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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.
Unusual compound of small molecular weight in the serum of horses with acute grass sickness. An unusual compound of small molecular weight has been detected in serum from horses with acute grass sickness by solvent extractions and thin-layer chromatography. The substance has not been detected in the serum of normal horses or cases of equine colic and apparently disappears from grass sickness serum after two to three days of clinical illness. Although this compound is found in sera which are known to possess in vivo neurotoxicity, this property could not be demonstrated in either the total chloroform extract which contains the compound or in the hydrophilic serum components remaining a...
Immunologically reactive proteins of Streptococcus equi. Immunologically reactive proteins in acid extracts and culture supernatants of Streptococcus equi were recognized through a combination of chromatographic and immunologic procedures. Both high- and low-molecular-weight components of each of these protein preparations were protective for mice and were, therefore, presumed to contain a variety of hydrolytic products or fragments of the M protein of S. equi. Convalescent horse sera that exhibited strong bactericidal activity for S. equi always reacted with polypeptides in the molecular weight range of 24,000 to 29,000, whereas preinfection sera d...
Cholesteryl sulfate: the major polar lipid of horse hoof. The lipids of horse hoof have been analyzed by quantitative thin-layer chromatography. The major components include cholesterol (37-40%), six groups of ceramides (10-15%), and cholesteryl sulfate (15-20%). Free fatty acids are abundant (15.8%) in the outer fully keratinized hoof, but are present at only low levels (3.1%) in the softer hyponychium. The material identified as cholesteryl sulfate was isolated by preparative thin-layer chromatography and characterized by a combination of chemical, chromatographic, and spectroscopic methods. The infrared spectrum of the isolated material had absorp...
Studies related to the metabolism of anabolic steroids in the horse: the phase I and phase II biotransformation of 19-nortestosterone in the equine castrate. The metabolism of 19-nor[4-14C]testosterone has been studied in the equine castrate. Following XAD-2 extraction of aliquots of the 0-24 h urine samples, the glucuronic acid and sulphate conjugates were separated by Sephadex LH-20 column chromatography. After hydrolysis of the conjugates, the neutral phase I metabolites of 19-nortestosterone were extracted, purified and identified by g.l.c.-mass spectrometry. In phase I metabolism stereospecificity was observed in the reduction of the A-ring with the formation of the 5 alpha, 3 beta-isomers of estranediol. Epimerization at C-17 and hydroxylatio...
Purification of lutropin and follitropin in high yield from horse pituitary glands. A method has been developed for the purification of equine lutropin (eLH) and equine follitropin (eFSH) from horse pituitary glands which attains high yields of both hormones in contrast to previous methods that were devoted to one or the other with inferior recovery of the hormones. Two-pass chromatography over CM-Sephadex was used to separate eLH from eFSH. Subsequent steps employing QAE (quaternary amino-ethyl)-Sephadex chromatography and gel filtration on Sephacryl S-200 produced highly purified hormone preparations. Yields of purified eLH and eFSH were 110 and 60 mg/kg of frozen pituitari...
Affinity chromatographic purification of horse muscle acylphosphatase: evidence of the existence of multiple molecular forms. Acylphosphatase was purified from horse muscle by a new procedure involving an affinity chromatography step and subsequent ion-exchange chromatography. This procedure was considerably milder than the preceding one, gave an overall yield of about 60% of activity and permitted isolation of three molecular forms with acylphosphatase activity. All these enzymatic forms are tightly bound to Sepharose 4B-linked anti-horse muscle acylphosphatase antibodies. Two of these forms (Ho1 and Ho3) are present in larger amounts: Ho1 corresponds to the enzyme purified according to the older procedure; this enz...
Identification of 3 beta-hydroxy-5,7-pregnadien-20-one and 3 beta-hydroxy-5,7-androstadien-17-one as endogenous steroids in the fetal horse gonad. The 5,7-dienes, 3 beta-hydroxy-5,7-pregnadien-20-one and 3 beta-hydroxy-5,7-androstadien-17-one were extracted from fetal horse gonads and purified by solvent partition, thin-layer chromatography and high performance liquid chromatography. The isolated steroids were identified by comparison with the synthetic steroids using ultraviolet and mass spectroscopy and by gas chromatography-mass spectroscopy. The identification of these compounds as endogenous steroids, together with the data on their biosynthesis reported previously, support the proposal that in the fetal horse gonad there is a 5,7-d...
