Ion-pair High-pressure Liquid Chromatography Research Articles

Using Engineered 2-O-Sulfotransferase to Determine the Activity of Heparan Sulfate C5-epimerase and Its Mutants

Heparan sulfate (HS) is involved in essential physiological and pathophysiological functions. HS is a highly sulfated polysaccharide consisting of glucuronic acid (or iduronic acid) linked to glucosamine carrying various sulfo groups. Biosynthesis of HS involves sulfotransferases and an epimerase. The HS C(5)-epimerase converts glucuronic acid to iduronic acid. The method for determining the activity has been cumbersome due to the use of a site-specifically (3)H-labeled polysaccharide substrate. Here, we report a two-enzyme coupling assay to determine the activity of C(5)-epimerase. HS 2-O-sulfotransferase (2OST) transfers the sulfo group to the 2-OH-position of glucuronic or iduronic acid. Unlike the wild type protein, 2-O-sulfotransferase mutant (2OST Y94I) transfers sulfate to the iduronic acid but not to the glucuronic acid. Thus, 2OST Y94I cannot sulfate N-sulfated heparosan, a polysaccharide containing glucuronic acid. Incubating N-sulfated heparosan with C(5)-epimerase converts some of the glucuronic acid to iduronic acid, thus becoming a substrate for 2OST Y94I. The susceptibility of the C(5)-epimerase-treated N-sulfated heparosan to 2OST Y94I modification directly correlates to the amount of the activity of C(5)-epimerase, proving that this two-enzyme coupling system can be used to assay for C(5)-epimerase. The method was further used to determine the activities of various C(5)-epimerase mutants. Our approach will significantly reduce the complexity for assaying the activity of C(5)-epimerase and facilitate the structural and functional analysis of C(5)-epimerase.

B-6 vitamer concentrations in mouse plasma, erythrocytes and tissues

The five major forms of vitamin B-6, pyridoxal 5'-phosphate (PLP), pyridoxal (PL), pyridoxamine (PM), pyridoxamine 5'-phosphate (PMP) and pyridoxine (PN) were measured in plasma, erythrocytes, liver and brain of A/Ibg and DBA/2Ibg mice by paired-ion High Pressure Liquid Chromatography. The mice were fed a non-purified diet that provided the bioavailable equivalent of approximately 3–4 mg PN-HCl/kg diet. In each tissue two of the vitamers accounted for nearly all of the total concentration of vitamin B-6. These were: plasma, PLP (14%) and PL (85%); erythrocytes, PLP (94%) and PL (3%); liver, PLP (63%) and PMP (36%); and brain PLP (55%) and PMP (43%). PM and PN contributed negligible amounts to the total vitamin B-6 content of these tissues. There were no significant differences in the concentrations of any of the vitamers between the two mouse strains, however, Pearson correlations between pairs of indices were different in the two strains. For example, plasma and liver PLP concentrations were uncorrelated in the A, but negatively correlated in the DBA mice. These data suggest that although plasma PLP may be a suitable index of dietary intake of vitamin B-6, genetic factors may be an important variable in the ability to predict tissue concentrations of the B-6 vitamers from plasma or erythrocyte concentrations.

Identification of 3,4-dihydroxyphenylalanine, 5,6-dihydroxyindole, and N-acetylarterenone during eumelanin formation in immune reactive larvae of Drosophila melanogaster.

3,4-Dihydroxyphenylalanine, 5-6-dihydroxyindole, and N-acetylarterenone were detected by electrochemical methods in the hemolymph of immune reactive larvae of Drosophila melanogaster following parasitization by the wasp Leptopilina boulardi. Determinations of the catechols were made after separation by reverse phase, ion-pairing high pressure liquid chromatography with electrochemical detection. The presence of 5,6-dihydroxyindole unequivocally establishes the eumelanin pathway in the defense response of Drosophila, and confirms previous investigations which have implicated certain catecholamine metabolizing enzymes in insect immunity. The occurrence of N-acetylarterenone, a derivative of the principal sclerotizing agent N-acetyldopamine, verifies the existence and proposed involvement of quinone methide isomerase in the regulation of catecholamine metabolism, and suggests that the cellular capsule formed by Drosophila in immune reactions against parasites is most likely a composite of both eumelanin and sclerotin. The absence of 3,4-dihydroxyphenylacetic acid in hemolymph samples from immune reactive hosts suggests that during parasitization certain catecholamines and metabolic precursors may be re-employed in alternate pathways, some of which may be used in defense reactions.

