Assay of inorganic sulfate in biologic fluids by nonsuppressed (single-column) ion chromatography

An assay using nonsuppressed (single-column) anion chromatography was developed to determine the concentration of inorganic sulfate in biologic fluids. A conventional HPLC system with an anion-exchange column and conductimetric detector interfaced with an automatic injector and integrator was used. The mobile phase for the chromatography of urine and serum samples is 4 mM potassium hydrogen phthalate, pH 4.5, and potassium iodide is used as the internal standard. For cerebrospinal fluid samples, the mobile phase is modified by addition of 10% of a 4 mM phthalic acid solution. Results of the HPLC assay were found to correlate well (r = 0.991 and 0.999) with those of two commonly used spectrophotometric methods for urine and serum inorganic sulfate determinations. However, the concentrations determined by ion chromatography were 2.5 to 10% lower, possibly due to less assay interference by other substances following chromatographic separation of sulfate. Anion chromatography using a single-column system is a convenient and relatively inexpensive method with sufficient sensitivity for the determination of inorganic sulfate concentrations in urine, serum, and cerebrospinal fluid.     

High-performance liquid chromatographic separation of heparin-derived oligosaccharides

Heparin has been enzymatically depolymerized with heparinase (heparin lyase (EC 4.2.2.7)) and then separated into di-, tetra-, hexa-, octa-, and decasaccharide mixtures by low-pressure gel-permeation chromatography (GPC). These sized mixtures were resolved by strong anion-exchange (SAX) HPLC into multiple components. The fractions from the SAX-HPLC were collected and characterized for size by GPC-HPLC and sulfate content by ion chromatography. This study provides detailed methodology for the separation of larger and more highly sulfated oligosaccharides than previously reported. It describes the first use of ion chromatography for the accurate determination of the sulfate content of heparin oligosaccharides, a method which can also be applied to heparin and other glycosaminoglycans.     

Gas chromatography—mass spectrometry of metabolites in hemodialysis fluid

An analytical method has been developed for the separation and identification of several metabolites in used hemodialysis fluid obtained during the treatment of a uremic patient on the artificial kidney. The procedure involves ion exchange, evaporation, and trimethylsilylation; the derivatized components were studied by combined gas chromatography—mass spectrometry.Twelve compounds were satisfactorily resolved; six were conclusively identified from mass spectral data. The identified components include phosphoric acid, glucopyranurono(6→1)-lactone, citric acid, d-gluconic acid-5-lactone, α-d-glucose, and β-d-glucose. A seventh component was tentatively identified as mannonic acid.     

Visualization method for sulfolipids and its application to the determination of nanomole quantities of lipid sulfur

A simple and sensitive visualization method for sulfolipids on a thin-layer chromatogram is described. By spraying with an acidic solution of azure A, a complex was formed between an anionic sulfolipid and a blue cationic compound. After the unbound dye was washed out by brief soaking in methanol, sulfolipids were visualized as clear dark-blue bands on a light-blue background. As little as 0.5 nmol could be detected. Sulfolipid-dye complex was estimated by densitometry or colorimetric measurement after extraction with chloroform/methanol. For the quantitative determination of sulfolipids having long sugar chains, it is necessary to treat thin-layer chromatography plates with acetic anhydride before color development. Of the other tissue lipids not containing sulfuric acid ester that were tested none were stained significantly. A linearity of quantitative determination was observed over the range of 1-8 nmol.     

Microsomal lauric acid 11- and 12-hydroxylation: a new assay method utilizing high pressure liquid chromatography.

A new assay method using high pressure liquid chromatography has been developed which permits the simultaneous isolation, determination, and quantitation of lauric acid and its hydroxylated products after methylation of extracts from kidney or liver microsomal incubation mixtures. The small differences in polarity between the lauric acid, 11-hydroxy- and 12-hydroxy-lauric acid after methylation permit their separation on reverse phase columns packed with octadecyltrichlorosilane bonded to silicone polymers. The total time required for the chromatography is less than 1 hr. Using this method, the formation of hydroxylated products was shown to have a linear dependence on protein concentration and time. The K_m for lauric acid and NADPH were determined to be 8 μm and 54 μm in kidney microsomes, respectively.     

