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.     

Enzymatic analysis with chondrosulfatases of constituent disaccharides of sulfated chondroitin sulfate and dermatan sulfate isomers by high-performance liquid chromatography

Various under-sulfated, monosulfated, and over-sulfated chondroitin sulfate and dermatan sulfate isomers were analyzed in terms of disaccharide units before or after desulfation with chondrosulfatases in addition to digestion with chondroitinases. The unsaturated disaccharides were separable by a high-performance liquid chromatography (HPLC) method using a resin made from a sulfonized styrene-divinylbenzene copolymer. The retention times of the parent sulfated unsaturated disaccharides and newly generated unsaturated mono- or nonsulfated disaccharides were reproducible. On desulfation of the parent sulfated unsaturated disaccharides with chondrosulfatases, almost all ΔDi-S showed the same retention times as those of standard ΔDi-S from known components. Following digestion of ΔDi-diS_B with chondro-4-sulfatase as well as ΔDi-diS_D or ΔDi-diS_G with chondro-6-sulfatase, three ΔDi-monoS with the same retention time were detected with the HPLC method. These newly generated ΔDi-monoS₂ showed that the structure is N-acetyl-d-galactosamine, uronic acid 2-sulfate.     

Dermatan sulfate isomers in human articular cartilage characterized by high-performance liquid chromatography

The Constituents of dermatan sulfate isomers in human articular cartilage were studied at the disaccharide level by high-performance liquid chromatography. Appreciable amounts of dermatan sulfate components, i.e., dermatan sulfate, chondroitin sulfate types G and B, could be detected after digestion with chondroitinases-B or -ABC. The oversulfated dermatan sulfate isomers were isolated only after digestion with chondroitinase-ABC but not with the AC-lyase. The dermatan sulfate isomers were found to be markedly increased in weight loading parts of articular cartilage. It is postulated that the dermatan sulfate isomers are formed as a result of the weight loading reaction, which may be responsible for the fibrosis of articular cartilage.     

Determination of stereo- and positional isomers of oxygenated triterpenoids by reversed phase high performance liquid chromatography

Twenty four oxygenated triterpenoids, including eight pairs of stereoisomers and five pairs of positional isomers, could be separated by reversed phase HPLC. The capacity factors obtained in methanol-water and acetonitrile-water solvent systems made it possible to correlate the molecular polarities due to the presence of multiple oxygenated functional groups in these compounds. It was found that the number and position of functional groups as well as the stereochemistry of these functional groups played important roles in governing the polarity of these lanostanoid acids. The polarity weighting factors were in the following order: 3 beta-OH greater than 3 alpha-OH greater than 3 alpha-OAc greater than 3 beta-OAc. The contribution to polarity due to 15 alpha-OAc and 22 beta-OAc was probably very similar. The unique stereochemical character and eluting sequences of the lanostanoid acids provide information to generate empirical rules for predicting the role of individual polar functional groups in the chromatographic behavior in reversed phase HPLC.     

Fluorometric analysis of pectenotoxin-2 in microalgal samples by high performance liquid chromatography

A rapid HPLC method with fluorescence detection of pectenotoxin-2 (PTX2), a polyether macrolide toxin, in microalgae is presented. A dienophile reagent, DMEQ-TAD, was used for precolumn fluorescence labeling. PTX2 could be quantitatively detected in the range 1-200 ng. This method confirmed the occurrence of PTX2 in net haul samples mostly composed of dinoflagellates Dinophysisspp. collected in the Adriatic Sea, Italy and Mutsu Bay, Japan.     

Advances in the analysis of amino acid phenylthiohydantoins by high performance liquid chromatography

All the organic soluble amino acid phenylthiohydantoins are eluted (but not totally resolved) in under 40 min on either a Zorbax ODS or Permaphase ETH column. The superior resolution of the Zorbax ODS column and the complimentary characteristic of the Permaphase ETH column allow the isocratic analysis of 15 phenylthiohydantoins in less than 20 min when the columns are operated simultaneously. The water soluble PTH-His and -Arg are analyzed separately in under 10 min with the Zorbax ODS column. Five picomoles of a derivative may be detected with a sample to noise ratio of 6, HPLC is the most sensitive, practical method for PTH analysis.     

Determination of methylputrescine oxidase by high performance liquid chromatography

N-Methyl-Δ¹-pyrrolinium chloride, the product of the title enzyme, was synthesized by methylation of aminobutyraldehyde diethylacetal followed by acidic cleavage. After purification to homogeneity, it was characterized by NMR and UV spectroscopy. The compound had an absorption maximum at 210 nm; previous data indicating a maximum at 267 nm were shown to arise from an impurity. An HPLC method for the assay of N-methylputrescine oxidase from plant material was developed based on the separation of N-methyl-Δ¹-pyrrolinium chloride on a cation exchange column and direct detection at 210 nm. The enzyme activity was measured in the protein fraction extracted from plant roots and treated by gel filtration on disposable PD 10 columns. A K_m value of 1.9 mM was determined for methylputrescine and the enzyme from tobacco roots. The enzyme activities from N. tabacum and Datura stramonium were compared.     

