chemotype XI was indicated by HVPE to be 3-deoxy-D-threo-hexulosonic acid which is a sugar constituent of Vibrio parahaemolyticus O7 and O12 LPS.

Lipopolysaccharide isolated from a new O-antigenic form (O13) of Vibrio parahaemolyticus.

The chemical properties of a lipopolysaccharide (LPS) isolated from a new O-antigenic form (O13) of Vibrio parahaemolyticus were investigated. The LPS contained glucose, galactose, L-glycero-D-manno-heptose and glucosamine. 2-Keto-3-deoxy-octonate (KDO) was not detected in the LPS by the periodate-thiobarbituric acid test (Weissbach's reaction) under conventional hydrolysis conditions. Instead, phosphorylated KDO (X1 and X2) was found in its strong-acid hydrolysate. This sugar composition was identical to that of V. parahaemolyticus O3, O5 and O11 LPS, indicating that, based on the sugar composition, O13 LPS belongs to Chemotype III to which O3, O5 and O11 belong. In addition, structural study demonstrated the presence of KDO 4-phosphate in its inner-core region.

Taxonomic implication of the apparent undetectability of 3-deoxy- [formula omitted]- manno-2-octulosonate (Kdo) in lipopolysaccharides of representatives of the family vibrionaceae and the occurrence of Kdo 4-phosphate in their inner-core regions

After conventional hydrolysis of lipopolysaccharides (LPSs), Kdo was not detectable by the periodate-thiobarbituric acid test in those of any member of Vibrionaceae except the genus Plesiomonas, but phosphorylated Kdo was demonstrated after strong-acid hydrolysis. dephosphorylation, periodate oxidation, and methylation analysis of LPS preparations from 7 strains selected from all genera of Vibrionaceae, except Plesiomonas, showed that the inner-core region (unlike that in enteric Gram-negative bacteria) contains only one molecule of Kdo 4-phosphate 5-substituted with heptose, a constituent of the distal part of the core region, as in enteric bacteria. The undetectability of Kdo in LPS after conventional hydrolysis and the occurrence of phosphorylated Kdo in strong-acid hydrolysates and of Kdo 4-phosphate in the inner-core region are taxonomic characteristics of the family Vibrinaceae.

Detection and Localisation of Sialic Acid inGardnerella vaginalis, Mobiluncusspp., and Other Vaginal Microorganisms

Sialic acid, n-acetylneuraminic acid, is found in the cell wall of all serotypes of group B Streptococcus and Escherichia coli serotypes K1 and K3. It has been most strongly associated with those strains which cause neonatal meningitis. The purpose of this study is to report the presence of sialic acid in some genital tract bacteria. Bacteria were grown to log phase. Mild-acid hydrolysis of whole organisms released a compound which reacted positively in the periodatethiobarbituric acid (PTA) assay, a colorimetric assay detecting n-acetylneuraminic acid. Surface localisation of sialic acid was supported by the observation of bacterial agglutination with an affinity-purified lectin ( Limulus polyphemus agglutinin) which binds sialic acid residues. Four vaginal isolates of Mobiluncus curtisii and three vaginal isolates of Mobiluncus mulieris strains tested contained a level of sialic acid equivalent to that of the group B Streptococcus control by PTA assay. Four Gardnerella vaginalis strains (three vaginal and two placental isolates) were positive for sialic acid by PTA assay. Nine clinical vaginal isolates of Peptostreptococcus : P. anaerobius (2), P. asaccharolyticus (3), P. magnus (2) and P. tetradius (2) were negative for sialic acid. Two clinical vaginal isolates of Enterococcus faecalis and four diphtheroid isolates were also negative. Eleven clinical vaginal isolates of viridans streptococci including S. intermedius (5) , S. constellatus (4), and S. acidominimus (2) were negative for sialic acid. One of three clinical vaginal isolates of S. acidominimus , one of four clinical vaginal isolates of coagulase negative staphylococci and two of seven clinical vaginal isolates of lactobacilli tested positive for low levels of sialic acid. All strains in which sialic acid was detected by PTA assay showed surface localisation by lectin agglutination. The presence of sialic acid on the surface of Gardnerella and Mobiluncus , two organisms associated with bacterial vaginosis, may be associated with their capacity to colonise or infect the genital tract. Keywords: Sialic acid; Mobiluncus spp.; Gardnerella vaginalis ; vaginal bacteria.

Alginase treatment of mucoid Pseudomonas aeruginosa enhances phagocytosis by human monocyte-derived macrophages.

