Capillary electrophoresis for the analysis of chondroitin sulfate- and dermatan sulfate-derived disaccharides

High-voltage capillary zone electrophoresis (CZE) has been used for the first time in the analysis of non-, mono-, di-, and trisulfated disaccharides derived from chondroitin sulfate, dermatan sulfate, and hyaluronic acid. These glycosaminoglycans are first depolymerized using polysaccharide lyases. The resulting unsaturated disaccharide products can be detected by their ultraviolet absorbance at 232 nm. Different retention times were obtained for each unsaturated disaccharide analyzed by CZE. The application of a constant voltage across a 70-cm fused silica capillary using a single, simple buffer system resolved an eight-component mixture within 40 min. Quantitation of disaccharides derived from chondroitin sulfate using chondroitin ABC lyase (EC 4.2.2.4) and mixtures of unsaturated disaccharide standards was possible requiring only picogram quantities of sample. The disaccharides examined had a net charge of from -1 to -4 and were resolved primarily on the basis of net charge and secondarily on the basis of charge distribution. Two unsulfated disaccharides both containing the same unsaturated uronic acid residue were analyzed. One was from chondroitin having an N-acetylgalactosyl residue and one from hyaluronate having an N-acetylglycosyl residue. Despite the fact that they differed only by the chirality at one center, these disaccharides were resolved by CZE. CZE is a fast and simple method that represents a powerful new tool for analysis and separation of acidic disaccharide components of glycosaminoglycans.     

Species-specific antibiotic-ribosome interactions: implications for drug development

In the cell, the protein synthetic machinery is a highly complex apparatus that offers many potential sites for functional interference and therefore represents a major target for antibiotics. The recent plethora of crystal structures of ribosomal subunits in complex with various antibiotics has provided unparalleled insight into their mode of interaction and inhibition. However, differences in the conformation, orientation and position of some of these drugs bound to ribosomal subunits of Deinococcus radiodurans (D50S) compared to Haloarcula marismortui (H50S) have raised questions regarding the species specificity of binding. Revisiting the structural data for the bacterial D50S-antibiotic complexes reveals that the mode of binding of the macrolides, ketolides, streptogramins and lincosamides is generally similar to that observed in the archaeal H50S structures. However, small discrepancies are observed, predominantly resulting from species-specific differences in the ribosomal proteins and rRNA constituting the drug-binding sites. Understanding how these small alterations at the binding site influence interaction with the drug will be essential for rational design of more potent inhibitors.     

Structures of Streptococcus pneumoniae hyaluronate lyase in complex with chondroitin and chondroitin sulfate disaccharides. Insights into specificity and mechanism of action

Streptococcus pneumoniae hyaluronate lyase is a surface enzyme of this Gram-positive bacterium. The enzyme degrades hyaluronan and chondroitin/chondroitin sulfates by cleaving the beta1,4-glycosidic linkage between the glycan units of these polymeric substrates. This degradation helps spreading of this bacterial organism throughout the host tissues and facilitates the disease process caused by pneumococci. The mechanism of this degradative process is based on beta-elimination, is termed proton acceptance and donation, and involves selected residues of a well defined catalytic site of the enzyme. The degradation of hyaluronan alone is thought to proceed through a processive mode of action. The structures of complexes between the enzyme and chondroitin as well as chondroitin sulfate disaccharides allowed for the first detailed insights into these interactions and the mechanism of action on chondroitins. This degradation of chondroitin/chondroitin sulfates is nonprocessive and is selective for the chondroitin sulfates only with certain sulfation patterns. Chondroitin sulfation at the 4-position on the nonreducing site of the linkage to be cleaved or 2-sulfation prevent degradation due to steric clashes with the enzyme. Evolutionary studies suggest that hyaluronate lyases evolved from chondroitin lyases and still retained chondroitin/chondroitin sulfate degradation abilities while being specialized in the degradation of hyaluronan. The more efficient processive degradation mechanism has come to be preferred for the unsulfated substrate hyaluronan.     

High pressure liquid chromatographic identification of hyaluronic acid and chondroitin sulphate disaccharides.