Regional amines levels in the rat brain following intra-accumbens cholecystokinin and intraperitoneal amphetamine pre-treatment.

Cholecystokinin octapeptide (CCK8) coexists with dopamine (DA) in a subpopulation of mesolimbic DA neurons including the projections to the nucleus accumbens. In this structure, CCK8 has been reported to exert agonist-like or antagonist-like effects on DA-mediated behaviours and on amphetamine's locomotor-activating effects in rodents. These findings raise the possibility that CCK8 plays a role in modulating the neurochemical mechanisms underlying the effects of DA and amphetamine in the mesolimbic DA system. The purpose of this study was to determine regional tissue monoamine levels in the rat brain and their modulation following injection of CCK8 in the nucleus accumbens. The same paradigm was used to determine the effects of this octapeptide on changes in amine levels induced by amphetamine administered intraperitoneally. DA, norepinephrine (NE), serotonin (5-HT) and their primary metabolites, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) were measured using a reversed-phase ion pair high-pressure liquid chromatography system with electrochemical detection (HPLC-ED). Frontal cortex displayed the highest DOPAC/DA ratio and the lowest DOPAC/HVA ratio in contrast to the olfactory tubercle. The intraperitoneal injection of amphetamine (1 mg/Kg) followed by the intra-accumbens administration of 0.15M saline decreased the levels of DOPAC and increased DA, 5-HT and 5-HIAA both in nucleus caudatus and nucleus accumbens. The DA agonist induced a decrease in the level of NE in olfactory tubercle and frontal cortex. The direct administration of CCK8 (300 pmol) into the nucleus accumbens decreased the level of DA, DOPAC and 5-HT mainly in olfactory tubercle and nucleus accumbens itself.(ABSTRACT TRUNCATED AT 250 WORDS)

High-pressure liquid chromatographic analysis of hemolymph plasma catecholamines in immune-reactive Aedes aegypti

Tyrosine and catecholamines have been implicated as substrates for the encapsulation reactions involved in the immune response of mosquitoes to microfilariae (mff). Identification and quantitation of tyrosine and catecholamines present in Aedes aegypti hemolymph plasma were accomplished by ion-pair high-pressure liquid chromatography with electrochemical detection at either +650 or +850 mV vs Ag/AgCl. Tyrosine, dopamine, and N-β-alanyldopamine were detected in the hemolymph plasma of naive A. aegypti. Although no differences in these compounds were observed in hemolymph plasma from A. aegypti inoculated with Dirofilaria immitis mff, the chromatogram howed a single major peak (PI) (65 μ m, expressed as dopamine equivalents) that was not present in naive hemolymph plasma. Saline-inoculated controls contained only 5% of the PI in immune reactive hemolymph plasma. A high concentration of PI (127 ± 39 μ m) was also detected after treatment of hemolymph plasma with mild alkaline conditions ( pH 9.0), indicating that it is normally present as an electrochemically inert form in naive mosquitoes. High concentrations of PI were also detected in the naive hemolymph plasma from three other mosquito species, but no PI was found in A. trivittatus under any conditions. PI did not cochromatograph with any of the catecholamines commonly thought to be involved in immune responses of dipterans against metazoan parasites, suggesting that it may be a unique substrate for these reactions. The biological relevance of PI was evidence by its appearance in the hemolymph plasma of two strains of D. immitis-inoculated A. aegypti.