Mucoid Pseudomonas aeruginosa colonizes and infects the respiratory tract of most older patients with cystic fibrosis. These bacteria resist both opsonin-dependent and -independent phagocytosis by human polymorphonuclear leukocytes and monocyte-derived macrophages. Resistance to phagocytosis is thought to be mediated in part by the mucoid exopolysaccharide associated with the bacterial surface. The purpose of this study was to determine whether degradation of the mucoid exopolysaccharide by alginase enhances bacterial susceptibility to nonopsonic phagocytosis by macrophages. Eight phagocytosis-resistant mucoid P. aeruginosa isolates from patients with cystic fibrosis were studied. The bacteria were treated with a preparation of alginase from Bacillus circulans, and phagocytosis by macrophages was measured by a visual inspection assay. Alginase degradation of mucoid exopolysaccharide was measured by the periodate-thiobarbituric acid assay and by indirect immunofluorescence with a mouse monoclonal antibody to the mucoid exopolysaccharide. Alginase degraded the mucoid exopolysaccharide of all eight mucoid strains tested. Phagocytosis was enhanced in five of the eight strains. Alginase-enhanced phagocytosis was magnesium dependent and heat labile. Alginase may be a useful tool for studying the biological properties of P. aeruginosa mucoid exopolysaccharide.

Occurrence of O-phosphorylated 2-keto-3-deoxyoctonate in the lipopolysaccharide ofBacteroides gingivalis

Abstract Periodate-thiobarbituric acid reaction-positive substances were found in the strong acid hydrolysates of the lipopolysaccharide (LPS) from Bacteroides gingivalis 381. They were not identical to 2-keto-3-deoxyoctonate (KDO) in high-voltage paper electrophoresis (HVPE), their electrophoretic mobilities relative to KDO being 1.54 and 1.80, respectively. Alkaline phosphatase treatment and HVPE demonstrated that they are some kind of O-phosphorylated derivatives of KDO; in particular, the slow-moving component is identical, at least in HVPE, to 5-O-phosphoryl-KDO isolated from the strong acid hydrolysates of Bordetella pertussis (phase I) LPS.

Surface localization of sialic acid on Actinomyces viscosus.

This study reports the presence of sialic acid in Actinomyces viscosus strains T14V and T14AV. Mild acid hydrolysis of whole organisms released a compound which reacted positively in the periodate-thiobarbituric acid, direct Ehrlich's and resorcinol assays, and which co-chromatographed on paper with authentic N-acetylneuraminic acid. Strain T14V contained 10-fold greater concentrations of sialic acid than did strain T14AV. Sialic acid content was dependent upon the stage of growth of the culture, reaching a maximum in early stationary phase. Epifluorescence microscopy of fluorescein isothiocyanate (FITC)-conjugated Limulus polyphemus agglutinin (LPA), a lectin specific for sialic acid, revealed a uniform distribution of bound lectin on the surfaces of strains T14V and T14AV. Additional evidence for surface localization was obtained by demonstration of whole-cell agglutination of both strains with LPA. All LPA interactions with A. viscosus were inhibited by the presence of 0.1 M-N-acetylneuraminic acid. Neuraminidases from Clostridium perfringens, Arthrobacter ureafaciens and Vibrio cholerae did not release detectable amounts of sialic acid, but the extracellular enzyme from A. viscosus cleaved amounts equivalent to those obtained by acid hydrolysis. Other laboratory strains (W1053, M100, W859, 5-5S, RC45, ATCC 19246, and 'binder') as well as recent clinical isolates of A. viscosus were agglutinated by LPA and released sialic acid upon mild acid hydrolysis. Surface-available sialic acid has been implicated in the inhibition of alternative complement pathway activation and subsequent opsonophagocytosis. Thus the occurrence of surface sialic acid in A. viscosus may represent a mechanism of pathogenesis for this oral bacterium.

An automated version of the periodate-thiobarbituric acid assay for analysis of Δ-4,5 unsaturated uronic acids and its application to the assay of hyaluronic acid and chondroitin sulfates

An automated periodate-thiobarbituric acid assay of Δ-4,5 unsaturated uronic acids which avoids extraction of chromogen has been developed and applied to the analysis of hyaluronic acid and chondroitin sulfates in standard glycosaminoglycan mixtures and in biological samples following digestion with eliminase enzymes. Assay of hyaluronic acid was linear between 0.1 and 2.5 μg of uronic acid, when digested with hyaluronidase from S. hyalurolyticus and use directly in the automated procedure. The measurement of unsaturated disaccharide standards (25–100 μ m) derived from chondroitin sulfates was also linear although the color yields were different. The proportions of chondroitin sulfate isomers were estimated by assay of the unsaturated chondroitin disaccharides which has been separated by thin-layer chromatography.

Micro Determination of Unsaturated Disaccharide Formed by the Action of Acidic Glycosaminoglycan-Endoeliminases

An improved method is described for the micro determination of acidic glycosaminoglycans after digestion with chondroitinase-ABC and -AC. The determination is based on the color production of D-gluco-4-enepyranosyluronic acid-containing disaccharides produced by the action of chondroitinase-ABC and -AC (acidic glycosaminoglycans-endoeliminase) when the periodate-thiobarbituric acid method is applied to the alpha,beta-unsaturated disaccharides. Suitable conditions for the quantitative assay are described.