High-performance liquid affinity chromatography on silica-bound alcohol dehydrogenase. Horse liver alcohol dehydrogenase was immobilized on glycerylpropyl-silica (10 micron, 1000-A pores) activated with 2,2,2-trifluoroethanesulfonyl chloride (tresyl chloride). The coupling and activity yield was almost 100%. The coenzyme-binding sites were equivalent and virtually unaffected by the immobilization process, as judged from Scatchard plots and active-site titrations. The silica-bound enzyme, packed in steel columns, was integrated with HPLC equipment and then successfully used for chromatography of adenine nucleosides, adenine nucleotides, and triazine dyes. Dissociation constants w...
The metabolism of fenclofenac in the horse. 14C-Fenclofenac (2-(2'-4'-dichlorophenoxy)-phenylacetic acid) was administered orally to horses, and urinary metabolites investigated by chromatography. Fenclofenac was rapidly absorbed and eliminated, with a plasma half-life (t1/2) of 2.3 h, with 83.2 and 85.8% of the dose being recovered in the urine in 0-24 h. The major urinary metabolite was the ester glucuronide (58.8, 70.0% dose), and evidence is presented that this metabolite undergoes a structural rearrangement to give beta-glucuronidase-resistant isomers. The other 14C-labelled components in horse urine were unchanged fenclofenac (13....
3-Hydroxy- and 3-keto-3-phenylpropionic acids: novel metabolites of benzoic acid in horse urine. The metabolism of benzoic acid has been examined in the horse, using 14C- and deuterium-labelled compounds. Chromatographic analysis of the urine showed the presence of hippuric acid, benzoyl glucuronide and benzoic acid and a discrete band which accounted for 2% of the dose administered. This material was isolated by solvent extraction and HPLC and, following treatment with diazomethane, examined by GC/MS. The major component of this fraction was 3-hydroxy-3-phenylpropionic acid methyl ester, which was accompanied by very much smaller amounts of cinnamic acid methyl ester and acetophenone. Th...
Isolation and identification of steroids from gonadal vein blood of the fetal horse. Direct connection of the artery of a fetal ovary to the carotid artery of the mare allowed collection of a large volume of blood over a 30-min period. Extraction of steroids and their fractionation was followed by separation of the steroids by alumina adsorption chromatography, and Sephadex LH-20 and Celite partition chromatography. Further resolution of the material by HPLC led to the identification of dehydroepiandrosterone (DHA) by nuclear magnetic resonance and mass spectrometry. Other compounds were isolated, which remain to be identified fully, but in the 8th month of pregnancy the princ...
Furosemide, Patella vulgata beta-glucuronidase and drug analysis: conditions for enhancement of the TLC detection of apomorphine, butorphanol, hydromorphone, nalbuphine, oxymorphone and pentazocine in equine urine. We have investigated the action of five sources of beta-glucuronidase enzymes on the hydrolysis of glucuronides of apomorphine, butorphanol, hydromorphone, nalbuphine, oxymorphone and pentazocine in equine urine. For all glucuronides tested, Patella vulgata beta-glucuronidase yielded the largest thin layer chromatographic (TLC) spots. For oxymorphone, P. vulgata was the only treatment to yield detectable TLC spots under test parameters. For these six drugs, TLC spot size and chromatographic quality were compared between control horses and horses pretreated with furosemide four hours earlier. F...
[Gangliosides of neural and extraneural tissues of various species of mammals]. The ganglioside patterns of the forebrain, cerebellum and brain stem from horse, donkey, mule and goat have been determined by thin-layer chromatography. GM1, GD1a, GD1b and GT1b are the four major brain gangliosides. N-acetylneuraminic acid as the predominant sialic acid (congruent to 97%) and traces of N-glycolyneuraminic acid were found. The four above mentioned major gangliosides were also found in the forebrain, cerebellum and brain stem of adult rats. This pattern is not modified in rats under stress situation (at 4 degrees C for 3 months). In other extraneural organs from rats such as l...
Gas/liquid chromatographic analysis of pemoline in biological fluids using electron capture detection. An analytical gas/liquid chromatographic (GLC) protocol is described for the quantitation of pemoline in biological fluids of the horse. Plasma samples containing known quantities of pemoline and its analog as an internal standard (IS) were deproteinized with 5-sulfosalicylic acid, heated at 80 degree C, and centrifuged. 5-Phenyl-2,4-oxazolidinedione, the hydrolytic product of pemoline in acid medium, was extracted with dichloromethane (DCM). The organic layer was in turn re-extracted with 1% NaHCO3. The aqueous layer was acidified with HCI, and re-extracted with DCM, which was evaporated to d...