Accumulation of sulfoglycolipids in hyperosmosis-resistant clones derived from the renal epithelial cell line MDCK (Madin-Darby canine kidney cell).

1. Two clones (osmR-A and osmR-B) resistant to hyperosmotic media of 700 and 800 mosmol/l, respectively, were selected from Madin-Darby canine kidney (MDCK) cells. 2. When cultured in isosmotic medium (300 mosmol/l), the concentration of galactosyl sulfatide and lactosyl sulfatide in these hyperosmosis-resistant clones was 3.4-5.9 times higher than in the wild-type MDCK. The rate of incorporation of [35S]sulfate into sulfolipids of osmR-A and osmR-B was 1.9-6.7 times higher than MDCK. 3. The stimulation of incorporation into sulfolipids by hyperosmotic culture was completely inhibited by cycloheximide. The pulse-chase studies indicated decreased turnover rate of sulfolipids in osmR-A.     

Alterations of renal cortex and medullary glycosaminoglycans in aging dog kidney

Age-related changes in renal function have been attributed to alterations in the chemical composition of the kidney tissues. Hence, the glycosaminoglycan composition of the renal cortex and medulla at varying age intervals was investigated. Glycosaminoglycans were isolated from the tissues by means of digestion with collagenase and pronase and purified by ethanol precipitation. Subsequent separation of various polyanions was accomplished by ion exchange chromatography on a Dowex 1-X2 column, using sodium chloride buffers of increasing ionic strengths. The glycosaminoglycans in each fraction were identified and quantitated by digestion with specific enzymes, including hyaluronidase, chondroitinase AC and ABC. The enzyme resistant material was separated and further digested with nitrous acid to quantitate the proportion of heparon sulfate. The results indicate that the glycosaminoglycan content of the renal medulla was much higher than the cortex at all the age intervals studied, and age-induced reduction was mainly cortical. There was a significant reduction in the heparan sulfate content of the cortex in aging. Interestingly, the major glycosaminoglycan content of the medulla was hyaluronic acid, which showed a sharp increase during aging, whereas heparan sulfate declined. Chondroitin sulfate was not altered due to age in either tissue. The molecular weight of hyaluronic acid was determined by column chromatography. Results indicate that the size of hyaluronate in the cortex was small and did not vary with age. In the medulla of the younger age group, a considerable amount of large size hyaluronate was observed. As age increased, the size decreased. The results strongly suggest that alteration in the renal glycosaminoglycans may be partly responsible for the age related protinuria and ionic imbalance.     

Identification and quantification of metabolite conjugates of activated cyclophosphamide and ifosfamide with mesna in urine by ion-pair extraction and fast atom bombardment mass spectrometry

The high bladder toxicity of the alkylating oxazaphosphorine anticancer drugs, cyclophosphamide and ifosfamide is effectively reduced by the concomitant administration of mesna (sodium 2-mercaptoethane sulphonate). The formation and rapid urinary excretion of conjugates of the activated (4-hydroxylated) oxazaphosphorine metabolites with mesna has been suggested as the pharmacological basis for the selective detoxification, but separation and identification of such metabolites in vivo have been extremely difficult due to their high polarity and chemical lability. In this study an ion-pair extraction procedure in combination with positive and negative ion fast atom bombardment mass spectrometry has been developed which enabled the identification and quantification of the conjugation products of activated oxazaphosphorine metabolites with mesna in urine. The conjugates extracted as the tetra-n-butylammonium salts are directly identified by their characteristic positive molecular ion adducts and fragment ions, and the corresponding abundant molecular anions. The pattern of molecular and fragment ion formation was established by comparison of the fast atom bombardment mass spectra of synthetic cyclophosphamide-mesna conjugates with various organic and inorganic counter ions. The ifosfamide-4-(2-thioethylsulphonate) (ifosfamide-mesna) conjugate was identified as a metabolite in the urine of rats, and in patients after administration of the combination, ifosfamide + mesna. By means of a two-step extraction and with the use of suitable analogues as internal standards, procedures for the quantification of parent oxazaphosphorine and of oxazaphosphorine-mesna conjugates by negative ion fast atom bombardment mass spectrometry have been developed, and first examples for the determination of excretion kinetics are described.     