Measurement of individual angiotensin peptides by HPLC-RIA

Immunoreactive measurements of Angiotensin II in plasma, relate to a variety of angiotensin peptides with different biological activities. A method is described to differentiate these individual angiotensin peptides. It involves extraction of the peptides from plasma by reversible adsorption to phenylsilyl silica cartridges, separation by an isocratic, ion pairing high-pressure liquid chromatography technique and measurement of the appropriate fractions by radioimmunoassay. In umbilical venous plasma molar concentrations of the smaller angiotensin fragments were found to range between 16 and 25% of the concentrations of the angiotensin II octapeptide. Because some angiotensin antisera show higher affinity for the smaller peptides than for the octapeptide, concentrations of angiotensin II, measured by radioimmunoassay, may be overestimated by up to 35% unless the various angiotensin peptides are adequately separated.

Analysis of cardiac membrane phospholipid peroxidation kinetics as malondialdehyde: nonspecificity of thiobarbituric acid-reactivity.

When exposed to xanthine oxidase (superoxide)-dependent, iron-promoted Fenton chemistry, purified cardiac membranes evidenced, by the thiobarbituric acid (TBA) test, a virtually instantaneous peroxidative response with a maximal linear rate of 5.8 nmol malondialdehyde (MDA)-equivalents/mEquivalents lipid ester reacted/min. Yet when the lipids purified from these same membranes and reconstituted into liposomes were peroxidized under identical reaction conditions, the TBA test indicated that a pronounced (approximately 20-min) lag period preceded a maximal peroxidation rate of only 2.1 nmol MDA-equivalents/mEquivalents lipid ester reacted/min. After 120 min of peroxidation, the cardiac membranes yielded some 300 nmol TBA-reactive MDA-equivalents/mEquivalent ester, whereas the isolated membrane lipids evidenced approximately 40% less TBA-reactivity. To verify that these quantitative and kinetic differences in membrane (phospho)-lipid peroxidation occurred with removal of the lipids from their membrane milieu, the MDA produced during both cardiac membrane peroxidation and the peroxidation of the lipids derived therefrom was isolated as its free anion by ion-pair high-pressure liquid chromatography. As quantified spectrophotometrically, true MDA production during myocardial membrane peroxidation was identical in kinetics and in amount to the production of TBA-reactive substance from the peroxidized isolated membrane lipids. These results demonstrate that significant non-MDA, TBA-reactive species are generated during the peroxidation of cardiac membranes, especially before the maximal rates of bona fide MDA production. As a direct consequence, artifactual levels and kinetics of membrane lipid peroxidation do result.

An Ion-Pairing High-Pressure Liquid Chromatography Assay for the Determination of Cefoperazone in Plasma and Urine

Abstract A high-performance liquid chromatographic assay for cefoperazone (cfp) in plasma and urine is described. For analysis, the internal standard ticarcillin (ticar) is solvated in acetonitrile, which is then added to plasma or urine. The supernatant is drawn off of the resulting protein precipitate and injected directly onto the reverse-phase C18 column, with detection at 254 nm. The mobile phase is composed of phosphate-acetonitrile-tetramethylammonium chloride (TMA). Coefficients of variation for reproducibility were less than 9% for extra-low, low, medium, and high controls. Limits of detection were 0.5 μG/mL for plasma and 1 μG/mL for urine. No interference from other cephalosporins or other antibiotics was found. This high-pressure liquid chromatographic ion-pairing assay is simple, accurate, inexpensive, and requires no extraction.

Analysis of oligosaccharides from heparin by reversed-phase ion-pairing high-performance liquid chromatography

An ion-pairing high-pressure liquid chromatography procedure was developed for analysis of mixtures of oligosaccharides generated by nitrous acid cleavage of heparin. Oligosaccharides were eluted from a Hi-Chrom 5S ODS (C18) column using mixtures of acetonitrile and buffers containing 40 m m ammonium phosphate and 1 m m tetrabutylammonium phosphate. Isocratic conditions were developed for optimal separation of a number of individual disaccharides and tetrasaccharides that were characterized previously ( M. J. Bienkowski and H. E. Conrad (1985) J. Biol. Chem. 260, 356–365 ). These isocratic conditions were then coupled to obtain gradient elution conditions for the ion-pairing separations of mixtures of disaccharides and mixtures of tetrasaccharides. A comparison of the elution profiles obtained in the ion-pairing chromatography procedure with profiles obtained by anion-exchange high-pressure liquid chromatography profiles showed markedly better overall resolution by the ion-pairing procedure. As a result of this improved resolution, the new procedure showed the presence of previously unidentified products in the heparin oligosaccharide mixtures.