In this report we describe a system capable of resolving all of the known unsaturated disaccharides derived from the chondroitin sulphates, dermatan sulphate and hyaluronic acid by chondroitinase digestion. This system is superior to others in that the non-sulphated and mono-, di- and tri-sulphated disaccharides can be separated with good resolution in approximately 40 min in an isocratic solvent. The system employs an amino-cyano silica gel column (Whatman Partisil 5 PAC, 25 cm) and is eluted with an isocratic solvent consisting of 48% (v/v) acetonitrile, 14% (v/v) methanol and 38% (v/v) aqueous buffer. This aqueous buffer contains 0.5 M Tris-HCl, 0.1 M boric acid, 23.4 mM sulphuric acid, pH 8.0. UV absorption is monitored at 229 nm and for most disaccharides as little as 150 ng can be reliably determined. The addition of boric acid to the eluent is essential for good resolution of all components and the addition of low concentrations of sulphuric acid is used to control the elution times of various components. The system was applied to the analysis of glycosaminoglycan standards and excellent agreement with previous compositional analyses was obtained.     

Effect of different monosaccharides and disaccharides on boar sperm quality after cryopreservation

The aim of the present study was to evaluate the cryoprotectant effect of different non-permeating sugars for boar sperm. Pooled semen from three boars was used for the experiments. In the first experiment, the sperm quality of boar sperm cryopreserved with an egg-yolk based extender supplemented with different monosaccharides (glucose, galactose or fructose) was compared to a control cryopreserved in lactose-egg yolk extender. In the second experiment, the effect of five disaccharides (lactose, sucrose, lactulose, trehalose or melibiose) on boar sperm cryosurvival was studied. Several sperm quality parameters were assessed by flow cytometry in samples incubated for 30 and 150 min at 37°C after thawing: percentages of sperm with intact plasma membrane (SIPM), sperm presenting high plasma membrane fluidity (HPMF), sperm with intracellular reactive oxygen substances production (IROSP) and apoptotic sperm (AS). In addition, the percentages of total motile (TMS) and progressively motile sperm (PMS) were assessed at the same incubation times with a computer-assisted sperm analysis system. Freezing extenders supplemented with each of the monosaccharide presented smaller cryoprotective effect than the control extender supplemented with lactose (P<0.05). However, from the three monosaccharides tested, glucose provided the best sperm quality after freezing-thawing. With respect to the disaccharides studied, samples frozen with the extender supplemented with lactulose exhibited in general the lowest sperm quality, except for the percentage of capacitated sperm, which was highest (P<0.05) in the samples cryopreserved with the trehalose extender. Our results suggest that disaccharides have higher cryoprotective effect than monosaccharides, although the monosaccharide composition of the disaccharides is also important, since the best results were obtained with those disaccharides presenting glucose in their composition.     

A high-performance liquid chromatography for constituent disaccharides of chondroitin sulfate and dermatan sulfate isomers

An improved high-performance liquid chromatography for unsaturated disaccharides prepared from chondroitin sulfate and dermatan sulfate isomers was developed using an ion-exchange resin made from a sulfonized styrene-divinylbenzene copolymer. By this newly devised method, it was found that the retention times of representative unsaturated disaccharides are very unique and appear in the following order: unsaturated 6-sulfated, nonsulfated, and 4-sulfated disaccharides. The content of the individual unsaturated disaccharides could be measured at similar sensitivities with ultraviolet absorbance. Sensitive and unique retention times as well as good resolution were found for various unsaturated disulfated disaccharides. The new microassay method by HPLC can be used to determine chondroitin sulfate and dermatan sulfate isomers in amounts as small as 100 ng to 8 micrograms. The practicality of this method was verified by application to the separation and quantitation of chondroitin sulfate and dermatan sulfate isomers from human coronary arteries.     

OTC analgesics and drug interactions: clinical implications

Background

Preventive care in the United States has been a priority, especially for children under 18 years of age. The objective of this analysis was to determine which predisposing, enabling, and need factors affect access to preventive health care for children.

Methods

Data were obtained from the National Survey of Children's Health (NSCH), a cross-sectional study of children in the United States. The current analysis examined whether predisposing, enabling, and need factors included in Andersen's Socio-Behavioral Model significantly affect having received preventive medical care among children 3–17 years of age. Logistic regression was used to compute odds ratios and 95% confidence intervals.

