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Topic:Chromatography
Chromatography is an analytical technique used to separate, identify, and quantify components within a mixture. In equine research, chromatography is applied to study various biological samples, such as blood, urine, and tissue, to investigate the metabolic and physiological processes in horses. This technique is instrumental in detecting and analyzing compounds like hormones, drugs, and nutrients, providing insights into equine health and performance. Different types of chromatography, including gas chromatography (GC) and high-performance liquid chromatography (HPLC), are utilized to achieve precise and reliable results. This page compiles peer-reviewed research studies and scholarly articles that explore the applications, methodologies, and findings of chromatography in equine science.
Purification of a plasminogen activator from Streptococcus uberis. A protein capable of activating bovine, equine and ovine plasminogen, but not that from human or porcine plasma, was purified from culture filtrates of Streptococcus uberis (strain 0140J). Purification was achieved by ammonium sulphate precipitation followed by molecular exclusion chromatography. The elution position of the native molecule was equivalent to a molecular mass of approximately 57 kDa. However, the molecular mass, as determined by SDS-PAGE, was 29 kDa, suggesting the existence of a dimeric structure. Purified immunoglobulin from three out of five monoclonal antibodies raised to th...
Molecular entrapment of small molecules within the interior of horse spleen ferritin. A procedure for trapping small molecules inside the interior of horse spleen ferritin (HoSF) and methods for characterizing HoSF and its small entrapped molecules are described. HoSF is first dissociated into subunits by adjustment to pH 2 in the presence of the small molecules to be trapped. The pH of the dissociated HoSF is then increased to 7 at which time the dissociated subunits reassemble reforming the 24-mer HoSF, thereby trapping solvent within its interior. HoSF is then separated from unbound molecules by dialysis, ultrafiltration, and/or ammonium sulfate precipitation. Sephadex G-25 ...
Comparison of immunoaffinity chromatography combined with gas chromatography-negative ion chemical ionisation mass spectrometry and radioimmunoassay for screening dexamethasone in equine urine. A comparison of the sensitive analytical methods of radioimmunoassay (RIA) and immunoaffinity chromatography (IAC) combined with gas chromatography-negative ion chemical ionisation mass spectrometry for the specific and reliable screening of dexamethasone in equine post-race urine is presented. Results from analyses of samples collected from a mare during 144 hours post administration of 26 mg of dexamethasone sodium phosphate are described.
Comparative studies of the Spi1 proteins of three equine alpha-1-proteinase inhibitor haplotypes following isolation by affinity chromatography. 1. Antiproteinase deficiency can result in excessive proteinase-induced tissue damage. The major anti-elastase (Spi1) protein of equine alpha 1-proteinase inhibitor (alpha 1-PI) was isolated from the plasma/serum of three common haplotypes (I, L and U). 2. The N-terminal amino acid sequences of the three inhibitors were identical, but were only approx 65-77% homologous with two other published equine Spi1 sequences. 3. All three inhibitors complexed quickly and irreversibly with equine leucocyte proteinase 2A (kass = 2 x 10(7) M-1 sec-1). They were also efficient inhibitors of chymase (rat mas...
Horse-liver glutathione reductase: purification and characterization. 1. Purification of horse-liver glutathione reductase was obtained by affinity chromatography on N6-(6-aminohexyl)-adenosine-1'5'-bisphosphate Sepharose (N6-2'5'-ADP-Sepharose) and Reactive Red-120-Agarose, and chromatography on DEAE-Sephadex and Sephacryl S-300. 2. The final preparation had 248 U/mg specific activity after 11,174-fold purification with 47% final recovery, and was homogeneous by SDS-electrophoresis. It showed charge heterogeneity in non-denaturing electrophoresis and chromatofocusing, with several peaks of pI between 5.7 and 6.7. 3. The enzyme was homodimeric (107,000 native MW...