Nucleic acid analogs for high performance liquid chromatography

HPLC resins containing nucleic acid base derivatives were successfully prepared. These resins were found to give excellent complementary separation of nucleic acid base derivatives, nucleosides, nucleotides, and oligonucleotides. These resins may be useful for separation of components of nucleic acids and polynucleotides as a specific separation system, while ion-exchange and reverse-phase systems are non-specific separation systems.     

Uptake and turnover of dopamine in rat mast cells studied by cytofluorometry and high performance liquid chromatography

Summary Uptake and turnover of dopamine (DA) in rat peritoneal mast cells were studied by a cytofluorometric technique. The main advantage of the method is that it permits the study of the distribution of amine content within populations of cells. Catecholamines and indolamines can be differentiated, but subtler structural differences in this group of compounds cannot be distinguished. We, therefore, combined the cytofluorometric measurements with a liquid chromatographic method based on reversed-phase chromatography followed by amperometric detection in a thin layer flow cell. Intraperitoneally injectedl-DOPA was rapidly decarboxylated to DA, which was accumulated in mast cell granules. The elimination of DA from the mast cells was much faster than previously published 5-hydroxytryptamine and histamine elimination rates. No evidence of intracellular conversion of DA before its elimination was found and simultaneous heparin quantitations gave no evidence of an elimination pathway due to exocytosis of granules. Electron microscopy disclosed no structural changes that could be related to exocytosis during the elimination phase of DA. The rapid elimination together with absence of inhibition of DA-uptake after storage of exogenous 5-hydroxytryptamine suggest that the mechanism of DA storage differs from the mechanism of storage of endogenous mast cell amines.     

Analysis of positional isomers of monounsaturated fatty acids by high performance liquid chromatography of 2,4-dinitrophenylhydrazones of reduced ozonides

A method is described for quantifying the positional isomers in monounsaturated fatty acid methyl ester (FAME) fractions. The procedure involves the preparation of 2,4-dinitrophenylhydrazones (DNPH) of the fragments generated during reductive ozonolysis of FAME, class isolation of the aldehyde and aldehyde ester DNPH, and separation of the aldehyde ester derivatives by high performance liquid chromatography (HPLC). The high extinction coefficient of the DNPH provides for a sensitive assay which is linear for a large range of components over a concentration range of 0.075-5 nmol/component, and the stability of the DNPH permits the independent analysis of the aldehyde and aldehyde ester fragments generated during reductive ozonolysis. The reductive ozonolysis-DNPH-HPLC method developed is as sensitive, reproducible, and accurate as reductive ozonolysis-gas-liquid chromatography and does not suffer from some of the drawbacks of the classical procedure.     

Rapid quantitative apolipoprotein analysis by gradient ultracentrifugation and reversed-phase high performance liquid chromatography

A new methodology for the analysis of lipoprotein composition using a combination of gradient ultracentrifugation and high performance liquid chromatography was used to determine the differences in lipoprotein composition between non-hyperlipidemic men and women. Lipoproteins from each subject were separated into six subfractions: VLDL, IDL, LDL, and three subfractions of HDL by a single gradient ultracentrifugation spin of less than 5 hr. The HDL subfractions were designated HDL-L (the lightest density subfraction, rich in apoCs and poor in apoA-II), HDL-M (the middle subfraction, rich in apoA-II), and HDL-D (the most dense, relatively poor in both the apoCs and apoA-II). The concentrations of the water-soluble apolipoproteins in each subfraction were determined using reversed-phase HPLC. The concentrations of apoB and the lipid components of the lipoproteins were determined by chemical and enzymatic methods. This methodology proved to be highly reproducible when performed on fresh plasma samples and we were able to identify many sex-associated differences in lipoprotein composition. This methodology is the only nonimmunological technique available for analyzing lipoprotein composition that offers such a combination of accuracy, speed, and completeness.     