Metabolic conjugation of some carboxylic acids in the horse. 1. 14C-Labelled benzoic acid, salicylic acid and 2-naphthylacetic acid were administered orally to horses, and urinary metabolites investigated by chromatographic and mass spectral techniques. 2. [14C]Benzoic acid (5 mg/kg) was eliminated rapidly in the urine, and quantitatively recovered in 24 h. The major urinary metabolite was hippuric acid (95% of dose) with much smaller amounts of benzoic acid, benzoyl glucuronide and 3-hydroxy-3-phenylpropionic acid. Administration of [ring-D5]benzoic acid together with [14C]benzoic acid to a pony permitted the mass spectral determination of metabolites ...
Studies related to the metabolism of anabolic steroids in the horse: the identification of some 16-oxygenated metabolites of testosterone and a study of the phase II metabolism. 1. Isomers of 3,17-dihydroxyandrostan-16-one, 3,16-dihydroxyandrostan-17-one and androstane-3,16,17-triol have been identified as urinary metabolites of testosterone in the horse. 2. Following XAD-2 extraction of urine samples, Sephadex LH-20 chromatography was used to separate the extract into conjugate groups. Metabolites obtained after hydrolysis of the conjugates have been investigated by g.l.c.-mass spectrometry. 3. Testosterone, 3,17-dihydroxyandrostan-16-one and 3,16-dihydroxyandrostan-17-one were found only in the sulphate fraction. 5 alpha-Androstane-3 beta,17 beta-diol, and two isome...
Fractionation and partial characterization of alpha-1-protease isoinhibitors of horse. The principal alpha-1-protease inhibitor of horse was fractionated by classical methods and analysed with a modified fibrinogen-agarose gel electrophoretic method of high sensitivity and resolving power. Starting with an electrophoretically homogeneous inhibitor in unfractionated serum, two isoinhibitor bands became apparent after fractionation with (NH4)2SO4 and DEAE-cellulose DE-52 ion-exchange chromatography. The isoinhibitors differed in electrophoretic migration and in the elution pattern from Sephadex G-100 gel filtration, but possessed identical antigenic determinants and enzyme specifi...
Horse erythrocyte gangliosides: preparation of the major hematoside NeuNG1-Lac-Cer. A simple method for the isolation of hematoside NeuNG1-Lac-Cer from horse erythrocytes is described. An aliquot of the crude ganglioside fraction was labeled by tritiated sodium borohydride after mild periodate oxidation. The compounds obtained were used as radioactive tracers in column chromatography. Gangliosides were applied onto a silicic acid column and eluted stepwise by solvents of steadily increasing polarity. The major ganglioside, NeuNG1-Lac-Cer, was eluted in a high yield by the solvent mixture chloroform/methanol/water (60:35:8, v/v/v).
Purification by affinity chromatography and characterization of a neutral alpha-glucosidase from horse kidney. A horse kidney neutral alpha-D-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) was purified about 580-fold with a yield of 33% by an affinity chromatography technique using the p-aminophenyl-beta-D-maltoside, a substrate derivative, as ligand. The purified enzyme, homogeneous in polyacrylamide gel electrophoresis, was a glycoprotein with a molecular weight of 280 000 as calculated by gel filtration and its isoelectric focusing points was found to be pH 4.1. The purified enzyme was able to hydrolyze various substrates having (alpha-1,2), (alpha-1,3), (alpha-1,4), and (alpha-1,6) glu...
A new method for the isolation of aminoacylase from mammalian kidneys. Aminoacylase (E.C. 3.5.1.14) was isolated from the kidneys of different mammalian species (horse, cattle, rabbit and pig) by extracting the organ with water and subjecting the extract to heat treatment at 70 degrees C for 10 min, then, after having removed denatured proteins by fractionating those remaining in the solution by ammonium sulfate. The enzyme obtained in this way can either be used directly for practical purposes (e.g. preparation of immobilized aminoacylase) or further purified by chromatography. For the further purification of porcine kidney aminoacylase we applied a combination ...
Purification of horse renal kallikrein and chemical relations with horse urinary kallikrein. Kallikrein was purified from horse kidney by several steps of chromatographic procedure and by affinity chromatography on Sepharose-Concanavaline. Horse urinary kallikrein was previously purified by DE-32 hydroxylapatite and by Sephadex G-100 gel filtration. On the purified final sample of renal and urinary kallikrein the aminoacid composition and the gel electrophoretic molecular weight were determined. The ratio in micronMoles between each aminoacid residue of both hydrolyzed renal and urinary kallikrein of horse is about 1,00 +/- 0,30. Except for Pro, 1/2 Cys and basic aminoacid residues a ...