Insulin-like growth factor binding proteins of equine serum. Ligand blotting analysis of serum from the horse using radiolabelled IGF-I revealed a protein at 96 kDa which was not present in serum from goat, cow, sheep, deer or donkey. These latter species all displayed five labelled bands in the range 24 to 41 kDa. Conversely, these were only weakly labelled in serum from the horse. Size exclusion chromatography of horse serum pre-incubated with radiolabelled IGF-I revealed reduced binding in the 130-kDa peak compared with goat plasma, and ligand blotting analysis indicated the 96-kDa protein was present in this peak. The 96-kDa protein from horse serum...
Isolation of a major form of pepsinogen from gastric mucosa of horses. In mammalian species studied previously, pepsinogen consisted of biochemically different groups of isozymogens. By use of gel filtration chromatography and electrophoresis, we isolated a predominant pepsinogen from the gastric mucosa of a horse. Peptide mapping with V8 protease revealed differences with its porcine homologue. However, porcine and equine pepsinogens, when activated to pepsin, had a similar pattern of activity when hemoglobin was used as substrate. Those results suggest that differences must exist in the primary structure of the pepsinogens of the 2 species.
Characterization of bromhexine and ambroxol in equine urine: effect of furosemide on identification and confirmation. The purpose of this study was two-fold: (1) to develop a simple and sensitive screening procedure for identifying and confirming bromhexine and ambroxol and, (2) to determine the effect of furosemide on the detection of bromhexine, ambroxol, or their metabolites in urine. Female horses (450-550 kg) treated with bromhexine or ambroxol (1 g, p.o.) were used. Urine samples were collected up to 48 h post-drug administration and analysed. Blind samples were used in evaluating the sensitivity of these methods and reproducibility of the results. Bromhexine and ambroxol were extensively metabolized in...
Chemical structures of three neutral oligosaccharides obtained from horse (thoroughbred) colostrum. 1. Three neutral oligosaccharides were obtained from horse colostrum by ion-exchange, activated charcoal column and preparative paper chromatographies. 2. The following structures were elucidated by methanolysis, methylation analysis and 75 MHz 13C-NMR spectroscopy; Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc (HM-3a), Gal beta 1-4GlcNAc beta 1-6Gal beta 1-4Glc (HM-3b) and Gal beta 1-4GlcNAc beta 1-3[Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4Glc (HM-5). 3. HM-3a and HM-5 have been found in human milk, named as lacto-N-neotetraose and lacto-N-neohexaose, respectively. HM-3b has been isolated from g...
Production of fumonisins by Fusarium moniliforme and Fusarium proliferatum isolates associated with equine leukoencephalomalacia and a pulmonary edema syndrome in swine. Fumonisin B1 (FB1) and FB2 were isolated from corn cultures of both Fusarium moniliforme and Fusarium proliferatum. Respective concentrations in culture materials of FB1 and FB2 ranged from 960 to 2,350 and 120 to 320 micrograms/g for F. moniliforme and from 1,670 to 2,790 and 150 to 320 micrograms/g for F. proliferatum. Thin-layer chromatography, gas chromatography-mass spectroscopy, high-performance liquid chromatography, and liquid secondary ion mass spectroscopy were used for detection. Fumonisins from F. proliferatum have not previously been reported.
Isolation, characterization, and quantitative analysis of C-reactive protein from horses. C-reactive protein (CRP) was isolated from equine serum by use of calcium-dependent affinity chromatography conjugated pneumococcal C-polysaccharide, anion exchange chromatography, and gel filtration. It was identified as genuine CRP by its immunochemical cross-reactivity with anti-human CRP, its homology with human CRP in amino acid composition, and its pentameric structure as revealed by electron microscopy. Purified equine CRP had a molecular weight of approximately 118,000 and was composed of 5 identical, nonglycosylated and noncovalently associated subunits with molecular weight of approx...