Ovothiols, a family of redox-active mercaptohistidine compounds from marine invertebrate eggs.

We have previously reported a novel thiol compound, 1-methyl-N alpha,N alpha-dimethyl-4-mercaptohistidine, or ovothiol, present at high concentration in the eggs of the sea urchin Strongylocentrotus purpuratus [Turner, E., Klevit, R., Hopkins, P. B., & Shapiro, B. M. (1986) J. Biol. Chem. 261, 13056-13063]. Here we report two related compounds, 1-methyl-N alpha-methyl-4-mercaptohistidine, or ovothiol B, from the scallop Chlamys hastata, and 1-methyl-4-mercaptohistidine, or ovothiol A, from the starfish Evasterias troschelii. These two compounds, as well as the S. purpuratus compound now designated ovothiol C, were isolated from eggs or ovarian tissue by S-carboxymethylation with [3H]iodoacetic acid, ion-exchange chromatography and ion-pairing high-pressure liquid chromatography. The structures of S-(carboxymethyl)ovothiols A and B were determined by 1H NMR, and that of ovothiol A was confirmed by comparison with authentic methylhistidine samples after desulfuration with Raney nickel. In the ovary of each species, the predominant methylation form of ovothiol accounts for at least 80% of the total 4-mercaptohistidine. The ovothiol concentration of the ovary far exceeds that of the testis or somatic tissues. The ovothiol C content of unfertilized S. purpuratus eggs is 1.14 mumol/10(6) eggs, equivalent to approximately 4.3 mM average concentration; the glutathione (GSH + GSSG) content is 0.9 mumol/10(6) eggs. In this species, high ovothiol levels persisted for the first 2 weeks of embryonic development. Ovothiol and glutathione account for virtually all of the trichloroacetic acid soluble-SH groups in the egg; these results are compared to several previous studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Polyamine metabolism in filarial worms

The human and animal filarial parasites Onchocerca volvulus, Dirofilaria immitis, Brugia patei and Litomosoides carinii contained low levels of putrescine but much higher levels of spermidine and spermine as estimated by ion-pair high pressure liquid chromatography; N-acetylated polyamines were present only in minute amounts. Enzyme activities of ornithine decarboxylase (EC 4.1.1.17) and arginine decarboxylase (EC 4.1.1.19), respectively, were not detectable. Experiments carried out with O. volvulus and D. immitis demonstrated the uptake and bioconversion of labeled polyamines. There is evidence for the existence of a complete reverse pathway generating putrescine from spermidine and spermine, respectively, in both worms. N-Acetylating enzyme activities were detected in 100 000 × g preparations of homogenates from D. immitis which were capable to acetylate putrescine, spermidine and spermine. Long term incubation of the worms in the presence of labeled polyamines resulted in the excretion of putrescine and N-acetylputrescine.

Determination of molybdenum, chromium and vanadium by ion-pair high-pressure liquid chromatography based on precolumn chelation with 4-(2-pyridylazo)resorcinol

A reversed-phase ion-pair liquid chromatography-spectrophotometric detection system for the separation and simultaneous determination of molybdenum, chromium and vanadium is described. The chelates of the metal ions with 4-(2-pyridylazo)resorcinol are separated on a Zorbax CN column with 1 × 10 −3 M tetrabutylammonium iodide and 0.01 M KH 2PO 4-Na 2HPO 4 buffer (pH 7.50) in 30:70 v v methanol-water mixture as the mobile phase, at a flow-rate of 1.0 ml min . The chelates are detected spectrophotometrically at 540 nm.