Results

63,924 out of 85,151 subjects were reported as having received preventive medical care. After stratifying by geographical region, the following factors were significant for predicting having received preventive care. Age was negatively associated with having received care in all four regions. Household education of less than a college degree and being white (compared to black) were negatively associated with having received care in the Northeast, Midwest, and South. Having fewer than 4 children was negatively associated in Northeast but positively associated in the West with having received care. Being male, having less than 3 children in the household, having less than 3 adults in the household, and being Hispanic were positively associated with having received care in the West only. Not having insurance and having a lower socioeconomic status were negatively associated with having received care; while, having a personal doctor or nurse was positively associated in all four regions. Primary language other than English was negatively associated with having received care in the Northeast only. Currently needing medicine was also positively associated with having received care in all four regions; while, having limited abilities to do things was positively associated in the West only.

Conclusion

Older children whose family resides in Northeast, Midwest, and South regions with low household education and poverty levels experience insufficient preventive health care. Medicaid or SCHIP coverage should be expanded for children who are still uninsured. For children in the West, gender, family size, ethnicity, and their ability to do things should also be considered when providing assistance for receiving preventive care.     

High performance liquid chromatography of unsaturated disaccharides produced from chondroitin sulfates by chondroitinase.

High performance liquid chromatography was performed by the ion pair method on unsaturated disaccharides produced from chondroitin sulfates by the action of chondroitinase. Completely separated peaks corresponding to deltaDi-0S, deltaDi-4S, and deltaDi-6S were obtained on a column of mu-bondapak-C18 with 0.035 M tetra-butylammonium phosphate (pH 7.54) as a mobile phase. There was a linear relationship between the ratio of the peak area and the amount of each standard tested.     

Amyloid-beta peptides interact with plasma proteins and erythrocytes: implications for their quantitation in plasma

Amyloid beta peptides are bound rapidly in the plasma complicating an accurate assessment of their in vivo abundance by immunoassay procedures. The extent of Abeta immunoassay interference was used to estimate the Abeta binding capacity of purified plasma proteins, erythrocytes and whole plasma. Human serum albumin bound Abeta peptides rapidly with a 1:1 stoichiometry and at physiological concentrations was capable of binding over 95% of an input of 5 ng/ml Abeta. Purified alpha2-macroglobulin was able to bind Abeta peptides and at physiological concentration bound 73% of 5 ng/ml of Abeta. Erythrocytes also sequestered the Abeta peptides, showing a preference for binding Abeta 1-42. Incubation of 5 ng/ml of Abeta in plasma revealed that about 30% of the peptides were still detectable by immunoassay, presumably reflecting the binding of Abeta peptides with albumin and other plasma molecules. Thus, our studies reveal that both the soluble and formed elements of the blood are capable of sequestering Abeta peptides. To avoid underestimating plasma Abeta values, we employed an improved column chromatography method under denaturing conditions to liberate Abeta from its associations with plasma proteins. Quantification of Abeta 40 and 42 levels in plasma from both normal and AD individuals after chromatography showed a large overlap between AD and control groups, despite the very large pool of Abeta present in the AD brains. The potential origins of the plasma Abeta pool are discussed.     

Effects of monosaccharides and disaccharides on the formation of food mutagens in model systems

The formation of the mutagenic imidazoquinoxalines (MeIQx, DiMeIQx) was studied using a modification of a previous model system. Creatine or creatinine (0.9 mmole) was heated together with glycine (0.9 mmole) and various sugars (0.45 mmole) dissolved in diethylene glycol and water (3 ml, 5 : 1) for up to 15 min at 180°C. This system produced the same amount of mutagenicity after 10 min at 180°C as a previous one during 2 h of reflux boiling at 12dgC. MeIQx (4 nmole/mmole creatin(in)e was the major mutagen produced together with minor amounts of DiMeIQx, both 4,8- and 7,8-DiMeIQx according to HPLC-MS. A few other mutagenic peaks were also separated on HPLC, but they were not identified. Varying the concentration (0–2.4 mmole) and type of monosaccharides and disaccharides greatly affected the yields of all the mutagenic compounds. Sugar in molar amounts lower than the creatin(in)e concentration increased the yield until an optimum was reached. In higher concentrations the formation of all the mutagens was markedly reduced. The same was found for glucose, fructose, sucrose, and lactose, though the monosaccharides showed the most pronounced inhibitory effects.