Comparison of the use of mass spectrometry and methylene unit values in the determination of the stereochemistry of estranediol, the major urinary metabolite of 19-nortestosterone in the horse. The stereochemistry of an isomer of 5-estrane-3,17 alpha-diol, the major metabolite of 19-nortestosterone in horse urine has been established by the use of methylene unit (MU) values. The empirical MU values of the bis-trimethylsilyl (TMS) derivatives of the eight available isomers of 5-androstane-3,17-diol and four isomers of 5-estrane-3,17 beta-diol were determined by capillary gas chromatography using three different columns. From this data the theoretical MU values for the bis-TMS derivatives of the four 5-estrane-3,17 alpha-diol isomers were predicted. Comparison of the experimentally det...
Comparative toxicity of the horse eosinophil peroxidase-H2O2-halide system and granule basic proteins. Stimulated eosinophils release cytotoxic granule constituents, including eosinophil peroxidase (EPO) and a group of granule basic proteins (GBP). EPO reacts with H2O2 formed by the respiratory burst and a halide to form cytotoxic oxidants. The relative potency of the EPO-H2O2-halide system and the GBP is considered here. Horse eosinophils were induced to degranulate, the degranulation products were separated by chromatography on Sephadex G-50 and comparable volumes of the column fractions were tested for toxicity to Escherichia coli and the schistosomula of Schistosoma mansoni in the presence ...
Steroids in equine testes: the identification of endogenous 19-hydroxy and 19-nor neutral steroids by gas chromatography–mass spectrometry. After homogenization of testicular tissue from stallions aged 1, 2 and 5 years, the unconjugated and conjugated steroids were isolated by a combined solvent-solid extraction procedure. The conjugates were further separated into glucuronides and sulphates by chromatography using Sephadex LH-20. After enzyme hydrolysis and solvolysis of the respective conjugate classes, the three extracts, unconjugated steroids, aglycones and solvolysed sulphates, were purified by chromatography using Kieselgel 60H columns. Five fractions were resolved from each extract; an aliquot of each fraction was derivatiz...
A growth-promoting factor for human myeloid leukemia cells from horse serum identified as horse serum transferrin. A growth-promoting factor for human myeloid cells was purified to apparent homogeneity from horse serum by a combination of gel filtration, blue Sepharose affinity chromatography, Mono Q anion-exchange chromatography, Mono P chromatofocusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The growth promoter was an iron-bound, single glycopolypeptide chain with a molecular weight of 84,000, an isoelectric point of 5.4 and an amino terminal sequence of Glu-Gln-Thr-Val-Arg-Trp-Cys-Thr-Val-Ser-Asn-His-Glu-Val-Ser-Lys-. According to the results of the amino acid sequence, iron bindi...
Use of a monoclonal antibody in the diagnosis of infection by Dermatophilus congolensis. A monoclonal antibody (McAb) to Dermatophilus congolensis was produced from murine hybridoma cultures and purified by affinity chromatography. Species specificity was demonstrated using indirect immunofluorescent staining; the McAb was shown to react with 10 D congolensis isolates but not with 10 Nocardia species isolates, a Rhodococcus and a Streptomyces species isolate. The McAb was used to demonstrate D congolensis in clinical material from confirmed bovine and ovine cases and presumptive equine cases of dermatophilosis by indirect immunofluorescent staining.
Purification of specific heterologous F(ab)2 fragments with DEAE-Zeta-Prep cartridges for ion-exchange chromatography. A simple two-step procedure for purifying F(ab)2 fragments of horse immunoglobulins is described. In the first step, the horse plasma is diluted, made up to 12% (w/v) with ammonium sulphate and digested with pepsin. In the second step, the previously dialyzed solution is chromatographed. Instead of a normal ion-exchange resin, a DEAE-cellulose, covalently linked to a synthetic vinyl polymer, was used (DEAE-Zeta-Prep). With this assembly it is possible to perform chromatography at a high flow-rate without the problems related to the use of large columns. The yield and purity of the final produc...