Application of thin-layer chromatography for identification of streptomycin.

A method for thin-layer chromatographic identification of streptomycin was investigated. To visualize streptomycin on a thin-layer chromatography plate, the periodate-thiobarbituric acid method was applied. Silica gel plate was better than ion-exchange cellulose or microcrystalline cellulose in giving a compact spot. Practical use of thin-layer chromatography for checking the purity of 3H-labeled streptomycin and preliminary studies on separation of streptomycin from biological material were also reported.

Colorimetric Determination of Streptomycin by Periodate-Thiobarbituric Acid Method

For the purpose of establishing a sensitive method for the determination of streptomycin in organic materials, the colorimetric method reported by Duda was investigated and the two main procedures in the original method were modified. With this modified method, 2.5-60 μg/ml of streptomycin in water was successfully determined and sensitivity of the method was twice that of the original method. The effect of protein coexisting with streptomycin on the colorimetry was examined. In lower concentration, protein suppressed the color development, and the absorbance in 1.5 mg/ml of albumin solution decreased the most and was as low as 80% of the control. On the contrary, in higher concentration of more than 6 mg/ml, protein promoted the reaction and the absorbance in 16 mg/ml of albumin solution was as high as 136% of the control. Thorough elimination of protein from the sample is therefore necessary to obtain an accurate streptomycin value in organic materials, but the technique is not yet established.

22] 2-Keto-3-deoxy-d-gluconate

This chapter discusses 2-keto-3-deoxy-D-gluconate (KDG). 2-Keto-3-deoxy-D-gluconate is prepared by enzymic dehydration of D-gluconate. This reaction can be carried out with a crude, enzyme preparation. The compound is purified on a Dowex 1 formate column and crystallized as K salt. The reaction mixture (350 ml) contains: 25 ml of sodium D-gluconate, 100 ml Tris HC1 buffer, 185 ml of water and 40 ml particle-free extract of Alcaligenes species M250 3 . Incubation is carried out at 30° with occasional shaking. The progress of the dehydration is followed by analyzing small samples for KDG formation by the periodate-thiobarbituric acid 5 or the semicarbazide assay methods. After 15–20 hr of incubation, more than 90% of the D-gluconate was converted to 2-keto-3-deoxy acid. The reaction mixture is then deproteinized by addition of 25 ml of 70% perchloric acid. After centrifugation, the supernatant is neutralized at 0 ° with 10 N KOH. The resulting precipitate of K perchlorate is removed by filtration. Crystallization of the K salt occurs overnight at 4°. The crystals are collected by filtration in the cold, washed with cold ether, recrystallized twice under similar conditions, and dried in vacuo (over CaCl 2 ) for 1 day at 4° and several days at room temperature. Alternatively, KDG can be crystallized as Ca salt. KDG can easily be detected on cellulose thin-layer chromatograms by spraying with either periodate-thiobarbituric acid or o -phenylenediamine. The K salt is probably a monohydrate 1 and is stable for several years when stored dry at ­–12°.

The mechanism of the periodate-thiobarbituric acid reaction of sialic acids.

1. The chromogen formation from N-acetylneuraminic acid in the periodate-thiobarbituric acid reaction was investigated. Measurement of periodate consumption showed an uptake of approx. 3moles/mole of substrate in neutral as well as in strongly acidic solution. Therefore the chromogen beta-formylpyruvic acid is not a direct product of the periodate oxidation; it is presumed to be formed from the true oxidation product, a hexos-5-uluronic acid, by aldol splitting during the reaction in hot acidic solution with thiobarbituric acid. 2. Methyl (methyl beta-l-threo-hexos-4-enepyranosid)uronate, an analogue of the pre-chromogen, has been shown to yield with thiobarbituric acid in acidic solution a pigment exhibiting an identical absorption spectrum and showing the same behaviour on paper chromatography as the pigment obtained from N-acetylneuraminic acid in the periodate-thiobarbituric acid assay. 3. The substitution at C-2 of methoxyneuraminic acid does not inhibit the periodate-thiobarbituric acid reaction. In neutral solution methoxyneuraminic acid is oxidized by periodate to a substance that reacts readily with thiobarbituric acid in acidic solution. When periodate oxidation is attempted in acidic solution, protonation of the amino group protects this group against oxidation, rendering methoxyneuraminic acid negative in the assay systems of Warren (1959a,b) and Aminoff (1959, 1961).

The Sialic Acid Content of Photographic Gelatin

The periodate-thiobarbituric acid method of Warren2 has been used to demonstrate the presence of sialic acid in gelatin. Amounts ranging from 54 to 304 p.p.m. were found in various specimens.