Occurrence of adenosine 2′,5′-bisphosphate in rat liver

Adenosine 2′,5′-bisphosphate (pAp) is present in liver from 2-day-fasted rats, at a concentration of around 1 μM. pAp was obtained through perchloric acid extraction of the liver followed by two successive DEAE-cellulose chromatographies and an ion-pair high-pressure liquid chromatography. Both pAp extracted from liver and that obtained from a commercial source showed the same pattern of hydrolysis by alkaline phosphatase, i.e., more 5′-AMP than 2′-AMP was obtained as an intermediate of the reaction.

Identification of N alpha-acetyl-alpha-endorphin and N alpha-acetyl-gamma-endorphin isolated from the neurointermediate lobe of the rat pituitary gland.

The peptides that represent the major components with alpha-endorphin- and gamma-endorphin-like immunoreactivity in the rat neurointermediate lobe were purified to homogeneity and chemically characterized. Rat neurointermediate lobes were extracted by boiling and homogenization in acetic acid. Peptide purification was based on gel filtration, followed by two high-pressure liquid chromatography steps. Pools containing peptides with the size and immunochemical properties of alpha- and gamma-endorphins were resolved by reverse-phase high-pressure liquid chromatography into multiple immunoreactive components. The major forms were finally purified by paired-ion high-pressure liquid chromatography. The amino acid compositions of these peptides fitted the beta-endorphin sequences 1-16 and 1-17. Tryptic mapping, aminopeptidase M digestion, chromatographic characterization, and immunoreactivity to an antiserum recognizing the N alpha-acetylated terminus of endorphins showed that these peptides were indistinguishable from N alpha-acetyl-alpha-endorphin (N alpha-acetyl-beta-endorphin 1-16), and N alpha-acetyl-gamma-endorphin (N alpha-acetyl-beta-endorphin 1-17). The NH2-terminal residue of the peptides was identified by mass spectrometry as N alpha-acetyltyrosine, substantiating the identity of the peptides. The results demonstrate the existence of N alpha-acetylated alpha- and gamma-endorphin as endogenous peptides in the neurointermediate lobe of the rat pituitary gland. In view of their occurrence and biological properties they should be considered significant members of the pro-opiomelanocortin family.

Characterization of aminosaccharins in commercial sodium saccharin produced by the Maumee process

The most abundant polar impurities detected by ion-pair high-pressure liquid chromatography of sodium saccharin produced by the Maumee process were identified as 5- and 6-aminosaccharin. The combined levels of these compounds in the samples analysed ranged from 99 to 152 mg/kg. Other less abundant polar impurities identified in Maumee process sodium saccharin were 7-aminosaccharin and o-sulphamoylbenzoic acid. Mutagenicity assays of 5-aminosaccharin, 6-aminosaccharin and a polar impurity fraction isolated from Maumee saccharin, gave negative results.

The Separation of Bilirubin, Photobilirubin and their Major Isomers by Ion-Pair High Pressure Liquid Chromatography

Abstract The separation of the three major isomers and the main photoproducts of bilirubin by Ion-Pair High Pressure Liquid Chromatography is described. Chromatographic procedures found in the literature proved difficult to reproduce, had unacceptably long elution times (over 2 hours), required complex gradients or two or more columns. As these reports provided no data to show why specific chromatographic conditions were chosen an optimization study was carried out. The separation is shown to be dependent on pH, organic content and buffer concentration of the mobile phase, ion-pairing agent concentration and injection solvent. A separation, carried out in less than thirty minutes on octadecyl silica using isocratic conditions with detection at 455 nm is demonstrated. This technique will be used in the elucidation of the mechanism involved in phototherapy treatment of neonatal jaundice.

Analysis of penicillin N ring expansion activity from Streptomyces clavuligerus by ion-pair high-pressure liquid chromatography.