Detection and identification of ketamine and its metabolites in horse urine. The possibility exists that ketamine, or ketamine in combination with xylazine, is being used illicitly to affect the performance of racehorses. This study was undertaken to identify the metabolites of ketamine in the urine of adult horses and to evaluate methods for detecting and confirming ketamine administration. Detection of ketamine and two ketamine metabolites is described using thin-layer chromatography (TLC) and their identities are confirmed by comparing their mass spectra and gas chromatographic retention times with those of authentic standards.
Covalently bound pyruvate in phosphopantothenoylcysteine decarboxylase from horse liver. Horse liver phosphopantothenoylcysteine decarboxylase (EC 4.1.1.36) incorporates nonexchangeable tritium from borotritide with a decrease of the activity. Substrate prevents both tritium incorporation and the decrease in activity. Acid and base hydrolysis of the tritiated protein releases labeled lactate identified by high-voltage paper electrophoresis, paper chromatography and silicic acid chromatography. These results indicate the presence of pyruvate covalently bound through an ester linkage to phosphopantothenoylcysteine decarboxylase which is then another example of a mammalian enzyme in ...
Acid-stable protease inhibiting polypeptides formed from denatured horse plasma by proteolysis. 1. Trypsin digestion of perchloric acid precipitated horse plasma yielded polypeptides with inhibitory properties for trypsin, chymotrypsin and, to a small extent, kallikrein. 2. The Mr of the inhibitory polypeptides were 73,000 and 24,000. 3. The number, enzyme specificity and Mr of the inhibitory polypeptides differed from the values known for the human being. 4. The inhibitory polypeptides were purified by affinity chromatography on Sepharose-trypsin and by gel filtration through Sephadex G-75. 5. Protease inhibitory polypeptides were generated in the same manner by chymotrypsin, elastase, ...
Purification of horse (Equus caballus) serum lecithin:cholesterol acyltransferase. 1. A method for the purification of horse serum lecithin:cholesterol acyltransferase has been established. 2. The method involves the adsorption of the enzyme from diluted horse serum on DEAE-Sephadex A-50, (NH4)2SO4 fractionation, 1-butanol treatment, and chromatographic techniques of DEAE-Sepharose CL-6B, DEAE-Sephadex A-50, Affi-Gel blue and hydroxylapatite. 3. The resultant enzyme preparation essentially formed a single main band when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. 4. The final purification of the enzyme was 20,000-fold with 7% yi...
Purification and characterization of equine relaxin. It has been previously determined that the equine placenta is the sole significant source of relaxin during pregnancy and that relaxin immunoactivity is also present in term placentas. Therefore, placentas obtained at the time of foaling were selected for starting material for purification of equine relaxin. Frozen whole placentas were ground and then extracted with 0.5 N HCl-85% acetone. Relaxin was precipitated by raising the acetone concentration to 97%. Equine relaxin was further purified by stepwise elution ion exchange, gel filtration, and gradient elution ion exchange chromatographies a...
Development of a gas chromatographic-mass spectrometric method using multiple analytes for the confirmatory analysis of anabolic steroids in horse urine. I. Detection of testosterone phenylpropionate administrations to equine male castrates. A gas chromatographic-mass spectrometric (GC-MS) method using three analytes to detect and confirm the administration to equine male castrates of veterinary pro-drugs based upon esters of testosterone is described. The method involves extraction of steroid conjugates from horse urine by C18-bonded cartridges and fractionation into glucuronic acid and sulpho-conjugates by Sephadex LH-20 column chromatography. After deconjugation, the free neutral steroids were partially purified by thin-layer chromatography and following derivatization (methyloxime-trimethylsilyl ether) were analysed by capilla...