The inhibition of the formation of the mutagenic compounds by an excess of sugars is proposed to be an effect of Maillard reaction products, which may block the formation of imidazoquinoxalines by attacking creatine. Support for this mechanism is given by data showing a lower recovery of unreacted creatine with increasing concentration of glucose and also by an inhibitory effect on the formation of these mutagens after adding a typical Maillard reaction product, 5-hydroxymethyl-2-furfural.     

Molecular interactions of neural chondroitin sulfate proteoglycans in the brain development

Aggrecan family proteoglycans, phosphacan/RPTPzeta/beta, and neuroglycan C (NGC) are the major classes of chondroitin sulfate proteoglycan in the developing mammalian brain. A multidomain is a common structural feature of these proteoglycans which can interact with various molecules including growth factors, cell adhesion molecules, and extracellular matrix molecules. Individual proteoglycans are distributed in the developing brain in a distinct temporal and spatial pattern, suggesting that they are involved in distinct phases of the brain development through multiple molecular interactions. This review mainly summarizes recent studies on the involvement of these three classes of proteoglycan in cell-cell and cell-substratum interactions during the brain development. Their expressions and proposed functional roles in injured brains are also mentioned. In addition, this review briefly covers potential functions of other neural chondroitin sulfate proteoglycans such as decorin, testican, NG2 proteoglycan, and amyloid precursor protein (APP) in developing and injured brains.     

High-performance liquid chromatographic determinations of disaccharides resulting from enzymatic degradation of isomeric chondroitin sulfates.

The use of Whatman Partisil-10 PAC and Altex LiChrosorb NH₂ bonded phase columns for the high-performance liquid chromatographic separation of the disaccharides obtained from digestion of isomeric chondroitin sulfates with chondroitinases has been investigated. The substituted unsaturated disaccharides in the enzymatic degradation mixtures undergo rapid isocratic separations on both bonded stationary phases. A complete separation can be established within 10 min. The development of these procedures has expedited enzymatic studies of isomeric chondroitin sulfates.The rate of depolymerization of chondroitin sulfates by the action of chondroitinases based on quantitative high-performance liquid chromatographic separations and detection of the disaccharide products was studied.     

The separation of chondroitin sulfate disaccharides and hyaluronan oligosaccharides by capillary zone electrophoresis

We have developed techniques for the separation of unsulfated (2-acetamido-2-deoxy-3-O-(4-deoxy-alpha-L-threo- hex-4-enopyranosyluronicacid)-D-galactose and -D-glucose), monosulfated (2-acetamido-2-deoxy-3- O-(4-deoxy-2-O-sulfo-alpha-L-threo-hex-4-enopyranosyluronic acid)-D-galactose and 2-acetamido-2-deoxy-3-O-(4-deoxy-alpha-L-threo-hex- 4-enopyranosyluronic acid)-4-sulfo-D-galactose and -6-sulfo-D-galactose),disulfated (2-acetamido-2-deoxy-3-O-(4-deoxy-2-O-sulfo-alpha-L-threo-hex-4- enopyranosyluronic acid)-4-sulfo-D-galactose and -6-sulfo-D-galactose and 2-acet-amido-2-deoxy-3-O-(4-deoxy-alpha-L-threo-hex-4-enopy- ranosyluronic acid)-4,6-di-O-sulfo-D-galactose), and trisulfated (2-acetamido-2-deoxy-3-O-(4-deoxy-2-O- sulfo-alpha-L-threo-hex-4-enopyranosyluronic acid)-4,6-di-O-sulfo-D-galactose) isomers of chondroitin using capillary zone electrophoresis. In addition, it is possible to separate oligomers of hyaluronan by similar protocols. These techniques represent a rapid, sensitive, and reproducible technique for the assay of these molecules from digests of connective tissues.     