Comparative purity of commercially available sodium dodecyl sulfates: A simple criterion

The purity of various commercially available sodium dodecyl sulfates was checked by gas chromatographic analysis of the fatty alcohols obtained from the acid hydrolysis of the alkyl sulfates. The content of tetradecyl sulfate in these samples was found to vary from 0 to 31% as impurity, while the content of the decyl sulfate homolog was ～2% in all the samples. Accurate critical micelle concentration measurements are a sensitive means of detecting impurities, especially those of higher chain-lengths. These measurements have indicated the presence of large amounts of tetradecyl sulfate impurity in one of the “pure” samples.In the course of work on the determination of the binding of large amounts of various detergents to proteins (1), we have discovered that some of the commercially available “pure” sodium dodecyl sulfates fall far short of any conceivable standards of purity. Although one might expect rather small amounts of inorganic salts and organic compounds such as unsulfated alcohols, the major impurities found are the homologous sodium decyl sulfate and sodium tetradecyl sulfate.In this communication, we will report only the degree of impurity arising from decyl and tetradecyl sulfates in some of the commercially available samples of “ 99 1/2% pure” sodium dodecyl sulfates. Such large contents of tetradecyl sulfates have been found that even methods simpler than the more sensitive ones (i.e., gas chromatography of the fatty alcohols after acid hydrolysis) reveal their presence.     

Assay methods and biological roles of labile sulfur in animal tissues

Sulfur is a chemically and biologically active element. Sulfur compounds in animal tissues can be present in two forms, namely stable and labile forms. Compounds such as methionine, cysteine, taurine and sulfuric acid are stable sulfur compounds. On the other hand, acid-labile sulfur and sulfane sulfur compounds are labile sulfur compounds. The sulfur atoms of labile sulfur compounds are liberated as inorganic sulfide by acid treatment or reduction. Therefore, the determination of sulfide is the basis for the determination of labile sulfur. Determination of sulfide has been performed by various methods, including spectrophotometry after derivatization, ion chromatography, high-performance liquid chromatography after derivatization, gas chromatography, and potentiometry with a sulfide ion-specific electrode. These methods were originally developed for the determination of sulfide in air and water samples and were then applied to biological samples. The metabolic origin of labile sulfur in animal tissues is cysteine. The pathways of cysteine metabolism leading to the formation of sulfane sulfur are discussed. Finally, reports on the physiological roles and pathological considerations of labile sulfur are reviewed.     

Determination of hydrogen sulfide and acid-labile sulfur in animal tissues by gas chromatography and ion chromatography

A sensitive and reliable method was developed for the determination of hydrogen sulfide and acid-labile sulfur (ALS) in animal tissues using gas chromatography with flame photometric detector (GC-FPD) and ion chromatography (IC). Hydrogen sulfide trapped in alkaline solution was determined by GC-FPD as hydrogen sulfide or by IC as sulfate after oxidation with hydrogen peroxide. Sodium sulfide used as a source of hydrogen sulfide was standardized by IC. Fresh rat liver and heart tissues contained 112.2±23.0 and 274.1±34.6 nmol/g of ALS respectively. Free hydrogen sulfide was not detected.     

Differentiation of mycobacterial species by investigation of petroleum ether-soluble sulfolipids using thin-layer chromatography after incubation with [35S]sulfate

The method of detecting petroleum ether-soluble sulfolipids by thin-layer chromatography after incubation with [35S]sulfate is useful for differentiation between mycobacterial species. Rapidly growing mycobacteria, including two subspecies of Mycobacterium chelonei, were differentiated by this method. Most species of slowly growing mycobacteria were characterized by the pattern of distribution of radioactive sulfolipids in the thin-layer chromatograms. Mycobacterium nonchromogenicum was clearly differentiated from Mycobacterium terrae and Mycobacterium triviale by the presence of a sulfolipid. The latter two did not contain any significant amount of sulfolipids. Mycobacterium avium contained a large amount of sulfolipids, whereas Mycobacterium scrofulaceum did not contain any detectable sulfolipid. Rapidly growing, scotochromogenic mycobacteria, except for Mycobacterium flavescens, did not contain any remarkable amounts of sulfolipids.     