Opiate-like and adrenocorticotrophin-like materials in equine pancreas. Equine pancreatic acetone powder was extracted with an acetone-water-HCl mixture. An acid acetone powder resulted by adding a copious volume of acetone to the extract. The powder was subjected to salt fractionation, gel filtration and chromatography on CM-cellulose. Steroidogenic activity, ACTH-like immunoreactivity and opiate receptor binding activity were distributed among the CM-cellulose chromatographic fractions derived from material unretarded as well as from material retarded on Sephadex G-25. The data indicates a separation of steroidogenic and opiate receptor binding activities, and t...
Isolation of meclofenamic acid and two metabolites from equine urine–a comparison between horse and man. Two metabolites of meclofenamic acid have been isolated from equine urine. Both metabolites are found to be monohydroxylated forms of meclofenamic acid by gas chromatography-mass spectrometry after extractive alkylation. The parent drug and the metabolites are separated by reversed-phase liquid chromatography on a Spherisorb ODS column, using methanol-phosphate buffer eluents and UV detection at 280 nm. The structure of the metabolites is discussed on the basis of LC, TLC and GC-MS data.
Lysosomal arylsulfatases A and B from horse blood leukocytes: purification and physico-chemical properties. Lysosomal arylsulfatases A and B (aryl-sulfate sulfohydrolases, EC 3.1.6.1) from horse leukocytes were purified about 680-fold and 70-fold, respectively, starting from a crude extract of the azurophil and specific granules of leukocytes, by affinity, ion exchange, and gel filtration chromatography. Purified arylsulfatase A displayed anomalous kinetics, a pH optimum at 5.2, an isoelectric point at 4.3, and a Km value for p-nitrocatechol sulfate (pNCS) of 0.37 mM. This enzyme was found to exist in two association states depending on pH: a high molecular weight form at pH 5.0 and a low molecular ...
Identification of betamethasone and a major metabolite in equine urine. Betamethasone and its major unconjugated metabolite, 6-beta-hydroxybetamethasone, were detected in equine urine by thin-layer chromatography and characterized by micro-liquid chromatography/mass spectrometry (micro-LC/MS). Their structures were confirmed by a combination of infrared spectroscopy and nuclear magnetic resonance spectroscopy.
Comparison of acetate and glucose incorporation into rat and horse skin lipids. The relative efficiency of acetate and glucose as substrates for the biosynthesis of lipids in the skin of the rat and horse was examined using in vivo pulse labelling of skin with [1-14C]acetate and [U-14C]glucose by intradermal injections. The resulting radiolabelled lipids were recovered in the rat by punch biopsy as well as by daily, long-term skin surface lipid collections and in the horse by punch biopsy of the injection sites. The lipids were examined by liquid scintillation and by a combination of thin-layer chromatography and autoradiography. In both species the recovery of radiolabel...
Identification of metabolites of methylprednisolone in equine urine. Methylprednisolone and three metabolites, 17,21-dihydroxy-6 alpha-methyl-1,4-pregnadiene-3,11,20-trione, 6 alpha-methyl-17,20 beta,21-trihydroxy-1,4-pregnadiene-3,11-dione, and 6 alpha-methyl-11 beta,17,20 beta,21-tetrahydroxy-1,4-pregnadien-3-one were detected in equine urine after intraarticular administration of methylprednisolone acetate. All four compounds were excreted both in the unconjugated form and as glucuronic acid conjugates. They were identified by comparing data obtained from analyses by high performance liquid chromatography, thin-layer chromatography, ultraviolet spectroscopy ...
Measurement of the time between biosynthesis and surface excretion of sebaceous lipids in the horse. The time between the biosynthesis and excretion of sebum to the skin surface of the horse was examined by in vivo intradermal injection of [1-14C]acetate followed by periodic surface lipid collections. The radiolabelling of the major neutral lipid classes, equolides (giant ring omega-lactones, C32-C36) and cholesteryl esters, was evaluated by thin-layer chromatography and autoradiography. The distribution of radioactivity within the monounsaturated equolides was examined by oxidative fragmentation and evaluation of the products. A peak of radioactivity in the equolides and cholesteryl esters o...