High-performance liquid chromatography of pyridylamino derivatives of unsaturated disaccharides produced from chondroitin sulfate isomers by chondroitinases

A sensitive method was developed for the separation and quantitation of four unsaturated disaccharides (delta Di-0S, delta Di-4S, delta Di-6S, and delta Di-diS) by high performance liquid chromatography. The unsaturated disaccharides were coupled with a fluorescent compound, 2-aminopyridine. Complete separation of the resulting pyridylamino derivatives was achieved on a column of muBondapak-C18 with 8 mM KH2PO4-Na2HPO4 (pH 6.0)/methanol (30/l, by volume) as a mobile phase. There was a linear relationship between the fluorescence emission (peak height), and the amount of each authentic disaccharide used for the coupling reaction. This method was applied to analyze commercially available chondroitin sulfates A and C, dermatan sulfate, and urinary glycosaminoglycans obtained from patients with mucopolysaccharidosis after digestion with chondroitinases. The data indicated that the present method is useful for the separation and quantitation of nmol-pmol levels of the unsaturated disaccharides produced from chondroitin sulfate isomers by chondroitinases and can be used for their structural characterization.     

Quantitative analysis of brain gangliosides by high performance liquid chromatography of their perbenzoyl derivatives

This report describes a convenient, highly sensitive, and reproducible HPLC procedure for the quantitative analysis of gangliosides from brain tissues. The procedure involves the conversion of gangliosides to their perbenzoyl derivatives, isolation of the derivatives on a C18-reversed-phase cartridge, separation of the derivatives on a column (3-micron silica) maintained at an elevated temperature, and UV detection of the derivatives at 230 nm. The convenience of the procedure, its sensitivity, reproducibility, and application to the analysis of gangliosides from tissue sources make it the method of choice for ganglioside quantification in our laboratories. Three aspects of the procedure contribute to its convenience: reaction conditions that lead to single products, a convenient isolation procedure for the derivatives, and chromatographic conditions that provide resolution of the derivatives.     

Subunit analysis of bovine cytochrome c oxidase by reverse phase high performance liquid chromatography

Bovine cytochrome c oxidase subunits were separated by reverse phase high performance liquid chromatography using a C4 column eluted with water and an acetonitrile gradient, both containing 0.1% trifluoroacetic acid. Subunits I and III precipitated in this solvent and could not be analyzed; the remaining eleven subunits were dissociated, denatured, soluble and could be resolved by elution from the column. The protein subunit eluting in each chromatographic peak was identified by a combination of polyacrylamide gel electrophoresis in sodium dodecyl sulfate, NH2-terminal amino acid sequencing, and amino acid analysis. Each subunit produced a single elution peak with the exception of subunit VIc (nomenclature of Kadenbach et al., 1983, Anal. Biochem. 129, 517-521), which eluted from the column as two well-resolved peaks. Sequence analysis showed that the two subunit VIc elution peaks resulted from partial chemical blockage of the alpha-amino serine residue of subunit VIc. The C4 reverse phase HPLC was used to document specific subunit removal from bovine cytochrome c oxidase either by tryptic digestion or by dodecyl maltoside extraction. The described HPLC method for separating cytochrome c oxidase subunits should be applicable for the analysis of other multisubunit proteins, especially other multisubunit membrane protein complexes.     

Analysis of bacterial menaquinone mixtures by high performance liquid chromatography

Menaquinone mixtures of microbial origin were separated according to the chain length and the degree of hydrogenation of the polyisoprenyl side-chain by reversephase partition high performance liquid chromatography. Menaquinones can be measured easily and sensitively by the chromatographic system described here.     

Analysis of protein-glutathione mixed disulfides by high performance liquid chromatography

After precipitation of proteins; serum, hepatocytes, or glutathione-derivatized bovine serum albumin, by perchloric acid, dithiotheritol was used to reduce glutathione-protein mixed disulfides in the ether-washed, resuspended pellet. Following neutralization and S-carboxymethylation of free sulfhydral groups in the acid soluble fraction by iodoacetic acid, 2,4-dinitrophenyl derivatives of released compounds were produced by addition of ethanolic fluorodinitrobenzene. The 2,4-dinitrophenyl derivative of S-carboxymethylglutathione was measured by high-performance liquid chromatography. The method was found to be reproducible and limited only by the sensitivity of the glutathione analysis (about 10 pmol/sample). Quantitation of protein-bound glutathione was shown to be indepedent of the ratio of bound to soluble glutathione as well as the protein concentration in the sample. This method was found to produce glutathione values identical to those measured after borohydride reduction without the problems of foaming, sample loss, and the need of continuous pH adjustment during reduction.     

Assay of bovine fibrinopeptides by high performance liquid chromatography.

A method for separating small amounts (<10^?5 mol) of bovine fibrinopeptides A and B employing high performance liquid chromatography has been developed. The limit of detectability of this method is about 10^?10 mol of fibrinopeptide. The separation was achieved within 20 min under reversed phase conditions using isocratic elution with aqueous buffer-acetonitrile solvent systems.