Determination of isomeric acid dopamine metabolites in human cerebrospinal fluid by mass fragmentography

A method for the quantitative determination of the isomers 4-hydroxy-3-methoxyphenylacetic acid (HVA) and 3-hydroxy-4-methoxyphenylacetic acid (iso-HVA) in cerebrospinal fluid (CSF) by mass fragmentography has been developed. The heptafluorobutyryl methyl ester derivations of the two compounds could not be separated by gas chromatography. The relative intensity of the base peak (m/e 333) and the molecular ion (m/e 392) in the mass spectra were, however, quite different and allowed a separate determination. Contradictory to a previous report, it was found that the concentration of iso-HVA was less than 2% of the HVA level in the investigated 12 CSF samples from patients with different diseases.     

Kinetics of ion exchange process for separation of glutamic acid

Kinetics of the separation of L-glutamic acid (GLU) by ion exchange has been studied with strongly acidic H⁺-type cation exchange resin Amberlite IR-122. Since glutamic acid is a trivalent ampholyte and dissociates according to three equilibrium reactions, separation of G⁺ ions by a cation exchange process is accompanied by the dominant reversible reaction, i.e. G⁺+H⁺ ? G⁰. Accompanying reversible reaction has an effect on the ion exchange rate, and decreases the performance of the process comparing with the ideal case that the exchanging ions retain their identity. The analysis was performed first with the ion exchange column, DIC (L/D=0.52); and then with the ion exchange column, IC (L/D=10.9). The data were collected with model glutamic acid solutions for both DIC and IC columns/reactors. IC experimental results were then compared with that of DIC and the effect of scale up on ion exchange process was investigated. The experimental results have provided an adequate basis for the design calculations, and the design parameters were determined. Rate coefficients for the liquid phase mass transfer controlled cation exchange process were calculated and interrelated with a plot of j _Mfactor versus Reynolds number.     

Improvements in the application of the 4,4-N,N-dimethylaminoazobenzene-4'-isothiocyanate micromethod to the sequence analysis of proteins

Different experimental conditions have been tested to improve the sequence determination of peptides and proteins by the DABITC (4,4-N,N-dimethylaminoazobenzene-4'-isothiocyanate) method and to facilitate automation in the analysis of the released 4,4-N,N-dimethylaminoazobenzene-4'-thiohydantoin derivatives (DABTHs). Conditions for a complete and rapid separation of all amino acid derivatives have been optimized by using different reversed-phase columns. The stability of the DABTHs in several water-organic solvent mixtures was determined by quantitative analysis and permitted the selection of the appropriate solvents for use in autosamplers. Also the amino acid side-products specific to individual residues which may be observed during thin-layer chromatography of DABTHs can be completely resolved by HPLC and are helpful for a safe assignment of the amino acid residues. The analytical procedures developed have been used to examine the influence of oxygen and detergents on the efficiency of the application of the DABITC manual micromethod on proteins. In the presence of oxygen the recovery of DABTHs is lower in most cases than when the operation is carried out in an inert atmosphere. The presence of a limited amount of detergents does not interfere in the HPLC analysis of DABTHs and, moreover, can increase the efficiency of the sequence analysis of proteins depending on their nature and concentration. In particular, it has been observed that sodium dodecyl sulfate at a concentration of 0.1% can in some cases produce a threefold increase in the recovery of DABTHs.     

Determination of DOPA,dopamine, DOPAC,epinephrine, norepinephrine, α-monofluoromethyldopa and α-difluoromethyldopa in various tissues of mice and rats using reversed-phase ion-pair liquid chromatography with electrochemical detection

A method for the determination of catecholic amino acids and amines by reversed-phase ion-pair high-performance liquid chromatography with electrochemical detection has been developed. By using octanesulfonic acid for ion pairing and by optimising ionic strength, pH and methanol concentration of the mobile phase, separation was achieved of 3,4-dihydroxyphenylalanine (DOPA), 3,4-dihydroxypehnylacetic acid (DOPAC), norepinephrine (NE), epinephrine (EPI), and dopamine (DA). α-Difluoromethyldopa (DFMD) and α-monofluoromethyldopa (MFMD), two potent enzyme-activated irreversible inhibitors of aromatic amino acid decarboxylase were also separated from the natural catechols. Concentrations of catechols and inhibitors were measured in brains, hearts and kidneys of mice treated with small repeated doses of MFMD. The method has also been applied to the determination of catechols in other organs such as prostates and seminal vesicles of rats and in smaller tissues like mesenteric arteries. A semi-automated procedure making use of an automatic sample processor and a digital integrator permitted the analysis of as many as sixty samples per day.     