An ion-pair, reversed-phase, high-pressure liquid chromatographic method for the analysis of penicillin N ring expansion activity has been developed which allows simultaneous measurement of both substrate and product. The high-pressure liquid chromatography conditions were as follows: stationary phase, C18; flow rate, 2 ml/min; detection, 220 nm. The stationary phase was preconditioned with 4.5 mM tetrabutylammonium bromide in 0.05 M KH2PO4 (pH 4.0)-methanol (85:15, vol/vol) and then equilibrated with 0.06 mM tetrabutylammonium bromide in 0.05 M KH2PO4 (pH 4.0)-methanol (95:5, vol/vol) for analysis of reaction mixtures. These conditions separated authentic samples of penicillin N and desacetoxycephalosporin C and allowed cell-free studies of the ring expansion of penicillin N to desacetoxycephalosporin C by a partially purified enzyme from Streptomyces clavuligerus to be followed conveniently.

Leukotriene formation by eosinophil leukocytes. Analysis with ion-pair high pressure liquid chromatography and effect of the respiratory burst.

In this paper, we describe ion-pair reverse-phase high pressure liquid chromatography, a novel, high-resolution method for the separation of leukotrienes. Using this technique, we have studied the production and release of leukotrienes from purified horse eosinophil leukocytes following stimulation with the ionophore A23187. At least 11 different metabolites with spectroscopical characteristics of leukotrienes were resolved. Four of them exhibited the biological activity of the slow-reacting substance an LTC 4/LTD 4 spectra with absorption maxima at 278/281 nm. The other metabolites were virtually devoid of slow-reacting substance activity and exhibited LTB 4 spectra with maxima at 269/271 nm. Since ionophore A23187, under our experimental conditions, induced a respiratory burst in the eosinophils, two modifies of this response, 2-deoxyglucose, an inhibitor of glucose metabolism and catalase, a scavenger of hydrogen peroxide, were used to investigate the influence of the burst on leukotriene generation. An overall inhibition of leukotriene release was induced by 2-deoxyglucose. Catalase strongly decreased the formation of LTB 4 and its stereoisomers and correspondingly enhanced the formation of LTC 4 and LTD 4. On the other hand, the increase of glucose in the medium augmented the production of B4-type leukotrienes while decreasing that of LTC 4 and LTD 4. These results indicate that the respiratory burst is involved in leukotriene synthesis by eosinophil leukocytes.

Benzidine-congener-based azo dyes: assays for purity and residues in feces from dosed rats.

Analytical methods were required to determine purities of benzidine-congener-based azo dyes and residues of the intact dyes in feces from rats before valid metabolism studies of such compounds could be conducted. A procedure is described for purity assays based on reduction of the dyes with stannous chloride followed by gas chromatography of the released free amine. Sixteen different samples of commercial dyes based on three benzidine congeners were assayed; purities ranged from 26.4 to 83.4%. Several dyes were also shown to be partially purified by cold water washes. A method to determine two intact dyes in feces from dosed rats, which consisted of extraction with dimethylformamide: water, clean-up by a rapid procedure using an octadecylsilane column, and quantification by ion-pair high-pressure liquid chromatography (HPLC) is reported. minimum detectable levels of both dyes in feces are 0.2 ppm. Excretion profiles based on parallel HPLC and radioassays of feces from rats dosed with 14C-labeled Direct Blue 15 and Direct Red 2 are presented. Based on radioassays, about 74% of each dose was excreted via the feces; however, HPLC assays showed that only about 11% of each dose was present as intact dye in the excrement.

A procedure for rapid extraction and high-pressure liquid chromatographic separation of the nucleotides and other small molecules from bacterial cells

A method for the extraction and separation of ribo- and deoxyribonucleosides and nucleotides from Salmonella typhimurium and Escherichia coli is described. Cells are harvested by a new rapid filtration method, and the small molecules are extracted with formic acid. The acid-extracted nucleotides and other uv-absorbing compounds are separated by boronate-affinity chromatography and by reverse-phase and ion-pair high-pressure liquid chromatography. The complete analysis uses the extract from 10 ml of log phase cells and separates the small molecule pool into about 170 components.