Physicochemical and biological characterizations of pregnant mare serum gonadotropin and its subunits. Pregnant mare serum gonadotropin and its subunits have been further characterized. Ultracentrifugation of the gonadotropin at pH 1.3 and 11.5 showed little evidence of dissociation compared to pH 8.2. Highly purified subunits are obtained by urea dissociation and ion-exchange chromatography followed by gel-filtration. Circular dichroism spectra of the gonadotropin and its subunits are much like those of ovine lutropin and its subunits in that there is little evidence for secondary structure and one or more tyrosine residues are inaccessible in the intact gonadotropin compared to the subunits. ...
Lactoperoxidase-catalyzed iodination of horse cytochrome c:monoiodotyrosyl 74 cytochrome c. Iodination of horse cytochrome c with the lactoperoxidase-hydrogen peroxide-iodide system results initially in the formation of the monoiodotyrosyl 74 derivative. This singly modified protein was obtained in pure form by ion exchange chromatography and preparative column electrophoresis. It shows an intact 695 nm absorption band, the midpoint potential of the native protein, a nuclear magnetic resonance spectrum which indicates an undisturbed heme crevice structure, a normal reaction with antibodies directed against native horse cytochrome c, and circular dichroic spectra in which the only cha...
Studies related to the metabolism of anabolic steroids in the horse: 19-nortestosterone. 1. The metabolism of 19-nortestosterone in a cross-bred horse has been studied using 14C-labelled material. 2. Two neutral metabolites isolated from urinary extracts by column chromatography were identified as isomers of 3-hydroxyestran-17-one and estrane-3,17-diol by g.l.c.-mass spectrometry. 3. The stereochemistry of the two metabolites has been investigated by comparison of the retention times of their trimethylsilyl derivatives with those of standard steroids of known configuration.
The antidoping control in horseraces in Italy. The results and the improvement of the analytical procedures adopted for the control of doping in horses will be reported. This control has been systematically carried out in Italy for about 10 years in the laboratories of Italian Federation of Sport and Medicine in which the biological samples for the control of doping in various sport activities (football, cycling, athletics etc.) are also examined. In this way it is possible to use the same instruments for all these similar problems and compare the results. The analytical procedure is based on the following steps: 1) Extraction of the sampl...
Doping control in Japan. An automated extraction procedure for the doping test. Horse racing in Japan consists of two systems, the National (10 racecourses) and the Regional public racing (32 racecourses) having about 2,500 racing meetings in total per year. Urine or saliva samples for dope testing are collected by the officials from thw winner, second and third, and transported to the laboratory in a frozen state. In 1975, 76, 117 samples were analyzed by this laboratory. The laboratory provides the following four methods of analysis, which are variously combined by request. (1) Method for detection of drugs extracted by chloroform from alkalinized sample. (2) Methods fo...
[Preparative isolation of alpha 2-macroglobulin, transferrin, albumin and study of their nonspecific gamma-inhibitory activity]. Profiles of distribution of non-specific gamma-inhibitors of influenza A2/Victoria/35/72 in donkey and horse sera were established by gel chromatography in Sephadex G-200. High and low molecular inhibitors were found in 19S and 4S serum fractions. Highly purified preparations of a2-macroglobulin, transferrine and albumin were isolated by a combination of methods of salt precipitation, gel chromatography on Sephadex G-100, G-200 and ion exchange on DEAE-Sephadex A-50. Heating sera resulted in a considerable increase of the antiviral activity of a2-macroglobulin and transferrine and a reduction ...
N-acetylserine in horse muscle acylphosphatase. A ninhydrin-negative peptide fraction obtained from tryptic digest of carboxymethyl acylphosphatase was isolated by chromatography on a column of PA 28 Beckman resin and analysed for the amino acid composition. Degradation with carboxypeptidase B and A indicated that the sequence of this peptide was: X-Thr-Ala-Arg. The amino-terminal residue was identified as N-acetylserine by high voltage electrophoresis. It is therefore suggested that the sequence of the NH2-terminal portion of CM-acylphosphatase is N-acetyl-Ser-Thr-Ala-Arg. Digestion with carboxypeptidase A and B indicated also that the COO...