Hyaluronic acid and chondroitin sulfate unsaturated disaccharides analysis by high-performance liquid chromatography and fluorimetric detection with dansylhydrazine

A system capable of resolving all the known unsaturated nonsulfated, mono- and disulfated disaccharides derived from chondroitin sulfate samples, dermatan sulfate, and hyaluronic acid after their derivatization with dansylhydrazine and separation by HPLC and fluorimetric detection is reported. This method was found superior to others in that unsaturated disaccharides can be separated with good resolution in about 50 min in an isocratic solvent with a sensitivity greater than about 50 pmol (approx 20-30 ng) and linearity from 50 to 500 pmol. The system was applied to the analysis of various chondroitin sulfate samples, including highly sulfated species and dermatan sulfate, and also to a defructosylated polysaccharide with a chondroitin backbone purified from Escherichia coli U1-41. Excellent agreement was obtained with traditional compositional analysis performed by anion-exchange HPLC separation and UV absorption at 230 nm.     

Tandem mass spectrometry for characterization of unsaturated disaccharides from chondroitin sulfate,dermatan sulfate and hyaluronan

Fast atom bombardment tandem mass spectrometry has been used in the characterization of non-, mono-, di- and trisulfated disaccharides from chondriotin sulfate, dermatan sulfate and hyaluronan. The positional isomers of the sulfate group of mono- and disulfated disaccharides were distinguished from each other by both positive- and negative-ion fast atom bombardment tandem mass spectra, which gave sufficient information characteristic of the isomers. The anomeric isomers of nonsulfated disaccharides were characterized by the technique in the positive-ion mode. This fast atom bombardment collision induced dissociation mass spectrometry/mass spectrometry technique was also applied successfully to the characterization of trisulfated disaccharide.Abbreviations FABMS fast atom bombardment mass spectrometry - MI metastable ion - CID collision induced dissociation - MIKE mass analysed ion kinetic energy - SIMS secondary ion mass spectrometry - MS/MS mass spectrometry/mass spectrometry - HPLC high performance liquid chromatography - GlcA d-gluco-4-enepyranosyluronic acid - CS chondroitin sulfate - DS dermatan sulfate - HA hyaluronan - UA-GalNAc 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-d-galactose - UA-GalNAc4S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose - UA-GalNAc6S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose - UA2S-GalNAc 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-galactose - UA2S-GalNAc4S 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose - UA2S-GalNAc6S 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose - UA-GalNAcDiS 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4,6-di-O-sulfo-d-galactose - UA2S-GalNAcDiS 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-4,6-di-O-sulfo-d-galactose - UA-GlcNAc 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-d-glucose     

A new electrophoretic method for the separation of disaccharides obtained by digestion of chondroitin sulfates and dermatan sulfate with chondroitinases

A new rapid and simple method has been developed for the separation of disaccharides obtained by chondroitinase digestion of chondroitin sulfates and dermatan sulfate using electrophoresis on cellulose acetate plates (Titan III cellulose acetate plates). Three disaccharides are completely separated by electrophoresis in barium acetate or calcium acetate in a short time, and less than 50 μg of glycosaminoglycan samples can be analyzed within 2 h.     

Spectroscopic studies of interactions of chondroitin sulfates with cisplatin

Complexation of cisplatin (CDDP) and chondroitin sulfate A (CSA) or C (CSC) has been reported to reduce the nephrotoxicity of CDDP. However the mechanism of interaction between CDDP and CSA or CSC was not known. In this study, spectroscopic analyses including NMR were carried out to examine the complexation interactions of CSA and CSC with CDDP. The time-dependent changes in the UV spectra indicate that CSA and CSC effectively complexes with CDDP in aqueous solution and that the reaction occurs subsequent to the hydrolysis of CDDP. The time-dependent change results measured by capillary electrophoresis showed that complexation of chondroitin sulfate (CS) followed first-order reaction kinetics and that the rate of CDDP hydrolysis in the complexation for both CSA and CSC was the same. These results suggested that the mechanism of complexation was a two-step process with monoaqua formation proved to be the first step, which was also the reaction rate controlling step. Moreover, NMR data suggested that the carboxylic and sulfate groups of CS played an important role in its interaction with CDDP.