Optimized extraction of glycosaminoglycans from normal and osteoarthritic cartilage for glycomics profiling

Articular cartilage is a highly specialized smooth connective tissue whose proper functioning depends on the maintenance of an extracellular matrix consisting of an integrated assembly of collagens, glycoproteins, proteoglycans (PG), and glycosaminoglycans. Isomeric chondroitin sulfate glycoforms differing in position and degree of sulfation and uronic acid epimerization play specific and distinct functional roles during development and disease onset. This work introduces a novel glycosaminoglycan extraction method for the quantification of mixtures of chondroitin sulfate oligosaccharides from intact cartilage tissue for mass spectral analysis. Glycosaminoglycans were extracted from intact cartilage samples using a combination of ethanol precipitation and enzymatic release followed by reversed-phase and strong anion exchange solid-phase extraction steps. Extracted chondroitin sulfate glycosaminoglycans were partially depolymerized using chondroitinases, labeled with 2-anthranilic acid-d(4) (2-AA) and subjected to size exclusion chromatography with online electrospray ionization mass spectrometric detection in the negative ion mode. The method presented herein enabled simultaneous determination of sulfate position and uronic acid epimerization in juvenile bovine and adult human cartilage samples. The method was applied to a series of 13 adult human cartilage explants. Standard deviation of the mean for the measurements was 1.6 on average. Coefficients of variation were approximately 4% for all compositions of 40% or greater. These results show that the new method has sufficient accuracy to allow determination of topographical distribution of glycoforms in connective tissue.     

Profiling of human body fluids in healthy and diseased states using gas chromatography and mass spectrometry,with special reference to organic acids

This review summarizes recent advances in the application of gas chromatography and mass spectrometry to the study of human diseases. Emphasis is placed upon the organic acid profiles of the various body fluids. Methods for sample work-up prior to separation and mass spectrometric analysis are reviewed, and artifacts and pitfalls are discussed. Organic acid profiles, obtained with packed or capillary columns attached to mass spectrometers with or without computer systems, have led to the discovery of new normal metabolites, new metabolic disorders, and to new knowledge about a number of other diseases. Stable isotopes and gas chromatography—mass spectrometry are suitable for quantitative analysis of many compounds in the body fluids, and well suited for investigation of metabolic pathways.     

Optimization and performance of a rapid gas chromatography–mass spectrometry analysis for methylmalonic acid determination in serum and plasma

We have developed a rapid and sensitive GC–MS assay for methylmalonic acid determination in serum and plasma utilizing an anion exchange solid-phase extraction and trimethylsilyl derivatization. Each step of the procedure was optimized by the experimental design methods to assure the assay reliable performance. The limit of detection and limit of quantitation were 0.025 and 0.1 μmol/l. The total coefficient of variation for the method was 9.8, 4.4, and 4.6% at the concentration of 0.2, 3.1, and 6.2 μmol/l methylmalonic acid concentration, respectively. The assay are linear up to 9.0 μmol/l, and showed good correlation with a reference method. The method has proven to be reliable in routine production, producing clean chromatography, unique ion fragments, and consistent ion mass ratio.     

间羟苯甲酸4－羟化酶纯化及部分性质研究

经超声破碎、硫酸铵分级沉淀、凝胶过滤、磷酸钙胶层析和离子交换层析等步骤, 从Comamonas testosteroni菌株中获得了SDS-PAGE单一条带, 相对分子质量为62×10³的间羟苯甲酸4-羟化酶比活提高21倍, 产率为30%.此酶为FAD加单氧酶, 催化间羟苯甲酸转变为3,4二羟苯甲酸.